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![• R. intestinalis is a small nonpathogenic flagellate found in
the large intestine.
• It also exists in two forms: (1) trophozoite, and (2) cyst.
• The trophozoite is elongated, pyriform in shape,
measuring 5- 10 mcm in length and 3-4 µmin breadth.
The cytoplasm is granular and vacuolated.
It has a cleft-like cytosome, spherical nucleus and
central karyosome.
- Two minute blepharoplasts are present near nucleus,
from which two flagella originate.
The trophozoite multiplies by binary fission.
• The cyst is ovoid or pyriform in shape, measuring 6 µm
in length and 3 µmin breadth.
• Water and food contaminated by cysts are the main
source of infection.
Diagnosis is made by identifying the cysts and trophozo-
ites in the direct wet mount and iron hematoxylin-stained
specimen ofstool.
• DIENTAMOEBA FRAG/L/S
D. Jragilis was previously considered as an amoeba but
has now been reclassified as an amoebojlagellate, based
on electron microscopic study and antigenic similarity to
Trichomonas.
• It is unique as it has only trophowile stage but no cyst
stage.
• The name Dien/amoeba Jragilis is derived from the
binucleate nature of trophozoite (Dien/amoeba) and the
fragmented appearance (fragilis) ofits nuclearchromatin.
• It is seen worldwide and is reported to be the most
common intestinal protozoan parasite in Canada.
• It lives in colonic mucosa] crypts, feeding on bacteria. It
does not invade tissues, but may rarely ingest red blood
cells (RBCs).
• The trophozoite is 7- 12 µm in diameter. It is motile
with broad hyaline leaf-like pseudopodia. They have
1-4 nuclei; the binucleace form being the most common
(Fig. 7). The nuclear chromatin is present as 3-5 granules
in the center, with no peripheral chromatin on the nuclear
membrane.
• In the absence of cyst stage, its mode of transmission
is not clear. Possibly, it is transmitted from person-to-
person by th e fecal-oral route or by the eggs ofEnterobius
vermicularis and other nematodes, which may serve as a
vector.
Formerly believed to be nonpathogenic, it has now been
associated with a variety of symptoms like intermittent
diarrhea, abdominal pain, flatulence, anorexia, nausea,
malaise a nd fatigue.
• High incidence is seen among children between 2 years
and 10 years of age.
Intestinal, Oral and Genital Flagellates
~ o l..• >.--~~lr-lngested
bacteria
' ...
Fig. 7: Trophozoite of Dientamoeba fragilis
Laboratory diagnosis is made by demonstration of
trophozoites in stool. At least three stool specimens
should be collected over a period of7 days.
Metronidazole, iodoquinol, paromomycin and tetracyc-
line have been used for treatment
REVIEW QUESTIONS
1. Describe briefly the life cycle and laboratory diagnosis of
Giardia Jamblia.
2. Write short notes on:
a. Trichomonas vagina/is
b. Dientamoeba fragilis
MULTIPLE CHOICE QUESTIONS
1. Normal habitat of Giardia is
a. Duodenum and jejunum
b. Stomach
c. Cecum
d. Ileum
2. All of the following protozoans are found in small intestine
except
a. Giardia lamblia
b. Balantidium coli
c. Cyclospora caytanensis
d. lsospora be/Ii
3. The following is true ofgiardiasis except
a. Feverand presence of blood and mucus in stool
b. Acute orchronic diarrhea
c. Duodenum and jejunum are the prime sites of involvement
d. Giardia cysts are resistant to dessication
4. Giardia lamblia was discovered by
a. Giard
b. Robert hook
c. Leeuwenhoek
d. L
osch](https://image.slidesharecdn.com/panikermedicalparasitology8e-210918093838/75/Paniker-medical-parasitology-8e-60-2048.jpg)
![Paniker'sTextbook of Medical Parasitology
Flow chart 2: Distribution and disease caused by Leishmania spp.
t
Leishmania I
I
•
Old world leishmaniasis
t
Visceral leishmaniasis
(Kala-azar)
L. donovanl complex
L. infantum
•
Cutaneous
leishmaniasis
L. Tropica complex
comprising
• L. tropica
• L. aethiopica
• L. major
Fig. 8: Geographical distribution of visceral leishmaniasis.
Endemic areas shaded; dots indicate sporadic cases
• Giemsa or Leishman-sL
ained films show pale blue
cytoplasm with a pink nucleus and bright red kinetoplast.
• Avacuole is present near the root of the flagellum.
• There is no undulating membrane.
• Promastigote forms, which develop in artificial cultures,
have the same morphology as in the sandtly.
Life cycle: l. donovani completes irs life cycle in two hosts
(Fig. 10):
1. Definitive host: Man, dog and other mammals.
2. Vector: Female sand.fly (Phlebolomus species) (Table 7).
Infective form: Promastigote form present in midgut of
female sandily.
Mode oftransmission:
• Humans acquire by bite ofan infected female sand.fly.
It can also be transmitted vertically from mother to fetus,
by blood transfusion and accidental inoculation in the
laboratory.
Incubation period: Usually 2-8 months, occasionally, it may
be as short as 10 days or as long as 2 years.
t
New world leishmaniasis j
I
•
Visceral leishmaniasis Cutaneous leishmaniasis
L. chagasi I
or mucocutaneous leishmaniasis
L. mexicana complex
L braziliensis complex
,__ _ __ Vacuole - - - ---ct't-
0 - -- 11--- Blepharoplast- - -r--v
c:::::.- - + -Parabasalbody- --!'-'~~,
Figs 9A and B: Morphology of Leishmania donovani. (A) Amastigote
[Leishman-Donovan (LO) body]; and (B) Promastigote
• The sandlly regurgitates the promastigotes in Lhe wound
caused by its proboscis.
• These are engulfed by the cells of reticuloendothelial
system (macrophages, monocytes and polymorphonu-
clear leukocytes) and change into amastigote (LD body)
within the cells.
The amastigote multiplies by binary fission, producing
numerous daughter cells that distend the macrophage
and rupture it. 1he liberated daughter cells are in turn,
phagocytosed by other macrophages and histiocytes.
Small number of LO bodies can be found in peripheral
blood inside neutrophils or monocytes (Fig. 10).
When a vector sandtly feeds on an infected person, the
amastigotes present in peripheral blood and tissue fluids
enter the insect along with its blood meal. In the midgut
(stomach) of the sandfly, the amastigote elongates and
develops into the promastigore form (Fig. 10).
The promastigore multiples by longitudinal binary fission
and reaches enormous numbers. They may be seen as
large roselles with their flagella entangled.](https://image.slidesharecdn.com/panikermedicalparasitology8e-210918093838/75/Paniker-medical-parasitology-8e-75-2048.jpg)
![remittent, or intermittent fever. True rigor is typically
presentin vivaxmalaria and is less common infalciparum
infection.
There can be both hypoglycemia or hyperglycemia in
malaria.
Sometimes, there may be hyperkalemia due to red cell
lysis and fall in blood pH.
infection with P. vivax usually follows a chronic course
with periodic relapses, whereas P. ovale malaria is
generallymild. Although P. malariaemalariaisless severe,
but it may lead to renal complications. Relapse mainly
occurs in inadequately treated cases after an interval of
8-40 weeks or more.
Malignant Tertian Malaria
incubation period: 8-14 days.
The most serious and fatal type of malaria is malignant
tertian malaria caused by PJalciparum. Falciparum malaria
if not treated timely or adequately, severe life-threatening
complications may develop. In severe Jalciparum malaria,
parasitic load is very high and more than 5% red cells are
affected. The term pernicious malaria also have been applied
to these conditions thar include cerebral malaria, blackwater
fever, algid malaria and septicemic malaria (Box 5).
• Cerebral malaria: It is the most common complication
ofmalignant malaria.
- The initial symptoms are nonspecific with fever,
headache, pain in back, anorexia and nausea.
- Anemia: The patient may be anemic and mildly
jaundiced.
- Hepatosplenomegaly: Liver and spleen are enlarged
and nomender.
Thrombocytopenia is common.
- After 4- 5 days of high fever, cerebral malaria is
manifested by features of diffuse symmetric
encephalopathy like headache, confusion, increased
muscle tone, seizures, paralysis, slowly lapsing to
coma.
Box 5: Complications of falciparum malaria
• Cerebral malaria
• Algid malaria
• Septicemic malaria
• Blackwater fever
• Pulmonary edema
• Acute renal failure
• Hypoglycemia (<40 mg/dl)
• Severe anemia (Hb<Sg/dl, PCV<l 5%)
• Hyperpyrexia
• Metabolic acidosis and shock
• Bleeding disturbances
• Hyperparasitemia.
Abbreviations: Hb, hemoglobin; PCV, packed cell volume
Malaria and Babesia
Retinal hemorrhages may be seen in 15% ofadults.
Hypoglycemia is common in patients following
quinine therapy or with) hyperparasitemia.
In 10% of cases renal dysfunction progressing to
acute renal failure may occur.
- Other complications include metabolic acidosis,
pulmonary edema and shock.
Even with treatment, death occurs in 15% ofchildren
and 20% ofadults who develop cerebral malaria.
This occurs particularly when nonimmune persons
have remained untreated or inadequately treated for
7-10 days after development ofthe primary fever.
- The basic pathogenesis of cerebral malaria is due to
erythrocyte sequestration in microvasculature of
various organs.
Late stage schizonts ofP.falciparum secrete a protein on
the surface ofRBCs to form knob-like deformities. This knob
produces specific adhesive proteins [Plasmodiumfalciparum
erythrocyte membrane protein-I (PfEMP-1)]. which promote
aggregation of infected RBCs to other noninfected RBCs and
receptors of capillary endothelial cells. These sequestrated
RBCs cause capillary plugging of cerebral microvasculature,
which results in anoxia, ischemia and hemorrhage in brain.
• Blackwater fever: A syndrome called blackwater
fever (malarial hemoglobinuria) is sometimes seen in
Jalciparum malaria, particularly in patients, who have
experienced repeated past infections and inadequate
treatment with quinine. An autoimmune mechanism has
been suggested.
Patients with glucose-6-phosphate dehydrogenase
(G6PD) deficiency may develop this condition after
taking oxidant drugs, even in the absence of malaria.
- Clinical manifestations include fever, prostration
and hemoglobinuria (black colored urine), bilious
vomiting and prostration, with passage ofdark red or
blackish urine.
The pathogenesis is believed to be massive
intravascular hemolysis caused by antierythrocyte
antibodies, lead ing to massive absorption of
hemoglobin by the renal tubules (hemoglobinuric
ne phrosis) producing blackwater fever.
Complications of blackwater fever include renal
failure, acute liver failure and circulatory collapse.
Algid malaria: This syndrome is characterized by
peripheral circulatory failure, rapid thready pulse with
low blood pressure and cold clammy skin. There may be
severe abdominal pain, vomiting, diarrhea and profound
shock.
Septicemic malaria: It is characterized by high
continuous fever with dissemination of the parasite to
various organs, leading to multiorgan failure. Death
occurs in 80% of the cases.](https://image.slidesharecdn.com/panikermedicalparasitology8e-210918093838/75/Paniker-medical-parasitology-8e-100-2048.jpg)
![Polar sac- -- - ~ +--=-
Exospore-- -"
Endospore
Plasma membrane
Fig. 1: Microsporidian spore
Box 1: Acid-fast parasitic organisms
• Microsporidia (spore)
• Cyclospora cayetanensis (oocyst)
• lsospora be/Ii (oocyst)
• Cryptosporidium parvum (oocyst)
• Spores are 2-4 µmin size and oval to cylindrical in shape,
with a polar filament or tubule {Fig. 1).
• The spores are the infective stage ofmicrosporidia and the
only stage of life cycle capable ofexisting outside the host
cell.
The polar tubule is an extrusion mechanism for injecting
infective spore contents into the host cell.
• Spores are surround by thick double-layered cyst wall:
Outer layer (exospore) is proteinaceous and
electron-dense
Inner layer (endospore) is ch itino us and
electronlucent.
• Spores are Gram-positive and acid-fast (Box l ).
• LIFE CYCLE
Infection in host is probably by ingestion or inhalation of
spores.
• In the duodenum, the spore with its nuclear material
is injected through the polar tubule into the host cell
(enterocyte).
• Inside the cell, the microsporidia multiply by repeated
binary fission (merogony) and produce large number of
spores (sporogony).
Microspora
Box 2: Parasites causing opportunistic infections in immuno-
compromised patients [Human immunodeficiency virus (HIV)-
positive cases]
• Microsporidia
• C
yclospora cayetanensis
• lsospora be/Ii
• Cryptosporidium parvum
• Toxoplasma gondii
• Strongyloides stercoralis
• Entamoeba histolytica
• During sporogony, a thick spore wall is formed that
provides environmental protection to the cyst.
The spores are then liberated free from the host cell and
infect other cells.
• CLINICAL FEATURES
They can cause wide range ofopportunistic illness in patients
with HIVand other immunocompromised diseases (Box 2).
• In patients with AIDS, Enterocytozoon bieneusi and
Encephalitozoon intestinalis lead to protracted and
debilitating diarrhea in 10-40% of cases.
E. intestinalis may also cause sinusitis, cholangitis and
bronchiolitis.
Infection with Pleistophora can lead to myositis and
E. he/Lem can cause superficial keratoconjunctivitis,
sinusitis, respiratory disease and disseminated infection.
Stromal keratitis associated with trauma has been
reported in infections with Nosema, Vittaforma and
Microsporidium in imrmmocompetent patients.
• LABORATORY DIAGNOSIS
Microscopy
Diagnosis of microsporidiosis is made by demonstration of
the spores in stool, urine, cerebrospinal fluid (CSF), or small
intestine biopsy specimen.
• The spores can be stained with Gram's stain, periodic
acid-Schiff (PAS) stain, or modified trichrome stain.
Note: Spores of microsporidia stain poorly with
hematoxylin and eosin stain.
Although intracellular spores can be visualized by light
microscopy, electron microscopy is the gold standard.
• Iden tification of species and genera of microsporidia is
based on electron microscopy of spore morphology.
• Direct fluorescent method using monoclonal antibody
is also used for detection of microsporidia in clinical
samples.](https://image.slidesharecdn.com/panikermedicalparasitology8e-210918093838/75/Paniker-medical-parasitology-8e-126-2048.jpg)
![Paniker'sTextbook ofMedical Parasitology
Cutaneous Manifestations
There may be dermatitis, with erythema and itching at the
site of penetration of the filariform larva, particularly when
large numbers of larvae enter the skin.
• In those sensitized by prior infection, there may be an
allergic response.
• Pruritus an d urticaria, particularly around the
perianal skin and buttocks, are sym ptoms of chronic
strongyloidiasis.
• the term larva currens (meaning racing larvae) has been
applied to the rapidly progressing linear or serpiginous
urticaria! tracks caused by migrating filariform larvae.
These often follow autoinfection and start perianally.
Pulmonary Manifestations
When the larva escape from the pulmonary capillaries into
the alveoli, small hemorrhages may occur in the alveoli and
bronchioles.
Bronchopneumonia may be present, which may progress
to chronic bronchitis and asthmatic symptoms in some
patients.
• Larva of Strongyloides may be found in the sputum of
these patients.
Intestinal Manifestations
The symptoms may resemble those of peptic ulcer or of
malabsorption syndrome.
• Mucus diarrhea is often present. In heavy infection, the
mucosa may be honeycombed with the worm and there
may be extensive sloughing, causing dysenteric stools.
• Other manifestations are protein-losing enteropathy and
paralytic Beus.
Hyperinfection
In debilitated individuals and particularly in those with
cellular immune defects, extensive internal reinfection takes
place, leading to an enormous number of adult worms in the
intestines and lungs and larvae in various tissues and organs.
This is known as hyperinfection.
• Severemalnutrition, lepromatousleprosy, lymphoreticular
malignancies, acquired immunodeficiency syndrome
(AIDS), immunosuppressive drugs and other situations,
in which cell-mediated immunity is defective, predispose
co this condition.
Hyperinfection is an important hazard of steroid therapy
and other instances of prolonged immunosuppression as
in transplant patients.
During hyperinfection, the filariform larvae may enter
in to arterial circulation and lodge in various organs, e.g.
heart, lungs, brain, kidney, pancreas, liver and lymph
nodes. Manifestations depend on the sites affected.
Brain abscess, meningitis and peritonitis are major fatal
complications.
It has been reported that circulating Strongyloides larvae
may carry intestinal bacteria, causing septicemia.
• LABORATORY DIAGNOSIS
Microscopy
Direct wet mount of stool: Demonstration of the
rhabditiform larvae in freshly passed stools is the most
important method of specific diagnosis. Larvae found in
stale stools have to be differentiated from larvae hatched
from hookworm eggs (Flowchart 2).
Concentration methods ofstoolexaminatum:Stool may
be concentrated by formol-ether concentration method
or Baermann's funnel gauze method and examined for
larvae more efficiently. Baermann's test requires a special
apparatus and relies on the principal that larva will
actively migrate out of the feces on a wire mesh covered
with several layers ofgauge.
Larvae maysometimes be presentin sputum or duodena]
asp.
iratcs and jejuna] biopsies.
Flow chart 2: Laboratory diagnosis of Strongyloides stercoralis
+
Microscopy
- Direcl wet mount of stool:
Demonstrates rhabditiform
larva (definitive diagnosis}
- Stool concentrations methods:
• Formol ether concentration
• Baermann's funnel gauze
- Demonstration of larva in
sputum or duodenal aspirates
or jejunal biopsies
Laboratory diagnosis
•
Stool culture
- Done when larvae are
scanty in stools
- Methods used:
• Agar plate culture
• Charcoal culture
method
I
Serology
- Done using
S/rongy/oides or
filarial antigens
- Methods used:
• Complement fixation
• Indirect hemagglutination
• ELISA
+
Radiological
imaging
Abbreviations: ELISA, enzyme-linked immunosorbent assay; lgE, immunoglobulin E
•Blood
examination
• Peripheral
eosinophilia
• Raised serum
lgE levels](https://image.slidesharecdn.com/panikermedicalparasitology8e-210918093838/75/Paniker-medical-parasitology-8e-199-2048.jpg)
![INDEX
Page numbers followed by b refer to box,frefer to figure.Jcrefer to flow chartand I refer to table
A
Abscess, splenic 19
Acanthamoeba 12, l3, IS, 26, 28,291
,231,
233, 244
culbertsoni, life cycle of 29f
keralicis 29, 30
Acanthocheilonema 223
Acanthopodia 28
Accidental host 2, 121
Acephalocysts 133
Acetabulum 14 l
Acid-fast
parasitic organisms 105b
stain IOOJ, 236
Acidosis, metabolic 79
Acquired immunodeficiencysyndrome 5,
13, 29, 93, 104, 184 243
Adenolymphangitis, acute 214
Adenophorea 166
Adoral cilia 107
Adult Trichuris rrichiura worms l76f
Adult worm 112, 144, 151, 154, 156, 160,
170, 175, 180, 181, 198, 203
African trypanosomiasis 42,46
Agar plate culture 239
Albendazole 128, 135
Alimentary canal, amebae of 13
Alphonse laveran 67
Amastigote 42, 48, 48f, 53, 54f
Ameba 244
classification of 151
drug sensitivity of 23
Amebapores 18
Amebiasis 20fc,246
cutaneous 19, 21
genitourinary 19, 21
hepatic 20
lesions of 22f
metastatic 2I
pulmonary 19, 21
Amebic
antigen detection 23
appendicitis 19
colitis 24
cysteine proteases 18
dysentery 13, L
S, 19, 20
encephalitis, granulomacous 26, 29
granuloma 30
hepatitis 19, 20
keratitis 26, 29
lectin 18
liver abscess 13, 15, 19, 20f, 21, 2lfc, 24
meningoencephalitis 13, 26-29
ulcer 18, 19f
flask-shaped 19/
Amebida 12
Ameboflagellate 27
Ameboma 19, 30
Amebostomes 27
American rrypanosomiasis 47
American visceral leishmaniasis 56
Amoeboflagellate 39
Amphotericin B 28, 61
Ampulla ofVater 155
Ancylostoma 6, 140, 165, 189
brazilie11se 165, 167
caninum 167
ceyla11iwm 193
duodenale 3, 7, 165, 176, 180, 187-189,
192,194,207,229, 243
adult worm of 188/
egg of 189]
life cycle of 190/
Anemia 46, 57
causesof 56b, 78b, 192b
dimorphic 192
severe 56, 87
Angiostrongyliasis, abdominal 231
Angiosrrongylus cantonensis 167, 230-233
Animal inoculation 8, 47, 50, 59, 94, 245
Anisakiasis 232, 233
Anisakis simplex 167, 232
Anodic antigen, circulating 147
Anopheles barbirostris 2LO, 219
Anthroponotic urban type 62
Anthropozoonoses 2
Antiamebic drugs 24 I
Antibody
demonstration of 217
detection 7, 23, 35, 51, 60, 95, 128, 147
Antigen 7
detectio11 7, 35, 47, 51, 59, 95, 128, 135,
146
tests, rapid 83
Anti-oocyst antibody 100
Apansporoblastina 12
Aphasmidia 165
Apicomplexa, phylum 66, 661
Appendicitis 178
Artcmisinin-based combination therapy 84
Ascariasis, ectopic 205
Ascaris 6, 8, 140, 207
eggs, types of 202f
fertilized egg of 202/
lumbricoirles 3, 7,112,165, 167, 176,180,
189, 194, 199-201, 20 If, 204, 206f c,
207, 243,248
life cycle of 204/
pneumonia 203
suum 167
unfertilized egg of 202/
Ascites 57
Aspirates, splenic 58
Aspiration 135, 135b
biopsies 59
Atovaquone 88
Autoimmune hemolysis 56
Axoneme 41, 42, 53
Azithromycin 88
B
Babesin 4, 12, 66
bovis 86
microti 13, 86, 86/
Babesiosis 87,871
Bachman intradermal test 174
Bacillary dysentery 20, 201
Bacterial infection, secondary 227
Baermann concenrration method 238, 238/
Balamuth's medium 23
composition of 244b
Balamuth's monophasic liquid medium 244
Balamuthia 26
mnndrillaris 15, 30
Balantidiasis I09
Balantidium 12, 109
coli 3, 7, 11, 13, 14, 39, 107, 107f, 109,
150,244
life cycle of 108/
Bancroftian filariasis 213, 247
Basal body 10
Basophilic stippling 73
Baylisascaris 207
procyonis 167
Bell's dilution-filtration count 238
Bentonite flocculation tests 247
Benznidazole 51
Bile
duct carcinoma 145
staining 123
Bilharziasis 143
Biliary
cirrhosis 156
obstruction, acute 205
passage 152
tract 142, 154](https://image.slidesharecdn.com/panikermedicalparasitology8e-210918093838/75/Paniker-medical-parasitology-8e-264-2048.jpg)
![Paniker'sTextbook ofMedical Parasitology
Binary fission 11, J6, 41
Binucleate cyst 16f, 25f
Biopsy 217
BiLhionol 153
Blackwater fever 79
Bladder
carcinoma 145
containing seeds 142
worm ll7, 123
Blastocyslis hominis 10 I, 10 If
Blastomeres 188
Blepharoplast 42, 53
Blinding filaria 221
Blister formation 227
Blood 13, 142
collection of 240
examination 6, 135
fluke 141-143
incubation infectivity test 47
loss 178
picture 87
smear 82b
transfusion malaria 801
urea nitrogen 88
Boeckand Drbohlav diphasic medium 244,
244b,
Bone marrow 56
aspirate 58
macrophage of 13
Bothriocephalus anemia 120
Bradyzoites 9 l, 93, 102
Brain 21, 104,232
parenchyma l28f
Bronchi 161
Brugia malayi 4, 7,165,208,210,218, 219f,
224,226
Brugia pahangi 167, 223
Brugia patei 167
Brugia Limori 165,208,210, 219,226
Buffy coat blood film 242
Bunostomum phlebotomum 167
C
Cachexia 57
Calabar swellings 219, 22 l
Calcofluor white staining 29
Candidate vaccine 61
Capil/aria a eropltila 16 1
Capillaria philippinensis 4, 165, 180, 231,
232
Card agglutination trypanosomiasis test
46, 47
Cardiac implantable electronic device 133
Cartwheel appearance 16
Casoni's inrradermal 1es1 134
Casoni's test 247
Cat liver fluke 156
Cathodic antigen, circulating 147
Caudal papillae l66
Cecum 18
Cellular exuda1
es 20,235
Cellulose acetate membrane precipitation
test 23
Central nervous system 13, 46, 129, 150, 171
infection 232t
Centrilobular necrosis 77
Cercarial dermatitis 148
Cerebral
amebiasis 19
angiostrongyliasis 230
malaria 79
paragonimiasis 161
Cerebrospinal fluid 6, 27-29, 45-47, 128,
230,231,245
Cestodes 4, 112, l l 5
classification of 115, l 16t
living 122b
Chagas disease 13, 42, 47
acute 49, 50
chronic 50
Chagas radioimmune precipitation assay 5 1
Chagoma 50
Chancre
painless 45
trypanosomal 45
Charcoal culture 240
Charcot-Leyden crystals 19, 20, 22, 22f, 235
Chemoprophylaxis 84, 85
Chiclero's ulcer 53, 63
Chilomastix 12
mesnili 32, 38
egg of 38f
Chinese liver fluke 154
Chocolate brown sputum 21
Cholangiocarcinoma 156
Cholangitis 156
Chopra's antimony test 60
Chromatoid bodies 10, 16
Chrysops 220, 221
Chylous urine 215/
Cilia 11
Ciliophora 11, 12
Cloaca 164
Clonorchis 113, 141,207
sinensis 4, 7, 143, 145, 151, 154, 172, 194,
201,232
egg of 154f
life cycle of 155
/
Coccidia 12, 66, 90
Coenurns 117, 129
Colon 13, 18
Complement fixation test 7, 46, 47, 58, 133,
135,216,246
Complete blood count 205
Congestive cardiac failure 87
Conjunctiva 165
Conjunctiva! biopsies 222
Conjunctiva] epithelium 104
Coproantigen, detection of 23
Copulatory spicules 200
Coracidium 118, 120
Cornea 29, 104
Corneal stroma 104
Counter-currem immunoelectrophoresis
23
Craig's medium 22
Creeping myiasis 167
Crescentic tachyzoites 90f
Crustacea 232
Cruzin 51
Cryptosporidiosis 247
Cryptosporidium 12, 14, 66, 100, 236
paruum 3, 4, 7, 13, 10, 97, 99, 100, 105,
199,204,248
life cycle of 99f
oocysrs of 98f, 100
/
Clenodactylusgundi 90
Cu/ex quinquefasciatus 2 10, 213
Culicoides 2!0, 223
Cutaneous leishmaniasis, diffuse 53, 62
cyclophyllidean 117, 1171
tapeworms 122
cyclops 118, 232
Cyclospora 66, 101,236
cayetanensis 3, 7,39,99, 100,105
Cylindrical esophagus 180
Cyst 14, 16, 17, 26, 27, 29, 30, 39, 107, 107
f
fluid 134
mature 96
/
uninucleate 16f
cysteine-pep1one-liver-maltose 37
cysticerci in muscles 124}
cysticercosis 126, 128, 140
Cysticercus 117, 123
bouis 123, 124f
cellulosae 123, 124f
cysts of I28
f
Cytoadherence 74
Cytolysis 18
Cytopharynx 107
Cytoplasm 10, 16
Cytopyge 107
Cytos10me 107
D
Deoxyribonucleic acid 8, 21, 35, 47, 58, 83,
127, 133,217,247
Dermatitis 184
Diamond's axenic medium 23
Diarrhea 13, 34b, 97
bloody 1501
1
Oichlorodiphenyltrichloroethane 85, 218
Dicrocoelium dendriticum 150, 153
Dienlamoeba 12, 39
fragilis 32, 39
trophozoite of 39{](https://image.slidesharecdn.com/panikermedicalparasitology8e-210918093838/75/Paniker-medical-parasitology-8e-265-2048.jpg)
![B !aniker'sTextbookofMedical Parasitology
cyst 90, 91, 93
hypoxia 77
necrosis 18
Toxic megacolon 19
Toxocara canis 167, 206, 232, 233
adult worms of 206/
Toxocara cati 167,206,232
Toxocariasis 247
Toxoplasma 11, 12, 14, 66, 90, 94
encephalitis 95, 96
gondii 1, 2, 4, 5, 10, 14, 46, 48, 90, 90{.
91
/, 93, 94/c,96, I 05, l 72, 248
lire cycle of 92
f
inrection 93, 94
pneumonia 94
Toxoplasmosis 94, 245, 246
acquired 93
acute 93
congenital 93, 95
Trachipleistophora I04
Transfusion malaria 80
Transovarian transmission 87
Transverse binary fission I08
Trau ma 5
Traveler' diarrhea 99/,
Trematodes 4, 112, 141, 143/,
classification of 141
zoological classification of 14 l/
Triatoma inrestans 48
Trichina worm 170
Trichinel/a 140, 164
cyst 171
Spira/is 4, 7, 46, 165, 170, 172, 173/< , 174,
180, 199,207, 226,232,243
adult worms or 170/
life cycle of 172
/, l73t
Trichinosis 247
Trichomonadida 12
Trichomonas 12, 13, 36, 39
hominis 32, 36
/, 38
tenax 32, 36
/. 37, 204
vagina/is 3, 6, 7, 13, 36, 36
); 95, 109, 217,
244
Trichostomatina 12
Trichostrongyliasis 193
Trichostrongylus orienlalis 7
Trichrome stain 235
modified LOS, 236
reagents of 236b
Trichuris 175,229
lrichiura 3, 7, l09, J50, 165, 175, J76,
178, 178
f c,189, 196,20l,207
egg of 176/
life cycle or L
77]
Triclabenda1ole 153
Tripartite 187
Trop-bio test 2 I7
Trophozoite 11, 14, 16, 16/; 27/ , 29, 33,33{,
38/, 39, 86/; 90, 91, 91{. 107, 107/
extracellular 90{
Tropical pulmonary eosinophilia 215, 2 17
Trypa11osoma 12, 13, 41, 233, 241, 244, 248
brucei 4, 13, 32, 42,231
brucei 43
lire cycle or 44
/
gambiense 5, 42, 50
rhodesiense 5, 43, 45, 46, 50
cruzi 4, 13, 32, 43, 46-48, 48{. 50, SO
J, 51,
93,226
life cycle or 49f
equiperdum 43
evansi 43
gambiense 43, 43/
lewisi 43
infections 95
rangeli 43, 51
rhodestense43f. 46[
Trypanosomatidae 41
Trypanosomatina 12
Trypanosomes 42, 5 1
classification or 42
Trypanosomiasis 43{. 46/< , 245, 246
Trypomastigote 42, 48, 48
]
Tsetse fly 45
Tubercles 147
Tubulina 12
Typhus-like examhema 94
u
Uncinaria stenoceplzala 167
United Nations Children's Fund 86
United ations Development Programme
86
Upper respiratory tract 29
Urethra 13
Urethritis 13
Urinary bladder 144
carcinoma 145
Urine 6, 7/J
V
Vaccination 5
Vacuole 53
Vagina 13
Vaginal sphincter, prominent 122
Vaginitis 13
Vascular cell adhesion molecule- I 74
Vector mosquito, eradication of 2 18
Vector transmission 4
Vermicules 87
Vertebrate host 44
Visceral larva migrans 167, 1671, 168, 1681,
206,207
Visceral leishmaniasis 52, 53, 54/, 61 h, 245
Viviparous nematodes 226/J
w
Water plants, ingestion of 159
Watsonius watsoni 153, 156, 159
West African trypanosomiasis 43, 451
Western blot 100
Wet saline mounts 235
Wheatley's lrichrome stain 235
Whip-like flagella 32
Whipworm 165, 175, 1761
White blood cell 29, 83, 242
Winterbonom's sign 45
Wolbachia 208,223
Wright's tain 241 ·
wuchereria 164
y
bancrofti 4, 7, 165, 199,208, 2 10, 211{.
212,213, 2 16/,, 2 17,218,224,226,
241
adult worm or 21J/
life cycle of 212/
Young erythrocytes 72
Young trophowites 69
z
Ziehl-Neelsen stain 100(
modified 97, 98, 236
Ziemann's stippling 75
Zinc sulfate noatation concentration
technique 237/
Zooa11throponoses 2
Zoomastigophorea 12
Zoonoses 2, 8
Zoonotic filariasis 223
Zoophilic nematode 167
Zygocotylidae 141
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Paniker medical parasitology 8e
- 2. J ANTONIE VAN LEEUWENHOEK Born:24.10.1632 - Died: 26.8.1723 Delft, Holland I .J This man, born poor, with little education, a draper in his hometown of Delft had surprising visitors! They included great men of science as well as the Royalty like the Tsar Peter the Great, Frederick the Great of Prussia and King James II of England.This was due to his hobby of grinding fine lenses through which he looked at various objects and brought forth the wonder world of small things that none had seen before. He kept clear descriptions and accurate drawings of what he saw and communicated them to the Royal Society in London. A strict check convinced the Society of their authenticity. The unlettered Antonie was elected a Fellow of the Royal Society! The papers sent by him over decades can still be seen in the Philosophical Transactions of the Royal Society. The discoveries he made are legion. He described the first protozoan pathogen Giardia. He also discovered many types of bacteria, human and animal spermatozoa, and eggs of various animals realizing their importance in reproduction. He could not recognize the significance of the different types of bacteria, and to him, they werejust'littleanimalcules'. His fault was in being much before the time, for it took two centuries more for people to accept the microbial origin of infectious diseases. But that should not deter us from acknowledging the great contributions made by Leeuwenhoek to Biology and many other branches of Science. He was truly the Founder of Microbiology. _J
- 4. Paniker's Textbook of MEDICALPARASITOLOGY
- 6. Paniker's Textbook of MEDICALPARASITOLOGY EIGHTH EDITION (late} CK Jayaram Paniker MD Formerly Director and Professor Department of Microbiology Principal Government Medical College, Kozhikode, Kerala Dean, Faculty of Medicine University of Calicut, Kerala, India Emeritus Medical Scientist Indian Council of Medical Research New Delhi, India Revised and Edited by Sougata Ghosh MD ocH Professor Department of Microbiology Government Medical College Kolkata, West Bengal, India Formerly Faculty Institute of Postgraduate Medical Education and Research (IPGMER) and Calcutta School ofTropical Medicine Kolkata, West Bengal, India Foreword Jagdish Chander t • The Health Sciences Publisher New Delhi ILondon I Panama
- 7. . !.Jaypee Brothers MedicalPublishers (Pl Ltd. Headquarters Jaypee Brothers Medical Publishers (P) Ltd 4838/24, Ansari Road, Daryaganj New Delhi 110 002, India Phone: +91-11-43574357 Fax: +91-1 1-43574314 EmaiI: [email protected] Overseas Offices J.P. Medical Ltd 83 Victoria Street, London SWlH0HW(UK) Phone: +44 20 3170 891 0 Fax: +44 (0)20 3008 6180 Email: info@ jpmedpub.com Jaypee Brothers Medical Publishers (P) Ltd 17/1 -B Babar Road, Block-B, Shaymali Mohammadpur, Dhaka-1207 Bangladesh Mobile: +08801912003485 Email:[email protected] Website: www.jaypeebrothers.com Website: www.jaypeedigital.com 10 2018, Jaypee Brothers Medical Publishers Jaypee-Highlights Medical Publishers Inc City of Knowledge, Bid. 235, 2nd Floor, Clayton Panama City, Panama Phone: + 1 507-301-0496 Fax:+1 507-301-0499 Email: [email protected] Jaypee BrothersMedical Publishers (Pl Ltd Bhotahity, Kathmandu Nepal Phone: +977-9741283608 Email: kathmandu@ jaypeebrothers.com The views and opinions expressed in this book are solely those of the original contributor(s)/author(s) and do not necessarily represent those of editor(s) of the book. All rights reserved. No part of this publication may be reproduced, stored or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior permission in writing ofthe publishers. All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The publisher is not associated with any product or vendor mentioned in this book. Medical knowledge and practice change constantly. This book is designed to provide accurate, authoritative information about the subject matter in question. However, readers are advised to check the most current information available on procedures included and check information from the manufacturer of each product to be administered, to verify the recommended dose, formula, method and duration of administration, adverse effectsand contraindications. It is the responsibility of the practitioner to take all appropriate safety precautions. Neither the publisher nor the author(s)/editor(s) assume any liability for any injury and/or damage to persons or property arising from or related to use of material in this book. This book is sold on the understanding that the publisher is not engaged in providing professional medical services.Ifsuch advice or services are required, the services of a competent medical professional should be sought. Every effort has been made where necessary to contact holders of copyright to obtain permission to reproduce copyright material. If any have been inadvertently overlooked, the publisher will be pleased to make the necessary arrangements at the first opportunity. The CD/DVD-ROM (if any) provided in the sealed envelope with this book is complimentary and free ofcost. Not meant for sale. Inquiriesfor bulk sales may be solicited at:jaypee@ jaypeebrothers.com Paniker'sTextbook ofMedical Parasitology First Edition: 1988 Second Edition: 1989, Reprint: 1991 Third Edition: 1993 Fourth Edition: 1997, Reprint: 1999 Fifth Edition: 2002, Reprint: 2003, 2004 Sixth Edition: 2007, Reprint: 2011 Seventh Edition: 2013 Eighth Edition: 2018 ISBN: 978-93-5270-186-5 Printedat: Ajanta Offset&Packagings Ltd., Faridabad, Haryana.
- 8. FOREWORD This is agreat pleasure to write the foreword to the eighth edition of Paniker's Textbook ofMedical Parasitology dealing with medically important parasites vis-a-vis human diseases caused by them. The parasitic infections (protozoa! and helminthic) are still major cause ofhigh morbidity as well as mortality of substantial number of population residing in the developing world of tropical and subtropical regions. The clinical presentations of parasitic diseases have also significantly evolved with the passage oftime. Malaria caused by Plasmodium vivax has never been life-threatening but now it is presenting with renal failure as well as acute respiratory distress syndrome (ARDS) thereby leading to fatal consequences. On the other hand, some of the infections such as dracunculiasis have been eradicated from India and others are the next targets being in the pipeline. There are a number of novel diagnostic techniques, which are being designed for rapid diagnosis ofvarious parasitic diseases and accurate identification of their causative pathogens. The non-invasive imaging techniques, both MRI and CT scans, are proving to be very useful tools for an early diagnosis thereby delineating the extent of disease in a particular patient.Therefore, to cope up with the changing epidemiological scenario and newer diagnostic modalities, medical students and professionals involved in the patient care need updates from time to time. Dr Sougata Ghosh (Editor), has done a remarkable job of going through the voluminous information and presenting it in a very lucid, concise and reproducible manner. This edition will ideally be suited for medical students and resident doctors, who are preparing for various examinations and entrance tests. I feel the present edition will also be appreciated by students and teaching faculties in all disciplines of medicine. The chapter on pneumocystosis has been removed, however, on sporozoa dealing with diseases caused by different species of microsporidia, traditionally retained in this edition, despite the fact that it hasalso been shifted now to the kingdom fungi like Pneumocystisjirovecii. The unique feature of the textbook is that it has many illustrations, photographs of clinical specimens and photomicrographs with an easy-to-read and understand format. This will help the students to memorize the information given in the texteasily as well as to usethe same in medical practice. Each chapter has key points with a set ofmultiple choice questions (MCQs), which will help a student for better understanding and preparation before the examination. Although it is meant for medical graduates, recent advances mentioned in this book will also be useful for the postgraduates. The original author, Professor CK Jayaram Paniker, was an experienced and enthusiastic medical teacher, and we recently lost him. Moreover, he was a legendary microbiologist and the author ofnumerous valuable textbooks, particularly co-author of Ananthanarayan's Textbook ofMicrobiology. His name has been retained as such in the title of the eighth edition ofthis textbook is a great honor and real tribute to him thereby continuing his legacy to attain more heights in the field of medical parasitology even in his physical absence. I hope that this textbook will continue to benefit the medical students and faculties for many years as it has done during the last three decades. Jagdish Chander Professor and Head Department ofMicrobiology Government Medical College and Hospital Chandigarh, India
- 10. PREFACE TO THEEIGHTH EDITION The previous editions of Paniker's Textbook ofMedical Parasitology have been widely accepted by the medical students and teachers across India and abroad for almost three decades. Medical science is not a static art. Methods ofdiagnosis and treatment of parasitic infections change constantly.To keep pace with these developments, all the chapters of present edition have been thoroughly revised and expanded, providing up-to-date epidemiological data, new diagnostic methods and recent treatment guidelines of parasitic infections. In the current edition, many newtables, flow chartsand photographs of specimens and microscopic view pictures have been added for better comprehension of the subject. Recent advances such as vaccinology of malaria and leishmaniasis, malarial drug resistance, new treatment protocols of different parasitic infections are the salient features of the book. The aim of the contents of the book remains same in this edition, that is compact yet informative and useful for both graduate and postgraduate students. Likethe last edition,the presentedition isalsodesigned in acolorful format,which can beeasily read and comprehended. Important points and terms have been highlighted by making them bold and italic. At the end of each chapter, the must-know facts are given as "Key Points" in box formats for quick recapitulation. Important multiple choice questions (MCQs) and review questions from various university examinations' papers have been added to test and reinforce understanding of the topics by the students. Sougata Ghosh
- 12. PREFACE TO THEFIRST EDITION Parasitic infections continue to account for a large part of human illness. Antimicrobial drugs and vaccines that have made possible the effective control of most bacterial and viral diseases have not been as successful against parasitic infections. The numbers of persons afflicted by parasites run into many millions. Malaria still affects over 500 millions, pinworm and whipworm 500 millions each, hookworm 800 millions and roundworm a billion persons. Filariasis, leishmaniasis and schistosomiasis remain serious public health problems. Infections due to opportunist parasites are becoming increasingly evident in the affluent countries. In recent years, there has been a resurgence in the study of parasitic infections. Much new knowledge has been gained making possible precise diagnosis and more effective control of parasites and the diseases, they cause. This textbook attempts to present the essential information on parasites and parasitic diseases, with emphasis on pathogenesis, epidemiology, diagnosis and control. Every effort has been made to incorporate recent advances in the subject. It is hoped that medical students, teachers and physicians will find the book useful. Their comments and suggestions for improvement ofthe book will be most welcome. SHANTHI, East Hill Road Kozhikode, Kerala-673 006 CK Jayaram Paniker
- 14. ACKNOWLEDGMENTS I gratefully acknowledgethe help of the Principal, Government Medical College, Kolkata; Director, Calcutta School of Tropical Medicine, Kolkata, West Bengal, India; and all my developmental colleagues for their valuable suggestions. Lastly, I want to thank my parents, wife and my son Anindya Ghosh, for their emotional support, whenever I needed during preparations ofthe manuscript. I solicit the comments and suggestions for the faculties and students for improvement of the book and many be e-mailed to [email protected] I owe my special thanks to Shri Jitendar P Vij (Group Chairman), Mr Ankit Vij (Group President) and Mr Sabyasachi Hazra (Commissioning Editor, Kolkata Branch) of M/s Jaypee Brothers Medical Publishers (P) Ltd, New Delhi, India, for their professional help and guidance to bring out the present edition of the book.
- 16. 1. General Introduction:Parasitology Parasites Host 7 Zoonosis 2 Host-parasite Relationships 2 Life Cycle of Parasites 3 Sources of Infection 3 Modes of Infection 4 Pathogenesis 4 Immunity in Parasitic Infection 5 Immune Evasion 5 Vaccination 5 Laboratory Diagnosis 6 2. Protozoa General Features 10 Structure 10 Cytoplasm 10 Nucleus 10 Terminologies Used in Protozoology 10 Reproduction 11 Life Cycle 11 Classification of Protozoa 11 CONTENTS 1 10 3. Amebae 15 Entamoeba histolytica 15 Nonpathogenic Intestinal Ameba 24 Pathogenic Free-living Amebae 26 4. Intestinal, Oral and Genital Flagellates Giardia lamblia 32 Trichomonas 36 Chilomastix mesnili 38 Enteromonas hominis 38 Retortamonas intestina/is 38 Dientamoeba fragilis 39 32
- 17. Paniker'sTextbook of MedicalParasitology 5. Hemoflagellates Zoological Classification of Flagellates 41 General Characteristics 41 Trypanosomes 42 Leishmania 52 6. Malaria and Babesia Malaria 66 Classification 66 Causative Agents of Human Malaria 66 Malaria Parasite 66 Babesia Species 86 Classification 86 History and Distribution 86 Habitat 86 Morphology 86 Life Cycle 86 Pathogenicity and Clinical Features 87 Laboratory Diagnosis 87 Treatment 88 Prophylaxis 88 7. Coccidia Toxoplasma gondii 90 /sospora be/Ii 96 Cryptosporidium parvum 97 Cyclospora cayetanensis 100 8/astocystis hominis 101 Sarcocystis 102 41 66 90 8. Microspora 104 History and Distribution 104 Morphology 704 Life Cycle 105 Clinical Features 105 Laboratory Diagnosis 105 Treatment 106 Prophylaxis 706
- 18. Contents 9. Balantidium coli107 History and Distribution 107 Habitat 107 Morphology 107 Life Cycle 108 Pathogenesis 108 Clinical Features 109 Laboratory Diagnosis 709 Treatment 709 Prophylaxis 709 10. Helminths: General Features Phylum Platyhelminthes 771 Phylum Nemathelminthes (Nematoda) 772 Important Features of Helminths 112 Zoological Classification of Helminths 773 111 11. Cestodes: Tapeworms 115 Classification of Cestodes 115 Tapeworms: General Characteristics 115 Pseudophyllidean Tapeworms 117 Cyclophyllidean Tapeworms 122 12. Trematodes: Flukes 141 Classification ofTrematodes 141 Flukes: General Characteristics 747 Life Cycle 142 Blood Flukes 743 Hermaphroditic Flukes: Liver Flukes 150 Intestinal Flukes 156 Lung Flukes 760 13. Nematodes: General Features General Characteristics 764 Life Cycle 764 Modes of Infection 765 Classification 765 Larva Migrans 765 164
- 19. Paniker'sTextbook ofMedical Parasitology 14.Trichinella spiralis Common Name 170 History and Distribution 170 Habitat 770 Morphology 110 Life Cycle 777 Pathogenicity and Clinical Features 172 Diagnosis 172 Treatment 174 Prophylaxis 174 1S. Trichuris trichiura Common Name 175 History and Distribution 175 Habitat 775 Morphology 175 Life Cycle 776 Pathogenicity and Clinical Features 177 Laboratory Diagnosis 778 Treatment 178 Prophylaxis 178 16. Strongyloides stercoralis History and Distribution 180 Habitat 180 Morphology 180 Life Cycle 182 Pathogenicity and Clinical Features 783 Laboratory Diagnosis 184 Treatment 185 Prophylaxis 185 170 175 180 17. Hookworm 187 History and Distribution 187 Ancylostoma duodenale 187 Necator americanus 189 Pathogenicity and Clinical Features of Hookworm Infection 190 Laboratory Diagnosis 797 Treatment 192
- 20. Prophylaxis 793 Other Hookworms193 Trichostrongyliasis 793 18. Enterobius vermicularis Common Name 795 History and Distribution 795 Habitat 795 Morphology 795 Life Cycle 796 Pathogenicity and Clinical Features 196 Laboratory Diagnosis 797 Treatment 798 Prophylaxis 799 19. Ascaris /umbricoides Common Name 200 History and Distribution 200 Habitat 200 Morphology 200 Life Cycle 201 Pathogenicity and Clinical Features 203 Laboratory Diagnosis 205 Treatment 205 Prophylaxis 205 20. Filarial Worms Lymphatic Filariasis 270 Subcutaneous Filariasis 2 79 21. Dracunculus medinensis Common Name 225 History and Distribution 225 Habitat 225 Morphology 225 Life Cycle 226 Pathogenicity and Clinical Features 227 Laboratory Diagnosis 227 Treatment 227 Prophylaxis 229 Contents 195 200 208 225
- 21. Paniker'sTextbook ofMedical Parasitology 22.Miscellaneous Nematodes Angiostrongylus cantonensis 230 Capillaria philippinensis 231 Gnathostoma spinigerum 231 Anisakiasis 232 23. Diagnostic Methods in Parasitology Examination of Stool 234 Examination of Blood 240 Sputum Examination 242 Urine or Body Fluids Examination 243 Tissue Biopsy 243 Muscle Biopsy 243 Duodenal CapsuleTechnique {Enterotest) 243 Sigmoidoscopy Material 244 Urogenital Specimen 244 Culture Methods 244 Animal Inoculation 245 Xenodiagnosis 245 Immunological Diagnosis 246 Skin Tests 247 Molecular Methods 247 230 234 Index 249 J
- 22. r CHAPTER 1 General Introduction: •INTRODUCTION Medical parasitology deals with the parasites, which cause human infections and the diseases they produce. • It is broadly divided into two parts: 1. Protozoology 2. Helminthology. • The pioneer Dutch microscopist, Antonie 11an Leeuwenhoek ofHolland in 1681, first introduced single lens microscope and observed Giardia in his own stools. • Louis Pastuer in 1870, first published scientific study on a protozoa( disease leading to its control and prevention during investigation of an epidemic silk worm disease in South Europe. • Aseminal discovery was made in 1878 by Patrick Manson about the role ofmosquitoes in filariasis. Thiswas the first evidence ofvector transmission. • Afterwards, Laveran in Algeria discovered the malarial parasite (1880), and Ronald Ross in Secunderabad and Calcuna in India, showed its transmission by mosquitoes (1897). Alarge nwnber ofvector-borne disease have since then been identified. • Bymid 20th century,with dramaticadvances in antibiotics and chemotherapy, insecticides and antiparasitic drugs, and improved lifestyles, all infectious diseases seemed amenable to control. • PARASITES Parasites are living organisms, which depend on a living host for their nourishment and survival. They mulriply or undergo development in the host. • The term "parasite" is usually applied to Protozoa (unicellular organisms) and Helminths (multicellular organisms) (Flow chart 1). • Parasites can also be classified as: - Ectoparasite: Ectoparasites inhabit only the body surface ofthe hostwithoutpenetratingthe tissue.Lice, ticks and mites are examples of ectoparasites. lhe Parasitology term infestation is often employed for parasitization with ectoparasites. - Endoparasite: A parasite, which lives within the body of the host and is said to cause an infection is called an endoparasite. Most of the protozoan and helminthic parasites causing human disease are endoparasites. - Free-living parasite: It refers to nonparasitic stages of active existence, which live independent of the host, e.g. cystic stage ofNaegleriafowleri. Endoparasites can further be classified as: Obligate parasite: The parasite, which cannot exist without a host, e.g. Toxoplasma gondii and Plasmodium. - Facultative para.site: Organism which may either live as parasitic form or as free-living form, e.g. Naegleriafowleri. - Accidental parasites: Parasites, which infect an unusual host are known as accidental parasites. Echinococcus granulosus infects man accidentally, giving rise to hydatid cysts. - Aberrant parasites: Parasites, which infect a host where they cannot develop further are known as aberrant or wandering parasites, e.g. Toxocara canis (dog roundworm) infecting lhwnans. • HOST Host is defined as an organism, which harbors the parasite and provides nourishment and shelter to latter and is relatively larger than the parasite. • The host may be ofthe following types: - Definitive host:The host, in which the adult parasite lives and undergoes sexual reproduction is called the definitive host, e.g. mosquito acts as definitive host in malaria. The definitive host may be a human or any other living being. However, in majority ofhuman parasitic infections, man is the definitive host (e.g. filaria, roundworm, hookworm).
- 23. Paniker'sTextbook of MedicalParasitology Flow chart 1: Types of parasites Parasite + Protozoa (unicellular) Kingdom-Protista + Helminths (multicellular) Kingdom-Animalia Amebae Flagellates Sporozoa Ciliates + + + • En/amoeba Naeglena Giardia Trichomonas Plasmodium Babesia Toxoplasma Balanlidium Nematodes • Ascaris Ancylostoma - Intermediate host: The host, in which the larval stage of the parasite lives or asexual multiplication takes place is called the intermediate host. In some parasites, two different intermediate hosts may be required to complete different larval stages. These are known as first and second intermediate hosts, respectively (Box 1). - Paratenic host: A host, in which larval stage of the parasite remains viable without further development is referred as a paratenic host. Such host transmits the infection to another host, e.g. fish for plerocercoid larva of D. lalum. Reservoir host: In an endemic area, a parasitic infection is continuously kept up by the presence of a host, which harbors the parasite and acts as an important source of infection to other susceptible hosts, e.g. dog is the reservoir host of hydatid disease. - Accidental host: The host, in which the parasite is not usually found, e.g. man is an accidental host for cystic echinococcosis. • ZOONOSIS The word zoonosis was introduced by RudolfVirchow in 1880 to include the diseases shared in nature by man and animals. • Later, in 1959, the World Health Organization (WHO) defined wonosis as thosediseasesand infeclions, which are naturallytransmittedbetween vertebrateanimals andman. Cestodes • Taenia Echinococcus Trematodes + Fasciola Schistosoma Box 1: Parasites with man as intermediate or secondary host • Plasmodium spp. • Babesia spp. • Toxoplasma gondii • Echinococcus granulosus • Echinococcus multilocu/aris • Taenia solium • Spirometra spp. • It is offollowing types: Protozoalzoonoses, e.g. toxoplasmosis, leishmaniasis, balanlidiasis and cryptosporidiosis. Ilelminthic zoonoses, e.g. hydatid disease, taeniasis. Anthropozoonoses: Infections transm itted to man from lower vertebrate animals, e.g. cystic echinococcosis. Zooanthroponoses: Infections transmitted from man to lower vertebrate animals, e.g. human n1berculosis to cattle. • HOST-PARASITE RELATIONSHIPS Hos t-parasite relationships are o f following types (Flow chart 2): Symbiosis • Commensalism • Parasitism.
- 24. General Introduction: Parasitology Flowchart 2: Host-parasite relationships Host-parasite relationships i 1 i Symbiosis Commensalism Parasitism • Both host and parasite are dependent upon each other • None of them suffers any harm from the association • Only the parasite derives benefit from the association without causing any injury to the host • The parasite derives benefits and the host is always harmed due to the association • A commensal is capable of living an independent life also • The parasite cannot hve an independent life • LIFE CYCLE OF PARASITES • Direct life cycle: When a parasite requires only single host to complete its development, it is called as direct life cycle, e.g. Entamoeba histolytica requires only a human host to complete its life cycle (Table 1). • Indirect lif e cycle: When a parasite requires two or more species of host to complete its development, the life cycle is called as indirect life cycle, e.g. malarial parasite requires both human host and mosquito to complete its life cycle (Tables 2 and 3). • SOURCES OF INFECTION Contaminatedsoiland water: Soil polluted with embryonated eggs (roundworm, whipworm) may be ingested or infected larvae in soil, may penetrate exposed skin (hookworm). lnfeclive forms of parasites present in water may be ingested (cyst ofameba and Giardia). Water containing the intermediate host may be swallowed (cyclops containing guinea worm larva). Infected larvae in water may enter by penetrating exposed skin (cercariae ofschisotosomes). - Free-living parasites in water may directly enter through vulnerable sites (Naegleria may enter through nasopharynx). Food: Ingestion of contaminated food or vegetables conraining infeclive stage of parasite (amebic cysts, Toxoplasma oocysts, Echinococcus eggs). Ingestion of raw or undercooked meat harboring infeclive larvae (measly pork containing cysticercus cellulosae, the larval stage of Taenia solium). Vectors: A vector is an agent, usually an arthropod that transmits an infection from man to man or from other animals to man, e.g. female Anopheles is the vector of malarial parasite. Vectors can be: - Biological vectors: The term biological vector refers to a vector, which not only assists in the transfer of Table 1: Parasites having direct life cycle (requiring no intermediate host) Protozoa Helminths • Entamoeba histalytica • Ascaris lumbricaides • Giardia lambfia • Enterobius vermicularis • Trichomonas vagina/is • Trichuris trichiura • Balantidium coli • Ancylostama duodenale • Cryptosporidium parvum • Necatoramericanus • Cyclospora cayeranensis • Hymenolepis nana • /sospara be/Ii • Microsporidia parasites but the parasites undergo development or multiplicaL ion in their body as well. They are also called as true vectors. Example oftrue vectors are: • Mosquito: Malaria, filariasis • Sandflies: Kala-azar • Tsetseflies: Sleeping sickness • Reduviicl bugs: Chagas disease • Ticks: Babesiosis. Mechanical vectors: The term mechanical vector refers to a vector, which assists in the transfer of parasitic form between hosts but is not essential in the life cycle of the parasite. Example of mechanical vectors is: • Housefly: Amebiasis In biological vectors, a certain period has to elapse after the parasite enters the vector, before it becomes infective.This is necessarybecause the vectorcan transmit the infecLion only after the parasite multiplies to a certain level or undergoes a developmental process in its body. This interval between the entry of the parasite into the vector and the L ime it takes to become capable of transmitting the infection is called the extrinsic incubation period. Animals: Domestic: • Cow, e.g. T. saginata, Sarcocystis
- 25. Paniker'sTextbook of MedicalParasitology Tab le 2: Parasites having indirect life cycle requiring one intermediate Box 2: Parasites causing autoinfection host and one definitive host Parasite Definitivehost Intermediatehost Protozoa Plasmodium spp. Female Anopheles Man mosquito Babesia Tick Man Leishmania Man. dog Sandfly Trypanosoma brucei Man Tsetse fly Trypanosoma cruzi Man Triatomine bug Toxoplasma gondii Cat Man Cestodes Taenia solium Man Pig T aenia saginara Man Cattle Echinococcus granulosus Dog Man Trematodes Fascia/a hepatica Man Snail Fascia/apsis buski Man, pig Snail Schistosoma spp. Man Snail Nematodes Trichinella spiralis Man Pig Wuchereria bancrofti Man Mosquito Brugia malayi Man Mosquito Dracunculus medinensis Man Cyclops Table 3: Parasites having indirect life cycle requiring two intermediate host and one definitive host Parasite lntermediate hosts Definitive host Fascia/a spp. Snail, plant Man Clonorchissinensis Snail, fish Man Diphyllobothrium latum Cyclops, fish Man Paragonimus westermani Snail, crustacean Man • Pig, e.g. T. solium, Trichinella spiralis • Dog, e.g. Echinococcus granulosus • Cat, e.g. Toxoplasma, Opisthorchis. Wild: • Wild game animals, e.g. trypanosomiasis • Wild felines, e.g. Paragonimus westermani • Fish, e.g. fish tapeworm • Molluscs, e.g. liver flukes • Copepods, e.g. guinea worm. Carrier: A person who is infected with parasite without any clinical or subclinical disease is known as carrier. He can transmit parasite to others. For example, all • Hymenolepisnana • Enterobiusvermicularis • Taenia solium • Strongyloidesstercoralis • Capillaria philippinensis • Cryptosporidium parvum anthroponotic infections, vertical transmission of congenital infections. • Self(autoinf ection) (Box 2): Finger-to-mouth transmission, e.g. pinworm Internal reinfection, e.g. Strongyloides. • MODES OF INFECTION Oral transmission: The most common method of transmission is through oral route by contaminated food, water, soiled fingers, or fomites. Many intestinal parasites enter the body in this manner; the infective stages being cysts, embryonated eggs, or larval forms. Infection with E. histolytica and other intestinal protozoa occurs when the infective cysts are swallowed. Skin transmission: Entry through skin is another important mode of transmission. Hookworm infection is acquired, when the larvae enter the skin of persons walkingbarefooted on contaminated soil. Schistosomiasis is acquired when the cercarial larvae in water penetrate the skin. Vector transmission: Many parasitic diseases are transmitted by insect bite, e.g. malaria is transmitted by bite offemaleAnophelesmosquito, filariasis is transmitted by bite of Culex mosquito. A vector could be a biological vector or a mechanicalvector. Direct transmission: Parasitic infection may be transmitted by person-to-person contact in some cases, e.g. by kissing in the case ofgingivaJ amebae and bysexual intercourse in trichomoniasis. Vertical transmission: Mother to fetus transmission may take place in malaria and toxoplasmosis. Iatrogenic transmission: It is seen in case of transfusion malaria and toxoplasmosis after organ transplantation. • PATHOGENESIS Parasitic infections may remain inapparcnt or give rise to clinical disease. A few organisms, such as E. histolytica may live as surface cornmensals, without invading the tissue. • Clinical infection produced by parasite may take many forms: acute, subacute, chronic, latent, or recurrent. • Pathogenic mechanisms, which can occur in parasitic infections are: - Lytic necrosis: Enzymes produced by some parasite can cause lyric necrosis. E. histolylica lyses intestinal cells and produces amebic ulcers.
- 26. - Trauma: Attachmentof hookworms on jejunal mucosa leads to traumatic damage of villi and bleeding at the site of attachment. Allergic manifestations:Clinical illness may be caused by host immune response to parasitic infection, e.g. eosinophilic pneumonia in Ascaris infection and anaphylactic shock in rupture ofhydatid cyst. Physical obstruction: Masses of roundworm cause intestinal obstruction. Plasmodium falciparum malaria may produce blockage of brain capillaries in cerebral malaria. inflammatoryreaction:Clinicalillness maybe caused by inflammatory changes and consequent fibrosis, e.g. lymphadenitis in filariasis and urinary bladder granuloma in Schistosoma haemalobium infection. Neoplasia: Afewparasitic infection have been shown to lead to malignancy. The liver fluke, Clonorchis may induce bile duct carcinoma, and S. haematobium may cause urinary bladder cancer. Space occupying lesions: Some parasites produce cystic lesion that may compress the surrounding tissue or organ, e.g. hydatid cyst. • IMMUNITY IN PARASITIC INFECTION Like other infectious agents, parasites also elicit immunoresponses in the host, both humoral as well as cellular (Fig. 1). But immunological protection against parasitic in fections is much less efficient, than it is against bacterial or viral infections. Several factors may contribute LO this: Compared to bacteria a nd viruses, parasites are enormously larger or more complex structurally and antigenically, so that immune system may not be able to focus attack on the protective anrigens. • Many protozoan parasites arc intracellular in location, and this protects them from immunological attack. Several protozoa and helminths live inside body cavities. 1h.is location limits the efficiencyofimmunologicalattack. • Once the parasitic infection is completely eliminated, the host becomes again susceptible to reinfection. This type ofimmunityto reinfection is dependent on the continued presence ofresidual parasite population and is known as "premunition". • Antibodies belonging to different immunoglobulin classes are produced in response to parasitic infections. Selective tests for immunoglobulin M (IgM) are helpful in differentiating current infections from old infections. Excessive IgE response occurs in helminthiasis. A characteristic cellular response in helminth parasite is eosinophilia both local and systemic {Fig. 1). • Parasites have evolved to be closely adapted to the host and most parasitic infections are chronic and show a degree ofhost specificity. For example, malarial parasites General Introduction: Parasitology Fig. 1: Eosinophils surroundingschistosomulum (an example of immune attack in bloodstream) Box 3: Parasites exhibiting antigenic variations • Trypanosoma bruceigombiense • Trypanosoma brucei rhodesiense • Plasmodium spp. • Giardia lamblia. of human, bird and rodents are confined to their own particular species. • Parasites like trypanosomes exhibit antigenic variation within the host. This genetic switch protects them from antibodies. Similar mechanism may be operative in the recrudescences in human malaria (Box 3). • Some parasites adopt antigenic disguise. Their surface antigens are so closely similar to host components that they are not recognized as foreign by the immune system. • Some infections may produce immunodeficiency due to extensive damage to the reticuloendothelial system, as in case ofvisceral leishmaniasis. The fact that immunity normally plays an important role in the containment ofparasitic infections is illustrated by the florid manifestations caused by opportunistic parasites such as Pneumocystis jirovecii and T. gondii, when the immune response is inadequate as in acquired immunodeficiency syndrome (AIDS) and other immunodeficiencies. • IMMUNE EVASION Allanimal pathogens, including parasitic protozoa and worms have evolved effective mechanism to avoid elimination by the host defense system as described in Table 4 . • VACCINATION No effective vaccine for humans has so far been developed against parasites d ue to their complex life cycles, adaptive responses and antigenic variation, great progress has been
- 27. Paniker'sTextbook ofMedical Parasitology Table4: Parasite escape mechanisms Parasiteescape mechanisms E xample Intracellular habitat Malarial parasite, Leishmania Encystment Toxop/asma Trypanosoma cruzi Resistance to microbial phagocytosis Leishmania Masking ofantigens Schistosomes Variation ofantigen Trypanosomes Plasmodium spp. Suppression of immune response Trichinella spirahs Schistosoma mansoni Malarial parasite Interference by polyclonal Trypanosomes activation Sharing ofantigens between parasite Schistosomes and host-molecular mimicry Continuous turnover and release of Schistosomes surface antigens ofparasite made in identifying protective antigens in malaria and some other infections, with a view to eventual development of prophylactic vaccines. • LABORATORY DIAGNOSIS Most of the parasitic infection cannot be conclusively diagnosed. On the basis of clinical features and physical examination laboratory diagnosis depends upon: • Microscopy • Culture . Serological test . Skin test . Molecular method . Animal inocuJation . Xenodiagnosis • Imaging . Hematology. Microscopy An appropriate clinical specimen should be collected for definitive diagnosis ofparasitic infections. • Following specimens are usually examined to establish a diagnosis: Stool Blood Urine Sputum Cerebrospinal fluid (CSF) Tissue and aspirates - Genital specimens. Stool Examination Examination of stool is very important for the detection of intestinal infections like Giardia, Enlamoeba, Ascaris, Ancylostoma, etc. Cysts and lrophozoites of E. histolytica, C. lamblia can be demonstrated in feces. Eggs of roundworm and tapeworm are also found in stool. The larvae are found in the feces in S. slercoralis infection (Table 5). For further details, refer to Chapter 23. Blood Examination Examination ofblood is ofvital importance for demonstrating parasites which circuJate in blood vessels (Table 6). Malarial parasite is confirmed by demonstration of its morphological stages in the blood. Urine Examination The characteristic lateral-spined eggs of S. haematobium and trophozoites of T. vagina/is can be detected in urine. Microfilaria of W bancrofti are often demonstrated in the chylous urine (Box 4). Sputum Examination lhe eggs ofP. westermani are commonly demonstrated in the sputum specimen. Occasionally, larvaJ stages ofS. s/ercoralis and A. lumbricoides may also be found in sputum. Cerebrospinal Fluid Examination Some protozoa like T. brucei, Naegleria, Acanthamoeba, Balamulhia and Angiostrongylus can be demonslrated in the CSF. Tissue and AspiratesExamination The larvae of Trichinella and eggs of Schistosoma can be demonstrated in the muscle biopsy specimens. By histopathological examination of brain, Naegleria and Acanthamoeba can be detected. In kala-azar, Leishman- Donovan (LO) bodies can be demonstrated in spleen and bone marrow aspirate. Trophozoites of Giardia can be demonstrated in intestinal aspirates. Trophozoites of E. histolytica can be detected in liver pus in cases of amebic liver abscess. Genital Specimen Examination Trophozoites of T. vagina.Lis are found in the vaginal and urethraldischarge. Eggs of E. vermicularis are found in anal swabs.
- 28. General Introduction: Parasitology Table5: Parasites and their developmental stages found in stool Cysts/Trophozoites • Entamoeba histolytica • Giardia lamblia • Balantidium coli • Sarcocystisspp. • lsospora be/Ii • C yclospora cayetanensis • Cryptosporidium parvum Eggs Cestodes • Taenia spp. • Hymenolepis nana • Hymenolepis diminuta • Oipy/idium caninum • Oiphylloborhrium /atum T rematodes • Schistosoma spp. • Fasciolopsis buski • Fascia/a hepatica • Fascia/a gigantica • Clonorchissinensis Table 6: Parasites found in peripheral blood film Protozoa • Plasmodium spp. • Babesia spp. • Trypanosoma spp. • Leishmania spp. Box 4: Parasites found in urine • Schistosoma haematobium • Wuchereria bancrofti • Trichomonasvagina/is Culture Nematodes • Wuchereria bancrafti , Brugia malayi • Loaloa • Mansonella spp. Larvae Adultworms • Gasrrodiscoides hominis Strongyloides stercoralis • T aeniasolium • Heterophyes heterophyes • Metagonimusyokogawai • Opisthorchisspp. Nematodes • Trichuris trichiura • Enterobius vermicularis • Ascaris lumbricoides • Ancylostoma duodenale • Necatoramericanus • T richosrrongylus orientalis • Taenia saginata • Oiphyllobothrium latum • Ascaris /umbricoides • Enrerobiusvermicularis • Tr/chine/la spiro/is Table 7: Antigen detection in parasitic diseases • Galactose lectin antigen • Giardia-specific antigen 65 • WKKand rk39 antigen • HRP-2 antigen • Vivax specific pLDH • 200 kDa Ag and OG4C3 antigen Entamoeba histo/ytica Giardia lamblia Leishmania donovani Plasmodium falciparum Plasmodium vivax Wuchereria bancrofti Abbreviations, Ag, antigen; HRP-2, histidine-rich protein 2; pLDH, P. folciparum lactate dehydrogenase; rk39, recombinant kinesin 39;WKK, Witebsky, Klingenstein and Kuhn by rapid immunochromatographic test. Filarial antigens are detected in current infection by enzyme-linked immunosorbcnt assay (ELISA) (Table 7). Some pa rasites like Leishmania, Entamoeba a nd Trypanosoma can be cultured in L he laboratory in various axenic and polyxenic media. Antibody Detection The following antibody detection procedures are useful in detecting various parasitic infections like amebiasis, echinococcosis and leishmaniasis in man: Serological Tests Serological tests are helpful for the detection and surveillance of many protozoa! and helminthic infections. These tests are basically of two types: 1. Tests for antigen detection 2. Tests for antibody detection. Antigen Detection Malaria antigen like P. falciparum lactate dehydrogenase (pLDI I) and histidine-rich protein 2 (HRP-2) are detected • Complement fixation test (CFT) • Indirect hemagglutination (IHA) • Indirect immunofluoresccnt antibody (IFA) test • Rapid immunochromatographic test (ICT) • Enzyme-linked immunosorbent assay test (ELISA). Skin Test Skin tests are performed by injecting parasitic an tigen intradermally and observing the reaction. In immediate hypersensitivity reaction, wheal and flare response is seen within 30 minutes of infection, whereas erythema and
- 29. Paniker'sTextbook of MedicalParasitology Box 5: Important skin tests done in parasitology • Casoni'stest done in hydatid disease • Montenegro test or leishmanin test done in kala-azar • Frenkel's test done in toxoplasmosis • Fairley·s test done In schistosomiasis • Bachman intradermal testdone in trichinellosis. induration seen after 48 hours ofinjection is called as delayed hypersensitivity reaction (Box 5). Molecular Diagnosis Molecular methods most frequently used to diagnose human parasitic infection are deoxyribonucleic acid (ONA) probes, polymerase chain reaction (PCR) and microarray technique. 1hese tests are very sensitive and specific. Animal Inoculation It is useful for the detection ofToxoplasma, Trypanosoma and Babesia from the blood and other specimens. Xenodiagnosis Some parasitic infection like Chagas disease caused by T. cruzi can be diagnosed by feeding the larvae ofreduviid bugs with patient's blood and then detection of amastigotes of T. cruzi in their feces. Imaging Imaging procedures like X-ray, ulcrasonography (USG), computed tomography (CT) scan and magnetic resonance imaging (MRI) are now being extensively used for diagnosing various parasitic infections like n eurocysticercosis and hydatid cyst disease. Hematology Anemia isfrequently seen in hookworm infection and malaria. Eosinophilia is frequently present in helminthic infections. HypergammaglobuJinemia occurs in visceral leishmaniasis. Leukocytosis is seen in am ebic liver abscess. KEY POINTS • Leeuwenhoek in 1681, first observed the parasite Giardia in stools. Laveran in 1880, discovered malarial parasite and Ronald Ross in 1897 showed the transmission of malaria by mosquitoes. • Protozoa belong to kingdom Protista and helminths belong to kingdom Animalia. • Definitive host: The host in which the adult stage lives or the sexual mode of reproduction takes place. • Intermediate host: The host in which the larval stage of the parasite lives or the asexual multiplication takes place. • Zoonoses: Diseases which can be transmitted to humans from animals, e.g. malaria, leishmaniasis, trypanosomiasis and echinococcosis. • Parasites like trypanosomes exhibit antigenic variation within the host. • Parasites like Ascaris and Echinococcus cause allergic manifestations in the host. • Innate immunity against parasite may be genetic or by nonspecific direct cell-mediated or by complement activation. • Acquired immunity in parasitic infections is by generating specific antibodies and effector T-cells against parasitic antigens. • Diagnosis of parasitic infections are made by direct identification of parasite in specimens like stool, blood, urine, bone marrow, CSF, sputum, etc. • Serological tests are also useful in diagnosis by detection of parasite-specific antibody and antigen. • Other diagnostic modalities include imaging, molecular methods like PCR, skin test and xenodiagnosis. REVIEW QUESTIONS 1. Write short notes on: a. Parasites b. Host c. Host-parasite relationship d. Zoonoses e. Immune evasion mechanism of the parasites. 2. Discuss briefly the laboratory diagnosis of parasites. 3. Describe immunity in parasitic infections. 4. Differentiate between: a. Direct and indirect life cycle b. Definitive host and intermediate hosts MULTIPLE CHOICE QUESTIONS 1. Definitive host isone a. In which sexual multiplication takes place and harbors adult form b. In which asexual multiplication takes place and harbors adult form c. In which sexual multiplication takes place and harbors larval form d. In which asexual multiplication takes place and harbors adult form 2. Autoinfection is seen in all except a. Hymeno/epis nano b. Enterabius vermicularis c. Taenia so/ium d. Ascaris lumbricoides
- 30. 3. Antigenic variationis exhibited by a. E ntamoeba b. Schistosoma c. T rypanosoma d. Leishmania 4. Which parasite enters, the body by piercing the skin a. Trichuris trichiura b. Ascaris c. Necatoramericanus d. Plasmodium 5. Which parasitic infection leadsto malignancy a. Babesiosis b. Clonorchis sinensis c. Trypanosoma cruzi d. Schistosoma haematobium 6. Xenodiagnosis is useful in a. Wuchereria bancrofti b. Trypanosoma cruzi c. Trichinella spiralis d. All of the above 7. The following are zoonotic disease except a. Leishmaniasis b. Balantidiasis c. Scabies d. Taeniasis General Introduction: Parasitology 8. Two hosts are required in a. Taenia solium b. Entamoeba histolytica c. Trichuris trichiura d. Giardia 9. Which of the following parasite passes its life cycle t hrough three hosts a. Fascia/a hepatica b. Fascia/a buski c. Schistosoma haematobium d. Clonorchis sinensis 10. Man is the intermediate host for a. Strongyloides stercoralis b. Plasmodium vivax c. Entamoeba histolytica d. Enterobius vermicularis Answer 1. a 2. d 8. a 9. d 3. C 10. b 4. C 5. b 6. d 7. C
- 31. CHAPTER 2 • INTRODUCTION •Single-celled eukaryotic microorganisms belonging to kingdom Protista are classified asProtozoa (Greekprotos: first; zoon: animal). • Parasitic protozoa are adapted to different host species. • Out of 10,000 species ofparasitic protozoa, man harbours only about 70 species. • GENERAL FEATURES • The single protozoa!cell performs all functions. • Most of the protozoa are completely nonpathogenic but few may cause major diseases such as malaria, leishmaniasis and sleepingsickness. • Protozoa like Cryptosporidium parvum and Toxoplasma gondii are being recognized as opportunistic pathogens in patients affected with human immunodeficiency virus (lllV) and in those undergoing immunosuppressive therapy. • Protozoa exhibit wide range ofsize (1- 150 µ111), shape and structure; yet all possess essential common features • The differences between protozoa and metazoa arc given in Table l . • STRUCTURE the typical protozoan cell is bounded by a trilaminar unit membrane, supported bya sheetofcontractile fibrils enabling the cell to move and change in shape. • CYTOPLASM It has two portions: 1. Ectoplasm: Outer homogeneous part that serves as the organ for locomotion and for engulfment of food by producing pseudopodia is called as the ectoplasm. It also helps in respiration, discharging waste material, and in providing a protective covering ofcell. 2. Endoplasm The inner granular portion of cyLOplasm that contains nucleus is called endoplasm. The Table 1: Differences between protozoa and metazoa Protozoa Morphology Unicellular; a single "cell-like unit" Physiology A single cell performs Example all the functions: reproduction, digestion, respiration, excretion. etc. Ameba Multicellular; a number of cells, making up a complex individual Each special cell performs a particular function Tapeworm endoplasm shows number ofstructures: the Golgi bodies, endoplasmic reticulum, food vacuoles and contractile vacuoles. Contractile vacuoles serve to regulate the osmotic pressure. • NUCLEUS The nucleus is usually single but may be double or multiple; some species having as many as 100 nuclei in a single cell. lhe nucleus contains one or more nucleoli or a central ka1yosome. • The chromatin may be distributed along periphery (peripheral chromatin) or as condensed mass around the karyosome. • TERMINOLOGIES USED IN PROTOZOOLOGY • Chromatoid body: Extranuclear chromatin material is called chromatoid body (e.g. as found in Entamoeba histolytica cyst). Karyosome: Itis a deoxyribonucleic acid (DNA)containing body, situated peripherally or centrally within the nucleus and found in intestinal ameba, e.g. E. histolytica E.coli. Kinetoplast: Nonnuclear DNA present in addition to nucleus is called kinetoplast. It is seen in trypanosomes. Flagellum originates near the kinetoplast. Point of origin offlagellum is called as basal body. 1
- 32. • Cilia: The heseare fine, needle-like filaments, covering the entire surface of the body and are found in ciliates, e.g. Balantidium coli. • Trophozoile (trophos: nourishment): Active feeding and growing stage of the protozoa is called the trophozoites. It derives nutrition from the environment by diffusion, pinocytosis and phagocytosis. • REPRODUCTION Reproduction can be: • Asexual reproduction • Sexual reproduction. Reproduction usually occurs asexually in protozoans; however, sexual reproduction occu rs in ciliates and sporozoans. Asexual Reproduction Binaryfission: It is a method of asexual reproduction, by which a single parasite divides either longitudinally or transversalJy into two or more equal number of parasites. Mitotic division of nucleus is followed by division of the cytoplasm. In amebae, division occurs along any plane, but in flagellates, division is along longitudinalaxis and in ciliates, in the transverse plane (Fig. 1). Multiple fission or schizogony: Plasmodium exhibits schizogony, in which nucleus undergoes several successive divisions within the schizont to produce large number ofmerozoiles (Fig. I). Endodyogeny: Some protozoa like Toxoplasma, multiply by internal budding, resulting in the formation of two daughter cells. Longitudinal binary fission (Flagellates) Binary fission (Ameba) Protozoa Sexual Reproduction • Conjugation: In ciliates,thesexual process isconjugation, in which two organisms join together and reciprocally exchange nuclear material (e.g. Balanlidium coli). • Gametogony or syngamy: In Sporozoa, male and female gametocytes are produced, which after fertilization form the zygote, which gives rise to numerous sporozoites by sporogony (e.g. Plasmodium). • LIFE CYCLE Single host: Protozoa like intestinal flagellates and ciliates require only one host, within which they multiply asexually in trophic stage and transfer from one host to another by the cysticform. • Second host: In some protozoa like Plasmodium, asexual method of reproduction occurs in one host (man) and sexual method of reproduction in another host (mosquito). • CLASSIFlCATION OF PROTOZOA Protozoan parasites of medical importance have been classified into kingdom Protista, subkingdom Protozoa which is further divided into the following four phyla (Table 2): l. arcomastigophora 2. Apicomplexa 3. Microspora 4. Ciliophora The important protozoan pathogens of human are summarized in Table 3. Multiple fission (schizogony) ( Transverse binary fission (Ciliates) Plasmod1um Red blood cell Daughter Nuclei Disrupts cell wall and is released Fig. 1: Asexual reproduction in protozoans
- 33. Table 2: Classificationof protozoa Phylum Subphylum Sarcomastigophora Mastigophora (having one or more flagella) Superclass class Zoomastigophorea Sarcodina Rhizopoda Lobosea Apicomplexa Clliophora Microspora (pseudopodia present) Sporozoea Kinetofragminophorea Microsporea Subclass Gymnamebia Coccidia Piroplasmia Vestibuliferia order Kinetoplastida Retortamonadida Diplomonadida Trichomonadida Amebida Schizopyrenida Eucoccidia Piroplasmida Trichostomastida Microsporidia Suborder - Trypanosomatina Enteromonadina Diplomonadina Tubulina Acanthopodina Eimeriina Hemosporina Trichostomatina Apansporoblastina Genus Trypanosoma Leishmania Rerortamonas Chllomastix Enteromonas Giardia Trichomonas • Dientamoeba • Entamoeba • Endolimax • /odamoeba Acanthamoeba Naegleria Cryptosporidium • /sospora • Sarcocystis • Toxoplasma Plasmodium Babesia Balantidium Enterocytozoon Encephalitozoon Microsporum ~ ::I ~ 11> ... .,.- ;t >< ... tr 0 0 ';It:" 0 - 3::: 11> Q. ;.· ~ ~ iil .,. ;:::.- 0 0 '° '<
- 34. Table 3: Principalprotozoan pathogens of man species Habitat Disease Entamoeba Large intestine Amebic dysentery, histolytica amebic liver abscess Naegleria fowleri CNS Amebic meningoencephalltis Acanthamoeba CNS, eye Encephalitis, keratitis Giardia lamblia Small intestine Malabsorption, diarrhea Trichomonos Vagina, urethra Vaginitis, urethritis vagina/is Trypanosoma brucei Blood, lymph node, Sleeping sickness CNS T rypanosomo cruzi Macrophage of bone Chagas disease marrow, nerves, heart, colon, etc. Leishman,a Reticuloendothelial Kala-azar, Postkala-azar donovani system dermal leishmaniasis Leishmania tropica Skin Cutaneous leishmaniasis (oriental sore) Leishmania Naso-oral mucosa Mucocutaneous braziliensis leishmaniasis (espundia, chiclero's ulcer) Plasmodium spp. RBC Malaria Babesia microti RBC Babesiosis lsospora be/Ii Intestine Diarrhea in AIDS Cryptosporidium Intestine Diarrhea in AIDS parvum Balantidium coli Large intestine Dysentery Protozoa from the nagellates by the loss of the flagella. Two groups of amebae are of medical importance: 1. Amebae of the alimentary canal: The most important of these is E. histolylica, which causes intestinal and extraintestinal arnebiasis. Amebae are aJso present in the mouth. 2. Potentially pathogenic free-living amebae: Several species ofsaprophytic arnebae are found in soil and water. Two of these, (I) Naegleria and (2) Acanthamoeba are of clinicalinterest because they can cause eye infections and fatal rneningoencephalitis. Flagellates These protozoa have whip-like appendages called flagella as the organs of locomotion. 1he fibriJJar structure of flagelJa is identical with that of spirochetes and it has been suggested that they may have been derived from symbiotic spirochetes, which have become endoparasites. In some species, the flagellum runs parallel to the body surface, to which it is connected by a membrane called the undulating membrane. FlageUates parasitic for man are divided into two groups: 1. Kinetoplastida: These possess a kinetoplast from which a single flagellum arises. They are the hemoflagellates comprising the trypanosomes and Leishmania, which are transmitted by blood-sucking insects and cause systemic or locaJ infections. 2. Flagellates without kinetoplast: These bear multiple flagella. Giardia, Trichomonas and other luminal flagellates belong to this group. Because most of them live in the intestine, they are generally called intestinal Abbreviations: AIDS, acquired immunodeficiency syndrome; CNS, central nervous flagellates. system; RSC, red blood cell Phylum Sarcomastigophora Phylum Sarcomastigophora has been subdivided into two subphyla based on their modes of locomotion: 1. Sarcodina (sarcos meaningflesh orbody): It includes those parasites, which have no permanent locomotory organs, but move about with the aid of temporary prolongations of the body called pseudopodia (e.g. amebae). 2. Masrigophora (mastix meaning whip or flagellum): It includes those protozoa which possess whip-like flagella (e.g. Trypanosoma and Trichomonas). Amebae These protean animalcules can assume any shape and crawl along surfaces by means of foot-like projections called pseudopodia (literally meaning false feet). They are structurally very simple and are believed to have evolved Phylum Apicomplexa Phylum Apicomplexa was formerly known as Sporozoa. Members of this group possess, at some stage in their life cycle, a structure called the apical complex serving as the organ of attachment to host cells. • 1hey are tissue parasites. • 1heyhave a complex life cycle with alternating sexual and asexualgenerations. • To this group, belongs the malarial parasites (Suborder: Hemosporina, Family: Plasm odiidae), Toxoplasma, Sarcocystis, lsospora, and Cryptosporidium (Under rhe Suborder: Eimeriina), Babesia (Under the Subclass: Piroplasma) and the unclassified Pneumocystisjiro11ecii. Phylum Ciliophora These protozoa are motile by means ofcilia, which cover their entire body surface. The onJy human parasite in this group is Balantidium coli, which rarely causes dysentery.
- 35. Paniker'sTextbook of MedicalParasitology Phylum Microspora Phylum Microspora contains many minute intracellular protozoan parasites, which frequently cause disease in immunodeficient subjects. They m ay also cause illness in the immunocompetent, rarely. The zoological classification of protozoa is complex and is subject to frequent revisions. The classification described in the chapter is an abridged version of the classification proposed in 1980 by the Commillee on Systematics and Evolution of the Society of Protozoologists, as applied to protozoa ofmedical importance. IMPORTANT POINTS TO REMEMBER • Only protozoan parasite found in lumen of human small intestine: Giardia /amblia. • Largest protozoa: Balantidium coli. • Most common protozoan parasite: Toxoplasma gondii. KEY POINTS OF PROTOZOA • Protozoa are single-celled, eukaryotic microorganisms consisting of cell membrane, cytoplasm and nucleus. • Some protozoa have kinetoplast and flagella or cilia. • Amebae move about with temporary prolongations of the body called pseudopodia. • Hemoflagellates comprising of Trypan osoma and Leishmania possess a single flagellum and kinetoplast. • Luminal flagellates like Giardia and Trichomonas bear multiple flagella without kinetoplast. • Balantidium coli belongs to the Phylum Ciliophora, which is motile by cilia that cover its entire body surface. • Trophozoites are active feeding and growing stage of protozoa. • Cysts are resting or resistant stage of protozoa bounded by tough cell wall. • Protozoa multiply by both asexual and sexual modes of reproduction. • Malaria parasite, Toxoplasma and Cryptosporidium belong to phylum Apicomplexa or Sporozoa, which possess apical complex at some stage of their life cycle and have a complex life cycle with alternating sexual and asexual generations. • Microspora are intracellular protozoan parasites, which cause disease in immunodeficient patients. REVIEW QUESTIONS 1. Define Protozoa and describe their general characteristics. 2. Writeshort noteson: a. Classification of Protozoa b. Reproduction in Protozoa 3. Differentiate between Protozoa and Metazoa. MULTIPLE CHOICE QUESTIONS 1. Protozoa belong to kingdom a. Monera b. Protista c. Plantae d. Animalia 2. All are intercellular parasitesexcept a. Leishmania b. Plasmodium c. Toxoplasma d. None of theabove 3. Non-nuclear DNA present in addition to nucleus in protozoan parasite is a. Chromatid body b. Karyosome c. Kinetoplast d. Basal body 4. Entamoeba histolytica trophozoitesmultiply by a. Binary fission b. Schizogony c. Gametogony d. All ofthe above 5. In humans, malarial parasites multiply by a. Binary fission b. Budding c. Gametogony d. Schizogony 6. Which of the following is not a flagellate a. Naegleria b. Leishmania c. Giardia d. Dientamoeba Answer 1. b 2. d 3. C 4. a 5. d 6. a .l
- 36. J I I 3 • INTRODUCTION The wordameba is derived from the Greek word "amibe" meaning change. Amebae are structurally simple protozoans which have no fixed shape. They are classified under Phylum: Sarcomastigophora, Subphylum: Sarcodina, Superclass: Rhizopoda and Order: Amebida. 1he cytoplasm of ameba is bounded by a membrane and can be differentiated into an outer ectoplasm and inner endoplasm. Pseudopodia are formed by the ameba by thrusting out ectoplasm, followed by endoplasm. TI1ese are employed for locomotion and engulfment offood by phagocytosis. • Reproduction occurs by fission and budding. Cyst is form ed in unfavorable conditions and is usually the infective form for vertebrate host (e.g. Entamoeba histolytica). • Amebae are classified as either free-living or inteslinaJ amebae (Table 1). • Afew ofthe free-living amebae occasionally act as human pathogens producing meningoencephalitis and other infections, e.g. Naegleria and Acanthamoeba The parasitic amebae inhabit the alimentary canal Table 1: Classification of amebae Intestinalamebae • Entamoeba histolytica • Entamoeba dispar • Entamoeba coli • Entamoebapolecki • Entamoeba hartmanni • E ntamoebagingivalis • Endolimax nana • /odamoeba butschlii Note: All intestinal amebae are nonpathogenic, except Entomoeba histolytica Free-living amebae • Naegleria fowleri • Acanthamoeba spp. • Balamuthia mondrillaris Note: All free-living amebae are opportunistic pathogens • ENTAMOEBA HISTOLYTICA History and Distribution £. histolytica was discovered by Losch in 1875, wh o demonstrated me parasite in the dysenteric feces of a patient in St. Petersburg in Russia. • In 1890, William Osler reported the case of a young man with dysentery, who later died of liver abscess. • Councilman an d Lafleur in 1891 established the pathogen esis of intestinal and hepa tic amebiasis and introduced the terms "amebic dysentery"and "amebic liverabscess''. E. histolytica is worldwide in prevalence, being m uch more common in the tropics than elsewhere. It has been found wherever sanitation is poor, in all climatic zones from Alaska (61°N) to Straits ofMagellan (52°S). • lt has been reported d1at about l 0% of world population and 50% of the inhabitants of developing countries may be infected with the parasite. • The infection is not uncommon even in affluent countries, about l %ofAmericans being reported to be infected. While the majority of infected humans (80-99%) are asymptomatic, invasive amebiasis causes disabling illness in an estimated 50 million of people and causes 50,000 deaths annually, mostly in the tropical belt ofAsia, Africa and Latin America. • It is the third leading parasitic cause of mortality, after malaria and schisL osomiasis. • Epidemiologically, India can bedivided into three regions, depending on the prevalence ofintestinal amebiasis: l. High prevalence states (>30%): Chandigarh, Tamil adu and Maharashtra. 2. Moderate prevalence s la tes (10-30%): Punjab, Rajasd1an, Uttar Pradesh, Delhi, Bihar, Assam, West Bengal, Andhra Pradesh, Karnataka and Kerala. 3. Low prevalence states (<10%): Haryana, Gujarat, Himachal Pradesh, Madhya Pradesh, Odisha, Sikkim and Puducherry.
- 37. Paniker'sTextbook of MedicalParasitology Morphology E. histolytica occurs in three forms (Figs IA to E): 1. Trophozoite 2. Precyst 3. Cyst. Trophozoite Trophozoite is the vegetative or growing stage of the parasite (Fig. IA). lt is the only form present in tissues. . • • . 1t is irregular in shape and varies in size from 12-60 µm ; average being 20 µrn. It is large and actively motile in freshly-passed dysenteric stool, while smaller in convalescents and carriers. The parasite, as it occurs free in thelumen as a commensal is generally smaller in size, about 15-20 µm and has been called the minuta form Cytoplasm: Outer ectoplasm is clear, transparent and refractile. Inner endoplasm is finely granular, having a ground glass appearance. The endoplasm contains nucleus, food vacuoles, erythrocytes, occasionally leukocytes and tissue debris. Pseudopodia are finger-like projections form ed by sudden jerky movements of ectoplasm in one direction, followed by the streaming in of the whole endoplasm. Typical ameboid motility is a crawling or gliding movement and not a free swimming one. The direction of movement may be changed suddenly, with another pseudopodium being formed at a different site, when the whole cytoplasm flows in the direction of the new pseudopodium. The cell has to be attached to some surface or particle for it to move. In culture tubes, the trophozoites may be seen crawling up the side of the glass tube. • Pseudopodia formation and motility are inhibited at low temperatures. • Nucleus is spherical 4-6 µm in size and contains central karyosome, surrounded by clear halo and anchored to the nuclear membrane by fine radiating fibrils called the Linin network, giving a cartwheel appearance. The nucleus is not clearly seen in the living trophozoites, but can be clearly demonstrated in preparations stained with iron hematoxylin. Ectoplasm Endoplasm Ingested erythrocytes • 111e nuclear membrane is lined by a rim of chromatin distributed evenly as small granules. • The trophozoites from acute dysenteric stools often contain phagocytosed erythrocytes. This feature is diagnostic as phagocytosed red cells are not found in any other commensaJ intestinal amebae. • The trophozoites divide by binaryfission in every8 hours. • Trophozoiles survive up to 5 hours at 37°C and are killed by drying, heat and chemical sterilization. Therefore, the infection is not transmitted by trophozoites. Even if live trophozoites from freshly-passed stools are ingested, they are rapidly destroyed in stomach and cannot initiate infection. Precystic Stage Trophozoites undergo encystm ent in the intestinal lumen. Encystment does not occur in the tissues nor in feces outside the body. • Before encystment, the trophozoite extrudes its food vacuoles and becomes round or oval, about 10-20 µmin size. This is the precystic stage ofthe parasite (Fig. 18). • It contains a large glycogen vacuole and two chromatid bars. • It then secretes a highly retractile cyst wall around it and becomes cyst. CysticStage The cyst is spherical in shape about 10-20 µmin size. . . . The early cyst contains a single nucleus and two other structures: (1) a mass of glycogen and (2) 1-4 chromatoid bodies or chromidial bars, which are cigar- shaped refractile rods with rounded ends (Fig. l C). The chromatoid bodies are so called because they stain with hematoxylin, like chromatin. As the cyst matures, the glycogen mass and chromidial bars disappear and the nucleus undergoes two successive mitotic divisions to form two (Fig. lD) and then four nuclei. 1he mature cyst is, thus quadrinucleate (Fig. IE). The cyst wall is a highly refractile membrane, which makes it highly resistant to gastric juice and unfavorable environmental conditions. Chromidial bar Glycogen mass a Nucleus . m m Figs 1A to E: Entamoeba histolytica. (A) Trophozoite;(B) Precystic stage; (C) Uninucleate c . D (D) Binucleate cyst; and (E) M ature quadrinucleate cyst yst; l I' I
- 38. • The nucleiand chromidial bodies can be made out in unstained films, but they appear more prominently in stained preparations. With iron hematoxylin stain, nuclear chromatin and chromaroid bodies appear deep blue or black, while the glycogen mass appears unstained. When stained with iodine, the glycogen mass appears golden brown, the nuclear chromatin and karyosome bright yellow, and the chromatoid bodies appear as clear space, being unstained. Life Cycle £. histolytica passes its life cycle only in one host man (Flowchart 1 and Fig. 2). InfectiveForm Mature quadrinucleate cyst passed in feces of convalescents and carriers. The cysts can remai n viable u nder moist conditions for about IOdays. Man acquires infection by ingestion of cysts in contaminated food and water Amebae Mode ofTransmission Man acquires infection by swallowing food and water contaminated with cysts. • As the cyst wall is resistant to action of gastric juice, the cysts pass through the stomach undamaged and enter the small intestine. • Excystation: When the cyst reaches cecum or lower part ofthe ileum, due to the alkaline medium, the cyst wall is damaged by trypsin, leading to excystation. Flow chart 1:Life cycle of Entamoeba histolytica (schematic) r···········• Trophozo1tes in colon Metacystic trophozoites I t Metacyst·in small intestine I t Cysts ingested t-----------• Precyst • Cysts 1- 4 nuclei ; • Passed in feces ~ ------- Cysts in contaminated food or water ~---) Ingested cyst from contaminated food or water ! Fig. 2: Life cycle of Entamoeba histolytica
- 39. Paniker'sTextbook of MedicalParasitology • The cytoplasm gets detached from the cyst wall a nd ameboid movements appear causing a tear in the cyst wall, through which quadrinucleate ameba is liberated. This stage is called the metacyst (Fig. 2). • Metacystic trophozoites: The nuclei in the metacyst immediately undergo division to form eight nuclei, each ofwhich gets surrounded by its own cytoplasm to become eight small amebulae or melacystic trophozoites. • if excystation takes place in the small intestine, the metacysric trophozoites do not colonize there, but are carried to the cecum. • The optimal habitat for the metacystic trophozoite is the submucosal tissue ofcecum and colon, where they lodge in the glandular crypts and grow by binary fission (Fig. 2). • Some develop inro precystic forms and cysts, which are passed in feces to repeat the cycle. • The entire life cycle is, thus completed in one host. rn m ostofthe cases, E. histolyticaremains as a commensal in the large intestine without causing any ill effects. Such per ons become carriers or asymptomatic cyst passers and are responsible for maintenance and spread of infection in the community. Sometimes, the infection may be activated and clinical disease ensues. Such latency and reactivation are the characteristics of amebiasis. Pathogenesis and Clinical Features • E. hislolytica causes intestinal a nd extraintestinal amebiasis. • incubationperiod is highly variable. On an average, it ranges from 4 days to 4 months. • Amebiasis can present in different forms and degree of everity, depending on the organ affected and the extent ofdamage caused. Intestinal Amebiasis The lumen-dwelling amebae do not cause any illness. They cause disease only when they invade the intestinal tissues. This happens only in about 10% of cases of infection, the remaining 90% being asymptomatic. Not all strain s of£. hislolylica are pathogenic or invasive. Differentiation between pathogenic and nonpathogenic strains can be made by susceptibility to complement- mediated lysis and phagocytic activity or by the use of genetic markers or monoclonal antibodies and zymodeme analysis. Adherence: Amebic lectins (Gal/ Gal Ac lectin, 260 kDa surface protein of E. histolytica) mediates adherence to glycogen receptors ofcolonic mucosa. - Cytolysis: The metacystic rrophozoites peneo·ate the columnar epithelial cells in the crypts oflieberkuhn in the colon. Penetration of the ameba is facilitated Box 1: Factors affectingvirulence of Entamoeba histolytica • Amebic cysteine proteinases, which inactivate complement factor C3 and degrade cellular matrix and lgA is an important virulence factor. • Amebic lectin (Gal/GalNAc lectin) and ionophore protein are other virulence factors. • Host factors such as stress, malnutrition, alcoholism, corticosteroid therapy and immunodeficiency influence the course of infection. • Glycoproteins in colonic mucus block the attachment of trophozoites to epithelial cells, therefore alteration in the nature and quality of colonic mucus may influence virulence. • Virulence may also be conditioned by the bacterial flora in the colon. • Based on electrophoretic mobility of six lsoenzymes (acetylgluco- saminidase, aldolase, hexokinase, NAD-diaphorase, peptidase and phosphoglucomutase), E. histolyrica strains can be classified into at least 22 zymodemes. Of these only nine are invasive and the rest are noninvasive commensals. • It has been proposed that pathogenic and nonpathogenic strains though morphologically identical may represent two distinct species: (1) the pathogenic strains being E. histalyticaand (2) the nonpathogenic strains reclassified as E .dispar. Trophozoites of£ dispar contain bacteria, bur no red blood cells (RBCs). by the motility of the trophozoites and the tissue lytic activity of the am ebic cysteine proteases like histolysi.n, cathepsin B, metallocollagenase. Cysteine proteases degrade the extracellular matrix (ECM) component ofhost cells and immunoglobulin A(lgA) (Box 1) and also inactivates complement C3. - Ameba,pores are ionophore proteins of ameba capable of inserting ion channels into liposomes causing lysis oftarget cell membrane ofhost cells. Tissue necrosis is also caused by the lysosomal enzymes of the in0ammatory cells surrounding the trophozoites and proinflammatory cytokines like interleukin-8 (IL-8) and tumor necrosis factor-a (T F-a) released from these cells. Mucosa! penetration by the ameba produces discrete ulcers with pinhead centerand raised edges.Sometimes, the invasion remains superficial and heals spontaneously. More often, the ameba penetrates to submucosaJ layer and multiplies rapidly, causing lytic necrosis and thus forming an abscess. The abscess breaks down to form an ulcer. • Amebic ulcer is the typical lesion seen in intestinal amebiasis (Fig. 3). The ulcers are multiple and are confined to the colon, being most numerous in the cecum and next in the sigmoidorectal region. the intervening mucous membrane between the ulcers remains healthy. • Ulcers appear initially on the mucosa as raised nodules with pouting edges measuring pinhead to l inch. They later break down discharging brownish necrotic material containing large numbers oftrophozoites.
- 40. i I l_ Amebae Figs 3A andB: (A) Intestinal amebiasis: Specimen showing amebic ulcer in colon; (B) Flask-shaped amebic ulcer • The typical amebic uJcer isflask-shaped in cross section, with mouth and neck being narrow and base large and rounded. Multiple uJcers may coalesce to form large necrotic lesions with ragged and undermined edges and are covered with brownish slough. Base is formed by muscular coat (Figs 3A and B). • The ulcers generally do not extend deeper than submucosal layer, but amebae spread laterally in the submucosa causing extensive undermining and patchy mucosa! loss. Amebae are seen at the periphery of the lesions and extending into the surrounding healthy tissues. Occasionally, the ulcers may involve the muscular and serous coats of the colon, causing perforation and peritonitis. Blood vessel erosion may cause hemorrhage. • The superficial lesions generally healwithoutscarring, but the deep ulcers form scars which may lead to strictures, partial obstruction and thickening ofthe gut wall. • Ameboma: Occasionally, a gran ulomatous pseudotumoral growth may develop on the intestinal wall by rapid invasion from a chronic ulcer. This amebic granuloma or ameboma may be mistaken for are maU gnant tumor. Amebomas are most frequent at cecum and rectosigmoid junction (Box 2). Systemicmanifestations ofamebomaare rectaltenesmus, high fever, abdominal discomfort, anorexia and nausea. Clinicalfeatures ofintestinalamebiasis:The clinical picture covers a wide spectrum from noninvasive carrier state to fulminant colitis (Box3). • The incubation period is highly variable from 1- 4 months. • l he clinical course is characterized by prolonged latency, relapses and intermissions. • The typical manifestation ofintestinal amebiasis is amebic dysentery. This may resemble bacillary dysentery, but can be differentiated on clinical and laboratory grounds. Box 2: Lesions in chronic intestinal amebiasis • Small superficial ulcers involving only the mucosa. • Round or oval-shaped with ragged and undermined margin and flask- shaped in cross section. • Marked scarring of intestinal wall with thinning, dilatation and sacculation. • Extensive adhesionswith the neighboring viscera. • Formation of tumor-like masses ofgranulation tissue (ameboma}. Box 3: Complications and sequelae of intestinal amebiasis • Fulminanramebic colitis: - Toxic megacolon - Perianal ulceration - Perforation and generalized peritonitis • Ameblc appendicitis • Ameboma • Extraintestinalamebiasis: - Amebic hepatitis - Amebic liver abscess - Pulmonary amebiasis - Cerebral amebiasis - Splenic abscess - Cutaneous amebiasis - Genitourinary amebiasis - Pericardia! amebiasis Compared to bacillary dysentery, it is usually insidious in onset and the abdominal tenderness is less and localized (Table 2). • 1he stools are large, foul-smelling and brownish black, often with blood streaked mucus intermingled with feces. The red blood cells (RBCs) in stools are clumped and reddish-brown in color. Cellular exudate is scanty. Charcot-Leyden crystals are often present. E. histolylica trophozoitescan beseen containingingestederythrocyces. • The patient is usually afebrile and nontoxic.
- 41. Paniker'sTextbook of MedicalParasitology Table 2: Differential features of amebic and bacillary dysentery features amebic Bacillarydysentery Clinical Onset Slow Acute Fever Absent Present Toxicity Absent Present Abdominal Localized Generalized tenderness Tenesmus Absent Present Stool Frequency 6-8 per day Over 10 per day Odor Offensive Nil Color Dark red Bright red Nature Feces mixed with blood Blood and mucus with and mucus Iittle or no feces Consistency Not adherent Adherent to container Reaction Acid Alkaline Microscopy Cellular exudates Scanty Abundant Red blood cells Clumped, yellowish Discrete or in rouleaux, brown bright red Macrophages Few Several, some with ingested red blood cells Eosinophils Present Absent Charcot-Leyden Present Absent crystals Motile bacteria Present Absent Ameba Motile trophozoites with Absent ingested red blood cells • In fulminant colitis, there is confluent ulceration and necrosis ofcolon. The patient is febrile and toxic. • Intestinal amebiasis does not always result in dysentery Quite often, there may be only diarrhea or vague abdominal symptoms popularly called "uncomfortable belly" or "growling abdomen''. • Chronic involvement of the cecum causes a condition simulating appendicitis. ExtraintestinalAmebiasis The various extraintestinal lesions in amebiasis have been summarized in Flow chart 2 and depicted in Figure 4. He11atic amebiasis: Hepatic involvement is the most common excraintestinal complication ofamebiasis. Although trophozoites reach the liver in mostca esofamebicdysentery, only in a small proportion do they manage to lodge and Flow chart 2: Sites affected in amebiasis Lungs + Subphrenic abscess Diaphragm j I Pericardium Peritoneum Skin Liver - ---+-• Stomach Portal circulation Intestine Inferior vena cava Spleen Peritoneum I- Primary infection 1------t• Suprarenal _ in colon Kidney General Perianal skin Genitals Fig. 4: Specimen showing amebic liver abscess multiply there. 1n the tropics, about 2- 10% of the individuals infected with E. histolytica suffer from hepatic complications. • The history of amebic dysentery is absent in more than 50% ofcases. Several patients wi1.h amebic colitis develop an enlarged tender liver without detectable impairment of liver function or fever.lhis acute hepatic involvement (amebic hepatitis) may be due to repeated invasion by amebae from an active colonic infection or to toxic substances from the colon reaching the liver. It is probable that liver damage may not be caused directly by the amebae, but by lysosomal enzyme of lysed polymorphonuclear neutrophils and monocytes and cytokines from the inflammatory cells surrounding the trophozoites.
- 42. Amebic liver abscess: •In about 5-10% of persons with intestinal amebiasis, liver abscesses may ensue (Fig. 4). The center of the abscess contains thick chocolate brown pus (anchovy sauce pus), which isliquefied necroticliverussue.ltis bacteriologically sterile and free ofameba. At the periphery, there is almost normal liver tissue, which contains invading ameba (Flow chart 3A). • Liver abscess may be multiple or more often solitary, usuallylocated in the upper right lobe ofthe liver.Cardinal signs of amebic liver abscess is painful hepatomegaly. Fever is present in most cases. Anorexia, nausea, weight loss and fatigue may also be present. About third-fourth cases ofamebic liver abscess have leukocytosis (>10,000/ µL) and increased serum transaminases. Jaundice develops only when lesions are multiple or when they press on the biliary tract. • Untreated abscesses tend to rupture into the adjacent tissues through the diaphragm into the lung or pleural cavity, pericardium, peritoneal cavity, stomach, intestine, or inferior vena cava orexternallythrough abdominal wall and skin. • Amebic liver abscess is 10 times more frequent in adults than in children and three times more frequent in males than in females. Pulmonary amebiasis: Very rarely, primary amebiasis of the lung may occur by direct hematogenous spread from the colon bypassing the liver, but it most often follow extension Amebae ofhepatic abscess through the diaphragm and therefore, the lower part ofthe right lung is the usual area affected (Fig. 5). • Jlepatobronchial fistula usually results with expectorauon of chocolate brown sputum. Amebic empyema develop less often. • The patient presents with severe pleuritic chest pain, dyspnea and nonproducuve cough. Metastatic amebiasis: Involvement of distant organs is by hematogenous spread and through lymphatics. Abscesses in kidney, brain, spleen and adrenals have been noticed. Spread to brain leads to severe destruction of brain tissue and is fatal. Cutaneous amebiasis: It occurs by direct extension around anus, colostomy site, or discharging sinuses from amebic abscesses. Extensive gangrenous destruction of the skin occurs. The lesion may be mistaken for condyloma or epithelioma. Genitourinary amebiasis: The prepuce and glans are affected in penile amebiasis which is acquired through anal intercourse. Similar lesions in females may occur on vulva, vagina, or cervix by spread from perineum. The destructive ulcerative le ions resemble carcinoma. Laboratory Diagnosis Diagnosis ofIntestinal Amebiasis Stool examination: Intestinal amebiasis has to be differentiated from bacillary dysentery (Table 2). The stool Flow charts 3A and B: (A) Laboratory diagnosis of amebic liver abscess; (BJLaboratory diagnosis of Entamoeba histolytica • Microscopy of pus or aspirate + Stool examination • Microscopy • Macroscopy • Iodine stained preparatron • Trichome stained preparations to demonstrate trophozoite or c st A. Laboratory diagnosis of amebic liver abscess l I • Histopatholoigical examination of pus or aspirate Amebic liver abscess I l Serodiagnosis • IHA • ELISA • Latex agglut1na11on test • Radiological examination • X-ray • USG •CT scan • MRI B. Laboratory diagnosis of Entamoeba histolytica Intestinal amebiasis I 1 ., + Stool culture Media used • Boeck and Drbohlav • NIH polygenlc • Craig's • Nelson's • Robinson's Mucosal scrapings Wet mount Stained preparation + Serodiagnosis • IHA • ELISA • Latex agglutination test • Stool examination • Molecular diagnosis • DNA probe Abbreviations: CT. computed tomography; DNA. deoxyribonucleic acid: ELISA, enzyme-linked immunosorbent assay; IHA, indirect hemagglut,nation: MRI. magnetic resonance imaging; USG, ultrasonography
- 43. Paniker'sTextbook ofMedical Parasitology Lung-+--...,.,..-+- abscess ~ - - - - - -~ Subdiaphragmatic- -- -+ abscess Liver- -- - abscess Amebic-- ---M, ulcers colon Periappendiceal-- - -- abscess Fig. 5: Lesions of amebiasis Ameboma colon shouldbecollectedintoa widemouthcontainerandexamined without delay. It should be inspected macroscopically as well as microscopically (Flow chart 38). • Macroscopic appearance: The stool is foul-smelling, copious, sem iliquid, brownish -black in color and intermingled with blood and mucus. It does not adhere to the container. • Microscopic appearance: saline preparation: • The cellular exudate is scantyand consists of only the nuclear masses pyknotic bodies) of a few pus cells, epithelial cells and macrophages. • The RBCs are in clumps and yellow or brown-red in color. • Charcot-Leyden crystals are often present. These are diamond-shaped, clear and refractilc crystals (Fig. 6). • Actively motile trophozoites throwing pseudo- podia can be demonstrated in freshly-passed stool. Presence of ingested RBCs clinches the identity ofE. hislolytica. ucleus is not visible but a faint outline may be detected. • Cyst has a smooth and thin cell wall and contains round refractile chromatoid bars. Glycogen mass is not visible. .. Fig. 6: Charcot-Leyden crystals Iodine preparation: • For the demonstration of cysts or dead trophozoites, stained preparations may be required for the study of the nuclear character. Iodine-stained preparation is comm only employed for this purpose. The trophozoite of E. histolytica stains yellow to light brown. ucleus is clearly visible witl1 a central karyosome. The cytoplasm of the cystic stage shows smooth and hyaline appearance. uclear chromatin and karyosome appear bright yellow. Glycogen masses stain golden brown and chromatoid bars are not stained. Trichrome stain is useful to demonstrate intracellular features of both trophozoites and cysts. • Since excretion of cysts in the stool is often interminent, at least three consecutive specimens should be examined (Fig. 7). Mucosal scrapings: Scraping obtained by sigmoidoscopy is often contributory. Examination method includes a direct wet mount and iron hematoxylin and immunofluorescent staining with anti-E.hislolytica antibodies. Stool culture: Stool culture is a more sensitive method in diagnosing chronic and asymptomatic intestinal amebiasis. Culture of stools yields higher positivity for E. histolytica as compared to direct examination. Polyxenic culture is done in enriched medium which contains bacteria, protozoa, serum, starch, etc. for nourishment ofthe ameba. Media used for polyxenic culture include: • Boeckand Drbohlav's biphasic medium • ill polygenic medium • Craig's medium
- 44. , ·~.. ~ .. Left-E. histolytica trophozoite, minuta form-smaller, no ingested erythrocytes. Right-Trophozoite, magna (tissue invading) form containing ingested erythrocytes. Left-E. hislo/ytica uninucleate cyst. Middle-Binucleate cyst Right-quadrinudeate cyst.(Heidenhain's hematoxytin stain. Magnification 2000X) Fig. 7: Entamoeba histolytica as it appears in laboratory specimen • Nelson's medium • Robinson's medium • Balamuth's medium. Axenic culture is done in medium that does not require presence of other microorganisms. Diamond's axenic medium is commonly used. Axenic cultures are used for: • Studies ofpathogenicity • Antigenic characterization • Drug sensitivity ofameba. To obtain growth in these media 50 mg of formed stools or 0.5 mL of liquid stool containing cyst or trophozoites of ameba is inoculated and incubated at37°C. Serodiagnosis: Serological tests become positive only in invasive amebiasis. Antibody detection: Amebic antibodies appear in serum only in late stages of intestinal amebiasis. Test for antibodies in scrum help in diagnosis ofmainly extraintestinal infections. Serological tests include indirect hemagglutination assay (IHA), indirect fluorescent antibody (IFA), enzyme- linked immunosorbent assay (ELISA), counter-current immunoelectrophoresis (CIEP) and latex agglutination tests. Serum with antibody titer of 1:256 or more by IHA and 1:200 by IFAare considered to be significant. Amebic a ntigen detectton: Amebic antigen in serum are detected onlyin patientswith active infections and disappears after clinical cure. Antigen like Lipophosphoglycan (LPG) amebic lectin, serine rich E. histolylica protein (SREHP) are detected using monoclonal antibodies by ELISA. Amebae Stool antigen detection: Detection of coproantigen of E. histolytica in stool by microwell EUSA is more sensitive than stool examination and culture. Commercially available ELISA tests like Techlab E. histolytica ll to detect Entamoeba antigen are more easily performed and are being used with increasing frequency. Moleculardiagnosis: Recently, deoxyribonucleic acid (DNA) probes and radioimmunoassay have been used to detect E. histolytica in stool. It is a rapid and specific method. Real-limepolymerasechainreaction (RTPCR)is asensitive test for detection ofE. histolytica from pus of liver abscess. Diagnosis ofExtraintestinalAmebiasis Microscopy: Microscopic examination of pus aspirated from liver abscess may demonstrate trophozoite ofE. histolytica in less than 20% cases. T n case ofliver abscess, when diagnostic aspiration is done, the pus obtained from the center of the abscess may not contain ameba as they are confined to the periphery. The fluid draining after a day or two is more likely to contain the trophozoite. Aspirates from the margins of the abscess would also show the trophozoites. Cysts are never seen in extraintestinal lesions. Liver biopsy: Trophozoite of E. histolytica may b e demonstrated in liver biopsy specimen, in case of hepatic amebiasis or amebic hepatitis. Serological test: Serological test, are ofimmense value in the diagnosis of hepatitis amebiasis. Craig (1928) was the first to report a complement.fixation test in amebiasis. Subsequently a number of different serological tests have been developed including: • indirect hemagglutination (IHA) • Latex agglutination (LA) • Gel diffusion precipitation (GDP) • Cellulose acetate membrane precipitation (CAP) test • Counter-current immunoelectrophoresis (CJE) • Enzyme linked immunosorbent assay (ELJSA) While IHA and LA are highly sensitive, they often give false-positive results. They remain positive for several years even after successful treatment. Gel precipitation tests are less sensitive, but more specific. ELISAs are both sensitive and specific and tests like GDP and CIE become negative within 6 months ofsuccessful treatment. Stool examination: It is not of much value as E. histolytica cyst can be detected in stool in less than 15%cases ofamebic hepatitis. Radiological examination: OnX-ray, the right lobe ofthe liver is generallyfound to be situated at a higher level.
- 45. Paniker'sTextbook of MedicalParasitology • Radioisotope scan of the liver may locate the space- occupying lesions. • Ultrasonography (USG), computed tomography (CT) scan, or magnetic resonance imaging (MRI) of liver may be found useful in detection ofamebic liver abscess (Plow chart 3A). The diagnosis of amebic liver abscess is based on the detection (generally by USG or CT) of one or more space- occupying lesions in the liver and a positive serologic test for antibodies against £. histolytica antigens. When a patient has a space-occupying lesion of the liver and a positive amebic serology, it is highly sensitive (>94%) and highly specific (>95%) for the diagnosis of amebic liver abscess {Flow chart 3A). Immunity Infection with invasive strains includes both humoral and cellular immune responses. Local and systemic antibodies can be demonstrated within a week of invasive infection. All classes of immunoglobulins are produced but IgG is predominant. Immunoglobulin A plays an important role in Immoral immunity to E. histolytica to resist Gal/GalNAc lectin. Infection confers some degree of protection as evidenced by the very low frequency of recurrence of invasive colitis and liver abscess in endemic areas. The course and severity of amebiasis does not seem to be a ffected by human immunodefi ciency virus (HIV) infection. Serological response i hardly ever seen in infection with noninvasive zymodemes. Treatment Three classes ofdrug are used in Lhe treatment of amebiasis: 1. Luminal amebicides: Diloxanide furoate, iodoquinol, paromomycin and tetracycline act in the intestinal lumen but not in tissues. 2. Tissue amebicides: Emetine, chloroquine, etc. are effecLive in systemic infection, but less effective in the intestine. Dosage of chloroquine in amebic liver abscess is l g for 2 days followed by 5 g daily for 3 weeks. 3. Bothluminal andtissue amebicides: Metronidazole and related compounds like rinidazole and om idazole act on both sites and are the drug of choice for treating amebic colitis and amebic liver abscess. Note: Although metronidazole and tinidazole act on both the sites but neither of them reach high levels in the gut lumen; therefore, patients with amebic colitis or amebic liver abscess should also receive treatment with a luminal agent (paromomycin or iodoquinol) to ensure eradication of infection (Table 3). Paromomycin is the preferred agent. • Asymptomatic individuals with docum ented E. histolytica infection should also be treated because of the risks of Table 3: Recommended dosages of antiamebic drugs Drug Dosage Duration (In days} Amebicco/iris oramebicliverabscess Tinidazole 2 g/day orally 3 Metronidazole 750 mg three times a day, 5-10 orally or intravenous (IV) Intestinalamebiasis Paromomycin 30 mg/kg four times a day, 5-10 orally in three divided doses lodoquinol 650 mg orally, three times 20 a day developing amebic colitis or amebic liver abscess in the future and risk of transmitting the infection to others. Paromomycin or iodoquinol in the doses listed in the Table 3 should be used in these cases. • Oral rehydration and electrolyte replacement should be done wherever necessary. Aspiration of liver abscess can be done as an adjunct to medical treatment in case of imminent rupture. Prophylaxis General prophylaxis is as for all fecal-oral infections. Food and water have to be protected from contamination with human excreta. Detection and treatment of carriers and their exclusion from food handling occupations will help in limiting the spread ofinfection. • Health education and inclusion ofhealthy personal habits helps in control. • NONPATHOGENIC INTESTINAL AMEBA Entamoeba Coli E. coli was first described by Lewis (1870) and Cunningham {1871) in Kolkata and its presence in healthy persons was reported by Grassi (1878). . . . It is worldwide in distribution and a nonpathogenic conunensal.intestinalameba. It is larger than£. histolytica about 20-50 µm with sluggish motility and contains ingested bacteria but no red cells. The nucleus is clearly visible in unstained films and has a large eccentric karyosome and thick nuclear membrane lined with coarse granules ofchromatin (Figs 8A and B). Cysts are large, 10- 30 µm in size, with a prominent glycogen mass in the early stage. The chromatoid bodies are splinter-Like and irregular. The mature cyst has eight nuclei (Fig. 8C). The life cycle is the same as in E. histolytica except that it remains a luminal commensal without tissue invasion and is nonpathogenic.
- 46. --- -~ -- Nucleus with eccentric nucleolus ....::.i'---+-- Phagocytosed bacteria Nucleus m Nucleus Chromatoid bodies Amebae Cyst membrane Figs 8A to C: Schematic diagram of the morphological forms of Entamoeba coli (Heidenhain's hematoxylin magnification 2000X). (A) Vegetative form; (B) Binucleate cyst; and (C) Eight-nucleate cyst Entamoeba Hartmanni E. hartmanni occurs wherever E. histolytica is found. le is now considered ro be a separate species of nonparhogenic commensal intestinal ameba. • It is much smaller than E. histolytica, the trophozoirc measuring 4- 12 µm and cyst 5-10 µmin size (Fig. 9). • Trophozoites do nor ingest red cells and their motility is less vigorous. • 1he cyst resembles that ofEndolimax nana. Differential features of cyst and trophozoites of E. coli, E. hartmanni and E. histolytica are shown in Table 4. Entamoeba Gingivalis E. gingivalis was the first ameba of humans, discovered by Gros in 1849. • It is global in disn·ibution. • Only the trophozoite is found; the cystic stage being apparently absent. The trophozoite is about 10-20 µm, actively motile with multiple pseudopodia. • The cytoplasm contains food vacuoles with ingested bacteria, leukocytes and epithelial cells. • Nucleus is round with central karyosome lined by coarse chromatin granules. • The ameba lives in gingival tissues and is abundant in unhygienic mouths. [t is a commensal and is not considered to cause any disease. • It is transmitted by direct oral contact. • E. gingivalis have been found in bronchial washings and vaginal and cervical smears, where it can be mistaken for E. histolytica. ___.....,.._ _ Nucleus with central nucleolus Fig. 9: Trophozoite of Entamoeba hartmanni Endolimax Nana This common commensal ameba is widely distributed. • It lives in rhe human intestine. • The trophozoite is small (nana: small), less than 10 µmin size with a sluggish motility (Fig. lOA). • The nucleus has conspicuous karyosome connected to nuclear membrane by one or none coarse strands. • The cyst is small, oval and quadrinucleate with glycogen mass and chromidial bars, which are inconspicuous or absent (Fig. 108). • It is nonpathogenic. lodamoeba Butsch/ii This is widely distributed, though less common than E. coli and E. nana. The trophozoice is small, 6- 12 µm, with conspicuous nucleus (Fig. llA). • The prominent karyosome is half the size of the nucleus, having bull's eye appearance.
- 47. Paniker'sTextbook ofMedical Parasitology Table4: Differential features of intestinal Entamoeba E. hlstolytica 12- 60 Active E . coll 20-50 Sluggish E.hartmanni ________....,. 4--12 Active Trophozoire Size (µm) Motility Pseudopodia Cytoplasm Finger-shaped, rapidly extruded Clearly defined into ectoplasm and endoplasm Short, blunt, slowly extruded Differentiation, not distinct Finger-shaped, rapidly extruded Clearly defined into ectoplasm and endoplasm Inclusions Red blood cells (RBCs) present, no bacteria Bacteria and other particles, no RBCs Bacteria and other particles, no RBCs Nucleus Karyosome Nuclear membrane Cyst Not clearly visible in unstained films Small, central Visible in unstained films Large, eccentric Not visible in unstained films Small, eccentric Delicate, with fine chromatin dots Thick, with coarse chromatin granules Coarse chromatin granules Size (µm) 10-15 4 10-30 8 5-10 4 Nuclei in mature cyst Glycogen mass Seen in uninucleate, but not in quadrinucleate stage Seen up to quadrinucleate stage Seen in uninucleate, but not in quadrinucleate stage Chromidial 1-4 with crounded ends Splinter-like with angular ends Many with irregular shape Figs 1OA and B: Endolimax nana. (A) Vegetative form: and (B) Quadrinucleate cyst • The cyst is oval, uninucleate and has a prominent iodine staining glycogen mass (iodophilic body). Hence, the name lodamoeba. It is nonpathogen.ic {Fig. llB). The comparative morphology of amebae infecting humans is illustrated in Figure 12. • PATHOGENIC FREE-LIVING AMEBAE Among the numerous types of free-living amebae found in water and soil, a few are potentially pathogenic and can cause human infections. • Primary amebic meningoencephalitis: It is caused by ameboflagellate Naegleria (the brain-eatingAmoeba). • Granulomatous amebic encephalitis and chronic amebic keratitis: It is caused by Acanthamoeba. A few instances of granulomatous amebic encephalitis (GAE) caused bylyptomyxid ameba likeBalamuthiahave also been reported. While primary amebic meningoencephalitis El B ~ ~ ~--Food vacuole containing bacteria Nucleus __::,,,-.-~ - - - Glycogen containing vacuole Figs 11A and B: lodamoeba butschlii. (A) Vegetative form: and (B) Cyst (PAM) and chronic amebic keratitis (CAK) occur in previously healthy individual, GAE has been associated with immunodeficient patients. The term amphiwic has been used for organisms, which can multiply both in the body ofa host (endozoic) and in free- living (exozoic) conditions. Naegleria Fowleri It is the only species ofgenus Naegleria, which infects man.
- 48. :::: :c: . (.) ... II) ~ .Q TrophozoiteNucleus @ 0 Fig. 12: Comparative morphology of amebae infecting humans. showing trophozoite and cyst stages, as well as enlarged representation of their nuclear structure N. Jowleri causes the disease primary amebic meningoencephalitis (PAM), a brain infection that leads to destruction ofbrain tissue. History and Distribution N. Jowleri is named after Malcolm Fowler, who along with Carter described it first from Australia in 1965. • N. fowleri is a heat-loving (thermophilic) ameba that thrives in warm water at low oxygen tension and is commonly found in warm freshwater (e.g. lakes, rivers, and springs) and soil. • It is worldwide in distribution. Amebae Morphology N.Jowleri occurs in three forms: 1. Cyst 2. Ameboicl trophozoite form 3. Flagellate trophozoite form. Trophozoite stage: The rrophozoites occur in two forms, (I) ameboid and (2)jlagellate. Ameboid form: The ameboid form is about 10-20 µm, showing rounded pseudopodia (lobopodia), a spherical nucleus with big endosome and pulsating vacuoles. With electron microscopy, vacuoles appear to be densely granular in contrast to highly vacuolated body of ameba and are called as ameboslomes. They are used for engulfing RBCs and white blood cells (WBCs) and vary in number, depending on the species. • Ameboid form is the feeding, growing, and replicating form of the parasite, seen on the surface of vegetation, mud and water. • It is the invasive stage of the parasite and the infective form of the parasite. Flagellate form: The biflagellate form occurs when trophozoites are transferred to distilled water. • This transformation of trophozoites co bijlagellate pear- shaped form occurs within a minute. • tje flagellate can revert to the ameboid form, hence N. Jowleri is classified as amebojlagellate. Cyststage:Trophozoitesencystdue co unfavorable conditions like food deprivation, desiccation, cold temperature, etc. The cyst is spherical 7-10 µm in diameter and has a smooth double wall. They are the resting or the dormant form and can resist unfavorable conditions, such as drying and chlorine up to SO ppm. The cyst can withstand moderate heat (45°C), but die at chlorine levels of2 ppm and salinity of0.7%. Cysts and flagellate forms of N. Jowleri have never been found in tissues ofcerebrospinal fluid {CSF). Life Cycle Typically, infection occurs when people go swimming or diving in warm freshwater river or ponds and poorly maintained swimming pools or nasal irrigation using contaminated tap water (Fig. 13). • The life cycle of N. Jowleri is completed in the external environment. • The ameboid form of trophozoite multiplies by binary fission. In the last 10 years from 2002 to 2011, 32 infections were reported in the United States (US), and in India, a total of • 17 cases have been reported so far. Under unfavorable conditions, it forms a cyst and which undergoes excystation in favorable conditions.
- 49. Paniker'sTextbook ofMedical Parasitology (~ Flagellateform Man acquires infection during swimming Fig. 13: Life cycle of Naegleria fowleri • Flagellate form of trophozoite helps in the spread of N. Jowleri to new water bodies. Since the ameboid form is the invasive stage, hence, the flagellate forms revert to ameboid forms to become infective to man. Pathogenicity and Clinical Features Patients are mostly previously healthy young adults or children. • Human infection comes from water containing the amebae and usuallyfollows swimmingor diving in ponds. • The amebae invade the nasal mucosa and pass through the olfactory nerve branches in the cribriform plate into the meninges, and brain to initiate an acute purulent meningitis and encephalitis, called as primary amebic meningoencephalitis (PAM). • the incubation period varies from 2 days to 2 weeks. • In theincubation period, thepatient experiencesanosmia. • TI1e disease advances rapidly, causing fever, headache, vomiting, stiffneck, ataxia, seizure and coma. • Cranial nerve palsies, especially of the third, fourth and sixth nerves have also been documented. • The disease almost always ends fatally within a week (average 5 days). LaboratoryDiagnosis The diagnosis of PAM is based on the find ing of motile Naegleria trophozoites in wet moums offreshly obtained CSE Cerebrospinal fluid examination: The CSF is cloudy to purulent, with prominent neutrophilic leukocytosis, elevated protein and low glucose, resembling pyogenic meningitis. • Wet film examination ofCSF may show trophozoites. • Cysts are not found in CSF or brain. • At autopsy, trophozoites can be demonstrated in brain histologicalJy by immunofluorescent staining. Culture: N. Jowleri can be grown in several kinds of liquid axenic media or nonnutrient agar plates coated with Escherichia coli. Both trophozoites and cysts occur in culture. Molecular diagnosis: Newer tests based on PCR technology are being developed. Treatment The drug of choice is amphotericin B intravenously. It can also be instilled directly into the brain. • Treatment combining miconazole and suJfadiazine has shown limited success, only when administered early. • More than95% cases ofPAM are fatal despite oftreatment . Acanthamoeba Species A. culbertsoni {formerly, HartmannelLa culbertsoni) is the species most often responsible for human infection but other species like A. polyphagia, A. castellaniiand A. astromyxhave also been reported. Distribution This isan opportunisticprotozoan pathogen found worldwide in the environment in water and soil. • Approximately, 400 cases have been reported worldwide. Morphology Acanthamoeba exists as active trophozoite form and a resistant cysUc form. • Thetrophozoiteislarge, 20-50µmin sizeand characterized by spine-like pseudopodia (acanthopodia). • It differs from Naegleria in not having a flagellate stage and in forming cysts in tissues (Table 5). • The polygonal double-walled cysts are highly resistant. • The cysts are present in all types ofenvironment, all over the world. Life Cycle • Both trophozoitcs and cysts are infecUve. Human beingsacquire byinhalation ofcystortrophozoite, ingestion of cysts, or through traumatized skin or eyes (Fig. 14).
- 50. Table 5: Differentialfeatures of Naegleria and Acanthamoeba AmnCNmol6o Disease Primary amebic Granulomatous amebic meningoencephalitis encephalitis (GAE) and (PAM) keratitis portalofentry Nose Upper respiratory tract, cornea Clinical course Acute Subacute or chronic Pathogenicity Acute suppurative Granulomatous inflammation inflammation Morphological Three stages: (1) Two stages: (1) forms trophozoite, (2) cyst and trophozoite and (2) cyst (3) nagellate form flagellate form absent Trophozoite 10-20 µm, with a single 20-S0 µm, with spine-like pseudopodia pseudopodia Cyst 7- 10 µm, round with 1S- 25 µm, polygonal smooth wall double-walled with wrinkled surface Nuclear By promitosis, nucleolus Nuclear membrane division divides, nuclear dissolves membrane persists WBCinCSF Predominantly Predominantly neutrophils lymphocytes Abbrtviorions:CSF,cerebrospinal fluid; WBC, white blood cell Man acquires infection by inhalation and ingestion of trophozoites and cysts Trophozoite showing spinous acanthopodia Fig. 14: Life cycle ofAcanthamoeba culbertsoni • After inhalation ofaerosol or dust containing trophozoites and cysts, the trophozoites reach the lungs and from there, they invade the central nervous system through the bloodstream, producing granulomatous encephalitis (GAE). Pathogenesis and Clinical Features • Infection usually occurs in patients with immuno- deficiency, diabetes, malignancies, malnutrition, systemic lupus erythematosus (SLE), or alcoholism. Amebae • Theparasitespreads hematogenouslyinto central nervous system. Subsequent invasion ofthe connective tissue and induction ofproinflammatory responses lead to neuronal damage that can be fatal within days. • A postmortem biopsy reveals severe edema and hemorrhagic necrosis. Clinical Disease It presents chiefly as two chronic conditions: (1) keratitis and (2) encephalitis. • Acanthamoeba keratitis: An infection of the eye that typically occurs in healthy persons and develops from the entry ofthe amebic cyst through abrasions on the cornea. - Majority ofsuch cases have been associated with the use ofcontact lenses. - The picture resembles that ofsevere herpetic keratitis with a slow relapsing course, but the eye is severely painful in the amebic infection. Unilateral photophobia, excessive tearing, redness and foreign body sensation are the earliest signs and symptoms; disease is bilateral in some contact lens users. - Keratitis and uveitis can result in permanent visual impairment or blindness. • Granulomatous amebic encephalitis: It is a serious infection ofthe brain and spinal cord that typically occurs in persons with a compromised immune system. Granulomatous amebic encephalitis is believed to follow inhalation ofthe dried cysts. The incubation period is longand the evolution of the illness is slow. Clinical picture is that of intracranial space- occupying lesions with seizures, pareses and mental deterioration. • Disseminated infection: In immunocompromised states like acquired immunodeficiency syndrome (AIDS), a widespread infection can affect skin lungs, sinuses, and other organs independentlyor in combination. Laboratory Diagnosis • Diagnosis ofamebic lceratitis is made by demonstration of the cyst in corneal scrapings by wet mount, histology and culture. Growth can be obtained from corneal scrapings inoculated on nutrient agar, overlaid with live or dead Escherichia coli and incubated at 30°C. Rapiddiagnosiscan be made by identification ofamebaor cyst in corneal scraping by fluorescent microscopy using calcofluor white staining and !FA Lest ([FAT) procedure. • Diagnosis of GAE is made by demonstration of trophozoites and cysts in brain biopsy, culture and immunofluorescence microscopy using monoclonal antibodies.
- 51. Paniker'sTextbook of MedicalParasitology - Cerebrospinal fluid shows lymphocytic pleocytosis, slightly elevated protein levels, and normal or slightly decrea ed glucose levels. - Computed LOmography scan of brain provides inconclusive findings. Treatment In acanlhamoeba keralitis, current Lherapy involves topical administration ofbiguanide or chlorhexidine with or without diamidine agent. In severe cases, where vision is Lhreatened, penetrating keratoplasty can be done. o effective trealmenl is available for "GAE~ Multidrug combinations including pentamidine, sulfadiazine, rifampicin and lluconazole are being used with limited success. Balamuthia Mandrillaris B. mandrillaris, a leptomixid free-living am eba, is a newly identified species reported to cause GAE. Morphology It exists in ameboid trophozoite stage. The flagellate stage is absent • It is relatively large (12-60 µm), irregul ar in shape and actively motile by broad pseudopodia. • Cyst of B. mandrillaris are usually spherical (6-20 µm), surrounded by a three-layered cyst wall: ( 1) outer irregular ectocyst, (2) a middle mesocyst and (3) an inner endocyst round wall. Under light microscopy, it appears to have two walls: (1) an outer irregular wall and (2) an inner smooth wall. • Infection is transmitled through respiratory tract, skin lesions, or eyes. • Life cycle is similar to that ofAcanthamoeba spp. Clinical Disease It causes granulomatous amebic encephalitis in both healthy and immunocompromi sed hosts particularly in children and elderly. Laboratory Diagnosis Laboratory diagnosis isdone by identifying trophozoites of B. mandrillaris in the CSF and trophozoites and cysts in brain tissue. Polymerase chain reaction also gives reliable diagnosis. KEY POINTS OF AMEBAE • E. histolytica is found in human colon and is mainly asymptomatic. • Cyst contains glycogen mass and 1-4 chromatid bars. • Pathogenic strains are identified by genetic markers and zymodeme analysis. • Stools: In amebic dysentery, stool is copious, foul-smelling, brownish black often with blood-streaked mucus. • Amebic ulcers: Typical ulcers are discrete, flask-shaped, with ragged undermined margin, found in cecum and sigmoidorectal region. • Amebic granuloma or ameboma may develop from chronic ulcers. • Extraintestinal complications: Amebic hepatitis and liver abscess are the most common. • Abscesses in other organs such as lung, brain, spleen and genitourinary tract may result from hematogenous spread or by direct spread from hepatic lesion. • Diagnosis: By demonstration of trophozoites and cyst in stool and also by serological tests and imaging techniques in hepatic amebias1s. • Treatment: By metronidazole or tinidazole along with parmomycin, d1loxanide furoate, or chloroquine. • E. hartmanni, E. coli, E. gingivalis, E. nana, and lodamoeba are commensals and nonpathogenic amebae. • Naeg/eria and Acanthamoeba are pathogenic free-living ameba. • N. fowleri occurs in three forms: (1) cyst, (2) trophozoite and (3) nagellate. It causes PAM. • Acanthamoeba species cause amebic keratltis and also GAE in immunocompromised subjects. REVIEW QUESTIONS 1. Describe briefly the life cycle and laboratory diagnosis of Entamoeba histolytica. 2. Write short notes on: a. Extraintestinal amebiasls b. Free-living amebae 3. Differentiate between: a. Amebic dysentery and bacillary dysentery b. Enramoeba histolytica and Entamoeba coli c. Naegleria and Acanthamoeba MULTIPLE CHOICE QUESTIONS 1. The main reservoir of Entamoeba histolytica is a. Man b. Dirty water c. Dog d. Monkey
- 52. 2. The infectiveform of Entamoeba histolytica is a. Trophozoite b. Binucleate cyst c. Quadrinucleate d. None of the above 3. The pathogenicity of Entamoeba histolytica is indicaded by a. Zymodeme pattern b. Size c. Nuclear pattern d. ELISA test 4. M/C site for extra intestinal amebiasis is a. Liver b. Lung c. Brain d. Spleen 5. Amoebic liver abscess can be diagnosed by demonstrating a. Cyst in the sterile pus b. Trophozoites in the pus c. Cyst in the intestine d. Trophozoites in the feces 6. Stool ofamoebicdysentry has all ofthe following characteristics except a. Charcot-Leyden crystals b. Pyknotic bodies c. RBCs d. Ghost cell 7. The term ameboma is used to describe a. Amebic liver abscess b. Skin lesion due to draining amebic abscess Amebae c. Granuloma at ileocecal junction d. None of the above 8. True statement regarding Entamoeba histolytica is a. The trophzoites are infective to man b. Mature cyst has eccenteric nucleolus c. It can cause primary amebic encephalitis d. Cyst are resistant to chlorine concentration used in drinking water 9. All are nonpathogenic ameba living in the lumen of large intestine except a. Entamoeba coli b. Entamoeba hartmanni c. Endolimax nana d. Entamoeba gingivalis 1O. Chronic amebic keratitis in seen in a. Entamoeba histolytica b. Acanthamoeba c. Naegleria fowleri d. Hemoflagellates 11 . Etiologic agent ofgranulomatous amebic encephalitis is a. Entamoeba histolytica b. Acanthamoeba c. Naegleria d. Dientamoeba fragillis Answer 1. a 8. d 2. C 9. d 3. a 10. b 4. a 11. b 5. b 6. d 7. C
- 53. CHAPTER 4 Intestinal, Oraland Genital Flagellates • INTRODUCTION Parasitic protozoa, which possess whip-likeflagella as their organs of locomotion are called as flagellates and classified as: Phylum: Sarcomasrigophora Subphylum: Mastigophora Class: Zoomastigophora (mastix: whip) Depending on their habitat, they can be considered under: Lumen-dwellingflagellates: Flagellates fow1d in the alimentary tract and urogenital tract (Table 1). Hemojlagellates: Flagellates found in blood and tissues (Table 1). Most luminal flagellates are nonpathogenic commensals. Two ofthem cause clinical diseases: (1) Giardia lamblia, which can cause diarrhea, and (2) Trichomonas vaginalis, which can produce vaginitis and urethritis. Table 1: Flagellates Group Lumen-dwelling flagellates • Giardia lamblia • Trichomonas vagina/is • Trichomonas tenax • Trichomonas hominis • Chilomastix mesnili • Enteromonas hominis • Retortamonas intestinalis • Dientamoeba fragilis Hemoflagellates • Leishmania spp. • Trypanosoma brucei • Trypanosoma cruzi Habitat • Duodenum and jejunum • Vagina and urethra • Mouth • Large intestine (cecum) • Large intestine (cecum) • Large intestine (colon) • Large intestine (colon) • Large intestine (cecum and colon) • Reticuloendothelial cells • Connective tissue and blood • Reticuloendothelial cells and blood • GIARD/A LAMBLIA History and Distribution It is one of the earliest protozoan parasites to have been recorded. • The flagellate was first observed by Dutch scientist Antonie van Leeuwenhoek (1681) in his own stools. • It is named Giardia after Professor Giard of Paris and lamblia after Professor Lambie of Prague, who gave a detailed description ofthe parasite. • It is the most common protozoan pathogen and is worldwide in distribution. Endemicity is very high in areas with low sanitation, especially tropics and subtropics. Visitors to such places frequentlydevelop traveler's diarrhea caused by giardiasis through contaminated water. Habitat G. lamblia lives in the duodenum and upperjejunum and is the only protozoan parasite found in the lumen ofthe human small intestine (Box 1). Morphology It exists in two forms: 1. Trophozoite (or vegetative form) 2. Cyst (or cystic form). Box 1: Protozoa found in small intestine • Giardia Iambi/a • lsospora be/Ji • Cyclospora cayeranensis • Cryptosporidium parvum • Sarcocystis hominis and suihominis
- 54. Trophozoite The trophozoite isin the shape of a tennis racket (heart- shaped or pyriform-shaped) and is rounded anteriorly and pointed posteriorly (Figs I and 2A and B). . . . . It measures 15 pm x 9 mcg wide and 4 mcg thick. Dorsally, it is convex; and ventrally, it has a concave sucking disk, which helps in its attachmem to the intestinal mucosa. It is bilaterally symmetrical and possesses: - One pair ofnuclei - Four pairs of flagella Blepharoplast, from which the flagella arise (four pairs) - One pair ofaxostyles, running along the midline - Two sausage-shaped parabasal or median bodies, lying transversely posterior to the sucking disk. 'The trophozoite is motile, with a slow oscillation about its long ax.is, often resemblingfalling leaf ~ Ventral aspect 0 ~ Lateral 5 aspect 0 Fig. 1: Giardia lamblia in duodenal fluid wet preparation. Magnification 1500X Sucking-- • ~ =-i"~ disc Basal bodies of flagella Nucleus Parabasal body m Flagella (4 pairs) Sucking disc Nucleus Flagella (4 pairs) m Intestinal, Oral and Genital Flagellates Cyst It is the infective form ofthe parasite (Fig. 2C). • The cyst is small and oval, measuring 12 mcgx 8 mcg and is surrounded by a hyaline cyst wall. • Its internal structure includes two pairs ofnuclei grouped at one end. Ayoung cyst contains one pair ofnuclei. • The axostyle lies diagonally, forming a dividing line within cyst wall. Remnants of the flagella and the sucking disc maybe seen in the young cyst. Life Cycle Giardia passes its life cycle in one host. Infective Form Mature cyst. Mode ofTransmission • Man acquires infection by ingestion of cysts in conta- minated water and food. • Ingestion ofas far as 10 cystsis sufficient to cause infection in a man. Children are commonly affected. • Direct person-to-person transmission may also occur in children, male homosexuals and mentally ill persons. Enhanced susceptibility to giardiasis is associated with blood group A, achlorhydria, use of Cannabis, chronic pancreatitis, malnutrition, and immune defects such as 19A deficiency and hypogammaglobulinemia. • Within half an hour of ingestion, the cyst hatches out into two trophozoites, which multiply successively by binary fission and colonize in the duodenum (Fig. 3). • The trophozoites live in the duodenum and upper part of jejunum, feeding bypinocytosis. c Axostyle Thick cyst wall Nuclei Nucleoli wv.- 4- Parabasal bodies Figs 2A to C: Trophozoite. (A) Ventral view; (B) Lateral view: and (Cl Quadrinucleate cyst
- 55. Paniker'sTextbook ofMedical Parasitology Manacquires infection by ingestion of cyst in contaminated food and waler Trophozo,tes multiply by binary fission.Remain adhered to duodenal mucosa Excyslalion occurs in the duodenum and 2 trophozoites hatch out Fig. 3: Life cycle of Giardia lamblia • During unfavorable conditions, encystment occurs usualJy in colon (Fig. 3). • Cysts are passed in stool and remain viable in soil and water for severalweeks. • There may be 200,000 cysts passed per gram offeces. • Injective dose is 10-100 cysts. Pathogenicity and Clinical Features G. lamblia is typically seen within the crypts of duodenal and jejuna! mucosa. It does not invade the tissue, but remains tightly adhered to intestinal epith elium by means of the sucking disk. • They may cause abnormalities of villous architecture by cell apoptosis and increased lymphatic infiltration of lamina propria. Loss of brush border epithelium of intestine leads to deficiency of enzymes including disaccharides. • Variant-specific surface proteins (VSSPs) of Giardia play an important role in virulence and infectivity of the parasite.Antigenic variation helps the parasite in evasion ofhost immune system. Box 2: Protozoan parasites causing diarrhea • Giard/a lamblia • Entamoeba histolytica • Cyclospora cayetanensis • Cryptosporidium parvum • lsospora be/Ii • Often they are asymptomatic, but in some cases, Giardia may lead to mucus diarrhea,fat malabsorption (stearorrhea), dull epigastric pain, belching and flatulence. The stool contains excess mucus and fat but no blood (Box 2). • Children may develop chronic diarrhea, malabsorption of fat, vitamin A, vitamin B12,folic acid, protein, sugars like xylose disaccharides, weight loss and sprue-like syndrome. Chronic giardiasis may be due to failure to develop irnmunoglobulin A (IgA) against specific Giardia antigen. • Occasionally, Giardia may colonize the gallbladder, causing biliary colic and jaundice. • Incubation period is variable, but is usuallyabout2 weeks.
- 56. L t Laboratory Diagnosis Stool Examination Giardiasiscan be diagnosed by identification of cysts of Giardia lamblia in the formed stools and the trophozoites and cysts ofthe parasite in diarrheal stools (Flow chart 1). • On macroscopicexamination, fecal specimenscontaining G. lamblia may have an offensive odor, are pale colored and fatty, and float in water. • On microscopic examination, cysts and trophozoites can be fow1d in diarrheal stools by saline and iodine wet preparations. • Often multiple specimens need to be examined and concentration techniques like formal etheror zinc acetate are used. In asymptomatic carriers, only the cysts are seen. Enterotest (String Test) A useful method for obtaining duodenal specimen is enterotest. A coiled thread inside a small weighted gelatin capsule is swallowed by the patient, after attaching the free end of the thread in the cheek. The capsule passes through the stomach to the duodenum. After 2 hours, the thread is withdrawn, placed in saline, and is mechanically shaken.1 he centrifuged deposit ofthe saline is examined for Giardia. The use of enterotest is not recommended because of the very high cost ofthe test. Serodiagnosis Antigen detection: Enzyme-linked immunosorbent assay (ELISA), immunochromatographic strip tests and indirect immunofluorescence (IIF) tests using monoclonal antibodies have been developed for detection of Giardia antigens in feces (Flow chart I). • The presence ofantigen indicates active infection. • Commercially available ELISA kits (ProSpecT/ Giardia kit) detects Giardia-specific antigen 65 (GSA 65). • 1l1e sensitivity of the test is 95% and specificity is I00%, when compared Lo conventional microscopy. Intestinal, Oral and Genital Flagellates • the test may be used for quantification of cysts and in epidemiological and control studies, but not for routine use. Antibody detection: Indirect immunofluorescence test and ELISA are used to detect antibodies against Giardia. • Demonstration of antibodies is useful in the epidemio- logical and pathophysiological studies. • lhese tests cannot differentiate between recent and past infection and lack sensitivity and specificity. Molecular Method Deoxyribonucleic acid (D A) probes and polymerase chain reaction (PCR) have been used to demonstrate parasitic genome in the stool specimen (Flow chart l ). Treatment Metronidazole (250 mg, thrice daily for 5-7 days) and tinidazole (2 g single dose) are the drugs of choice. • Cure rates with metronidazole are more than 90%. • Tinidazole is more effective than metronidazole. • Furazolidone (100 mg QID x 7 days) and nitazoxanide (500 mg BO x 3 days) are preferred in children, as they have fewer adverse effects. • Paromomycin, an oral aminoglycoside, can be given to symptomatic pregnant females (500 mgTDS x 5 days). Note: Only symptomatic cases need treatment. Prophylaxis Giardiasis can be prevented by following measures: • Proper disposal ofwaste water and feces. • Practice of personal hygiene like handwashing before eating and proper disposal ofdiapers. • Prevention offood and water contamination. Community chJorination of water is ineffective for inactivating cysts. Boiling of water and filtration by membrane filters are required. Flow chart 1: Laboratory diagnosis of Giardia tamblia • Stool examination · Macroscopic examination • Microscopic examination of stained prepration • Laboratory diagnosis I j • Enterotest (string test) Serological test • Antigen detection -ELISA • IIF test • Antibody detection -ELISA - IIF test • Molecular diagnosis • DNA probe · PCR Abbreviations: DNA, deoxyribonucleic acid; ELISA. enzyme-linked immunosorbent assay: IIF, indirect immunofluorescence; PCR, polymerase chain reaction
- 57. Paniker'sTextbook of MedicalParasitology KEY POINTS OF GIARD/A LAMBLIA • Giardia is the only protozoan parasite found in the lumen of the human small intestine (duodenum and jejunum). • Trophozoites are pear-shaped, bilaterally symmetrical with two nuclei, four pairs of flagella and a ventral concave sucking disk. They exhibit motility resembling a "falling leaf". • Ellipsoid cysts contain four nuclei with remnants of flagella. • Infective form: Ellipsoid cysts. • Clinical features: Mostly asymptomatic but in some cases may cause diarrhea, dull epigastric pain and malabsorption. Stool contains excess mucus but no blood. • Diagnosis: By microscopic demonstration of trophozoites or cysts in stool, enterotest a nd serodiagnosis by ELISA (ProSpecT/Giardia antigen assay). • Treatment Metronidazole and tinidazole are the drugs of choice. • TRICHOMONAS Trichomonas differs from other flagellates, as they exist only in rrophozoite stage. Cystic stage is not seen. Genus trichomonas has three species, which occur in humans (Figs 4A to C): l. T. vaginalis (Fig. 4A) 2. T. hominis (Fig. 4B) 3. T. tenax (Fig. 4C) Trichomonas Vagina/is History andDistribution T: vaginalis was fi rst observed by Donne (1836) in vaginal secretion. Prevalence of trichomoniasis varies from 5% patients at hospitals to 75% in sexual workers. Morphology It is pear-shaped or ovoid and measures 10-30 µm in length and 5-10 µmin breadth with a short undulating membrane reaching up to the m iddle of the body (Fig. 4A). • It has four anterior flagella and fifth running along the outer margin of the undulating membrane, which is supported at its base by a flexible rod, costa. • A prom inent axostyle runs throughout the length of the body and projects posteriorly like a tail. • the cytoplasm shows prominent siderophilic granules, which are most numerous alongside the axostyle and costa. • It is motile with a rapidjerkyortwitchingtype movement. Habitat In females, it lives in vagina and cervix and may also be found in Bartholin 's glands, urethra and urinary bladder. L n males, it occurs mainly in the anterior urethra, but may also be found in the prostate and preputial sac. Life Cycle Life cycle of T. vaginalis is com pleted in a single host either male or female. Mode oftransmission: • The trophozoite cannot survive outside and so infection has to be transmitted directly from person-to-person. Sexual transmission is the usual mode of infection (Box 3). • Trichomoniasis often coexists wilh other sexually trans- mitted diseases like candidiasis, gonorrhea, syph ilis, or human immunodeficiency virus (HIV). • Babies may get infected during birth. • Vaginal pH ofmore than 4.5 facilitates infection. Figs 4A to C: Trichomonas species. (A) T. vagina/is; (B) T. hominis; and (C) T. tenax
- 58. Box 3: Protozoatransmitted by sexual contact , Trichomonas vagina/is , Giardia Iambi/a , Entamoeba histolytica Fomites such as towels have been implicated in trans- mission. • Trophozoites divide by binaryfission. As cysts are not formed, the lrophozoite itself is the infectiveform. • Incubation period is roughly 10 days. Pathogenesis T. vaginalis particularly infects squam ous epithelium and not columnarepithelium.Itsecretes cysteine proteases, adhesins, lactic acid and acetic acid, which disrupt the glycogen levels and lower the pH of Lhe vaginal fluid. It is an obligate parasite and cannot live without close association with the vaginal, urethral, or prostatic tissues. Parasite causes petechial hemorrhage and mucosa! capillary dilation (strawberry mucosa), metaplastic changes and desquamation of the vaginal epithelium. Intracellular edema and so called chicken-like epithelium, is the characteristic feature oftrichomoniasis. Clinical Features Infection is often asymptomatic, particularly in males, although some may develop urethritis, epididymitis and prostatitis. • In females, it may produce severe pruritic vaginitis with an offensive, yellowish green, often frothy dis- charge, dysuria and dyspareunia. Cervical erosion is common. Endometritis and pyosalpingitis are infrequent complications. • Rarely, neonatal pneumonia and conjunctivitis have been reported in infants born to infected mothers. The incubation period of trichomoniasis is 4 days to 4 weeks. LaboratoryDiagnosis Microscopic examination Wet mount: Vaginal or urethral discharge is examined microscopically in saline wet mount preparation for characteristic jerky and twitching motility and shape. In males, trophozoites may be found in urine or prostatic secretions. An abundance ofleukocytes is seen. Permanent stain: Fixed smears may be stained with acridine orange, Papanicolaou and Giemsa stains. Intestinal, Oral and Genital Flagellates Direct fluorescent antibody: • Direct fluorescent antibody (DFA) is another method of detection of parasite and is more sensitive than the wet mount. Culture: Culture is recommended when direct microscopy is negative and is considered as a "gold standard" as well as the most sensitive (95%) method for the diagnosis of T. vagina/is infection. • It grows best at 35-37°C under anaerobic conditions. The optimal pl Ifor growth is 5.5-6.0. • It can be grown in a variety of solid or liquid media, tissue culture and eggs. Cysteine-peptone-liver-maltose (CPLM) medium and plastic envelope medium (PEM) are often used. Serology: Enzyme-linked immunosorbenl assay is used for demonstration of T. vagina/is antigen in vaginal smear using a monoclonal antibody for 65 kDA surface polypeptide of T. vaginalis. Rapid immunochromatographic tests (lCTs) are now available for detection of Antigen like OSOM Trichomonas rapid test, Xenostrip-Tv. Molecular method: Deoxyribonucleic acid hybridization and PCR are also highly sensitive (97%) and specific (98%) tests for the diagnosis of trichomoniasis. Sensitive and specific commercially available ucleic acid amplification test (NAAT) has been developed (Aptima Trichomonas vagina/is assay). Treatment Simultaneous treatment of both partners is recommended as it is an STD. Metronidazole 2 g orally as a single dose or 500 mg orally twice a day for 7 days is the drug of choice. In patients not responding to treatment with standard regime, the dose ofmetronidazole may be increased or it may be administered parenterally. • In pregnancy, metronidazole is safe in 2nd and 3rd trimesters. Prophylaxis Prevention is same as for other sexually transmitted diseases. • Avoidance of sexual contact with infected partners and use of barrier method during intercourse prevent the disease. Patient's sexual partner should be tested for T. vagina/is when necessary. Trichomonas Tenax T. tenax, also known as T. buccalis, is a harmless commensal which lives in mouth, in the periodontal pockets, carious tooth cavities and, less often, in tonsillar crypts.
- 59. Paniker'sTextbook of MedicalParasitology • It is smaller(S-10 µm) than T. vagina/is. • It is transmitted by kissing, through salivary droplets and fomites. There are sporadic reports of its involvement in respiratory infections and thoracic absce ses. • Beller oral hygiene rapidly eliminates the infection and no therapy is indicated. Trichomonas Hominis T. hominis measures 8- 12 µm, pyriform-shaped, and carries five anterior flagella and an undulating m embrane that extends the full length of the body. • It is a very harmless commensal ofthe cecum. • Microscopic examination of stool will reveal motile trophozoite of T. hominis. • Transmission occurs in rrophic form by fecal-oral route. KEY POINTS OF TRICHOMONAS • Trichomonas occurs only in trophozoite form, which is pear- shaped, with five flagella and an undulating membrane. • The motility is rapid jerky or twitching type. • Habitat: Vagina and cervix in female and urethra in males. • Clinical features: Often asymptomatic in males. In females, it leads to pruritic vaginitis with greenish yellow discharge, strawberry mucosa and dysuria. • Diagnosis: By wet mount microscopy of vaginal or urethral discharge, culture(gold standard), PCR and bydemonstration of antigen in vaginal smear by ELISA. • Treatment: Metronidazole is the drug of choice and simulta- neous treatment of both partners is recommended. • CH/LOMASTIX MESNILI This occurs as trophozoites and cysts (Fig. 5). • The trophozoite is pear-shaped measuring 5-20 µm in length and 5- 10 µmin breadth. Flagella Cytostome Trophozoite Cyst Clear hyaline knob Cytostome Nucleus Fig. 5:Trophozoite and egg of Ch//omastix mesnifi • At the anterior end, it has a spherical nucleus. • A distinct spiral groove is seen on one side of the nucleus. • The cysts are lemom-shaped having a spiral projection at the anterior end. lt measures 5-10 µmin length and 4-6 µm in breadth and is surrotmded by a thick cyst wall. • Both rrophozoites and cysts are demonstrated in the semi-formed srool. • It is a harmless commensal of cecum where the organism feeds on bacteria and food debris. Since infection is acquired through ingestion of cysts, prevention depends on improved personal hygiene. • ENTEROMONAS HOMINIS £. hominis is a nonpathogenic commensal that lives in the large inte tine, mainly in the cecum. . . It exists in two forms: (1) trophozoile, and (2) cyst (Fig. 6). the trophozoite is pear-shaped, with three anterior and one posterior flagella. It measures 5-10 µmin length and3-6 µmin breadth. - 1he cytoplasm contains numerous bacteria and an anteriorly placed nucleus but no cytostoma. - It shows jerky forward movements. The cyst is oval in shape, measuring 5-8 µm in length and 4- 6 µm in breadth. It contains 2-4 nuclei. The cyst of E. fzominis may mimic a two-nucleated cyst of E. nana. lnfection occurs through fecal-oral route by ingestion of cysts in contaminated food and water. Diagnosis is made by identification of trophozoites or cysts in the stool by iron hematoxylin stain. • RETORTAMONAS INTESTINALIS Wenyon and O'Connor first observed the parasite in stool in Egypt. Nucleus __:,n-r-3 anterior flagella - -----Posterior flagella Trophozoite Nuclei Cyst Fig. 6:Trophozoite and cyst of Enteromonas hominis 1 I
- 60. • R. intestinalisis a small nonpathogenic flagellate found in the large intestine. • It also exists in two forms: (1) trophozoite, and (2) cyst. • The trophozoite is elongated, pyriform in shape, measuring 5- 10 mcm in length and 3-4 µmin breadth. The cytoplasm is granular and vacuolated. It has a cleft-like cytosome, spherical nucleus and central karyosome. - Two minute blepharoplasts are present near nucleus, from which two flagella originate. The trophozoite multiplies by binary fission. • The cyst is ovoid or pyriform in shape, measuring 6 µm in length and 3 µmin breadth. • Water and food contaminated by cysts are the main source of infection. Diagnosis is made by identifying the cysts and trophozo- ites in the direct wet mount and iron hematoxylin-stained specimen ofstool. • DIENTAMOEBA FRAG/L/S D. Jragilis was previously considered as an amoeba but has now been reclassified as an amoebojlagellate, based on electron microscopic study and antigenic similarity to Trichomonas. • It is unique as it has only trophowile stage but no cyst stage. • The name Dien/amoeba Jragilis is derived from the binucleate nature of trophozoite (Dien/amoeba) and the fragmented appearance (fragilis) ofits nuclearchromatin. • It is seen worldwide and is reported to be the most common intestinal protozoan parasite in Canada. • It lives in colonic mucosa] crypts, feeding on bacteria. It does not invade tissues, but may rarely ingest red blood cells (RBCs). • The trophozoite is 7- 12 µm in diameter. It is motile with broad hyaline leaf-like pseudopodia. They have 1-4 nuclei; the binucleace form being the most common (Fig. 7). The nuclear chromatin is present as 3-5 granules in the center, with no peripheral chromatin on the nuclear membrane. • In the absence of cyst stage, its mode of transmission is not clear. Possibly, it is transmitted from person-to- person by th e fecal-oral route or by the eggs ofEnterobius vermicularis and other nematodes, which may serve as a vector. Formerly believed to be nonpathogenic, it has now been associated with a variety of symptoms like intermittent diarrhea, abdominal pain, flatulence, anorexia, nausea, malaise a nd fatigue. • High incidence is seen among children between 2 years and 10 years of age. Intestinal, Oral and Genital Flagellates ~ o l..• >.--~~lr-lngested bacteria ' ... Fig. 7: Trophozoite of Dientamoeba fragilis Laboratory diagnosis is made by demonstration of trophozoites in stool. At least three stool specimens should be collected over a period of7 days. Metronidazole, iodoquinol, paromomycin and tetracyc- line have been used for treatment REVIEW QUESTIONS 1. Describe briefly the life cycle and laboratory diagnosis of Giardia Jamblia. 2. Write short notes on: a. Trichomonas vagina/is b. Dientamoeba fragilis MULTIPLE CHOICE QUESTIONS 1. Normal habitat of Giardia is a. Duodenum and jejunum b. Stomach c. Cecum d. Ileum 2. All of the following protozoans are found in small intestine except a. Giardia lamblia b. Balantidium coli c. Cyclospora caytanensis d. lsospora be/Ii 3. The following is true ofgiardiasis except a. Feverand presence of blood and mucus in stool b. Acute orchronic diarrhea c. Duodenum and jejunum are the prime sites of involvement d. Giardia cysts are resistant to dessication 4. Giardia lamblia was discovered by a. Giard b. Robert hook c. Leeuwenhoek d. L osch
- 61. Paniker'sTextbookofMedical Parasitology S. Drugofchoice in giardia.sis is a. Metronidazole b. Albendazole c. Thiabendazole d. Diloxanide furoate 6. True about Giardia is a. Maycause traveller's diarrhea b. Giardia inhabits ileum c. Trophozoites are infective to man d. Encystment oftrophozoitesoccur injejunum 7. Which onefollowing test is usedfordiagnosisofGiardia lamblia infections a. Enterotest b. Casoni's test c. Parasight Ftest d. Napier's test 8. MotilityofTrichomonas vagina/is is describedas a. Amoeboid b. Jerky c. Falling leaf d. Lashing 9. Vaginal discharge in Trichomonas vaginitis is a. Colorless b. Yellow c. Curd- white d. Blood stained 10. All of the following protozoan can be transmitted by sexual contact except a. Trichomonas vagina/is b. Entamoeba histolytica c. Enteromonas hominis d. Giardia lamblia Answer 1. a 2. b 8. b 9. b 3. a 10. C 4. C 5. a 6. a 7. a
- 62. I CHAPTER 5 • INTRODUCTION• The blood and tissue flagellates belong to the family Trypanosomatidae. The family consists of six genera, of which two genera Trypanosoma and Leishmania are pathogenic to humans. • ZOOLOGICAL CLASSIFICATION OF FLAGELLATES Phylum: Sarcomastigophora Subphylum: Mastigophora Class: Kinetoplastidea Order: Trypanosomatida Family: Trypanosomatidae Genera: Leishmania and Trypanosoma • GENERAL CHARACTERISTICS • They live in the blood and tissues of man and other vertebrate hosts and in the gut ofthe insect vectors. • Members ofthisfamilyhave asingle nucleus, a kinetoplast and a single flagellum (Fig. I). • Nucleus is round or ovaland is situated in the central part of the body. • Kineloplast consists ofa deeply staining parabasal body and adjacent dot-Like blepharoplast. The parabasal body and blepharoplast are connected by one or more thin fibrils (Fig. l ). • Flagellum is a thin, hair-like structure, which originates from the blepharoplast. The portion of the flagellum, which is inside the body ofthe parasite and extends from the blepharoplasl to surface of the body is known as axoneme. A free flagellum at the anterior end traverses on the surface of the parasite as a narrow undulating membrane (Fig. 1). • Hemoflagellates exist in two or more of four morpho- logical stages. These forms were formerly called the Blepharoplast Undulating Flagellum membrane Fig. 1: Basic morphology of hemoflagellates Note: Parabasal body and blepharoplast together constitute the klnetoplast. leishmanial, leptomonad, crithidial and trypanosomal stages. But as these names are also given to different genera within the family, they were changed to amasti- gote, promastigote, epimasligote and trypomastigote. The names of the stages are formed by the suffix mastigote, combined with various prefixes, referring to the arrangement of the Oagella in relation to the position of the nucleus and its point of emergence from the celJs (Table 1). • Stainingcharacteristicsoftrypanosomes: For smears of body fluids, Romanowsky's Wrights stain, Giemsa stain and Leishman's stain are suitable for identifying internal structures. The cytoplasm appears blue, the nucleus and flagellum appear pink, and the kinetoplast appears deep red. For tissue section, hematoxylin-eosin staining is done for demonstrating structures ofthe parasite. • AU members ofthe family have similar life cycles. They all require an insect vector as an intermediate host. • Multiplication in both the vertebrate and invertebrate host is by binaryfission. No sexual cycle is known.
- 63. Paniker'sTextbook ofMedical Parasitology Table1: Differences between various morphological stages of hemoflagellates Morphological characteristics Amastlgote Promastigote Lanceolate in shape. Kinetoplast is anterior to Epimastigote Trypomastlgote This stage is elongated, spindle· shaped with a central nucleus. Rounded or ovoid, without any external flagellum. The nucleus, kinetoplast and axial filaments can be seen. The axoneme extends up to the anterior end of the cell the nucleus (antinuclear kinetoplast) near the anterior end of the cell, from which flagellum emerges. There is no undulating membrane Elongated, with the kinetoplast placed more posteriorly, though close to and in front of the nucleus (juxtanuclear kinetoplast).The flagellum runs alongside the body as ashort undulating membrane, before emerging from the anterior end The kinetoplast is posterior to the nucleus (postnuclear kinetoplast) and situated at t he posterior end of the body. The flagellum runs alongside the entire length of the cell to form a long undulating membrane before emerging as a free flagellum from the anterior end Seen in Schematic illust ration T rypanosoma cruzi and Leishmania as intracellular form in vertebrate host It is t he infective stage of Leishmania, found in the insect vector as well as in cultures in vitro It isthe form in which Trypanosoma bruce/ occur in salivary gland of the vector tsetse fly and Trypanosoma cruzi in the midgut of the vector reduviid bug. Note:This stage is lacking in Leishmania This is the infective stage of trypanosomes found in arthropod vector and in the blood of infected vertebrate. Note: This stage is lacking in Leishmania Abbreviations:A, axoneme; B, blepharoplast; F , flagellum; K, kinetoplast; N, nucleus; P, parabasal body; U, undulating membrane Note: Besides the stages described in the table, some transitional stages have been recognized. These include the spheromostigote, a motile round form with free Oagellum, which isa transitional stage from amastigoteto promastigote, seen in the genus Trypanosoma and the paramastigote, a transitional form leading to the infective promastlgore in Leishmania. • TRYPANOSOMES General Characters All members of the genus Trypanosoma (trypanes: to bore, soma: body), exist al sometime in their life cycle, as trypomastigotestagewith an elongated spindle-shaped body, central nucleus, a posterior kinetoplast and long-undulating membrane. Volutin granules are found in cytoplasm. Some trypanosomes such as T. cruzi assume amastigote forms in vertebrate hosts. In addition to the typical forms, cells with atypical features are frequently found, a condition known as polymorphism. • Trypanosomapasstheirlifecyclein twohosts:(l) vertebrate hosts (definitive hosts) and (2) insect vectors (intermediate hosts). 1herefore called as digenetic parasites. The vector becomes infective to the vertebrate host only after an extrinsic incubation period, during which the parasite undergoes development and multiplication. • In the vector, the trypanosomes follow one or two modes of development and are accordingly classified into two groups: (1) Salivaria and (2) Stercoraria. l. Salivaria (anterior station): In salivaria, the trypano- somes migrate to mouth parts of the vectors, so that infection is transmined by their bite (inoculative transmission). Examples are T. gambiense and T. rhodesiensecausingAfrican trypanosomiasis, which are transmitted by the bite of tselse flies. 2. Stercoraria (posterior station): In stercoraria, the trypanosomes migrate to the hindgut and are passed in feces (stercorarian transmission), e.g. T. cruzi causing Chagas disease, which is acquired by rubbing the feces of the vector bug into the wound caused by its bite and T. lewisi, the rat trypanosome, which is transmitted by ingestion of feces ofinfected rat fleas. • Distribution: Human trypanosomiasis is strictly restricted to certain geographical regions; the African and South American trypanosomiasis being seen only in ll1e respective continents. This is due to the vector being confined to these places alone. - African trypanosomiasis (sleeping sickness) - South American trypanosomiasis (Chagas disease). Classification ofTrypanosomes Trypanosomes Infecting Man • Trypanosoma brucei complex, causing African trypano- somiasis or sleeping sickness, subspecies are: Trypanosoma brucei gambiense: It causing West African sleeping sickness.
- 64. Trypnnosoma brucei rhodesiense:It causing East African sleeping sickness. • Trypanosoma cruzi, causing South American trypano- somiasis or Chagas disease. Trypanosoma rangeli, a nonpathogenic trypanosome causing human infection in South America. Trypanosomes ofAnimals • Trypanosoma brucei brucei, causing the economically important disease "nagana"in African cattle. Trypanosoma evansi, causing the disease "surra" in horses, camels and elephants. It is transmitted mechanically by biting flies and also byvampire bats. This infection is found in India. • Trypanosoma equiperdum, causing "stallion's disease" in horses and mules. It is transmitted by sexual contact, without the need for an insect vector. • Trypanosoma lewisi, causing harmless infection of rats all over the world. The vector is rat flea. A trypanosome resembling Trypanosoma lewisi was reported from Madhya Pradesh in India in peripheral blood of two persons with short-term fever. Trypanosoma Brucei Gambiense (West African Trypanosomiasis) History and Distribution Trypanosomiasis is believed to have been existing in tropical Africa from antiquity (Fig. 2). Fig. 2: Geographical distribution of trypanosomiasis In Africa. Lines indicate areas endemic for Trypanosoma gambiense and dots represent Trypanosoma rhodeslense Hemoflagellates • Trypanosome was first isolated from the blood of a steamboat captain on the Gambia river in 1901 (hence, the nan1e gambiense) by Forde. • Dutton, in 1902, proposed the name Trypanosoma gambiense. • It is endemic in scattered foci in West and Central Africa between 15° land 18°S latin1des. Habitat Trypanosomes live in man and other vertebrate host. They are essentially a parasite of connective tissue, where they multiply rapidly and then invade regional lymph nodes, blood and finally may involve central nervous system. Morphology Vertebrate forms: In the blood of vertebrate host, T. brucei gambiense exists as trypomastigoce form, which is highly pleomorphic. It occursasa longslenderform, a stumpyshortbroadform with anenuated or absent flagellum and an intermediate form. • The trypomastigotes are about 15-40 µm long and 1.5- 3.5 µm broad. • In fresh blood films, trypornastigotes are seen as colorless, spindle-shaped bodies that move rapidly, spinning around the red cells. • In smears stained with Giemsa or other Romanowsky's stain, the cytoplasm appears pale blue and the nucleus appears red. The kinetoplast appears as a deep red dot and volutin granules stain deep blue. The undulating membrane appears pale blue and the flagellum red. Insectforms: In insects, it occurs in two forms: I. Epimastigotes 2. Metacyclic trypomastigore forms. Antigenic Variation Trypanosomes exhibit unique antigenic variation of their glycoproteins. • There is a cyclical fluctuation in the trypanosomes in the blood ofinfected vertebrates after every7- 10 days. • Each successive wave represents a varinnt antigenic type (VAT) of trypomastigote possessing variant-specific surface antigens (VSSAs) or variant surface glycoprotein (VSG) coat antigen. It is estimated that a single trypanosome may have as many as 1,000 or more VSG genes that help to evade immune response. Besides this, trypanosomes have other mechanisms also that help them to evade host immune respon es.
- 65. Paniker'sTextbook of MedicalParasitology Life Cycle Host: T. bruceigambiense passes its life cycle in two hosts: l . Vertebrate host: Man, game animals and other domestic animals. 2. Invertebrate host: Tsetse fly. Both male and female tsetse fly of Glossina species (G. palpalis) are capable of transmitting the disease ro humans. These flies dwell on the banks of shaded streams, wooded Savanna and agricultural areas. /11/ectiveform: Meracyclic trypomastigote forms are infective ro humans. Mode oftransmission: • By bite oftsetse fly. • Congenital transmission has also been recorded. Reservoirs: Man is the only reservoir host, although pigs and others domestic animals can act as chronic asymptomatic carriers ofthe parasite. Development in man and other vertebrale hosts: • Metacyclic slage (infective form) of L rypomastigotcs arc inoculated into a man (definitive host) through skin when an infected tsetse fly takes a blood meal (Fig. 3). Epimastigote form Short stumpy form ingested by Tsetse fly during blood meal Invades blood- stream Short stumpy form Tsetse fly (Vector) Man (Definitive host) Intermediate form Fig. 3: Ufe cycle of Trypanosoma brucei Metacyclic trypomatigote form infective f Transferred to man by bite of infected Tsetse fly Metacychc trypomastigote form
- 66. • l he parasite transforms into slender forms that multiply asexua lly for l -2 days before entering th e peripheral blood and lymphatic circulation. • These becom e "stumpy" via intermediate forms and enter the bloodstream. • In chronic infection, the parasite invades the central nervous system. • Trypomastigotes (short plumpy form) are ingested by tsetse fly (male or female) during blood meal. Development in tsetsefly: • In the midgut of the fly, short stumpy trypomastigotes develop into long, slender forms and multiply. • After2-3 weeks, they migrate to the salivaryglands, where they develop into epimastigotes, which multiply and fill the cavity of the gland and eventually transform into the infective metacyclic trypomastigotes (Fig. 3). • Development of the infective stage within the tsetse fly requires 25-50 days (extrinsic incubation period). • l hereafter, the fly remains infective throughout its life of about 6 months. Pathogenicity and Clinical Features T. brucei gambiense causes African trypanosomiasis (West African sleeping sickness). The illness is chronic and can persist for many years. • There is an initial period of parasitemia, following which parasite is localized predominantly in the lymph nodes. • A painless chancre (trypanosomal chancre) appears on skin at the site ofbite by tsetse fly, followed by intermittent fever, chills, rash, anemia, weight loss and headache. • Systemic trypanosomiasis w ithout central nervous system involvement is referred to as stage 1 disease. In this stage, there is hepatosplenomegaly and lymphadeno- pathy, particularly in the posterior cervical region (Wi11terbottom's sign). • Myocarditis develops frequently in patients with stage I disease and is especially common in T. brucei rhodesiense infections. • Hematological manifestations seen in stage I include anemia, moderate leukocytosis and thrombocytopenia. High levels of immunoglobulins mainly immunoglobulin M (lgM) are a constant feature. • Stage Tl disease involves invasion of central nervous system.With theinvasion ofcentral nervoussystem,which occurs afterseveral months, the "sleeping sickness" tarts. This is marked by increasing headache, mental dullness, apathy and day time sleepiness. The patient falls into profound coma followed by death from asthenia (Box 1). • Histopathology shows chronic meningoencephalitis. The meninges are heavily infiltrated with lymphocytes, plasma cells and rnorula cells, which are atypical plasma cells containing mulberry-shaped masses oflgA. Brain vessels Hemoflagellates show perivascular cuffing. This is followed by infiltration of the brain and spinal cord, neuronal degeneration and microglial proliferation. Abnormalities in cerebrospinal fluid (CSF) include raised intracranial pressure, pleocytosis and raised total protein concentrations. Trypanosoma Brucei Rhodesiense (East African Trypanosomiasis) • It is found in Eastern and Central Africa (Uganda, Tanzania, Zambia and Mozambique) (Fig. 2). • Stephens and Fantham discovered T brucei rhodesiense in 1910 from the blood of a patient in Rhodesia sufferin g from sleeping sickness. • The principal vector is G. morsitans, G. palpalis and G. swynr1ertoni, which live in tl1e open savannah cow1tries. • Although the disease is usually transmitted by the vector from man-to-man, the disease is actually a zoonosis, with the reservoir being wild game animals like bush buck, antelope and domestic animals like cattle. Its morphology, habitat and lifecycle is similar lo T. brucei gambiense (Fig. 3). • 1he difference between T. brucei gambiense and T. brucei rhodesiense are detailed in Table 2. Box 1: Clinical staging of human African trypanosomiasis (HAT) • Stage/: Characterized by hematogenousand lymphatic dissemination ofthe disease. • StageII: Characterized by central nervous system involvement. Table 2: Differences between West African and East African trypano- somiasis Characteristics West African EastAfrican Organism T. bruceigambiense T. bruceirhodesiense Distribution West and Central East and Central Africa Africa Vector Tsetse ny (Glossina Tsetse fly(Glossina pa/pa/is group) morsitans group) Reservoir Mainly humans Wild and domestic animals Virulence Less More Course ofdisease Chronic (latecentral Acute {early central nervoussystem nervous system invasions);months invasion); less than to years 9 months Parasitemia Low High and appearsearly Lymphadenopathy Early, prominent Less common Isolation in rodents No Yes Mortality Low High
- 67. Paniker'sTextbook of MedicalParasitology Pathogenesisand ClinicalFeatures T. brucei rhodesiense causes East African sleeping sickness (Table 2). • East African trypanosomiasis is more acute than rhe Gambian form and appears after an incubation period of4 weeks. • It may end fatally within an year of onset, before the involvement ofcentral nervous sysrem develops. • Pathological features are similar in both diseases with some variations: Ede ma, myocarditis and weakness a re more prominent in East African sickness (Box 2) . Headache, diffuse muscle and joint pain are present in majority ofthe patients. Lymphadenitis is less prominent. Febrile paroxysms arc more frequent and severe. 1here is a larger quantity ofparasite in the peripheral blood. Central nervous system involvement occurs early. Mania and delusions may occur but the marked somnolence, which occurs in T. brucei garnbiense infection is lacking. LaboratoryDiagnosis The diagnosis of both types of African trypanosomiasis is similar (Flow chart 1). Box 2: Parasites causmg myocarditis • Trypanosoma bruceirhodesiense • Trypanosoma cruzi • Toxoplasma gondii • Echinocaccus granulosus • Trichinella spiralis Nonspecificfindings: • Anemia and monocytosis. • Raised erythrocyte sedimentation rate (E R) due to rise in gamma globulin levels. • Reversal ofalbumin:globulin ratio. • Increased CSF pressure and raised cell count and proteins inCSF. Specific findings: Definitive diagnosis of sleeping sickness is established by the demonstration of trypanosomes in peripheral blood, bone marrow, lymph node, CSF and chancre Ouid. Microscopy: • Wet mount preparation of lymph node aspirates and chancre fluid are used a a rapid method for demonstra- tion oftrypano omes. These specimens are aJso examined for parasites after fixing and stainingwith Giemsa stain. • Examination of Giemsa-stained thick peripheral blood smears reveals the presence of the trypomastigotes (Fig. 4). Thrombocyte fragments Erythrocyte undulating membrane Nucleus Fig. 4: Trypanosoma rhodes/ense, blood smear Giemsa stain, magnification 1100X Flow chart 1: Laboratory diagnosis of trypanosomiasis • Microscopy Detection of Trypanosomes 1n- • Wet mount preparation of lymph node aspirate • Giemsa-stained thick peripheral blood smear or concentrated blood smear • Wet mount, stained smear of CSF • Culture In Weinman's or Tobie's medium Laboratory diagnosis I I Imaging CT scan Shows cerebral edema MRI Shows white matter enhancement • Serodiagnosis Antibody detection IHA IIF ELISA CATT CFT Antigen detection ELISA • • Molecular diagnosis Others PCR • animal Inoculation • Blood examination reveals anemia, monocytosis, raised ESR and reversed albumin globulin ratio Abbreviations: CATT. card agglutination trypanosomlasis test: CT, computed tomography: CFT. complement fixation test: CSF, cerebrospinal fluid: ELISA, enzyme- linked immunosorbent assay; ESR, erythrocyte sedimentation rate: IHA, indirect hemagglutination: IIF, indirect immunofluorescence; MRI, magnetic resonance Imaging: PCR, polymerase chain reaction
- 68. • If parasitemiais low, then examination of concentrated blood smear is a highly sensitive method. Different con- centration techniques employed are buffy coat examina- tion, differential centrifugation, membrane filtration and ion exchange column chromatography. • Examination ofwet mount and stained smear of the CSF may also showtrypanosomes (Flowchart l). Culture: The organisms are difficult to grow, hence culture is not routinely used for primaryisolation of the parasite. How- ever, it can be cultivated in Weinman's or Tobie's medium. Animal inoculation: Inoculation of specimens from sus- pected cases to white rat or white mice is a highly sensitive procedure for detection of T. brucei rhodesiense infection. Serodiag11osis: Antibody detection: Almost all patientswith African trypano- somiasis have very high levels of total serum IgM antibodies and later, CSF IgM antibodies. Various serological methods have been developed to detect these antibodies and are as follows: • Indirect hemagglutination (IHA) • Indirect immunotluorescence (llF) • Enzyme-linked immunosorbent assay (ELISA) • Card agglutination trypanosomiasis test (CATT) • Complementfixation test (CFT) Specific antibodies are detected by these tests in serum within 2-3 weeks of infection. Specific antibodies in CSF are demonstrated by UF and ELISA. These serological tests are useful for field use and mass screening (Flow chart 1). Antigen detection: Antigens from serum and CSF can be detected by ELISA. Molecular diagnosis: Polymerase chain reaction (PCR) assays for detecting African trypanosomes in humans have been developed, but none is commercially available. Imaging: Computed tomography (CT) scan of the brain shows cerebral edema and magnetic resonance imaging (MRI) shows white matter enhancement in patients with late stage central nervous systems involvement {Flow chart 1). Blood incubation infectiuitytest: For differentiation between the"hwnanstrains" and "animalstrains" ofT. brucei, the blood incubation infectivity test {BUT} had been widely used. • The strain is incubated with oxalated human blood and then inoculated into the multimammate rat or other Hemotlagellates Table 3: Treatment of human African trypanosomiasis Causativeorganism T. bruceigambiense (WestAfrican) T. brucei rhodesiense (East African) clinicalstage I (normalCSFJ II(abnormalCSF} Pentamidlne Eflornithine Suramin Melarsoprol Abbreviation: CSF,cerebrospinal fluid lsoenzyme study: More recently their differentiation is based on isoenzymes, deoxyribonucleicacid (DNA) and ribonucleic acid (RNA) characterisLics (Flow chart 1). Treatment In the initial stages, when central nervous system is not involved, i.e. stage I, pentamidine is the drug of choice for gambiense human African trypanosomiasis (HAT) and suramin is the drug ofchoice for rhodesiense HAT. Dose: • Pentamidine: Dose 3-4 mg/kg of body weight, intra- muscularly daily for 7- 10 days. Suramin: Dose 20 mg/kg of body weight in a course of five injections intravenously, at an interval of 5- 7 days. Suramin does not cross blood-brain barrier but it is nephrotoxic. • In patients with central nervous system involvement, melarsoprol (Mel-B) is the drug of choice, as it can cross the blood-brain barrier. Dose: 2-3 mg/kg/ per day (maximwn 40 mg) for 3-4 days (Table 3). Prophylaxis Control is based on early diagnosis and treatment ofcases to reduce the reservoir ofinfection. • Control of tsetse fly population (most important pre- ventive measure) by wide spraying of insecticides, traps and baits impregnated with insecticides. • No vaccine is available. Trypanosoma Cruzi T. cruzi is the causative organism ofChagas disease or South American trypanosomiasis. susceptible rodents. History and Distribution • lhe infectivity of "animal strains" will be neutralized by human blood, while "human strains" retain infectivity It is a zoonotic disease and is limited to South and Central after incubation with human blood. America. • In vitro culture systems are now employed instead of • Carlos Chagas, investigating malaria in Brazil in 1909, rodents for testing infectivity. accidentally found this trypanosome in the intestine of a
- 69. Paniker's Textbook ofMedical Parasitology triatomine bug and then in the blood of a monkey bitten by the infected bugs. • Chagas named the parasite T cruzi after his mentor Oswaldo Cruz and the disease was named as Chagas disease in his honor. Habitat • In humans, T cruzi exists in both amastigote and trypomastigote forms: Amastigotes are the intracellular parasites. They are found in muscular tissue, nervous tissue and reticuloendothelial system (Box 3). - Trypomastigotes are found in the peripheral blood. • In reduviid bugs, epimastigote forms are found in the midgut and metacyclic trypomastigote forms are present in hindgut and feces. Morphology Amastigote: Amastigotes are oval bodies measuring 2-4 µm in diameter having a nucleus and kinetoplast (Fig. 5A). • Flagellum is absenc. Morphologically, it resembles the amastigote of Leishmania spp., hence, it is frequently called as leishmanialform. • Multiplication ofthe parasite occurs in this stage. • This form is fo und in muscles, nerve cells and reticuloenodothelial systems. Trypomastigote: Trypomastigotes are nonmultiplying forms found in the peripheral blood of man and other mammalian hosts (Fig. 5B). • In the blood, they appear either as long, thin flagellates about (20 mcm long) or short stumpy form (15 µm long). • Posterior end is wedge-shaped. • In stained blood smears, they are shaped-like alphabet "C'';"U'';or "S'; having a free flagellum of about one-third the length of the body. • These forms do not multiply in humans and are taken up by the insect vectors. Epimastigote form: Epimasrigote forms are found in the insect vector, the reduviid bug and in culture also (Pig. 5C). • It has a kinetoplast adjacent to the nucleus. • An undulating membrane runs along the anterior half o f the parasite. • Epimastigotes divide by binary fission in hindgut of the vector. Life Cycle Host: 1: cruzi passes its life cycle in two hosts (Pig. 6): 1. Definitive host: Man. 2. .Intermediate host (vector}: Reduviid bug or triaLOmine bugs. Box 3: Obligate intracellular parasites • Trypanosoma cruzi • Leishmaniaspp. • Plasmodium spp. • Babesia spp. • Toxoplasma gondii • Microsporidia Nucleus Parabasal body m FigsSA to C: Trypanosoma cruzi. (A) Amastigote; (B) Trypomastigote;and (C) Epimastigote Reservoirhost: Armadillo, cat, dog and pigs. Infectiveform: Metacyclic trypomastigotes forms are the infective forms found in feces ofreduviid bugs. • The parasite occurs in three different but overlapping infection cycles, a sylvatic zoonosis in wild animals such as armadillos and opossums, peridomestic cycle in dogs, cats, and other domestic animals and domestic cycle in humans. Different vector species are active in these infection cycles. The vectors importantinhwnan infection are thereduviid bugs adapted to living in human habitations, mainly Triatoma infestans, Rhodnius prolix.us and Panstrongylus megistus. These are large (up to 3 cm long) night-biting bugs, which typically defecate while feeding. The feces of infected bugs contain the metacyclic trypomastigote. Mode oftransmission: • Transmission of infection to man and other reservoir hosts takes place when mucus membranes, conjunctiva, or wound on the surface of the skin is contaminated by feces ofthe bug containing metacyclic trypomastigotes. • T. cruzi can also be transmitted by the blood transfusion, organ transplantation and vertical transmission, i.e. from mother LO fetusorveryrarelybyingestion ofcontaminated food or drink.
- 70. Trypomastigote ingested by reduviidbug during blood meal Reduviid bug (Vector) Hemoflagellates Metacyclic trypomastigote (Infective form to man) Shed in feces ,,______________ Man acquires Man (Definitive host) infection by rubbing the bug feces Trypomastigote formed and released in blood bloodstream (Infective form to reduviid bug) Amast1gote passes through promastigote and epimastigote stages Trypomastigote h : ~ (<) Transforms into amastigote form Fig. 6: Life cycle of Trypanosoma cruzi Development in man: • The metacyclic trypomastigotes introduced in human body by bite of reduviid bugs invade the reticuloendo- thelial system and spread to other tissues. • After passing through promastigote and epimastigote forms, they again become trypomastigotes, which are released into the bloodstream and are the infective stage for triatomine bug. No multiplication occurs in this stage. Multiplication takes place only intracellularly in the amastigote form and to some extent as promastigote or epimastigotes (Pig. 6). Development in reduviid bugs: Bugs acquire infection by feeding on an infected mammalian host. • Most triatomine bugs are nocturnal. • The trypomastigotes are transformed into epimastigotes in the midgut, from where they migrate to the hindgut and multiply. These, in turn, develop into nondividing metacyclic trypomastigotes (infective form), which are excreted in feces (stercorarian transmission). • The development of T cruzi in the vector takes 8- 10 days, which constitutes the extrinsic incubation period. Pathogenicity and Clinical Features The incubation period of T. cruzi in man is 1-2 weeks. The disease manifests in acute and chronic form. Acute chagas disease:Acute phase occurssoon afterinfection and may last for 1- 4 months.
- 71. Paniker'sTextbook of MedicalParasitology • lt is seen often in children under 2 years ofage. • First sign appears within a week afterinvasion ofparasite. • "Chagoma" is the typical subcutaneous lesion occurring at the site of inoculation. Inoculation of the parasite in conjunctiva causes unilateral, painless edema of periocular tissues in the eye called as Romana'ssign,.This is a classical finding ofthe acute Chagas disease. • In few patients, there may be generalized infection with fever, lymphadenopathy and hepatosplenomegaly. • The patient may die of acute myocarditis and meningoencephalitis. • Usually within 4-8 weeks, acute signs and symptoms resolve spontaneously and patients, then enter the asymptomatic or indeterminate phase of chronic T. cruzi infection. Chronic chagas disease: The chronic form is found in adults and older children and becomes apparent years or even decades after the initialinfection. • ln chronic phase, T. cruzi produces inflammatory response, cellular destruction and fibrosis of muscles and nerves that control tone of hollow organs like heart, esophagus, colon, etc. Thus, it can lead to cardiac myo- pathy and megaesophagu.s and megacolon (dilatation of esophagus and colon). Congenital infection: Congenital transmission is possible in both acute and chronic phase of the disease causing myocardial and neurological damage in the fetus. Laboratory Diagnosis Diagnosis is done by demonstration of T. cruzi in blood or tissues or byserology. Microscopy: • The diagnosis of acute Chagas disease requires detection ofparasites. • Microscopic examination of fresh anticoagulated blood or the buffy coat is the simplest way to see motile organisms. • In wet mount, trypomastigotes are faintly visible but their snake-like motion against red blood cells (RBCs) makes their presence apparent. • Trypomastigotes can also be seen in thick and thin peripheral blood smear, stained with Giemsa stain (Box 4) (Fig. 7). • Microhematocrit containing acridine orange as a stain can also be used. • When used by experienced personnel, all these methods yield positive results in a high proportion ofcases ofacute Chagas disease. Note: Serologic testing plays no role in diagnosing acute Chagas disease. Culture: ovy, MacNeal and nicolle(NNN) medium or its modifications are used for growing T. cruzi. • This medium is inoculated with blood and other specimens and incubated at 22-24°C. • The fluid from the culture is examined microscopically by 4th day and then every week for 6 weeks. • Epimastigotes and trypomastigotes are found in the culture. • Culture is more sensitive than smear microscopy. Animal inoculation: Guinea pig or mice inoculation may be done with blood, CSF, lymph node aspirate, or any other tissue material and the trypomastigote is looked for in its blood smears in a few days after successful inoculation. Xenodiagnosis: This is the method of choice in suspected Chagas disease, ifother examinations are negative, especially during the early phase of the disease onset. The reduviid bugs are reared in a trypanosome-free laboratory and starved for 2 weeks. They are then fed on patient's blood. If trypomastigotes are ingested, they will multiply and develop into epimastigotes and trypomastigotes, which can be found in the feces of the bug 2 weeks later. Histopathology: Biopsy examination of lymph nodes and skeletal muscles and aspirate from chagoma may reveal amastigotes of T. cruzi. Box 4: Protozoan parasites detected in peripheral blood film • Trypanosoma cruzi • Trypanosoma brucei rhodesiense • Trypanosoma bruceigambiense • Leishmania spp. , Plasmodium spp. • Babesiaspp. Blepharoplast (large) Fig. 7: Trypanosoma cruzi, blood smear Giemsa stain, magnification 1100X
- 72. Serology: Antigen detection: T.cruzi antigen can be detected in urine and sera in patients with chronic Chagas disease. ELISA has been developed for detection ofantigens. Antibody detection:Antibodies (IgG) against T. cruzi may be detected by the following tests: • Indirect hemagglutination • Complementfixation test (Machado-Guerreiro test) • Enzyme-linked immunosorbent assay • Indirect immunofluorescence • Direct agglutination test (DAT): It is a simple test being recommended for field use. • Chagas radioimmune precipitation assay (RIPA) is a highly sensitive and specific confirmatory method for detecting antibodies of T. cruzi. The disadvantage of the antibody based rests is that they may be false positive with other disease like leishmaniasis and syphilis. fntradermal test: The antigen "cruzin" is prepared from T. cruzi culture and used for the intradermal test. A delayed hypersensitivity reaction is seen. Molecular diagnosis: Polymerase chain reaction is available that detects specific primers, which have been developed against T. cruzi kinetoplastic or nuclear DNA. The disadvantage ofthe testis that it is not commercially available. Other tests: • Electrocardiography (ECG) and chest X-ray are useful for diagnosis and prognosis of cardiomyopathy seen in chronic Chagas disease. the combination ofright bundle branch block (RBBB) and left anterior fascicular block is a typical feature ofChagas heart disease. • Endoscopy helps in visualization of megaesophagus in Chagas disease. Treatment o effective specific treatment is available for treating Chagas disease. Nifurtimox and benznidazole have been used with some success in both acute and chronic Chagas disease. These drugs kill only the extracellular trypanosomes but not the intracellular forms. Dose: Nifurtimox: 8-10 mg/kg for adults and 15 mg/kg for children. The drug should be given orally in four divided doses each day for 90- 120 days. Benznidazole: 5- 10 mg/day orally for 60 days. Prophylaxis • Application ofinsecticide to control the vector bug. • Personal protection using insect repellant and mosquito net. Hemoflagellates Table 4: Differences between T. cruzi and T. rangeli Trypanosomacruzi • Pathogenic • 15-20 µm long • Cor U-shaped • Kinecoplast: Large and terminal • Primary reservoirs: Opossums, dog,cats and wild rodents Trypanosomarangeli ___-.1 Nonpathogenic 30µmlong, more slenderand longer Not Cor U-shaped Kinteoplast: Small and subterminal • Primary reservoir: W ild rodents • Improvement in rural housing and environment to eliminate breeding places ofbugs. Trypanosoma Rangeli T. rangeli was first described by Tejera in 1920 while examin- ing the intestinal content ofreduviid bug (R. proli.xus). . . . . It is nonpathogenic. T. rangeli infections are encountered in most areas where T. cruzi infection also occurs (Mexico, Central America and northern South America). Morphologically, it is similar to T. cruzi, except that it is slender and long (26-36 µm long) and has a smaller kinetoplast (Table 4). It is commonly found in dogs, cars and humans. Infection is transrnined by botb bite of triatomine bug and fecal contamination from reduviid bug. T. rangeli multiplies in human blood by binary fission. Intracellular stage is typically absent. • • . T. rangeli can circulate in blood of infected animals for a long period, unlike T. cruzi. Although T. rangeli appears to be a normal commensal, they do reduce the life span ofreduviid bug. Diagnostic methods are similar to that of T. cruzi. KEY POINTS OF TRYPANOSOMES • Trypanosomes follow one of the two developmental modes in vectors. In Sa/ivaria: The trypanosomes migrate to mouth parts ofvector tsetse fly, e.g. T. gambiense, T. rhodesiense. In Stercoraria: The trypanosomes migrate to hindgut of vector bug, e.g. T. cruzi. • T. brucei gambiense causes West African sleeping sickness manifested by fever, hepatosplenomegaly and posterior cervical lymphadenopathy with chronic central nervous system invasion. • T. brucei rhodesiense causes East African sleeping sickness manifested by fever, early and acute central nervous system invasion, with loss of weight and myocarditis. • Diagnosis: By detection of trypanosomes in wet mount preparations of lymph node aspirates or blood or by serology and PCR.
- 73. Paniker'sTextbook of MedicalParasitology • Drug of choice: For stage I, HAT by T. brucei gambiense is pentamidine and byT. brucei rhodesiense is suramin. In stage II, the drugof choice is melarsoprol in both cases. • South American trypanosomiasis (Chagas disease) is caused by T. cruzi. • It is transmitted by wound or conjunctiva! contamination of feces of the reduviid bugs. • Clinical features: "Chagoma· is the typical subcutaneous lesion commonlyon face (Romana's sign) in Chagas disease. Damageto nerve cells and muscles leads to megaesophagus, megacolon and cardiac myopathy. • Diagnosis: By demonstration of T. cruzi in blood or tissue or by serology and xenodiagnosis. • Treatment: Nifurtimox and benznidazole. • LEISHMAN/A General Characteristics The genus Leishmania is named after Sir william Leishman, who discovered the flagellate protozoa causing kala-azar, the Indian visceral leishmaniasis (VL). • All members of the genus Leishmania are obligate intracellularparasitesthat pass theirlife cycle in two hosts: {l) The mammalian host, and (2) the insect vector, female sandfly. • ln humans and other mammalian hoses, they multiply within macrophages, in which they occur exclusively in the amastlgote form, having an ovoid body containing a nucleus and kinetoplast. • In the sand.fly, they occur in the promasligote form, with a spindle-shaped body and a single flagellum arising from anterior end. • Leishmaniasis has an immense geographical distribution in the tropics and subtropics of the world, extending through most of the Central and South America, part of NorthAmerica, Central and South-EastAsia, India, China, the Mediterranean region and Africa. • The disease affects the low socioeconomic group of people. Overcrowding, poor ventilation and collection of organic material inside house facilitate its transmission. • Across the tropics, three different diseases are caused by various species ofgenus Leishmania. These are: I. Visceral leishmaniasis: The species L. donouani complex infecting internal organs {liver, spleen and bone marrow) ofhuman is the causative parasite. 2. Cutaneous leishmaniasis: The species L. tropica complex, L. aethiopica, L. major and L. mexicana complexare the causative parasite. 3. Mucocutaneous leishmaniasis: It is caused by the L. braziliensiscomplex. Classification The genus Leishmania includes a number of different varieties and subspecies, which differ in several features such as antigenic structure, isoenzymes, and other biochemical characteristics, growth properties, host specificity, etc. (Table 5). Leishmania species can also be classified on the basis of geographical distribution as given in Tables sand 6. The various manifestations of leishmaniasis and Leishmania species causing them have been summarized in Flow chart 2. Old World Leishmaniasis Leishmania Donovani L. donouani causes VL or kala-azar. It also causes the condition, Post-kala-azar dermal leishmaniasis (PKDL). History a nd distribution: Sir William Leishman in 1900 observed the parasite in spleen smears of a soldier who died of "dumdum fever" or kala-azar contracted at Dum Dum Calcutta. Leishman reported this finding from London 1903. In the same year, Donovan also reported the same parasite in spleen smears of patients from Madras. The name Leishmania donouani was, therefore given to chis parasite. The amastigote forms of the parasite as seen in smears from patients are called Leishman-Donovan (LD) bodies. • Visceral leishmaniasis or kala-azar is a major public health problem in many parts ofworld. According to the World Health Organization (Wl-1O), a total of 500,000 cases ofVL occur every year. Of these new cases, 90% are fow1d in the Indian subcontinent and Sudan and Brazil. • The disease occurs in endemic, epidemic, or sporadic forms. Major epidemics ofthe disease are currendy found in India, Brazil and Sudan {Fig. 8). • lhe resurgence of kala-azar in India, beginning in the mid 1970s, assumed epidemic proportions in 1977 and involved over 110,000 cases in humans. Initially, the disease was confined to Bihar (Muzaffarpur, Samastipur, Vaishali and Sitamarhi). Since then, the cases are increasing and involving newer areas. The epidemic extended to West Bengal and first outbreak occurred in 1980in Malda district. • At present, the disease has established its endemicity in 31 _districts in Bihar, 11 districts in West Bengal, five districts in Jharkhand and three districts in Uttar Pradesh. Sporadic cases have been reported from Tamil Nadu, Maharashtra, Karnataka and Andhra Pradesh. Habitat: The amastigote (LD body) ofL. donouani is found in the reticuloendothelial system. They are found mostly within
- 74. Table 5: Leishmaniaspecies involved in human disease species Disease Geographical distribution Leishman/a donovani Visceral leishmaniasis Middle East, Africa and (kala-azar or dumdum Indian subcontinent fever) Leishmania infantum Visceral leishmaniasis, Mediterranean coast, cutaneous Middle East and China leishmaniasis Leishman/a chagasi Visceral leishmaniasis Tropical Sout h America Leishmania tropica Cutaneous Middle East and leishmaniasis (oriental Central Asia sore, Baghdad boil) Leishmania major Cutaneous Africa, Indian leishmaniasis subcontinent and Central Asia Leishman/a aethiopica Cutaneous and Ethiopia and Kenya diffuse cutaneous leishmaniasis Leishmania braziliensis Mucocutaneous Tropical South complex leishmaniasis America (Espundla) Leishmania mexicana Mucocutaneous Central America and complex leishmaniasis Amazon basin (Chiclero's ulcer) Table 6: Classification of Leishman/a based on geographical distribution Oldworldleishmanlasis Newworldleishmanlasis • Leishman/a donovani • Leishman/a braziliensis complex • Leishman/a infancum • Leishmania mexicana complex • Leishmania tropica • Leishmania chagasi • Leishmania major • Leishmaniaperuviana • Leishmania aethiopica the macrophages in the spleen, liver, bone marrow and less often in other locations such as skin intestinal mucosa and mesenteric lymph nodes. Morphology:The parasite exists in two forms (Figs9A and B): 1. Amastigoteform: In humans and other mammals. 2. Promastigoteform: In the sandOy and in artificial culture. Amastigote: The amastigote form (LD body) is an ovoid or rounded cell, about 2- 4 µmin size (Fig. 9A). • It is typically intracellular, being found inside macro- phages, monocytes, neutrophils, or endothelial cells. They are also known as LD bodies. Hemoflagellates Vector Reservoir Transmission Phlebotomus Humans Anthroponotic, argentipes, occasionally zoonotic Phlebotomus orientalis Phlebotomus Dog, fox, jackal and Zoonotic pemiciosus, wolf Phlebotomus ariasi, Phlebotomus paporasi Lutzomyia longipalpis Fox and wild canines Zoonotic Phlebotomus sergenti Humans Anthroponotic Phlebotomus papatosi, Gerbil Zoonotic Phlebotomus duboscqi Phlebotomus longipes, Hydraxes Zoonotic Phlebotomus pedifer Lutzomyia umbratilis Forest rodents and Zoonotic peridomestic animals Lutzomyia olmeca, Forest rodents and Zoonotic Lutzomyia marsupials flaviscutellata • Smears stained with Leishman, Giemsa, or Wright's stain show a pale blue cytoplasm enclosed by a limiting membrane. • The large oval nucleus is stained red. Lying at the right angles to nucleus, is the red or purple-stained kinetoplast • In well-stained preparations, the kinetoplast can be seen consisting of a parabasal body and a dot-like blepharoplastwith a delicate thread connecting the two. The axoneme arising from the blepharoplast extends to the anterior tip ofthe cell. • Alongside the kinetoplast a clear unstained vacuole can be seen. Flagellum is absent. Promastigote: It is a flagellar stage and is present in insect vector, sandfly and in cultures. • The promastigotes, which are initially short, oval or pear- shaped forms, subsequently become long spindle-shaped cells, 15- 25 µm in length and 1.5-3.5 µm in breadth (Fig. 9B). • A single nucleus is situated at the center. lhe kinetoplast lies transversely near the anterior end. lhe flagellum is single, delicate and measures 15- 28 µm.
- 75. Paniker'sTextbook of MedicalParasitology Flow chart 2: Distribution and disease caused by Leishmania spp. t Leishmania I I • Old world leishmaniasis t Visceral leishmaniasis (Kala-azar) L. donovanl complex L. infantum • Cutaneous leishmaniasis L. Tropica complex comprising • L. tropica • L. aethiopica • L. major Fig. 8: Geographical distribution of visceral leishmaniasis. Endemic areas shaded; dots indicate sporadic cases • Giemsa or Leishman-sL ained films show pale blue cytoplasm with a pink nucleus and bright red kinetoplast. • Avacuole is present near the root of the flagellum. • There is no undulating membrane. • Promastigote forms, which develop in artificial cultures, have the same morphology as in the sandtly. Life cycle: l. donovani completes irs life cycle in two hosts (Fig. 10): 1. Definitive host: Man, dog and other mammals. 2. Vector: Female sand.fly (Phlebolomus species) (Table 7). Infective form: Promastigote form present in midgut of female sandily. Mode oftransmission: • Humans acquire by bite ofan infected female sand.fly. It can also be transmitted vertically from mother to fetus, by blood transfusion and accidental inoculation in the laboratory. Incubation period: Usually 2-8 months, occasionally, it may be as short as 10 days or as long as 2 years. t New world leishmaniasis j I • Visceral leishmaniasis Cutaneous leishmaniasis L. chagasi I or mucocutaneous leishmaniasis L. mexicana complex L braziliensis complex ,__ _ __ Vacuole - - - ---ct't- 0 - -- 11--- Blepharoplast- - -r--v c:::::.- - + -Parabasalbody- --!'-'~~, Figs 9A and B: Morphology of Leishmania donovani. (A) Amastigote [Leishman-Donovan (LO) body]; and (B) Promastigote • The sandlly regurgitates the promastigotes in Lhe wound caused by its proboscis. • These are engulfed by the cells of reticuloendothelial system (macrophages, monocytes and polymorphonu- clear leukocytes) and change into amastigote (LD body) within the cells. The amastigote multiplies by binary fission, producing numerous daughter cells that distend the macrophage and rupture it. 1he liberated daughter cells are in turn, phagocytosed by other macrophages and histiocytes. Small number of LO bodies can be found in peripheral blood inside neutrophils or monocytes (Fig. 10). When a vector sandtly feeds on an infected person, the amastigotes present in peripheral blood and tissue fluids enter the insect along with its blood meal. In the midgut (stomach) of the sandfly, the amastigote elongates and develops into the promastigore form (Fig. 10). The promastigore multiples by longitudinal binary fission and reaches enormous numbers. They may be seen as large roselles with their flagella entangled.
- 76. Hemoflagellates Stomach '°'""'--- ..,.__~ Amastigotes become promastigotes accumulatein which multiply pharynx and block it 7·~'-" Sandfly (Intermediate host Man acquires infection by bite of female sandfly Amastigote ingested ey~r Man (Definitive host) eeeee eoeee e0 eP Amastigotes in peripheral blood 0 I Promastigote deposited in punctured wound Phagocytosed by macrophage Fig. 10: Life cycle of Leishmania donovani • In the sandfly, they migrate from the midgut to the pharynx and hypostome, where they accumulate and block the passage. • Such blocked sandflies have difficulty in sucking blood. Wh en they bite a person and attempt to suck blood, plugs ofadherent parasites may get dislodged from the pharynx and they are deposited in the punctured wound. It cakes about 10 days for the promastigotes to reach adequate numbers after ingestion of the amastigotes, so as to block the buccal cavity and pharynx of the sandfly. This is, therefore, the duration ofextrinsic incubation period. This period is also synchronous with the gonadotropic cycle of the vector, so that amastigotes ingested during a single blood meal, are ready to be transmitted when the sandlly takes the next blood meal after its eggs have been laid. Pathogenicity:L. donovani causes VL or kala-azar. • Kala-azar is a reticuloendotheliosis resulting from the invasion of reticuloendothelial system by L. donovani. • The parasitized macrophages disseminate the infection to all parts of the body. • Three major surface membrane proteins of Leishmania, namely (1) gp63, (2) lipophosphoglycan (LPG) and
- 77. Paniker'sTextbook of MedicalParasitology Table 7: Vector species responsible for transmission of Leishmania Box 5: Causes of anemia in kala-azar donovani Coun - - - - ~ - - Phlebotomus species India • P. argentipes China, Bangladesh • P. chineses P. sergenti Sudan and Africa Mediterranean countries Middle East and Russia Central Asia South America • P. pernicious P. orientalis {Sudan) P. longicuspis P. sergenti • P. pernicious P.paparasii P. major P. tobbi • P. perfulievi P. papatasii • P. papatasii • P. longipalpis P. intermudias P. lutzi (3) glycosylphosphatidylinositols (CP!s) give pro- tection against hydrolytic enzymes of macrophage phagolysosome. In the spleen, liver and bone marrow particularly, the amastigotes multiply enormously in the fixed macro- phages to produce a "blockade" ofthe reticuloendothelial system. thisleads to a marked proliferation and destruc- tion ofreticuloendothelial tissue in these organs. Spleen: The spleen is the most affected organ. It is grossly enlarged and the capsule is thickened due to perisplenitis. - Spleen is soft and friable and cuts easily due to absence offibrosis. The cut section is red or chocolate in color due to the dilated and engorged vascular spaces. The trabeculae are thin and atrophic. Microscopically, the reticulum cells are greatly increased in numbers and are loaded with LD bodies. Lymphocytic infiltration is scanty, but plasma cells are numerous. Liver: the liver is enlarged. - 11,e Kupffer cells and vascular endothelial cells are heavily parasitized, but hepatocytes are not affected. - Liver function is, therefore, not seriously affected, although prothrombin production is commonly decreased. The sinusoidalcapiJlaries are dilated and engorged. Some degree of fatty degeneration is seen. The cut surface may show a "nutmeg" appearance. • Splenic sequestration of red blood cells (RBCs) • Decreased erythropoiesis due to replacement of bone marrow with parasitized macrophages • Autoimmune hemolysis • Hemorrhage • Marrow suppression by cytokines. • Bone marrow: - The bone marrowis heavilyinfiltrated with parasirized macrophages, which may crowd the hematopoielic tissues. • Peripheral lymph nodes and lymphoid tissues of the nasopharynx and intestine are hypertrophic, although this is not seen in Indian cases. • Severe anemia with hemoglobin levels of 5-10 g/dL may occur in kala-azar, as a result of infiltration of the bone marrow as well as by the increased destruction of erythrocytes due to hypersplenism. Autoantibodies to red cells may contribute to hemolysis (Box 5). • Leukopeniawith marked neutropeniaand thrombocyto- penia are frequentlyseen. Antibodies againstwhite blood cells (WBCs) and platelets suggest an autoimmune basis for the pancytopenia observed in kala-azar. Ecological types: the epidemiology and clinical features ofVL and the ecology of the parasite are very different in different geographical areas. The different clinical syndromes have, therefore been considered to be distinct entities and the parasite causing them have been given separate species or subspecies status, as listed here: • Indian visceral leishmaniasis: Caused by L. donouani producing the anthroponotic disease kala-azar and its sequel PKDL. The disease is not zoonotic; human beings being the only host and reservoir. Vector is the sandfly, P. argentipes. • Mediterranean leishmania.sis: Middle Eastern leishmaniasis caused by L. donovani infantum affecting mostly young children. It is a zoonolic disease; the reservoir being dogand wild caninessuch as foxes, jackals and wolves. Vectors are P. pernicious and P. papatasii. • American (New World) visceral leishmaniasis: Caused by L. chagasi. It is present is most parts of Lalin America and resembles the disease caused by L. infanlum. The main vector is L. longipalpis. Clinicalfeatures ofkala-azar: • 11,e onset is typically insidious. The clinical illness begins with high-grade fever which may be remittent with twice dailyspikes or intermittent or less commonly continuous. Splenomegaly starts early and is progressive and massive (Fig. I l). It is usually soft and nontender. Hepatomegaly is moderate.
- 78. Fig. 11 :Kala-azar spleen showing a greatly enlarged organ • l ymphadenopathy is common in most endem ic areas except Indian subcontinent. • Skin becomes dry, rough and darkly pigmented (hence, the name kala-azar). • The hair becomes thin and brittle. • Cachexia with marked anemia, emaciation and loss of weight is seen. • Hematologicalabnormalities: Anemia is most always present and is usually severe leukopenia 111rombocytopenia is associated with epistaxis, gum bleeding, gastrointestinal (GI) bleeding. • Asciles and edema may occur due to hypoalbum inemia. • Renal involvement is also common. • In late stage of human immunodeficiency virus (IIIV) infection VL can present as opportunistic infection. HIV coinfection rate is5%in India and 20%in African countries. • Secondary inf ections such as herpes, measles, pneumo- nia, tuberculosis, bacillary dysentery may occur. • Most untreated patients die in about 2 years, due to some intercurrent disease such as dysentery, diarrhea and tuberculosis. Post-kala-azar dermal leishmariiasis: About 3- 10% cases of palients of VL in endemic areas develop PKDL, about an year or 2 after recovery from the systemic illness. • Post-kala-azar dermal leishmaniasis is seen mainly in lndia and East Africa and not seen elsewhere. The Indian and African diseases differ in several aspects; important features of PKDL. in these two regions are listed in Table 8. Post-kala-azar dermal leishmaniasis is a nonulcerative lesion of skin. lhe lesions are of three types: 1. Depigmented or hypopigmented macules: These commonly appear on the face, the trunk and extremities and resemble tuberculoid leprosy. Hemoflagellates Fig. 12: Erythematous patches (Butterfly distribution) Table 8: Differences between post-kala-azar dermal leishmaniasis (PKDL) of India and East Africa Characreristics India East Africa Incidence 5% 50% Time interval between Occurs after visceral Occurs during visceral visceral leishmaniasis leishmaniasis. May leishmaniasis and PKDL take 3- 5 years Age group affected Any age Mostly children Appearance of rash Rashes appear Rashes may appear after visceral during visceral leishmaniasis leishmaniasis Spontaneous cure Not seen Seen Duration of treatment 60-120 days 60 days with sodium stibogluconate 2. Erythemalous patches: These are distributed on the face in a "butterfly distribution" (Fig. 12). 3. Nodular lesion: Both of the earlier mentioned lesions may develop into painless yellowish pink nonulcerating granulomatous nodules. • The parasite can be demonsrrated in the lesions. Diagnosis ofpost-kala-azar dermal leishmaniasis: • The nodular lesions are biopsied and amastigote forms are demonstrated in stained sections. Th e biopsy materialcan be cultured or animal inoculation can be done. • lmmunodiagnosis has no role in the diagnosis of PKDL. Treatment ofpost-kala-azardermal leishmaniasis: • Liposomal amphotericin-8 (AmBisome) 2.5 mg/ kg/ day for 20 days or sodium stibogluconate (SSG) 20 mg/ kg/day for 40- 60 days are given.
- 79. Paniker'sTextbookofMedical Parasitology Immunity: • Theimmune response in VL is very complex. • There is increased production of proinflammatory cytokines and chemokines. Interleukin-IO (IL-10) and transforming growth factor-B (TGF-B) are the dominant cytokines. • The most important immunological feature in kala-azar is the marked suppression of cell-mediated immunity to leishmanial antigens. This makes unrestricted intra- cellular multiplication of the parasite possible. Cellular responses to tuberculin and other antigens are also suppressed and may be regained some 6 weeks after recovery from the disease. • 1n contrast, there is an overproduction of immunoglobu- lins, both specific antileishmanial antibodies as well as nonspecific polyclonal IgG and lgM. Circulating immune complexes are demonstrable in serum. Laboratory diagnosis: Laboratory diagnosis of kala-azar depends upon direct and indirect evidences {Flow chart 3). Direct evidence: Microscopy: • Demonstration of amastigotes in smears of tissue aspirates is the gold standard for diagnosis ofVL. • For microscopic demonstration of the parasite, the materials collected are: Peripheral blood Bone marrow - Splenic aspirate Enlarged lymph node. • The smears are stained by Leishman, Giemsa, or Wright's stains and examined under oil immersion objective. • Amastigote parasite can be seen within the macrophages, often in large numbers. Afew extracellular forms can also be seen. • Peripheral blood smear: Peripheral blood contains the amastigotes present inside circulating monocytes and less often in neutrophils, but the numbers are so scanty that a direct blood smear may not show them. - Chances of detecting them are somewhat improved by examination ofa thick blood film. - It is best to examine huffy coatsmear, although even these are not often found positive. Buffy coat smears show a diurnal periodicity, more smears being positive when collected during the day than at night. • Bone marrowaspirate: Bone marrow aspirate is the most common diag- nostic specimen collected. - Generally, the sternal marrow is aspirated by puncturing the sternum at the level of the 2nd or 3rd intercostal space, using a sternal puncture needle. Bone marrow samples can also be obtained by puncturing the Iliac crest. • Splenic aspirates: - Splenic aspirates are richer in parasites and therefore, are more valuable for diagnosis. Flow chart 3: Laboratory diagnosis of kala-azar Laboratory diagnosis • Direct evidence I l • Demonstration of Culture LO bodies In NNN medium In stained or Schneider's smears of thick blood liquid medium film, splenic, bone to demonstrate marrow, and promastigote lymph node aspirate form • Indirect evidence I l • t • Animal Serodiagnosis Molecular Nonspecific Inoculation In hamster or mice Detection of antigen ELISA --------' diagnosis serum test • DNA probe • Aldehyde test • PCR • Chopra's antimony test. The tests are positive in hypergammaglobulinemia Detection of antibody • CFT using WKK antigen •DAT • IFAT • CIEP • DOT-ELISA • ICT using rK39 antigen • • Skin test Blood picture Leishmanln • Anemia or • Progressive Montenegro leukopenia test • Reverse albumin: globulin ratio Abbreviations: CFT, complement fixation test: CIEP. counter immunoelectrophoresis: DAT, direct agglutination test; DNA, deoxyribonucleic acid; ELISA, enzyme- linked ,mmunosorbent assay; ICT, immunochromatographlc test; !FAT, indirect immunofluorescent antibody test: LD, Leishman-Donovan; NNN, Novy, MacNeal and Nicolle; PCR, polymerase chain reaction; rK39, recombinant kinesin 39
- 80. Hemoflagellates Volutin granules Nucleus Lymphocyte Flagella Parasite from disrupted cell Nucleus Characteristic clustersin ' 0 cultures Dividing parasite ) Ingested - ~ -..:c_--''--- Nucleus of a liver parenchymal cell Commencing parasites division Figs 13A and B: Leishmania donovani. (A) Culture form (Giemsa stain, magnification 1100X); and (B) Liver smear (Giemsa stain. magnification 1100X) But, the procedure can sometimes cause dangerous bleeding and therefore, should be done carefully and only when a marrow examination is inconclusive. • Lymph node aspirates: Lymph node aspirates are not useful in the diagnosis of Indian kala-azar, although it is employed in VLin some other countries. Comparison ofaspiration biopsies: Although splenic aspiration is the most sensitive method (98% positive), bone marrow puncture (50-85%, positive) is a safer procedure when compared to spleen puncture,as there is risk of hemorrhage in splenic puncture particularly in patients with advanced stage ofdiseasewith soft enlarged spleen. Splenic aspiration is contraindicated in patients with prolonged prothrombin time, or if platelet count is less than 40,000/mm3 • Liver biopsy is also not a safe procedure and carries a risk of hemorrhage. Lymph node aspiration is positive in 65% ofcases of African kala-azar, but not useful in cases ofIndian kala-azar. Culture: Different tissue materials or blood are cultured on N N medium (described by Novy, MacNeal and Nicolle). this is a rabbit blood agar slope consisting of two parts of salt agar and one part of defibrinated rabbit blood. The material is inoculated into the water of condensation and culture is incubated at 22- 24°C for 1-4 weeks. At the end of each week, a drop of culture fluid is examined for promastigotes under high power objective or phase contrast illumination (Figs 13A and B). Other biphasic medium, like Schneider's drosophila tissue culture medium with added 30%fetal calf serum can also be used. Animal inoculation: Animal inoculation is not used for routine diagnosis. - ' •• •• ••• ' . • ••• ...,. ••J•• ···-· •••• •• ,, Fig. 14: Leishman-Donovan (LD) body in spleen smear of experimentally infected animal (Giemsa stain) • When necessary, Chinese golden hamster is the animal employed. The material is inoculated intraperitoneally or intra- dermally into the skin of nose and feet. the inoculated animals are kepl at 23- 26°C. • In positive cases, the amastigote can be demonstrated in smears taken from ulcers or nodules developing at the sites of inoculation or from the spleen (Fig. 14). • Animal inoculation is a very sensitive method, but takes several weeks to become positive. Indirect evidences: Serodiagnosis: • Detection ofantigen: The concentration of antigen in the serum or other body fluids is very low. ELISA and PCR have been developed for detection of leishmanial antigen.
- 81. Paniker'sTextbook of MedicalParasitology • Two noninvasive antigen detection test in urine for VL are under evaluation. Detection ofantibodies: Complement fixation test was the first serological test used to detect serum antibodies in VL. The antigen originally used, was prepared from human tubercle bacillus by Witebsky, Klingenstein and Kuhn (hence, called WKK antigen). CFT using WKK antigen becomes positive early in the disease, within weeks of infection. Positive reaction also occurs in other conditions, including tuberculosis, leprosy and tropical eosinophilia. Specific leishmanial antigens prepared from cultures have been used in a number of tests to demonstrate specific antibodies. These tests include: • Indirect immunofluorescent antibody test (!FAT) • Counter immunoelectrophoresis (CIEP) • ELISA and DOT-ELISA • Direct agglutination test (DAT) rk 39 test: A specific rapid immunchromatographic test (JCT) method for antibody has been developed using a recombinant leishmanial antigen rk 39 consisting of 39 amino acids conserved in kinesin region of L. infantum. The sensitivity ofthe test is 98% and specificity is 90%. Note: The direct aggl utination test for antileishmanial antibody has been found to be highly specific and sensitive for diagnosis ofkala-azar. However, rk39 antibody test is more useful and easy to perform and recommended by National Vector Borne Disease Control Programme (NVBDCP) in India. Molecular diagnosis: A number of molecular diagnosis methods have been developed, which help in species identification of Leishmania. The methods include Western blot and PCR. The use of PCR is confined to specialized laboratories and is yet to be used for routine diagnosis ofVL in endemic areas. Nonspecific serum tests: These tests are based on the greatly increased globulin content ofserum in the disease. • 11,e two tests widely used are: J. Napier's aldehyde orJormogel test 2. Chopra's antimony test. • Napier aldehyde test: l mL of clear serum from the patient is taken in a small test tube, a drop offormalin (40% formaldehyde) is added, shaken and kept in a rack at room temperature. A control tube with normal serum is also set up. A positive reaction is jellification and opacification ofthe test serum, resembling the coagulated white of egg appearing within 3-30 minutes. - About 85% of patients with disease of 4 months or more give positive reaction. Aldehyde test is always negative in cutaneous leishmaniasis (CL). The test merely indicates greatly increased serum gamma-globulin and thus, is nonspecific. • Chopra's antimony test: It is done by taking 0.2 mL of serum diluted 1:10 with distilled water in a Dreyer's tube and overlaying with few d rops of 4% solution of urea stibamine. Formation of tlocculcnt precipitate indicates positive test. - The reaction is said to be more sensitive than the aldehyde test. • Both the tests give false-positive reactions in several other disease such as multiple myeloma, cirrhosis of liver, tuberculosis, leprosy, schistosomiasis, African trypanosomiasis, etc. where hypergammaglobulinemia exists. Skin test: • Lelshmanin skin test (Montenegro test): It is delayed hypersensitivity test. This was first discovered by Montenegro in South America and hence, named after him. 0.1 mL ofkilled promastigote suspension (l 06 washed promastigotes/ mL) is injected intradermally on the dorsoventral aspect of forearm. Positive result is indicated by an induration and erythcma of5 mm or more after48-72 hours. - Positive resultindicates prior exposure to leishmanial parasite. In active kala-azar, this test is negative and becomes positive usually 6-8 weeks after cure from the disease. Blood picture: • Complete blood countshows normocytic normochromic anemia and thrombocytopenia. Leukocyte count reveals leukopenia accompanied by a relative increase of lymphocytes and monocytes. Eosinophil granulocytes are absent. During the course of disease, there is a progressive diminution of leukocyte count falling to l ,000/mm3 of blood or even below that. • The ratio of leukocyte to erythrocyte is greatly altered and may be about 1:200 to 1:100 (normal 1:750). • Serum shows hypergammaglobulinemia and a reversal ofthe albumin: globulin ratio. • Liver function tests show mild elevations ofliverenzymes. • Erythrocyte sedimentation rate is elevated. Treatment: Kala-azar responds to Lreatrnentbetterthanother forms of VL. The standard treatment consists of pentavalent antimonial compound, which is the drug of choice in most of the endemic regions of the world, but there is resistance to antimony in Bihar in India, where amphotericin-B- deoxycholate or miltefosine is preferred.
- 82. Pentavalent antimonial compound:Two pentavalent anti- monial (Sbv) preparations are available: 1. Sodium stibogluconate (100 mg ofSbv/mL) (SSG) 2. Meglumine antimoniate (85 mg of Sb"/mL). Dosage: The daily dose is 20 mg/kg by rapid intravenous (TV) infusion or intramuscular (IM) injection for 20-30 days. Cure rates exceed 90% in most of the old world, except in Bihar (India) due to resistance (cure rate 36%). Amphotericin-B: • Amphotericin-B is currently used as a first-line drug in Bihar. ln other parts of the world, it is used when initial antimonia1 treatment fails. • Dosage: 0.75-1.0 mg/kg on alternate days for a total of 15 infusions. Note: Fever with chills is almost seen in all patients, using amphotericin-8 infusions. • Liposomal amphotericin-B (AmBisome): It has been developed and used extensively to treat VL in all parts of the world. It is the only drug approved by the US Food and Drug Administration (FDA) for the tream1ent of VL; dose being 3 mg/kg daily. By using liposoma1 amphotericin-B, higher doses can be given, improving the cure, without toxicity (Box 6). • Current recommendation in India isl0 mg/kg single dose. Paromomycin: Paromomycin is an intramuscular amino- glycoside antibiotic with anrileishmanial activity. Dosage: It is given in a dose of 11 mg/kg daily for 21 days. Millefosine: Milcefosine is the first oral drug, approved for the treatment ofleishmaniasis. Dosage: 50 mg dailyfor 28 days for patients weighing less than 25 kg, and twice daily for patients weighing more than 25 kg. Prophylaxis: • Early detection and treatment ofall cases. • Integrated insecticidal spraying to reduce sandfly population. • Destruclion of animal reservoir host in cases or zoonotic kala-azar. Box 6:Advantages of drugcoadministrations in visceral leishmanias,s • Increase activity by additive and synergistic effect. • Reduce length of treatment, toxicity. drug-dose burden. • Reduce resistant cases and improve patient compliance. • Improve success in treating human immunodeficiency virus (HIV)• leishmanlasis coinfected cases. • Regime of coadministrated drug include: - AmBisome + Paromomycin - AmBisome + Miltefosine - Paromomycin + Miltefosine Hemoflagellates • Personal prophylaxis by using anrisandfly measures like, using thick clothes, bed nets, window mesh, or insect repellants and keeping the environment clean. • No vaccine is available at present against kala-azar. • Candidate vaccine: Many 2nd generation subunit vac- cines are under trial in rodent models, e.g. hydrophilic acctylated surface protein Bl (HASBl), kinetoplastid membrane protein JI (KMPII) and Leishlll. Leishmanla tropica Complex • lt includes three species: 1. Leishmania tropica 2. Leislzmania major 3. Leishmania aethiopica. • All these species cause old world cutaneous leishmanlasis. The disease is also known as oriental sore, Delhi boil, Bagdad boil, or Aleppo button. History and distribution: Cunningham (1885) first observed the parasite in the tissues ofa Delhi boil in Calcutta. • Russian military surgeon, Borovsky (1891) gave an accurate description of its morphology and Luhc (1906) gave the name L. tropica. • L. tropica and L. major arc found in Middle-East, India, Afghanistan, Eastern Mediterranean countries and North Africa. • L. aethiopica occurs in Ethiopia and Kenya. • In India, CL is restricted to the dry western hair of the lndo-Gangetic plains including dry areas bordering Pakistan, extending from Amritsar to Kutch and Gujarat plains. To the East, the cases have been reported from Delhi and Varanasi in uttar Pradesh. Habitat: L. tropica causing CL(old world CL) are essentially the parasite of skin. The amastigote forms occur in the rcticulocndothelial cells of the skin, whereas promastigote forms arc seen in sandflyvector. Morphology: Morphology of L. tropica complex is indistin- guishable from that ofL. donovani. Lif e cycle: The life cycle of L. tropica is similar to that of L. donouani except: Vectors: The vectors of L. tropica complex are Phlebotomus sandflies.The following species ofsandflies acr as vector: • P. sergenti-L. tropica • P. papatasi- L. major • P. longipes- L. aethiopica Mode oftransmission: • The most common mode of infection is through bite of sandflies. • Infection may also sometimes occur by direct contact. • Infection may be transmitted from man-to-man or animal-to-man by direct inoculation oramasligotes.
- 83. Paniker's Textbook ofMedical Parasitology • infection may also occur by autoinoculation. • The amastigotes are present in the skin, within large mononuclear cells, neutrophils, inside capillary endo- thelial cells, and also free in the tissues. • They are ingested by sandflies feeding near the skin lesions. • In the midgut ofthe sand.fly, the amastigotes develop into promastigotes, which replicate profusely. • These are in turn transmitted to the skin of persons bitten by sandflies in the skin, the promastigotes are phagocytosed by mononuclear cells, in which they become amastigotes and multiply. • However, they remain confined to the skin, without being transported to the internal organs, as is the case in VL. lncubation period:Incubationperiod varies from 2-8 months. Pathology: Amastigote forms are found in histiocytes and endothelial cells. there is an inflammatory granulomatous reaction with infiltration of lymphocyte and plasma cells. Early lesions are papular, followed by ulceration necrosis. Papule and ulcer are the main pathological lesions. They heal over months to years, leaving scars. clinical features: L. tropica causes old world cutaneous leishmaniasis. • Features of the disease vary with epidemiological pattern from region-to-region. • Three distinct patterns of old world CL have been recognized. • The anthroponotic urban type causing painless dry ulcerating lesions, leading to disfiguring scars, caused by the species L. tropica. This is prevalent from the Middle East to North- Western India. The mostimportant vectoris P. sergenti. - IL is seen mainly in children in endemic areas and is called as orientalsore or Delhi boil. It begins as a raised papule, which grows into a nodule that ulcerates over some weeks. Lesions may be single or multiple and vary in size from 0.5 to more than 3 cm. Lymphatic spread and lymph gland involvement may be palpable and may precede the appearance ofthe skin lesion. - The margins of the ulcer are raised and indurated. - The ulcer is usually painless unless secondary bacterial infection occurs. - There may be satellite lesions, especially in L. major and L. tropica infections. - The dry ulcers usually heal spontaneously in about an year. • Thezoonotic rural type causing moist ' which are inflamed, often multiple, caused by L. major. - The incubation period is usually less than 4 months. - Lesions due to L. major heal more rapidly than L. tropica This is seen in the lowland zones ofAsia, Middle East and Africa. - Gerbils, rats and other rodents are the reservoirs. - P. papatasi is the most important vector. • Diffuse cutaneous leishmaniasis: The nonulcerative and often diffuse lesions caused by L. aethiopica and seen in the highlands of Ethiopia and Kenya are known as diffuse cutaneous leishmaniasis (DCL). - P. /ongipes is the usual vector. - It is a rare form of disease, where nodular lesions although restricted to skin are disseminated on the face and extremities from initial localized papule. - It is characterized by low humoral as well as cell- mediated immunity. - the lesions last for years or even for entire age. - It is difficult to treat. Leishmaniasis recidiuans is a type of lesion seen in persons with a high degree of cell-mediated immunity to the parasite. The lesions are chronic with alternating periods of activity and healing, characterized by a central scar with peripheral activity. The lesions resemble those of lupus or tuberculoid leprosy. Parasites are very scanty in the lesions. Leishmanin test is strongly positive. Chemotherapy is not very useful. Better results follow local application of heat. Laboratorydiagnosis: Microscopy: • Smear is made from the material obtained from the indurated edge of nodule or sore and stained by Giemsa or Leishman stain. • Amastigotes are found in large numbers inside the macrophages. • Definitive diagnosis is made by demonstration of amastigote in the smear collected from the lesion. Culture: Promastigote forms can be isolated by culture of the aspirate material in NN 1 medium. Skin test: Leishman.in skin test is helpful. Positive leishmanin test in children under 10 years of age from endemic areas is highly suggestive of the disease. The skin test is negative in diffuse CL. Serology: these are of limited value as the patient shows no detectable levels ofcirculating antibodies. Treatment: The specific treatment ofCLis same asVL. • Antimony-resistant diffuse CL can be treated with pentamidine. • Topical treatment consists of a paste of 10% charcoal in sulfuric acid or liquid nitrogen.
- 84. Prophylaxis: Control of sandflypopulation by insecticides and sanitation measures. Personal protection by use ofprotective clothing and use of insect repeUants. Elimination of mammalian reservoir. New World Leishmaniasis L. BraziliensisComplex and L. Mexicana Complex History and distribution: Lindenberg and ParanJ10s (1909) first described amascigotes in the ulcers of skin in a man in Brazil. Vianna (1911) named the species as L. braziliensis. • L. braziliensis complex and L. mexicana complex cause new world leishmaniasis in Central and South America. Habitat: These occur as intracellular parasite. The amastigote form is seen inside the macrophages of skin and mucous membrane of the nose and buccal cavity. The promastigote form occurs in vector species Lutzomyia. Morphology: Morphology of amastigoce and promascigote forms of both the parasites is same as that of the other two species of Leishmania. Life cycle: The life cycle of Leishmania species causing the new world cutaneous and mucocutaneous leishmaniasis is similar to that ofL. donovani except: • Amastigotes are found in the reticuloendothelial cells and lymphoid tissues ofskin, but not in the internal organs. • The infection is transmitted to man from animals by bite ofsandfly vectors ofgenus Lutzomyia. • Sylvatic rodents and domestic animals are the common sources and reservoir of infection. • Direct transmission and aucoinfection also occurs man-co-man. Clinicalfeatures: L. mexicana complex leads to cutaneous leishmaniasis which closely resembles the old world CL. However a specific lesion ofcaused by L. mexicana is chiclero ulcerwhich is characterized by ulcerations in pinna. • Chiclero ulcer is also called as selfhealingsore ofMexico. • L. braziliensis complex causes both mucocutaneous leishmaniasis (espundia) and "CL''. • L. braziliensis causes the most severe and destructive form ofcutaneous lesion. • It involves the nose, mouth andlarynx. • The patient experiences a nodule at the site ofsandfly bite with symptoms consistentwith oriental sore. • Subsequent mucocutaneous involvement leads co nodules inside the nose, perforation of the nasal septum, and enlargement ofthe nose and lips (espundia). Hemoflagellates • Ifthe larynx is involved, the voice changes as well. • Ulcerated lesions may lead to scarring and tissue destruction that can be disfiguring. • The disease occurs predominantly in Bolivia, Brazil and Peru. • L. mexicana, L. amazonensis also cause DCL similar to chat of L. aethiopica in individuals with defective cell- mediated immunity. Montenegro skin test is negative. Pian bois: It is also known as "forest yaws''. It is caused by L. braziliensis guyanensis and is characterized byappearance ofsingle or multiplepainless dry persistent ulcers appear all. Laboratorydiagnosis: Microscopy: Amastigotes are demonstrated in smears taken from lesions of skin and mucous membrane. L. mexicana amastigotes are larger than those of L. braziliensis and their k:inetoplast is more centrally placed. Biopsy: Amastigotes can also be demonstrated from slit-skin biopsy. Culture: Culturing material obtained from ulcers in N N medium demonstrates promastigotes. L. mexicana grows well in comparison to L. braziliensis, which grows slowly. Serology: Antibodies can be detected in serum by IFA test, which is positive in 89-95% of cases. ELISA is also a sensitive method to detect antibody; being positive in 85% ofcases. Skin test: Leishmanin test is positive in cutaneous and mucocutaneous leishmaniasis. Treatment: Treatment with a pentavalent antimonial com- pound is moderately effective for mild mucocutaneous leishmaniasis. Amphotericin-B is the best alternative drug currently available. In case of respiratory complications, glucocorticoids can be used. Prophylaxis: • Due to sylvatic and rural nature of the disease, control is often difficult. • Use of insect repellants, spraying of insecticides and screening are advisable. • Forest workers should use protective clothing and other protective measures. • A recently developed polyvalent vaccine using five Leishmania strains has been reported to be successful in reducing the incidence ofCL in Brazil.
- 85. Paniker'sTextbook of MedicalParasitology KEY POINTS OF LEISHMAN/A • Visceral leishmaniasis (kala-azar) is caused by L. donovani and L. intantum. • Vector of kala-azar is sandfly (argentipes). • Amastigote forms (LD body) are found in macrophages and monocytes in human. • Promastigote forms with a single flagellum is found in vector sandfly and artificial culture. • Clinical features: Kala-azar: Fever, hepatosplenomegaly, marked anemia, darkly pigmented skin, weight loss, cachexia, etc. • Post-kala-azar dermal leishmaniasis: Seen after 1 - 2 years of treatment in 3-10% cases and is a nonulcerative lesion of skin. • Diagnosis: By demonstrations of LO bodies in peripheral blood, bone marrow aspirate, splenic aspirate and lymph node aspirate; culture done in NNN medium; aldehyde test; detection of specific antigen and antibody by IIF, ELISA, DAT and rapid rk 39 antibody detection test. • Blood picture: Anemia, thrombocytopenia, leukopenia with relative lymphocytosis and hypergammaglobulinemia. • Treatment: Sodium stibogluconate, amphotericin-B and oral miltefosine. • Old world CL (oriental sore) is caused by L. tropica and the vectors are P. sergenti and P. papatasi. • New world mucocutaneous (espundia) and CL are caused by L. brazifiensis and L. mexicana. Vector is sandfly of genus Lutzomyia. REVIEW QUESTIONS 1. Describe briefly the life cycle and laboratory diagnosis of: a. Trypanosoma bruceigambiense b. Trypanosoma cruzi c. Leishmania donovani 2. Write short notes on: a. Sleeping sickness b. Chagas disease c. Antigenic variations of Trypanosoma bruceigambiense d. Morphological stages ofhemoflagellates e. Trypanosoma rangeli f. Kala-azar g. Post-kala-azar dermal leishmaniasis h. Cutaneous leishmaniasis i. Diffuse cutaneous leishmaniasis 3. Differentiate between: a. East African trypanosomiasisand West African trypanosomiasis b. Trypanosoma cruzi and Trypanosoma rangeli MULTIPLE CHOICE QUESTIONS 1. Vector for Trypanosoma cruzi is a. Reduviid bug b. Tsetse fly c. Sandfly d. Hard tick 2. All of the following are obligate intracellular parasite except a. Plasmodium b. Trypanosoma cruzi c. Toxoplasma gondii d. Trypanosoma bruceigambiense 3. Romana's sign occursin a. Babesiosis b. Leishmaniasis c. Trypanosomiasis d. Schisotosomiasis 4. Vector for T. brucei gambiense is a. Sandfly b. Reduviid bug c. Tsetse fly d. House fly S. Winterbottom sign in sleeping sicknens refers to a. Unilateral conjunctivitis b. Posterior cervical lymphadenitis c. Narcolepsy d. Trasient erythema 6. The drug that can clear trypanosomes from blood and lymph nodes and is active in late nervous system stages of African sleeping sickness is a. Emetine b. Melarsoprol c. Nifurtimox d. Suramin 7. Which of the following is not true about West African trypanosomiasis. a. Primary reservoirs are human b. Low parasitemia c. Illness is usually chronic d. Minimal lymphadenopathy 8. Chronic infections with which of the following hemoflagellates may be associated with megaesophagus or megacolon a. Trypanosoma gambiense b. Trypanosoma cruzi c. Leishmania donovani d. Leishmania tropica 9. True about visceral leishmaniasis is/are a. Caused by Leishmania tropica b. Post leishmaniasisdermatitisdevelops in 20% ofpatients
- 86. c. Antimonial compoundsare useful d. Vector is tsetse fly 10. Which ofthe following is most severely affected in kala-azar a. Spleen b. Liver c. Lymph nodes d. Bone marrow 11. LD bodies are a. Amastigotes ofLeishmania donovani inside RBCs b. Giant cells seen in leishmaniasis c. Degenerative lesions seen in leishmaniasis d. Amastigotes ofLeishmania donovani inside macrophages 12. In a case of kala-azar, aldehyde test becomes positive after a. 2 weeks b. 4weeks c. 8 weeks d. 12 weeks 13. Mucocutaneous leishmaniasis is caused by a. Leishmania braziliensis b. Leishmania donovani c. Leishmania tropica d. None of the above 14. Chiclero's ulcer is caused by a. Leishmania mexicana complex b. Leishmania braziliensis complex c. Leishmania trapica d. Leishmania infantum Answer 1. a 8. b 2. d 9. C 3. C 10. a 4. C 11. d 5. b 12. d Hemoflagellates 6. b 13. a 7. d 14. a
- 87. CHAPTER 6 MALARIA • INTRODUCTION Procozoanparasites characterized by the production ofspore- like oocysts containing sporozoites were known as sporozoa. . • • . They live inlracellularly, at least during part of their life cycle. At some stages in their life cycle, they possess a structure called the apical complex, by means ofwhich they anach co and penetrate host cells. These protozoa are therefore grouped under the Phylum Apicomplexa. The medically important parasites in this group are the malaria parasites, Coccidia, and Babesia. The Phylum Apicomplexa includes two classes viz. (1) hematozoa and (2) coccidia and three orders- (1) eimeriida, (2) hemosporida and (3) piroplasmida (Table l). Note: Many minute intracellular protozoa formerly grouped as sporozoa have been reclassified because of some strucrural differences. These are now called microspora. they infect a large spectrum of hosts including vertebrates and invertebrates. Infection is mostly asymptomatic, but clinical illness is often seen in the immunodeficient. Table 1: Phylum Apicomplexa (Sporozoa) Class Hematozoa Coccidia Order Hemosporida Piroplasmida Eimeriida Genera • Plasmodium • Babesia • Toxoplasma • Cyclospora • Cryptosporidium • lsospora • Sarcocystis • CLASSIFICATION Malaria parasite belongs to: Phylum: Apicomplexa Class: Sporozoa Order: Hemosporida Genus: Plasmodium. • The genus Plasmodium is classified into two subgenera: (1) P. vivax, (2) P. malariae and P. ovale belong to the subgenus Plasmodium while P. falciparum belongs to subgenus Laverania because it differs in a number of aspects from tl1e other three species. • P. vivax, P. malariaeandP. ovalearecloselyrelated to other primate malaria parasites. P.falciparum is more related to bird malaria parasites and appears to be a recent parasite ofhumans, in evolutionary terms. Perhaps for this reason, falciparum infection causes the most severe form of malaria and is responsible for nearly all fatal cases. • P. knowlesi, a parasite of long-tailed Macaque monkeys may also affect man. • CAUSATIVE AGENTS OF HUMAN MALARIA • Plasmodium vivax: Benign tertian malaria • Plasmodiumfalciparum: Malignant tertian malaria • Plasmodium malariae: Benign quartan malaria Plasmodium ovate: Benign tertian malaria. • MALARIA PARASITE History and Distribution Malaria has been known from ancient times. Seasonal intermittent fevers with chills and shivering, recorded in the religious and medical texts of ancient Indian, Chinese and Assyrian civilizations, are believed to have been malaria (Fig. I).
- 88. • The namemalaria (mal: bad, aria: air) was given in the 18thcenturyin Italy, as itwas thoughtto be caused by foul emissions from marshy soil. • The specific agent of malaria was discovered in red blood cells (RBCs) of a patient in 1880 by Alphonse Laueran, a French army surgeon in Algeria. • ln 1886, Golgi in Italy described the asexual development of the parasite in RBCs (erythrocytic schizogony), which therefore came to be called as Golgi cycle. • three different species of malaria parasite infecting man: (1) P. vivax, (2) P. malariae, and (3) P. falciparum were described in Italy between 1886 and 1890. The fourth species, P. ovale was identified only in 1922. • The mode of transmission of the disease was established in 1897, when Ronald Ross in Secunderabad, India identified the developing stages of malaria parasites in mosquitoes. This led to various measures for the control and possible eradication of malaria by mosquito control. Both Ross (1902} and Laveran (1907) won the Nobel Prize for their discoveries in malaria. • Incidence of malaria is more in poor population in rural areas, also in urban areas having bad sanitary condition. An epidemic can develop when there are changes in environmental, economic and social conditions such as migrations and heavy rains following draughts. • The relative prevalence of the four species of malaria parasites varies in different geographical regions (Fig. 1): l. P. uiuax is the most widely distributed, being most common in Asia, NorthAfrica, and Centraland South America. 2. P. Jalciparum, the predominant species in Africa, Papua New Guinea and Haiti, is rapidly spreading in Southeast Asia and India. 3. P. malariae is present in most places but is rare, except in Africa. 4. P. ouale is virtually confined to West Africa where it ranks second after P.falciparum (Fig. 1). D Areas where Areas where malaria Is absent malaria Is present • Areas with limited risk of malaria Fig. 1: Global distribution of malaria Malaria and Babesia • Malaria may occur in endemic as well as epidemic patterns. It is described as endemic, when it occurs constantly in an area over a period of several successive years and as epidemic, when periodic or occasional sharp rises occur in its incidence. Th e World Health Organization (WHO} has rec01mnended the classification ofendemicity depending on the spleen or parasite rate in a statistically significant sample in the populations of children (2-9 years) and adults. According to this: - Hypoendemic (transmission is low): Spleen or parasite rate less than 10% Mesoendemic (transmission is moderate): Spleen or parasite rate 11-50% - Hyperendemic (transmission is intense butseasonal): Spleen or parasite rate 51-75% - Holoendemic (transmission ofhigh intensity):Spleen or parasite rate more than 75%. In India, malaria is a major public health threat. In India, about 27% population lives in high transmission (>l case/ 1,000 population) andabout58%in lowtransmission (0- 1 case)/1,000 population) area. • In spite of decline of total number of malaria cases, the number of cases ofP.Jalciparum malaria has increased. Vectors Human malaria is transmitted by over 60 species of female Anopheles mosquito. • The male mosquito feeds exclusively on fruits and juices, but the female needs at least two blood meals, before the first batch of eggs can be laid. • Out of 45 species of Anopheles mosquito in India, only few are regarded as the vectors of malaria. These are An. culicifacies,An.jluviatilis, An. stephensi, An. minimus, An. philippinensis, An. sundaicus, etc. Life Cycle Malaria parasite passes its life cycle in two hosts: 1. Definitive host: Female Anopheles mosquito. 2. Intermediate host: Man. • The life cycle of malarial parasite comprises of two stages-(1) an asexual phase occurring in humans, which act as the intermediate host and (2) a sexualphase occurring in mosquito, which serves as a definitive host for the parasite (Fig. 2). Asexual Phase • In this stage, the malaria parasite multiplies by division or splitting a process designated to as schizogony (from schizo: to split, and gone:generation).
- 89. Paniker'sTextbook of MedicalParasitology Mosquito injects sporozoites during blood meal Mature oocyst ruptures I~~ sporozoites, which reach ./ " the salivary gland of mosquito • l I I I I I / / ______r ... ' ' ' , Ookinete penetrates ' B the epithelial lining of , mosquito stomach Ookinete wall ' Fertilization--Q , occurs, I Sporozoites infect liver cell Sch1zont formed - ~ ___:__.__ Ruptured schizont merozoites liberated Merozoites invade RBC , zygote formed 1 ', ~ - 0 1 ' - ~.:.. I ' , Microgamete Macrogamete..- 1 "'- •• ,-p.1 ~~11~ - - ----'-Late trophozoite Early trophozoite formed .., --. ___ -.. ,, - J ~ :r'- - -- - -l.Mature schizont Gametogony Midgut of mosquito . ~~ Female Male Mature schizont burst releasing merozoites Fig. 2: Life cycle of the Plasmodium vivax Abbreviation: RBC, red blood cell Because this asexual phase occurs in man, it is also called the vertebrate, intrinsic, or endogenous phase. • In humans, schizogony occurs in two locations- (!) in the red blood cell (erythrocyticschizogony)and (2) in the liver cells (exoerythrocytic schizogony or the tissue phase). • Because schizogony in the liver is an essential step before the parasites can invade erythrocytes, it is called pre- erythrocyticschizogony. • The products of schizogony, whether erythrocytic or exoerythrocytic, are called merozoites (meros: a part, zoon:animal). Sexual Phase • Female Anopheles mosquito represents definitive host, in which sexual forms takes place. Although the sexual forms ofthe parasite (gametocytes) originate in human RBCs. • Maturation and fertilization take place in the mosquito, giving rise to a large number ofsporozoites (from sporos: seed). Hence, this phase of sexual multiplication is called sporogony. It is also called the invertebrate, extrinsic, or exogenous phase. Thus, there is an alternation ofhosts as the asexual phase takes place in humans followed by sexual phase in mosquito. Human Cycle (Schizogony) Human infection comes through the bite of the infective fem ale Anopheles mosquito (Fig. 2). • The sporozoites, which are infective forms of the parasite are present in the salivary gland of the mosquito. • theyare injected into blood capillaries when the mosquito feeds on blood after piercing the skin. • Usually, 10- 15 sporozoites are injected at a time, but occasionally, many hundreds may be introduced. The sporozoites pass into the bloodstream, where many are destroyed by the phagocytes, but some reach the liver and enter the parenchymal cells (hepatocytes).
- 90. Pre-erythrocytic (tissue) stageor exoerythrocytic stage: Withinan hourofbeinginjectedinto the bodyby the mosquito, the sporozoites reach the liver and enter the hepatocytes to initiate thestage ofpre-erythrocytic schizogony or merogony. • The sporozoites, which are elongated spindle-shaped bodies, become rounded inside the liver cells. • They enlarge in size and undergo repeated nuclear division to form several daughter nuclei; each ofwhich is surrounded by cytoplasm. • this stage of the parasite is called the pre-erythrocytic or exoerythrocylicschizont or meront. • the hepatocyte is distended by the enlarging schizont and the liver cell nucleus is pushed co the periphery. • Mature liver stage schizonts arc spherical (45-60 µm), multinucleate and contain 2,000-50,000 uninucleate merozoites. • Unlike erythrocytic schizogony, there is no pigment in liver schizonts. These normally ruprure in 6-15 days and release thousands ofmerozoites into the bloodstream. • The merozoites infect the erythrocytes by a process of invagination. • Prepatent period: The interval between the entry of the sporozoites into the body and the first appearance of the parasites in blood is called the prepatent period. • The duration ofthe pre-erythrocytic phase in the liver, the size of the mature schizont and the number ofmerozoites produced vary with the species ofthe parasite (Table 2). Latent stage: In P. vivax and P. ovate, two ki nds of sporozoites are seen, some of which multiply inside hepatic cells to form schizonts and others persist and remain dormant (resting phase). • Relapse: The resting forms are called hypnozoites (hypnos: sleep). From time to time, some are activated to become schizonts and release merozoites, which go on infecting RBCs producing clinical relapse. • Recrudescence: In P. falciparum and P. malariae, initial tissue phase disappears completely, and no hypnozoites are found. However, small numbers of erythrocytic parasites persist in the bloodstream and in due course of time, they multiply to reach significant numbers resulting in clinical disease (short-term relapse or recrudescence). Erythrocyticstage: t hemerozoilesreleased bypre-erythrocytic schizonts invade the RBCs. the receptor for merozoites is glycophorin, which is a major glycoprotcin on the red cells. The differences in the glycophorins of red cells ofdifferent species may account for the species specificiry of malaria parasites. Merozoitesare pear-shapedbodies, about 1.5µmin length, possessing an apical complex (rhoptery). They attach to the erythrocytes by their apex and then the merozoites lie within an intraerythrocytic parasitophorous vacuole formed by red cell membraneby a process ofinvagination. Malaria and Babesia • In the erythrocyte, the merozoite loses its internal organelles and appears as a rounded body having a vacuole in the center with the cytoplasm pushed to the periphery and the nucleus at one pole. These young parasites are, therefore called the ringforms or young trophowites. • The parasite feeds on the hemoglobin of the erythrocyte. it does not metabolize hemoglobin completely and therefore, leaves behind a hematin-globin pigment called the malaria pigment or hemozoin pigment, as residue (Box 1). • The malaria pigment released when the parasitized cells rupture is taken up by reticuloendothelial cells. Such pigment-laden cells in the internal organs provide histological evidence of previous malaria infection. • As the ring form develops, it enlarges in size becoming irregular in shape and shows ameboid motility. This is called the ameboidform or late trophozoiteform. When the ameboid form reaches a certain stage of development, its nucleus starts dividing by m itosis followed by a division of cytoplasm to become mature schizonts ormeronts. • A mature sch izont contains 8-32 merozoites and hemozoin. The mature schizont bursts releasing the merozoites into the circulation. The merozoites invade fresh erythrocytes within which they go through the same process of development. 1h is cycle of erythrocytic schizogony or merogony is repeated sequentially, leading to progressive increase in the parasitemia, till it is arrested by the development of host immune response. Table 2: Features of pre-erythrocytic schizogony in human malaria parasites P. vivax P. falciparum P. malariae P. ovale Pre-erythrocytic 8 6 15 9 stage (days) Diameter of 45 60 55 60 pre-erythrocytic schizont(µm) No. of merozoites 10,000 30,000 15,000 15,000 in pre-erythrocytic schizont Box 1:Appearance of malaria pigments in different species • P. vivax: Numerous fine golden-brown dust-like particles • P. falciparum: Few 1-3 solid blocks ofblack pigment • P. malariae: Numerous coarsedark-brown particles • P. ovale: Numerous blackish-brown particles.
- 91. Paniker'sTextbookofMedical Parasitology • Therupture of the mature schizont releases large quantities of pyrogens. This is responsible for the febrile paroxysms characterizing malaria. • The interval between the entry of sporozoites into the host and the earliest manifestation of clinical illness is the incubation period (Box 4). This is different from prepatentperiod, which is the time taken from entry ofthe sporozoites to the first appearance of malaria parasite in peripheral blood. V, ~ ·o N 0 .t: 0.. e t- V, 'E 0 N :c 0 (J) V, ., >. 0 .9 ., E "' (!) >- 't:: (l) w <I) ~ _J <I) <ii E <I) u.. P. vivax P falciparum @ ~~ . . • • • In P. falciparum, erythrocyric schizogony always takes place inside the capillaries and vascular beds of internal organs. Therefore, in P. falciparum infections, schizonrs and merozoites are usually not seen in the peripheral blood. • The erythrocytic stages of all the four species of Plasmodium arc shown in Figure 3. P malariae P ovate V ..- .,.... ! .· ... ·:.; .: • • - • • • I .' . , Fig. 3: Malaria parasites-Erythrocytic stages of the four species (Giemsa stain. Magnification 2000X)
- 92. Gametogony After a fewerythrocytic cycles, some of the merozoites that infect RBCs do not proceed to become trophozoites or schizonts but instead, develop into sexually differentiated forms, the gametocytes. • They grow in size till they almost fill the RBC, but the nucleus remains undivided. • Development ofgametocytes generally takes place within the internal organs and only the mature forms appear in circulation. • The mature gametocytes are round in shape, except in P. Jalciparum, in which they are crescent-shaped. • In all species, the female gametocyte is larger (macrogametocyte)and has cytoplasm stainingdarkblue with a compact nucleus staining deep red. In the smaller male gametocyte (microgametocyte), the cytoplasm stains pale blue or pink and the nucleus is larger, pale stained and diffuse. Pigment granules are prominent. • Female gametocytes are generally more numerous than the male. • Gametocyte appears in circulation 4-5 days after the first appearance ofasexual form in case ofP. vivax and 10-12 days in P.falciparum. • A person with gametocytes in blood is a carrier or reservoir. • the gametocytes do not cause any clinical illness in the host, but are essential for transmission of the infection. • A gametocyte concentration of 12 or more per mm3 of blood in the human host is necessary for mosquitoes to become infected. The Mosquito Cycle (Sporogony) When a female Anopheles mosquito ingests parasitized erythrocytes along with its blood meal, the asexual forms of malaria parasite are digested, but the gametocytes are set free in the midgut (stomach) of mosquito and undergo further development. The nuclear material and cytoplasm of the male gametocytes divides to produce eight microgametes with long, actively motile, whip-like filaments (ex.flagellating malegametocytes) (Fig. 4). • At 25°C, the cxflagellation is complete in 15 minutes for P. vivax and P. ovale and 15-30 minutes for P.falciparum. 1he female gametocyte does not divide but undergoes a process of maturation to become the female gamete or macrogamete. It is fertilized by one of the microgametes to produce the zygote (Fig. 4). • Fertilization occurs in 0.5-2 hours after the blood meal. The zygote, which is initially a motionless round body, gradually elongates and within 18-24 hours, becomes a vermicular motile form with an apical complex anteriorly. This is called the ookinete (travellingvermicule). Malaria and Babesia Female gametocyte Male gametocyte ~ l l 0 flagellation I - ' Macrogamete Microgamete Fig. 4: Schematic diagram showing formation of microgamete and macrogamete • It penetrates the epithelial lining of the mosquito stomach wall and comes to lie just beneath the basement membrane. • It becomes rounded into a sphere with an elastic membrane. 1his stage is called the oocyst, which is yet another multiplicatory phase, within which numerous sporozoites are formed. • tje mature oocyst, which may be about 500 µm in size, bulges into body cavity of mosquito and when it ruptures, the sporozoites enter into the hemocele or body cavity, from where some sporozoites move to the salivary glands. The mosquito is now infective and when it feeds on humans, the sporozoites are injected into skin capillaries to initiate human infection. • Extrinsic incubation period: The time taken for completion of sporogony in the mosquito is about 1-4 weeks (extrinsic incubation period), depending on the environmental temperature and the species. Types of Malarial Parasites Plasmodium Vivax P. vivax has the widest geographical distribution, extending through the tropics, subtropics and temperate regions. It is believed to account for 80% of all malaria infections. lt is the most common species of malaria parasite in Asia and America, but is much less common in Africa. It causes benign tertian malaria with frequent relapses. • The sporozoites ofP. vivaxare narrow and slightly curved. Onenteringthe livercells,thesporozoitesinitiate two types of infection. Some develop promptly into exoerythrocytic schizonts, while others persist in the dormant state for varyingperiods as hypnozoites.There may be two distinct types of sporozoites: (1) the tachysporozoites (tachy: fast), which develops into the primary exoerythrocytic schizont and (2) the bradysporozoite (brady: slow) which becomes the hypnozoite.
- 93. Paniker'sTextbook of MedicalParasitology • The pre-erythrocytic schizogony lasts for 8 days and the average number of merozoites per tissue schizont is 10,000. • Merozoites of P. vivax preferentially infect reticulocytes and young erythrocytes. • All stages of erythrocytic schizogony can be seen in peripheral smears (Fig. 5). • The degree of parasitization is not generally heavy, each infected red cell usually having only one trophozoite and not more than 2-5% of the red cells being affected. Reticulocytes are preferentially infected. • The rrophozoite is actively motile, as indicted by its name vivax. The ring form is well-defined, with a prominent central vacuole. One side of the ring is thicker and the other side thin . Nucleus is situated on the thin side of the ring (Signet ring appearance). The ring is about 2.5-3 µm in diameter, about a third of the size of an erythrocyte. The cytoplasm is blue and the nucleus red in stained films. 1l1e ring develops rapidJy to the ameboid form and accumulates malarial pigment (Figs 6 and 7). Erythrocyte ...........: .. .. ........ ... . . .., . :. - I .· Commencing chromatin division ........ ....... • Young ring stage Further chromatin division • The infected erythrocytes are enlarged and show red granules known as Schujfner's dots on the surface. They become irregular in shape, lose their red color and present a washed out appearance.Afew ofthe parasitized erythrocytes retreat into the blood spaces of the internal organs. • The schizont appears in about 36-40 hours. It occupies virtually the whole of the enlarged red cell. The schizont matures in the next 6-8 hours, with the development of merozoites, each with its centralnucleus and surrounding cytoplasm. The pigment granules agglomerate into a few dark brown collections at the center, and with the merozoites around it, this stage presents a rosette appearance.There areabout 12-24 (usually 16) merozoites per schizont. Erythrocytic schizogony takes approximately 48 hours. The red cell, which now measures about 10 µm in diameter is heavily stippled and often distorted. It bursts to liberate the merozoites and pigment. 1l1e pigment is phagocytosed by reticuloendothelial cells. .. • . ........ . . Older ring stage with Schuffner's dots .... ·.:- .....~=::.. ·..::~t~·;., .~ ."• .•"' ~.•.:. . Schizont Adult ring in enlarged cell, Schuffner's dots marked Schizont mature form prior to merozoite liberation .... :..,....;;-·..-~·. .. .. .. ... .... .. .. .... ....:..:.,.·..:.:_.:..=·~.. ,. ............... ., :...::..::..:.:....,. Female gametocyte early stage Female gametocyte mature Male gametocyte Fig. 5: Plasmodium vlvax (Giemsa stain, magnification 2000X)
- 94. • Leishman's, X1000 Oil Fig.6: Malarial parasite in blood film-Ring stage of P. vivax Source: Mohan H. Textbook of Pathology, 6th edition. New Delhi: Jaypee Brothers Medical Publishers; 2010. p. 189. • The merozoites measure about 1.5 µm and have no pigment. • Gametocytes appear early, usually within 4 days after the trophozoites first appear. Both male and female gametocytes are large, nearly filling the enlarged red cell. Themacrogametocyte hasdensecytoplasm stainingdeep blue and a small compact nucleus. lhe microgametocyte has pale-staining cytoplasm and a large diffuse nucleus. Pigment granules are prominent in the gametocytes. Plasmodium Falciparum The name Jalciparum comes from the characceristic sickle shape ofthegametocytes ofthis species (Jalx: sickle, parere: to bringforth). 1his is the highly pathogenic ofall the plasmodia and hence, the name malignant tertian or pernicious malaria for its infection. • The disease has a high rate of complications and unless treated, is often fatal. The species is responsible for almost all deaths caused by malaria. Schizogony:The sporozoites are sickle-shaped. the tissue phase consists of only a single cycle of pre-erythrocytic schizogony. No hypnozoites occur. The mature liver schizont releases about 30,000 merozoites. • They attack both young and mature erythrocytes and so the population of cells affected is very large. Infected erythrocytes present a brassy coloration. Ringform: The early ring form in the erythrocyte is very delicate and tiny, measuring only a one-sixth of the red cell diameter. Rings are often seen attached along the margin of the red cell, the so-called form applique or accole. Binucleate rings (double chromatin) are Malaria and Babesia Lelshman·s, X1000 Oil Fig. 7: Malarial parasite in blood film- Ameboid form of P. vivax Source: Mohan H. Textbook of Pathology, 6th edition. New Delhi: Jaypee Brothers Medical Publishers; 2010. p. 189. common resembling stereo headphones in appearance. Several rings may be seen within a single erythrocyte. In course of time, the rings become larger, about a third of the size of the red cell and may have I or 2 grains of pigment in its cytoplasm (Figs 8 and 9). • The subsequent stages of the asexual cycle- late trophozoite, early and mature schizoncs- a re not ordinarily seen in peripheral blood, except in very severe or pernicious malaria. The presence of P. falciparum schizonts in peripheral smears indicates a grave prognosis (Box 2). • The mature schizont is smaller than in any other species and has 8-24 (usually 16) merozoites. The erythrocytic schizogony takes about 48 hours or less, so that the periodicity offebrile paroxysms is 36-48 hours. Very high intensity of parasitization is seen in Jalciparum malaria. In very severe infections, the rate of parasitized cells may even be up to 50%. • The infected erythrocytes are of normal size. They show a few (6- 12)coarse brick-red dotswhich are called Maurer's clefts. Some red cells show basophilic stippling. Gametogony: It begins after several generations of schizogony. Gametocytes are seen in circulation about 10 days after the ring stage first appears. The early gametocytes seldom appear in peripheral circulation.The mature gametocytes, which are seen in peripheral smears are curved oblong structures, described as crescentic, sickle, sausage, or banana-shaped. They are usually referred co as crescents (Fig. 10). • The male gamecocytes are broad and sausage-shaped or kidney-shaped, with blunt rounded ends as compared to the female gametocytes, which are thinner and more
- 95. Paniker'sTextbook of MedicalParasitology Erythrocyte .. ' .~ Mature ring and Maurer's dots Advanced merozoite development with commencing pigmentation • ' Marginal ring form .. ' .. .. Trophozoite amoeboid stage commencing chromatin division Schizont mature with centralized pigment Rarely seen in peripheral circulation t) Young ring stage Nuclear division • u t) • Ring forms with double chromation dots Merozoite development Rarely seen in peripheral circulation Female gametocyte (crescent) Male gametocyte (crescent) Fig. 8: Plasmodium falciparum (Giemsa stain, magnification 2000X) Fig. 9: Malarial parasite in blood film-Ring stage of P. falciparum Source: Mohan H. Textbook of Pathology, 6th edition. New Delhi: Jaypee Brothers Medical Publishers; 2010. p. 189. Box 2: Pathogenesis of malignant malaria • Late stage schizonts of P. falciparum secrete protein on the surface of RBCs to form knob-like protuberances in erythrocyte's cell membrane. These knobs produce specific adhesive Plasmodlum falciparum erythrocyte membrane protein-1 (PfEMP-1 ) so that infected RBCs become sticky. • Sometime Inflammatory cytokines particularly IFN-y produced by the malaria parasite upregulate the expression of endothelial cytoadherence receptors like thrombospondin, E-selectin, VCAM-1, ICAM-1 in capillaries in the brain, chondroitin sulfate B in placenta and (D36 in most other organs. The infected RBCs stick inside and eventually block capillaries and venules. This phenomenon is called cytoadherence. At the same stage these P. falciparum infected RBCs adhere to uninfected RBCs to form rosettes. • This process of cytoadherence and rosetting causes capillary plugging and decrease microclrculatory flow in vital organs like brain, kidney, lungs, spleen, intestine, bone marrow and placenta resulting in serious complications such as cerebral malaria. • Other virulence factors of P. falciparum are histidine-rich protein II (HRPII) and glycosylphosphatidylinositol (GPIJ. Abbreviations: ICAM-1, intercellular adhesion molecule-1; IFN-y, interferon gamma; RBCs, red blood cells; VCAM-1 , vascular cell adhesion molecule-1
- 96. Leishman's, X1000 Oil Fig.1O: Malarial parasite in blood film-Gametocytes of P. falciparum Source: Mohan H. Textbook of Pathology, 6th edition. New Delhi: Jaypee Brothers Medical Publishers; 2010. p. 189. typicallycrescentic, with sharply rounded or pointed ends. The mature gametocyte is longer than the diameter of the red cell and so produces gross distortion and sometimes even apparent disappearance of the infected red cell. The red cell is often seen as a rim on the concave side of the gametocyte. The cytoplasm in the female gametocyte is deep blue, while in the male it is pale blue or pink. The nucleus is deep red and compact in the female, with the pigment granules closely aggregated around it, while in the male, it is pink, large and diffuse, with the pigment granules scattered in the cytoplasm. • Falciparum crescents can survive in circulation for up to 60 days, much longer than in other species. Gametocytes are most numerous in the blood of young children, 9 months to 2 years old. They, therefore serve as the most effective source ofinfection to mosquitoes. Plasmodium Malariae This was the species of malaria parasite first discovered by Laveran in 1880 and the name malariae is the one given by him. It causes quartan malaria, in which febrile paroxysms occur every 4th day, with 72 hours interval between the bouts. The disease is generally mild, but is notorious for its long persistence in circulation in undetectable levels, for 50 years or more. Recrudescence may be provoked by splenectomy or immunosuppression. • The development ofthe parasite, in man and mosquito is much slower than with other species. Chimpanzees may be naturally infected with P. malariae and may constitute a natural reservoir for quartan malaria. • P. malariae occurs in tropical Africa, Sri Lanka, Burma an d parts oflndia, but its diso·ibution is patchy. Malariaand Babesia • The sporozoites are relatively thick. Pre-erythrocytic schizogony takes about 15 days, much longer than in other species. Each schizont releases about 15,000 merozoites. Hypnozoites do not occur. The long latency of the infection is believed to be due to long time survival of few erythrocytiv forms in some internal organs. • P. malariae preferentially infects older erythrocytes and the degree ofparasitization is low. The ring forms resemble those of P. vivax, although thicker and more intensely stained. The old rrophozoites are sometimes seen stretched across the erythrocyte as a broad band. These bandforms are a unique feature of P. malariae. Numerous large pigment granules are seen (Fig. 11). • The schizonts appear in about 50 hours and mature during the next 18 hours. The mature schizont has an average of eight merozoites, which usually present a rosette appearance. • The infected erythrocytes may be of the normal size or slightly smaller. Fine stippling, called Ziemann's stippling, may be seen with special stains. The degree of parasitization is lowest in P. malariae. • Erythrocytic schizogony takes 72 hours. • The gametocytes develop in the internal organs and appear in the peripheral circulation when fully grown. Gametocytes occupy nearly the entire red cell. The male has pale blue cytoplasm with a large diffuse nucleus, while the female has deep blue cytoplasm and a small compact nucleus. Plasmodium Ova/e This parasite produces a tertian fever resembling vivax malaria, but with milder symptoms, prolonged latency and fewer relapses. • It is the rarest ofall plasmodia infecting humans and is seen mostly in tropical Africa, particularly along the West Coast. • Thepre-erythrocytic stage extends for 9 days. Hepatocytes containing schizonts usually have enlarged nuclei. The mature liver schizont releases about 15,000 merozoites. Hypnozoites are present. • The trophozoites resemble those in vivax malaria, but are usually more compact, with less ameboid appearance. Schuffner's dots appear earlier and are more abundant and prominent than in vivax infection (Fig. 12). • The infected erythrocytes are slightly enlarged. In thin films, many ofthem presentan oval shape with fimbriated margins. This oval appearance ofthe infected erythrocyte is the reason for the name ovate given to this species. • The schizonts resemble those of P. malariae, except that the pigment is darker and the erythrocyte is usually oval, with prominent Schuffner's dots.
- 97. Paniker'sTextbook ofMedical Parasitology Erythrocyte Schizont, commencing daisyform Ring form with eccentric nucleus Schizont. mature pigment centrally clumped daisy form ,, - ·~ .-- Commencement of band form dividing chromatin pigment accumulation Female gametocyte compact chromatin Band form Note: Chromatin on one side or band Male gametocyte diffuse chromatin Fig. 11: P/asmodium malariae stages of erythrocytic schizogony (Giemsa stain, magnification 2000X) Erythrocyte Commencing chromatin division Daisy form of the parasite • 0 Young ring stage Older ring stage Further chromatin division Schizont oval form or erythrocyte persisting .. + ••" ;,•••••• • .. ,... . >· ••• .:;. .... . . . ~ ..., ..... :-:!,., , ' • ._;~-· -<·· ,." •• •• • .,. .,, ..,. ."""' ., ..,. ..,.. • • - I I • I · • Female gametocyte Adult ring in enlarged oval erythrocyte Schuffner's erythrocyte .. <f . .... .. Merozoite development Note: Continued oval form and Schuffner's dots Male gametocyte Fig. 12: Plasmodium ovate stages of erythrocytic schizogony (Giemsa stain, magnification 2000X)
- 98. Malaria and Babesia MixedInfections Pathogenesis ln endemic areas it is not uncommon to find mixed infections with two or more species of malaria parasites in the same individual. Clinical manifestations in malaria are caused by products of erythrocytic schizogony and the host's reaction to them. • Toe disease process in malaria occurs due to the local or systemic response of the host to parasite antigens and tissue hypoxia caused by reduced oxygen delivery because of obstruction of blood flow by the parasitized erythrocytes. • Mixed infection with P. vivax and P. falciparum is the most common combination with a tendency for one or the other to predominate. • The clinical picture may be atypical with bouts of fever occurring daily. • Diagnosis may be made by demonstrating the characteristic parasitic forms in thin blood smears. The characteristics of the four species of plasmodia infecting man are listed in Table 3. Liver is enlarged and congested. Kupffer cells are increased and filled with parasites. Hemozoin pigments are also found in the parenchymal cells (Fig. 13). Parenchymal cells show fatty degeneration, atrophy and centrilobular necrosis. Table 3: Comparison of the characteristics of plasmodia causing human malaria P. vivax P. lalclparum P.malariae P. ovale Hypnozoites Yes No No Yes Erythrocyte preference Reticulocytes Young erythrocytes, but can Old erythrocytes Reticulocytes infect all stages Stages found in peripheral blood Rings, trophozoites, Only rings and gametocytes As in vivax As In vivax schizonts, gametocytes Ring stage Large, 2.5 µm, usually single, Delicate, small, 1.5 µm, double Similar to vivox, but Similar to vivax, more prominent chromatin chromatin, and multiple rings thicker compact common, accole formsfound Late trophozoite Large irregular, actively Compact, seldom seen in Band form Compact, coarse ameboid, prominent vacuole blood smear characteristic pigment Schizont Large filling red cell Small, compact, seldom seen Medium size Medium size in blood smear Number of merozoites 12- 24 in irregular grape-like 8-24 grape-like cluster 6-12 in daisy-head or 6-12 irregularly cluster rosette pattern arranged Microgametocyte Spherical, compact, pale blue Sausage or banana-shaped As in vivax As in vivax (male gametocyte) cytoplasm, diffuse nucleus pale blue or pink cytoplasm, large diffuse nucleus Macrogametocyte Large, spherical, deep blue Crescentic, deep blue As in vivax As in vivax (female gametocyte) cytoplasm, compact nucleus cytoplasm, compact nucleus Infected erythrocyte Enlarged, pale, with Normal size, Maurer's clefts, Normal, occasionally Enlarged, oval Schuffner's dots sometimes basophilic Ziemann's stippling fimbriated, prominent stippling Schuffner's dots Duration of schizogony (days) 2 2 3 2 Prepatent period (days) 8 5 13 9 Average incubation period (days) 14 12 30 14 Appearance ofgametocyte after 4-5 10 12 11- 14 5-6 parasite patency (days) Duration of sporogony in 9-10 10-12 25- 28 14-16 mosquito (25°CJ (days) Average duration of untreated 4 2 40 4 infection (years)
- 99. Paniker'sTextbook of MedicalParasitology ,....,_...- - - -Brain Liver - ---- - -1 (Encephalopathy) Heart (Congestive heart failure) (Hepatomegaly) 't:t-,--..,.--,--- Spleen (Splenomegaly) Kidneys (Hemoglobinunc nephrosis) Fig. 13: Major pathological changes in organs in malaria Box 3: causes of anemia in malaria • Destruction of large number of RBCs by complement-mediated and autoimmune hemolysis. • Suppression oferythropoiesis in the bone marrow. • Increased clearance of both parasitized and nonparasitized RBCs by the spleen. • Failure ofthe host to recycle the iron bound in hemozoin pigment. • Antimalarial therapy in G6PD deficient patients. Abbreviations: G6PD, glucose-6-phosphate dehydrogenase; RBCs, red blood cells • Spleen is soft, moderately enlarged and congested in acute infection. In chronic cases, spleen is hard with a thick capsule and slate gray or dark brown or even black in color due to dilated sinusoids, pigment accumulation and fibrosis (Fig. 13). Kidneys are enlarged and congested. Glomeruli frequently contain malarial pigments and tubules may contain hemoglobin casts (Fig. 13). • The brain in P. Jalciparum infectio n is congested. Capillaries ofthe brain are plugged with parasilized RBCs. The cul surface of the brain shows slate gray cortex with multiple punctiform hemorrhage in subcortical white matter. • Anemia: After few paroxysms of fever, normocytic and normochromic anemia develops. Anemia is caused by destrucrion of large number of red cells by complement- mediated autoimmune hemolysis. Spleen also plays an active role by phagocytic removal of a large number of both infected and uninfected RBCs. Excess removal of uninfected RBCs mayaccount for up co 90% oferythrocyte lo s (Box3). Box4: Incubation period , It is the time interval between the bite of infective mosquito and the first appearance of clinical symptom s. The duration of incubation period varies with the species of the parasite. • The average incubation periods of different speciesofPlasmodium are as follows: - P. vivax: 14 (12- 17) days - P. falc,parum: 12 (8- 14) days - P. ovale: 14 (8-31) days - P. malarioe: 28 (18-40) days. The incubation period is to be distinguished from the prepatent period, which is the interval between the entry of the parasites into the host and the time when they first become detectable in blood. There is also decreased erythropoiesis in bone marrow due to rumor necrosis factor (T F) toxiciry and failure of the host to recycle the iron bound in hemozoin pigments. • Cytokines like T F, interleukin {IL)-1 and interferon (IFN)-gamma play an important role in the pathogenesis ofend-organ disease ofmalaria. Clinical Features Benign Malaria • Incubation period: 12- 17 days {Box4). the typical clinical feature of malaria consists of periodic bouts offever with chill and rigor, followed by anemia, splenomegaly and hepatomegaly. • The classic febrile paroxysm comprises of three distinct stages- {!) coldstage, (2) hot stage and (3) sweatingstage. I . Cold stage: The patient feels incense cold with chill and rigor along with lassitude, headache and nausea. This stage lasts for 15 minutes to I hour. 2. Hot stage: The patient feels intensely hot. The temperarure mounts to 4 1•c or higher. Headache persists but nausea commonly diminishes.This stage lasts for 2- 6 hours. 3. Sweating stage: Profuse sweating follows the hot stage and the temperature comes down to normal. The skin is cool and moist. The patient usually falls asleep to wake up refreshed. The paroxysm usually begins in the early afternoon and lasts for 8-12 hours. lhe febrile paroxysm synchronizes with the erythrocytic schizogony. The periodicity is approximately 48 hours in tertian malaria (in P. uiuax, P. falciparum and P. ovale) and 72 hours in quanan malaria {in P. malariae). Quotidian periodiciry, with fever occurring at 24 hour intervals may be due to two broods of tertian parasites maturing on successive days or due to mixed infection. Regular periodiciry is seldom seen in primary attack, but is established usually only after a few days ofcontinuous,
- 100. remittent, or intermittentfever. True rigor is typically presentin vivaxmalaria and is less common infalciparum infection. There can be both hypoglycemia or hyperglycemia in malaria. Sometimes, there may be hyperkalemia due to red cell lysis and fall in blood pH. infection with P. vivax usually follows a chronic course with periodic relapses, whereas P. ovale malaria is generallymild. Although P. malariaemalariaisless severe, but it may lead to renal complications. Relapse mainly occurs in inadequately treated cases after an interval of 8-40 weeks or more. Malignant Tertian Malaria incubation period: 8-14 days. The most serious and fatal type of malaria is malignant tertian malaria caused by PJalciparum. Falciparum malaria if not treated timely or adequately, severe life-threatening complications may develop. In severe Jalciparum malaria, parasitic load is very high and more than 5% red cells are affected. The term pernicious malaria also have been applied to these conditions thar include cerebral malaria, blackwater fever, algid malaria and septicemic malaria (Box 5). • Cerebral malaria: It is the most common complication ofmalignant malaria. - The initial symptoms are nonspecific with fever, headache, pain in back, anorexia and nausea. - Anemia: The patient may be anemic and mildly jaundiced. - Hepatosplenomegaly: Liver and spleen are enlarged and nomender. Thrombocytopenia is common. - After 4- 5 days of high fever, cerebral malaria is manifested by features of diffuse symmetric encephalopathy like headache, confusion, increased muscle tone, seizures, paralysis, slowly lapsing to coma. Box 5: Complications of falciparum malaria • Cerebral malaria • Algid malaria • Septicemic malaria • Blackwater fever • Pulmonary edema • Acute renal failure • Hypoglycemia (<40 mg/dl) • Severe anemia (Hb<Sg/dl, PCV<l 5%) • Hyperpyrexia • Metabolic acidosis and shock • Bleeding disturbances • Hyperparasitemia. Abbreviations: Hb, hemoglobin; PCV, packed cell volume Malaria and Babesia Retinal hemorrhages may be seen in 15% ofadults. Hypoglycemia is common in patients following quinine therapy or with) hyperparasitemia. In 10% of cases renal dysfunction progressing to acute renal failure may occur. - Other complications include metabolic acidosis, pulmonary edema and shock. Even with treatment, death occurs in 15% ofchildren and 20% ofadults who develop cerebral malaria. This occurs particularly when nonimmune persons have remained untreated or inadequately treated for 7-10 days after development ofthe primary fever. - The basic pathogenesis of cerebral malaria is due to erythrocyte sequestration in microvasculature of various organs. Late stage schizonts ofP.falciparum secrete a protein on the surface ofRBCs to form knob-like deformities. This knob produces specific adhesive proteins [Plasmodiumfalciparum erythrocyte membrane protein-I (PfEMP-1)]. which promote aggregation of infected RBCs to other noninfected RBCs and receptors of capillary endothelial cells. These sequestrated RBCs cause capillary plugging of cerebral microvasculature, which results in anoxia, ischemia and hemorrhage in brain. • Blackwater fever: A syndrome called blackwater fever (malarial hemoglobinuria) is sometimes seen in Jalciparum malaria, particularly in patients, who have experienced repeated past infections and inadequate treatment with quinine. An autoimmune mechanism has been suggested. Patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency may develop this condition after taking oxidant drugs, even in the absence of malaria. - Clinical manifestations include fever, prostration and hemoglobinuria (black colored urine), bilious vomiting and prostration, with passage ofdark red or blackish urine. The pathogenesis is believed to be massive intravascular hemolysis caused by antierythrocyte antibodies, lead ing to massive absorption of hemoglobin by the renal tubules (hemoglobinuric ne phrosis) producing blackwater fever. Complications of blackwater fever include renal failure, acute liver failure and circulatory collapse. Algid malaria: This syndrome is characterized by peripheral circulatory failure, rapid thready pulse with low blood pressure and cold clammy skin. There may be severe abdominal pain, vomiting, diarrhea and profound shock. Septicemic malaria: It is characterized by high continuous fever with dissemination of the parasite to various organs, leading to multiorgan failure. Death occurs in 80% of the cases.
- 101. Paniker's Textbook ofMedical Parasitology Merozoite-induced Malaria Natural malaria is sporozoite-induced, the infection being transmitted by sporozoites introduced through the bite of vector mosquitoes. Injection of merozoites can lead to direct infection ofred cells and erythrocytic schizogonywith clinical illness. Such merozoite-induced malaria may occur in the following situations: • Tra11sfusio11 malaria: Blood transfusion can accidentally transmit malaria, if the donor is infected with malaria. The parasites may remain viable in blood bank for 1-2 weeks. As this condition is induced by direct infection of red cells by the merozoites, pre-erythrocytic schizogony and hypnozoites are absent. Relapse does not occur and incubation period is short. Table 4 enumerates the differences between mosquito- borne malaria and blood transfusion malaria. Congenital malaria: A natural form of merozoite- induced malaria, where the parasite is transmitted transplacentally from mother to fetus. • Renal transplantation may lead to malaria if the donor had parasitemia. • Shared syringes among drug addicts may be responsible. Tropical Splenomegaly Syndrome Tropical splenomegaly syndrome (TSS) or hyper-reactive malarial splenomegaly (HMS) is a benign condition seen in people of malaria endemic areas mainly tropical Africa, new Guinea and Vietnam. ft happens from abnormal immunological response to repeated malaria infection. • Tropical splenomegalysyndrome is characterized by high level of immunoglobulin M (IgM) against malaria due to polyclonalactivation of8-cells, decreased C3 and massive splenomegaly. Malaria parasite is absent in peripheral blood. Table 4: Difference between mosquito-borne malaria and blood transfusion malaria Mosquito-borne mo/aria Mode oftransmission Mosquito bite Infective stage Sporozolte Incubation period Long Pre-erythrocytic Present schizogony Hypnozoites Maybe present Bloodtransfusion malaria Blood or blood products transfusion Trophozoite Short Absent Absent Severity Relapse Comparatively less More complications seen May occur Does not occur Radical treatment Required Not required • A normocytic normochromic anemia is present which does not respond to hematinics or antihelminthics. Spleen and liver are enlarged, congested, with dilated sinusoids and marked lymphocytic infiltration. umerous pigment-laden Kupffer cells dot the liver. Changes are also seen in bone marrow, kidneys and adrenals. Tropical splenomegaly syndrome differs from various other types of splenomegalies seen in the tropics in its response to antimalarial treatment. Immunity Immunity in malaria could be two types: (1) innate immunity and (2) acquired immunity. Innate Immunity It is the inherent, nonimmune mechanism of host resistance against malarial parasite. innate immunity could be due ro: Duffy negative red blood cells: The invasion of red cells by merozoites requires the presence of specific glycoprotein receptors on the erythrocyte surface. It has been found duffy blood group negative persons are protected from P. vivax infection. Duffy blood group is absent in West Africa where P. vivax malaria is not prevalent. Nature of hemoglobin: Hemoglobin E provides natural protection against P. vivax. P.falciparum docs not multiply properly in sickled red cells containing lfbS. icklecell anemia trait is very common in Africa, where falciparum malaria is hyperendemic and offers a survival advantage. HbF present in neonates protects them against all Plasmodium species. Glucose-6-phosphale dehydrogenasedeficiency: Innate immunity to malaria has also been related to G6PD deficiency found in Mediterranean coast, Africa, Middle East and India. - Human leukocyte antigen-B53: Human leukocyte antigen-B53 (HL/-853) is protected from cerebral malaria associated with protection from malaria. r utritional status: Patients with iron deficiency and severe matnutrition are relativelyresistantto malaria. - Pregnancy: Falciparum malaria is more severe in pregnancy, particularly in primigravida and may be enhanced by iron supplementation. Splenectomy: The spleen appears to play an important role in immunity against malaria. Splenectomy enhances susceptibility to malaria. Acquired Immunity Infection with malaria parasite induces specific immunity involving both humoral and cellular immunity, which can
- 102. bring about clinicalcure but cannot eliminate parasites from the body. It can prevent superinfection, but is not powerful enough to defend against reinfection. This type of resistance in an infected host, which is associated with continued asymptomatic parasite infection is called premunition. This type of immunity disappears once the infection is eliminated. Humoral immunity: Circulating antibodies (IgM, lgG and IgA) against asexual forms give protection by inhibiting red cell invasion and antibodies against sexual forms reduce transmission of malaria parasite. • Acquired antibody-mediated immunity is transferred from mother to fetus across the placenta and is evident in endemic areas where infants below the age of 3 months are protected by passive maternal antibodies. Young children are highly susceptible to malaria. As they grow up, they acquire immunity by subclinical or clinical infections, so that incidence of malaria is low in older children and adults. Cellular immunity: Sensitized T cells release cytokines that regu.late macrophage activation and stimulate B cells to produce antibodies. The activated macrophages inside liver, spleen and bone marrow phagocytose both parasitized and nonparasitized RBCs. Clinical note: Protective immunity against malaria is species specific, stage specific and strain specific. Recrudescence and Relapse Recrudescence In P. falciparum and P. malariae infections after the primary attack, sometimes there is a period of latency, during which there is no clinical illness. But some parasites persist in some erythrocytes, although the level of parasitemia is below the fever threshold or sometimes below the microscopic threshold. Erythrocytic schizogony is repeated at a low level in the body when the number of parasites attain a significant level, fresh malarial attackdevelops. This recurrence ofclinical malaria caused by persisting P. Jalciparum and P. malariae is called recrudescence. Recrudescence may be due to waning immunity of the host or possibly due to antigenic variation. In P.Jalciparu.m infections, recrudescences are seen for 1-2 years, while in P. malariae infection, they may last for long periods, even up to 50 years (Table 5). Relapse It is seen in inadequately treated P. vivax and P. ovale infections. In both these species, two kinds ofsporozoites are seen, some of which multiply inside heparocytes promptly Malaria and Babesia Table 5: Differences between recrudescence and relapse Recrudescence Seen in P. falciparum and P. malariae Due to persistence of the parasite at a subclinical level in circulation Occurs within a few weeks or months of a previous attack Can be prevented by adequate drug therapy or use of newer antimalarial drugs in case of drug resistance Relapse Seen in P. vivax and P. avale Due to reactivation of hypnozoites present in liver cells Occurs usually 24 weeks to 5 years after the primary attack Can be prevented by giving primaquine to eradicate hypnozoites to form schizonts and others which remain dormant. These latter forms are called hypnozoites (from hypnos: sleep). Hypnozoites remain inside the hepatocytes as uninucleated forms, 4-5 µm in diameter, for long periods. Reactivation of hypnozoites leads to initiation of fresh erythrocytic cycles and new anacks ofmalarial fever. Such newattacks ofmalaria, caused by dormant ex:oerythrocytic forms, reactivated usually from 24 weeks to 5 years after the primary attack are called relapses (Table 5). Laboratory Diagnosis Demonstration ofParasite byMicroscopy Diagnosis of malaria can be made by demonstration of malarial parasite in the blood (Box 6). Two types of smears are prepared from the peripheral blood. One is called thin smear and the other is called thick smear. 1. Thin smears: 1hey are prepared from capillary blood of finger tip and spread over a good quality slide by a second slide held at an angle of30-45° from the horizontal such that a tail is formed. • A properly made thin film will consist of an unbroken smearofa single layerofredcells, ending in a tongue, which stops a little short ofthe edge ofthe slide. • Thins smears are air dried rapidly, fixed in alcohol and stained by one of the Romanowsky stains such as Leishman, Giemsa, Field's, or JSB stain (named after Jaswant Singh and Bhattacharjee). • Thins smears are used for detecting the parasites and determining the species. 2. thick smears:thhey can be made on the same slide ofthin smear or separately. • In a thick film, usually three drops ofblood are spread over a small area (about 10 mm). • The amount of blood in thin smear is about 1- 1.5 µL, while in a thick smear it is 3-4 µL. • The thick film is dried and kept in a Koplin jar for 5-10 minutes for dehemoglobinization.
- 103. Paniker'sTextbook ofMedical Parasitology Box6: Morphological feature of malaria parasites in blood smear . In P. vivax, P. ovate and P. matariae all asexual forms and gametocytes can be seen in peripheral blood. In P. fatciparum infection, only ring form alone or with gametocytes can be seen. • Ring forms of all species appear as streaks of blue cytoplasm with detached nuclear dots.Theyare large and compact in P. vivax, P. ovate, and P. matariae and fine delicate with double chromatin (head-phone appearance). In P. fa/ciparum, multiple rings with •accole"forms are seen. • Gametocytes are banana-shaped (crescents) in P. falciparum and round in P. vivax, P. ovate andP. matariae. • Enlarged red blood cells (RBCs) with intracellular coarse brick-red stippling (Schuffner'sdots) are characteristic in P. vivax. In P. falciparum, RBCs are normal in size with large red dots (Maurer's dots) and sometimes, with basophilic stippling. Careful search in blood should be made for mixed infections. Box 7: Quantification of parasites Quantification of parasites can be done by thick smear.The counting of parasites are done to an approximate number in the following method: • + = 1- 1Oparasite per 100 thick film fields • ++ = 11- 100 parasite per 100 thick film • +++= 1- 1Oparasite per thick film field • ++++= More than 10 parasite per thick film field. • It is not fixed in m ethan ol. • 1hick film is stained similar to thin film. • 1he stained film is examined under the oil immersion m icroscope. • 1he thick film is more sensitive, when examined by an experienced person, because it concentrates 20- 30 layers of blood cells in a small area. • Thick film is more suitable for rapid d etection of malarial parasite, particularly wh en they are few (as low as 20 parasites/ µL) (Box 7). • 1he dehemoglobinized and stained thick film does nor show any red cells, but only leukocytes, and, when present, the parasites. But the parasites are o ften distorted in form, and as the diagnostic changes in blood cells such as enlargement and stippling cannot be made out, species identification is difficult. • Thin film is examined first at the tail end and if parasites are found, there is n o need for examining thick film. If parasites are not detected in thin film, then thick film should be examined. • leis recommended that200oil immersion fields should be examined before a thick film is declared negative (Fig. 14). Quantitative Buffy Coat, Smear The quantitative buffy coal {QBC) test is a novel method for diagnosing malaria, wherein a small quantity ofblood (50- 110 µL) of blood is spun in QBC centrifuge at 12,000 revolutions per minutes for 5 minutes. Multiple rings Erythroblast Gametocyte Fig. 14: Malarial parasite, Ptasmodium fatciparum, in the peripheral blood showing numerous ring stages and a crescent of gametocyte. The background shows a normoblast Source: Mohan H. Textbook of Pathology, 6th edition. New Delhi: Jaypee Brothers Medical Publishers; 2010. p. 314. • Red blood cell containing malaria p arasites are less d ense than normal RBCs and concentrate just below the buffy coat of leukocytes at the top of the erythrocycic column. • Precoating of the tube with acridine orange induces a fluorescen ce on the parasites, which can then be readily visualized u nder the oil immersion microscope because the parasite contains deoxyribonucleic acid (D A), but the m ature RBCs do not contain DNAand ribonucleic acid (RNA). The nucleus of the parasite is d etected by acridine orange stains and appears as fluorescing greenish-yellow against red background. • The advantage of QBC is that it is faster and more sens itive than thick blood sm ear. • The disadvantage of the test is that it is less sensitive than thick film and is expens ive. • A careful sm ear examination s till remains as the "go ld standard " in m alaria diagnosis. Microconcentration Technique In microconcentralion technique, blo od sample is collected in microh ematocrit tube and centrifuged at high s p eed. The sediment is m ixed with normal serum a nd sm ear is prepared . Though it increases the positivity rate, it changes the m orphology of the parasite. Culture ofMalaria Parasites Th e o riginal m ethod of p etridish culture employed a candle jar to provide an atmosphere of 3% oxygen and
- 104. 10% carbon dioxideand a relatively simple self-culture medium (RPMl1640) supplemented with human, rabbit, or calf serum Lo maintain infected erythrocytes. Fresh red cells were added periodically for continuation of the growth and multiplication of plasmodia. The continuous flow method devised by Trager enables the prolonged maintenance ofstock cultures. • Computer-controlled culture sysL ems, introduced subsequently, provide a steady abundant supply of parasites. Several culture lines have been established from blood of infected Aotus monkey or directly from human patients. • Schizogony proceeds normally in culture. Gametocytes are formed infrequently. Pre-erythrocytic stages of some species have been obtained in tissue cultures. Plasmodia retain their infectivity in culture. • Culture ofplasmodia provides a source ofthe parasites for study of their antigenic structure, in seroepidemiologic surveys, drug sensitivity tests and studies in immunoprophylaxis. Serodiagnosis Serodiagnosis is not helpful in clinical diagnosis because they will not differentiate between an active and past infection. It is used mainly for seroepidemiologicaJ survey and to identify the infected donors in transfusion malaria. The tests used are indirect hemagglutination (IHA), indirect fluorescent antibody (!FA) test and enzyme-linked immunosorbent assay (ELISA). Newer Methods of Diagnosis (Box 8) Fluorescence microscopy: Kawamoto technique: Fluorescent dyes like acridine orange or benzothiocarboxy purine are used, which stain tl1e parasites enteringthe RBCs but notwhite blood cells (WBCs). This is a method ofdifferentialstaining. • Acridine orange stains DNJ as fluorescent green and cytoplasmic RNA as red. Box 8: Laboratory diagnosis of malaria • Demonstration of malarial parasites in thick and thin blood smear examination by Leishman, Giemsa, or JSB stain. • lmmunofluorescence staining and QBC smear. • Rapid immunochromatographic test (ICT) for detection of malaria antigen (PfHRP-2 and pLDH). • Moleculardiagnosis: DNA probe and PCR. • Routine blood examination for Hb, PCV and blood sugar. Abbreviations: DNA, deoxyribonucleic acid; Hb, hemoglobin; JSB, Jaswant Singh and Bhattacharjee; PCR, polymerase chain reaction; PCV, packed cell volume; PfHRP-2; P/asmodium falciparum histidine rich protein-2; pLDH, parasite lactate dehydrogenase; QBC, quantitative buffy coat Malaria and Babesia • The stained slide is examined under fluorescent microscope. • The method is mainly used for mass screening in field laboratory. Rapid antigendetectiontests :Rapid diagnostictest are based on the detection of antigens using immunochromatographic methods. These rapid antigen detection tests have been developed in different test formats like the dipstick, card and cassette bearing monoclonal antibody, directed against tje parasite antigens. Several kits are available commercially, which can detect Plasmodium in 15 minutes (Fig. 15). Parasite-F test: This test is based on detection of histidine rich protein-2 (HRP-2) antigen produced by the asexual stages ofP. falciparum expressed on the surface ofred cells. • Monoclonal antibody produced against HRP-2 antigen (Pf band) is employed in the test strip. • Advantage: It is widely popular and has high sensitivity (98%) and specificity. - The test is said to detect low asexual parasitemia of more than 40 parasites/µL. - The test can be performed within IO minutes. • Disadvantage: Plasmodium falciparum HRP-2 (PfHRP- 2) antigen detection test cannot detect the other three malaria species. - T t remains positive up to 2 weeks after cure. - In P.falciparum infection, PfHRP-2 is not secreted in gametogony stage. Hence in "carriers'; the Pf band may be absent. Dual antigen test: The test detects parasite lactate dehydrogenase (pLDH) produced by trophozoites a nd gametocytes ofall plasmodimn species and PfHRP-2 antigen produced by P.falciparum simultaneously. • Thus, one band (Pv band) is genus specific (Plasmodium specific) and other is PlasmodiumJalciparum specific (Pf band). Fig. 15: Rapid ICT Kit for dual antigen
- 105. Paniker's Textbook ofMedical Parasitology • This test is a rapid two-site sandwich immunoassay used for specific detection and differentiation of P. falciparum and P vivax. malaria in areas with high rates of mixed infection. • The "Pv" band can be used for monitoring success of antimalarial therapy in case of stained alone P. vivax infection as the test will detect only live parasites and therefore will be negative, if the parasite has been kiU ed by the treatment. • The disadvantage of the test is that it is expensive and cannot differentiate between P. vivax, P ovale and P. malariae. Molecular Diagnosis Deoxyribonucleic acid probe: Deoxyribonucleic acid probe is a highly sensitive method for the diagnosis of malaria. 1t can detect less than IO parasites/ µL of blood. Polymerase chain reaction: Polymerase chain reaction {PCR) is increasingly used now for species specification and for detection of drug resistance in malaria. • Chloroquine resistancein P.Jalciparumis due to mutation in the Plasmodium Jalciparum chloroquine resistance transporter (PfCRT),a transporter gene in the parasite. • Point mutation in another gene Plasmodium falciparum multidrug resistance protein l (PfMDRI) is responsible for resistance in vitro. • Pyrimethamine andsulfadoxine resistances are associated with point mutations in dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS)genes, respectively. • Mutation in PfATPase gene is associated with reduced susceptibility to artemisinin derivatives. Other Tests • Measurement of hemoglobin and packed cell volume (PCV), in case of heavy parasitemia, particularly in children and pregnant woman. • Total WBC and platelet count in severe Jalciparum malaria. • Measurement of blood glucose to detect hypoglycemia, particularly in young children and pregnant women with severefalciparum malaria and patients receiving quinine. • Coagulation tests like measurement of antithrombin TI! level, plasma fibrinogen, fibrin degradation products (FDPs), partial thromboplastin time (PTT), if abnormal bleeding is suspected in falciparum malaria. • Urineforfree hemoglobin, ifblackwaterfever issuspected. • Blood urea and serum creatinine to monitor renal failure. • Glucose-6-phosphate dehydrogenase screening before treatment with an antioxidant drug like primaquine. Treatment Antimalarial drugs are used with various objectives like clinical cure, prevention ofrelapse,prevention oftransmission and prophylaxis. Therapeutic Objective is to eradicate the erythrocytic cycle and clinical cure. Radical Cure Objective is to eradicate the exoerythrocytic cycle in liver to prevent relapse. Gametocidal Objective is to destroy gametocytes to prevent mosquito transmission and thereby reducing human reservoir. Chemoprophy/axis Objective is to prevent infections in nonimmune person visiting endemic areas. The most commonly used antimalarials are chloroquine, amodiaquine, quinine, pyrimethamine, doxycycline, sulfadoxine, proguanil and primaquine. Newer antimalarial like artemisinin, lumefantrine, mefloquine, halofantrine are now commonly used for multidrug-resistant P. Jalciparurn infections. Treatment of Uncomplicated Malaria Positive P. vivax, P. ovate and P malariae cases are treated with chloroquine 25 mg/kg divided over 3 days. Vivax malaria relapses due to the presence ofhypnozoites in the liver. The relapse rate of vivax malaria in India is about 30%. • For prevention of relapse, primaquine is given in a dose of0.25 mg/ kg daily for 14 days under supervision. • Primaquine is contraindicated in G6PD deficiency patients, infants and pregnant women. • In case ofchloroquine resistance: Quinine is given in a dose of600 mg 8 hourly for 7 days along with doxycycline 100 mg/day. Treatment ofComplicated (Falciparum) Malaria Due to emergence of drug resistance offalciparum malaria is based on area resistant or sensitive antimalarial drugs. • Artemisinin-basedcombination therapy: According to revised malaria drug policy in India arremisinin-based
- 106. combination therapy (ACT)(artemisinin + sulfadoxine - pyrimethamine) should be given to all microscopically positive Jalciparum cases for 3 days in all over India except North-eastern states. This is accompanied by single dose of primaquine 45 mg (0.75 mg/kg) on day 2 as gametocidal drug. In North-eastern states considering resistant to sulfadoxine - pyrimethamine drugs, Technical Advisory Committee on Malaria recommended artemether (20 mg+ lumefantrine) as per age specific dose schedule. Note: According to revised Malaria Drug Policy 2013, there is no scope for presumptive treatment. Production and sale of artemisinin as monotherapy has been banned in India as it can lead to developmenr ofparasite resistance to the drug. Drug resi-Stance of malarial parasite: • A drug resistant parasite is defined as a parasite that will survive and multiply in a dosage that normally cures the infection. Such resistance may be relative (yielding to increased doses of the drug tolerated by the host) or complete (withstanding a maximum dose tolerated by the host). Malaria and Babesia Malaria Vaccine Malaria vaccine is an area of intensive research. Over past decades, there has been a significant progress in malaria vaccine development. A completely effective vaccine is not yet available for malaria, although several vaccines are under development. SPf66 (a cocktail of four antigens, three asexual blood stage antigens + circumsporozoite of Pf) was te ted extensively in endemic areas in the 1990s, but clinical trials showed it to be insufficiently effective. Other vaccine candidates targeting the blood stage of parasite's life cycle using merozoite surface protein 1 (MSP1), MSP2, MSP13 and ring-infected erythrocyte surface antigens (RESAs) have also been in insufficient on their own. Several potential vaccines targeting the pre-erythrocytic stage are being developed,witl1 RTS,S/ASOl showing the most promising results. The RTS,S/ ASOl(commercial name, mosquirix) was engineered using genes from the outer protein ofP. falciparam and a portion ofhepatitis B virus, plus a chemical adjuvant (ASOl) to boost irrunune response. Vector Control Strategies • Resistance arises from spontaneous point mutations in the genome or gene duplications. The emergence of • resistance can be prevented by use of combination of drugs with different mechanisms of action and different drug target. Residual spraying: Spraying of residual in secticides, e.g. dichlorodiphenyltrichloroethane (DDT), malathion and fenitrothin in the indoor surfaces of the house is highly effective against adult mosquitos. Space application: Insecticidal formulation is sprayed into the atmosphere by ultra-low volume in the form of mist or fog to kill insects (pyrethrwn extracts). Individual protection: Man-vector contact can be reduced by other preventive measures such as the use of repellants, protective clothing, bed net, preferably impregnated with long-acting repellant, mosquito coils and screening of house. • Three levels ofresistance (R) are defined by the WHO: 1. RI: Following treatment, parasitemia clears but recrudescence occurs. 2. RII: Following treatment, there is a reduction but not • a clearance of parasitemia. 3. R11/: Following treatment, there is no reduction of parasiternia. the earlier method of classifying resistance is based on counting trophozoites in blood film daily for 7 days after tJeatment and monitoring the patient for any subsequent AntilarvalMeasures recrudescence. All patients with afalciparum parasitemia of more than one rrophozoite per high power field (+++or over} • in areas ofsuspected drug resistance, should be checked for a decrease and clearing ofparasites following treatment. Prophylaxis Chemoprophylaxis It is recommended for travelers going to endemic areas as short-term measure. Chloroquine (300 mg) or mefloquine (400 mg) weekly should be given 1week and 2 weeks before travel to endemic area respectively. Alternatively doxycycline (100 mg) daily can be given from day l before travel. Old antilarval measures such as oiling the collection of standing water or dusting them with Paris green have now become promising with the increase of insecticide resistance. Source reduction: Mosquito breeding sites can be reduced by proper drainage, filling of land, water level management, intermittent irrigation, etc. Integrated Control In order to reduce too much dependence on residual insecticides, increasing emphasis is being put on integrated vector control methodology, which includes bioenvironmental and personal protection measures.
- 107. Paniker'sTextbook of MedicalParasitology Malaria Control Programs In India, the National Malaria Control Programme was introduced in 1953, with the objective of the ultimate eradication of the disease and operated successfully for 5 years, bringing down the annual incidence of malaria from 75 million in 1958 to 2 milJion. • By 1961, the incidence dropped to an all time low of 50,000 cases and no deaths. However, there have been setbacks from 1970 and by 1976, the incidence rose to 6.4 million cases. With the implementation of modified plan of operation in 1977, the upsurge of malaria cases dropped down to 2.1 million cases in 1984. Since then, the epidemiological situation has not shown any improvement. • Malaria control added impetus as "roll-back malaria initiative" launched jointly by WHO, United Nations Children's Fund (UNICEF), United Nations Development Programme (UNDP) and the World Bank in 1998. Accordingly, National Vector Borne Disease Control Programme (NVBDCP) is implemented by Directorate of Health Services jointly with Mission Directorate and National Rural Health Mission (NRHM). ational goal established under the program is to reduce the number of cases and deaths recorded in 2000, by 50% or more in 2010 and by75% or more by 2015. BABESIA SPECIES • INTRODUCTION Babesia is intraerythrocytic sporozoan parasites that morphologically resemble Plasmodium and cause tick-borne malaria like illness in domestic and wild animals. It causes opportunistic infection in humans. • CLASSIFICATION Order: Piroplasmida Family: Babesiidae Species: Medically important Babesia species are: • B. rnicroti (rodent strain) • B. clivergen.s (cattle strain) • 8. hovis (cattle strain) • HISTORY AND DISTRIBUTION Babesia is so named aJter Babes, who in 1888 described the intraerythrocytic parasite in the blood of cattle and sheep in Romania. • In 1893, the parasite was shown to cause the tick-borne disease, Texas fever, an acute hemolytic disease of cattle in southern United States ofAmerica (USA). • This was the first arthropod-borne disease to have been identified. • In 2009, more than 700 cases were reported from endemic state ofUSA. • Prevalence ofB. rnicroti is underestimated because young healthy individuals typically experience a mild self- limiting disease and may not seek medical attention. • HABITAT The parasite is present in erythrocytes and resembles the ring stage ofP.Jalciparum. • MORPHOLOGY Trophozoites are pleomorphic 2-5 µm in diameter found inside the red cells. The shape may be pyriform, ameboid, or spindle-Like, usually in pairs and are often mistaken as ring form ofPlasmodiu:m (Fig. 16). Merozoites may be spherical or oval or pyriform bodies, found in pairs. • LIFE CYCLE Definitive Host lxodid ticks. Intermediate Host Man or other mammals. Infective Form Sporozoites are the infective form for humans. Mode ofTransmission Infection in vertebrate occurs through bite of the nymphal stage of T xodid ticks. Transmission occurs during May to Fig. 16: Trophozoites of Babesia microti in human blood smear
- 108. September. Incubation periodis 1-6 weeks. Babesiosis can also be transmitted via blood transfusion. Transovarian transmission in ticks also occurs. • In their life cycle, merogony takes place in vertebrate hosts and sporogony in the invertebrates. • Man acquires infection by bite of the infected ticks (definitive host). • Sporozoites present in the salivary glands of tick are introduced in man or other mammals (intermediate host). • Sporozoites change to trophozoites in the circulation, which then invade the RBCs and multiply asexually by binary fission or schizogony to form four or more trophozoites. ewly formed trophozoites are released by rupturing erythrocytes and invade new erythrocytes. • Some of the sporozoites grow slowly inside red cells and become folded like an accordion. These are thought be gametocytes. • Female ticks become infected by feeding the host blood. • In the digestive tract of tick, the gametocytes multiply sexually and later migrate to the salivary glands where they divide by multiple fission into smaller forms known as "vcrmicules''. • Vermicules undergo secondary schizogony to produce sporozoites, which are the infective forms for human. • PATHOGENICITY AND CLINICAL FEATURES Hemolysisofthe infectederythrocytes is primarilyresponsible for many clinical manifestations. • Thereis accumulation ofparasites in the capillaries ofliver, spleen and kidneys which leads to cellular degeneration and necrosis. • The illness develops 1-6 weeks after the tick bite. • This may be subclinical or mild self-limiting or acute illness, resembling malaria. • In acute disease, there is malaise, fatigue, fever, myalgia, arthralgia, dry cough and anorexia. Fever exceeds 38°C and can reach 40.6°C accompanied by chill and sweat. • Less common syndromes are neck stiffness, sore throat, abdominal pain, jaundice and anemia. • Severe babesiosis is associated with parasitemia levels of more than 4% infected RBCs and requires hospitalization. Fatality rate is 5% among hospitalized cases but is higher (20%) among immunocompromised patients. • Complications of acute babesiosis are renal failure, disseminated intravascular coagulation (DIC), acute respiratory distress syndrome (ARDS) and congestive cardiac failure (CCF). • Risk factors for complication are severe anemia (<10 g%) and high levels ofparasitemia. • LABORATORY DIAGNOSIS Microscopy Malaria and Babesia Diagnosis of babesiosis is primarily done by examination of blood films stained with Leishman or Giemsa stain. • Babesia appears as intraerythrocytic round or pyriform, or ring form simulating P.falciparum (Fig. 16). • The ring forms are the most common and lacks the central hemozoin deposit, typical ofP.falciparum. Other distinguishing features are the absence ofschizonts and gametocytes and presence of tetrads (maltose crosses), which are pathognomonic of B. microti or B. duncani (Table 6). Polymerase Chain Reaction If parasite cannot be identified by microscopy, amplificarion ofbabesia1 18S rRNA by PCRis recommended. Serology It is useful to confirm the diagnosis. An !FA for B. microti is available. Immunoglobulin M titer of more than 1:64 and IgG titer more than 1:1024, signify active or recent infection. Titer declines over 6-12 months. Blood Picture Parasitemia levels typically range from 1% to 20% in immunocompetent patients but can reach up to 85% in asplenic patients. Table 6: Differential features of malaria and babesiosis Characteristics Malaria Babesiosis Distribution Worldwide North America and Europe Vector Anopheles Tick mosquito Reservoir Man Rodent and cattle No. of parasites per 1-3 1-12 red blood cell (RBC) Schizont Present Absent Gametocyte Present Absent Pigment in Present Absent trophozoite Antigenic variation None Profound Level of parasltemia Correlate with Does not correlate with severity of disease severity of disease Animal inoculation Negative Positive
- 109. Paniker'sTextbook of MedicalParasitology • Reticulocyte count is elevated. • Thrombocytopenia is common. • White blood cell count may be normal or slightly decreased. OtherTests Liver function tests such as serum glutamic pyruvate transaminase(SGPT) and alkaline phosphatase yield elevated value. • Urine analysis m ay detect hemoglobi nuria, excess urobilinogen and proteimuia. • In renal complications, increased blood urea nitrogen (BUN) and serum crealinine are found. • TREATMENT B. microti infection appears to be mild and self-limiting. Most of the patients recover without any specific chemoLherapy, with only symptomatic treatment. • In acute cases chemotherapy is required. • Atovaquone 750 mg twice daily, along with azithromycin 500 mg- 1 g/day for a period of 7- 10 days is effective. Alternatively, clindamycin (300- 600 m g, 6 hourly) along with quinine (650 mg 6-8 h ourly) may be given intravenously. • lnfulminantcases,exchangetransfusionisrecommended. • PROPHYLAXIS No vaccine is available at present. There is no role of chemotherapy. Individuals who reside or travel in endemic areas, should wear protective clothing and apply tick repellents. Individuals with history of symptomatic babesiosis or with positive antibody titer should be indefinitely deferred from donating blood. KEY POINTS OF PLASMODIUM AND BABES/A • Malaria parasite belongs to the genus Plasmodium. • Four species of Plasmodium cause malaria in man- (1) P. vivax, (2) P. falciparum, (3) P. malariae and (4) P. ovale. • Definitive host: Anopheles mosquito (sexual phase of life cycle). • Intermediate host: Man (asexual phase of life cycle). • Infective form: Sporozoites present in salivary gland of mosquito. • P. vivax and P. ovate cause benign tertian malaria, P. falciparum causes malignant tertian malaria and P. matariae causes benign quartan malaria. • Acute falciparum malaria is the most dangerous and fatal form and is due to heavy parasitization of RBCs which cause blockage of capillary and venules by cytoadherence. • Clinical features: Typical picture of malaria consist of periodic bouts of fever with rigor followed by anemia and splenomegaly. Febrile paroxysms comprise of cold stage, hot stage and the sweating stage. • Tropical splenomegalysyndromeis a chronicbenign condition resulting from abnormal immunological response to malaria. • Relapse of malaria occurs in P. vivax and P. ovate infection due to persistence of dormant stage hypnozoites in liver. Recrudescence occurs commonly in P. falciparum and P. matariae due to persistence of parasite in circulation at a subclinical level. • Diagnosis: By demonstration of parasite in thick and thin smear of peripheral blood and also by detection of malaria antigen by rapid ICT. • Treatment: Chloroquine, sulfadoxine and pyrimethamine along with primaquine. In chloroquine resistance, quinine or artemisinin are used. • Babesia spec/es comprising 8. microti, 8. divergens and 8. bovis, are intraerythrocytic sporozoan parasite resembling plasmodia. They cause opportunistic infections in humans. • Mode of transmission: Through bite of lxodid ticks. • Reservoirs: Rodents and cattle. • Clinical features: Mild and self-limiting. In immuno- compromised patients, it causes anemia, jaundice, hemoglobinuria, respiratory failure. etc. • Diagnosis: By examination of stained blood films for intraerythrocytic parasites, reticulocytosis, increased SGPT, alkaline phosphatase, hemoglobinuria. • Treatment: Atovaquone + azithromycin. Alternatively, clindamycin and quinine may be given. REVIEW QUESTIONS 1. Describe briefly the life cycle and laboratory diagnosis of: a. Plasmodium vivax b. Plasmadium falciparum 2. Write short noteson: a. Clinical features of malaria b. Cerebral malaria c. Blackwater fever d. Malignanttertian malaria e. Prophylaxis of malaria f. Treatment of malaria g. Rapid detection test h. Babesiosis 3. Differentiate between: a. Different malarial parasites b. Recrudescence and relapse c. Malaria and Babesiosis
- 110. MULTIPLE CHOICE QUESTIONS 1.Old RBCsare preferentially infected by a. Plasmodium falciparum b. P/asmodium malariae c. Plasmodium vivax d. Plasmodium ova/e 2. The infective form of the malaria parasite is a. Oocyst b. Sporozoite c. Bradyzoite d. Tachyzoite 3. Prolonged parasitism in malaria isdue to a. Antigenic variation b, lntracellularity of parasite c. lmmunosuppression d. Sequestration 4. Malaria pigment is formed by a. Parasite b. Bilirubin c. Hemoglobin d. All of the above 5. Schuffner'sdot in RBCsare sesen in infection with a. Plasmodium vivax b. Plasmodium falciparum c. Plasmodium malariae d. Plasmodium ovale 6. Quartan malaria is caused by a. Plasmodium vivax b. Plasmodium falciparum c. Plasmodium malariae d. Plasmodium ovale Malaria and Babesia 9. Malaria is not seen in patientswith a. G6PD deficiency b. Sickle cell trait c. Duffy negative blood group d. All of the above 10. Which plasmodial infection is more often associated with nephritic syndrome a. Plasmodium vivax b. Plasmodium falciparum c. Plasmodium malariae d. Plasmodium ovale 11. Which is the t reatment of choice for benign tertian malaria a. Sulfamethoxazole - pyrimethamine b. Quinine c. Mefloquine d. Chloroquine 12. Gametocidal pernicious malaria may occur in a. Plasmodium vivax b. Plasmodium falciparum c. Plasmodium malariae d. Plasmodium ovale 13. Babesiosis is transmitted by a. Ticks b. Mites c. Flea d. Mosquito 14. Maltose cross is a characteristic feature of a. Cryptococcus neoformans b. Babesia microti c. 8/astomycosis d. Micrococcus 7. Schlzonts ofPlasmodium fa/ciparum are not found in peripheral Answer blood because a. Schizontsare absent in the life cycle b. Schizonts are killed by antibodies c. Schizonts develop only in capillaries of internal organs d. None ofthe above 8. Crescent-shaped or banana-shaped gametocytes are seen in infection with a. Plasmodium vivax b. Plasmodium falciparum c. Plasmodium malariae d. Plasmodium ovale 1. b 8. b 2. b 9. d 3. b 10. C 4. C 11. d 5. a 12 b 6. C 13. a 7. C 14. b
- 111. CHAPTER 7 • INTRODUCTION Thecoccidia are unicellular protozoa and belong to the Phylum Apicomplexa. . • They live intraceUularly, at least duringa part of their life cycle, and at some stage in their life cycle, they possess a structure called the apical complex, by means of which they attach to and penetrate host cells; hence included in Phylum Apicomplexa. All coccidian have a sexual sporogonic phase and an asexual schizogonicphase. Many of them also show an alteration of hosts-a definitive host and an intermediate host. Many parasites considered in this chapter have acquired great prominence due to their frequent association with human immunodeficiency virus (I-ITV) infection. • TOXOPLASMA GONDII History and Distribution Toxoplasma gondii is an obligate intracellular coccidian parasite, first described in 1908 by Nicolle and Manceaux in B a small orth American rodent called gundi (Ctenodactylus gundi). • Its importance as a human pathogen was recognized much later, when Janku in 1923 observed the cyst in the retina ofa chi!d with hydrocephalus and microphthalmia. • The name Toxoplasma is derived from the Greek word Toxon meaning arc or brow referring to the curved shape of the trophowite. • Toxoplasma is now recognized as the most common protozoan parasite globally, with the widest range ofhosts spread over 200 species of birds, reptiles and mammals, including humans. Morphology T. gondii occurs in three forms (Figs IA to C): l. Trophozoite 2. Tissue cyst 3. Oocyst. • The trophozoite and tissue cyst representstages in asexual multiplication (schizogony), while the oocyst is formed by sexual reproduction (gametogony or sporogony). Figs 1Ato C: Toxoplasma gondii. (A) Smear from peritoneal fluid of infected mouse, showing crescentic tachyzoites-extracellular trophozoites and intracellular form within macrophage; (B) Thick-walled tissue cyst containing rounded forms bradyzoites; and (C) Oocyst containing two sporocysts with sporozoites inside
- 112. • All threeforms occur in domestic cats and other felines, which are the definitive hosts and support both schizogony and gametogony. • Only the asexual forms, trophozoites and tissue cysts are present in other animals, including humans and birds, which are the intermediate hosts. • All the three forms are infectious roman. Trophozoites (Tachyzoites) The trophozoite is crescent-shaped, with one end pointed and the other end rounded. • It measures 3- 7 µm in length. The nucleus is ovoid and is situated at the blunt end ofthe parasite. • Electron microscopy reveals an apical complex at the pointed end (Fig. 2). • The trophozoite stains well with Giemsa stain, the cytoplasm appearing azure blue and the nucleus red (Fig. 3). • The activelymultiplying trophozoite is seen intracellularly in various tissues during early acute phase of infection. Extracellular trophozoites can also be seen in impression smears. • It can invade any nucleated cell and replicate within cytoplasmic vacuoles by a process called endogony (internal budding), wherein two daughter trophozoites are formed, each surrounded by a membrane, while still within the parent cell. When the host cell becomes distended with the parasite, it disintegrates, releasing the trophozoites that infect other cells. • During acute infection, the proliferating trophozoite within host cell may appear rounded and enclosed by the host cell membrane. This is called pseudocyst or colony and can be differentiated from tissue cysts by staining reactions. Fig. 2: Toxopfasma gondii. Trophozoite (tachyzoite), fine structure seen by electron microscopy Coccidia The rapidly proliferating trophozoites in acute infection are called tachyzoites. The trophozoites are susceptible to drying, freeze-thawing and gastric digestion. Tissue Cyst Tissue cysts are the resting form of the parasite. • They are found during chronic stage of the infection and can be found in the brain (most common site), skeletal muscles and various other organs. • The cyst wall is eosinophilic and stains with silver, in contrast to the pseudocyst. • With periodic acid-Schiff (PAS) stain, the cyst wall stains weakly, and the parasites inside are stained deeply. The slowly multiplying parasites within the cyst are called bradyzoites. • The cyst is round or oval, 10-20 µmin size and contains numerous bradyzoites. Cysts remain viable in tissue for several years. • In immunologically normal hosts, the cysts remain silent, but in the immunodeficient subjects, they may get reactivated, leading to clinical disease. • It is relatively resistant and when the raw or undercooked meat containing the cysts is eaten, infection occurs. • The cyst wall is disrupted by peptic or tryptic digestion and the released parasites initiate infection by invading intestinal epithelial cells. • lhey reach various tissues and organs through blood and lymphatic dissemination. • Cysts aresusceptible to desiccation, freezing, and thawing, and heat above 60°C. -~'' :;:>'I ~' ~ .... .,~ ~·· J ... - ~,~ II II_, -,- Fig. 3: Toxopfasma gondii. Trophozoite grows in tissue culture. Smear shows trophozoites arranged in different patterns-singly, in cluster, or as rosette (Giemsa stain)
- 113. Paniker'sTextbook ofMedical Parasitology Oocyst Oocystsdevelop only in definitive hosts- in the intestine of cats and other felines but not in humans. • lt is oval in shape and measures 10-12 µm in diameter. Each cyst is surrounded by a thick resistantwall. • The oocysts are form ed by sexual reproduction (gametogony). • Cats shed millions of oocysts per day in feces for about 2 weeks during the primary infection. The freshly passed oocyst is not infectious. • 1hey undergo sporulation in the soil with formation of two sporocysts, each containing four sporozoites. The sporulaced oocyscis infective. • Oocyst is very resistant co environmental conditions and can remain infective in soil for about a year. • When the infective oocyst is ingested, it releases sporozoites in the intestine, which initiates infection. Life Cycle Host: T. gondii completes its life cycle in two hosts (Fig. 4). l. Definitive hosts: Cats and other felines, in which both sexual and asexual cycles take place. Cat acquires infection by ingestion of rodent meat containing tissue cyst Tissue cyst formed in birds, rats, etc ~ _____. Undergo schizogony l,I.J,.Aol (asexual cycle) In Bradyzoites mucosal cells released Enteric cycle Contaminated soil containing sporutated oocyst ingested by rats, birds, etc 2. Intermediate hosts: Man and other mammals, in which only the asexual cycle takes place. T. gondii has 1'•vo types oflife cycles: 1. Enteric cycle 2. Exoenteric cycle. Enteric Cycle (Feline Cycle) Enteric cycle occurs in cat and other definitive hosts (Fig. 4). • Both sexual reproduction (gametogony) and asexual reproduction (schizogony) occur within the mucosa! epithelial cells of the small intestine of the cat. • Cat acquires infection by ingestion of tissue cysts in the meat of rats and other animals or by ingestion of oocysts passed in its feces. • The bradyzoites are released in the small intestine and they undergo asexual multiplication (schizogony) leading to formation of merozoites. • Some merozoites enter extrainrestinal tissues resulting in the formation oftissue cysts in other organs of the body. • Other merozoites transform into male and female gametocytes and sexual cycle (gametogony) begins, with the formation ofmicrogamete and macrogamete. n Excys1a1;01J on srna111 0cc ~~ II.Ian acquires Infection Bradyzoite released by ingestion of from tissue cyst contaminated food ~ and water containing 'VI sporulated oocyst or Sporozoite released by ingestion of undercooked from oocyst meat containing tissue cyst 1 - Man-<lead end (cycle end) ~ Exoenteric ~ cycle ~i !!!. Fig. 4: Life cycle of Toxoplasma gondii
- 114. • A macrogameteis fertilized by motile microgamete resulting in the formation of an oocyst, which passes through maturation stages (sporulation) in the soil after being excreted from host through feces. • A mature oocyst containing eight sporozoites is the infective form which may be ingested by rats or other mammals to repeat the cycle. Exoenteric Cycle (Human Cycle) Exoenteric cycle occurs in humans, mice, rats, sheep, cattle, pigs and birds, which are the intermediate hosts. • . . . • . • . . Humans acquire infection after: - Eating uncooked or undercooked infected meat, particularly lamb and pork containing tissue cysts. Ingestion of mature oocysts through food, water, or fingers contaminated with cat feces directly or indirectly. Intrauterine infection from mother to fetus (congenital loxoplasmosis). Blood transfusion or transplantation from infected donors. Sporozoites from the oocysts and bradyzoites from the tissue cysts enter into the intestinal mucosa and multiply asexually and tachyzoiles are formed (endodyogeny). Tachyzoites continue to multiply and spread locally by lymphatic system and blood. Some tachyzoites also spread to distant extraintestinal organs like brain, eye, liver, spleen, lung and skeletal muscles and form tissue cysts. The slowly multiplying forms inside the tissue cysts are known as bradyzoiles, which remain viable for years. The dormant bradyzoites inside the cyst may be reactivated in immune suppression causing renewed infection in the host. Human infection is a dead end for the parasite (Pig. 4). Human roxoplasmosis is a zoonosis. The full natural cycle is maintained predominantly by cats and mice. Mice eat materials contaminated with oocysts shed in cat's feces. Tissue cysts develop in mice. When such mice are eaten by cats, they get infected and again shed oocysts in feces. Pathogenicity and Clinical Features The outcome of Toxoplasma infection depends on the immune status ofthe infected person. • Active progression of infection is more likely in immunocompromised individuals. Toxoplasmosis has acquired great importance as one of the major fatal Coccidia complications in acquired immunodeficiency syndrome (AIDS). Most human infections are asymptomatic. Clinical toxoplasmosis may be congenital or acquired. Congenital Toxoplasmosis Congenital toxoplasmosisresultswhen T gondiiistransmitted transplacentally from mother to fetus (Box 1). . • . . . . This occurs when the mother gets primary toxoplasma infection, whether clinical or asymptomatic, during the pregnancy. The risk of fetal infection rises with progress ofgestation; from 25%, when the mother acquires primary infection in 1st trimester to 65% in the 3rd trimester. Conversely, the severity of feral damage is highest, when infection is transmitted in early pregnancy. Mothers with chronic or latent Toxoplasma infection, acquired earlier, do not ordinarily infect their babies. But in some women with latent or chronic infection, the tissue cyst may be reactivated during pregnancy and liberate trophozoites, which may infect the fetus in utero. Most infected newborns are asymptomatic at birth and may remain so throughout. Some (0.3-1%) develop clinical manifestations of toxoplasmosis within weeks, months and even years after birth. The manifestations of congenital toxoplasmosis include chorioretinitis, cerebral calcifications, convulsions, strabismus, deafness, blindness, mental retardation, microccphaly and hydrocephalus. A few children are born with manifestations of acute toxoplasmosis, which may include fever, jaundice, petechial rashes, microphthalmia, cataract, glaucoma, lymphadenopathy, hepatosplenomegaly, myocarditis, cerebral calcifications and chorioretinitis. Acquired Toxoplasmosis infection acquired postnatally is mostly asymptomatic. • The most common manifestation of acute acquired toxoplasmosis is lymphadenopathy; the cervical lymph nodes being most frequently affected. • Fever, headache, myalgia and splenomegaly are often present. the illness may resemble m ildflu and is self- limited, although the lymphadenopathy may persist. Box 1: Parasites which can be transmitted from mother to fetus • Toxoplasma gondii • Plasmodium spp. • Trypanosoma cruzi.
- 115. Paniker'sTextbookof Medical Parasitology •In some cases, there may be a typhus-like exanthema with pneumonitis, myocarditis and meningoencephalitis, which may be fatal. Ocular Toxoplasmosis Another type of toxoplasmosis is ocular. • It may present as uveitis, choroiditis, or chorioretinitis. • Some casesmay besosevere thattheyrequire enucleation. Toxoplasmosis in lmmunocompromised Patients Toxoplasmosis is the most serious and often fatal in immunocompromised patients, particularly in AIDS, whether it may be due to reactivation of latent infection or new acquisition ofinfections. • In these patients, involvement ofbrain is most common. • Clinical manifestations include encephalitis, altered mental state, seizures, cerebellar signs, meningismus and neuropsychiatric manifestations. • Besides central nervous system involvement, other organs involved are lungs, pancreas, gastrointestinal tract, eyes, heart and liver. • Toxoplasma pneu monia can be confused with Pneumocystis pneumonia. Host Immunity Host defense against Toxoplasma infection involves both humoral (antibody-mediated) andcellularresponses. Specific immunoglobulin G (lgG) antibody can lyse extracellular trophozoites, but activated T cells and natural killer cells appear to be more important in containing the infection and preventing clinical disease. Laboratory Diagnosis The diagnosis of acute toxoplasmosis is made mainly by demonstration of rrophozoites and cysts in tissue and body fluids and by serology (Flow chart 1). Microscopy Tachyzoites and tissue cysts can be detected in various specimens like blood, sputum, bone marrow aspirate, cerebrospinal fluid (CSF), amniotic fluid, and biopsy material from lymph node, spleen and brain. • Smear made from earlier specimens is stained by Giemsa, PAS, or Gomori methenamine silver (GMS) stain. • Tachyzoites appear as crescent-shaped structures with blue cytoplasm and dark nucleus. • Tachyzoites or cyst can also be demonstrated effectively by fluorescent conjugated antibody technique in tissue biopsy or impression smear. • Presence of only tissue cysts does not differentiate between active and chronic infection. • The presence of cysts in placenta or tissues of newborn establishes congenital Toxoplasma infection. Animal Inoculation Toxoplasmacan be isolated by inoculating body fluids, blood, or tissue specimens by inrraperitoneal inoculation in mice or Flow chart 1: Laboratory diagnosis of Toxop/asma gondii Laboratory diagnosis t Microscopy Tachyzoites and tissue cysts detected in blood, sputum and bone marrow aspirates Stains used: -Giemsa - PAS -GMS t Serodiagnosis • Antibody detection: Test for detecting lgG antibody: • ELISA • IFAT • Latex agglutination test • Sabin-Feldman dye test Test for detecting lgM antibody: • Double sandwich lgM ELISA • lgM-ISAGA Test for detecting lgA antibody: • Double sandwich lgA ELISA • Antigen detection: by ELISA Molecular diagnosis • PCR t Imaging • MRI and CT scan for central nervous system involvement • USG for congenital toxoplasmosis • Others • Animal inoculation • Skin test of Frenkel Abbreviations: CT. computed tomography: ELISA, enzyme-linked lmmunosorbent assay: GMS. Gomorl methenamme silver: IFAT. indirect fluorescent antibody test; lgM-ISAGA, immunoglobulin M-immunosorbent agglutination assay; MRI. magnetic resonance imaging; PAS, periodic acid-Schiff; PCR. polymerase chain reaction: USG. ultrasonography
- 116. in tissue culture.Mice should be examined for Toxoplasma in d1eir peritoneal exudate after 7-10 days of inoculation. Serodiagnosis Serology is the mainstayfor diagnosis oftoxoplasmosis. Antibody detection: Diagnosis of acute infection with T. gondiican bemade bydetection of thesimultaneouspresence oflgM and lgG antibodies. • Tests for detecting IgG antibody include: - Enzyme-linked immunosorbent assay (ELISA) - Sabin-Feldman dye test - Indirect fluorescent antibody test (!FAT) Latex agglutination test. • Positive lgG titer (>l:10) can be detected as early as 2-3 weeks after infection. Peak level of antibody is observed in blood 4-8 weeks after infection. • A positive IgM antibody titer indicates an early primary infection. The serum lgM titer can be measured by double-sandwich lgM ELISA or IgM-im munosorbent agglutination assay (lgM-ISAGA). Both assays are equally specific and sensitive. egative IgM titer and positive IgG titer indicate distantinfection. • The double-sandwich lgA EUSA test is used for detecting congenital infection in newborns. Antigen detection: Detection of antigen by ELISA indicates recent Toxoplasma infection. • In AIDS and other immunocompromised patients, an tigen detection is very useful. • Detection of antigen in amniotic fl uid is helpful to diagnose congenital toxoplasmosis. Skin Test ofFrenkel Diluted toxoplasmin is injected intradermally and delayed positive reaction appears after 48 hours. This test is not very reliable for diagnosis of ToxopLasma. Sabin-Feldman Dye Test This was the first serological test for Toxoplasma antibody to be described by Sabin and Feldman (1948). Principal:The testis based on specific inhibition by antibody, ofthe staining oftrophozoites byalkaline methylene blue dye. Technique: Equal volumes of diluted patient's serum are incubated with live trophozoites and normal human serum (accessory factor) for an hour at 37°C. Later, a drop ofalkaline methylene blue dye is added to each tube and is examined under microscope. If less than 50% of the tachyzoites first take up stain and the cytoplasm remains colorless, the test is considered to be positive. lhe presence of 90- 100% tachyzoites, deeplyswollen andstainedwith blue color, shows Coccidia me test to be negative. It denotes the absence of Toxoplasma antibodies. The highest dilution of the serum, which inhibits staining up to 50%, is the titer. Limitation: The test is reported to give false-positive reaction in Sarcocystis, Tr ichomonas vaginalis and Trypanosoma lewisi infections. It cannot differentiate between recent and past infection. MolecularMethods Deoxyribonucleic acid (DNA) hybridization techniques and polymerase chain reaction (PCR) are increasingly used to detect Toxoplasma from different tissues and body fluids. • B,gene ofT. gondii can bedetected by PCRofthe amniotic fluid in case of congenital toxoplasmosis. Imaging Magnetic resonance imaging (MRl) and compu ted tomography (CT) scan are used to diagnose toxoplasmosis with central nervous system involvement. • Ultrasonography (USG) of the fetus in utero at 20-24 weeks of pregnancy is useful for diagnosis of congenital toxoplasmosis. Treatment Congenital Toxoplasmosis eonates with congenital infection are treated the oral pyrimethamine (1 mg/kg) daily and sulfadiazine (100 mg/ kg) with folinic acid for 1year. Systemic corticosteroid may be added to reduce chorioretinitis. lmmunocompetent Patients Immunologically competent adults and older children, who have only lymphadenopathy, do not require specific therapy unless they have persistent severe symptoms. • Patients with ocular toxoplasmosis are treated for 1 month with pyrimethamine plus either sulfadiazine or clindamycin (600 mg QID). • Folinic acid should be administered concomitantly to avoid marrow suppressive effect of pyrimethamine. lmmunocompromised Patients Acquired immunodeficiency syndrome patients who are seropositive for T. gondii and have a CD4€ T-lymphocyte count below less than 100/ µL, should receive primary prophylaxis against Toxoplasma encephalitis. • Trimemoprim-sulfamemoxazole is the drug of choice. If trimeilioprim-sulfamemoxazole cannot be tolerated by patients, dapsone-pyrimethamine is the recommended alternative drug ofchoice.
- 117. Paniker'sTextbook of MedicalParasitology • Prophylaxis against Toxoplasma encephalitis should be discontinued in pa tients who have responded to antiretroviral therapy (ART) and whose cd4€ T-lymphocyte count has been above 200/µL for 3 months. Prophylaxis Individuals at risk, particularly pregnant women, children and immunocompromised persons should avoid contact with cat and its feces. • Proper cooking ofmeal. • Proper washing of hands and washing of vegetables and fruits before eating. • Blood or blood products from seropositivepersons should not be given and screening for T. gondii antibody should be done in all blood banks. Control It is difficult to control roxoplasmosis because of wide range of animal reservoirs. Currently, there is no effective vaccine available for humans. A genetically engineered vaccine is under development for use in cats. KEY POINTS OF TOXOPLASMA GOND/1 • Obligate intracellular parasite. • Exists in three forms: (1) trophozoite, (2) tissue cyst, and (3) oocyst. • Definitive host: Cat family (enteric cycle). • Intermediate host: Human (exoenteric cycle). • Human infection occurs by ingestion of food containing oocyst and tissue cyst. • Congenital infection can also occur. • Clinical features: Acute encephalopathy, fever, chorioretinitis, lymphadenopathy, myocarditis, hepatosplenomegaly. • Disseminated infection in AIDS. • Diagnosis: By demonstration of parasite in tissue specimen, ELISA, IFAT, Sabin-Feldman dye test, lgM-ISAGA. • Treatment: Congenitalinfection istreated with pyrimethamine and sulfadiazine. For primary prophylaxis. trimethoprim- sulfamethoxazole is the drug of choice. • ISOSPORA BELLI History and Distribution lsospora belliis a coccidian parasite which can cause diarrhea in humans. • It was originally described by Virchow in 1860 but it was named in 1923. • The name belli (from bellium meaning war) was given for its association with war, because several cases ofinfection with this parasite were seen among troops stationed in Middle East during the First World War. • It is more common in tropical and subtropical coumries. Morphology Oocysts of I. belli are elongated-ovoid and measure 25 µm x 15 µm. . . . Each oocyst is surrounded by a thin smooth two-layered cyst waU (Figs SA and B). Immature oocysts seen in the feces of patients contain two sporoblasts. The oocysts mature outside the body. On maturation, the sporoblasr convert into sporocysts. Each sporocyst contains four crescent-shaped sporozoites (Figs 6A and B). The sporulated oocyst containing eight sporowiles is the infective stage ofthe parasite. Sporoblast a Figs SA and B: Oocysts of lsospora be/Ii. (A ) Immature cyst: and (B) Mature cyst m Sporocyst Figs 6A and B: Oocysts of lsospora be/Ii. (A) Oocyst showing two sporoblasts; and (B) Mature oocyst with two sporocysts containing sporozoites
- 118. Life Cycle I. bellicompletes its life cycle in one host. . . • • . Man gets infection by ingestion of food and water contaminated with sporulated oocyst. When a sporulated oocyst is swallowed, eight sporozoites are released from the two sporocysts in the small intestine and invade the intestinal epithelial cells. In the epithelium, the sporozoites transform into trophozoites, which multiply asexually (schizogony) to produce a number of (merowites). tje merozoites invade adjacent epithelial cells to repeat asexual cycle. Some of the trophozoites undergo sexual cycle (gametogony) in the cytoplasm of enterocytes and transform into macrogametocyles and microgametocytes. After fertilization, a zygoteis formed, which secretes a cyst wall and develops into an immatureoocyst. These immature oocysts are excreted with feces and mature in the soil. Incubation period: 1- 4 days. Clinical Features Infection is usually asymptomatic. • Clinical illness includes abdominal discomfort, mild fever, diarrhea and malabsorption. • The diarrhea is usually watery and does not contain blood or pus and is self-limiting. However, protracted diarrhea, lasting for several years can be seen in immunocompromised persons, particularly in the HIV infected. Laboratory Diagnosis Stool Examination Indirect evidence: • High fecal fat content. • Presence of fatty acid crystals in stool. • Presence ofCharcot-Leyden crystals in stool. Direct evidence: It may be difficult to demonstrate the transparent oocyst in saline preparation of stool. • Stool concentration techniques may be required when direct wet mount ofstools are negative. • the staining techniques used are modified Ziehl-Neelsen (ZN) stain or Kinyoun acid-fast staining ofstool smear. In these methods, pink-colored acid-fast large oocyst (>25 µm) can be demonstrated. the stool smear can also be stained by auramine-rhodaminc and Giemsa stains. Duodenal Aspirates After repeatedly negative stool examinations, duodenal aspirate examination or enterotest can be performed to demonstrate oocyst. Coccidia Intestinal Biopsy Upper gastrointestinal endoscopy may provide biopsy specimens for demonstration of oocysts. Others Eosinophilia, which is generally not seen with other enteric protozoan infections, is detectable in case of isosporiasis. Treatment • o treaunent is indicated in self-limiting infection in immunocompetenc persons. Immunodeficient patients with diarrhea and excreting oocysts in the feces should be treated with cotrimoxazole (trimethoprim-suIfamethoxazole) in a dose oftwo tablets, four times a day for 10 days followed by two tablets two times a day for 3 weeks. • For patients intolerant to sulfonamides, pyrimethamine 50-75 mg/day is given. Relapses can occur in persons with AIDS and necessitate maintenance therapy with cotrimoxazole one tablet thrice a week. • CRYPTOSPORIDIUM PARVUM History and Distribution Cryptosporidia were first observed in the gastric mucosa! crypts oflaboratory mice byTyzzer in 1907. • Its importance as a pathogen causing diarrhea in animals was recognized in 1971 and the first case of human infection was reported in 1976. • Cryptosporidium has assumed great importance as a frequent cause of intractable diarrhea, in AIDS patients and immunocompromised subjects. • It is worldwide in distribution. • Two species of Cryptosporidium, C. hominis and C. parvum mostly cause human infections. Habitat C. parvum inhabits the small intestine. It may also be found in stomach, appendix, colon, rectum and pulmonary tree. Morphology The infective form of Lhe parasite is oocyst. • The oocyst is spherical or oval and measures about 5 µm in diameter. • Oocyst does not stain with iodine and is acid-fast. • TI1e wall of the oocysts is thick, but in 20%cases, wall may be thin. These thin-walled oocysts are responsible for autoinfeclion.
- 119. Paniker'sTextbook ofMedical Parasitology Figs7Aand B: Oocysts of Cryptosporidium parvum. (A) Thick-walled oocyst; and (B) Thin-walled oocyst • Both thin-walled and thjck-walled oocyst contain four crescent-shaped sporozoites (Figs 7A and B). • Oocyst can remain viable in the environment for long periods, as it is very hard and resistant to most disinfectants and temperature up to 60°C. • Itcan survivechlorinated water,butsequentialapplication of ozone and chlorine has been found effective in eliminating the cysts. Life Cycle The parasite complete its life cycle, sexual and asexual phases in a single host (monoxenous) (Fig. 8). Suitable Host Man. Reservoirs Man, cattle, cat and dog. Mode of Transmission Man acquires infection by: • Ingestion of food and water contaminated with feces containing oocysts. • Autoinfection. Infective Form Sporulated oocysts. • The oocyst contains four sporozoites, which are released in the intestine. • The sporozoites develop into trophozoites within parasitophorous vacuoles in the brush border of the intestine. • The trophozoites undergo asexual multip lication (schizogony) to produce type I meronts. • Eight merozoites are released from each type l meront. These merozoites enter adjacent epithelial cells to repeat schizogony or form type II meronts, which undergo gametogony. • Four merozoires are released from each type II meront. The merozoites enter host cell to form sexual stages- microgamele and macrogamete. • After fertilization, the zygote formed develops into the oocyst. The oocyst undergoes sporogony to form sporulated oocyst, which contains four sporozoites. Sporulated oocystsare released into the feces and transmit the infection from one person to another. Some of the oocysts have a thin wall surrounding four sporozoites and are called as thin-walled oocysts. These oocysts infect the same host and maintajn the cycle ofautoinfection. • The oocysts are fully mature on release and are infective immediately without further development {Fig. 8). Pathogenicity and Clinical Features • Humans get infection either by ingestion ofcontaminated food and water with feces or by direct contact with infected animals. Human-to-human transmission can also occur lncubation period is 2-14 days. • Clinical manifestations of C. parvum infection vary depending upon the immune status of the host. - Infection in healthy immunocompetent persons may be asym ptomatic or cause a self-limiting febrile illness, with watery diarrhea in conjunction with abdominal pain, nausea and weight loss. It can also cause childhood and traveler's diarrhea, as well as waterborne outbreaks (Box 2). - In immunocomprom ised hosts, especially those with AIDS and cd4€T-cell counts below 100/mcL, diarrhea can be chronic, persistent, and remarkably profuse, causing significant fluid and electrolyte depletion, weight loss, emaciation and abdominal pain. Stool volume may range from I L/day to 25 L/ day. Biliary tract involvement can manifest as right upper quadrant pain, sclerosing cholangitis, or cholecystitis. Laboratory Diagnosis Stool Examination Diagnosis is made by demonstration of the oocysts in feces. • Adirect wet mount reveaJs colorless, spherical oocyst of 4-5 µm, contajning large and small granules. • The oocysts are difficult to visualize in unstained wet preparations. • A number of staining techniques have been employed for demonstration of oocysts of C. parvum in th e stool specimen. Modified ZNstaining is the method of choice
- 120. Coccidia -- @Ji / --- - - - Autoinfection ('.(!R ff Sporozoites ~~" ~ released Thick-walled Thin-walled sporulated oocyst oocyst in / in feces feces 00 Thick-walled unsporulated oocyst ' Zygote Thin-walled unsporulated oocyst Mlcrogamete Macrogamete 'z~ q;- ~ v: 4 merozoites released .,,,,. Sporozoite attaches to brush border epithelium of ;mM""' Sporozoite develops into trophozoite o9e ~,~al'I ~--., ~cfo ~, ;,,,<:'a o<:< J Undergoes schlzogony (asexual cycle) in mucosal cells ,._rt> 0 #' ~ I::,~ ~ ;::-0 {f- ~q; -~§ t$' Ci ~ ~ Type I meront . ..,__.---D~~ relaa~d Type II meront Fig. 8: Life cycle of Cryptosporidium parvum Box 2: Parasites causing traveler's diarrhea • Cryptosporidlum parvum • Entamoeba histolyt/ca Giardia Iambi/a Cyc/ospora cayetanensis and by this method oocysts appear as red acid-fast spheres, against a blue background (Figs 9A and B).Yeast closely resembles oocysts of C. parvum in shape and size but can be differentiated by using acid-fast stain, as they are not acid-fast and appear blue in color. The staining can also be used for demonstration of oocysts in other specimens like sputum, bronchial washing, etc. • If oocysts, load is less and cannot be demonstrated even after examination of three wet mounts ofstool specimen, concentration techniques like Sheather's sugar floatation technique and zinc sulfate tloatation technique can be applied.
- 121. Paniker's Textbook ofMedical Parasitology Figs9A and B: Oocysts of Cryptosporidium parvum. (A) Acid-fast stain; and (B) Ziehl-Neelsen stain • Fluorescent staining with auramine-phenol or acridine orange has also been reported to be a useful technique. • Definitive identification can be made by indirect immunofluorescence microscopy using specific monoclonal antibody. Histopathological Examination Cryptosporidium can also be identified by light and electron microscopy at the apical surface of intestinal epithelium from biopsy specimen of the small bowel Uejunum being the preferred site). Serodiagnosis Antibody specific to C. parvum can be demonstrated within 2 months ofacute infection. • Anti-oocyst antibody persists for at least one year and can be demonstrated by ELISA or immunofluorescence. • An ELISA for detection of Cryptosporidium antigens in stools using monoclonal antibody has also been developed and is highly sensitive and specific. Molecular Diagnosis For seroepidemiological srudy, western blot technique is employed by using a 17 kDa and 27 kDa sporozoite antigen. • Polymerase chain reaction technique has also been applied to detect viable cysts. Treatment ochemotherapeuticagenteffectiveagainst Crypt.osporidium has been identified, although nitazoxanide (500 mg BO x 3 days) or paromomycin may be partially effective in few patients with AlOS. Improvement in immune status with ART can lead toamelioration ofcryptosporidiosis. Other treatment methods include supportive therapy with fluid, electrolytes and nutrient replacement. KEY POINTSOF CRYPTOSPOR/0/UM PARVUM • Sexual and asexual cycle in a single host. • Infective form: Sporulated oocyst in food and water. • Clinical features: Self-limited diarrhea with abdominal pain in healthy persons. Chronic persistent watery diarrhea in immunocompromised hosts. • Diagnosis: Demonstration of round oocyst in stool by direct microscopy, fluorescent microscopy and modified acid-fast stain. • Treatment: Supportive therapy with electrolytes and fluids and early ART in AIDS patients. • CYCLOSPORA CAYETANENSIS • It is a coccidian parasite. • It was first reported from Nepal, where it caused seasonal outbreaks ofprolonged diarrhea, with peak prevalence in the warm rainy months. Morphology The morphological form found in the feces is an oocyst. • The oocyst is a nonrefractile sphere, measuring 8- 10 µm in diameter. • It contains two sporocysts. • Each sporocyst contains two sporozoites. Hence, each sporulated oocyst contains four sporozoites. Life Cycle Oocyst shed in feces sporulares outside the host. • The sporulated oocysts are infectious to humans.
- 122. • Man acquiresinfection by ingestion of food and water contaminated with feces-containing oocysts. • Excystation of the sporocyst releases crcscentic sporozoites measuring 9 µm x 1.2 µm. • The sporozoites infect enterocytes in the small intestine. • The sporozoites develop into unsporulated oocysts, which are excreted in feces. Pathogenicity and Clinical Features Infection is through fecal-oral route by ingestion of contaminated water and vegetables. • Incubation period is of 1-7 days. • Histopathological examination of the enterocytes shows features of acute and chronic inflammation with blunting and atrophy ofvilli and hyperplasia ofcrypts. • It causes prolonged diarrhea with abdominal pain, low- grade fever and fatigue. • Like other coccidian parasites the infection is more severe in immunocompromised hosts, especiallywith AlOS. Diagnosis Stool Examination Diagnosis is by direct wet mount demonstration of oocysts in feces. • The oocysts can be stained by ZN stain. Oocysts of Cyclospora are acid-fast and stain red in color. • Under ultraviolet illumination, unstained oocysts of C. cayetanensis are autofluorescent. Histopathology Biopsy specimen from jejunum shows villous atrophy and blunting ofvilli along with other inflammatory changes. • The parasite can also be seen in small bowel biopsy material by electron microscopy. Treatment Cyclosporiasis is treated with cotrimoxazole (trimethoprim 160 mg/sulfamethoxazole 800 mg) twice daily for 7 days. HIV-infected patients may require long-term suppressive maintenance therapy. • BLASTOCYSTIS HOMINIS Blastocystis hominis was previously considered a yeast, but recently it has been reclassified as a protozoan (Fig. 10). Habitat It is a strict anaerobic protozoa found in large intestine of humans. Coccidia Fig. 1O: Blastocystis hominis Morphology B. hominis has three morphological forms: 1. Vacuolated form is usually seen in stool specimen. It measures 8 µm in diameter and is characterized by its large central vacuole, which pushes the cytoplasm and the nucleus to the periphery. It multiplies by binary fission. 2. Ameboidform is a polymorphous cell slightly larger than the vacuolated form occasionally seen in the feces. Ir multiplies by sporulation. 3. Granularf orm measures 10-60 µm in diameter and is seen exclusively in old cultures. Pathogenicity and Clinical Features The parhogenicity of B. hominis is doubtful. However, recent studies have shown the parasite to be associated with diarrhea. • Clinical manifestations include diarrhea, abdominal pain, nausea, vomiting, fever and chills. More than half of the patients suffering from infection with B. hominis has been found to be immunologically compromised. Diagnosis The condition is diagnosed by demonstration ofthe organism in stool smear stained by Giemsa or iron hematoxylin or trichrome stains. Treatment If diarrheal symptoms are prominent, either metronidazole (750 mg thrice a day for 10 days) or iodoquinol (650 mg thrice a day for 20 days) can be used.
- 123. Paniker's Textbook ofMedical Parasitology • SARCOCYSTIS Three species ofgenus Sarcocystis can infect humans: 1. S. hominis (transmitted through cattle) 2. S. suihominis (transmitted through pig) 3. S. Lindemanni. • Humans are the definitive host of S. hominis and S. suihominis and the intermediate host for S. lindemanni. • Sarcocyslis species produce cyst in the muscle of the intermediate hoses. These cysts, called sarcocysts, contain numerous merozoites (bradyzoiles) (Fig. 11). • When sarcocyst is eaten by the definitive host, the merozoites are released in the intestine, where they develop inro male and female gametes. • After fertilization, the zygote develops into an oocyst conraining two sporocysts, each having four sporozoites (Fig. 12). • These oocysts are shed in feces and are ingested by intermediate host. • ln the intermediate hosts, the sporozoites invade rhe bowel wall and reach the vascular endothelial walls, where they undergo schizogony producing merozoites (tachywites). • Thesespread to muscle fibers and develop into sarcocysts. • Cow is the intermediate host for S. hominis. Human infection is acquired by eating raw or undercooked beef. Oocysts are shed in human feces, which contaminate grass and fodder eaten bycows. Bradyzoites Fig. 11: Sarcocyst Fig. 12: Oocyst of Sarcocystis hominis • In the case of S. suihominis, the pig is the intermediate hosr and human infection is obtained through eating contaminated pork. Human infection with S. hominis and S. suihominis is related to food habits. • Humans are the intermediate host in S. lindemanni; the definitive hostofwhich is noryet known. It is believed that S. lindemanni maynot be a singlespecies but a group ofas yet unidentified species. Humans apparently get infected by ingestion ofoocysts. Sarcocysts develop in the human skeletal muscles and myocardium. Clinical Features • Intestinalsarwcystosisis usuallyasymptomatic. Patients may have nausea, abdominal pain and diarrhea. • Muscular sarcocystosis is also usually asymptomatic but maycause muscle pain, weakness, or myositis, depending on the size ofthe cyst. Laboratory Diagnosis Stool Examination Characteristically sporocysts or occasionally oocysts can be demonstrated infeces ofhuman beings. Species identification is not possible with microscopy. Muscular Sarcocystosis Diagnosis can be made by demonstration of sarcocysts in the skeletal muscle and cardiac muscle by biopsy or during autopsy. Treatment 1o specific treatment is available for sarcocystosis. Prophylaxis • By avoiding eating raw or w1dercooked beefor pork. • By avoidance of contamination of food and drink with feces ofcat, dog, or other carnivorous animals. REVIEW QUESTIONS 1. Describe the life cycle, clinical features and laboratory diagnosis ofToxoplasma gondii. 2. Discuss in brief life cycle of Cryptosporidium parvum. 3. Write short notes on: a. Congenital toxoplasmosis b. Cryptosporidium parvum c. Sabin-Feldman dye test d. Sarcocyst
- 124. MULTIPLE CHOICE QUESTIONS 1.Route oftransmission of Toxoplasma a. Blood b. Feces c. Urine d. None 2. Toxoplasma gondii lives inside the a. Lumen of small intestine b. Lumen of large intestine c. Reticuloendothelial cell and many other nucleated cell d. RBC 3. Oocyst of toxoplasma is found in a. Cat b. Dog c. Mosquito d. Cow 4. Toxoplasmosis in the fetus can be best confirmed by a. lgM antibodies in the mother b. lgM antibodies in the fetus c. lgG antibodies in the mother d. lgG antibodies in the fetus 5. Intermediate hosts of toxoplasmosis are a. Sheep b. Cattle c. Pigs d. All of the above 6. The following statements regarding congenital toxoplasmosis are correct except a. Most severe form of congenital infection occurs, ifit is acquired in 1st trimester b. Chorioretinitis and hydrocephalus are common manifestations in congenital infections c. Presence of Toxoplasma-specific lgM antibodies in an infant are suggestive of congenital infection d. Most severe form of congenital infection occur if it is acquired in 3rd trimester Coccidia 7. Frenkels' skin test is positive in a. Spinal cord compression b. Toxoplasmosis c. Pemphigus d. Pemphigoid 8. In humans, cryptosporidiosis presents as a. Meningitis b. Diarrhea c. Pneumonia d. Asymptomatic infection 9. Which stain demonstrates the oocyst of Cryptosporidium best a. Hematoxylin-eosin b. Gram's stain c. Kinyoun modified acid fast stain d. Modified trichrome stain 1O. All of the following cause diarrhea except a. Entamoeba histolytica b. Giardia lamblia c. Naegleria fowleri d. Cyc/ospora caytanensis 11. The oval oocyst of /sospora be/Iifound in human feces measures a. 1-3 µm x 5- 7 µm b. 3-5 µm x 8-10 µm c. 5-8 µm x 10- 15 µm d. 22- 33 µm x 10-15 µm 12. Stool in lsospora belfi infection may contain all except a. High fecal content b. Blood c. Fatty acid crystals d. Charcot-Leyden crystals Answer 1. a 8. b 2. C 9. C 3. a 10. C 4. b 11. d 5. d 12. b 6. d 7. b
- 125. CHAPTER 8 • INTRODUCTION Microsporidiaare classified under Phylum Microspora. They are minute, intracellular, Gram-positive, spore-forming protozoa. • Microsporidia are also classified based on their habitat and the infections caused by them (Table 1). • HISTORY AND DISTRIBUTION Microsporidia are of historical interest as they are the first protozoan parasite to have been successfully studied and controlled byLouis Pasteur in 1863,duringan investigation of silkworm disease epidemic in France. It was this experience, which led Pasteur to his epochal work on human and animal diseases that formed the foundation of microbiology. The Table 1: Classification of Microsporidia Species Habitat andinfectioncaused causative agent of the silkworm disease (pebrine) is Nosema bombycis, a microsporidian parasite. • Microsporidia had been known as animal parasite for long, but their role as human pad1ogens was recognized only in the mid 1980s with the spreading of acquired immunodeficiency syndrome (AlDS). • Som e nine genera and 13 species are associated with human disease, particularly in the human immunodeficiency virus (HIV) infected and other immunocompromised subjects. • MORPHOLOGY Microsporidia are unicellular, obligate intracellularparasite. • They reproduce in host cells by producing spores (sporogony). Genus Enterocytozoon E. bieneusi Small intestine epithelium (leading to diarrhea and wasting). Also found in biliary tract of patients with cholecystitis. Rarely spreads to respiratory epithelium Encephalitozoon E. intestinalis E.hellem E. cuniculi Small intestine epithelium (causing diarrhea and wasting). Also causes sinusit is, cholangitis and bronchiolitis Conjunctiva! and corneal epithelium (causing keratoconjunctivitis). Also causes sinusitis, respiratory tract disease and disseminated infection • Small intestine epithelium (causing diarrhea) • Corneal and conjunctivaI epithelium (causing keratoconjunctivitis). Rarely, may cause hepatitisand renal infection P/eistophora P. ronneafier Skeletal muscle (causing myositis) Brachia/a , 8. vesicularum • Skeletal muscle (causing myositis) • 8. conori • Muscles (smooth and cardiac) Trachipleistophora • T. hominis • Cornea and conjunctivaI epithelium (leading to keratoconjunctivitis). Also causes myositis • T. anthropophtheria , Brain Vittaforma V. corneae Corneal stroma (causing stromal keratitis) Nosema N. ocularum Corneal stroma (causing stromal keratitis) Microsporidium • M. ceylonensis Corneal stroma (causing stromal keratitis) • M. africanum
- 126. Polar sac- --- ~ +--=- Exospore-- -" Endospore Plasma membrane Fig. 1: Microsporidian spore Box 1: Acid-fast parasitic organisms • Microsporidia (spore) • Cyclospora cayetanensis (oocyst) • lsospora be/Ii (oocyst) • Cryptosporidium parvum (oocyst) • Spores are 2-4 µmin size and oval to cylindrical in shape, with a polar filament or tubule {Fig. 1). • The spores are the infective stage ofmicrosporidia and the only stage of life cycle capable ofexisting outside the host cell. The polar tubule is an extrusion mechanism for injecting infective spore contents into the host cell. • Spores are surround by thick double-layered cyst wall: Outer layer (exospore) is proteinaceous and electron-dense Inner layer (endospore) is ch itino us and electronlucent. • Spores are Gram-positive and acid-fast (Box l ). • LIFE CYCLE Infection in host is probably by ingestion or inhalation of spores. • In the duodenum, the spore with its nuclear material is injected through the polar tubule into the host cell (enterocyte). • Inside the cell, the microsporidia multiply by repeated binary fission (merogony) and produce large number of spores (sporogony). Microspora Box 2: Parasites causing opportunistic infections in immuno- compromised patients [Human immunodeficiency virus (HIV)- positive cases] • Microsporidia • C yclospora cayetanensis • lsospora be/Ii • Cryptosporidium parvum • Toxoplasma gondii • Strongyloides stercoralis • Entamoeba histolytica • During sporogony, a thick spore wall is formed that provides environmental protection to the cyst. The spores are then liberated free from the host cell and infect other cells. • CLINICAL FEATURES They can cause wide range ofopportunistic illness in patients with HIVand other immunocompromised diseases (Box 2). • In patients with AIDS, Enterocytozoon bieneusi and Encephalitozoon intestinalis lead to protracted and debilitating diarrhea in 10-40% of cases. E. intestinalis may also cause sinusitis, cholangitis and bronchiolitis. Infection with Pleistophora can lead to myositis and E. he/Lem can cause superficial keratoconjunctivitis, sinusitis, respiratory disease and disseminated infection. Stromal keratitis associated with trauma has been reported in infections with Nosema, Vittaforma and Microsporidium in imrmmocompetent patients. • LABORATORY DIAGNOSIS Microscopy Diagnosis of microsporidiosis is made by demonstration of the spores in stool, urine, cerebrospinal fluid (CSF), or small intestine biopsy specimen. • The spores can be stained with Gram's stain, periodic acid-Schiff (PAS) stain, or modified trichrome stain. Note: Spores of microsporidia stain poorly with hematoxylin and eosin stain. Although intracellular spores can be visualized by light microscopy, electron microscopy is the gold standard. • Iden tification of species and genera of microsporidia is based on electron microscopy of spore morphology. • Direct fluorescent method using monoclonal antibody is also used for detection of microsporidia in clinical samples.
- 127. Paniker'sTextbook ofMedical Parasitology CellCulture Microsporidia spores can be cultured in monkey and rabbit kidney cells and human fetal lung fibroblast. Molecular Diagnosis Microsporidial deoxyribonucleic acid (DNA) can be amplified an d detected by polymerase chain reaction (PCR). • TREATMENT There is no specific and effective drug for microsporidia. • Intestinal microsporidia may be treated wi th m eu·onidazole and albendazole. • For superficial keratoconjunctivitis, topical therapy with fumagillin suspension can be used. • PROPHYLAXIS Improved personal hygi ene and sanitation, especially in immunocompromised persons can prevent microsporidia. KEY POINTS OF MICROSPORIDIA • Microsporidia are intracellular spore-forming protozoa, which belong to Phylum Microspora. • Spores of microsporidia are oval or cylindrical in shape with polar filaments or tubules. • Mode of infection: By ingestion or inhalation of spores. • Reproduction: Microsporidia multiply by both merogony and sporogony. • Clinical features: Protracted and debilitating diarrhea and disseminated infection in eyes, muscles and lungs. • Diagnosis: By demonstration of spores in stool, urine and CSF by Gram·s, PAS, or modified trichrome stains. Serological diagnosis includes direct fluorescent antibody test. PCR is also very useful. Electron microscopy is useful in species in identification of microsporidia. • Treatment: There is no specific and effective treatment. Intestinal microsporidia can be treated with metronidazole and albendazole. Topical therapy with fumagillin suspension is used for superficial keratoconjunctivitis. REVIEW QUESTIONS 1. Describe briefly the laboratory diagnosisofMicrosporidia. 2. Write short note on the morphology ofMicrosporidia species. MULTIPLE CHOICE QUESTIONS 1. All are true about Microsporidia except a. First protozoan parasite studied by Louis Pasteur b. Causative agent of silk worm disease c. Extracellularspore-forming protozoa d. Cause infection in immunocompromised subjects 2. Laboratory diagnosis of Microsporidia can be done by all except a. Modified trichrome stain b. Hematoxylin and eosin-stain c. Direct fluorescent antibody d. Electron microscopy 3. Enterocytozoon bieneusi preferentially infects a. Brain b. Conjunctiva c. Kidneys d. Small intestine 4. Microsporidial keratoconjunctivitis is commonly caused by a. Enrerocytozoon bieneusi b. Vittaforma c. Encephalitozoon hellem d. Encepha/itozoon intestinalis Answer 1. C 2. b 3. d 4. C
- 128. CHAPTER 9 • INTRODUCTION Balantidiumcoli belongs to Lhe Phylum Ciliophora and Family Balantiididae. • It is the only ciliate protozoan parasite of humans. • It is the largest protozoan parasite ofhumans. • Largest protozoan parasite residing in the large intestine ofman: Balantidium coli. • HISTORY AND DISTRIBUTION lt was first described by Malmsten in 1857, in the feces of dysenteric patients. • It is presentworldwide, butthe prevalence ofthe infection is very low. • lhe most endemic area is 1ew Guinea, where there is a close association between man and pigs. • HABITAT 8. coli resides in the large lntestlneofman, pigs and monkeys. • MORPHOLOGY B. coli occurs in two stages: (1) trophozoite and (2) cyst (Figs IA and B). Trophozoite The trophozoite lives in the large intestine, feeding on cell debris, bacteria, starch grains and other particles. • . . . The trophozoite is actively motile and is invasive stage of the parasite found in dysenteric stool. ft is a large ovoidcell, about 60- 70 µmin length and 40- 50 µm in breadth. Very large cells, measuring up to 200 µm are sometimes seen. The cell is enclosed within a delicate pellicle showing longitudinal striations. The motility of trophozoite is due to the presence of short delicate cilia over the entire surface of the body. ,,,,_,....,..--Cytostome 1=--',--- Cytopharynx .i.='--'o--Food vacuole Contractile--+~ ~ .I vacuole --- - .;.--Micronucleus--lL--- ----; t--J..--Macronucleus~~-"'111--"II h---Cilia ~~---Cytopyge Figs 1A and B: Morphology of Balantidium coli. (A) Trophozoites: and (B) Cyst • Its anterior end is narrow and posterior end is broad. • At the anterior end, there is a groove (peristome) leading to the moulh (cytostome), and a short funnel-shaped gullet (cytopharynx). • Posteriorly, there is a small anal pore (cytopyge). • The cilia around the mouth are larger (adoral cilia). • The cell has two nuclei: (1) a large kidney-shaped macronucleus, and (2) lying in its concavity a small micronucleus. • The cytoplasm has one or two contractile vacuoles and several food vacuoles. Cyst The cyst is spherical in shape and measures 40-60 µm in diameter. It issurrounded bya thick and transparentdouble-layered wall. • The cytoplasm is granular. Macronucleus, micronucleus and vacuoles are also present in the cyst. • 1he cyst is the infective stage of8. coli. It is found in chronic cases and carriers.
- 129. Paniker'sTextbook ofMedical Parasitology •LIFE CYCLE B. coli passes its life cycle in one host only (monoxenous). Natural Host Pig. Accidental Host Man. Reservoirs Pig, monkey and rat. Infective Form Cyst. Mode ofTransmission • Balantidiasis is a zoonosis. Human beings acquire infection by ingestion of food and water contaminated with feces containing the cysts of 8. coli. Reservoir:Pig • Infection is acquiredfrom pigs and otheranimal reservoirs or from human carriers. Once the cyst is ingested, excystation occurs in the small intestine (Fig. 2). • From each cyst, a single trophozoite is produced which migrates to large intestine. • Liberated trophozoites multiply in the large intestine by transverse binaryfission. Sexual union by conjugation also occurs infrequently, during which reciprocal exchange of nuclear material takes place between two trophozoites enclosed within a single cyst wall. • Encystation occurs as the trophozoite passes down the colon or in the evacuated stool. In this process, the cell rounds up and secretes a tough cyst wall around it. The cysts remain viable in feces for a day or 2 and may contaminate food and water, thus it is transmitted to other human or animals. • PATHOGENESIS In a healthy individual, B. coli lives as lumen commensal and is asymptomatic. • Clinicaldisease occurs onlywhen the resistance ofhost is lowered by predisposing factors such as malnourishment, Fig. 2: Life cycle of Balantidium coli
- 130. alcoh olism, achlorhydria,concurrent infection by Trichuris trichiura, or any bacterial infection. • Clinical disease results when the trophozoites burrow into the intestinal mucosa, set up colonies and initiate inflammatory reaction. This leads to mucosal ulcers and submucosal abscesses, resembling lesions in amebiasis. • Unlike E. histolytica, B. coli does not invade liver or any other extraintestinal sites. • CLINICAL FEATURES Most infections are asymptomaric. • Symptomatic disease or balantidiasis resembles amebiasis causing diarrhea or frank dysentery with abdominal colic, tenesmus, nausea and vomiting. • Balantidium ulcers may be secondarily infected by bacteria. • Occasionally, intesti nal perforation peritonitis and even death may occur. • Rarely, there may be involvement of genital and urinary tracts. • In chronicbalantictiasis, patientshavediarrhea alternating with constipation. • LABORATORY DIAGNOSIS Stool Examination Diagnosis of B. coli infection is established by demonstration oftrophozoites and cysts in feces. • Motile trophozoites occur in diarrheic feces and cysts are found in formed stools. • The trophozoites can be easily recognized by their large size, macronucleus and rapid-revolving motility. • The cysts can also be recognized in the formed stools by their round shape and presence oflarge macronucleus. Biopsy When stool examination is negative, biopsy specimens and scrapings from intestinal ulcers can be examined for presence of trophozoites and cysts. Culture B. coli can also be cultured in vitro in Locke's egg albumin medium or IH polyxenic medium such as Entamoeba histolytica, but it is rarely necessa1 y (Box 1). • TREATMENT Tetracycline is the drug of choice a nd is given 500 mg, four times daily for 10 days. Alternatively, doxycycline can be Balantidium Coli Box 1: Parasites which can be cultured in laboratory • Balantidium coli • Entamoeba hlstolytica • Acanthamoeba spp. • Giardia lamblia • Trichomonas vagina/is , Trypanosoma spp. • Leishman/a spp. given. Metronidazole and ni troimidazole have also been reported to be useful in some cases. • PROPHYLAXIS • Avoidance of contamination of food a nd water with human or animal feces. Prevention of human-pig contact. • Treatment of infected pigs. • Treatment of individuals shedding 8. coli cysts. KEY POINTS OF BALANTIDIUM COLI • It is the only ciliate parasite of humans. • Largest protozoan parasite residing in large intestine. • It occurs in two stages: (1) trophozoite and (2) cyst. • Trophozoite is oval-shaped with a slightly pointed anterior end with a groove, peristome leading to the mouth, cytostome. Rounded posterior end has a small anal pore, cytopyge and has a large kidney-shaped macronucleus and small micronucleus. • Cyst: It is the infective stage of the parasite. • Mode of infection: Infection is acquired from pigs and other animals by ingestion of cysts in contaminated food and drink. • Infection leads to mucosa! ulcers and submucosal abscess in intestine. • Clinical features: Most infections are asymptomatic. In mild infections, it causes diarrhea, abdominal colic, tenesmus, nausea and vomiting. • Diagnosis: Based on demonstration of trophozoites and cysts in feces and examination of biopsy specimens and scrapings from intestinal ulcers. • Treatment: Tetracycline is the drug of choice. • Prophylaxis: Avoiding contamination of food and water and treatment of infected pigs and persons. REVIEW QUESTIONS 1. Write short notes on the morphology of Balantidium coli along with suitable illustration. 2. Discuss briefly the life cycle and laboratory diagnosis of Balantidium coli.
- 131. Paniker'sTextbook of MedicalParasitology MULTIPLE CHOICE QUESTIONS 1. Largest protozoa! parasite is a. Entamoeba histolytica b. Trichomonas vagina/is c. Leishmania donovani d. Balantidium coli 2. The infective form of Balantidium coli is a. Tachyzoites b. Cyst c. Sporozoite d. Trophozoite 3. Which ofthe following acts as the main reservoir of Balantidium coli infection a. Man b. Monkey c. Pig d.Cow 4. Drug of choice for treating balantidiasis a. Doxycycline b. Tetracycline c. Metronidazole d. Pentamidine Answer 1. d 2. b 3. C 4. b
- 132. CHAPTER 10 • INTRODUCTION Thehelminth ic parasites are multicellular (metazoa) bilaterally symmetrical animals having three germ layers (triploblaslic melazoa) and belong to the kingdom Metazoa. • The term helminth (Greek helmins-worm) originally referred to intestinal worms, but now comprises many other worms, including tissue parasites as well as many free-living species. Helminths, which occur as parasite in humans belong to two phyla (Table 1): 1. Phylum Platyhelminthes (flatworms): Tt includes two classes: i. Class: Cestoda (tapeworms) ii. Class: Trematoda (flukes or digeneans) 2. Phylum Nemathelminthes: lt includes class Nematoda and two subclasses: i. Subclass: Adenophorea (Aphasmidia) ii. Subclass: Secernenrea (Phasmidia). • The differences between cestodes, trematodes and nematodes have been summarized in Table 2. Table 2: D ifferences between cestodes, trematodes and nematodes • PHYLUM PLATYHELMINTHES The Platyhelminthes are tape-like, dorsoventrally flattened worms. • They either lack alimentary canal (as in cestodes) or their alimentary canal is incomplete, lacking an anus (as in trematodes). Table 1: General features of helminths Nematohelminthes (Nematode) Helminths Platyhelminthes (cestode. trematode) • Body Elongated, cylindrical, Dorsoventrally flated leaf like unsegmented or tape like segmented or unsegmented • Sex Separate (diecious) • Body cavity Present • Alimentary Complete canal Mostly hermaphrodite except schistosomes (diecious) Absent Incomplete or absent Cestodes Trematodes Nematodes Shape Head end Alimentarycanal Body cavity Sex Lifecycle Tape-like, segmented Suckers present; some have attached hooks Absent Absent, but inside is filled with spongy undifferentiated mesenchymatous cells, in the midst ofwhich lie the viscera Not separate: Hermaphrodite (monoecious) Requires two host except Hymenolepis (one host) and Diphyllobothrium (three host) Leaf-like unsegmented Suckers are present but no hooks Present but incomplete, no anus Same as cestodes Not separate: Hermaphrodite except Schistosoma Requires three host except schistosomes (two host) Elongated, cylindrical, unsegmented Hooks and sucker absent. Well-developed buccal capsule with teeth or cutting plates seen in some species Complete w ith anus Present and known as pseudocele. Viscera remains suspended in the pseudocele Separate (diecious) Requires one host except filarial worms (two host) and Dracunculus (two host)
- 133. Paniker'sTextbook ofMedical Parasitology •Body cavity is absent, viscera is suspended in gelatinous matrix. • They are mosdy hermaphrodites (monoecious). • Phylwn platyhelminthes includes two classes: 1. Class:Cestoda 2. Class: Trematoda. Class Cestoda Cestodes have tape-like, dorsoventrally flattened, segmented bodies. • They do not possess an alimentary system. • The head carries suckers and some also have hooks. • They possess scolex, neck and proglottids. • They are monoecious and body cavity is absent. • They are oviparous. ClassTrematoda Trematodes have flat or fleshy, leaf-like unsegmented bodies. • The alimentary canal is present but is incomplete, i.e. without an anus. • Theypossess suckers but no hooks. • The sexes are separate in the schistosomes, while the other flukes are hermaphroditic. • They are oviparous. • PHYLUM NEMATHELMINTHES (NEMATODA) ematodes are elongated, cylindrical worms with an unsegmented body. • • • • • They possess a relatively well-developed complete alimentary canal, with an anus. Body cavity is present. The head does not have suckers or hooks, but may have a buccal capsule with teeth or cutting plates. The sexes are separate (diecious). Theyare either oviparous or larviparous. • IMPORTANT FEATURES OF HELMINTHS Adult Worms Helminths have an outer protective covering, the cuticle or Integument, which may be tough and armed with spines or hooks. The cuticle of live helminths is resistant to intestinal digestion. • The mouth may be provided with teeth or cutting plates. Many helminths possess suckers or hooks for attachment to host tissues. • They do not possess organs of locomotion, but in some species the suckers assist in movement. • Locomotion is generally by muscular contraction and relaxation. • Many helmjnths have a primitive nervous system. • The excretory system is better developed. • The greatest development is seen in the reproductive system. Helminths may be monoecious (with functioning male and female sex organs in the same individual) or diecious (the two sexes, male and female, separate). ln the hermaphroditic helminths, both male and female reproductive systems are present in the same worm and self-fertiljzation as well as cross-fertilization takes place (e.g. Taenia solium). In the diecious species, males and females are separate, the male being smaller than the female (e.g. Ascaris lumbricoides). Rarely, the female is parthenogenic, being able to produce fertile eggs or larvae without matingwith males (e.g. Strongyloides). Eggs The eggs or larvae are produced in enormous nwnbers-as many as 200,000 or more per female per day. Various helminths have distinct morphology of eggs, whlch can be used to differentiate the helminths (discussed in the respective chapters). Larval Forms There are various larval forms ofhelminths found in man and other hosts. These forms are as follows: • Cestodes: Th e various larval forms are cysticercus, coenurus, coracidiwn, cysticercoid, procercoid, hydatid cyst and plerocercoid forms. • Trematodes: The various larval forms are miracidium, cercaria, redia, metacercaria and sporocyst • Nematodes: The various larval forms are microfilaria, filariform larva and rhabditiform larva. Multiplication Helminths differ from protozoans in their inability to multiply in the body of the host. Protozoans multiply in the infected person, so that disease could result from a single infection. But helminths, apart from very rare exceptions, do not multiply in the human body, therefore, a single infection does not generally leads to disease. Heavy worm load follows multiple infections. Sometimes, multiplication occurs within larval forms in Platyhelminthes. Life Cycle • Cestodes: They complete their life cycle in two different hosts, except Hymenolepis nana, which completes its life cycle in a single host and Diphyllobothrium latum which completes its life cycle in three hoses. • Trematodes: They complete their life cycle in one de finitive host (man) and two intermediate hosts.
- 134. Fresh water snailor mollusc act as first intermediate host and fish or crab act as second intermediate host except schistosomes which require two hosts: (I) one definitive host (man) and (2) other intermediate host (snail). • Nematodes: Nematodes require only one host to complete their life cycle except filarial nematodes and Dracunculus medinensis, which complete their Life cycle in two hosts. Pathogenecity: The pathological lesions in helminthic diseases are due to direct damage caused by helminths or due to indirect damage by host response, for example allergic response of the host to the helminths. Many helmmths cause malnutrition of the host. Malnutrition interferes with antibody production. • ZOOLOGICAL CLASSIFICATION OF HELMINTHS Phylum Platyhelminthes Class Trematoda • B1 ood fl ukes (sexes separate, infection by cercarial penetration). Family: Schistosomatidae (schistosomes) • Hermaphroditic flukes (bisexual, infection by ingestion ofcercariae). - family Fasciolidae (large flukes, cercariae encyst on aquatic vegetation) • Genus: Fasciola, Fasciolopsis - Family: Paramphistomatidae (large ventral sucker posteriorly) • Genus: Gastrodiscoides Family: Echinostomatidae (coUar of spines behind oral sucker, cercariae encyst in mollusc or fish) • Genus: Echinostoma - Family: Triglotrematidae (testes side-by-side behind ovary, cercariae encyst in Crustacea) • Genus: Paragonimus Family: Opisthorchidae (testes in tandem behind ovary, cercariae encyst in fish) • Genus: Clonorchis, Opisthorchis Family: Dicrocoelida (testes in front of ovary, cercariae encyst in insects) • Genus: Dicrocoelium Family: Heterophyidae (minute flukes, cercarial encyst in fish) • Genus: Helerophyes, Metagonimus. Helminths:General Features Class Cestoda Order: Pseudophyllidea (scolex has grooves) - Genus: Diphyllobothrium Order: Cyclophyllidea (scolex has suckers) - Family: Taeniidae (proglottid longer than broad, numerous testes, one genital pore, larva in vertebrates) • Genus: Taenia, Multiceps, Echinococcus - Family: Hymenolepididae (transverse proglottids, one genital pore, larva in insects) • Genus: Hymenolepis - Family: Dilepidiidae (two genital pores) • Genus: Dipylidium. Phylum Nemathelminthes It includes class Nematoda which is further divided into: • Subclass: Adenophorea or Aphasmidia (no phasmids, no caudal papillae in male) Subclass: Secernentea or Phasmidia (phasmids present, numerous caudal papilJae). Detailed classification of class Nematodes is given in Chapter 13. KEY POINTS OF HELMINTHS • Helminths are multicellular and bilateral symmetrical parasite. • Helminths are divided into two broad phyla-the cylindrical worms belonging to phylum Nematohelminthes (class Nematoda) and flat tape or leaf like helminths belonging to phylum platyhelminthes (class Cestoda and Trematoda). • Sexes are separate in Nematodes. Cestodes and trematodes are hermaphrodites. • Trematodes are cestodes require two or three hosts. Nematodes requires one host except filarial worms which require two host. REVIEW QUESTIONS 1. Short notes on: a. Generalfeatures ofhelminths b. Phylum Nematoda 2. Differentiate between: a. Trematodes and nematodes b. Cestodes and nematodes
- 135. Paniker's Textbook ofMedical Parasitology MULTIPLE CHOICE QUESTIONS 1. Digestive tract is completely absent in a. Trematodes b. Cestodes c. Nematodes d. All of the above 2. Sexesare always separate in a. Cestodes b. Trematodes c. Nematodes d. None of the above 3. Nematodes are different iated from other worms by the following except a. Absent fragmentation b. Flat or fleshy leaf-like worm c. Separate sexes d. Cylindrical body 4. Which ofthe following worm requires two intermediate host a. Taenia saginata b. Oiphyllobothrium /arum c. Hymenolepis nana d. Echinococcus granulosus 5. Whichof thefollowing statementistrue in respectto trematodes a. Dorsoventrally flattened b. Intermediate host is snail c. Hermaphrodite except schistosomes d. All of the above Answer 1. b 2. C 3. b 4. b 5. d
- 136. CHAPTER 11 l~- • INTRODUCTION Cestodes(Greek kestos-girdle or ribbon) are multi- segm ented, dorsoventrally flattened tape-like worms whose sizes vary from a few millimeters to several meters. The adult worms are found in the small intestine of humans. • CLASSIFICATION OF CESTODES Systemic Classification Cestodes belong to Phylum Platyhelminthes and class Cestoidea. The class Cestoidea includes two orders: 1. Pseudophyllidea 2. Cyclophyllidea For detailed classification see Table l. Classification ofCestodes Based on the Form of Parasite Important to Man the detailed classification is given in Table 2. • TAPEWORMS: GENERAL CHARACTERISTICS Adult Worms • The adult worm consists of three parts: Head (scolex) eek - Trunk (strobila) {Figs IA to D). Head (Seo/ex) It is the organ of attachment to the intestinal m ucosa of the definitive host, human or animal (Figs lA to D). • In parasites of the order Cyclophyllidea, the scolex possesses four suckers (or acetabula). In some Cyclophyllidea like Taenia solium, scolex has an apical Table 1: Classification of medically important Cestodes order Family_ Genus Pseudophyllidea Diphyllobothriidae • Diphyllobothrium • Spirometra Cyclophyllidea Taeniidae • Taenia • Echinococcus Hymenolepididae Hymeno/epis Dipylidiidae Dipylidium C I ' A - ) D - - - B - - - - - ) - - - Figs 1A to D: Tapeworm. (A) Scolex or head; (B) Neck, leading to the region of growth below, showing immature segments; (C) Mature segments; and (D) Gravid segments filled with eggs protrusion called as the rostellum. The rostellum may or may not be armed with hooks. • In parasites of the order Pseudophyllidea, the scolex does not possess suckers but possesses a pair of longitudinal grooves called as bothria, by which it attaches to the intestine of the host.
- 137. Paniker'sTextbook of MedicalParasitology Table 2: Classification of Cestodes based on the form of parasite important to man order Pseudophyllidea Cyclophyllidea Heads Proglottld Adultwonn-,,in humanintestine Diphyllobothrium latum the fish tapeworm • Taenia saginata, the beeftapeworm • Taenia solium, the pork tapeworm • Hymenolepis nana, the dwarf tapeworm • Hymenolepis diminuta, the rat tapeworm (rare) • Dipylidium caninum, the double-pored dog tapeworm (rare) Taenia solium 4 suckers 2 rows or hooks Longer than broad 7-12 uterine branches on each side Taenia saginata 4 suckers No hooks Longer than broad 15-30 uterine branches on each side Larvalstageseen In humans • Spirometra mansoni • Spirometra theileri • Spirometra erinacei (larval stage causing sparganosis) • Taenia solium, the pork tapeworm (larval form can cause cysticercus cellulosae) • Echinococcus granulosus, the dog tapeworm (larval form causes hydatid disease in man) • Echinococcus mulrlloculoris (larval stage causes alveolar or multilocular hydatid disease) • Mulriceps mulriceps and other species (larval stage may cause coenurosis in man) Hymenolepis nana 4 suckers single row of 20- 30 hooks Broader than long Hymenolepis diminuta 4 suckers No hooks Broader than long Diphyllobothrium latum 2 Suctorial grooves or bothria, no suckers, No hooks Broader than long Uterus coiled Echinococcus granulosus 4 suckers 2 rows of hooks Longer than broad Fig. 2: Differences between heads and proglottids of various Cestodes Neck ILis the part, immediately behind the head and is the region of growth from where the segments ofthe body (proglottids) are being generated continuously. Trunk (Strobila) The trunk also called as strobila is composed of a chain of proglottids or segments (Figs IA to D). • The proglonids near the neck, are the young immature segments, behind them are the mature segments, and at the hind end, are the gravid segments. • Tapeworms are hermaphrodites (monoecious) and every mature segment contains both male and female sex organs. In the immature segments, the reproductive organs are notwell-developed.Theyare well-developed in the mature segments. The gravid segments are completely occupied by the uterus filled with eggs. • Tapeworm do not have a body cavity or alime ntary canal. • Rudimentary excretory and nervous systems are present. 'The differences between heads and proglottids ofvarious Cestodes have been illustrated in Figure 2. Eggs The eggs ofCyclophyllidea and Pseudophyllidea are different from each other (Table 3).
- 138. Cestodes:Tapeworms Table 3: Differencesbetween eggs of Orders Cyclophyllidea and • PSEUDOPHYLLIDEAN TAPEWORMS Pseudophyllidea Cyclophyllidean egg • Covered by two layers: (1) egg shell and (2) embryophore • Spherical • Embryonated from the beginning • Eggsare not operculated and the embryo is not ciliated Pseudophyllidean egg • Covered by one layer: egg shell • Ovoid in shape • Freshly-passed eggs in feces are unembryonated • E ggs are operculated and the embryo is ciliated • The embryo inside the egg is called the oncosphere (meaning hooked ball) because it is spherical and has hooklets. • Oncospheres of human tapeworms typically have three pairs of hooklets and so, are called hexacanth (meaning six-hooked) embryos. Life Cycle Cestodes complete their life cycle in two hosts: (1) definitive host and (2) intermediate host. • Humans are the definitive host for most tapeworms, which cause human infection. An important exception is the dog tapeworm, Echinococcus granulosus, for which dog is the definitive host and man is the intermediate host. In Taenia solium, man is ordinarily the definitive host, but its larval stages can also develop in the human body. Cestodes complete their life cycle in two different hosts. Exceptions are: - 1/ymenolepis that requires only one host, man and Diphyllobothrium that requires three hosts, (I) definitive host: man; (2) first intermediate host: Cyclops; and (3) second intermediate host: fish. • Clinical disease can be caused by the adult worm or the larval form. In general, adult worm causes only minimal disturbance, while the larvae can produce serious illness, particularlywhen they lodge in critical areas like the brain or the eyes. • Pseudophyllidean tapeworms have a central unbranched convoluted uterus, which opens through a pore, possess ventrally situated genital pores, and produce operculated eggs that give rise to ciliated larvae. In Cyclophyllidean tapeworms, the uterus is branched and does not have an opening. They have lateral genital pores and produce nonoperculated eggs that yield larvae, which are not ciliated. Their larvae are called "bladder worms" and occur in four varieties: (1) cysticercus, (2) cysticercoid, (3) coenurus and (4) Echinococcus. Diphyllobothrium Latum Common Name Fish tapeworm/ Broad tapeworm. History and Distribution The head of the worm was found by Bonnet in 1777, and its life cycle was worked out by Janicki and Rosen in 1917. Diphyllobothriasis (infection with Diphyllobothrium) occurs in Central and orthern Europe, particularly in the Scandinavian countries. It is also found in Siberia, Japan, orth America and Central Africa. In countries like India, where fish is eaten only after cooking, the infection does not occur. Longest cestode infecting man: Diphyllobothrium latum • Smallest cestode infecting man: Hymenolepis nana. Habitat The adult worm is found in the small intestine, usually in the ileum, where it lies folded in several loops with the scolex embedded in the mucosa. Morphology Adull worm: It is ivory-colored and very long, measuring up to 10 meters or more. It is the largest tapeworm inhabiting the small intestine of man. • As in all cestodes, the adult worm has three parts: (1) scolex, (2) neck and (3) strobila. Scolex(head) is spatulateor spoon-shaped, about 2-3 mm long and l mm broad. It carries two slit-like longitudinal sucking grooves (bothria), one dorsal and the other ventral. the scolex lacks suckers and hooks ( Fig. 3A). • eek is thin, unsegmented and is much more longer than the head. • Strobila consists of 3,000-4,000 proglottids, consisting of immature, mature and gravid segments in that order from front to backwards. • The mature proglottid is broader than long, about 2-4 mm long and l 0-20 mm broad and is practically filled with male and female reproductive organs (Fig. 3B). • The testes are represented by numerous minute follicles situated laterally in the dorsal plane. • The female reproductive organs are arranged along the midline, lying ventrally. The ovary is bilobed . The large rosette-like uterus Lies convoluted in the center. 1hree genital openings are present ventrally along the midline-the openings of the vas deferens, vagina and uterus in that order, from front to backwards.
- 139. Paniker's Textbook ofMedical Parasitology ........--........ / ' ,' ' 2 Suctorial Neck 1 ' grooves or ,' bothria I ', .,' ___.... Strobila m Figs 3A and B: Diphyllobothrium latum. (A) Adult worm showing spatulate scolex, neck and strobila; and (B) Mature proglottid ......,,,..._----Knob Fig. 4: Operculated egg of Diphyllobothrium latum • The fertilized ova develop in the uterusand aredischarged periodically through the uterine pore. • The terminal segments become dried up after delivering many eggs and are discharged in strands of varying lengths in the feces. Egg: D. la.tum is a prolific egg layer and a single worm may pass about a million eggs in a day. • Egg is broadly ovoid, about 65 µm by 45 µm, with a thick, light brown shell (Fig. 4). • It has an operculum at one end and often a small knob at the other. • The freshly-passed egg contains an immature embryo surrounded by yolk granules. The eggs are resistant to chemicals but are killed by drying. The embryo with six hooklets inside the egg is called the oncosphere. • The egg does not.fl.oatin saturated salt solution and is bile stained. • They are not infective to humans. Larval stages: There are three stages oflarval development: l. First stage larva (coracidium) 2. Second stage larva (procercoid) 3. Third stage larva (plerocercoid). LifeCycle Definitive hosts: Man, dog and cat. Man is the optimal host. First intermediate host: Freshwater copepod, mainly of genera Cyclops or Diaptomus. Second intermediate host: Freshwater fish (salmon, trout, etc.). Tnfectiveform to human: Third stage plerocercoid larva. • The adult worm lives in the small intestine. It lays operculated eggs which are passed along with the feces in water (Fig. 5). • The freshly-passed egg contains an immature embryo surrounded by yolk granules. The embryo with six hooklets (hexacanth embryo) inside the egg is called the oncosphere. • In water, it matures in about l 0- 15 days and ciliated first stage larva, called coracidium emerges through the operculum. • Coracidium (first stage larva) can survive in water for about 12 hours, bywhich time itshould be ingested by the fresh water crustacean copepod Cyclops, which is the first intermediate host (Fig. 5). • In the midgut of the Cyclops, the coracidium casts off its ciliated coat and by means of its six hooklets, penetrates into the hemocele (body cavity). In about 3 weeks, it becomes transformed into the elongated second stage larva about 550 µm long, which is called the procercoid larva. • Procerco id larva has a rounded caudal appendage (cercomer) which bears the now useless hooklets. • If the infected Cyclops is now eaten by a freshwater fish (second intermediate host), the procercoid larva penetrates the intestine ofthe fish and grows. In the fish, procercoid larva looses its caudal appendage and develops into the third stage larva called the plerocercoid larva or sparganum (Fig. 5). • Plerocercoid larva has a glistening white flattened unsegmented vermicule, with a wrinkled surface, is about 1-2 cm long, and possesses rudim entary scolex. lhis is the stage infective for humans. Man gets infection by eating raw or undercooked fish containing plerocercoid larva. • The larva develops into adult worm in the small intestine. • The worm attains maturity in about 5-6 weeks and starts laying eggs, which are passed along with the feces. The cycle is thus repeated. The adult worm may Live for about 10 years or more.
- 140. Plerocercoid larva / ,...,,,,'"fish Infectedcyclops eaten by fresh -·~1''""-""'host) Man acquires infection by ingestion of infected freshwater fishes Cestodes:Tapeworms Fresh water fish 2nd intermediate host Adult worm lives in Man small intestine of man Procercoid larva ,,~Ops fCyct""' Definitive host LIFE CYCLE OF DIPHYLLOBOTHRIUM LATUM Cyclops Feces 1st intermediate host Oncosphere penetrates intestine of cyclops (l g, tt. %.% ~? i.c;- £, ~ I>) <JI ,. 0 ~ Coracidium ingested by cyclops (1st intermediate host) Water -~t::klets ~ ~ Coracidium {ciliated 1st } . . stage larva) emerging Ciliated through the operculum epithelium Coracidium Fig. 5: Life cycle of Diphyl/obothrium latum
- 141. Paniker'sTextbook of MedicalParasitology Pathogenicity and Clinical Features The pathogenic effects of diphyllobothriasis depend on the mass ofthe worm, absorption ofits byproducts bythe host and deprivation of the host's essential metabolic intermediates. • In some persons, infection may be entirely asymptomatic, while in others there may be an evidence of mechanical obstruction. • Transient abdominal discomfort, diarrhea, nausea, weakness, weight loss and anemia are the usual manifestations. Patients m ay be frightened by noticing the strands ofproglottids passed in their feces. • A kind of pernicious anemia, sometimes caused by the infection, is called bothriocephalus anemia. This was forme rly believed to be racia lly determined, being com mon in Finland and rare elsewhere. The anemia develops because the tapeworm absorbs large quantity of vitamin 812 and interfereswith its ilea! absorption, leading to vitamin B12 deficiency. • In severe cases, patients may exhibit neurologic sequelae ofvitamin 8 12 deficiency. Laboratory Diagnosis Stool microscopy: Eggs are passed in very large number in feces, and therefore, their demonstration in feces offers an easy method ofdiagnosis. The proglottids passed in feces can also be identified by their morphology. Serodiagnosis: A coproantigen detection test is available to diagnose diphyllobothriasis. Treatment • Praziquantel in a single dose of10 mg/kg is effective. • Eggs are oval, operculated, bile stained and not infective to man. • Infective stage: Plerocercoid larva. • Mode of transmission: Man gets infection by consuming uncooked or undercooked fish containing third stage plerocercoid larva. • Clinical features: Abdominal discomfort, nausea and megaloblastic anemia. • Diagnosis: Stool microscopy for egg and coproantigen test. • Treatment: Praziquantel and if required, vitamin B12 • Spirometra Genus Spirometra belongs to Diphyllobothriidae fam ily. Species of this genera which are medically important are- S. mansoni, S. theileri and S. erinacei. • Spirometraalong with otherDiphyllobothriumtapeworms that are not normal human parasite, can accidentally infect man and cause disease called as sparganosis. • The disease is so named because itis caused bysparganum (plerocercoid larva) ofthe parasite. Distribution Sparganosis has been reported mostly from Japan and Southeast Asia; less often from America and Australia. A few cases have been reported from India also. Habitat Adult worms live in the intestinal tract ofcats and dogs. • Parenteral vitamin 812 should be given, ifB12 deficiency is Life Cycle present. Prophylaxis Infection can be prevented by: • Proper cooking offish. • Deep freezing (- 10°C for 24-48 hours) of fish, if it is to be consumed raw. Prevention offecal pollution of narural waters. • Periodical deworming ofpet dogs and cats. KEY POINTS OF 0/PHYLLOBOTHRIUM LATUM • Longest tapeworm found in man. • Adult worm up to 10 meters in length having spoon-shaped head with two slit-like grooves (bothria). • Definitive host: Man (optimal host), dogs and cats. • First intermediate host: Cyclops. • Second intermediate host: Freshwater fish. Definitive host: Dog and cat. First intermediate host: Cyclops. Second intermediate host: Snakes, frogs and fishes. • Adult worms live in the intestinal tract of dogs and cats and produce large number of eggs which pass out along with feces in water (Fig. 6). • Eggs hatch in fresh water to release ciliated first stage larva called as coracidium. • The coracidium is ingested by Cyclops (fir L intermediate host), where it develops into second stage larva called as procercoid larva. • When the infected Cyclops is ingested by fish, snakes, amphibians (second intermediate host), the procercoid larva migrates to various organs of the body and develops into plerocercoid larva (sparganum Larva). This is the inf ective stage of the larva for dogs and cats (definitive host) (Fig. 6).
- 142. Infected fish, ~' ingested by dogs, cats Adult worms I in intestine of cats and dogs (Definitive host) Larva migrate to tissues, and form plerocercoid (sparganum) larva '_ _..,,., Infected cyclops eaten by fresh water fish, frog (2nd intermediate host) I Eggs, passed ''" ® Eggs hatch in freshwater to Cestodes:Tapeworms Infection occurs due to 1. Ingestion of infected Cyclops 2. Ingestion of raw, infected fish 3. Local application of raw, infective flesh to skin, conjunctiva, or vagina (Used as a poultice) Sparganosis Sparganum larvae (plerocercoid larva) develops in tissues ''"' / "''°osl s Man-dead end (cycle ends) Fig. 6: Life cycle of Spirometra • When a cat or dog eats the second intermediate host, the plerocercoid larva develops into adult worms in the intestine. • Man acts as an accidental host and gets infection by: - Ingestion ofCyclops containing procercoid larva. - Ingestion of plerocercoid larva present in uncooked meat ofanimals or birds, frogs. - Local application of raw flesh of infected animals on skin or mucosa. 1l1e last method follows the practice prevalent among the Chinese, of applying split frogs on skin or eye sores as a poultice. Sparganosis: The term sparganosi.s is used for ectopic infection by sparganum (plerocercoid larva) of Spirometra and some Diphyllobothrium species. • The sparganum (L3 larva) are liberated from the Cyclops in the human intestin e. Theypenetrate the intestinal wall and migrate to subcutaneous tissue, where they become encysted and develop into spargana. • The sparganum is usually found in the subcutaneous tissues in various pans of the body, but may also be present in the peritoneum, abdominal viscera, or brain. Laboratory Diagnosis Diagnosis is usually possible only after surgical removal ofthe nodules and demonstration ofthe worm. Treatment Definitive treatment is surgical removal ofthe nodule. Prophylaxis Human's sparganosis is prevented by: • Properly filtering and boiling drinking water. • Eating properly cooked flesh.
- 143. Paniker'sTextbook ofMedical Parasitology •CYCLOPHYLLIDEAN TAPEWORMS Taenia Saginata and Taenia Solium Common Name • Taenia saginata: Beeftapeworm • Taenia solium: Pork tapeworm. History andDistribution T. saginata has been known as an intestjnal parasite of man from very ancient times. But it was only in 1782 when Goeze differentiated itfrom the porktapeworm, T. solium. ltslife cycle was elucidated when Leuckart, in 1861, first experimentally demonstrated that cattle serve as the intermediate host for the worm. • The name Taenia is derived from the Greek word meaning tape or band. It was originally used to refer to most tapeworms, but is now restricted to the members of the Genus Taenia. • 1: saginata is worldwide in distribution, but the infection is not found in vegetarians and those who do not eat beef. • T. solium is also worldwide in distribution except in the countries and communities, which proscribe pork as taboo. Habitat The adultworms ofboth T. saginata and T. solium(Fig. 7) live in the human small intestine, commonlyin the jejunum (Box I). Morphology Adult worm of T. saginata: The adult 7: saginata worm is opalescent white in color, ribbon-like, dorsoventrally flattened and segmented, measuring 5- 10 meters in length. • The adult worm consists of head (scolex), neck and strobila (body). The general features of adult worm are similar to any cyclophyllidean cestodes. • Scolex: The scolex (head) of T. saginata is about 1-2 mm in diameter, quadrate in cross-section, bearing four hemisphericalsuckers situated atits four angles.Theymay be pigmented. The scolex has no rostellum or booklets (which are present in T. solium). T. saginata is, therefore called the unarmed tapeworm. the suckers serve as the sole organ for attachment (Fig. 8). • TI1e neck is long and narrow. The strobila (trunk) consists of 1,000-2,000 proglottids or segmen ts- immature, mature and gravid. • The gravid segments are nearlyfour times long as they are broad, about 20 mm long and 5 mm broad. The segment contains male and female reproductive structures. The testes are numerous, 300-400 (twice as many as in T solium). The gravid segment has 15-30 lateral branches Box 1: Cestodes living in small intestine • Diphyl/oborhrium latum • Toenia solium • Taenia saginara saginara • T aenia saginara asiacica • Hymenolepis nano Fig. 7: Adult worm of T. so/ium Hooklets --=:::::--: (2 rows) Suckers Taenia saginata Taenia solium Fig. 8: Scolex of Taenia saginata and Taenia solium (as against 7-13 in T. solium). It differs from 1: solium also in having a prominent vaginal sphincter and in lacking the accessory ovarian lobe. The common genital pore opens on the lateral wall of the segments. • The gravid segments break away and are expelled singly, actively forcing their way out through the anal sphincter. As there is no uterine opening, the eggs escape from the uterus through its ruptured wall. Adult worm ofT. solium: • The adult worm is usually 2- 3 meters long. The scolex of T solium is small and globuJar about l mm in diameter, with four large cup-like suckers (0.5 mm in
- 144. Table 4: Differencebetween Taenia saginata and Taenia so/ium Taenia saginata Taenia solium Length 5-10 meter 2- 3 meter Seo/ex Large quadrate Small and globular Rostellum and hooks Rostellum and hooks are are absent present Suckers may be Suckers not pigmented pigmented Neck Long Short Proglottids 1,000-2,000 Below 1,000 Measurement 20mmx 5mm 12mmx6mm (gravid segment) Expulsion Expelled singly Expelled passively in chains of 5 or 6 Uterus Lateral branches 15-30 Lateral branches 5- 10 on each side; thin and on each side; thick and dichotomous dendritic Vagina Present Absent Accessory lobe of Absent Present ovary Testes 300-400 follicles 150- 200 follicles Larva Cysticercusbovis; Cysticercus cellulosae; present in cow not in present in pig and also man in man Egg Not infective to man Infective to man Definitive host Man Man Intermediate Cow Pig, occasionally man host Disease Causes intestinal Causes intestinal taeniasis taeniasis and cysticercosis diameter), and a conspicuous rounded rostellum, armed with a double row ofalternating round and small dagger- shaped hooks, 20- 50 in number. • 111e neck is short and halfas thick as the head. • The proglottids number less than a 1,000. They resemble those of T. saginata in general. 1he gravid segments are twice as long as broad, 12 mm by 6 mm. The testes are composed of 150-200 follicles.1here is an accessory lobe for the ovary. The vaginal sphincter is absent. The uterus has only 5-10 (under 13) thick lateral branches. A lateral thick-lipped genital pore is present, alternating between the right and left sides ofadjacent segments. • The gravid segments are not expelled singly, but pass passively out as short chains. The eggs escape from the ruptured wall ofthe uterus. The other differentiating features of T sagina/a and T. so/ium are given in Table 4. Cestodes: Tapeworms Eggs: Eggs ofboth species are indistinguishable. • 1he egg is spherical, measuring 30-40 mcm in diameter. • lt has a thin hyaline embryonic membrane around it, which soon disappears after release. The inner embryophore is radially striated and is yellow- brown due to bilestaining (Figs 9A and B). • In the center is a fully-developed embryo (oncosphere) with three pairs ofhooklets (hexacanth embryo). • The eggs do not float in saturated salt solution. • 1l1e eggs of T. saginata are infective only to cattle and not to humans, whereas the eggs of T. solium are infective to pigs and humans too. Larva: The larval stage of Taenia is called as cysticercus. • Cysticercus bovis is the larva ofT. saginal.a (Fig. IO). • Cysticercus cellulosae is the larva of T. solium (Fig. 12). Cysticercus bovis: • It is the larval form of T. saginala. 1l1e name cysticercus in derived from the Greek, kystis- b/adderand kerkos- tail. The larva (cysticercus bovis) is infectivestage for humans. • 1l1e cysricercus is an ovoid, milky-white opalescent fluid- filled vesicle measuring about 5 mm x 10 mm in diameter, and contains a single invaginated scolex (bladder worm). • the cysticerci are found in the muscles of mastication, cardiac muscles, diaphragm and tongue ofinfected cattle (Fig. IO). They can be seen on visual inspection as shiny white dots in the infected beef(measly beef) (Fig. 11). • Cysricercus bovis is unknown in humans. Cysticercus cellulosae: • lt is d1e larval form of T. solium and also the inf ectiveform of the parasite. • It can develop in various organs of pig as well as in man. • the cysticercus cellulosae or "bladder worm" is ovoid opalescent milky-white, measuring 8-10 mm in breadth and 5 mm in length. The scolex of the larva, with its suckers, lies invaginated within the bladder and can be seen as a d1ick white spot. It remains viable for several months (Fig. 12). Life Cycle ofTaenia Saginata T. saginata passes its life cycle in two' hosts (Fig. 13): 1. Definitive host: Humans are the definitive hosts and harbor the adult worm. 2. Intermediate host: Cattle (cow or buffalo) are the intermediate host and harbor the larvalstage ofthe worm. Infective stage: Cysticercus bovis (larval stage) is the infective stage to man, while eggs are infective to cattle. • 1l1e adult worm lives in the small intestine of man. The gravid segments from the adult worm break away and are expelled singly. They actively force their way out through the analsphincter.
- 145. Paniker'sTextbook ofMedical Parasitology Eggor gravid proglottid passed in feces man '"'"'"" iafeolioo by ingestion of unde __ r_coo_k_ed_p_o_ rk ___________________________rl Pig (Intermediate host) ~ Ingested by pig O~e,l> (''('I F;; :of-lo"~e~ e~ 'i>~ ..__ _.-=-.oe{u~• ~eie Oncosphere penetrates In 01.10 0 { co,;,9 ruptures, the wall of intestine Fig. 14: Life cycle of Taenia solium They are filtered out principally in the muscles, where they develop into the larval stage, cysticercus cellulosae in about 60-70 days. • ln humans, it is a dead end and the larvae die without funher development. Intestinal infection with 1: solium occurs only in persons eating undercooked pork and usually in persons of low socioeconomic condition with poor sanitation. It is uncommon in Jews and Mohammedans, who are not generally pork eaters. But cysticercosis may occur in any person residing in endemic areas, even in vegetarians because the mode ofinfection is contamination offood or drink with egg deposited in soil. Eggs of T. solium are infective to pigs as well as to man. Pathogenicity and Clinical Features Intestinal taeniasis: It can be caused by both T. saginata and T solium. • Theadultworm, inspiteofitslargesize, causessurprisingly little inconvenience to the patient. • When the infection is symptomatic, vague abdominal discomfort, indigestion, nausea, diarrhea and weight loss may be present. Occasional cases oracute intestinal obstruction, acute appendicitis and pancreatitis have also been reported. Cysticercosis: It is caused by larval stage (cysticercus cellulosae) of T solium. • Cysticercus cellulosae may be solitary or more often multiple. • Any organ or tissue may be involved, the most common being subcutaneous tissues and muscles. It may also affect the eyes, brain, and less often the heart, liver, lungs, abdominal cavity and spinal cord. • The cysticercus is surrounded by a fibrous capsule except in the eye and ventricles of rhe brain. • The larvae evoke a cellular reaction starting with infiltration of neutrophils, eosinophils, lymphocytes, plasma cells, and at times, giant cells. This is followed by fibrosis and death ofthe larva with eventual calcification. • The clinical features depend on the site affected: - Subcutaneous nodules are mostly asymptomatic. Muscular cystlcercosis may cause acute myositis. Neurocysticercosis (cysticercosis of brain) is the most common and most serious form of
- 146. cysticercosis. About 70%of adult-onset epilepsy is due to neurocysticercosis. Other clinical features of neurocysticercosis are increased intracranial tension, hydrocephalus, psychiatric disturbances, meningoencephalitis, transient paresis, behavioral disorders, aphasia and visual disturbances. 1t is considered as the second most common cause of intracranial space occupying lesion (lCSOL) after tuberculosis in India. - In ocular cysticercosis, cysts are found in vitreous humor, subretinal space and conjunctiva. The condition may present as blurred vision or loss of vision, iritis, uveitis and palpebral conjunctivitis. Laboratory Diagnosis Stool examination: Eggs: • Microscopic examination of feces shows characteristic eggs ofTaenia in 20- 80% ofpatients. • Formol-ether sedimentation method of stool concentration is useful. • Eggs can also be detected by cellophane swab method (NIH swab) in 85-95% patients. • Species identification cannot be made from the eggs, since the eggs of T. saginata and T. solium are similar (Flowchart 1). Cestodes:Tapeworms Proglonids: Species identification can be done by examining with a hand lens, the gravid proglortid pressed between two slides, when branching can be made out (15- 20 lateral branches in T. saginata; under 13 in T. solium). Scolex: Definitive diagnosis can also be established bydemonstration of unarmed scolex in case of T. saginala after anthelmintic treatment. Detection of Taenia antigen in feces: Antigen capture enzyme-linked immunosorbenl assay (ELISA) using polyclonal antisera against Taenia are employed to detect coproantigen in feces since 1990 and is more sensitive than microscopy (specificity 100% and sensitivity 98%). The drawback of the test is that it cannot differentiate between T. saginata and T.solium (Flow chart 1). Serodiagnosis: Specific antibodies to adult stage antigen in serum can be d emonstrated by ELISA, indirect immunofluorescence test and indirect hemagglutination (IHA) test (Flowchart I). Molecular diagnosis: Both deoxyribonucleic acid (D A) probes and polymerase chain reaction (PCR) technique are used to detect and differentiate between eggs and proglottids of T. saginata and T. solium (Flow chart 1). It can also differentiate between the two subspecies ofT. saginata, viz. T. saginata saginata and T. saginata asiatica. Flow chart 1: Laboratory diagnosis of Taenia spp. + Taeniasis Stool examination a) Eggs: - Shows characteristic eggs of Taenia but species identification cannot be done - Concentration method: Formol ether sedimentation method b) Proglottids: - Species identification possible by examining proglottids c) Taenia antigen (Coproantigen) - More sensitive than microscopy. - Cannot differentiate between Taenia so/ium and Taenia saginata Laboratory diagnosis Serodiagnosis can be done by • ELISA • IHA Molecular diagnosis • Done by DNA probes and PCR Biopsy Definitive method of diagnosis • Species and subspecies identification possible + C sticercosis Serodiagnosis • Antibody detection by - ELISA - EITB • Antigen detection by ELISA using monoclonal antibodies Imaging methods • X-ray • CT scan • MRI scan AbbreviaUons: CT. computed tomography; DNA. deoxyribonucleic acid: EITB, enzyme-linked immunoelectrotransfer blot: ELISA. enzyme-linked immunosorbent assay: IHA. indirect hemagglutlnal.lon; MRI. magnetic resonance imaging; PCR, polymerase chain reaction
- 147. Paniker'sTextbook ofMedical Parasitology LaboratoryDiagnosis ofCysticercosis Diagnosis of cysticercosis is based on the following {Flow chart 1): • Biopsy: Definitive diagnosis of cysticercosis is by biopsy ofthe lesion and its microscopic examination to show the invaginated scolex with suckers and hooks. • Imaging methods: - X-ra.y: Calcified cysticerci can be detected by radiography of subcutaneous tissue and muscles particularly in the buttocks and thigh. X-ray of the skull may demonstrate cerebral calcified cyst. - Computed tomography (CT) scan of brain is the best method for detecting dead calcified cysts. The cysticcrcal lesions appear as small hypodensities (ring or disk-like) with a bright central spot (Figs 15A and B). - Magnetic resonance imaging (MRI) scan of the brain is more helpful in detection of noncalcified cysts and ventricular cysts. It also demonstrates spinal cysticerci. • Serology: - Antibody detection: Anticysticercus antibodies in serum or cerebrospinal fluid (CSF) can be detected by "ELISA" and enzyme-linked immunoelectrotrasfer blot (EITB) tests. Antigen detection:Antigen can be detected in serum and CSF by ELISA, using monoclonal antibodies and indicate recent infection. • Others: - Ocularcysticercosiscan be made out by ophthalmo- scopy. Eosinophilia: Usually occurs in early stage of cysticercosis, but is not constant. Figs 15A and B: (A ) Computed tomography (CT) scan shows multiple calcified cysts of cysticercus cellulosae in the brain parenchyma; and (B) CT scan of brain shows clear cyst wall in a cyst,cercal lesion Treatment Intestinal taeniasis: Single dose of praziquantel (10-20 mg/ kg) is the drug of choice. • iclosamide (2 g), single dose, is another effective drug. • Purgation is not considered necessary. Cysticercosis: • For cysticercosis, excision is the best method, wherever possible. • Asymptomatic neurocysticercosis requires no treatment. • For symptomatic cerebral cysticercosis, praziquantel in a dose of 50 mg/kg in three divided doses for 20-30 days and albendazole in a dose of 400 mg twice daily for 30 days may be administered. • Corticosteroids may be given along with praziquantel or albendazole to reduce the inflammatory reactions caused by the dead cysticerci. • In addition, antiepileptic drugs should be given until the reaction of Lhe brain has subsided. • Operative intervention is indicated for hydrocephalus. Prophylaxis • Beef and pork to be eaten by man should be subjected to effective inspection for cysticerci in slaughter house. • Avoidance of eating raw or undercooked beef and pork. The critical thermal point of cysticercus is 56°C for 5 minutes. • Maintenance of clean personal habits and general sanitary measures. • For control of cysticercosis, prevention of fecal contamination of soil, proper disposal of sewage and avoidance of eating raw vegetables grown in polluted soil are useful measures. • Detection and treatmentofpersonsharboringadult worm, as they can develop cysticercosis due to autoinfection. KEY POINTS OF TAENIA SAG/NATA • Most common, large ribbon-like tapeworm. • Rostellum and hooks absent (unarmed tapeworm). • 1,000- 2,000 proglottids with 15-30 dichotomously branched uterus. • Definitive host: Man. • Intermediate host: Cow. • Mode of infection: Undercooked (measly) beef containing cysticercus bovis • Eggs are not infective to human. • Asymptomatic, clinicalfeaturesoccuroccasionally- abdominal discomfort, indigestion. • Diagnosis: Eggs or proglottids in stool, serodiagnosis, molecular diagnosis. • Treatment: Praziquantel is the drug of choice and excision in case of cysticercosis. • Prophylaxis: By avoidance of eating undercooked beef.
- 148. KEY POINTS OFTAENIA SOLIUM • Smaller than T. saginata with rostellum and hooks (armed tapeworm). • Less than 1,000 proglottids with 5- 10 thick dend ritic branched uterus. • Definitive host: Man. • Intermediate host: Pig, occasionally man (in case of cysticercosis). • Mode of infection: Undercooked (measly) pork containing cysticercuscellulosae; autoinfection and eggin contaminated vegetable, food and water. • Eggs are infective to human. • Clinical features: Adult worm is asymptomatic. Larval forms cause cystic lesion in subcutaneous tissue, muscle, brain (neurocysticercosis) and eye. • Diagnosis: Intestinal taeniasis-egg or proglottids in stool; cysticercosis- biopsy, X -ray, CT scan, MRI and serology. • Treatment: Praziquantel, albendazole, antiepileptics in neurocysticercosis. • Prophylaxis: By avoidance of eating undercooked pork and raw vegetables. Taenia Saginata Asiatica T. saginala asiatica is closely relared to T. saginata and is found mainly in Asia. • It is morphologically similar to T. saginata except: - Tt is smaller than T. saginata. Intermediate host is pig (not cow). Its cysticerci are located primarily in liver of rhe pig (not muscle). • Clinical features, diagnosis and treatment are similar to that of T. saginata. Multiceps Multiceps (Taenia M ulticeps) Tapeworms of the Genus Multiceps (M. multiceps, M. serialis, M. glomeralus, etc.) are widespread natural parasites of dogs and other canines. Definitive host: Dog, wolfand fox. Intermediate host:Sheep, cattle, horsesand otherruminants. • Humans act as accidental intermediare host. • Humans get infected by ingesting food or water contaminated with dogs feces containing eggs. Oncospheres hatch out from the eggs, penetrate the intestine and migrate to various organs, usually central nervous system (CNS) where it transforms into the larval stage called as coenurus. Coenurus is a roughly spherical or ovoid bladder worm, up to 3 cm in size, and bearing multiple invaginated protoscolices (hence, the name multiceps). Cestodes:Tapeworms • In sheep, coenurus is typically seenin the brain and spinal cord. Affected sheep develop cerebellar ataxia, giving the disease its name "staggers''. • Human coenurosis has been reported from Africa, Europe and the United States ofAmerica (USA).The sites affected mainly are the orbit, brain and subcutaneous tissue. • Clinical disease is due to pressure effects, symptoms being headache, vomiting, paresis and seizures and also due to allergic reactions. • Surgical removal, where feasible is the only mode of treatment. Echinococcus Granulosus Common Name Dog tapeworm. History and Distribution Hydatid cysts had been described by Hippocrates and other ancient physicians. • Adult £. granulosus was described by Hartmann in the small intestine ofdog in 1695 and the larval form (hydatid cysts) was recognized in 1782 by Goeze. • The disease is prevalent in most parts ofthe world, though it is most extensive in the sheep and cattle-raising areas of Australia, Africa and South America. It is also common in Europe, China and the Middle East. It is a significant health problem in India. It is seen more often in temperate than in tropical regions. Habitat • lhe adult worm lives in the jejunum and duodenum of dogs and other canine carnivora (wolfand fox). • The larval stage (hydatid cyst) is found in humans and herbivorous animals (sheep, goat, cattle and horse). Morphology Adult worm: It is a small tapeworm, measuring only 3-6 mm in length. It consists ofa scolex, a short neck and strobUa. The scolex is pyriform, with four suckers and a prominent rostellwn bearing two circular rows ofhooklets (25-30). The neck is shortthan the restof the worm (3 mm x 6 mm). The strobila is composed of only three proglottids: (1) the anterior immature, (2) the middle mature and (3) the posterior gravid segment {Figs 16A to C). The terminal proglottid is longer and wider than the rest of the worm and contains a branched uterus filled with eggs. The adult worm lives for 6-30 months.
- 149. Paniker'sTextbook ofMedical Parasitology m Hooklets(2 rows) Scolex Neck ....=....---- Immature proglottid -••a..-- Mature proglottid Strobila Gravid proglottid m ' Figs 16A to C: Echinococcus granutosus. (A) Schematic diagram of adult worm; (B) Microscopic appearance of scolex of Echinococcus; and (C) Microscopic appearance of scolex in tongue Egg: • The eggs ofEchinococcus are indistinguishable from those ofTaenia species. • It is ovoid in shape and brown in color. • It contains an embryo with three pairs ofhookJets. Larvalform:The larval form is found within the hydalid cyst developing inside various organs ofthe intermediate host. • lt represents the structure of the scolex ofadult worm and remains invaginated within a vesicular body. • After entering the definitive host, the scolex with suckers and rostellar hookJets, becomesexuaginatedand develops into adult worm. Life Cycle The worm completes its life cycle in tvvo hosts {Fig. 17): 1. Definitive hosts: Dog (optimal host), wolf, jackal and fox. 2. Intermed iate host: Sheep and cattle. Sheep is the ideal intermediate host. • Man acts as an accidental intermediate host (dead end). • The larval stage of the parasite is passed in intermediate hosts, including man, giving rise to hydatid cyst. The adult worm lives in the small intestine of dogs and other canine animals.These animals discharge numerous eggs in the feces. • Intermediate hosts (sheep and cattle) ingest them while grazing. Human infection follows ingestion of the eggs due to intimate handling of infected dogs or by eating raw vegetables or other food items contaminated with dog feces. • The ova ingested by man or by sheep and cattle are liberated from the chitinous wall by gastricjuice liberating the hexacanth embryos which penetrate the intestinal wall and enter the portal uenules, to be carried to the liver along the portal circulation. • These are trapped in hepatic sinusoids, where they eventually develop into hydatid cyst. About 75% of hydatid cyst develops in liver, which acts as the first filter for embryo. • However, some embryo which pass through the liver, enter the right side of heart and are caught in pulmonary capillaries (forming pulmonary hydatid cysts), so that the lung acts as the second.filler. • A few enter the systemic circulation and get lodged in various other organs and tissues such as the spleen, kidneys, eyes, brain, or bones. When sheep or cattle harboring hydatid cysts die or are slaughtered, dogs may feed on the carcass or offal. Inside the intestine of dogs, the scolices develop into lhe adult worms that mature in about 6-7 weeks and produce eggs to repeat the life cycle. When infection occurs in humans accidentally, the cycle comes to a dead end because the human hydatid cysts are unlikely to be eaten by dogs.
- 150. Cestodes:Tapeworms .... Carcasses of infectedsheep with hydatid cyst ingested by dog (Definitive host) ' Adult worm in small intestine Egg passed in feces ~ ~ ~_ ) -✓))f~ n' , - - - Egg ingested by sheep (intermediate host) J Hexacanth embryos hatch in the duodenum Fig. 17: Life cycle of Echinococcus granulosus Man (accidental host) Hydatid cyst forms in liver, lungs, etc. Pathogenesis Evolution of hydatid cyst: At the site of deposition, the embryo slowly develops into a hollow bladder or cyst filled with fluid (Figs 18 to 20). This becomes the hydatid cyst (Greek hydatis: a drop ofwater). Pericyst (outer) • It enlarges slowly and reaches a diameter of 0.5- 1 cm in about 6 months. The growing cyst evokes host tissue reaction leading to the deposition of fi brous capsule around it. • The cyst wall secreted by the embryo consists of three indistinguishable layers (Figs 18 and 19): l. Pericyst is the outer host inflammatory reaction consisting of fibroblastic proliferation, mononuclear cells, eosinophils and giants cells, eventually Ectocyst (Intermediate) Hydatid sand Fig. 18: Hydatid cyst in the liver Brood capsules Scolex
- 151. Paniker's Textbook ofMedical Parasitology Brood capsule Hooklets ·' Fig. 19: Microscopy shows three layers in the wall of hydatid cyst. lnbox in the right photomicrograph shows a scolex with a row of hooklets Source: Mohan H. Textbook of Pathology. 6th edition. New Delhi: Jaypee Brothers Medical Publishers: 2010. p. 617. Figs20A to C: Hydatid cyst of the liver- typical look Source: Bhat S. SRB's Manual of Surgery, 4th edition. New Delhi: Jaypee Brothers Medical Publishers: 2012. p. 639. developing into dense fibrous capsule which may even calcify. 2. Ectocyst is the intermediate layer composed of characteristic acellular, chitinous, laminated hyaline material. It has the appearance of the white of a hard boiled egg. 3. Endocyst is the inner germinal layer which is cellular and consists of number of nuclei embedded in a protoplasmic mass and is extremely thin (22- 25 µm). The germinal layer is the vital layer of the cyst and is the site of asexual reproduction giving rise to brood capsules with scolices. It also secretes hydatid fluid, which fills the cyst. Hydatidfluid:The interior ofthe cyst is filled with a clear colorless or pale yellow fluid called as hydatidfluid. pH ofthe fluid is 6.7 (acidic). Composition: It contains salts (sodium chloride 0.5%, sodium sulfate, sodium phosphate, and salts of succinic acid) and proteins. It is antigenic and highly toxic so that its liberation into circulation gives rise to pronounced eosinophilia or may even cause anaphylaxis. - The fluid was used as the antigen for Casoni's intradermal test. • A granular deposit or hydatid sand is found at the bottom of the cyst, consisting offree brood capsules and protoscolices and loose hook.lets. Brood capsules: From the germinal layer, small knob-like excrescences or gemmules protrude into the lumen of the cyst. 1hese enlarge, become vacuolated, and arc filled with fluid. These are called as brood capsules.
- 152. • They areinitially attached to the germinal layer by a stalk, but later escape free into the fluid-filled cyst cavity. • From the inner wall of the brood capsules, protoscolices (new larvae) develop, which represent the head of the potential worm, complete with invaginated scolex, bearing suckers and hook.lets. • Several thousands of protoscolices develop into a mature hydatid cyst, so that this represents an asexual reproduction ofgreat magnitude. • Inside mature hydatid cysts, further generation of cyst, daughter cysts and granddaughter cysts may develop. The cyst grows slowly often taking 20 years or more to become big enough to cause clinical illness and is therefore, particularly seen in man. Acephalocysts:Some cysts are sterile and may never produce brood capsules, while some brood capsule may nut produce scolices. -These are called acephalocysts. Fate of hydatid cysts: The cyst may get calcified or spontaneously evacuated following inflammatory reaction. Hydatid cyst of liver may rupture into lung or other body cavity producing disseminated hydatid lesions. Clinical Features • Most of the times infection is asymptomatic and accidentally discovered. • Clinical disease develops only when the hydatid cyst has grown big enough to cause obstructive symptoms. Disease results mainly from pressure effects caused by the enlarging cysts. • In about half the cases, the primary hydatid cyst occurs in liver (63%) {Figs 20A to C), mostly in the right lobe. Cestodes:Tapeworms Hepatomegaly, pain and obstructive jaundice are the usual manifestations. The next common site is the lung (25%) (most common beingthe lower lobe ofthe rightlung). Cough, hemoptysis, chest pain, pneumothorax and dyspnea constitute the clinical picture. • In the kidney (2%), hydatid cyst causes pain and hematuria. • Other sites affected include spleen (1%), brain ( I%), pelvic organs, orbit and bones (3%). - Cerebral hydatid cysts may present as focal epilepsy. - When hydatid cyst is formed inside the bones, the laminated layer is not well-developed because of confinement by dense osseous tissues. The parasite migrates along the bony canals as naked excrescences that erode the bone tissue. This is called osseous hydatid cyst. Erosion of bone may lead to pathological fractures. • Apart from pressure effects, another pathogenic mechanism in hydatid disease is hypersensitivity to the echinococcalantigen.The host is sensitized to the antigen by minute amounts of hydatid fluid seeping through the capsule. Ilypersensitivity may cause urticaria. But if a hydatid cyst ruptures spontaneously or during surgical interference, massive release of hydatid fluid may cause severe, even fatal anaphylaxis. Laboratory Diagnosis Imaging: Radiological examinations and other imaging techniques such as ultrasonography (USG), CT scan and MRI reveal the diagnosis in most cases of cystic echinococcosis (Flow chart 2). Flow chart 2: Laboratory diagnosis of Echinococcus granulosus Laboratory Diagnosis • Imaging techniques • USG: Diagnostic procedure of choice • CT scan: For extrahepatic disease · MRI: For cysts in spinal vertebrae and cardiac cysts • X-ray: For cysts of bones and lungs • IV pyelogram: For renal cysts • Examination of cyst fluid • Reveals-Scolices, brood capsules and hooklets • Diagnostic puncture of cyst is not recommended Casoni's test • Immediate hypersensitivity skin test • Abandoned due to nonspecificity • Serodiagnosis 1) Antibody detection Tests detecting antibody against antigen B (8 and 16 KDA) • IHA • Indirect immunofluorescence • ELISA Tests detecting antibody against hydatid fluid fraction 5 antigen • CFT • Precipitation test 2) Antigen detection • Double diffusion • CIED Others • Blood-shows eosinophilia • Molecular diagnosis by DNA probes and PCR Abbreviations: CT, computed tomography; CFT. complement fixation test; CIED, cardiac implantable electronic device; DNA, deoxyribonucleic acid; ELISA, enzyme-linked immunosorbent assay; IHA, indirect hemagglutlnatlon; IV, Intravenous; MRI, magnetic resonance imaging; PCR. polymerase chain reaction; USG, ultrasonography
- 153. Paniker'sTextbook ofMedical Parasitology Fig.21: Computed tomography (CT) scan shows a large noncalcified hydatid cyst in right hepatic lobe Source: Dr Soma Sarkar • Ultrasonography is the diagnostic procedure of choice. Cystwall typicallyshowsdoubleechogeniclinesseparated by a hypoechoic layer (double contour). Pathogenic findings include daughter cysts and the "water-lily" sign due to detached endocyst floating within the cavity. • Computed tomography scan is superior for the detection ofextrahepatic disease (Figs 21 and 22). • Magnetic resonance ima. ging appears to add diagnostic benefit for cysts, especially at difficult sites such as spinal vertebrae and cardiac cysts. • Plain X-rays permit the detection of hydatid cyst in lung and bones. In cases where long bones are involved, a mottled appearan ce is seen in the sk.iagram (Fig. 23). • Intravenous (IV) pyelogram is often helpfulfor detection of renal hydatid cyst. Examination of cyst fluid: Examination of aspirated cyst fluid under microscope after trichome staining reveals scolices, brood capsules and hooklets. Exploratory puncture of the cyst to obtain cystic fluid should be avoided as it may cause escape of hydatid fluid and consequent anaphylaxis. lherefore, fluid aspirated from surgically removed cyst should only be examined (Flow chart 2). Casoni's intradermal test: Itis an immediate hypersensitivity {Type 1) skin test introduced by Casoni in 1911, using fresh sterile hydatid fluid. The antigen in hydatid fluid is collected from animal or human cysts and is sterilized by Seitz or membrane filtration. The fluid is injected (0.2 mL) intradermally in one arm and an equal volume of saline as control is injected in the other arm. In a positive reaction, a large wheal of about 5 cm in diameter with multiple pseudopodia like projections appears within half an hour at Fig. 22: Computed tomography (CT) scan showing a hydatid cyst with noncalcified wall in right lower lobe of lung Source: Dr Himanshu Roy Fig. 23: Chest X-ray shows homogenous radiopaque opacity involving right lower lung with costophrenic angle Source: Dr Soma Sarkar the test side and fades in about an hour. Asecondary reaction consisting of edema and induration appears after 8 hours. lhe test is almost abandoned now due to nonspecificity and has been supplemented by serological tests (Flow chart 2). Serology: Antibody detection: • Detection of serum antibodies using specific antigens {8 and 16 kDa) from hydatid fluid are frequently used to support the clinical diagnosis of cystic echinococcosis. The tests include indirect hemagglutination (IHA),
- 154. indirect immunol1uorescence andELISA. In hepatic cysts, the sensitivity of test is relatively superior (85-98%) than pulmonary cyst (50-60%). • The slide latex agglutination test a n d immu ne electroph oresis using hydatid fluid fraction 5 antigen are also widely used. Precipitin test a nd complement fixation test (CFT) with hydatid antigen have also been found to be positive. CFT is not very sensitive and false-positive reaction is seen in those receiving neural antirabic vaccine. CFT is useful after surgical removal of cysts, when a negative test has a better progn ostic value (Plow chart 2). Antigen detection: Specific echinococcal antigen in sera and in CSF can be detected by double diffusion and counter immunoelectrophoresis (CIEP) technique (Flow chart 2). Blood examination: It may reveal a generalized eosinophilia of20-25%. Excretion of the scolices: Excretion of scolices into the sputum or urine may be observed in pulmonary or renal cyst, respectively and can be demonstrated by acid-fast staining or lactophenol cotton blue (LPCB) staining. Specific molecular diagnostic: Specific molecular diagnostic me thods have been developed involving DNA probes and PCR, but their applica tion is lim ited by their technical complexity. Treatment Traditionally surgical removal was considered as the best mode of treatment of cysts. Currenlly, ultrasound staging is recommended and management depends on the stage. In early stages, the treatment of choice is puncture, aspiration, injection and reaspiration (PAIR). Puncture, aspiration, injection and reaspiration, considered as a controversial procedure earlier, is now widely used in early stages of the disease (Box 2). • Th e basic steps involved in PAlR include: - Ultrasound or CT-guided puncture ofthe cyst. Aspiration ofcyst fluid. Infusion of scolicidal agent (usually 95% ethanol; alternatively, hypertonic saline) (Box 3). - Reaspiration ofthe fluid after 5 minutes. • Great care is taken to avoid spillage and cavities are sterilized with 0.5% silver nitrate or 2.7% sodium chloride for prophylaxis of secondary peritoneal echinococcosis due to inadvertent spillage offluid during PAIR (Box 4). Albendazole (15 mg/ kg in rwo divided doses) is initiated 4 days before the procedure and continued for 4 weeks afterwards. Surgery: It is the treatment of choice for complicated E. granulosus cysts like those communicating with the biliary tract and in those cysts where PAIR is not possible. Cestodes:Tapeworms Box 2: Indications of puncture, aspiration, injection and reaspiration (PAIR) • Cystswith internal echoeson ultrasound (snowflakesign) multiple cysts, cystswith detached laminar membrane. . Contraindications of PAIRfor superficially located cysts, cysts with multiple thickinternal septal divisions (honeycombing pattern), cysts communicating with biliarytree. Box 3: Scolicidal agents and their complications • Cetrimide: Itcan cause acidosis • Alcohol 95%: Itcan cause cholangitis • Hypertonic saline: Hypernatremia • Sodium hypochlorite: Hypernatremia • Hydrogen peroxide. Note: Incases with biliary communicationonly hypertonic saline (1 5- 20%) is used. Box 4: Echinococcus species and the diseases caused by them • Echinococcusgranulosus: Hydatid disease • Echinococcus multilocularis: Alveolarormultilocularhydatid disease • Echinococcus vogeli and Echinococcus o/igarthrus: Polycystic hydatid disease The preferred surgical approach is pericystectomy. For pulmonary cyst, treatment consists ofwedge resection or lobectomy. Recurrence after surgery is common. Pre and postoperative chemotherapy with albendazole for 2 years after curative surgery is recommended. • Positron emission tomography (PET) scanning can be used to follow disease activity. • Other new treatment modalities include laparoscopic hydatid liver surgery and percutaneous thermal ablation (PTA)ofthegermin al layerofthe cyst usingradiofrequency ablation device. Chemotherapy: Chem otherapy with benzimidazole agents are restricted to resid ual, postsu rgical and inoperable cysts. Albendazole (400 mg BO for 3 months) and praziquantel (20 mg/ kg/ day for 2 weeks) have proved beneficial. Prophylaxis E. granulosus infection can be prevented by: Ensuring pet dogs do nor eat animal carcass or offal. • Periodical deworming ofpet dogs. • Destruction ofstray and infected dogs. • Maintaining personal hygiene such as washing of hands after touching dogs and avoidance ofkissing pet dogs.
- 155. Paniker'sTextbook of MedicalParasitology KEY POINTS OF ECHINOCOCCUS GRANULOSUS • Echinococcus causes hydatid cyst in man. • Smaller than other cestodes • It measures 3-6 mm and consists of pyriform shaped, scolex, short neck and strobila consists of 3 proglottids. • Eggs are similar to taenia • Larval form is called hydatid cyst which develops inside various organs of the intermediate host • Hydatid cyst consists of three layers-pericyst, ectocyst and endocyst and filled with hydatid fluid • Hydatid cyst may be a symptomatic or may cause pressure effect and anaphylactic reactions. • Laboratory diagnosis by USG, CT scan, MRI and rays. • Treatment option includes surgery, PAIR and chemotherapy with albendazole praziquantel. Echinococcus Multilocularis This causes the rare but serious condition of alveolar or multilocular hydatid disease in humans (Box 5). • It is found in the northern parts of the world, from Siberia in the East to Canada in theWest. The adult worm is smaller than E. granuLosus and lives in the intestines of foxes, dogs and cats which are the definitive host. Rodents are the main intermediate hosts. Human infection develops from eatingfruits or vegetables contaminated with their feces. E. multilocularis leads to multilocular hydatid cyst. The liver is the most commonly affected organ. The multilocular infiltrating lesion appears like a grossly invasive growth, without any fluid or free brood capsule or scolices which can be mistaken for a malignant tumor. Patients present with upper quadrant and epigastric pain. Liver enlargement and obstructive jaundice may also be present. It may also metastasize to the spleen, lungs and brain in 2%cases. The prognosis is very grave and if untreated, 70% cases progress to dealt. Surgical resection, when possible, is the best method of treatment. Albendazole therapy is recommended for 2 years after curative surgery. In those cases, where surgery is not possible, indefinite treatment with albendazole is recommended. Hymenolepis Nana Common Name Dwarf tapeworm. Box 5: Malignant hydatid disease • It is a misnomer, as it is a benign condition. • It is caused by Echinococcus multilocu/aris (alveolaris). It presents with multiple small cysts in both lobes of the liver. • It is difficult to treat and mimics clinically and prognosis wise to malignancy; hence the name. • Patients die of liverfailure. History and Distribution The name Ilymenolepis refers to the thin membrane covering the egg (Greek hymen-membrane, lepis-rind or covering) and nana to its small size (nan.us-dwarf). It was first discovered by Bilharz in 1857. • It is cosmopolitan in distribution bur is more common in warm than in cold climates. • Infection is most common in school children and institutional populations. • Ilymenolepis nan.a is the smallest and the most common tapeworm found in the human intestine. It is unique that it is the only cestode which completes its life cycle in one host-humans. Habitat The adult worm lives in the proximal ileum of man. H. nan.a var.jraterna is found in rodents like mice and rats, where they are found in the posterior part of the ileum. Morphology Adult worm: H. nan.a is the smallest intestinal cesrode that infects man. • It is 5-45 mm in length and less than l mm thick. The scoLex has four suckers and a retractile rostellum with a single row ofhook.lets (Fig. 24). • The long slender neckis followed by the strobila consisting of 200 or more proglottids, which are much broader than long. • Genital pores are situated on the same side along the margins. • The uterus has lobulated walls and the testis is round and three in nwnber. Eggs are released in the intestine by disintegration of the distal gravid segments. Egg: The egg is roughly spherical or ovoid, 30-40 µmin size. • It has a thin colorless outer membrane and inner embryopfwre enclosing the hexacanth oncosphere (Figs 25Aand B).
- 156. Cestodes: Tapeworms Fig.24: Adultworm of Hymenolepis nana Figs 25A and B: Egg of Hymenolepis nana. (A) As seen under microscope; and (B) Schematic diagram • The space between two membranes contains yolk granules and 4-8 thread like polar.filaments arising from two knobs on the embryophore. • The eggs float in saturated solution ofsalt and are nonbile stained. • They are immediately infective and unable to survive for more than lOdays in external environment. Life Cycle Host: Man. • There is no intermediate host. Mode oftransmission: Infection occurs by ingestion of the food and water contaminated with eggs. Internal autoinfection may also occur when the eggs released in the intestine hatch there itself (Fig. 26). - External a.utoinfection occurs when a person ingest own eggs by fecal oral route. H. nana is unusual in that it undergoes multiplication in the body of the definitive host. When the eggs are swallowed, or in internal autoinfection, they hatch in the small intestine. the hexacanth embryo penetrates the intestinal villus and develops into the cysticercoid larva.
- 157. Paniker's Textbook ofMedicalParasitology 1 Man Ingestion of contaminated food and water causes infection Eggs ingested by rat Oncosphere is liberated and it penetrates intestinal wall Internal autoinfection (in children) or external autoinfection Cysticercoid larva in rat flea LIFE CYCLE OF HYMENOLEPIS NANA INFECTING RODENTS ' Rat flea ingest eggs of Hymenolepis nana LIFE CYCLE OF HYMENOLEPIS NANA INFECTING MAN No intermediate host required Adult worm in small intestine Egg in feces Fig. 26: Life cycle of Hymeno/epis nana • This is a solid pyriform structure, with the vesicular anterior end containing the invaginated scolex and a short conical posterior end. • After about 4 days, the mature larva emerging out of the villus evaginates its scolex and attaches to the mucosae. • It startsstrobilization, to become the mature worm,which begins producing eggs in about 25 days. Adifferent strain ofH. nana infects rats and mice. The eggs passed in rodent feces are ingested by rat fleas (Xenopsylla cheopisandothers), which actsastheintermediatehost.The eggs develop into cysticercoid larvae in the hemocele ofthese insects. Rodents get infected when they eat these insects. The murine strain does not appear to infectman. However, the humanstrain may infect rodents,which may, therefore,constitute a subsidiary reservoir ofinfection for the human parasite.
- 158. Clinical Features Hymenolepiasis occursmore commonly in children. • There are usua!Jy no symptoms but in heavy infections, there is nausea, anorexia, abdominal pain, diarrhea and irritability. • Sometimespruritus mayoccurdue roan allergic response. Laboratory Diagnosis The diagnosis is made bydemonstration ofcharacteristic eggs in feces by direct microscopy. Concentration methods like salt flotation and formalin ether may be readily used. ELJSA test has been developed with 80% sensitivity. Treatment Praziquantel (single dose of 25 mg/kg) is the drug of choice, since it acts both against theadultworms and the cysticercoids in the intestinal villi. • Nitazoxanide 500 mg BD for 3 days may be used as alternative. Prophylaxis • Maintenance of good personal hygiene and sanitary improvements. • Avoidingofconsumption ofcontaminated foodand water. • Rodent control. Hymenolepis Diminuta This is called the rat tapeworm and is a common parasite of rats and mice. • The name diminuta is a misnomer, as it is larger than H. nana being 10-60 cm in length. • Its life cycle is similar to that of the murine strain of H. nana. • Rarely, human infection follows accidental ingestion of infected rat fleas. Human infection is asymptomatic. Dipylidium Caninum This common tapeworm of dogs and cats, it may accidentally cause human infection, mainly in children. Morphology • The adult worm in the intestine is about l0-70 cm long. • The scolex has four prominent suckers and a retractile rostellum with up to seven rows ofspines {Figs 27A to C). • The mature proglortid has two genital pores, one on either side, hence the name Dipylidium (dipylos-two entrances). C estodes:Tapeworms m Figs 27A to C: Dipylidium caninum. (A) Scolex showing four suckers and rostellum with multiple rows of hooklets; (B) Mature proglottid showing two genital pores, one on either side; and (C) Eggs found in clusters enclosed in a membrane Box 6: Parasites requiring as intermediate host • Hymenolepis diminuta , Dipylidium caninum • Hymenolepis nano (murinestrain) • Gravid proglottids are passed out of the anus of the host singly or in groups. Life Cycle Definitive host: Dogs, cats and rarely man. Intermediate host: Fleas (Box 6). • Man acquires infection by ingestion of flea harboring cysticercoid larva. • 1he eggs or proglottids passed in feces ofdogs and cats are eaten by larval stagesofdogand cat fleas, Ctenocephalides canis and C.felis. • The embryo develops into a tailed cysticercoid larva. • When the adult fleas containing the larvae are eaten by dogs, cats, or rarely humans, infection is transmitted. Clinical Features Human infection is generally asymptomatic, but the actively motile proglottids passed in srools may raise an alarm. Diagnosis the diagnosis is made by detection of proglortids or eggs in stool. Treatment the drug ofchoice is praziquantel.
- 159. Paniker'sTextbook ofMedical Parasitology REVIEWQUESTIONS 1. Describe briefly: a. General characters of cestodes b. Classification ofcestodes 2. Short notes on: a. Echinococcus granulosus b. Hymenolepis nana c. Diphyllobothrium /atum d. Hydatid cyst e. Casoni's test f. Sparganosis g. Coenurosis h. Dipylidium caninum i. Cysticercuscellulosae j. Neurocysticercosis 3. Describe morphology, life cycle and laboratory diagnosis of: a. Taenia solium b. Taenia saginata c. Echinococcus granulosus 4. Differentiate between: a. Taenia solium and Taenia saginata b. Taenia saginata saginata and Taenia saginata asiatica MULTIPLE CHOICE QUESTIONS 1. Autoinfection is a mode of transmission in a. Trichinella b. Cysticercosis c. Ancylostoma d. Ascaris 2. Pigs are reservoir for a. Taenia solium b. Diphyllobothrium latum c. Trichinella spiralis d. Ancyclostoma 3. On microscopic examination, eggs are seen, but on saturation with salt solution eggs are not seen.The eggsare likely to be of a. Trichuris trichiura b. Taenia solium c. Ascaris lumbricoides d. Ancylostoma duodenale 4. Which ofthe following is not a cestodes a. Diphyllobothrium latum b. Taenia saginata c. Schistosoma mansoni d. Echinococcus granulosus 5. Consumption of uncooked pork is likely to cause which of the following helminthic disease a. Taenia saginata b. Taenia so/ium c. Hydatid cyst d. Trichuris trichiura 6. All of the following are true about neurocysticerosis, except a. Not acquired by eating contaminated vegetables b. Caused by regurgitation of larva c. Acquired by orofecal route d. Acquired by eating pork 7. The longest tapeworm found in man a. Diphyllobothrium /atum b. Taenia saginata c. Taenia solium d. Echinococus granulosus 8. Second intermediate host of Diphyl/obothrium latum is a. Cyclops b. Man c. Snail d. Fresh water fish 9. Dwarf tapeworm refers to a. Echinococcus granulosus b. Loa/oa c. Hymenolepis nano d. Schistosoma mansoni 10. The egg of which of the following parasites consists of polar filaments arising from either end ofthe embryophore a. Taenia saginata b. Taenia solium c. Echinococcus granulosus d. Hymenolepisnana 11 . Coenurus is the larval form of a. Taenia solium b. Taenia multiceps c. Echinococcus granulosus d. Echinococcus multilocularis 12. Larval form of Echinococcus granulosus is seen in a. Dog b. Man c. Wolf d. Fox 13. The adult worm of Echinococcus granulosus contains a. 3- 4 segments b. 50- 100 segments c. 100- 200 segments d. 1000-2000 segments 14. Which skin test is useful for diagnosis of hydatid disease a. Casoni's test b. Schick test c. Dick'stest d. Tuberculin test Answer 1. b 8. d 2. a 9. C 3. b 10. d 4. C 11. b 5. b 12. b 6. a 13. a 7. a 14. a
- 160. CHAPTER 12 • INTRODUCTION Trematodesare leaf-shaped unsegmented, flat and broad helminths (hence the namefluke, from the Anglo-Saxon word floe meaningflatfish). The name trematode comes from their having large prominent suckers with a hole in the middle (Greek trema: hole, eidos: appearance). • CLASSIFICATION OF TREMATODES Systemic Classification Trematodes belong to: Phylum: Platyhelminthes Class: Trematoda The detailed systemic classification has been given in Table I . Table 1: Zoological classification of trematodes Superfamily Schistosomatoidea Paramphistomatoidea Echinostomatoidea Opisthorchioidea Plagiorchioidea Family Schistosomatidae Zygocotylidae Fasciolidae • Opisthorchiidae • Heterophyidae Paragonimidae Classification Based on Habitat Based on habitat, trematodes can be classified as (Table 2): • Blood flukes Liver flukes • Intestinal llukes • Lung llukes. • FLUKES: GENERAL CHARACTERISTICS They vary in size from 1 mm to several centimeters. Males are shorter and stouter than females. • The unique feature of flukes is the presence of two muscular cup-shaped suckers (hence called distomata)- the oral sucker surrounding the mouth at the anterior end and the ventral sucker or acetabulum in the middle, ventrally (Fig. 1). Genus Schistosomo • Gastrodiscoides • Waisonius • Fasciola • Fasciolopsis • Opisthorchis • Clonorchis • Heterophyes • Metagonimus Paragon/mus Species • S. haemarobium • S. mansoni • S.japonicum • S. mekongi • S. intercalatum • G.hominis • W. watsoni • F. hepatica • F. buski • 0. felineus • 0. viverrini • C. slnensis • H. heierophyes • M. yokogawai P. westermani
- 161. Paniker's Textbook ofMedical Parasitology All schistosomes live in venous plexuses in the body of the definitive host, the location varying with the species (urinary bladder in S. haematobium, sigmoidorectaJ region in S. mansoniand UeocecaJ region in S.japonicum). Schistosoma Haematobium History and Distribution This vesical blood fluke, formerly known as bilharzia haematobium, has been endemic in the Nile valley in Egypt for millenia. Its eggs have been found in the renal pelvis of an Egyptian mummy dating from l ,250-1,000 BC. Schistosome antigens have been identified by enzyme-linked immunosorbent assay (ELlSA) in Egyptian mummies of the Predynastic period, 3,100 BC. • The adult worm was described in 1851 by Bilharz in Cairo. Its life cycle, including the larval stage in the snail, was worked out by Leiper in 1915 in Egypt. • Although maximally entrenched in the Nile valley, S. haematobium is also endemic in most parts ofAfrica and in West Asia. • An isolated focus ofendemicity in India exists in Ratnagiri district ofMaharashtra. • About 200 million persons are at a risk ofinfection and 90 million arc infected by S. haematobiumglobally. Habitat The adult worms live in the vcsicaJ and pelvic plexuses of veins. Morphology Adult worm: • 1he male is 15 mm long by 0.9 mm thick and covered by a thick tuberculate tegument. • It has two muscular suckers: (1) the oral sucker being small and (2) the ventral sucker large and prominent. Beginning immediately behind the ventral sucker and extending to the caudal end is the gynecophoric canal, in which the female worm is held (Fig. 3). • the adult female is long and slender(20 mm by0.25 mm). • 1h e gravid worm contains 20-30 eggs in its uterus at one time and may pass up to 300 eggs a day. Egg: The eggs are elongated, brownish yellow (about 150 µm by 50 µm) and nonoperculated. the eggs have characteristic terminal spine at one pole (Fig. 4). Mechanism of egg expulsion: The eggs are laid usually in the small venules of the vesical and pelvic plexuses, though sometimes they are laid in the mesenteric portal system, pulmonary arterioles and other ectopic sites. • 1he eggs are laid one behind the other with the spine pointing posteriorly. Bifurcated alimentary canal Tubercles on back----:v!"tr of male parasite ~ ii.'~ Oviduct==="Jttf'lfJ ~ IJ Ovary Cecum Schistosoma mansoni Coupled worms Giemsa staining, magnification 25X Fig. 3: Structural details of Schistosoma (coupled) Fig. 4: Egg of Schistosoma haematobium • From the vcnules, the eggs make their way through the vesical wall by the piercing action of the spine, assisted by the mounting pressure within the venules and a lytic substance released by the eggs. • The eggs pass into the lumen of the urinary bladder together with some extravasated blood. • TI1cy are discharged in the urine, particularly towards the end of micturition. • For some unknown reasons, the eggs are passed in urine more during midday than at any other time ofthe day. • The eggs laid in ectopic sites generally die and evoke local tissue reactions. They may be found, for instance in rectal biopsies, but are seldom passed live in feces.
- 162. Life Cycle S. haematobiumpasses its life cycle in rwo hosts: 1. Definitive host: Humans are the only natural definitive hosts. o animal reservoir is known. 2. Intermediate host: Freshwater snails (snail of the genus Bulinus). Infectiveform: Cercaria larva. • The eggs that are passed in urine are embryonated and hatch in water under suitable conditions to release the free-living ciliated miracidia. • Miracidia swim about in water and on encountering a suitable interm ediate host, penetrate into its tissues and reach its liver (Fig. 6).The intermediate hosts are snails of Bulinusspecies in Africa. In India, the intermediate host is the limpet, Ferrissia tenuis. Development insnail: Inside the snail, the miracidia lose their cilia and in about 4-8 weeks, successively pass through the stages of the first and second generation sporocysts (Fig. 6). • Large numbers of cercariae are produced by asexual reproduction within the second generation sporocyst. The cercaria has an elongated ovoid body and forked tail (furcocercous cercaria) (Fig. 5). • The cercariae escape from the snail into water. • Swarms of cercariae swim about in water for 1-3 days. Persons become infected bycontact with watercontaining cercariae during bathing. Suckers and lytic substances secreted bycercariae helps them to penetrated intact skin. Development in man: After penetrating the skin, the cercariae loss their tails and become schistosomulae which travel via peripheral venules to systemic circulation (Fig. 6). • They then start a long migration, through the vena cava into the right heart, the pulmonary circulation, the left heart and the systemic circulation, ultimately reaching the liver. • In the intrahepatic portal veins, the schistosomulae grow and become sexually differentiated adolescents about 20 days after skin penetration. • They then startmigrating against the bloodstream into the inferior mesenteric veins, ultimately reaching the vesical Anterior sucker Ventral sucker Forked tail Fig. 5: Cercaria larva of Schistosoma spp. Trematodes: Flukes and pelvic venous plexuses, where they mature, mate and begin laying eggs. Eggs start appearing in urine usually 10-12 weeks after cercarial penetration. The adult worms may live for 20-30 years. Pathogenicity and Clinical Features Clinical illness caused by schistosomes can be classified as acute and chronic based on the stages in the evolution of the parasite. Acute schistosomiasis: Duringskinpenetration ofcercariae, intense irritation and skin rash may develop at the side ofcercarial penetration (swimmer's itch). It is particularly severe when infection occurs with cercariae of nonhuman schistosomes. Anaphylactic or toxic symptoms may develop during incubation period due to liberation of toxic metabolites by schistosomules. Migration ofschistosomulae into lungs may cause cough and mild fever. Chronic schistosomiasis: Egg deposition in urinary bladder causes mucosa! damages leading to painless hematuria, dysuria and proteinuria, particularly in children in endemic areas. There is innammation of the urinary bladder due to release of soluble antigens from the eggs causing pseudoabscesses in the surrounding tissues. Initially the trigone is involved but ultimately the whole mucosa is inflamed, ulcerated and thickened. There is heavy infi ltration of macrophages, lymphocytes, eosinophils and fibroblasts. Many of the eggs die and become calcified eventually producing fibrosis ofvesical mucosa and formation ofegg granulomas (sandy patches). Fibrosis may cause obstructive uropathies like hydronephrosis and hydroureter. Chronic schistosomiasis has been associated with urinary bladder carcinoma (Box3). Chronic cystitis may develop due to secondary bacterial infection. Chronic infection may result in calculusformation. Involvement ofotherorgans during schistosomiasis: Lungs and central nervous system (spinal cord), skin and genital organs may be involved. Box 3: Parasites associated with malignancy • Schisrosoma haematobium: Bladder carcinoma • Clonorchis sinensis: Bile duct carcinoma • Opisthorchis viverrini: Bile duct carcinoma
- 163. Paniker'sTextbook ofMedical Parasitology ~ ,;s <J<YMature in mtrahepatic portal veins Adult worms in venous plexus Cercaria sheds its tail ro""/= _,_,,. MAN (Definitive host) S. haematobium S. mansoni S. japonicum WATER Penetrate skin of man (Definitive host) Free-living ciliated miracidium hatches In water (16 hours) Development within snail (Intermediate host) In 4-8 weeks 1. Primary sporocysts 2. Secondary sporocysts 3. Developing cercariae within secondary sporocysts Fig. 6: Life cycle of Schistosoma spp. • Ectopic lesions in the spinal cord produce a transverse myelitis-like syndrome. Schistosomiasis favors urinary carriage oftyphoid bacilli. Laboratory Diagnosis Urine microscopy: The eggs with characteristic terminal spines can be demonstrated by microscopic examination of centrifuged deposits of urine or by filtration of a known volume ofurine through nucleopore filters (Flow chart 1). • Eggs are more abundant in the blood and pus passed by patients at the end ofmicturition. • Nucleopore filtration method provides quantitative data on the intensity ofinfection. Eggs can also be seen in the seminal fluid in males and occasionally in feces. Histopathology: Schistosome infection may also be diagnosed by demonstrating its eggs in bladder mucosa! biopsy and rectal biopsy. Detection of antigen: Another diagnostic method is by detection of specific schistosome antigens in serum or urine. Two circulating antigens related to gut ofadult schistosomes: (1) circulating anodic antigen (CAA) and (2) circulating
- 164. Trematodes:Flukes Flow chart 1:Laboratory diagnosis of Schistosoma haematobium laboratory diagnosis ' ' l ' • Demonstration of Detection of antigens Detection of antibody lntradermal skin test Imaging characteristic egg • Urine microscopy (CAA and CCA) by ELISA • Complement fixallon test (CFT) (Fairley·s test) The test is group specific and gives positive result in all schistosomiasis • X-ray to demonstrate bladder and ureteral calc1ficat1 on • Bladder mucosal biopsy • Bentonite flocculation test • Indirect hemagglutinat,on • USG, !VP and cystoscopy for indirect diagnosis (IHA) • lmmunofluorescence • FAST/ELISA • Enzyme-linked ,mmunoelectrotransfer blol(EITB) Abbreviations: CAA, circulating anodic antigen; CCA, circulating cathodic antigen; ELISA. enzyme-linked immunosorbent assay; FAST, falcon assay screening test; IVP, intravenous pyelogram; USG, ultrasonography cathodic antigens (CCAs) can be demonstrated by dipstick assay and ELISA. The test is very sensitive and specific, but is available only in specialized laboratories. Soluble egg antigens (SEAs) can be demonstrated in serum (Flow chart 1). Detection ofantibody: Several serological tests have been described for detection of specific antibody, but are not very useful as they cannot differentiate between present and past infection. These include complement fixation test (CFT), bentonite flocculation test, indirect hemagglutination (IHA), immunofiuorescence and gel diffusion tests. Two serological tests for detection of antibodies against Schistosoma haematobium adult worm microsomal antigen (HAMA)are:(l )thefalronassayscreeningtest(HAMAFAST)/ ELISA and (2) HAMA enzyme-linked immunoelectrotransfer blot (EITB). Both these tests are highly sensitive and specific (95% sensitive and 99% specific) (Flow chart 1). Intradermal skin test (Fairley's test): 11,csc allergic skin tests are group-specific. The test uses antigen from larvae, adult forms and eggs of schistosomes from artificially infected snails and infected laboratory animals. Imaging: • X-ray of the abdomen may show bladder and ureteral calcification. • Ultrasonography (USG) is also useful in diagnosing S. haematobium infection. USG may show hydroureter and hydronephrosis. • Intravenous pyelogram (TVP) and cystoscopy are also useful in indirect diagnosis ofthe disease. Treatment Prazjquantel (40-60 mg per kg in divided doses in a single day) is the drug ofchoice. Metriphonate is the alternative drug of choice in schistosomiasis due to S. haematobium (7.5 mg/kg weekly for 3 weeks). Prophylaxis Prophylactic measures include: • Eradication ofthe intermediate molluscan hosts by using molluscicides. • Prevention of environmental pollution with urine and feces. • Effective treatment ofinfected persons. • Avoid swimming, bathing and washing in infected water. Schistosoma Mansoni History and Distribution Tn 1902, Manson discovered eggs with lateral spines in the feces of a West Indian patient that led to the recognition of this second species of human schistosomes. It was, therefore named S. mansoni. • It is widely distributed in Africa, South America and the Caribbean islands. Habitat Adult worm lives in the inferior mesenteric uein. Morphology S. mansoni resembles S. haematobium in morphology and life cycle, except: • The adult worms are smaller and their integuments studded with prominent coarse tubercles. • In the gravid female, the uterus contains very few eggs, usually 1-3 only.
- 165. Paniker'sTextbook of MedicalParasitology S. mansoni Ova with a lateral spine (obtained from stool) S. haematobium Ova with a terminal spine (obtained from urine) S. japonicum Ova with a lateral knob (obtained from stool) Note: The characteristic surround of tissue particles Fig. 7: Schematic diagram to show distinguishing features of eggs of S. mansoni, S. haematobium and S. japonicum • The prepatent period (the interval between cercarial penetration and beginning ofegg laying) is 4-5 weeks. • The egg has a characteristic lateral spine (Fig. 7), more near to the rounded posterior end. The eggs are nonoperculated and yellowish brown. Life Cycle Definitivehost: Humans are the only natural definitive hosts, though in endemic areas monkeys and baboons have also been found infected. Intermediate host: Planorbid freshwater snails of the genus Biomphalaria. Infectiveform: Fork-tailed cercaria. In humans, the schistosomulae mature in the liver and the adult worms move against the bloodstream into the venules ofthe inferior mesenteric group in the sigmoidorectal area.Eggs penetrate the gutwall, reach the colonic lumen and are shed in feces. Pathogenicity and Clinical Features • Cercarial dermatitis: - Following skin penetration by cercariae: A pruritic rash called as cercarial dermatitis or swimmers itch may develop locally. It is a self-limiting disease. • Katayama/ever: - A.fter4-8 weeks orcercarial invasion a serum sickness like illness may happened during production ofeggs. - lt results from high worm load andeggantigen stimuli which leads to formation ofimmune complexes. Sign and symptoms include high fever, rash, arthralgia, hepatosplenomegaly, lymphadenopathy and eosinophilia. • Intestinalbilharziasis: - During the stage of egg deposition in small intestine, patients may develop pain in abdomen and bloody dysentery, which may go on intermittently for many years. The eggs deposited in the intestinal wall may cause microabscesses, granulomas, hyperplasia and eventual fibrosis. Egg granulomas are found in the distal part of the colon and rectum. Ectopic lesions include hepatosplenomegaly and periportal fibrosis, portal hypertension, as some of the eggs are carried through portal circulation into liver. - Portal hypertension may cause gastrointestinal hemorrhage. Laboratory Diagnosis Stool microscopy: Eggs with lateral spines may be demonstrated microscopically in stools. Kato-Katz thick smear or otl1er concentration methods may be required when infection is light. Kato-Katz thick smear provides quantitative data on tl1e intensityofinfection,which is ofvalue in assessing the degree of tissue damage and monitoring the effect of chemotherapy. Rectal biopsy: Proctoscopic biopsy of rectal mucosa may reveal eggs when examined as fresh squash preparation between two slides. Serological diagnosis: Serological diagnosis by detecting schistosomal antigen and antibody is similar to that of S. haematobium. Imaging: Ultrasonography is useful to detect hepato- splenomegaly and periportal fibrosis. Blood examination: Blood examination may reveal eosinophilia and increased levels ofalkaline phosphatase. Treatment Praziquantel (single oral dose 40mg/kg) is the drug ofchoice. Oxamniquine (single oral dose 15 mg/ kg) is also effective. It damages the tegument of male worm and thereby, makes
- 166. the worm moresusceptible to lethal action of the immune system. Prophylaxis Same as S. haematobium. Schistosoma Japonicum Common Name Oriental blood Duke. Distribution S. japonicum is found in the Far East, Japan, China, Taiwan, Philippines and Sulawesi. Habitat The adult worms are seen typically in the venules of the superior mesenteric vein draining the ileocecal region. They are also seen in the intrahepatic portal venules and hemorrhoidal plexus ofveins. Morphology Morphologically, they are similar to the schistosomes described earlier except: The adult male is comparatively slender (0.5 mm thick) and does not have cuticular tuberculations. Trematodes:Flukes • In the gravid female, the uterus contains as many as JOO eggs at one time and up to 3,500 eggs may be passed daily by a single worm. • The prepatent period is 4-5 weeks. • The eggs are smaller and more spherical than those of S. haematobium and S. mansoni. The egg has no spine, but shows a lateral small rudimentary knob (Fig. 7). Differentiating features between the three species of Schislosoma are illustrated in Table 3. Life Cycle Life cycle of S. japonicum is similar to S. haematobium with the following exceptions: Definitive host: Man is the definitive host but in endemic areas, natural infection occurs widely in several domestic animals and rodents, which act as reservoirs ofinfection. lritermed iate host: Amphibian snails of the genus Oncomelania. lnfectiveformfor humans: Fork-tailed cercaria. • Eggs deposited in the superior mesenteric venules penetrate the gut wall and are passed in feces. • They hatch in water and the miracidia. infect the intermediate hosts, amphibian snails of the genus Oncomelania. • The fork-tailed cercaria, which escapes from the snails is the infectiveform for men and other definitive hosts. Table 3: Differentiating features of S. haematobium, S. mansoni and S. japonicum Habitat Morphology Size: Male Female Integument Number of testes Ovary Uterus Egg Cephalic glands in cercariae Distribution Definitive host Intermediate host Schlstosoma haematobium Veins ofthe vesical and pelvic plexuses, less commonly in portal vein and its mesenteric branches • 1.Scmx 1 mm • 2 cm x 0.22 mm • Finely tuberculated • 4 5 in groups • In the posterior one-third ofthe body • Contains 20-30 eggs Elongated with terminal spine Two pairs oxyphilic and three pairs basophilic Africa, Near East, Middle East and India Man Snail ofgenus Bulinus Schistosoma mansonl Inferior mesenteric vein and its branches • 1 cmx 1 mm • 1.4 cm x0.25 mm • Grossly tuberculated • 8-9 in a zigzag row • In the anterior half ofthe body • 1-3 eggs Elongated with lateral spine Two pairs oxyphilic and four pairs basophilic Africa and South America Man Snail of genus Biomphaloria Schistosomajaponlcum Superior mesenterlc vein and its branches • 1.2- 2cm x0.5 mm • 2.6cm x 0.3mm • Nontubercular • 6-7 in a single file • In the middle of the body • SO or more eggs Round with small lateral knob Five pairs oxyphilic, no basophilic China, Japan and Far East (oriental) Man (mainly) domestic animals and rodents (which act as reservoir of infection) Amphibian snail ofgenus Oncomelania
- 167. Paniker's Textbook ofMedical Parasitology Pathogenicity and Clinical Features Disease caused by S. japonicum is also known as oriental schistosomiasis or Katayama disease. • Pathogenesis is almost sim ilar to that of S. mansoni. But the disease is more severe due to higher egg production. • During the acute phase of the disease, Katayama/ever is similar to that seen in S. mansoni. • Chronic illness is characterized by intestinal m ucosa! h yp erp lasia, h epatosplenomegaly an d portal hypertension. Liver is hard and shows periportal fibrosis (clay pipestem fibrosis). Portal hypertension leads to esophageal varices and gastrointestinal bleeding. Intestinal disease manifests as colicky abdominal pain, bloody diarrhea and anemia (Box 4). • Central nervous system and lung involvem ent (cor pulmonale) m ay occur in 2-4% of cases. Parietal lobe of the brain and spine are commonly affected. Severe epileptic seizures may be observed in these patients. Laboratory Diagnosis Similar to that ofS. mansoni. Treatment S. japonicum infection is more resistant to treatment than other schistosomiasis. A prolonged course of intravenous tartar emetic gives good results. Praziquantel is the drug of choice. Prophylaxis Same as S. haematobium. Schistosoma lntercalatum S. intercalatum was first noted in 1934 in West-Central Africa. • The eggs are fully embryonated without any opercu.lum having terminal spines, but are passed exclusively in stools. The eggs are acid-fast. • It produces few symptoms involving the mesenteric portal system. Box 4: Parasites leading to bloody diarrhea • IntestinalSchistosomo species: - S.japonicum - S. mansoni - S.intercalarum - S.mekongi. • Trichuris trichiura • Entamoeba histolytica • Balantidium coli. • Diagnosis is established by detection of the egg in feces and rectal biopsy. • Praziquantel is the drug ofchoice. KEY POINTS OF SCHISTOSOMES • Schistosomes are dioecious, sexes are separate. • Habitat: In the mesenteric venous plexus (S. mansoni and S. japonicum) and vesical, and prostatic venous plexus (S. haematobium). • Leaf-like unsegmented body with two cup-like suckers with delicate spines. • Intestine is bifurcated (inverted Y-shaped}. • Male is broader than female. • They produce elongated nonoperculated eggs containing ciliated embryo, miracidium. • Definitive host: Man. • Intermediate host: Freshwater snails. • Infective form: Fork-tailed cercariae. • Clinical features: Swimmer's itch, Katayama fever, hematuria and portal hypertension. • Diagnosis: Detection of eggs in urine or stool, biopsy, imaging, and detection ofantigen and antibody. • Treatment: Praziquantel is the drug of choice. • Prophylaxis: Avoidance of bathing in infected water and eradication of snail. Schistosoma Mekongi this species first recognized in 1978 is found in Thailand and Cambodia, along the Mekong river. • lt is closely related to S. japonicum but is slightly smaller and round. • Man and dog are the definitive host. • Man acquires infection in the same wayas in S.japonicum. • HepatosplenomegaJy and asci.tes are the common clinical finding. • HERMAPHRODITIC FLUKES: LIVER FLUKES The adult forms of all hermaphroditic flukes infecting man reside in the lumen of the biliary, intestinal, or respiratory tracts. This location gives the flukes suitable protection from host defense mechanisms and also facilitates dispersal of eggs to the environment. • Flukes inhabiting the h uman biliary tract are Clonorchis sinensis, Fasciola hepatica, less often Opisthorchisspecies, and rarely, Dicrocoelium dendriticum. Fascio/a Hepatica Common Name Sheep liver fluke.
- 168. History and Distribution F.hepatica was the first trematode that was discovered more than 600 years ago in 1379 by Jehan de Brie. • It was named by Linnaeus in 1758. • It is the largest and most common liver fluke found in man, however its primary host is the sheep and to a less extent, cattle. • It causes the economically important disease, "liver rot'; in sheep. • It is worldwide in distribution, being found mainly in sheep-rearing areas. • In India, few cases reported from North India and North Eastern part of India including Uttar Pradesh (UP), Bihar and Assam. • F. gigantica is more prevalent in India than F hepatica. Habitat The parasite resides in the liver and biliary passages of the definitive host. Morphology Adult worm: • It is large in size, flat leaf-shaped fluke measuring 30 mm long and 15 mm broad, gray or brown in color. • lt has a conical projection anteriorly containing an oral sucker and is rounded posteriorly (Figs BA and B). • The adult worm lives in the biliary tract of the definitive host for many years-about 5 years in sheep and 10 years in humans. • Like all other trematodes, it is hermaphrodite. Egg:The eggs are large, ovoid, operculated, bile-stained and about 140 µm by 80 µmi n size (Box 5 and Fig. 9). Trematodes: Flukes Eggs contain an immature larva, the miracidium. Eggs do not float in saturated solution ofcommon salt. Eggs of F. hepatica and Fasciolopsis buski cannot be differentiated. • Eggs are unembryonated when freshly passed. Box 5: Parasites with operculate eggs • Fascia/a hepatica • Fascia/a gigantica • Fascia/apsis buski • C/anarchis sinensis • Paraganimus westermani • Gastradiscaides haminis • Opistharchis felineus • Opistharchis viverrini • Heteraphyes heteraphyes • Diphy/Jabathrium /atum. Fig. 9: Egg of Fasciola hepatica Bf"f<ie-- - - - Oral sucker /~ ·•,.,,'4'"-<----- Intestinal cecum Ventral sucker - -"'~r=--?-~ Uterus- -F.;'.:~ · ..,.- _,j - - - Vitellaria Ovary Figs 8A and B: (A) Fasciola hepatica; and (B) Specimen showing Fasciola hepatica
- 169. Paniker'sTextbook ofMedical Parasitology LifeCycle Migrates t @~ · /.,.,oo~,ru,o,cy,ts 11)1.•-.= in duodenum Adult worm in bile ducts Man and other herbivores get infection by eating aquatic plants eocysi wlth metace,~,;,. Man (Definitive host) Metacercarfa Water plants (2nd intermediate host) r ll encysts on aquatic vegetations lo become metacercaria Water Snail (1st intermediate host) Development within snail (First intermediate host) 1. Sporocyst 2. First generation redia 3. Second generation redia 4. Cercariae Fig. 10: Life cycle of Fasciola hepatica Egg embryonates in water and miracidium escapes out I Miracidium ingested / bysoan F. hepatica passes its life cycle in one definitive host and two intermediate hosts. Mode ofinfection: 'TI1e definitive host, sheep and man, get infection by ingestion of metacercariae encysted on aquatic vegetation. Adult worm lives in the biliary passage of sheep or man. Eggs are laid in the biliary passages and are shed in feces. Definitive host:Sheep, goat, cattle and man. Intermediate host: Snails of the genus Lymnaea and Succinea. Encystment occurs on aquatic plants, which act as second intermediate host. • lhe embryo matures in water in about 10 days and the miracidium escapes. It penetrates the tissues of first intermediate host, snails of the genus Lymnaea (Fig. 10).
- 170. Box 6: Parasiteswith aquatic vegetations as the source of infection • Fascia/a hepatica • Fasciolopsis buski • Gastrodiscoideshominis • Watsonius watsoni. • In snail, the miracidium progressesthrough the sporocyst and the first and second generation redia stages to become the cercariae in about 1-2 months. • the cercariae escape into the water and encyst on aquatic vegetation or blades of grass to become metacercariae, which can survive for long periods (Box 6). • Sheep, cattle, or humans eating watercress or other water vegetation containing the melacercaria become infected. themetacercariae excystin theduodenumofthedefinitive host and pierce the gut wall to enter the peritoneal cavity. TI1ey penetrate the Glisson's capsule, traverse the liver parenchyma, and reach the biliary passages, where they matureinto the adult worms in about3-4months (Fig. 10). Pathogenicity Fascioliasis differs from clonorchiasis in that F. hepatica is larger and so causes more mechanical damage. In traversing the liver tissue, it causes parenchymal injury. As humans are not its primary host, it causes more severe inflammatory response. Some larvae penetrate right through the liver and diaphragm ending up in the lung. • In acute phase during the migration ofthe larva, patients presentwithfever, right upper quadrant pain, eosinophilia and tender hepatomegaly. In chronic phase, patients may develop biliary obstruction, biliary cirrhosis, obstructive jaundice, cholelithiasis and anemia. No association to hepatic malignancy has been ascribed toJascioliasis. Occasionally, ingestion of raw liver of infected sheep results in a condition called halzoun (meaning suffocation). The adult worms in the liver attach to the pharyngeal mucosa, causing edematous congestion of the pharynx and surrounding areas, leading to dyspnea, acute dysphagia, deafness and rarely, asphyxiation. However, this condition is more oflen due to pentastome larvae. Halzoun is particularly common in Lebanon and other parts ofthe Middle East and North Africa. Diagnosis Stool microscopy:Demonstration ofeggs in feces or aspirated bile from duodenum is the best method of diagnosis. Eggs of E hepatica and F. buski are indistinguishable. Blood picture: It reveals eosinophilia. Trematodes: Flukes Serodiagnosis:Serological testssuchasimmunofluorescence, ELISA, immunoelectrophoresis and complement fixation are helpful in lightly infected individuals for detection ofspecific antibody. ELISA becomes positive within 2 weeks ofinfection and is negative after treatm ent. In chronic fascioliasis, Fasciola coproantigen may be detected in stool. Imaging: Ultrasonography, computed tomography (CT) scan, endoscopic retrograde cholangiopancreatography (ERCP) and percutaneous cholangiography may be helpful in diagnosis. Treatment Oral triclabendazole (10 mg/kg once) is the treatment of choice. Alternative drug is bithionol (30-50 mg for 10- 15 days). Prednisolone at a dose of 10- 20 mg/ kg is used to control toxemia. Prophylaxis Fascioliasis can be prevented by: Health education. Control ofsnails. Proper disposal of human, sheep and cattle feces. Proper disinfection of watercresses and other water vegetations before consumption. KEY POINTS OF FASCIOLA HEPATICA • Largest and most common liver fluke. • Large leaf-shaped with a dorsoventrallyflattened body. • Hermaphroditic parasite. • Eggs are ovoid, operculated and bile-stained. • Definitive host Primary definitive host is sheep, but it is also found in biliary tract of man. • first intermediate host Fresh water snails (Lymnaea). • Second intermediate host Aquatic vegetations. • Infective form: Metacercariae encysted on raw aquatic vegetations. • Clinical features: Acute phase-fever, right upper quadrant pain and hepatomegaly. Chronic phase-biliary obstruction, obstructive jaundice, cholelithiasis and anemia. • Diagnosis: Detection of eggs in stool and aspirated bile, USG, ERCP and ELISA. • Treatment: Oral triclabendazole or bithional. • Prophylaxis: Preventing pollution of water with feces and proper disinfection. Dicrocoelium Dendriticum Also known as the "lancet Duke" because of its shape, D. dendriticum is a very common biliary parasite of sheep and other herbivores in Europe, North Africa, Northern Asia and parts of the Far East.
- 171. Paniker'sTextbook of MedicalParasitology Definitive Host Sheep and other herbivores. First Intermediate Host Snails. Second Intermediate Host Ants ofgenus Formica. • Eggs passed in feces ofsheep are ingested by land snails. • Cercariae appear in slime balls secreted by the snails and are eaten by ants of the genus Formica, in which metacercariae develop. • Herbivores get infected when they accidentally eat the ants while grazing. • Reports of human infection have come from Europe, Middle East and China. • However, spurious infection is more common. ln the latter, the eggs can be passed in feces for several days by persons eating infected sheep liver. • Eurytrema pancreaticum, a related fl uke is commonly present in the pancreatic duct of cattle, sheep and monkeys. Occasional human infection has been noticed in China and Japan. Clonorchis Sinensis Common Name The Chinese liver fluke and oriental liver fluke. History and Distribution C. sinensis was first described in 1875 by McConnell in the biliary tract of a Chinese carpenter in Calcutta Medical College Hospital. • Complete life cycle of Clonorchis was worked out by Faust and Khaw in 1927. • Human clonorchiasis occurs in Japan, Korea, Taiwan, China and Vietnam, affecting about 10 million persons. Habitat Adult worm lives in the biliary tract and sometimes in the pancreatic duct. Morphology Adultworm: It has a flat, transparent, spatulate body; pointed anteriorly and rounded posteriorly {Fig. 11). • It is 10- 25 mm long and 3-5 mm broad. • The adult worm can survive in the biliary tract for 15years or more. • The hermaphroditic worm discharges eggs into the bile duct. / t-""l't--t-::::,--- Intestinal ceca ,-,;;.a~r-,.;...:..:,.:.,i,_ Testes (2) Fig. 11: Adult worm and egg of Clonorchis sinensis Eggs:Eggs are flask-shaped, 35 µm by20 µmwith a yellov.rish- brown (bile-stained) shell. • It is operculated at one pole and possesses a tiny knob at the other pole and a small hook-like spine at the other (Fig. 11). • Eggs do not float in saturated solution ofcommon salt. • The eggs passed in feces contain the ciliated miracidia. Life Cycle Definitive host: Humans are the principal definitive host, but dogs and other fish-eating canines act as reservoir hosts. Intermediate hosts: Two intermediate hosts are required to complete its life cycle, the first being snail and the second being.fish. Infectiveform: Metacercaria larva. Mode of infection: Man acquires infection by eating undercooked freshwater fish carrying metacercariae larvae. Clonorchis eggs although embryonated do not hatch in water, but only when ingested by suitable species of operculate snails (first intermediate host), such as Parafossarulus, Bulimus, orAlocinma species. The miracidium develops through the sporocyst and redia stages to become the lophocercus cercaria with a large fluted tail in about 3 weeks {Fig. 12). The cercariae escape from the snail and swim about in water, waiting to get attached to the second intermediate host, suitable freshwater fish ofthe Carp family. The cercariae shed their tails and encyst under the scales or in the flesh of the fish to become metacercariae, in about 3 weeks, which are the infective stage for humans.
- 172. Trematodes: Flukes Metacercaria excystsin duodenum Adult worm in bile ducts Man (definitive host) Infected fish ingested by man t Ingested by snail Fish (2nd intermediate host) Snail ~ (1st intermediate host) • Cercaria penetrates under scales of fresh-water fish and develops into metacercaria Miracidium hatches out in the midgut of snail Development within snail (First intermediate host) 1. Sporocyst 2. First generation redia 3. Second generation redia 4. Cercariae ~ Fig. 12: Life cycle of Clonorchis sinensis Infection occurs when such fish are eaten raw or inadequately processed by h uman or other definitive hosts. Frozen, dried, or pickled fish may act as source of infection (Fig. 12). Infection may also occur through fingers or cooking utensils contaminated with the metacercariae during preparation ofthe fish for cooking. • The metacercariae excyst in the duodenum of the definitive host. • The adolescaria that come out, enter the common bile duct through the ampulla of Vater and proceed to the distal bile capillaries, where theymarure in about a month and assume the adult form (Fig. 11). • Adult worms produce an average of 10, 000 eggs per day, which exit the bile ducts and are excreted in the feces. The cycle is then repeated. Pathogenicity The m igration of the larva up the bile duct induces desquamation, followed by hyperplasia, and sometimes, adenomatous changes. The smaller bile ducts undergo cystic dilatation.
- 173. Paniker'sTextbook of MedicalParasitology • The adult worms may obstruct and block the common bile duct leading to cholangitis. • Patients in the early stage have fever, epigastric pain, diarrhea and tender hepatomegaly. This is followed by biliary colic, jaundice and progressive liver enlargement. Many infections are asymptomatic. • Chronic infection may result in calculusformation. • A few cases go on to biliary cirrhosis and portal hypertension. • Some patients with chronic clonorchiasis tend to become biliary carriers of typhoid bacilli. • Chronic infection has also been linked with cholangiocarcinoma. Diagnosis 1he eggs may be demonstrated in feces (stool microscopy) or aspirated bile. They do not float in concentrated saline. • Several serological tests have been described including complement fixation and gel precipitation but extensive cross-reactions limit their utility. !HA with a saline extract ofetherized worms has been reported to be sensitive and specific. • Intradermal allergic tests have also been described. Treatment Drug ofchoice is praziquantel 25 mg/kg, three doses in l day. Surgical intervention may become necessary in cases with obstructive jaundice. Prophylaxis Clonorchiasis can be prevented by: • Proper cooking of fish. • Proper disposal of feces. • Control ofsnails. Opisthorchis Species Some species of Opisth.orchis, which resemble C. sinesis can cause human infection. • O.Jelineus, the cat liver nuke, which is common in Europe and the erstwhile Soviet Union, may infect humans. • Infection is usually asymptomatic but may sometimes cause liver disease resembling clonorchiasis. • 0. viverrini is common in Thailand, where the civet cat is the reservoir host. Chandler found that 60% of cats in Calcutta, were infected with the parasite and human cases have also been reported from India. • Most of the infected patients have a low worm burden, so they are asymptomatic. • Cholangiocarcinoma is epidemiologically related 10 C. sinensis infection in China and to 0. viverrini infection in ortheastThailand. • the life cycle and other features of Opisthorchis are same as those ofClonorchis. • INTESTINAL FLUKES A number of flukes parasitize the human small intestine. These include Fasciolopsis buski, 1-/eterophyes, Metagonimus yokogawai, Watsonius watsoni and Echinostoma. Only one fluke Gastrodiscoides hominis, parasitizes the hwnan large intestine. Fasciolopsis Buski Common Name Giant intestinal fluke History and Distribution It was first described by Busk in 1843 in the duodenum ofan East Indian sailor, who died in London. • Ir is the largest and most common intestinal fluke ofman and pigs. • Mainly found in China and in Southeast Asian countries. • In India it occurs in Assam, Bengal, Bihar and Odisha. • Prevalence rate is as high as 22.4%in India. • Children are more prone to infection than adults as they enjoy playing in water. Habitat The adult worm lives in the duodenum or jejunum of pigs and man. Morphology Adult worm: The adult is a large fleshy worm, 20-75 mm long and 8-20 mm broad (Fig. 13) and 0.5-3 mm in thickness. • Largest trematode infecting humans: Fasciolopsis buski • Smallest trematode infecting humans: 1-/eterophyes • It is elongated ovoid in shape, with a small oral sucker and a large acetabulum. lt has no cephalic cone as in F. hepatica (Fig. 14). • The adult worm has a lifespan ofabout 6 months. • The two intestinal caeca do not bear any branches (Fig. 14). Eggs: • The operculated eggs are similar 10 those of F. hepatica (Fig. 15). • Eggs are laid in the lumen of the intestine in large numbers, about 25,000 per day.
- 174. Fig. 13: Specimenshowing Fascio/opsis buski Life Cycle F. buski passes its life cycle in one definitive host and two intermediate host. Definitive host: Man and pigs. Pigs serve as a reservoir of infection for man. First intermediate host:Snails ofthe genus Segmentina. Second intermediate host: Encystment occurs on aquatic plants, roots ofthe lotus, bulb ofthe water chestnut which act as second intermediate host. Infectiveform: Encystedmetacercariae on aquaticvegetation. • The eggs passed in feces ofdefinitive hosthatch in walerin about 6 weeks, releasing the miracidia which swim about. • On coming in contact wilh a suitable molluscan intermediate host, snails of the genus Segmentina, miracidia penetrates its tissues to undergo development in the next few weeks as sporocyst, first and second generation rediae and cercariae (Fig. 16). • The cercariae, which escape from the snail, encyst on the roots of the lotus, bulb of the water chestnut, water hyacinth and on other aquatic vegetations. • When they are eaten by man, the metacercariae excysts in the duodenum, become allached to the mucosa and develop into adults in about 3 months (Fig. 16). Pathogenesis The pathogenesis of fasciolopsiasis is due to traumatic, mechanicaland toxic effects. • Larvae that attach to the duodenal and jejuna! mucosa cause inflammation and local ulceration. Intoxication and sensitization also account for clinical illness. Trematodes: Flukes mlf---"<----Oral sucker Pharynx .::=~7--'~+-Uterus cr-CTT~ -+--~ Ovary Vilellaria - -et-- Intestinal cecum Fig. 14: Fasciolopsis buski Fig. 15: Egg of Fasciolopsis buski • In heavy infections, the adult worms cause partial obstruction of the bowel, malabsorption, protein-losing enteropathy and impaired vitamin 812 absorption. • ·n,e initial symptoms are diarrhea and abdominal pain. • Toxic and allergic symptoms appear usually as edema, asciL es, anemia, prostration and persistent diarrhea. • Paralytic ileus is a rare complication. Laboratory Diagnosis History ofresidence in endemic areas suggests the diagnosis, which is con.fumed by demonstration ofthe egg in feces or of the worms after administration ofa purgative or anthelmintic drug.
- 175. Paniker'sTextbook ofMedical Parasitology Metacercariaexcysts in duodenum and attaches to intestinal wall in small intestine I Man (Definitive host) Operculated Man and other herbivores eats aquatic plants with encysted metacercariae ' Water plants (2nd intermediate host) Metacercaria I encysts on aquatics vegetations beco metacercaria Free swimming cercaria escapes from snail into water Water Snail (1st intermediate host) egg in feces Egg embryonates In water and miracidium ""T'"' Miracidium ingested by snail Development within snail (First intermediate host) 1. Sporocyst 2. First generation redia 3. Second generation redia 4. Cercariae Fig. 16: Life cycle of Fasciolopsis buski Treatment Drug of choice is praziquantel. • Hexylresorcinol and tetrachloroerh ylene have also been fow1d useful. Prophylaxis • Treatment of infected persons. • Proper disinfection ofwater vegetables, by hot water. • Prevention of polution of water resources from human and pig feces. • Community-based praziquantel treatment can be used to control infection. • Control ofsnails. Heterophyes heterophyes This is the smallest trematode parasite of man. • 1he infection is prevalent in the Nile delta, Turkey and in the Far East. • The worm has been reported in a dog in India. • The adult worm lives in the small intestine and has a lifespan ofabout 2 months.
- 176. Definitive Hosts Humans, cats,dogs, foxes and other fish-eating mammals. First Intermediate Host Snails of the genera Pirone/la and Cerithidea. Second Intermediate Host Fishes, such as the mullet and tilapia; encystment occurs in fishes. • Man acquires infection by eating raw or undercooked fishes containing metacercaria. • In the small intestine, it can induce mucous diarrhea and colicky pains. • Ectopic lesions may occur as granulomas in myocardium, brain and spinal cord. • Diagnosis is based on the finding ofa minute operculated egg in the stool. Drug ofChoice Praziquantel. Metagonimus Yokogawai It is found in the Far East, Northern Siberia, Balkan states and Spain. Definitive Hosts Humans, pigs, dogs, cats and pelicans. First Intermediate Host Freshwater snail. Second IntermediateHost Fish. • Definitive hosts are infected by eating raw fish containing the metacercariae. • Pathogenic effects consist ofmucous diarrhea and ectopic lesions in myocardium and central nervous system as in heterophyasis. Drug ofChoice Praziquantel. Watsonius Watsoni • This trematode infects various primates inAsia andAfrica. onnal host is the monkey. • Eggs are operculated. Trematodes: Flukes • Infection occurs by ingestion ofwater plants containing metacercariae. • Diagnosis, clinical features, treatment and prophylaxis is same as that ofHeterophyes. Echinostoma Echinostomes are medium-sized fl ukes causing small intestinal infection ofrats and dogs. • Seen in Japan, Philippines and all along the Far East. • The characteristic feature is a crown of spines on a disc surrounding the oral sucker, justifying its name Echinostoma which means "spiny mouth''. • Its eggs resemble those ofPasciolopsis.Mild infections are asymptomatic, but diarrhea and abdominal pain follow heavy infection. • E. ilocanum is the species usually seen in human infections. Gastrodiscoides Hominis C. hominis is the only fluke inhabiting the human large intestine (Fig. 17). lt was discovered by Lewis and McConnell in 1876 in the cecum ofan Indian patient. lt is a common human parasite in Assam. Cases have also been reported from Bengal, Bihar and Odisha. lt also occurs in Viemam, Philippines and some parts of erstwhile Union ofSoviet Socialist Republics (USSR). The adult worm is pyriform, with a conical anterior end and a discoidal posterior part. It is about 5- 14 mm long and 4-6 mm broad. • The eggs are operculated and measure 150 µm by 70 µm. Fig. 17: Specimen showing Gastrodiscoides hominis
- 177. Paniker'sTextbook ofMedical Parasitology DefinitiveHost Man, pigs and monkey. Pigs are the reservoir hosts. First Intermediate Host Snails. Second Intermediate Host Aquatic plants. • The miracidia invade the tissues of the intermediate molluscan host. • The cercariae encyst on water plants. Infected persons develop mucoid diarrhea. • Man and animals become infected by feeding upon vegetations harboring the metacercaria. Drug ofChoice Praziquamel. Tetrachloroethylene is also useful in treatment. • LUNG FLUKES Paragonimus Westermani Common Name Oriental lung fluke. History and Distribution P. westermani was discovered in 1878 by Kerbert in the Iungs of a Bengal tiger captured in India that died in the zoological gardens at Amsterdam. • The parasite is endemic in the Far East-Japan, Korea, Taiwan, China and South EastAsia- Sri Lanka and India. • There are about 40 species of Paragonimus that infect mammals. • ln India, cases have been reported from Assam, Bengal, TamiI Nadu, Kerala, Manipur, Sikkim, Arunachal Pradesh and Nagaland. • P. westermani is the most common species infecting human. • Endemic foci of P. westermani and P heterotremus are present in Manipur. • lt is an important hwnan pathogen in Central and South America. Morphology Adult worm: The adult worm is egg-shaped about 10 mm long, 5 mm broad and 4 mm thick and reddish-brown in color (Fig. 18). • The integument is covered with scale-like spines. Fig. 18: Paragonimus westermani morphology lntestinal---<c::,:..,;;.s,;;..;_, Spine cecum ~ ::r--H'T"r:~ 5-.:-- Ventral sucker Uterus ---'""""r..:,.-++-~ ~ ,...,...__ ...,_ N~~~- Ovary Testes --.;::-::,,"61;=+-i---:-,r- (two) Fig. 19: Paragonimus westermani • It has an oral suckerplaced anteriorly and a ventral sucker located towaJds the middle ofthe body (Fig.19). • It has two unbranched intestinal caeca which end blindly in the caudal area. • They have a lifespan ofup to 20 years in humans. Egg: The eggs are opercu!ated, golden-brown in color and about 100 µm by 50 µmin size (Fig. 20). • They are unemb1yonated when freshly laid. Habitat Adults worms live in the lungs, usually in pairs in cystic spaces that communicate with bronchi (Table 4). Life Cycle Definitive host: Man. Besides humans, other definitive hosts include cats, tigers, leopards, foxes, dogs, pigs, beavers, mongoose, and many other crab-eating mammals and domestic animals.
- 178. Fig. 20: Eggof Paragonimus westermani Table 4: Helminths present in lung Trematode Paragonimus westermani Cestode Echinococcus granulosus Dirolilario immitis Nematode Capillaria aerophila First intermediate host: Freshwater snail, belonging lo the genera Semisulcospira and Brotia. Second intermediate host: Freshwater crab or crayfish. Infectiveform: Metacercariae encysted in crab or crayfish. Mode of infection: Man acquires infection by eating undercooked crab or crayfish containing metacercariae. • The adull worms live in the respiratory tract of the definitive host. • Unembryonated eggs escape into the bronchi and are coughed up and voided in sputum or swallowed and passed in feces {Fig. 21). • The eggs mature in about 2 weeks and hatch to release free-swimmingmiracidia. • These infect the.firstintermediate molluscan host, snails belonging to the genera Semisulcospira and Brotia. • Cercariae that are released from the snails after several weeks are microcercus, having a short stumpy tail. • The cercariae that swim about in streams are drawn into the gill chambers of the second intermediate crustacean host, crabs or crayfish (Fig. 21). • lhey encyst in the gills or muscles as metacercariae. • Definitive hosts are infected when they eat such crabs or crayfish raw or inadequately cooked. • The metacercariae excyst in the duodenum and the adolescariae penetrate the gut wall, reaching the abdominal cavity in a few hours. Trematodes: Flukes They then migrate up through the diaphragm into the pleural cavity and lungs finally reaching in the vicinity of the bronchi, where they develop into adult worms in 2-3 months {Fig. 21). The worm is hermaphroditic but usually it takes 2 for fertilization. Sometimes, the migrating larvae lose their way and reach ectopic sites such as the mesentery, groin and brain. Pathogenicity and Clinical Features Pulmonary features: In the lungs, the worms lie in cystic spaces surrounded by a fibrous capsule formed by the host tissues. The cysts, about a centimeter in diameter are usually in communication with a bronchus. Inflammatoryreaction to the worms and theireggs lead to peribronchial granulomatous lesions, cystic dilatation ofthe bronchi, abscesses, pneumonitis and eosinophilia. • Patients presentwith cough, chest pain and hemoptysis. The viscous sputum is speckled with the golden-brown eggs. Occasionally, the hemoptysis may be profuse. • Chronic cases may resemble pulmonary tuberculosis. Extrapulmonary features: The clinical features depend on the site ofinvolvement. Extrapulmonary infections are more common in P. mexicanus, P. heterolremus and rare in P. westermani. • Abdomi1tal paragonimiasis: Occasionally the fluke migrates to liver and intestinal wall resulting in enlarge liver, abdominal tenderness and bloody diarrhea. • Cerebral paragonimiasis: Encapsulated cyst of Paragonimus is found in brain and spinal cord. Symptoms include headache, fever, paralysis, visual disturbances and convulses seizures. Laboratory Diagnosis Microscopy: Demonstration of the eggs in sputum or feces provides definitive evidence. Sputum examination should be repeated for 7 consecutive days. Serology: Complement fixation test is positive only during and shortly after active infection, while the intradermal test remains positive for much longer periods. Parasite-specifi c immunoglobulin E ( lgE) and antiparagonimus antibodies can be detected in serum. • Indirect hemagglutination and ELISA tests are highly sensitive. they become negative within 3-4 months after successful treatment. • Serology is ofparticular importance in egg-negative cases and in cerebral paragonimiasis. Imaging: Chest X-ray reveals abnormal shadows (nodular, cystic, ring infiltrative) in the middle and lower lung field.
- 179. Paniker'sTextbook of MedicalParasitology Metacercaria excysts in duodenum Man gets infected by ingestion of ray ' 0 0 y-<00k e<Jc,ab Man Crab (2nd intermediat host) Metacercaria develops inside the viscera, muscles. and gills or crab Water Cercaria penetrates crab Free-swimming cercaria escape from snail into water Snail (1st intermediate host) Development within snail (First intermediate host) 1. Sporocyst 2 First generation redia 3. Second generation redia 4. Cercariae Fig. 21: Life cycle or Paragonimus westermani Prophylaxis Egg embryonates in water and free-swimming miracidium released • Computed tomographyscan ofchestalso helpsin diagnosis of pulmonary lesions and cerebral lesions. "Soap-bubble'' like appearance may be seen in cerebralcysts. Adequate cooking of crabs and crayfish and washing lhe hands after preparing them for food. Treatment • Praziquantel (25 mg/ kg TDS for 1-2 days) is the drug of choice. • Bithionol and niclofolan are also effective in treatment. • Treatment of infected persons. • Disinfection of sputum and feces. • Eradication of molluscan hosts.
- 180. KEY POINTS OFPARAGONIMUS WESTERMAN( • Adult worm is egg-shaped, reddish, brown and covered with scale-like spine. • Habitat: Cystic spaces in the lung. • Eggs are oval, operculated and golden brown. • Definitive hosts: Man and domestic animals. • First intermediate host: Snails of genera Semisu/cospira (Melania species). • Second intermediate host Crab or crayfish. • Infective form: Encysted metacercaria in crab or crayfish. • Clinical features: Peribronchial granuloma and cystic dilation of bronchi. Dyspnea, hemoptysis, pneumonitis, bronchiectasis, abscess and pneumothorax. Extrapulmonary lesions in brain and intestine. • Diagnosis: Ova in sputum, X-ray and CT scan of chest, CFT, IHA and ELISA. • Treatment: Praziquantel is the drug of choice. • Prophylaxis: Adequate cooking of crabs and crayfish, eradication of molluscan hosts and t reatment of infected persons. REVIEW QUESTIONS 1. Describe briefly: a. General characters oftrematodes b. Classification of trematodes c. General charactersof schistosomes 2. Short notes on: a. Clonorchis sinensis b. Fasciolopsis buski c. Paragonimus d. Opisthorchis species 3. Describe morphology, life cycle and laboratory diagnosis of a. Fascia/a hepatica b. Schistosoma haematobium 4. Differentiate between Schistosoma haematobium, S. mansoni and S.japonium. MULTIPLE CHOICE QUESTIONS 1. Which of the following flukes is carcinogenic a. Fascia/a b. Clonorchis c. Paragonimus d. Gastrodiscoides Trematodes:Flukes 2. Organism causing biliary tract obstruction a. Ancylostoma duodenale b. Clonorchis sinensis c. Strongyloides stercoralis d. Enterobius vermicularis 3. All float in a saturated salt solution except a. Clonorchis sinensis b. Fertilized eggs of Ascaris c. Larva of Strongyloides d. Trichuris trichiura 4. Terminal spined eggs are seen in a. Schistosoma haematobium b. Schistosoma mansoni c. Schistosomajaponicum d. Clonorchis sinensis 5. Largest trematode infecting humans a. Fascia/a hepatica b. Fasciolopsis buski c. Schistosoma haematobium d. Paragonimus westermani 6. The second intermediate host of Fasciola hepatica is a. Snail b. Fresh water fish c. Crab d. Aquatic plants 7. Schistosomajaponicum resides in a. Superior mesenteric vein b. Inferior mesenteric vein c. Small intestine d. Gallbladder 8. All of the following lead to bloody diarrhea except a. Schistosomajaponicum b. Entamoeba histolytica c. Schistosoma mansoni d. Schistosoma haematobium Answer 1. b 5. b 2. b 6. d 3. a 7. a 4. a 8. d
- 181. CHAPTER 13 Nematodes: General Features •INTRODUCTION Nematodes are said to be the most worm-like of all hel- minths. This is because they generally resemble the common earthworm in appearance, which is considered to be the prototype of "worms''. However, taxonomically earthworms are not nematodes as they are segmented worms of the Phylum Annelida. • ematodes are elongated, cylindrical, unsegmented worms with tapering ends. The name "nematode" means "thread-like'; from "nema" meaning "thread''. Unlike u·ematodes and cestodes, all ofwhich are parasitic, most nematodes arefree-living forms found in soil and water. • Several species are parasites of plants and are of great economic importance. Many nematodes parasitize invertebrate and vertebrate animals. • The largest nwnber of helminthic parasites of humans belong to the class of nematodes. There are an estimated 500,000 species of nematodes. • GENERAL CHARACTERISTICS They are cylindrical, or filariform in shape, bilaterally symmetrical with a secondary triradiate symmetry al the anterior end. The adults vary greatly in size, from about a millimeter (Strongyloides stercoralis) to a meter (Dracuncu.lus medinensis) in length. Male is generally smaller than female and its posterior end is curved or coiled ventrally. Their body is covered wirh a tough outer cuticle, which may he smooth, striated, bossed, or spiny. 1he middle layer is hypodermis and the inner layer is the somatic muscular layer. They move by sinuousjlexion ofthe body. • The body cavity is a pseudocele, in which all the viscera are suspended. The digestive system is complete, consisting of an anteriorly placed mouth leading to the esophagus, which characteristically varies in shape and structure in differentgroups. The intestineislined with a single layerof Box 1: Types of female nematodes • Oviparous (laying eggs): - Unsegmented eggs: Ascaris, Trichuris - Segmented eggs:Ancy/ostoma, Necator - Eggs containing larvae: Enterobius • Viviparous (producing larvae): Trichinella, Wuchereria, Brugia, Dracunculus. • Ovoviviparous (laying eggs containing fully formed larvae, which hatch outimmediately): Strongylaides. columnar cells and leads to the rectum, opening through the anus. In the male, the rectum and the ejaculatory duct open into the cloaca. • Nematodes have simple excret01y and nervous systems. • The nematodes are diecious, i.e. the sexes are separate. • The male reproductive system consists ofa single delicate tubule differentiated into testis, vas deferens, seminal vesicle and ejaculatory duct, which opens into the cloaca. It also includes copulatory structures such as spicules or bursa or both. • The female reproductive system consists of the ovary, oviduct, seminal receptacle, uterus and vagina. • Female nematodes may produce eggs (oviparous) or larvae (viviparous). Some lay eggs containing larvae, which immediatelyhatch out (ovoviviparous) (Box l). • LIFE CYCLE The life cycle of nematodes consists typically offour larval stages and the aduJt form. The cuticle is shed while passing from one stage to the other. • Man is Lhe optimum host for all. the nematodes. They pass their life cycle in one host, except the superfamilies Filarioidea and Dracunculoidea, where two hosts are required. Insect vectors and Cyclops constitute the second hosts in these superfamilies, respectively. cmatodes localize in rhe intestinal tract and their eggs pass our with the feces of the host. They undergo few developmental changes before they enter new host.
- 182. • MODES OFINFECTION • By ingestion of: - eggs Ascaris, Enterobius, Trichuris - Larvae within intermediate host: Dracunculus - Encysted larvae in muscle: Trichinella • By penetration ofskin: Ancylostoma, Necator, Strongyloides • By blood-sucking insects: Filariae • By inhalation ofdust containingeggs: Ascaris, Enterohius. • CLASSIFICATION Nematodes can be classified on the basis of the habitat of the adult worm (Table 1) and zoologically (Table 2). Zoological Classification • Phylum:Nemathelminthes (Nematoda) • Class: Nematoda which is divided into two subclasses based on the absenceorpresenceof"phasmids'; which are caudal chemoreceptors. The two subclasses were earlier called Aphasmidia and Phasmidia, but now have been renamed as Adenophorea and Secernentea, respectively (Table 3). Detailed zoological classification of nematodes is given in Table 2. Table 1: Classification of nematodes on the basis of the habitat of adult worms Intestinal human nematodes Smallintestine • Ascaris /umbricoides (common roundworm) • Ancylostomaduodenale (Old World hookworm) • Necatoramericanus (American or New World hookworm) • Strongyloides stercoralis • Trichinella spiralis • Capil/aria philippinensis Large Intestine • Trichuris trichiura (whipworm) • Enterobius vermicularis (thread or pinworm) Somatic human nematodes Lymphatics • Wuchereria bancrofti • Brugia malayi • Brugia timori Skin/subcutaneous tissue • Loa Joa • Onchocerca volvulus • Dracunculus medinensis (guinea worm) Mysentery • Mansonella ozzardi • Mansonella perstans Conjunctiva • Loa loa Nematodes: General Features • LARVA MIGRANS The life cycles of most nematodes parasitizing humans include larval migration through various tissues and organs of the body. Sometimes the larvae appear to lose their way and wander around aimlessly. This condition is known as larva migrans. • This is generally seen when human infection occurs with nonhuman species of nematodes. In such infections, the worm is unable to w1dergo normal development and complete its life cycle. • Abnormal or arrested larval migration may also some- times occur when human parasitic nematodes infect immune persons. The immunity is sufficient to prevent the normal progression of infection. • Larva migrans can be classified into cutaneous or vis- ceral types, depending on whether the larval migration takes place in the skin or in deeper tissues (Table 4). Cutaneous Larva Migrans This condition also known as creeping eruption (also called ground itch) is caused bynematode larvae that infect byskin penetration. Etiology The most common cause is nonhuman species of hookworm (Ancylostoma braziliense and A. caninum) (Table 5). Pathogenesis Parasite eggs are passed in the feces of infected animals into the soil, where the larvae hatch out. • Infection with these hookworms of dogs and cats is acquired from soil contaminated with excreta of these animals. • On coming in contact with human skin, the larvae penetrate the skin to cause infection. • Between a few days and a few months after the initial infection, the larvae migrate beneath the skin. • In normal animal host, the larvae are able to penetrate the deeper layers of the skin by reaching there via circulation. • Once they enter intestine, they mature sexually and lay more eggs that are then excreted to repeat the cycle. • However, in a human host, which is an accidental host for the parasite, the larvae are unable to penetrate the basement membrane to invade the dermis, so that the disease remains confined to the outer layers ofthe skin. Clinical Features • The larvae produce itching papules, which develop into serpiginous tunnels in the epidermis. With the
- 183. Paniker'sTextbook ofMedical Parasitology Table2: Zoological classificat ion of nematodes Subclass Order Su erfamily Family Genus Species Adenophorea/ Enoplida Trichinelloidea (anterior part of Trichinellidae • Trichinefla • T. spiralis Aphasmidia (no body narrower than posterior) Trichuridae • Trichuris T. trichiura phasmids, no caudal • Capillaria C. philippinensis papillae in male, eggs C. aerophila usually unsegmented C. hepatica w ith polar plugs or hatching in uterus) S ecernentea/ Rhabditida Rhabditoidea (alternation Strongyloididae St rongyloides S. stercoralis Phasmidia (phasmids of free-living and parasit ic present, numerous generations, parasitic females caudal papillae) parthenogenetic) Strongylida • Ancylostomatoidea (prominent • Ancylostomatidae • Ancylostoma • A. duodenale buccal capsule with teeth or • Metastrongylidae • Necator • N. americanus cutting plates) • Angiostrongylus • A. cantonensis • Metastrongyloidea (tissue parasites, inconspicuous buccal capsule, have intermediate hosts) Ascaridida Ascaridoidea (large wormsof gut • Ascarididae • Ascaris • A. lumbricoides lumen, mouth has three lips) Oxyurida Oxyuroidea (male has no caudal bursa, short stout body, esophagus has prominent bulb, eggs planoconvex, embryonate in uterus) Spirurida • Filarioidea (tissue parasites, viviparous, insect vector) • Dracunculoidea (very long female and small male, viviparous, larvae escape from ruptured uterus) • Gnat hostomatoidea (spiny body w ith bulbous head) Table 3: Differences in subclass adenophorea and secernentea Adenophorea Phasmid (sensory st ructure) Excretory system Caudal papillae Infective stage of larva Absent Without lateral canals Absent or few First larval stage movements of the larva in the skin, the lesion also shifts, hence the name "creeping eruption''. Scratching may lead to secondary bacterial infection. • Transient creeping eruptions may be produced some- times by the human hookworm, Necator americanus. Gnathostomiasis and sparganosis may produce larva • Anisakidae Oxyuridae • Onchocercidae • Dracunculidae • Gnathostomatidae • Anisakis Enterobius • Wuchereria • Brugia • Dirofilaria • Loa • Mansonefla • Onchocerca • Dracunculus • Gnathostoma Secernentea Present With lateral canals Numerous Third larval stage • A. simplex E. vermicularis • W. bancrofti • 8.malayi • D. conjunctivae • D.immitis • L. loa • M. perstans • M. ozzardi • M. streptacerca • 0. volvulus • D. medinensis • G.spinigerum migrans, where the lesions are deeper, subcutaneous or in the muscles. Loeffler's syndrome may occur in one- fourth to one-halfof the cases. • A rapidly moving lesion is produced by Strongyloides stercoralis particularly in immune persons. This is known as larva currens.
- 184. Table 4: Animalnematodes infecting man Viscerallarvamigrans It is a syndrome caused by nematodes that are normally parasitic for nonhuman host species In human, these nematode larvae do not develop into adult worms, but, instead, migrate through host tissues and elicit eosinophilic inflammation Common causes: • T oxocara canis(dog roundworm)- most common • Taxocara cati (cat roundworm) • Ascarissuum (pig ascaris) • Angiosrrongylus cantonensis • Gnathostoma spinigerum • Anisakissimplex • Baylisascarisprocyonis Table 5: Etiological agents (cutaneous larva m igrans) Zoophilic nematode • Ancylosroma braziliense • Ancylostoma caninum • Gnathostomaspinigerum • Dirofrlaria • Spirometra • Uncinaria stenocephala • Bunostomum phlebotomum Human nematode • Strongy/oides srercoralis • Necatoramericanus • Loa loo Human trematode • Ectopic infection with Fasciola and Paragonimus Nonhelmenthic agents • Flies of genus Hypoderma and Gastrophilus Nematodes: General Features Cutaneous larvamigrans • It isa serpiginous skin eruption caused by burrowing larvae ofanimal hookworms (usually the cat and the cat hookworm) • The larvae hatch from eggs passed in dog and cat feces and mature in the soil. Humans become infected after skin contact with contaminated soil. After larvae penetrate the skin, erythematous lesionsform along the tortuous tracksof their migration. It isalso known as creeping eruption Common causes: • Ancylosroma braziliense (hookworm of wild and domestic dogs and cats) • Ancylosroma caninum (dog hookworm found in Australia) • Uncinaria srenocephala (dog hookworm found in Europe) • Bunostomum phlebotomum (cattle hookworm) Table 6: Etiological agents (visceral larva migrans) Zoophilicnematode Nonhuman nematode • Taxocara canis • Filariaspp. • Toxocara cat/ • Dirofrlariaimmitis • Angiostrongylus cantonensis • Brug/apahangi • Brugiapatei • Angiostrongylus costaricensis • Anisakis • Gnathostoma spinigerum Human nematode • Ascaris lumbricoides • Strongyloides stercoralis Visceral Larva Migrans This condition is caused by the migration of larvae of nonhuman speciesofnematodes that infect by the oral route. • Creeping myiasis is caused by flies of the genus Hypoderma and Eastrophilus. Etiology The most common cause is the dog ascarid, Toxocara canis and less often the cat ascarid, T. cati. Visceral larva migrans may also be caused by Anisakis, which are large ascarid parasites of marine animals and also by Gnathostoma spinigerum, Angiostrongylus cantonensis. Human nematodes like A. lumbricoides and S. stercoralis may produce visceral larva migrans, when they get lost in ectopic sites (Table 6). • Ectopic infections with Fasciola and Paragonimus may produce creeping lesions on abdominal wall. Diagnosis Eosinophilia is rare and occurs only when Loefler's syn- drome develop. • Serological tests are not developed. • On biopsy, larvae are rarely found in the skin lesion. 11iagnosis is based mainly on clinical features. Treatment Th iabendazole is useful in treatment. When the lesions are few, freezing the advancing part of the eruption with ethyl chloride is effective. Pathogenesis When the infective eggs present in the soil contaminated by dog and cat feces are ingested, the larvae hatch in the small intestine, penetrate the gut wall, and migrate to the liver. • They may remain there or migrate to other organs such as lungs, brain, or eyes. • In humans they do not develop into adults, but induce granulomatous lesions, which cause local damage.
- 185. Paniker'sTextbook ofMedical Parasitology Table7: Difference between cutaneous and visceral larva migrans Cutaneous larvamigrans S kin Viscerallarvamigrans Tissue involved Infecting organism Portal ofentry E osinophilia Serodiagnosis Treatment Mostly bynonhuman nematodes Penetration of skin Various organs of body like liver, lungs and eyes Mainly by dog and cat (Toxocara spp.) Ingestion ofinfected eggs Clinical Features Mild Notdeveloped Thiabendazole Clinical manifestations depend on the sites affected and the degree and duration ofinfection. • As children are more likely to swallow dirt, this condition is much more frequent in them. • Fever, hepatomegaly, pneumonitis, hyperglobulinemia and pica are th e common findings. • Patients may develop neurological disturbances (neural larva migrans) and endophthalmitis (ophthalmic larva migrans). • Marked leukocytosis occurs with persistently high eosinophilia. Diagnosis Serological tests, such as passive hemagglutination, bentonite nocculation, microprecipitation, and more specifically, enzyme-linked immunosorbent assay (ELISA) have been developed for Lhe diagnosis of toxocariasis (visceral larva migrans). Treatment Diethylcarbamazine (DEC), 100 mg TDS for 3 weeks in an adult, killsthe larva and arrest thedisease. Thiabendazole may be useful in treatment/ Prednisolone should be administered concurrently either topically or systemically. Prophylaxis Deworming ofhousehold petshelps in prevention by limiting the contamination ofsoil. Differences between cutaneous and visceral lar va migrans are given in Table 7. Persistent high Well developed Diethylcarbamazine and prednisolone KEY POINTS OF CUTANEOUS AND VISCERAL LARVA MIGRANS • Sometimes larvae lose their way and wander around aimlessly in human body, this condition is known as larva migrans (cutaneous or visceral). • Mainlycaused by nonhuman species of nematodes (zoophilic helminths), but occasionally by nonhelminthic agents like mite and larvae of fly (myiasis). • Man acquires the infection as an accidental host. • Abnormal migrations also occur sometimes in human nematodes. • The helminths are unable to complete their development and life cycle in man and are arrested at some level in skin or other organs like lung, liver, etc. • Pathogenesis: Due to mechanical damage and host's inflammatory response against parasitic antigen. • Clinical manifestations: Depend on route of entrance, sites affected, and degree and duration of infection. • Diagnosis: Based mainly on clinical features, skin biopsy and serology. • Treatment: Symptomatic and specific therapy with antihel- minthics. REVIEW QUESTIONS 1. Describe briefly: a. General characters of Phylum Nematoda b. Systematic classification of nematodes 2. Short notes on: a. Classification of nematodes based on habitat b. Cutaneouslarva migrans c. Visceral larva migrans d. Viviparous nematodes e. Larva currens 3. Differentiate between class Adenophorea and Secernentea. 4 . Enumerate the etiological agents of cutaneous and visceral larva migrans.
- 186. MULTIPLE CHOICE QUESTIONS 1.All of the following nematodes are oviparous except a. Ascaris b. Ancylostoma c. Trichinella d. Enterobius 2. Nematoda residing in large intestine a. Necatar b. Trichinella c. Strongyloides d. Trichuris 3. All of the following are somatic nematodes except a. Loaloa b. Capillaria phi/ippinensis c. Onchacerca vo/vulus d. Brugia malayi 4. Most common cause of visceral larva migrans a. Ancylostoma braziliensis b. Anisakis simplex c. Strongyloides stercora/is d. Toxocara canis Nematodes: General Features 5. Cutaneous larva migrans isdue to a. Ancyclostoma braziliensis b. Wuchereria bancrofti c. Brugia malayi d. Dracuncu/us medinensis 6. A teenager who plays with dogs developed skin rash, eosinophilia, and an enlarged liver and spleen for 1 year. The most likely cause of this infection is a. Trichinosis b. Schistosomiasis c. Toxoplasmosis d. Visceral larva migrans Answer 1. c 2. d 3. b 4. d 5. a 6. d
- 187. CHAPTER 14 • INTRODUCTION •Trichinella spiralis, tissue nematode, is the causative agent of trichinosis. • The name Trichinella is derived from the minute size of the adult (Greek trichos-hair, ella suffix for diminutive, spiralis refers to the spirally coiled appearance of larvae in muscles). • COMMON NAME Trichina worm. • HISTORY AND DISTRIBUTION • It was first observed in 1821 in the muscles of a patient at autopsy by James Paget, who was then a first year medical student at St Bartholomew's Hospital, London. • Owen, in 1835, described the encysted larval form in muscles and named it Trichinella spiralis. • Virchow discovered its life cycle in 1859. • The major source ofhuman infection was shown to be the consumption ofinadequatelycooked pork. • Trichinosis is recognized as an important public health problem in Europe and America, but is much less common in the tropics and oriental countries. • Human trichinosis had not been recorded in India till 1996, when the first case was reported from Punjab. • HABITAT Adult worms live deeply buried in the mucosa of small intestine (duodenum orjejunum) ofpig, bear, rat, or man.The encysted larvae are present in the striated muscles of these hosts. There are no free-living stages. • MORPHOLOGY Adult Worm The adult T. spiralis, a smallwhiteworm justvisible to the naked eye, is one of the smallestnematodes infecting humans. The male measures about 1.5 mm by 0.04 mm and the female about3 mm by0.06 mm (twice the length ofmale). The anterior halfof the body is thin and pointed, well- adapted for burrowing into the mucosa! epithelium (Fig. 1). 1he posterior end of the male has a pair of pear-shaped clasping papillae (termed as claspers), one on each side of the cloaca/ orifice that it uses to hold the female worm during mating (Fig. 1). Fig. 1: Adult worms of Trichinel/a spiralis (male and female)
- 188. • The femaleworm is viviparous and discharges larva instead ofeggs. • The lifespan of the adult worm is very short. The male worm dies soon after fertilizing the female and the female dies after 4 weeks to 4 months (16 weeks), the time required for discharging the larvae. Larvae The larva becomes encysted in the striated muscle fiber (Fig. 2) and at the time of encystment measures 1 mm in length by 36 µm in diameter. The larva in the cyst is coiled and hence, the name spiralis. Trichinella Cyst • Cysts are ovoid 400 mcm by 250 mcm in size. • The cyst is formed by the tissue reaction around the encapsulated larvae. • Cysts develop preferentially in muscles relatively poor in glycogen and in hypoxic environment. Therefore, the diaphragm, biceps, muscles of jaw, ex:traocular muscles, neck, and lower back, which are constantly active, are the ones mostly affected. Cysts are more abundant near the sites of attachment of muscles to tendons and bones than in otherparts. They lie longitudinally along the muscle fibers. The deltoid being easily accessible, is chosen for taking diagnostic muscle biopsies. • The larva remains infective inside the cyst for years and eventually, most become calcified and die. • LIFE CYCLE TrichinelLa is a parasite that has a direct life cycle, which means it completes all stages of development in one host. But only a single cycle occurs in one host and for continuation of the cycle and maintenance of the species, it is necessary for the infection to be transmitted to another host ofthe same species or ofdifferent species (Fig. 3). • Optimum host: Pig. • Alternate host: Man. • Infection can pass from-pig-to-pig (facilitated by the custom offeeding pigswith untreated household garbage, which may contain bits of pork with infective cysts), rat- to-rat and pig-to-rat (Table 1). • Man is the dead-end of the parasite, as the cysts in human muscles are unlikely to be eaten byanother host. • Inf ectiveform: Encysted larva found in the muscles of pigs and other animals (Fig. 2). Mode of infection: Man acquires infection mainly by eating raw or undercooked pork or inadequately Trichinella Spira/is Fig. 2: Encysted larva in muscles; infective stage processed sausages or other meat products containing the viable larvae. • When such meat is eaten without adequate cooking, the cysts are digested by the gastric juice and viable larvae are released (excystation) in the stomach, duodenum and jejunum. • The larvae immediately penetrate the mucosal epithelium • They m oult four times and rapidly develop into adults, either male or female, by the 2nd day of infection. withnin 5 days, they become sexually mature. • The male dies after fertilizing the female. The fertilized females start releasing motile larvae by the 6th day of infection. Larvae continue to be discharged during the remaining part of the lifespan of the female worm, which ranges from 4 weeks to 4 months. • Each female gives birth to approximately 1,000 larvae. • " These larvae enter the intestinal lymphatics or mesenteric venules and are transported in circulation to different parts ofthe body. • They get deposited in the muscles, central nervous system and other sites. The larva dies in most other situations, except the skeletal muscles, where it grows. Deposition in the muscles occurs mostly during the 2nd week of infection. Larval development in muscles takes place during the next 3 or 4 weeks. • Within 20 days after entering the muscle celJs, the larvae become encysted. Amuscle cell carryinglarva of T. spiralis is called as a nurse cell. Encysted larvae lie parallel to the muscles ofhost. • Encysted larva can survive for months to years. In man, die life cycle ends here (Fig. 3). • Smoking, salting or drying the meat does not destroy the infective larvae. Prolonged freezing (20 days in a normal freezer or at -20°C for 3 days) decontaminates the meat.
- 189. Paniker'sTextbook ofMedical Parasitology ~?"8. ~ Larvae released from due to the action of ;g digestive enzymes ;? ~ " _ _,__ Striated muscle Man Pig Adult worms in small intestine Larvae die in other tissues, except striated Encysted larva in striated muscle muscles Man dead-end (cycle ends) ~ Larva undergoes encystment in muscle and nurse cell-larva complex formed t Fig. 3: Life cycle of Trichinella spiratis Table 1: Parasites with source of infection Pork Fish • Taenia solium • Diphyllobathrium latum • Trichine/Ja spiralis • C/onorchis sinensis • Sarcocystis suihominis • Metagonimus yokogawai • Heterophyes spp. • Gnathostoma spp. Beef • Taenia saginata • Sarcocystis hominis • Toxoplasma gondii • PATHOGENICITY AND CLINICAL FEATURES The disease caused by T. spiralis is called trichinosis. • The manifestations vary from asymptomatic infection, which is very common, to an acute fatal illness, which is extremely rare. • The pathology and clinical features vary according to the stage in the life cycle ofthe worm (Table 2). • DIAGNOSIS Diagnosis of trichinosis can be made by direct and indirect methods. Direct Methods • Detection of spiral larvae in muscle tissue by performing muscle biopsy. Deltoid, biceps, gastrocnemius, or pectoralis muscles are usually selected for biopsy (Box 1). • Detection of adult worms and larvae in the stool during the diarrheic stage. • Xenodiagnosis: For xenodiagnosis, biopsy bits are fed to laboratory rats, which are killed in a month or so, later. The larvae can be demonstrated more easily in the muscles ofsuch infected rats (Flow chart 1).
- 190. Trichinella Spira/is Table 2:Stages in the life cycle of Trichinella spiralis (in man) Stage of intestinalinvasion: Stage ofmuscle invasion: Stage ofencystation: First stage Second stage Fina/stage Pathology The stage begins with the ingestion of raw pork containing Infective larvae and ends with the larvae invading the intestinal epithelium and developing into adult The stage begins when new infective larvae are released from the adult female and ends with the deposition of the larvae in the muscles. Myositis and basophilic granular degeneration of muscles occurs in this stage This stage occurs only in striated muscle. The infective larvae become encysted in t his stage Clinical Malaise, nausea, vomiting, diarrhea, Fever, myalgia, periorbital edema, weakness of affected muscle, hemorrhage in subconjunctiva and new beds (splinter hemorrhages), myocarditis (if heart musclesare involved) and encephalitis (if central nervoust issue is involved). Eosinophilia is a constant feat ure of this stage. The stage isseen 1-4 weeks after infection All symptoms subside features abdominal cramps. Onset within 2- 30 hours of ingestion of infective food Box 1: Muscle biopsy • Muscle biopsy specimen iscollected for demonstration ofspiral larvae. • Specimen: Deltoid, biceps, gastrocnemius, or pectoralis. • At least 1 gram of muscle should be taken for biopsy, preferably near tendon insertion. • Examination technique: Muscle fibersare digested with trypsin and mounted on a glass slide and examined under microscope. Young larvae may be digested and missed during such examination. - A teased preparation of muscle tissue isprepared in a drop of saline solution and it is squeezed between two glass slides. - Muscle tissue is stained with safranin. + Direct methods Flow chart 1: Laboratory diagnosis of Trichinella spiralis Laboratory diagnosis I + Indirect methodsI ... Muscle biopsy Alternative method for definitive diagnosis. Demonstrates larva in muscle tissue Xenodiagnosis History History of consumption of raw or inadequately cooked pork- • Serology • Radiological examination Calcified cysts can be detected on X-ray Stool examination May demonstrate adult worms and larvae Indirect Methods Detection of antibody by: • ELISA (Confirmatory test) 2 weeks earlier • Bentonite flocculation test • Latex fixation test Blood examination Differential blood count shows eosinophilia (20-95%) • Raised levels of muscle enzymes, including creatine phosphokinase • Serology: Bachman intradermal test The test remains positive for years after infection PCR Uistoty of consumption of raw or inadequately cooked or processed pork, about 2 weeks earlier along with a recent episode ofgastroenteritis. There is massive hypergammaglobulinemia with elevated serum imm unoglobulin E (IgE). Blood examination: It shows eosinophilia (20-95%). T. spiralis antibody can be detected by enzyme- linked immunosorbenc assay (ELISA) test using TSL-1 secreting antigens obtained from the infective
- 191. Paniker'sTextbook ofMedical Parasitology stagelarvae. Bentonite flocculation test and latex fixation test for demonstration of antibodies have also been widely used. Apositive test indicates recent infection. • Bachman intra.dermal test: It uses a 1:5,000 or 1:10,000 dilution of the larval antigen. An erythematous wheal appears in positive cases within 15- 20 minutes. The rest remains positive for years after infection. • Radiological examination: Calcified cysts may be demonstrated on X-ray examination. • Molecularmethodslike multiplexpolymerase chain reac- tion (PCR) are now being used for species identification of Trichinetla (Flow chart 1). • TREATMENT • Mild cases: Supportive treatment consisting of bedrest, analgesics and antipyretics. • Moderate cases: Albendazole 400 mg BID for 8 days or mebendazole 200-400 mg TID for 3 days, then 400 mg TlD for 8 days. • Severe cases: Add glucocorticoids like prednisolone to albendazole or mebendazole. Note: Mebendazole and albendazole are active against enteric stage of the parasite, but their efficacy against encysted larva has notyet been completely demonstrated. • PROPHYLAXIS • Proper cooking ofporkand other meat likely to be infected. • The most effectivemethod is to stop the practice offeeding pigs with raw garbage. Extermination of rats from pig farms-the spread of infection. KEY POINTS OF TR/CH/NELLA SP/RAUS • One of the smallest nematodes infecting humans (1.5-3 mm). • Entire life cycle is passed in one host. • The fema le worm is viviparous. • Optimum host: Pig. • Alternate host: Man. Man is the dead-end for parasite. • Infective form: Encysted larvae in the striated muscles of pigs and other animals. • Larvae remain encysted tightly coiled in striated muscles in human body. • Muse/es commonly involved: Diaphragm, pectoralis, deltoid, biceps and gastrocnemius. • Pathogenesis: Myositis and basophilic degeneration of the muscles. • Clinical features: Malaise, diarrhea, periorbital edema, muscle weakness, myocarditis, encephalitis. • Diagnosis: Muscle biopsy for larvae, stool examination for adult worm or larvae, xenodiagnosis, Bachman intradermal test, ELISA, X-ray for calcified cyst, PCR. • Treatment: Albendazole and mebendazole along with corticosteroids (in case of severe infection). REVIEW QUESTIONS 1. Name the various intestinal nematodes and describe briefly the life cycle of Trichinella. 2. Write short notes on: a. Trichinella cysts b. Laboratory diagnosis of Trichinel/a spiralis MULTIPLE CHOICE QUESTIONS 1. Larva found in muscle is a. Trichinella spiralis b. Ancylostoma duodena/e c. Trichuris trichiura d. Enterobius vermiculoris 2. Which of the following is not a neuroparasite a. Taenia solium b. Acanthamoeba c. Naegleria d. Trichinel/a spiralis 3. Which of the following is viviparous a. Strongyloides stercoralis b. Trichinella spiralis c. Enterobius d. Ascaris 4. Best site for taking biopsy for diagnosis of trichinellosis is a. Deltoid muscle b. Diaphragm c. Pectoralis major d. Liver 5. Bachman's test is done to diagnose infectionswith a. Schistosomajaponicum b. Trichinella spiralis c. Trichuris trichiura d. Ancylostoma duodenale 6. The larval form of Trichinella can be destroyed by a. Smoking of meat b. Deep freezing of meat c. Drying of meat d. Salting of meat Answer 1. a 2. d 3. b 4. a 5. b 6. b
- 192. CHAPTER 15 • INTRODUCTION •The name Trichuris means a "hair-like tail" (Greek trichos- hair, oura-tail). lhis name is not quite correct because it is the anterior end of the worm that is hair-like and not the tail. the name whipworm is more apt as the thick posterior part resembles the stock and thin anterior end resembles the lash of a whip. • The helminth causes trichiuris in humans, an intestinal infection caused by invasion of colonic mucosa. • COMMON NAME Whipworm. • HISTORY AND DISTRIBUTION • Trichuris trichiura, the human whipworm, was first described by Linnaeus in 1771. • The antiquity of the whipworm as a human parasite is indicated by the demonstration of its eggs in colonic contents of a young man, who died on the Alps some 5,300 years ago and whose well-preserved body was discovered in 1990. • It is worldwide in distribution, but is much more common in the tropics. The infection is widespread in tropical Afri ca, South America and South-cast Asia. Trichuris infection is found throughout India. • Some 800 million people are estimated to be infected with this worm. • While whipworm infection is extremely frequen t, whipworm disease is relatively rare. • HABITAT T. trichiura lives in the large intestine (Box 1). The adult worms are found attached to the wall of the cecum and less commonly to the vermiform appendix, colon and anal canal. • MORPHOLOGY Adult Worm The male worm is 30- 45 mm long, while the female is slightly larger, about 40-50 mm. . . . . The worm is flesh-colored. In shape, it resembles a whip, with the anterior three-fifth (3/ 5) thin and thread-like and the posterior two-fifth (2/ 5) th ick and fleshy, appearing like the handle of a whip (Figs l Aand B). The anenuated anterior portion, which contains the capillary esophagus, is embedded in the mucosa. The posterior part contains the intestines and reproductive organs. The posterior end of the male is coiled ventrally, while the hind end of the female is straight, blunt and rounded (Figs IAand B). 1l1e worm has a lifespan of5-10 years. Egg the egg has a characteristic appearance. It is brown in color being bile-stained. • It has a triple shell, the outermost layer of which is stained brown. • It is barrel-shaped and about 50 mcm long and 25 mcm wide in the middle, with a projecting mucus plug at each pole containing an unsegmented ovum (Figs 2A and B). The plugs are colorless. • The egg floats in saturated salt solution (Boxes 2 and 3). Box 1: Nematodes present in large intestine • Enterobius vermicularis • Trichuris trichiura • Oesophagosromum spp.
- 193. Paniker'sTextbook ofMedical Parasitology 5 ~E!i!i:•==S!!!!!!!!~i!!t~•~,i~11 ~~ ~ ~ ~ Comma or ;::> ~ ~~~t~hri~eednd Very thin IP.I anterior Iii portion Figs lA and B: (A) Adult Trichuris trichiura worms (male and female); and (B) Specimens of male and female whipworm Mucous plug • Figs 2A and B: Egg of Trichuris trichiura. (A) As seen under microscope; and (B) Schematic diagram Box 2: Helminths whose eggs float in saturated salt solution • Enterobius vermicularis • Ancylostoma duodenale • Necatoramericanus • Ascaris lumbricoides • Trichuris trichiura • When freshly passed, the egg contains an unsegmented ovum. At this stage, it is not infective for humans. • The fertilized female lays about 5,000 eggs per day. • LIFE CYCLE Natural host: Man. No intermediate host is required. Box 3: Helminths whose eggs do not float in the saturated solution • Eggs of Taenia solium and Taenia saginata • Eggs of all intestinal flukes • Unfertilized eggsof Ascaris lumbricoides Infectiveform: Embryonated eggs containing rhabditiform larva. • Adult female worm lives in large intestine, worm lays eggs which are discharged in feces. • The egg undergoes development in soil, optimally under warm, moist, shady conditions, when the infective rhabditiform larva develops within the egg in 3-4 weeks. At lower temperatures, this may be delayed for 3 months
- 194. Trichuris Trichiura ~ Developsinto adult worms Passed down to cecum I liberated larva in small intestine Larva liberated through one of the poles of egg in small intestine Man Egg passed in feces ---------------------- ,,,_--- Ingested -- embryonated egg Man acquires infection by with infective consuming food and water rhabditiform larva contaminated with embryonated egg Fig. 3: Life cycle of Trichuris trichiura or more {Fig. 3). These embryonated eggs are infective to man. Mode of transmission:Infection occurs in humans when the mature embryonaled eggs containing the infective larvae are swallowed in contaminatedfood or waler. The eggs hatch in the small intestine and Lhe larva, which emerges through the pole of Lhe egg, passes down into the cecum. In about 2-3 months, they become man1re adults and lie embedded in the cecal wall, with the thread-like anterior portion piercing the mucosa and the thick posterior end projecting out. the gravid adult female lays eggs, which are discharged in feces and the cycle is repeated (Fig. 3). The entire life cycle can be passed in one host, from the ingested infective egg to the development of the adults and the release of their eggs in feces. But for transmission ofinfection to other hosts and perpetuation of the species, the egg has to undergo development in the soil and then infect another person. • Humans are the only natural host for T. trichiura, but morphologically similar worms are found to infect pigs and some monkeys. • Eggs start appearing in feces usually about 3 months after infection. • PATHOGENICITY AND CLINICAL FEATURES Infection with T. trichiura (trichuriasis, whipworm infection, or trichocephnliasis) is asymptomatic, except when the worm load is heavy. Disease may result either due L o mechanical effects or allergic reaction.
- 195. CHAPTER 16 • INTRODUCTION Normand(1876) observed minute cylindrical worms in the diarrheic feces and intestinal walls of some French soldiers in Cochinchina. 111ese were named Strongyloides stercoralis (strongylus- round, eidos-resembling, stercoralis-fecal). • HISTORY AND DISTRIBUTION • It is found mainly in the warm moist tropics, but may also occur in the temperate regions. It is common in Brazil, Columbia, and in the Far East-Myanmar, Thailand, Vietnam, Malaysia and Philippines. • Another species S. fullerborni is widely prevalent in African monkeys. It infects pygmies in the forests ofZaire and Zambia. It also causes human infection in Papua New Guinea. Trichostrongylus, a parasite of sheep and goats, seen in Africa and Asia (including India), may cause human infection, which is usually asymptomatic (Table 1). • HABITAT The adult worm is found in the small intestine (duodenum and jejunum) of man (Box l). • Largest nematode known to cause human infection: Ascaris lumbricoides. • Smallest nematode known to cause human infection: Strongyloides stercoralis. • MORPHOLOGY Adult Worms Female Worm 111e female worm is thin, transparent, about 2.5 mm long and 0.05 mm wide (Fig. 1). • It has a cylindrical esophagus occupying the anterior one-third of the body and the intestines in the posterior Table 1: Difference between filariform larva of hookworm and Strongyloides Hookworm Strongy/oides • Esophagus extended up to 25% • Esophagus extended up to 40% of the total body length ofthe total body • Sheathed • Nonsheathed • Tail: Pointed • Tail: Forked Box 1: Nematodes present in small intestine • Strangyloides stercoralis • Ascaris lumbricoides • Ancylostoma duodenale • Necator americanus • Trichinella spiralis • Trichostrongylus spp. • Capillaria philippinensis. two-thirds, opening through the anus situated ventrally, a little in front ofthe pointed tail tip. • The reproductive system contains paired uteri, vagina and vulva. The paired uteri lead to the vulva situated at the junction of the middle and posterior thirds of the body. In the gravid female, the uteri contain thin-walled transparent ovoid eggs, 50 µm by30 µmin size. • the worm is ovoviviparous. • the individual worm has a lifespan of 3 or 4 months, but b eca use it can cause a utoinfection, the infection m ay persist for years. Male Worm the male worm is shorterand broader than female measuring 0.6- 1 mm in length and 40-50 mcmin width. • The copulatory spicules, which penetrate the female during copulation, are located on each side of the gubernaculum (Fig. 1).
- 196. • They arenot seen in human infection because they do not have penetrating power, therefore do not invade the intestinal wall. Eggs Eggs are conspicuous within the uterus ofgravid female. • Each uteruscontains8- 10eggs arranged anteroposteriorly in a single row (Fig. 1). • They are oval and measure 50-60 mcm in length and 30-35 µrn in breadth (Fig. 2). • As soon as the eggs are laid, theyhatch out to rhabditiform larva (first stage larva). Thus, it is the larva and not the egg, which is excreted in feces and detected on stool examination and not egg. Larva Rhabditiform Larva (L1Stage) (Fig. 3A) t his is d1e first stage of larva. Eggs hatch ' 10 form Ll larva in the smaJI intestine. • It is the most common form of the parasite found in the feces. • It measures 0.25 mm in length, with a relatively short muscular double bulb esophagus (fig. 3B). • The Ll larva migrates into the lumen of the intestine and passes down the gut to be released in feces. Filariform Larva (L3 Stage) This is the third stage of larva. • Ll larva moults twice to become the L3 larva. • It is long and slender and measures 0.55 mm in lengd1 with a long esophagus of uniform width and notched tail (Fig. 3C). • It is the infective stage of the parasite to man. Strongyloides Stercoralis Anal opening f o' Vulval Esophagus Fig. 1: Adult worm (male and female) Rhabditiform larva Fig. 2: Egg of Strongyloides stercoralis "Double bulb" esophagus Long, slender esophagus ll Notched tail Figs 3A to C: Larvae of Strongyloides stercoralis. (A and B) Rhabditiform Larva (Courtney Dr Anita Nandi); (C) Filariform larva
- 197. Paniker'sTextbook of MedicalParasitology Filariform larva penetrates skin of man (Definitive host • LIFE CYCLE In the small intestine, ~ larvae mature into / adult worms Larva enters circulation and via heart, lungs, respiratory tree, and esophagus, reach small intestine I ~ .... ........ ........ "'lt,t, o,;, Penetrates the ~Ct,· perianal skin 1 01) Man Soil Direct cycle Rhabditiform larva directly metamorphose to infective filariform larva in soil "Double bulb" esophagus Indirect cycle Egg in soil Fig.4: Life cycle of Strongy/oides stercoralis Infective Form Filariform larva. • Mode ofinfecti.on: ,, Adult female embedded in the mucosa of small intestine .,, ,,, ,,,. / ,,, ~ Egg containing the larva J 0) habditiform larva immediately released and passes out in feces The life cycle of S. stercoralis is complex because of the multiplicity of pathways through which it can develop. It is unique among human nematodes as it has a parasitic cycle and a Cree-living soil cycle, in which it can persist for long periods in soil by feeding on soil bacteria, passing through severalgenerations (Fig. 4 and Flow chart 1). Penetration ofskin by the third stage filariform larva, when a person walks barefoot Natural Host Man, although dogs and cats are found infected with morphologically indistinguishable strains. - Autoinfection (Box 2). • The adult female worm is found in the human intestine embedded in the mucosa of the duodenum and upper jejunum. • Since only the female worms are seen in the intestine, it was earlier believed that they are parthenogenetic and
- 198. Flow chart 1:Life cycle of Strongyloides stercoralis Female worm in intestine lays eggs l Rhabditiform larvae hatch out Develop into filariform larvae in ut Pass through feces into soil Passed in feces Direct cycle Develop into filariform larvae in soil Indirect cycle Develop into free-living males and females Penetrate gut wall "internal reinfection" Autoinfection by piercing perianal and perinea! skin Parasitic phaseI Box 2: Autoinfection Penetrate skin of feet to infect another host Females lay eggs! l Rhabditiform larvae hatch out Become Develop into filariform free-living larvae which males and infect humans females --...--.... Free-living phase I • External autoinfection: S. srercora/1s has a cycle of autoinfection. Here the rhabditiform larvae mature into the infective third stage larvae during their passage down the gut. These filariform larvae cause reinfection by piercing the perianal and perinea! skin during defecation. The larvae wander in the dermis of the perianal region for sometime, causing a radiating perianal creeping eruption, a form of cutaneous larva migrans. They ultimately enter the lymphatics or venules and are carried to the right heart and the lungs to complete the life cycle as earlier. • Internal autoinfection: In this type of autoinfection, seen typically in immunodeficient hosts, the rhabditiform larvae mature into the infective filariform larvae in the bowel itself. The filarlform larvae penetrate the deeper layers of the intestine, to reach the mesenteric venules and are carried in circulation to complete the life cycle. This mode of autoinfection is called internal reinfection. It may lead to very heavy infection causing serious and sometimeseven fatal illness. can produce offsprings without being fertilized by the male. But, it has nowbeen established that parasitic males do exist. They can be demonstrated in experimentally infected dogs. They are not seen in human infections because they do not invade the intestinal wall and so are eliminated from the bowel soon after the females begin to oviposit. However, the majority of females are probably parthenogenetic. • The eggs laid in the mucosa hatch immediately, releasing rhabditiform larva. Strongy/oides Stercoralis The rhabditiform larva migrates into the lumen of the intestine and passes down the gut to be released in feces. • The rhabditiform larva may even metamorphose into .filariform larva during passage through the bowel. These filariform larvae may penetrate colonic mucosa or perianal skin without leaving the host and going to the soil, thus providing a source of autoinfection. 1l1is ability to cause autoinfection explains the persistence of the infection in patients for long periods, even 30-40 years, after leaving the endemic areas. The rhabditiform larva voided with the feces mayundergo two types of development in the soil (Flow chart 1): 1. Directdevelopment 2. Indirect development. Direct development: l he rhabditiform larva on reaching the soil moults twice to become the infective filariform larva. - Each rhabditiform larva gives rise to one filariform larva. When apersonwalks barefooton soilcontaining the infective filariform larvae, they penen·ate the skin and enter the circulation. The larvae are carried along the venous circulation to the right side of the heart and to the lungs. Here, they escape from the pulmonary capillaries into the alveoli, migrate up the respiratory tract to the pharynx, and are swallowed, reaching their final destination, small intestine. - In the intestine, they mature into adult parasitic females and males in 15-20 days. Female worms then burrow into the mucosa ofthe intestine and lays eggs. - 1he rhabditiform larvae hatch out immediately and enter into lumen of the bowel. They are excreted in the feces and thus, the life cycle is repeated. • Free-living phase/ indirect development: The rhabditiform larva passed in stools develop in moist soil into free-living males and females. They mate in soil. The fertilized female lays eggs, which hatch to release the next generation of rhabditiform larvae. - These may repeat the free-living cycle or may develop into the filariform larvae, which infect humans and initiate the parasitic phase. • PATHOGENICITY AND CLINICAL FEATURES Strongyloidiasis (infection caused byS. stercoralis) is generally benign and asymptomatic. Blood eosinophilia and larvae in stool being the only indications ofinfection. Sometimes it may cause clinical manifestations, which may be severe and even fatal, particularly in those with defective immune response. • The clinical disease may have cutaneous, pulmonary and intestinal manifestations.
- 199. Paniker'sTextbook ofMedical Parasitology CutaneousManifestations There may be dermatitis, with erythema and itching at the site of penetration of the filariform larva, particularly when large numbers of larvae enter the skin. • In those sensitized by prior infection, there may be an allergic response. • Pruritus an d urticaria, particularly around the perianal skin and buttocks, are sym ptoms of chronic strongyloidiasis. • the term larva currens (meaning racing larvae) has been applied to the rapidly progressing linear or serpiginous urticaria! tracks caused by migrating filariform larvae. These often follow autoinfection and start perianally. Pulmonary Manifestations When the larva escape from the pulmonary capillaries into the alveoli, small hemorrhages may occur in the alveoli and bronchioles. Bronchopneumonia may be present, which may progress to chronic bronchitis and asthmatic symptoms in some patients. • Larva of Strongyloides may be found in the sputum of these patients. Intestinal Manifestations The symptoms may resemble those of peptic ulcer or of malabsorption syndrome. • Mucus diarrhea is often present. In heavy infection, the mucosa may be honeycombed with the worm and there may be extensive sloughing, causing dysenteric stools. • Other manifestations are protein-losing enteropathy and paralytic Beus. Hyperinfection In debilitated individuals and particularly in those with cellular immune defects, extensive internal reinfection takes place, leading to an enormous number of adult worms in the intestines and lungs and larvae in various tissues and organs. This is known as hyperinfection. • Severemalnutrition, lepromatousleprosy, lymphoreticular malignancies, acquired immunodeficiency syndrome (AIDS), immunosuppressive drugs and other situations, in which cell-mediated immunity is defective, predispose co this condition. Hyperinfection is an important hazard of steroid therapy and other instances of prolonged immunosuppression as in transplant patients. During hyperinfection, the filariform larvae may enter in to arterial circulation and lodge in various organs, e.g. heart, lungs, brain, kidney, pancreas, liver and lymph nodes. Manifestations depend on the sites affected. Brain abscess, meningitis and peritonitis are major fatal complications. It has been reported that circulating Strongyloides larvae may carry intestinal bacteria, causing septicemia. • LABORATORY DIAGNOSIS Microscopy Direct wet mount of stool: Demonstration of the rhabditiform larvae in freshly passed stools is the most important method of specific diagnosis. Larvae found in stale stools have to be differentiated from larvae hatched from hookworm eggs (Flowchart 2). Concentration methods ofstoolexaminatum:Stool may be concentrated by formol-ether concentration method or Baermann's funnel gauze method and examined for larvae more efficiently. Baermann's test requires a special apparatus and relies on the principal that larva will actively migrate out of the feces on a wire mesh covered with several layers ofgauge. Larvae maysometimes be presentin sputum or duodena] asp. iratcs and jejuna] biopsies. Flow chart 2: Laboratory diagnosis of Strongyloides stercoralis + Microscopy - Direcl wet mount of stool: Demonstrates rhabditiform larva (definitive diagnosis} - Stool concentrations methods: • Formol ether concentration • Baermann's funnel gauze - Demonstration of larva in sputum or duodenal aspirates or jejunal biopsies Laboratory diagnosis • Stool culture - Done when larvae are scanty in stools - Methods used: • Agar plate culture • Charcoal culture method I Serology - Done using S/rongy/oides or filarial antigens - Methods used: • Complement fixation • Indirect hemagglutination • ELISA + Radiological imaging Abbreviations: ELISA, enzyme-linked immunosorbent assay; lgE, immunoglobulin E •Blood examination • Peripheral eosinophilia • Raised serum lgE levels
- 200. Stool Culture When larvaeare scanty in stools, diagnosis may be facilitated by stool culture. Culture techniques used: • Agar plate culture • Charcoal culture method. • The larvae develop into free-living forms and m ultiply in charcoal cultures set up with stools. Large number of free- living larvae and adult worms can be seen after 7-1Odays. • Serial examinations and the use of agar plate detection method improves the sensitivity ofstool diagnosis. Serology Serological tests have been described, using Strongyloides or filarial antigens. • Complement fixaLion, indirect hemagglutination and enzyme-linked immunosorbent assay (ELISA) have been reported. • Enzyme-linked immunosorbent assay has a sensitivity of 95% and should be used when microscopic examinations are negative. • Limitations ofserological tests: - Larval antigens are not freely available. - There is extensive cross-reactions with other helminthic infections. Imaging Radiological appearances in intestinal and pulmonary infection are said to be characteristic and helpful in diagnosis. Others • Peripheral eosinophilia (>500/ cu mL of blood) is a constant finding. However, in severe hyperinfection, eosinophilia may sometimes be absent. Total serum immunoglobulin (lg) E antibody level is elevated in more than halfofthe patients (Flow chart 2). • TREATMENT All cases of strongyloidiasis, whether symptomatic or not, should be treated to prevent severe invasive disease. • lvermcctin (200 mg/ kg daily for 2 days) is more effective than albendazole (400 mg daily for 3 days). • For disseminated srrongyloidiasis, treatment with ivermectin should be extended for at least 5-7 days. • PROPHYLAXIS Strongyloidiasis can be prevented by: • Prevention ofcontamination ofsoil with feces. StrongyloidesStercoralis Avoiding contact with infective soil and contaminated surface waters. • Treatment ofall cases. KEY POINTS OF STRONGYLOIDES STERCORAL/5 • It is the smallest nematode infecting man. • Adult worm lives in duodenum and jejunum of man. • Females are ovoviviparous. • Egg is ovoid, thin-walled and transparent. • Natural host: Man (optimal host). • Infective form: Third stage filariform larva. • Mode of transmission: Penetration through the skin by the filariform larva in soil. Autoinfection can occur. • Clinical features: Generally benign and asymptomatic, but may cause cutaneous, pulmonary and intestinal manifestations. • Diagnosis: By demonstrating larva or adult females in stool or by demonstrating larval antigen by serological methods like ELISA. • Technique for stool concentration: Baermann's technique and formal-ether concentration. • Techniques for stool culture: Agar plate culture, charcoal culture. • Treatment: Drug of choice is ivermectin or albendazole. REVIEW QUESTIONS 1. Classify intestinal nematodes and describe briefly the life cycle ofStrongyloides. 2. Short notes on: a. Strongyloides b. Hyperlnfection c. Larva currens 3. Differentiate between filariform larvae of hookworm and Strongyloides. MULTIPLE CHOICE QUESTIONS 1. Parasites penetrating through skin for entry into the body are a. Trichinella b. Strongyloides c. Roundworm d. Trichuristrichiura 2. Larval form ofthe following parasites is found in stool except a. Strongyloides stercoralis b. Ancylostoma duodenale c. Ascaris lumbricoides d. Necator americanus 3. Autolnfection is seen with a. Cryptosporidium b. Strongyloides c. Giardia d. Gnathostoma
- 201. Paniker'sTextbook ofMedical Parasitology 4.The term larva currens is used for migrating larva of a. Stronglyloidesstercoralis b. Necatoramericanus c. Ancylostoma duodonale d. Hymeno/epis nano 5. Smallest nematode known to cause infection in man is a. Trichinella spiralis b. Strongyloides stercoralis c. Ancy/ostoma duodenale d. Trichuris trichiura 6. Infective form ofStrongyloides is a. Eggs b. Rhabditiform larva c. Filariform larva d. Cercaria larva 7. Baermann's funnel gauze method is used for detection of larva of a. Necator b. Strongyloides c. Ancy/ostoma d. Ascaris 8. Strongyloides can be cultured in /by a. NNN medium b. Harada Mori method of stool culture c. Agar plate culture d. Hockmeyer's medium Answer 1. b 5. b 2. C 6. C 3. b 7. b 4. a 8. C
- 202. CHAPTER 17 • HISTORYAND DISTRIBUTION Hookworms have been known since very ancient times. 11,ey have been referred to in the Ebers Papyrus (Circa 1600 BC). • Two species of hookworms are human parasites: (l) Ancylostomaduodenaleand(2)Necatoramericanus. • Ancylostoma duodenale (Greekankylos-hooked,stoma- mouth) was originally described by Dubini in 1843 in Italy. ·n,e life cycle of the worm was worked out by Looss in 1898 in Egypt. • lhe second species Necator americanus was identified by Stiles in 1902 in specimens obtained from Texas, United States of America (USA). lhe name literally means the "American murderer" (Latin neca.tor-murderer). It is called the American or the " ew World" hookworm and A. duodenale the "Old World" hookworm. But, it is believed that N. americanus actually originated in Africa and was transported to America with the slave trade. • Hookworm disease is prevalent throughout the tropics and subtropics. Even though it has been controlled in the advanced countries, it is estimated that it still affects some 900 million people, causing the loss of about 9 million liters ofblood overall each day (Box 1). • A. duodenale was prevalent along the Mediterranean coast of Europe and Africa, in northern India, China and Japan, while N. americanus was prevalent in Central and South America, Central and Southern Africa, Southern India, the Far East and the Southern Pacific region. Box 1: Conditions favoring hookworm infection • Presence of infected persons. • Dispersal of eggs in soil due to indiscriminate defecation and inadequate processing of excreta. • Appropriate environmental factors facilitating development of eggs in soil, and opportunity for the larva to infect people through their exposed skin surfaces. Note: These conditions prevail throughout the year in most parts of the tropics, but in subtropical areas, these conditions exist only seasonally, being limited to the warmer months. • However, in more recent times, movement of infected persons has blurred the geographic differences in distribution of the two species. For example, A. duodenale is nowcommonly seen alongwith N. americanus in South India and South EastAsia. • ANCYLOSTOMA DUODENAL£ Habitat The adult worms live in the small intestines of infected persons, mostly in the jejunum less often in tl1e duodenum, and infrequently in rhe ileum. Morphology Adult Worm They are relatively stout cylindroidal worms. • They are pale pink or greyish white, but may appear reddish-brown due to ingested blood. • the body is curved with the dorsal aspect concave and the ventral aspect convex. The anterior end is somewhat conslricted and bent dorsally in the same direction of general body curvature. this cervical curvature gave it the name hookworm (Fig. 1). • The mouth is not at the Lip but directed dorsally. The prominent buccal capsule, reinforced with a hard chitin- like substance carries six teeth, four hook-like teeth ventrally and two knob-like with a median cleft dorsally. Male worm: The male worm is smaller than female worm- 8- 11 mm in lengtl1and 0.4 mm thick. The posterior end of 1hc male is expanded into a copulat0ry bursa which consists oflhree lobes, one dorsal and two lateral. there are 13fleshy chitinous rays, five each in lateral lobes and three in dorsal lobe. TI1e dorsal ray is partially divided al the tip and each division is tripartite. The pattern of the rays helps in distinguishing between different species.
- 203. Paniker'sTextbook of MedicalParasitology Buccal---- capsule Esophagus Vulva----1.:z. opening Anal pore &>1r----Buccal capsule Esophagus "'llt--c;;,-Copulatory spicules Copulatory bursa Fig. 1: Adult worm of Ancylostoma duodenale (male and female) Table 1: Distinguishing features of male and female worms of Egg 'The egg ofhookworm is: Oval or elliptical, measuring 60 µm by 40 mcm • Colorless, not bilestained. • Surrounded bya thin transparent hyaJineshell membrane. • Floats in saturated salt solution. • When released by the worm in the intestine, the egg contains an unsegmented ovum. During its passage down the intestine, the ovum develops. When passed in feces, the egg contains a segmented ovum, usually with four oreightblastomeres. • There is a clear space between the segmented ovum and the egg shell (Figs 2A and B). • A single female worm lays about 25,000-30,000 eggs in a day and some 18-54 million during its life time. Life Cycle Life cycleofAncylostoma is completed in a singlehost(Fig. 3). Ancylostoma duodenale--~-- ____ Definitive Host Male Female Size Smaller, about 8-11 mm Larger, 10-13 mm in in length length Copulatory Present Absent bursa Genital opening Opens in cloaca along Opens at the junction of with anus the middle and posterior third of body Posterior end Expanded in like Tapering umbrella • The cloaca into which the rectum and genital canal open is situated within the copulatory bursa. • There are two long retractile bristle-like copulatory spicules, the tips ofwhich project from the bursa. Female worm:The female worm is larger, 10- 13 mm long and 0.6 mm thick. • Its hind end is conoid, with a subterminal anus situated ventrally. • The vulva opens ventrally at the junction of the middle and posterior thirds ofthe body. • The vagina leads to two intricately coiled ovarian tubes which occupy the hind and middle pan s ofthe worm. • During copulation the male attaches its copulatory bursa to the vulva. The copulali ng pair therefore presents a Y-shaped appearance. • Sexes are easily differentiated by d1eir size, the shape of the posterior end and the position of the genital opening (Table 1). Humans are the only natural host. No intermediate host is required like other helminths (Box 2). Infective Form Third-stage filariform larva. • Adult worm inhabiting the small intestine of man attach themselves to the mucous membrane by means of their mouth parts. The female worm lays eggs. • The eggscontaining segmented ovawith four blastomeres, are passed out in the feces of infected person (Fig. 3). Eggs freshly passed in feces are not infective for humans. • When deposited in the soil, the embryo develops inside the eggs. Its development takes place optimally in sandy loamy soil with decaying vegetation under a moist, warm, shady environm ent. • In about 2 days, a rhabditiform larva, measuring 250 mcm in length hatches out of the egg. It feeds on bacteria and other organic matter in the soil and grows in size (Fig. 3). • It moults twice, on the 3rd and 5th days after hatching to become the third-stage inf ectivefilariform larva (Fig. 3). • Filariform larva is about 500-600 µm long, with a sharp pointed tail. The filariform larva is nonfeeding. They can live in the soil for 5-6 weeks, with their heads waving in the air, waiting for their hosts. They can also ascend on blades of grass or other vegetation, being carried in capillary water films on their surface. Direct sunlight, d1 ying, or salt water can kill the larva. • Mode ofinfection: - When a person walks barefooted on soil conta ining the filariform larva, they penetrate the skin and enter
- 204. Hookworm • Figs 2A andB: Egg of Ancy/ostoma duodenale. (A) As seen under microscope; and (B) Schematic diagram Box 2: Helminths requiring no intermediate host • Ancylostoma duodenale • Necatoramericanus • Ascaris lumbricoides • Trichuris trichiura • Enterobius vermicularis • Hymenolepis nano the subcutaneous tissue. The common sites of entry are the skin between t.he toes, the dorsum of the foot and the medial aspect of the sole. In farm workers and miners, the larvae may penetrate the skin of the hands. - Rarely, infection may take place by the oral route, the filariform larva being carried on contaminated vegetables or fruits. The larvae may penetrate the buccal mucosa co reach the venous circulation and complete their migration via the lungs. - Transmammary and transplacental transmission has been also reported for Ancylostoma, but not for Necator. Inside the human body, the larvae are carried along the venous circulation to the right side of the heart and to the lungs. Here, they escape from the pulmonary capillaries into the alveoli, migrate up the respiratory tract to the pharynx, and are swallowed, reaching their final destination, small intestine. During migration or on reaching the esophagus, they undergo third moulting. They feed, grow in size, and undergo a fourth and.final moulting in the small intestine and develop the buccal capsule, by which they attach themselves to the small intestine and grow into adults. • There is no multiplication in the host and a single infective larva develops into a single adult, male or female. It takes usually about 6 weeks from the time of infection for the adult worms to become sexually mature and start laying eggs. Bur sometimes, there may be an arrest in development and the process may take much longer, 6 months or more. Alternatively, the larvae may be swallowed and may develop directly into adults in the small intestine without a tissue phase. • NEGATOR AMER/GANUS Morphology The adult worms are slightly smaller than A. duodenale, the male being 7-9 mm by 0.3 mm and the female 9-11 mm by 0.4mm. • the anterior end is bent in a direction opposite to the general curvature of the body, while in A. duodenale the bend is in the same direction. • They have a smaller buccal capsule with two pairs of semilunar cutting plates instead of teeth as in A. duodenale. The copulatory bursa of the male is long and wide. The copulatory spicules are fused at the ends to form a barbed tip. In female, the vulva is placed in the middle of the body or anterior to it (Figs 4A to C). The eggs of N. americanus are identical with those of A. duodenale.1beir life cycle is similar to that ofA. duodenale. The lifespan ofNecator is much longer being abour 4-20 years than in Ancylostoma, where it is of2-7 years.
- 205. Paniker'sTextbook of MedicalParasitology Settle in small intestine and larvae reach pharynx and are ultimately develop into .....__ adult worms ......_ I swallowed Man (Definitive host) 7 Penetrates skin of man (Definitive host) Soil J 'g @ ·S ~ ;/' ~ ... ?s ~ I lt q;f'- ~ 0 re ·-$" Q / Rhabditifo,:'--1 ---® larva hatches out Egg containing rhabditiform larva Fig. 3: life cycle of Ancylostoma duodena/e The differentiating features of A. duodenale and N. americanushave been discussed in Table2and differentiating features between filariform larva of both species has been discussed in Table 3. • PATHOGENICITY AND CLINICAL FEATURES OF HOOKWORM INFECTION Effects Due to Migrating Larva Ground itch: Larvae may give rise to severe itching at the site of penetration. It is more common in N. americanus than in A. duodena/e. • Creeping eruption: ft is formed due to subcutaneous migration of filariform larvae. There is reddish itchy papule along the path traversed by them. Respiratory system: Mild transient pneumonitis, or bronchitis occurs when larvae break out of pulmonary capillaries into alveoli. Effect Due to Adult Worm • Early hookworm infection: Adult worms produce epigastric pain, dyspepsia, nausea, vomiting and diarrhea. Chronic hookworm inf ection: It leads to iron deficiency anemia and protein energy malnutrition resulting from
- 206. A.duodenale N.americanus ~~R ~DR ~ s ~ cs Figs 4A to C: Major distinguishing features between Ancylostoma duodenale and N. americanus. (A) Adult female in Ancy/ostoma- anterior curvature uniform with body curve; in Necator anterior curvature in opposite direction to body curve. Vulva opens atjunction of middle and posterior thirds in Ancy/ostoma; in (Necator) it opens a little in front of the middle; (B) Buccal capsule, (Ancy/ostoma) has two pairs of hook-like teeth ventrally and a dental plate with median cleft dorsally; (Necator) has two pairs ofsemilunar cutting plates instead of teeth; and (C) Copulatory bursa. In (Ancy/ostoma), the dorsal ray (DR) is single with a split end, making a total of 13 rays; (Necator) has a paired dorsal ray, making a total of 14 rays. Copulatory spicules (CS) separate in (Ancylostoma); they are fused at the tip in (Necator) blood loss. Adult worms attach themselves to intestinal wall by buccal capsule and teeth and suck blood. A duodenale ingests 0.15- 0.25 mL of blood and N. americanus 0.03 ml of blood per day. 1hey also secrete anticoagulants at the attachment site so that bleeding from these sites continue. There is also interference of absorption of iron, vitamin B12 and folic acid. The pathogenesis and clinical features has been described in Flow chart 1. • LABORATORY DIAGNOSIS Direct Methods • Demonstration of characteristic oval segmented hookworm eggs in feces by direct wet microscopy or by Hookworm Table 2: Differentiating features of two species of hookworm Ancylostomaduodenale Necatoramerlcanus Adultworms Size Large and thicker Small and slender Shape Head bent in same Head bent in opposite direction as body direction Buccal capsule Four ventral teeth and Two ventral and two dorsal knob-like two dorsal chitinous teeth cutting plates Copulatory bursa 13 rays, two separate 14 rays, two spicules spicules, dorsal ray single fused at the tip, dorsal ray split Caudal spine in Present Absent female Vulval opening Situated behind the Situated in anterior to middle of the body middle part of body Pathogenicity More Comparatively less Eggs Similar Similar First and second Similar Similar stage larva Egg/day 15,000-20,000 6,000-11,000 Rate of Faster Slower development Pulmonary More common Less common reaction Blood loss/worm 0.2 mUday 0.03 ml/day Iron loss (mg/day) 0.76 mg 0.45mg Male:female ratio 1:1 1.5:1 Life span 2-7 years 4-20 years Table 3: Differential features of filariform larva (third-stage larva) Ancylostomaduodenale Necatoramericanus Size 720µm Head Slightly conical Buccal cavity Short, lumen larger Sheath Faint culticular striations Intestine No gap between esophagus and intestine Posterior end A small retractile body is of intestine present Esophageal Not prominent spears Tail Long and blunt 660µm Rounded Larger, lumen shorter Prominent striation A gap is present between esophagus and intestine No retractile body Prominent Short and pointed
- 207. Paniker'sTextbook of MedicalParasitology Flow chart 1: Clinical disease in hookworm Clinical disease j I + Larva + Ground itch • When the filariform larvae enter the skin, they cause severe local itching •An erythematous papular rash develops which later becomes vesicular. It occurs when large number of larvae penetrate the skin • More common in infection with Necator than with Ancylostoma • Self-limiting condition, lasting for 2-4 weeks i Creeping eruption (cutaneous larva migrans) ----- • It is a condition in which the filariform larvae wander about the skin and produce a reddish itchy papule along the path traversed by them • More common in infections with animal hookworms than with human hookworms concentration methods is the best method ofdiagnosis. In stool samples examined 24 hours or more after collection, the eggs may have hatched and rhabditiform larvae may be present. These h ave to be differentiated from Strongyloides larvae. • Egg counts give a m easure of the intensity of infection. Modified Kato-Katz smear technique is a useful m ethod for quantitative estimation of eggs in stool. A count of less than five eggs per mg of feces seldom causes chmcal disease, while coun ts of 20 eggs or more are associated with significant anemi a (Box 3). Egg cow1ts of SO or more represent m assive infection. • Adulthookwormsm ay sometimes beseen in feces. Eggs of A. duodenale and N. americanus cannot be differentiated by m orphology. Thus specific diagnosis can only be made by studyin g morphology of adult worms. • Duodenal contents may reveal eggs or adult worms. Stool culture: Ha rada-Mori m ethod of stool culture is carried out 10 dem on sn·ate third-stage filariform larvae which helps in distinguishing A. duodenale and N. americanus (Flow chart 2). Indirect Methods • Blood examination reveals microcytic, hypochromic anemia and eosinophilia. l Respiratory I manifestations • Occurs when larvae break out of the pulmonary capillaries and enter the alveoli • Manifests as bronchitis and bronchopneumonia • Rarely, Loeffler syndrome can be seen • Adult worm ! I • It is responsible for hook worm disease Adult worm sucks blood leading to microcytic hypochromic anemia • Patient develops epigastric pain, dyspepsia, vomiting and diarrhea. The stool becomes reddish or black in color • Symptoms and signs of anemia are present, viz. exertional dyspnea, palpitation, dizziness, generalized puffy edema, dry brittle hair and koilonychia • Severe hookworm anemia commonly leads to cardiac failure Box 3: Causes of anemia in hookworm infection • Blood sucking by the parasite for their food. • Chronic hemorrhages from the punctured sites fromjejuna! mucosa. • Deficient absorption of vitamin Bl 2 and folic acid. • Depression ofhematopoietic system by deficient intake ofproteins. • Average blood loss by the host per worm per day is 0.03 ml with N. americanus and 0.2 ml with A. duodenale. • With iron deficiency, hypochromic microcytic anemia is caused and with deficiency of both iron and v1tam1n Bl 2 or fohc acid, d1morph1c anemia is caused. • Secretion of anticoagulants at the site of attachment. • Stool examination may show occult blood and Charcot- Leyden crystals (Plow chart 2). • Ch est X-ray may sh ow pulmonary infiltrates in the migratory phase. • TREATMENT • For specific anlihelminthic treatment, the most practical and effective drug is alben dazole (400 mg single dose) or mebendazole (500 mg once). Pyrantel pamoate (11 mg/ kg x 3 days) is also effective and can be used in pregnancy. Thiabendazole is less effective. The old drug tetrachloroethylene is active, but toxic. Beph en ium
- 208. Hookworm Flow chart 2:Laboratorydiagnosis of hookworm Direct methods Laboratory diagnosis I + Blood examination Microcytic hypochromic anemia and eosinophilia + Indirect methods l Stool examination To demonstrate presence of occult blood and Charcot-Leyden crystals Chest X-ray Demonstration of eggs In feces by direct Demonstration of adult Worm in feces Stool culture By Harada-Mori method wet microscopy or by concentration method or in duodenal aspirate or duodenal aspirate (specific diagnosis) hydroxynaphthoate is active against Ancylostoma but not against Necator. • Treatment of hookworm disease also includes relief of anemia. In hookworm disease, the intestinal absorption of iron is apparently normal so that oral administration of iron can correct the anemia, but in severe cases, a preliminary packed cell transfusion may be needed. When the hemoglobin level is very low, antihelminthic drugs should not be used before correcting the anemia. • PROPHYLAXIS • Prevention ofsoil pollution with feces and properdisposal of night soil and use ofsanitary lan-ines. • Use of footwear to prevent entry of larva through the skin of the foot. Gloves give similar protection to the hands of farm workers. Treatment of patients and carriers, preferably all at the same time, limit to the source ofinfection. • OTHER HOOKWORMS Ancylostoma ceylanicum naturally parasitizes cats and wild felines in South-East Asia, but can occasionally infect man. A.braziliense, a parasite of cats and dogs and some other species of animal ancylostomes have been reported to infect man, but rhey tend to cause creepingeruption (Jarva m igrans) rather than intestinal infection. • TRICHOSTRONGYLIASIS • Trichostrongylus species, normally parasitic in sheep and goats, can also cause human infections. This is particularly likely, where the use of night soil as manure is prevalent. The infection is present in some parts ofIndia. 1he life cycle is similar to that ofhookworms. • Human infection is usually acquired by ingestion of leafy vegetables carrying the third-stage larva. • Adults attach themselves to small intestinal mucosa, suck blood and live for long periods. Infection is mostly asymptomatic but epigastric discomfort and anemia with marked eosinophilia occur in massive infections. • The eggs passed in feces resemble hookworm eggs, but are larger, with more pointed ends and show greater segmentation with 16-32 blastomeres. Metronidazole is effective in treatment. KEY POINTS OF HOOKWORM • A. duodenale is the Old World hookworm and N. americanus is the New World hookworm. • Adult worm live in small intestine Uejunum and duodenum). • In A. duodenale, the anterior end is bent dorsally in the same direction of body curvature, hence the name hookworm. The mouth contains six teeth, four hook-like teeth ventrally and two knob-like dorsally. Posteriorend of male has a copulatory bursa. • Female is longer than male with tapering end. • Eggs are oval, colorless. not bile-sta ined, and float in saturated salt solution a nd contain segmented ovum with four blastomeres. • Natural host: Humans. life cycle is completed in a single host. • Infective form: Third-stage filariform larva. • Portal of entry: Penetration of skin. Contd...
- 209. Paniker'sTextbook of MedicalParasitology Contd... • Clinical features: Ground itch, creeping eruption (cutaneous larva migrans), bronchitis and bronchopneumonia in lung, hypochromic microcytic or dimorphic anemia and intestinal symptoms like epigastric pain, dyspepsia, nausea and pica. • Diagnosis: Done by demonstration of characteristic egg in the feces by direct microscopy or by concentration methods or by demonstration of adult worms in stool or duodenal aspirate. • Treatment: Albendazole, mebendazole and pyrantel pamoate. Oral iron in anemia. REVIEW QUESTIONS 1. Name the helminths that do not require any intermediate host and describe briefly the life cycle of Ancylostomaduodenale. 2. Short notes on: a. Causes of anemia in hookworm infection b. Clinical disease in hookworm infection c. Trichostrongyliasis d. Prevention of hookworm infection 3. Differentiate between: a. Male and female ofAncylosromaduodenale b. Ancylostoma duodenaleand Necatoramericanus c. Filariform larvae ofAncylosroma and Necaror MULTIPLE CHOICE QUESTIONS 1. Highest incidence of anemia in the tropics is due to a. Hookworm b. Thread worm c. Ascaris d. Guinea worm 2. The average blood loss per worm in ancylostomiasis is a. 0.2 ml/day b. 2 ml/day c. 0.33 ml/day d. 1 ml/day 3. Which of the following does not cause biliary tract obstruction a. Ascaris lumbricoides b. Ancy/ostoma duodenale c. Clonorchis sinensis d. Fascia/a hepatica 4. Which of the following stages of Ancylostoma duodenale is infective to human beings a. Rhabditiform larva b. Filariform larva c. Eggs d. Adult worm 5. A 6-year-old girl is emaciated with a hemoglobin level of 6 g/dl. Her face appears puffy with swollen eyelids and edema over feet and ankles. There are no laboratory facilities available. The most likely cause ofthechild's condition is a. Schistosomiasis b. Cercarial dermatitis c. Ascariasis d. Hookworm disease 6. All of the following are characteristics ofAncylostoma except a. Its copulatory bursa has 13 rays b. Caudal spine is present in females c. Head is bent in a direction opposite to body d. Vulval opening is situated in the middle of the body. Answer 1. a 2. a 3. b 4. b 5. d 6. C
- 210. CHAPTER 18 • INTRODUCTION Thename Enterobius 11ermicularis means a tiny worm living in the intestine (Greek enteron-intestine, bias-life and vermiculus-small worm). The term Oxyuris means "sharp tail'; a feature of the female worm, from which the name "pinworm" is also derived. • COMMON NAME Pinworm, seatworm, threadworm. • HISTORY AND DISTRIBUTION Enterobius vermicularis, formerly called Oxyuris vermicularis has been known from ancient times. • Leuckart (1865) first described the complete life cycle of the parasite. • lt is worldwide in distribution. Unlike the usual situation, where helminthic infections are more prevalent in the poor people of the tropics, E. vermicularis is one worm infestation which is far more common in the affluent nations in the cold and temperate regions (cosmopolitan). • Enterobius uermicularis is considered to be world's most common parasite, which specially affects the children. • HABITAT Adult worms are found in the cecum, appendix and adjacent portion ofascending colon. • MORPHOLOGY Adult Worm 1l1e adults are short, white, fusiform worms with pointed ends, .lookinglike bits ofwhite thread. • The mouth is surrounded by three wing-like curicular expansions (cervical alae), which are transversely striated. A ~ r--- - Cervical alae Double bulb esophagus 00:::.-r::tH - Eggs in uterus ,____ Posterior and straight Cervical alae Posterior one-third Fig. 1: Adult worm of Enterobius vermicularis (male and female) • The esophagus has a double bulb structure, a feature unique to this worm (Fig. 1). Female Worm The female is 8-13 mm long and 0.3-0.5 mm thick. • Its posterior third is drawn into a thin pointed pin-like tail (Fig. 1). • The vulva is located just in front of the middle third of the body and opens into the single vagina, which leads to the paired uteri, oviducts and ovaries. In the gravid femaJe, virtually the whole body is filled by the distended uteri carrying thousands ofeggs. • The worm is oviparous. • Females survive for 5- 12 weeks.
- 211. Paniker'sTextbook ofMedical Parasitology MaleWorm The mal e worm is 2-5 mm long and 0.1-0.2 mm thick. • Its posterior end is tightly curved ventrally, sharply truncated and carries a prominent copulatory spicule (Fig. 1). • Males live for about 7- 8 weeks. Egg The eggis colorless and not bile-stained. • It floats in saturated salt solution. • It has a characteristic shape, being elongated ovoid, flattened on one side and convex on the other (plano- convex), measuring 50-60 µm by 20-30 µm (Fig. 2). • The eggshell is double-layered and relativelythick, though transparent. The outer albuminous layer makes the eggs stick to each other and to clothing and other objects. • The egg contains a tadpole-shaped coiled embryo, which is fully formed, but becomes infectious only 6 hours after beingdeposited on the skin. Under cool moist conditions, the egg remains viable for about 2 weeks (Fig. 2). • Asingle female worm lays 5,000-17,000 eggs. • LIFE CYCLE Enterobius vermicuLaris is monoxenous, passing its entire life cycle in the human host. It has no intermediate host and does not undergo any systemic migration (Box 1). Natural Host Man. Fig. 2: Planoconvex egg of Enterobius vermicularis containing tadpole-shaped embryo Box 1: Nematodes not showing systemic migration in man • Enterobiusvermiculoris • Trichuris trichiuro. Infective Form Embryonated Eggs • Mode of infection: Man acquires infection by ingesting embryonated eggs containing larva by means of: - Contaminated fingers - Autoinfection. • Eggs laid on perianal skin containing infective larvae are swallowed and hatch out in the intestine. • They moult in the ileum and enter the cecum, where they mature into adults. • It takes from 2 weeks to 2 months from the time the eggs are ingested, to the development of the gravid female, ready to lay eggs. • The gravid female migrates down the colon to the rectum. At night, when the host is in bed, the worm comes out through the anus and crawls about on the perianal and perineal skin to lay its sticky eggs. The worm may retreat into the anal canal and come out again to lay more eggs. • The female worm may wander into the vulva, vagina and even into the uterus and fallopian tubes, sometimes reaching the peritoneum. • the male is seldom seen as it does not migrate. It usually dies after mating and is passed in the feces. • When all the eggs are laid, the female worm dies or gets crushed by the host during scratching. The worm may often be seen on the feces, having been passively carried from the rectum. The eggs, however, are only infrequently found in feces, as the female worm lays eggs in the perianal area and not the rectum. • Crawling of the gravid female worm leads to pruritus and the patient scratches the affected perianal area. These patients have eggs ofE. vermicularis on fingers and under nails leading to autoinfection (Fig. 3). • Autoinfection: Ingestion of eggs due to scratching of perianal area with fingers leading to deposition of eggs under the nails. This type of infection is mostly common in children. This mode of infection occurs from anus to mouth. • Retroinfection:In this process, theeggs laid on theperianal skin immediately hatch into the infective stage larva and migrate through the anus to develop into worms in the colon. This mode ofinfection occurs from anus to colon. • PATHOGENICITY AND CLINICAL FEATURES Enterobiasis occurs mostly in children. It is more common in females than in males. About one-third of infections are asymptomatic. • The worm produces intense irritation and pruritus of the perianal and perinea! area (pruritus ani), when it crawls out of the anus to lay eggs. This leads to scratching and excoriation ofthe skin around the anus.
- 212. Enterobius Vermicularis V """"''''lato''"" woon Liberated larva migrate towards cecum Man Adult worms in ( The egg shell is dissolved by the digestive juices large intestine Soil Eggs laid at the perianal skin by the gravid female Freshly laid and larva liberated In small intestine .~- i '"'""' ~ eo,~"""''""'""· ll"iJ Egg (with infective larva) food, ~~~~-~~?lhin 1 / 'Y swallowed by man~ Embryonated egg (definitive host) with infective larva in soil Fig. 3: Life cycle of Enterobius vermicularis • As the worm migrates out at night, it disturbs sleep. Nocturnal enuresis is sometimes seen. • The worm crawling into the vulva and vagina causes irritation and a mucoid discharge. lt may migrate up to the uterus, fallopian tubes and into the peritoneum. This may cause symptoms of chronic salpingitis, cervicitis, peritonitis and recurrent urinary tract infections. • The worm is sometimes found in surgically removed appendix and has been claimed to be responsible for appendicitis. • LABORATORY DIAGNOSIS Pinworm infestation can be suspected from the history of perianal pruritus. Diagnosis depends on the demonstration ofthe eggs or adult worms (Flowchart 1). Demonstration of Eggs • Eggs are present in the feces only in a small proportion of patients and so feces examination is not useful in diagnosis. • They are deposited in large numbers on the perianal and perineal skin at night and can be demonstrated in swabs collected from the sites early morning, before going to the toilet or bathing. Swabs from perianal folds are most often positive. • The eggs may sometimes be demonstrated in the dirt collected from beneath the finger nails in infected children. NIH Swab Method The NIH swab [named after National Institutes of Health, United States of America (USA)) has been widely used for
- 213. Paniker'sTextbookof Medical Parasitology Flowchart 1: Laboratory diagnosis of Enteroblus vermicu/aris Laboratory diagnosis Under finger nails Detection of egg I NIH swab method collection of specimens. This consists of a glass rod at one end of which a piece of transparent cellophane is attached with a rubber band. The glass rod is fixed on a rubber stopper and kept in a wide test tube. The cellophane part is used for swabbing by rolling over the perianal area (Fig. 4). It is returned to the test tube and sent to the laboratory, where the cellophane piece is detached, spread over a glass side and examined microscopically. Scotch Tape Method Another method for collection of specimens is with scotch tape (adhesive transparent cellophane tape) held sticky side out, on a wooden tongue depressor. The mounted tape is firmly pressed against the anal margin, covering all sides (Fig. 5). The tape is transferred to a glass slide, sticky side down, with a drop of toluene for clearing and examined under the microscope. Demonstration of Adult Worm The adult worms may sometimes be noticed on the surface ofstools. • Tuey may occasionally be found crawling out ofthe anus while the children are asleep. • They may be detected in stools collected after an enema and may be in the appendix during appendectomy (Box2). Note: Unlike the other intestinal nematodes, Enterobius infection is not associated with eosinophilia or with elevated immunoglobulin E(lgE). • TREATMENT Pyrantelpamoate(l l mg/kgonce, maximumlg),albendazole (400 mgonce) or mebendazole (100 mgonce) can be used for single dose therapy, while piperazine has to be given daily for 1week. l Scotch tape method I Rubber stopper Test tube Rubber band Detection of adult worm Stool sample Fig. 4:National Institutes of Health (NIH) swab. A piece of transparent cellophane is attached with rubber band to one end of a glass rod, which is fixed on a rubber stopper and kept in a wide test tube Fig. S: Scotch tape method (press the sticky side of the tape against the skin across the anal opening)
- 214. Box 2: Infectiousparasites which may be present in a fecal sample • Enterobius vermicularis • Strongyloides stercoralis • Taenia solium • Hymenolepis nano • Entamoeba histolytica • Giardia lamblia • Cryptosporidiumparvum • It is necessary to repeat the treatment after 2 weeks to take care of autochthonous infections and ensure elimination ofall worms. • As pinworm infection usually affects a group, it is advisable to treat the whole family or group of children, as the case may be. • PROPHYLAXIS • Maintenance ofpersonal and community hygiene such as frequent hand washing, finger nail cleaning and regular bathing. • Frequent washing ofnight clothes and bed linen. KEY POINTS OF ENTEROBIUS VERMICULARIS • Adult worm lives in cecum and appendix. • Mouth is surrounded by three wing-like cervical alae. Esophagus has a double bulb structure. • Worm is oviparous. • Eggs are colorless, not bile-stained; plano-<:onvex in shape. • Natural host: Humans. E. vermicularis passes its entire life cycle in human host. No intermediate host is required. • Infective form: Embryonated egg containing infective larva. • Mode of infection: By ingestion of eggs or autoinfection. Seen mostly in children and among family members. • Clinical features: Pruritus ani, nocturnal enuresis. Sometimes, salpingitis, peritonitis, appendicitis, etc. may be seen. • Diagnosis: Detection of eggs by NIH swab and cellophane scotch tape method. Detection of adult worm in finger nails or from stool after enema. • Treatment: Mebendazole, albendazole, or pyrantel pamoate. Enterobius Vermicularis REVIEW QUESTIONS 1. List the parasites causing autoinfection and describe briefly the life cycle of Enterobius vermicularis. 2. Short notes on: a. Egg ofEnterobius vermicularis b. Laboratory diagnosis ofEnterobius vermicularis c. NIH swab MULTIPLE CHOICE QUESTIONS 1. Most common presenting symptom of thread worm infection amongst the following is a. Abdominal pain b. Rectal prolapse c. Urticaria d. Vaginitis 2. Which one of the following does not pass through the lungs a. Hookworm b. Ascaris c. Strongyloides d. Enterobius vermicularis 3. Infection with which of the following parasites may cause enuresis a. Ascaris lumbricoides b. Enterobius vermicularis c. Trichinella spiralis d. Wuchereria bancrofti 4. History of mild intestinal distress, sleeplessness, itching, and anxiety is seen in preschool child attending play school. Possible parasite agent causing these manifestations is a. Trichomonas vagina/is b. Enterobius vermicu/aris c. Ascaris lumbricoides d. Necator americanus 5. The common name for Enterobius vermicularis is a. Threadworm b. Pinworm c. Seatworm d. Whipworm 6. Which ofthe following nematodes lays eggs contaning larvae a. Trichinella spiralis b. Enterobius vermicularis c. Brugia malayi d. Ascarislumbricoides Answer 1. a 2. d 3. b 4. b 5. C 6. b
- 215. CHAPTER 19 • COMMONNAME Roundworm. • HISTORY AND DISTRIBUTION Ascaris lumbricoides has been observed and described from very ancient times, when itwas sometimes confused with the earthworm. • Its specific name lumbricoides is derived from its resemblance with earthworm (Lumbricus, meaning earthworm in Latin). • It is the most common of human helminths and is distributed worldwide. A billion people are estimated to be infected with roundworms. The individual worm burden could be very high, even up to over a thousand. An editorial in the Lancet in 1989 observed that if all the roundworms in all the people worldwide were placed end-to-end theywould encircle the world 50 times. • The incidence may be as high as 80- 100% in rural areas with poor sanitation. • HABITAT Adultworms live in the small intestine (85% in jejunum and 15% in ileum). The roundworm, Ascaris lumbricoides is the largest nematode parasite in the human intestine. • MORPHOLOGY Adult Worm They are large cylindrical worms, with tapering ends, the anterior end beingmore pointed than the posterior (Fig. 1). • They are pale pink or flesh colored when freshly passed in stools, but become white outside the body. • The mouth at the anterior end has three finely toothed lips, one dorsal and two ventrolateral (Figs 2A to E). Fig. 1: Specimen ofAscaris /umbricoides Male Worm • The adult male worm is little smaller than female. It measures 15-30 cm in length and 2-4 mm in thickness (Figs 2A to E). • Its posterior end is curved ventrally to form a hook and carries two copulatory spicules (Figs 2A to E). Female Worm The female is larger than male, measuring 20-40 cm in length and 3-6 mm in thickness. • Its posterior extremity is straight and conical. • The vulva is situated mid-ventrally, near the junction of the anterior and middle thirds of the body. A distinct groove is often seen surrounding the worm at the level of the vulvar opening. This is called the vulvar waist or genital girdle and is believed to facilitate mating (Figs 2A to E). The vulva leads to a single vagina, which branches
- 216. Ascaris Lumbricoides ~ Dorsalllp (one) ....,...,---Paplllla '-.:"¥:;:,-<--r==--ventrallips Vulvar waist cl (two) .-ll!E""--tt- - Anal El Copulatory spicules opening II Figs 2A to E: Ascaris lumbrlco/des. (A) Adult female and male worms; (B) Anterior end of worm. Head-on view, showing one dorsal and two ventral lips with papillae; (C) Posterior end of female, showing anal opening, a little above the conical tip; (D) Posterior end of male, showing two protruding copulatory spicules(s); and (E) Specimen showing Ascaris lumbricoides, male and female into a pair of genital tubules that lie convoluted through much of the posterior two-thirds ofthe body. The genital tubules ofthe gravid worm contain an enormous number ofeggs as many as 27 million at a time (Box 1). • A single worm lays up to 200,000 eggs per day. The eggs are passed in feces. Egg Two types ofeggs are passed by the worms: (1) fertilized and (2) unfertilized. 1. The fertilized eggs, laid by females, inseminated by mating with a male, are embryonated and develop into the infective eggs (Figs 3A to C). 2. The unfertilized eggs, are laid by uninseminated female. These are nonembryonated and cannot become infective (Fig. 3D). Note:Stool samples may show both fertilized and unfertilized eggs, or either type alone (Table 1). • LIFE CYCLE Life cycle ofAscarisinvolves only one host. Box 1: Parasites with bile-stained eggs • Ascaris lumbricoldes • Clonorchis sinensis • Trichuris trichiura • Fasc/ola hepatlca • Taenia solium • Fasclo/opsls busk/ • Taenia saginata. Natural Host Man.There is no intermediate host. Infective Form Embryonated eggs. • Mode of transmtsston: - Infection occurs when the egg containing the infective rhabditiform larva Is swallowed. Afrequent mode of transmission is through fresh vegetables grown in fields manured with human feces (night soil). Infection may also be transmitted through contaminated drinkingwater.
- 217. Paniker'sTextbook of MedicalParasitology c Figs 3A to D: Types of Ascaris eggs found in stools. (A) Fertilized egg surface focus. showing outer mamillary coat; (8) Fertilized egg, median focus, showing unsegmented ovum surrounded by three layers of coats; (C) Decorticated fertilized egg, the mamillary coat is absent; and (D) Unfertilized egg, elongated, with atrophic ovum Table 1: Features of roundworm egg Typeofegg Unfertilized egg (Fig. 4A) Fertilized eggs (Fig.48) Main feature • Elliptical in shape • Narrower and longer • 80 µm x 55 µm in size • Has a thinner shell with an irregular coating of albumin • Contains a small atrophied ovum with a mass ofdisorganized highly refractile granulesofvarious size • Does not float in salt solution • Round or oval in shape • Size 60-75 µm x 40-45 µm • Always bile-stained • Golden brown in color • Surrounded by thick smooth translucent shell with an outer coarsely mammillated albuminous coat. a thick transparent middle layer and the inner lipoidal vitelline membrane • Some eggs are found in feces without the outer mammillated coatThey are called the decorticated eggs (Fig. 3C) In the middle ofthe egg is a large unsegmented ovum, containing a mass ofcoarse lecithin granules. It nearly fills the egg, except for a clear crescentic area at either poles Floats in saturated solution ofcommon salt m Figs 4A and B: (A) Unfertilized egg of Ascaris; and (B) Fertilized egg of Ascaris
- 218. Children playing aboutin mud can transmit eggs to their mouth through dirty fingers (geophage). Where soil contamination is heavy due to indiscriminate defecation, the eggs sometimes get airborne along with windswept dust and are inhaled. The inhaled eggs get swallowed. Development in Soil The fertilized egg passed in feces is not immediately infective. It has to undergo a period of incubation in soil before acquiring infectivity. • The eggs are resistant to adverse conditions and can survive for several years. • The development ofthe egg in soil depends on the nature of the soil and various environmental factors. A heavy clayey soil and moist shady location, with temperature between 20°c and 30°Care optimal for rapid development ofthe embryo. • The development usually takes from J0-40 days, during which time the embryo moults twice and becomes the infective rhabditiforrn larva, coiled up within the egg. Development in Man When the swallowed eggs reach the duodenum, the larvae hatch out. • . • • • • The rhabditlform larva, about 250 µm in length and 14 µm in diameter, are actively motile. They penetrate the intestinal mucosa, enter the portal vessels and are carried to the liver. They then pass via the hepatic vein, inferior vena cava, and the right side ofthe heart and in about 4 days reach the lungs, where theygrow and moult twice. After development in the lungs, in about 10-15 days, the larvae pierce the lung capillaries and reach the alveoli. They crawl up or are carried up the respiratory passage to the throat and are swallowed. The larvae moult.finally and develop into adults in the upper part of the small intestine. They become sexually mature in about 6-12 weeks and the gravid females start laying eggs to repeat the cycle (Fig. 5). The adult worm has a lifespan of12-20 months. • PATHOGENICITY AND CLINICAL FEATURES Disease caused byA. lumbricoides is called as ascariasis. • Clinical manifestations in ascariasis can be caused either by the migrating larvae or by the adult worms. Symptoms Due to the Migrating Larvae The pathogenic effects oflarval migration are due to allergic reaction and not the presence of larvae as such. Therefore, AscarisLumbricoides the initial exposure to larvae is usually asymptomatic, except when the larval load is very heavy. • When reinfection occurs subsequently, there may be intense cellular reaction to the migrating larvae in the lungs, with infiltration of eosinophils, macrophages and epithelioid cells. • This Ascaris pneumonia is characterized by low- grade fever, dry cough, asthmatic wheezing, urticaria, eosinophilia and mottled lung infiltration in the chest radiograph. • The sputum is often blood-tinged and may contain Charcot-Leyden crystals. The larvae may occasionally be found in the sputum, but are seen more often in gastric washings. This condition is called Loejfler's syndrome. • The clinical features generally clear in 1 or 2 weeks, though it may sometimes be severe and rarely, even fatal. Loeffler'ssyndromecan also becaused byhypersensitivity to other agents, both living and nonliving (Box 2). Symptoms Due to the Adult Worm Clinical manifestations due to adult worm vary from asymptomaticinfectiontosevereandevenfatalconsequences. • Asymptomatic infection: Generally seen in mildly infected cases; however, it is not unusual to find children apparently unaffected in spite of heavy infestation with the worms. • The pathological effects, when present, are caused by spoliative action, toxic action, mechanical effects and wandering effects. - The spoliative ornutritional effects are usually seen when the worm burden is heavy. The worms may be present in enormous numbers, sometimes exceeding 500, in small children, occupying a large part of the intestinal tract. This interferes with proper digestion and absorption of food. Ascariasis may contribute to protein-energy malnutrition and vitamin A deficiency. Patients have loss ofappetite and are often listless. Abnormalities ofthe jejuna! mucosa are often present, includingbroadeningand shortening ofvilli, elongation of crypts and round cell infiltration of lamina propria. These changes are reversed when the worms are eliminated. - the toxic effects are due to hypersensitivity to the worm antigens and may be manifested as fever, urticaria, angioneurotic edema, wheezing and conjunctivitis. These are more often seen in persons who comeinto contactwith theworm occupationally, as in laboratory technicians and abattoir workers {who become sensitive to the pig ascarid, A. suum), than in children having intestinal infestation. - The mechanical effects are the most important manifestations of ascariasis. Mechanical effects can
- 219. Paniker'sTextbook ofMedical Parasitology Larvaburrows through the mucous membrane of the small intestine Rhabditiform larva liberated in the duodenum Man acquires infection by Ingestion of food and water contaminated with embryonated eggs Reach the lungs, trachea and pharynx. From here they are swallowed and reach small intestine. Man Soil Contamination of vegetables Adult worms in small intestine of man Fertilized egg containing Unfertilized unsegmented ovum egg passed in feces Rhabditiform larva develops in soil within the egg Fig. 5: Life cycle ofAscaris lumbricoides Box 2:Parasites causing pneumonitis or Loeffler's syndrome • Migrating larvae of: - Ascaris lumbricoides - Strongyloides stercoralis - Ancylostomaduodena/e - Necatoramericanus • Echinococcus granulosus • Eggs of Paragonimus westermani • Cryptosporidium parvum • Trichomonas tenax • Entamoeba histolytica. be due to masses ofwormscausing luminal occlusion or even a single worm infiltrating into a vital area. The adult worms Jive in the upper part of the small intestine, where they maintain their position due to their body muscle tone, spanning the lumen. They may stimulate reflex peristalsis, causing r ecurr en t and often severe colicky pain in the abdomen. The worms may be clumped together into a mass, filling the lumen, leading to volvulus, intussusception, or intestinal obstruction and intestinal perforation.
- 220. - Ectopic ascariasis(Wanderlust): The worms are restless wanderers, apparently showing great inquisitiveness, in that they tend to probe and insinuate themselves into any aperture they find on theway. The wandering is enhanced when the host is ill, particularly when febrile, with temperature above 39°C. The male worm is more responsive to illness of the host, than the female. The worm may wander up or down along the gut. Going up, it may enter the opening of the biliary or pancreatic duct causing acute biliary obstruction or pancreatitis. It may enter the liver parenchyma, where it may lead to liver abscesses. The worm may go up the esophagus and come out through the mouth or nose. It may crawl into the trachea and the lung causing respiratory obstruction or lung abscesses. Migrating downwards, the worm may cause obstructive appendicitis. It may lead to peritonitis when it perforates the intestine, generally at weak spots such as typhoid or tuberculous ulcers or through suture lines. This tendency makes preoperative deworming necessary before gastrointestinal surgery in endemic areas. The wandering worm may also reach kidneys. • LABORATORY DIAGNOSIS Detection of Parasite Adult Worm The adult worm can occasionally be detected in stool or sputum ofpatient by naked eye. Barium meal may reveal the presence of adult worm in the smalJ intestine. A plain abdominal film may reveal masses of worms in gas-filled loops of bowel in patients with intestinal obstruction. PancreaticobiJiary worms can be detected by ultrasound (more than 50% sensitive) and endoscopic retrograde cholangiopancreatography (ERCP; 90%sensitive). Larvae In the early stages of infection, when migrating larvae cause Loeffler's syndrome, the diagnosis may be made by demonstrating the larvae in sputum, or more often in gastric washings. • Presence of Charcot-Leyden crystals in sputum and an attendant eosinophilia supports the diagnosis. At this stage, no eggs are seen in feces. • Chest X-ray may show patchy pulmonary infiltrates. Eggs Definitive diagnosis of ascariasis is made by demonstration ofeggs infeces. Ascaris Lumbricoides • Ascarids are prolific egg layers. A single female may account for about three eggs per mg of feces. At this concentration, the eggscan be readilyseenbymicroscopic examination of a saline emulsion of feces. Both fertilized and unfertilized eggs are usually present. Occasionally, only one type is seen. The fertilized eggs may sometimes appear decorticated. The unfertilized eggs are not detectable by salt floatation. • Rarelywhen the infestationislight, eggs are demonstrable only by concentration methods. • Eggs may not be seen if only male worms are present, as may occasionally be the case. Fecal films often contain many artifacts resembling Ascaris eggs and care must be taken to differentiate them. • Eggs may be demonstrative in the bile obtained by duodenal aspirates (Flow chart 1). Serological Tests Ascaris antibody can be detected by: • Indirect hemagglutination (IHA) • Indirect fluorescent antibody (IFA) • Enzyme-linked irnmunosorbent assay (ELISA). • Serodiagnosis is helpful in extraintestinal ascariasis like Loeffler's syndrome (Flow chart 1). Blood Examination Complete blood countmay show eosinophilia in early stage of invasion (Flowchart 1). • TREATMENT Several safe and effective drugs are now available for treatment of ascariasis. These include pyrantel pamoate (11 mg/kg once; maximum 1 g), albendazole (400 mg once), mebendazole (100 g twice daily for 3 days or 500 mg once), or ivermectin (150-200 mg/kg once). These medications are contraindicated in pregnancy; however, pyrantel pamoate is safe in pregnancy. • Partial intestinal obstruction should be managed with nasogastric suction, intravenous fluid administration and instillation ofpiperazine through the nasogastric tube. • Complete obstruction requires immediate surgical intervention. • PROPHYLAXIS • Ascariasis can be eliminated by preventing fecal contamination of soil. The Ascaris egg is highly resistant. Therefore, the use of night soil as manure will lead to spread of the infection, unless destruction of the eggs is ensured by proper composting. Treatment of vegetables and other garden crops with water containing iodine 200
- 221. Paniker'sTextbook of MedicalParasitology i Eggs + • Definitive diagnosis of ascariasis is made by demonstration of eggs in feces • Rarely, when the infestation is light, eggs are demonstrable only by concentration methods Flow chart 1: Laboratory diagnosis of Ascaris lumbricoides Laboratory Diagnosis ! Detection of Parasite l Larva + In lhe early stage of infection,when migrating larva cause Loeffler's syndrome, the diagnosis may be made by demonstrating the larvae in sputum, or in gastric washings Adult worm + • Can occasionally be seen by naked eye in stool or sputum of patient • X-ray, Barium meals and ultrasound imaging may help in diagnosis l Serodiagnosis Ascans antibodies can be detected by •ELISA • IHA • IFA Serodiagnosis is helpful in extraintestinal ascariasis like Loeffler's syndrome l Blood examination Eosinophilia may be seen in early stages of infection Abbreviations: ELISA, enzyme-linked immunosorbent assay; IFA, indirect fluorescent antibody; IHA, indirect hemagglutination ppm for 15 minutes kills the eggs and larvae ofAscarisand other helminths. • Avoid eating raw vegetables. • Improvement ofpersonal hygiene. • Treatment ofinfected persons especially the children. KEY POINTS OF ASCARIS LUMBRICOIDES • A. lumbricoides is the largest nematode infecting human. • Adult worm is cylindrical resembling an earthworm. • Male is little smaller than female. Posterior end of male is curved ventrally to form a hook with two copulatory spicules. Posterior end of female is conical and straight. • Fertilized eggs are bile-stained, round or oval, surrounded by a thin translucent wall with outer mammillated coat containing a large unsegmented ovum. Unfertilized eggs are elliptical, longer with an outer thinner irregular mammillated coat, containing a small atrophied ovum and retractile granules. • Natural host: Man. • Infective form: Embryonated egg containing rhabditiform larva. • Clinical features: Spoliative action-protein and vitamin A deficiency. Toxic action-utricaria and angioneurotic edema. Mechanical action- intestinal obstruction, intussusception, volvulus, intestinal perforation. In lungs- Ascaris can cause pneumonia (Loeffler's syndrome). • Diagnosis: Demonstration of eggs in stool, finding of larvae in sputum, finding of adult worm in stool or sputum. • Treatment: Albendazole, mebendazole, ivermectin, or pyrantel pamoate. Fig. 6: Adult worms of Toxocara canis • OTHER ROUNDWORMS Toxocara Toxocara canis and T. cati, natural parasites ofdogs and cats (Fig. 6), respectively can cause aberrant infection in human s leading to visceral larva migrans. • Infection is acquired in puppies by transmission oflarvae transplacentally or lactogenically (through breast milk), but in kittens, only Jactogenic transmission is recorded.
- 222. Ascaris Lumbricoides Box 3:Geohelminths • Soil-transmitted intestinal nematodes are called Geohelminths. In all of them, eggs passed in feces undergo maturation in soil. They are classified into three categories based on their life cycle: 1. Direct: Ingested infective eggs directly develop into adults in the intestine, e.g. whipworms. 2. Modified direct: Larvae from ingested eggs penetrate intestinal mucosa enter bloodstream and through the liver, heart, lungs, bronchus and esophagus, reach the gut to develop into adults, e.g. roundworms. 3. Skin penetrating: Infective larvae in soil penetrate host skin, reach the lung, and proceed to the gut as in the modified direct method, e.g. hookworms. • Geohelminths posea serious health problem in poor countries, particularly among children.Their control requires general measures such as personal hygiene, sanitation and health education, besides provision of diagnostic and treatment facilities. • Older animals are infected by ingestion of mature eggs in soil or oflarvae by eating infected rodents, birds, or other paratenic hosts. • Eggs are shed in feces and become infective in 2-3 weeks. • Human infection is by ingestion ofeggs. • Larvae hatch out in the small intestine, penetrate the mucosa, and reach the liver, lungs, or other viscera. They do not develop any further. • Mostinfectionsareasymptomatic,butinsome,particularly in young children, visceral larva migrans develops, characterized by fever, hepatomegaly, cough, pulmonary infiltrates, high eosinophilia and hyperglobulinemia. In some, the eye is affected (ophthalmic larva migrans). Baylisascaris Baylisascaris procyonis, an ascarid parasite of raccoons in North America, is known to cause serious zoonotic infections leading to visceral larva migrans, ophthalmic larva migrans and neural larva migrans.Complications include blindness and central nervous system lesions ranging from minor neuropsychiatric conditions to seizures, coma and death (Box3). REVIEW QUESTIONS 1. Name the parasites causing pneumonitis and describe briefly the life cycle and laboratory diagnosisof Ascaris lumbricoides. 2. Short notes on: a. Clinical manifestations of ascariasis b. Loeffler's syndrome c. Surgical complications ofascariasis d. Toxocariasis e. Geohelminths 3. Differentiate between fertilized and unfertilized egg of Ascaris lumbricoides. MULTIPLE CHOICE QUESTIONS 1. Which of the following parasites does not penetrat e human skin a. Ascaris Jumbricoides b. Ancylostoma duodenale c. Strongyloidesstercora/is d. Schistosoma haematobium 2. The common nam e for Ascarislumbricoides is a. Roundworm b. Hookworm c. Threadworm d. None of the above 3 . The largest intestinal nematode infecting humans is a. Necator americanus b. Ascaris lumbricoides c. Enterobius vermicularis d. None of the above 4. All of the following are correct regarding fertilized egg of Ascaris except a. It is always bile-stained b. Covered by an outer mamilliated coat c. Floats in saturated solution of salt d. Does not float in saturated solution of salt 5. All of the following parasites have bile-stained eggsexcept a. Ascaris b. Clonorchis c. Taenia so/ium d. Enterobius 6. Loeffier's syndrome may b e seen in infection with a. Ancy/ostomaduodena/e b. Ascaris lumbricoides c. Trichinella spiralis d. Trichuris trichiura Answer 1. a 2. a 3. b 4. d 5. d 6. b
- 223. CHAPTER 20 • INTRODUCTION Nematodesbelonging to the superfamily Filarioidea are slender thread-like worms(Latin,filum and thread), which are transmitted by the bite ofblood-sucking insects. . • • • . • The filarial worms reside in the subcutaneous tissues, lymphatic system, or body cavities ofhumans (Table 1). The adult worm generally measures 80-100 mm in length and 0.25- 0.30 mm in breadth; the female worm being longer than the males. The tail of the male worm has perianal papillae and unequal spicules butno caudal bursa. The female worms are viviparous and give birth to larvae known as microfilariae. The microfilariae released by the female worm, can be detected in the peripheral blood or cutaneous tissues, depending on the species. In some species, the microfilariae retain their egg membranes which envelop them as sheath. They are known as sheathed microfilariae. In some other species of filarial nematodes, the egg membrane is ruptured and is known as unsheathed microfilariae. Once themicrofilariae are classifiedon the basis ofsheath as "sheathed" or"unsheathed'; theirfurther differentiation can be done on the characteristic arrangement of nuclei (Flowchart 1 and Table 2). • Periodicity: Depending on when the largest number of microfilariae occur in blood, filarial worms can exhibit nocturnal, dJurnal perlodJcity or no periodicity at all (Box 1). The basis of periodicity is unknown but it may be an adaptation to the biting habits ofthe vector. • The life cycle offilarial nematodes is passed in two hosts: (1) definitive host is man and (2) intermediate host are the blood-sucking arthropods. • The microfilariae complete their development in the arthropod host to produce the infective larval stages. Table 1: Classification of filarial worm based on location in body Lymphatkfilarlasls Subcutaneous serous cavityfilarlasis • Wuchereria bancrofti • Brugia malayi • Brugia timari filarlasls • Loaloa • Onchocerca volvulus • Mansonella streptocerca • Mansonella perstans • Mansonella ozzardi(They are virtually nonpathogenic) These are transmitted to humans by arthropod, which are their vectors also during the next feed. Adult worms live for manyyears whereas microfilariae survive for 3-36 months. • Eight species of filarial worms infect humans, of them six are pathogenic-(1) Wuchereria bancrofti, (2) Brugia malayi and (3) B. timori cause lymphatic filariasis; (4) Loa loa causes malabarswellings and allergic lesions; (5) Onchocerca volvulus causes eye lesions and dermatitis; (6) Mansonella streptocerca leads to skin diseases; and two of them, (7) M. ozzardi and (8) M. perstans are virtually nonpathogenic (Table 3). • Infection with any of the filarial worms may be called .filariasis, but traditionally, the term filariasis refers to lymphatic filariasis caused by Wuchereria or Brugia species. • Adult filarial worm contains an endosymbiotic Rickettsia- like a-proteobacterium ofthe genus Wolbachia spp. This has got definite role in the pathogenesis of filariasis and has become a target for antifilarial chemotherapy. • Wolbachia spp. along with filarial antigen activates the release of proinflammatory and chemotactic cytokines. These include cellular infiltration and amplification of inflammatory processes. Toll-like receptors (TLRs) play an important role in the process.
- 224. Filarial Worms Flow chart1: Differentiating features of various microfilariae on the basis of presence of nuclei in tail end + Sheathed microfilariae I Nuclei do not extend up to the tail ti Tail end Nuclei extend up to the tail tip Microfilariae I + Unsheathed microfilariae Nuclei extend up to the tail tip Tail end I Nuclei do not extend up to the tail tip Nuclei present in a row up to the tail tip Two nuclei at the tip of tail Mansonella perstans Mansonella streptocerca Wuchereria bancrofti Loaloa Brugia malayi Table 2: Head and tail ends of microfilariae found in humans Species Wuchereria Brugia malayi Loaloa bancrofti Shape (5;' ~ ~ Posterior end ~ ~ ~ Tail nuclei Nuclei do not 2 nuclei at the tip Nuclei form extend to the tip of the tail continuous row of tail in the tip ofthe tail Anterior end ~ ~ ~ Size 300 x 8 µm 220 x6 µm 270 x 8 µm Sheathed/unsheathed Sheathed Sheathed Sheathed Habitat Blood Blood Blood Mansonella ozzardi Mansonella Mansonella perstans ozzardi Onchocerca volvu/us Onchocerca volvulus ~ ~ _,.r I Nuclei extend to Nuclei do not Nuclei do not the tip of the tail extend to the tip extend to the tip of of the tail the tail -- - 180 X 4 µm 220 X 4 µm 200 x 360 µm Unsheathed Unsheathed Unsheathed Blood Blood Skin, eye
- 225. Paniker'sTextbookofMedical Parasitology Box 1:Different types of periodicity exhibited by m,crofilariae • Nocturnalperiodicity:When the largest number ofmicrofilariae occur in blood at night, e.g. Wuchereria bancrofti • Diurnalperiodicity:When the largest number of microfilariae occur in blood during day, e.g. Loa loa • Nonperiodic:When the microfilariae circulate at constant levels during the day and night, e.g. Onchocerca volvulus • Subperiodic or nocturnally subperiodic: When the microfilariae can be detected in the blood throughout the day but are detected in higher numbers during the late afternoon or at night. Note:The microfilariae are found in capillaries and blood vessels of lungs during the period when they are not present in the peripheral blood. Table 3: Filarial nematodes infecting humans Parasite I. Lymphatic filariasis Wuchereria bancrofti Brugia malayi Brugia timori II. Subcutaneous filariasis Loaloa Onchocerca volvulus Mansonella streptocerca Ill. Serous cavityfilariasis Mansonella ozzardi Mansonella perstans Location In body adult Lymphatics Lymphatics Lymphatics Connective tissue, conjunctiva Mlcrofilaria Blood Blood Blood Blood Subcutaneous nodules Skin, eyes Subcutaneous Skin Peritoneum and pleura Blood Peritoneum and pleura Blood • LYMPHATIC FILARIASIS Wuchereria Bancrofti History and Distribution Filariasis has been known from antiquity. Elephantiasis had been described in India by Sushruta and in Persia by Rhazes and Avicenna. • Elephantiasis-painful, disfiguring swelling of the legs and genital organs-is a classic sign oflate-stage disease. • The term Malabar leg was applied to the condition by Clarke in 1709 in Cochin. • Microfilaria was first observed by Demarquay (1863) in the hydrocele fluid of a patient from Havana, Cuba. The genus is named after Wucherer, a Brazilian physician who reported microfilariae in chylous urine in 1868. Charocterlstlcs ofmlcrofilarla Periodicityof micrafilaria Sheathed, pointed tail tip free Nocturnal of nuclei Sheathed, blunt tail tip with Nocturnal two terminal nuclei Sheathed, longer than Nocturnal Mf.malayi Sheathed, nuclei extending Diurnal up to pointed tail tip Unsheathed, blunt tail tip free Nonperiodic of nuclei Unsheathed blunt tail tip Nonperiodic with nuclei Unsheathed, pointed tail tip Nonperiodic without nuclei Unsheathed, pointed tail tip Nonperiodic with nuclei Principal vector Cu/exqumquefasciatus Manson/a spp. Anopheles barbirostris Chrysops spp. Simulium spp. Culicoides Culicoides Culicoides Microfilaria was first demonstrated in human blood in Calcutta by Lewis (1872). • In 1876, Bancroft first reported and described adult female worm and in 1888, adult male wormwas described by Bourne. • Manson (1878) in China identified the Culex mosquito as the vector. This was the first discovery of insect transmission of a human disease. Manson (1879) also demonstrated the nocturnal periodicity of microfilariae in peripheral blood. • W. bancrofti is distributed widely in the tropics and subtropics of sub-Saharan Africa, South-East Asia, India and the Pacific islands. The largest number of cases of filariasis occurs in India (Fig. 1). • In India, the endemic areas are mainly along the sea coast and along the banks of the large rivers, though infection occurs virtually in all states, except in the north-west.
- 226. Fig. 1: Geographicaldistribution of Wucherer/a bancrofti Habitat The adult worms reside in the lymphatic system of man. The microfilariae are found in blood. Morphology Adult worm: The adults are whitish, translucent, thread-like worms with smooth cuticle and tapering ends. • • • • • The female is larger (70-100 x 0.25 mm) than the male (25-40 X 0.1 mm). The posterior end of the female worm is straight, while that of the male is curved vertically and contains two spicules ofunequal length. Males and females remain coiled together usually in the abdominal and inguinal lymphatics and in the testicular tissues (Fig. 2). The female worm is viviparous and directly liberates sheathed microfilariae into lymph. The adult wormslivefor manyyears, probably 10-15years or more. Mtcro.filariae: The microfilaria has a colorless, translucent body with a blunt head, and pointed tail {Fig. 3). • It measures 250-300 µm in length and 6-10 µm in thickness. It can move forwards and backwards within the sheath which is much longer than the embryo. • It is covered by a hyaline sheath, within which it can actively move forwards and backwards as sheath is much longer than the embryo. • WhenstainedwithLeishmanorother Romanowskystains, structural details can be made out. Along the central axis of the microfilaria, a column of granuJes can be seen, which are called somatic cells or nuclei.The granules are absent atcertain specific locations-a feature which helps in the identification of the species. The specific locations are as following (Fig. 3): Filarial Worms Fig. 2: Adult worm of Wucherer/a bancrofti ---...--Sheath _,...,__.....---.,..---,,,,,__ Stylet Anterior V-spot Fig. 3: Morphology of Microfilaria bancrofti - At the head end is a clear space devoid of granules, called the cephalic space. In Micro.ft/aria bancrofti, the cephalic space is as Jong as it is broad, while in Micro.ft/aria malayi, it is longer than its breadth. With vital stains, a stylet can be demonstrated projecting from the cephalic space (see Fig. 9). In the anterior half of the microfilaria, is an oblique area devoid ofgranules called the nerve ring. Approximately midway along the length of the microfilaria is the anterior V-spot, which represents the rudimentary excretory system. The posterior V-spot (tail spot) represents the cloaca or anal pore.
- 227. Paniker'sTextbook ofMedical Parasitology -The genital cells (G-cells) are situated anterior to the anal pore. - The internal(central) bodyofManson extending from the anterior V-spot to G-cell one, representing the rudimentary alimentary system. - The tail tip, devoid of nuclei in Mf bancrofti (distinguishing feature), bears two distinct nuclei in Mf malayi (see Fig. 9). • Microfilariae do not multiply or undergo any further development in the human body. Ifthey are not taken up by a female vector mosquito, they die. • Their lifespan is believed to be about 2- 3 months. • Tt is estimated that a microfilarial density ofat least 15 per drop of blood is necessary for infecting mosquitoes. Periodicity • The microfilariae circulate in the bloodstream. • In India, China and many other Asian countries, they show a nocturnal periodicity in peripheral circulation; being seen in large numbers in peripheral blood only at night (between 10pm and 4 am). • This correlates with the night biting habit of the vector mosquito. Infective larva • Periodicity may also be related to the sleeping habits of the hosts. lt has been reported that if the sleeping habits of the hosts are reversed over a period, the microfilariae change their periodicity from nocturnal to diurnal. • Nocturnal periodicrnicrofilariae are believed to spend the day time mainly in the capillaries ofthe lung and kidneys or in the heart and great vessels. • In the Pacific islands and some parts of the Malaysian archipelago, the microfilariae are nonperiodic or diurnal subperiodic, such that they occur in peripheral circulation at all times, with a slight peak during the late afternoon or evening. This is related to the day-biting habits of the local vector mosquitoes (some authors separate the subperiodic Pacific type of W. bancrofti as a distinct species designated W. pacifica, but this is not widely accepted). Life Cycle Wuchereria bancrofti passes its life cycle in two hosts (Fig. 4): 1. Definitive host: Man. No animal host or reservoir is known for W. bancrofti. 2. Intermediate host: Female mosquito, ofdifferent species acts as vectors in different geographic areas. The major deposited on the skinl_,,,___._ of man when mosquito bites ( Infective 3rd-stage of mosquito Mosquito lymphatic system and lymph nodes X larva lying in the proboscis sheath (intermediate host) Man (definitive host) 2nd-stage larva Short 1st-stage larva Ingested by female mosquito during blood meal Fig. 4: life cycle of Wuchereria bancrofti
- 228. Box 2: Parasiteswith mosquito as intermediate host • Wuchereria bancrofti • Brugia spp. • Mansonella spp. • Dirofilaria spp. vector in India and most other parts of Asia is Cu/ex quinquefasciatus(C.fatigans) (Box 2). Infectiveform: Actively motile third-stage filariform larva is infective to man. Mode of transmission: Humans get infection by bite of mosquito carrying filariform larva. Development in mosquito:When a vector mosquito feeds on a carrier, the microfilariae are taken in with the blood meal and reach the stomach ofthe mosquito. • Within 2-6hours,theycastofftheirsheaths (exsheathing), penetrate the stomach wall and within 4- 17 hours migrate to the thoracic muscles where they undergo further development. • During the next 2 days, they metamorphose into thefirst- stage larva, which is a sausage-shaped with a spiky tail, measuring 125-250 x 10-15 µm (Fig. 4). • Within a week, it moults once or twice, increases in size and becomes the second-stage larva, measuring 225-325 x 15-30 µm {Fig. 4). • In another week, it develops its internal structures and becomes the elongated third-stage filariform larva, measuring 1,500-2,000 x 15-25 µm. It is actively motile and is the infectiveform {Fig. 4). • It enters the proboscis sheath of the mosquito, awaiting opportunity for infecting humans on whom the mosquito feeds. • There is no multiplication of the microfilaria in the mosquito and one rnicrofilaria develops into one infective larva only. • The time taken from the entry of the microfilaria into the mosquito till the development of the infective third- stage larva located in its proboscis sheath, constitutes the extrinsic incubation period. Its duration varies with environmental factors such as temperature and humidity, as well as with the vector species. Under optimal conditions, its duration is 10-20 days. • When a mosquito with infective larvae in its proboscis feeds on a person, the larvae get deposited, usually in pairs, on the skin near the puncture site. Developmentin man:The larvae enter through the puncture wound or penetrate the skin by themselves. • The infective dose for man is not known, but many larvae fail to penetrate the skin by themselves and many more are destroyed in the tissues by immunological and other Filarial Worms Table 4: Differences between classical and occult filariasis Cause Basic lesion Organs involved Classkal filariasis Due to adult and developing worms Lymphangitis, lymphadenitis Lymphatic vessels and lymph node Occult filariasis Hypersensitivity to mlcrofilarial antigen Eosinophilic granuloma formation Lymphatic system, lung, liver, spleen,joints Microfilaria Present in blood Present in tissues but not in blood Serological Complement fixation Complement fixation test test test not so sensitive highly sensitive Therapeutic No response Prompt response to response diethylcarbamazine (DEC) defense mechanisms. A very large number of infected mosquito bites are required to ensure transmission to man, perhaps as manyas 15,000infective bitesperperson. • After penetrating the skin, the third-stage larvae enter the lymphatic vessels and are carried usually to abdominal or inguinal lymph nodes, where they develop into adult forms {Fig. 4). • There is no multiplication at this stage and onlyone adult develops from one larva, male or female. • They become sexually mature in about 6 months and mate. • The gravid female worm releases large numbers of microfilariae, as many as 50,000 per day. They pass through the thoracic duct and pulmonary capillaries to enter the peripheral circulation. • The microfilariae are ingested with the blood meal by mosquito and the cycle is repeated. Prepatent period: The period from the entry ofthe infective third-stage larvae into the humanhosttill the first appearance of microfilariae in circulation is called the biological incubation period or the prepatent period. This is usually about 8-12 months. Clinical incubation period:The period from the entry of the infective larvae, till the development of the earliest clinical manifestation is called the clinical incubation period. This is veryvariable, butis usually8-16months, though itmayoften be much longer. Pathogenesis Infection caused by W. bancrofti is termed as wuchereriasisor bancroftian filariasis. The disease can present as (Table 4): • Classical filariasis • Occult filariasis.
- 229. Paniker'sTextbook of MedicalParasitology Classicalfilariasis: Pathogenesis: • It occurs due to blockage of lymph vessels and lymph nodes by the adult worms. The blockage could be due to mechanicalfactors or allergic inflammatory reaction to worm antigens and secretions. The affected lymph nodes and vessels are infiltrated with macrophages, eosinophils, lymphocytes and plasma cells. The vessel walls get thickened and the lumen narrowed or occluded, leading to lymph stasis and dilatation of lymph vessels. The worms inside lymph nodes and vessels may cause granuloma formation, with subsequent scarring and even calcification. Inflammatory changes damage the valves in lymph vessels, further aggravating lymph stasis. Increased permeability of lymph vessel walls lead to leakage of protein-rich lymph into the tissues. This produces the typical hard pitting or brawny edema of filariasis. Fibroblasts invade the edematous tissues, laying down fibrous tissue, producing the nonpitting gross edema of elephantiasis. Recurrent secondary bacterial infections cause further damage. • Animal models have been developed, such as experi- mental filarial infection in cats with Brugia pahangi or Br. malayi. These have helped in understanding the pathogenesis of the disease, but in cats and other animals, filarial infection does not cause elephantiasis. Elephantiasis is a feature unique to human filariasis, apparently caused by human erect posture and consequent hydrodynamic factors affecting lymph flow. Clinical manifestations: The most common presentations of lymphatic filariasis are asymptomatic (subclinical) microfilaremia, acute adenolymphangitis (AOL) and chronic lymphatic disease. • Most of the patienLs appear clinically asymptomatic but virtually all of them have subclinical disease including microscopic hematuria or proteinuria, dilated lymphatics (visualized by imaging) and in men with W. bancrofli infection, scrotal Iymphangiectasia (detected by ultrasound). • Acute adenolymphangitis is characterized by high fever, lymphatic inflammation (lymphangitis and lymphadenitis) and transient local edema. - Fever is of high grade, sudden in onset, associated with rigors and last for 2 or 3 days. Lymphangitis is inflamed lymph vessels seen as red streaks underneath the skin. Lymphatics of the testes and spermatic cord are frequently involved, with epididymo-orchitis and funiculitis. Acute lymphangitis is usually caused by allergic or inflammatory reaction to filarial infection, but may often be associated with streptococcal infection also. - Lymphadenitis: Inflammation oflymph nodes. Most common affected lymph nodes being inguinal nodes followed by axillary nodes. The lymph nodes become enlarged, painful and tender. - Lymphedema: This follows successive attacks of lymphangitis and usually starts as swelling around the ankle, spreading to the back of Lhe foot and leg. It may also affect the arms, breast, scrotum, vulva, or any other part of body. Initially, the edema is pitting in nature, but in course of time, becomes hard and nonpitting. Lymphangiovarix: Dilatation of lymph vessels commonly occurs in the inguinal, scrotal, testicular and abdominal sites. The lymphangitisandlymphadenitiscaninvolve both the upper and lower extremities in both bancroftian and brugian filariasis but involvement of genital lymphatics occurs exclusively with W. bancrofti infection. The genital involvement can be in the form offuniculitis, epididymitis and hydrocele formation. • Hydrocele: This is a very common manifestation of filariasis. Accumulation offluid occurs due to obstruction of lymph vessels of the spermatic cord and also by exudation from the inflamed testes and epididymis. The fluid isusuallyclearand strawcolored but maysometimes be cloudy, milky, or hemorrhagic. The hydrocele may be unilateral or bilateral and is generally small in size in the early stage, but may occasionally assume enormous proportions in association with elephantiasis of the scrotum. The largestreported hydrocele weighed over 100 kilograms. • Lymphorrhagia: Rupture of lymph varices leading to release of lymph or chyle and resulting in chyluria (Fig. 5), chylousdiarrhea, chylousascitesand chylothorax, depending on the involved site. • Elephantiasis: This is a delayed sequel to repeated lymphangitis, obstruction and lymphedema. Repeated leakage oflymph into tissues first results in lymphedema, then to elephantiasis, in which there is nonpitting brawny edema with growth of new adventitious tissue and thickened skin, cracks, and fissures with secondary bacterial and fungal infections, commonly seen in leg but may also involve other parts ofbody {Fig. 6). Clinical features of filarlasis • Asymptomaticmicrofilaremia,acuteadenolymphangitis, lymphadenitis , Lymphedema, lymphangiovarix, chronic funiculitis, epldidymiltis hydrocele, elephantiasis, chylothorax, chyluria Occultfllariasis: • It occurs as a result of hypersensitivity reaction to microfilarial antigens, not directly due to lymphatic involvement.
- 230. Fig. 5: Chylousurine • Microfilariae are not found in blood, as they are destroyed by the allergic inflammation in the tissues. • Clinical manifestations: - Massive eosinophilia (30-80%) - Hepatosplenomegaly - Pulmonary symptoms like dry nocturnal cough, dyspnea and asthmatic wheezing. - Occult filariasis has also been reported to cause arthritis, glomerulonephritis, thrombophlebitis, tenosynovitis, etc. - Classical features oflymphatic filariasis are absent. • Meyers Kouwenaar syndrome is a synonym for occult filariasis. • Tropical pulmonaryeosinophilia: - This is a manifestation of occult filariasis which presents with low-grade fever, loss of weight, and pulmonary symptoms such as dry nocturnal cough, dyspnea and asthmatic wheezing. - Children and young adults are more commonly affected in areas of endemic filariasis including the Indian subcontinent. - There is a marked increase in eosinophil count (>3000 µm which may go up to 50,000 or more). - Chest X-ray shows mottled shadows similar to miliarytuberculosis. - It is associated with a high level of serum immunoglobulin E (IgE) and filarial antibodies. - Serological tests with filarial antigen are usu ally strongly positive. - The condition responds to treatment with diethylcarbamazine (DEC), which acts on microfilariae. FilarialWorms Fig. 6: Elephantiasis of the legs LaboratoryDiagnosis The diagnosis of filariasis depends on the clinical features, history of exposure in endemic areas and on laboratory findings. The laboratory tests that can be used for diagnosis has been described in Flow chart 2. Demonstrationofmicrofilaria: • Microfilaria can be demonstrated in blood, chylous urine (Fig. 6) exudate of lymph varix and hydrocele fluid. Peripheral blood is the specimen ofchoice. • The method has the advantage that the species of the infecting filaria can be identified from the morphology of the microfilaria seen. It is also the method used for carrier surveys. • In India and other areas, where the prevalent filarial species is nocturnally periodic, it is best to collect "night blood" samples between 10 pm and 4 am. • Microfilaria can be demonstrated in unstained as well as stained preparations and in thick as well as thin smears (Fig. 7). Unstainedfilm: • Examination under the low power microscope shows the actively motile microfilariae lashing the blood cells around. • The timing of blood collection is critical and should be based on the periodicity ofthe microfilariae. • The examination may be conveniently made the next morning as microfilariae retain theirviability and motility for a day or 2 at room temperature. Stained.film:A "thickand thin" blood smear is prepared on a clean glass slide and dried.
- 231. Paniker'sTextbook of MedicalParasitology Flow chart 2: Laboratory diagnosis of Wuchereria bancrofti laboratory diagnosis I l I I l Direct evidence Detection of microfllariae By examination of a thick and thin blood smear, stained with Giemsa stain By examination of unstained mount of blood under microscope By acridlne orange - microhematocrit tube technique Indirect evidence • Eosinophilia in blood • Elevated serum lgE levels Detection of adult worm • Lymph node biopsy • On X-ray (if worms are calcified) • High frequency ultrasound and Doppler within the scrotum Note: Adult worms have a distinctive pattern of movement (termed the ti/aria dance sign) within the lymphatic vessel lmmunodiagnosis Antigen detection ELISA • ICT • Both tests have sensitivity of 93-100% and specificity of 100% and sample can be collected during day time Antibody detection • CFT • IHA • IFA These test have low sensitivity and specificity Molecular diagnosis • Done by PCR • The test is positive only when microfilaria are present in peripheral blood. Negative in chronic filariasis Abbreviations: CFT, complement fixation test; ELISA, enzyme-linked immunosorbent assay; ICT, immunochromatographic test; IFA, indirect fluorescent antibody; lgE, immunoglobulin E; IHA, indirect hemagglutination; PCR, polymerase chain reaction Fig. 7: Microfilaria in blood film Source: Mohan H. Textbook of Pathology, 6th edition. New Delhi: Jaypee Brothers Medical Publishers; 2010. p. 190. • The thick part of the smear is dehemoglobinized by applying distilled water. The smear is fixed in methanol and stained with Giemsa, Leishman, or polychrome methylene blue stains. Microfilariae may be seen under the low power microscope in the thick film. • The morphology of microfilariae can be studied in thin film. The microfilaria of W. bancrofti are sheathed and appear as smooth curves in stained smear and are 298 µm long and 7.5-10 µmin diameter (Fig. 7). • By using a micropipette for taking a known quantity of blood (20-60 rnm3 ) for preparing the smear and counting the number of microfilariae in the entire stained smear, microfilaria counts can be obtained. Concentration techniques: When the microfilaria density is low, concentration techniques are used: • Knott's concentration technique: Anticoagulated blood (1 mL) is placed in 9 mL of 2% formalin and centrifuged 500 x g for 1 minute. The sediment is spread on a slide to drythoroughly. Theslide is stained with Wright or Giemsa stain and examined microscopically for microfilariae. • Nucleopore filtration: In the filtration methods used at present, larger volumes of blood, up to 5 mL, can be filtered through millipore or nucleopore membranes (3 µm diameter). The membranes may be examined as such or after staining, for microfilariae. The filter membrane technique is much more sensitive, so that blood can be collected even duringdaytime for screening. The disadvantages of the technique are the cost and the need for venipuncture. • Dtethylcarbamazine provocation test: A small dose of DEC (2 mg per kg body weight) induces microfilariae to
- 232. Box 3:Parasites foundin urine • Wuchereria bancrofti • Schistosoma hematabium • Trichamanas vagina/is. appear in peripheral blood even during day time. For surveys, blood samples can be collected 20-50 minutes after the administration of one 100 mg tablet of DEC to adults. • Other specimens: Microfilaria may be demonstrated in centrifuged deposits of lymph, hydrocele fluid, chylous urine or other appropriate specimens. Usually 10-20 mL ofthefirst earlymorningurineiscollectedforexamination and demonstration (Box 3). Biopsy:Adultfilarialwormscan be seeninsectionsofbiopsied lymph nodes, but this is not employed in routine diagnosis. Skin test:Intradermal injection offilarial antigens (extracts of microfilariae, adult worms and third-stage larvae of8. malayi or ofthe dog filaria Dirofilaria irnrnitis) induce an immediate hypersensitivity reaction. But, the diagnostic value ofthe skin test is very limited due to the high rate of false-positive and negative reactions. Imaging techniques: mtrasonography: High frequency ultrasonography (USG) ofscrotum and female breast coupled with Doppler imaging may result in identification of motile adult worm (filaria dance sign) within the dilated lymphatics. • Adult worm may be visualized in the lymphatics of the spermatic cord in up to 80% of the infected men with microfilaria associated with W. bancrofti. Radiology: • Dead and calcified worms can be detected occasionally by X-ray. • In tropical pulmonary eosinophilia (TPE), chest X-ray shows mottled appearance resembling miJiary tuberculosis. • Intravenous urography, retrograde pyelography, lymphangiography and lymphoscintigraphy may be used to demonstrate abnormal lymphatic urinaryfistula. Serodiagnosis: Demonstration of antibody: Several serological tests, including complement fixation, indirect hemagglutination (IHA), indirect fluorescent antibody (IPA), immunodiffusion and immunoenzyme tests have been described. • Indirect immunofluorescence and enzyme-linked irnmunosorbent assay (ELISA) detect antibodies in over 95% of active cases and 70% ofestablished elephantiasis. Disadvantages: Antibody detection test cannot differentiate between current and past infections. Filarial Worms Demonstration of circulating antigen: Highly sensitive and specific test for detection of specific circulating filarial antigen (CFA) have been developed for detection of recent bancroftian filariasis. • The Trop-bio test is a semiquantitative sandwich ELISA for detection ofCPA in serum or plasma specimen. • Imrnunochromatographic test (JCT) is a new and rapid filarial antigen test that detects soluble W. bancrofti antigens using monoclonal antibody (AD/2) in the serum ofinfected humans. • Both assay have sensitivities of93-100% and specificities approaching 100%. • Specific IgG4 antibody against W. bancrofti antigen WbSXP-1 have been used to develop ELISA for detecting circulating filarialantigen in sera ofpatientswith filariasis. • There is however, extensive cross-reactivity between filarial antigens and antigens of other helminths, including intestinal roundworm, thus interpretation of serological findings can be difficult. Advantages: Antigen detection tests are more sensitive than microscopy and can differentiate between current and past infections. Molecular diagnostic technique: Polymerase chain reaction (PCR) can detect filarial deoxyribonucleic acid (DNA) from patient's blood, onlywhen circulatingmicrofilaria are present in peripheral blood but not in chronic carrier state. • Usually the testprovides sensitivities that are up to tenfold greater than parasitic detection by direct examination and is 100% specific. Indirectevidences:Eosinophilia (5-15%) isa commonfinding in filariasis. Elevated serum IgE levels can also be seen. Treatment Diethylcarbamazine is the drug of choice. It is given orally in a dose of6 mg/ kg body weight daily for a period of12 days amounting to a total of 72 mg of DEC per kg of body weight. It has both macro and microfilaricidal properties. Following treatment with DEC severe allergic reaction (Mazzotti reaction) may occur due to death ofmicrofilariae. It kills both microfilaria and adult worm. Antihistamines or corticosteroids may require to control the allergic phenomenon. The administration of DEC can be carried oul in three ways: l. Mass therapy: In this approach, DEC is given to almost everyone in community irrespective of whether they have rnicrofilarernia disease manifestation or no signs of infection except those under 2 years of age, pregnant womenandseriously-ill patients. The dose recommended is 6 mg/kg body weight. In some countries it is used alone and in some, with albendazole or ivermectin. Mass therapy is indicated in highly endemic areas.
- 233. Paniker'sTextbook of MedicalParasitology 2. Selective treatment: Diethylcarbamazine is given only to those who are microfilaria-positive. In India, the current strategy is based on detection and treatmelll of human carriers and filarial cases. The recommended dose in the Indian program is DEC 6 mg/ kg of body weight daily for 12 doses, to be completed in 2 weeks. In endemic areas, treatment must be repeated every 2 years. 3. Diethylcarbamazine medicated salts: Common salt medicated with 1-4 gram ofDEC per kg has been used for filariasis conLrol in Lakshadweep island, after an initial reduction in prevalence had been achieved by mass or selective treatment ofmicrofilaria carriers. Ivermectin: In doses of 200 µg/kg can kill the microfilariae but has no effect on adults. It is not used in India. It is used in regions ofAfrica. Tetracyclines or doxycycline for 4-8 weeks also have an effectin the treatmentoffilariasis byinhibitingendosymbiotic bacteria (Wolbachia species) that are essential for the fertility ofthe worm. Supportive treatment: • Chroniccondition may not be curableby antifilarial drugs and require other measures like elevation of the affected limb, use of elastic bandage and local foot care reduce some ofthe symptoms ofelephantiasis. • Surgery is required for hydrocele. • Medical management of chyluria includes bed rest, high protein diet with exclusion of fat, drug therapy with DEC and use of abdominal binders. • Surgical management of refractory case includes endoscopic sclerotherapy using silver nitrate. Prophylaxis The two major measures in prevention and control offilariasis are: l. Eradication ofthe vector mosquito. 2. Detection and treatment ofcarriers. Eradication ofvector mosquito: • Antilarval measures: The ideal method of vector control would be elimination of breeding places by providing adequate sanitation and underground waste water disposal system. However, this involves a lot of expenditure, hence current approach in India is to restrict the antilarval measures to urban areas by: Chemicalcontrol: Using antilarval chemicals like: • Mosquito larvicidal oil • Pyrosene oil-E • Organophosphorous larvicides like temephos, fenthion, etc. - Removal ofPistia plant: Mainly restricted to control of Mansonia mosquitoes leading to brugian filariasis. • Anliadultmeasures:Adultmosquitoes can berestricted by use of dichlorodiphenyltrichloroethane (DDT), dieldrin and pyrethrum. However, vector mosquitoes of filariasis have become resistant to DDT and dieldrin. Pyrethrum, as a space spray, is still being used. • Personal prophylaxis: Using mosquito nets and mosquito repcllants is the best method. KEY POINTS OF WUCHERER/A BANCROFT/ • Adult worm is white, thread-like with smooth cuticle and tapering end. • The female worm is viviparous. The embryo (microfilaria) is colorless, sheathed, with tail-tip free of nuclei and actively motile. • Microfilaria in blood shows nocturnal periodicity (10 pm to 4 am). • Definitive host: Man. • Intermediate host: Cu/ex quinquefasciatus (C. fatigans). • Microfilaria do not multiply in man. When taken up by vector mosquito, it undergoes stages of development and become third-stage filariform larva which is the infective form. • Pathogenesis: Adult worm causes mechanical blockage of lymphatic system and allergic manifestations. • Clinical features: Early stage-fever, malaise, urticaria, fugitive swelling, lymphangitis. Chronic stage-lymphadenitis, lymphangiovarix, chyluria, hydrocele and elephantiasis. Tropical pulmonary eosinophilia occurs due to hypersensitivity reaction to filarial antigen. • Diagnosis: Demonstration of microfilaria in peripheral blood or chylous urine. Demonstration ofadult worm in biopsy, Doppler USG and X-ray. Demonstration of filarial antigen and antibody. • Treatment: Drug of choice is DEC and ivermectin. Supportive and surgical management in some cases. Detection and treatment of carriers: The recommended treatment is DEC 6 mg per kg body weight daily for 12 days, the drug being given for 2 weeks, 6 days in a week. Brugia Malayi History and Distribution • the genus Brugia was named after Brug, who in 1927 described a new type ofmicrofilaria in the blood ofnatives in Sumatra. • The adult worm of 8. malayi was described by Rao and Maplestone in India (1940). • Besides 8. malayi, the genus includes B. timori, which parasitizes humans in Timor, Indonesia and a number of animal species, such as B. pahangi and 8. patei infecting dogs and cats. • The geographical distribution of B. malayi is much more restricted than that of W. bancrofti. It occurs in India and Far-East, Indonesia, Philippines, Malaysia, Thailand, Vietnam, China, South Korea and Japan.
- 234. Fig. 8: Geographicaldistribution of Brugia malayi In India, Kerala is the largest endemic area, particularly the districts of Quilon, Alleppey, Konayam, Emakulam and Trichur. Endemic pockets occur in Assam, Orissa, Madhya Pradesh and West Bengal. B. malayi and W. bancrofti may be present together in the same endemic area, as in Kerala. In such places, B. malayi tends to be predominantly rural and W. bancrofti urban in distribution (Fig. 8). Morphology Adult worms: • The adult worms of B. malayi are generally similar to those ofW. bancrofti, though smaller in size. Microfilariae: The microfilariae of B. malayi, although sheathed are different in a number of respects from Micro.ft/aria bancrofti. • Mf malayi is smaller in size, shows kinks and secondary curves, its cephalic space is longer, carries double stylets at the anterior end, the nuclear column appears blurred in Giemsa-stained films and the tail tip carries two d istinct nuclei, one terminal and the other subterminal (Fig. 9 and Table 5). Life Cycle the life cycle of 8. malayi is similar to that or W. bancrofli; however, the intermediate host ofBrugia are vectors ofgenera Mansonia, Anopheles and Aedes. In India, main vectors are Mansonia annulifera and M. uniformis. • Pathogenicity, clinical features, laboratory diagnosis and treatment are similar to W. bancrofti. Filarial Worms Table S: Distinguishing features of Mf. bancrofti and Mt. malayi Features Mf. bancrofti Mf.malayi Length 250-300 µm 175- 230µm Appearance Graceful, sweeping Kinky, with secondary curves curves Cephalic space Length and breadth Almost twice as long as equal broad Stylet at anterior Single Double end Excretory pore Not prominent' Prominent Nuclear column Discrete nuclei Blurred Tail tip Pointed, free of nuclei Two distinct nuclei, are at tip, the other subterminal Sheath Faintly-stained Well stained • Prevention: The breeding of Mansonia mosquito is associated with certain plants such as Pistia. In absence ofthese plants, mosquito cannot breed. Thus in countries like Sri Lanka and India where M. annulifera is the chief vectorofB. malayi, the transmission ofthe parasite can be effectively reduced by removal ofthese plants in addition to the antilarval, antiadult and self prophylaxis methods described in W. bancrofti. Brugia Timori Brugia timori is limited to Timor and some other islands of Eastern Indonesia. • The vector of B. timori is Anopheles barbirostris, which breeds in rice fields and is a night feeder. • Definitive host: Man. No animal reservoir is known. • The microfilaria is larger than Mf malayi. The sheath of Mf timori fails to take Giemsa stain with 5-8 nuclei present in the tail. • 11,e lesions produced by 8. timoriare milder than those of bancroftian or malayan filariasis. Acharacteristic lesion is the development of draining abscesses caused by worms in lymph nodes and vessels along the saphenous vein, leading to scarring. • SUBCUTANEOUS FILARIASIS Loa Loa Common Name African eyeworm. History and Distribution Loa Loa, causing loiasis, "fugitive swellings'' or "Calabar swellings'; was first detected in the eye of a patient in West
- 235. Paniker'sTextbook ofMedical Parasitology Largeregular~ and smooth body waves Single-- -~H style! Length and breadth of cephalic space equal Sheath faintly ____ stained Body nuclei---- discrete Tail up,-- - --+tP pointed Microfilaria bancrofti Kinky, small and irregular body waves Length of cephalic space more than breadth 1 - - - - Well-stained sheath I-Body nuclei blurred and squeezed 11-+.~+--Tail tip rounded with two nuclei at tail-tip Mlcrofl/aria malayi Fig. 9: Schematic diagram showing distinguishing features of Microfilaria bancrofti and Microfi/aria ma/ayi Indies in 1770. But at present, it is limited to its primary endemic areas in the forests ofWest and Central Africa, where about 10 million people are affected. Life Cycle Life cycle is completed in two hosts: 1. Morphology 2 · Adult worm: The adult worm is thin and transparent, measuring about 30-70 mm in length and 0.3-0.5 mm in thickness. • • In infected persons, they live in the subcutaneous tissues, through which they wander. They may also occur in the • subconjunctival tissue. • Adults live for 4-l 7 years. Micro.filaria: The microfilariae are sheathed with column of • nuclei extending completely to the tip ofthe tail. • They appear in peripheral circulation only during the day • from 12 noon to 2 pm diurnal periodicity). Definitive host: Man Intermediate host or vectors: Day-biting flies (mango flies) ofthe genus Chrysops, (C. dimidiata, C. silacea and other species) in which the microfilariae develop into the infective third-stage larvae. Infection is transmitted to manthrough the bite ofinfected Chrysops during their blood meal. The infective third-stage larvae enter the subcutaneous tissue, moult, and develop into mature adult worm over 6-12 months and migrate in subcutaneous tissues. Femaleworms produce sheathed microfilaria which have diurnal periodicity. The microfilaria is ingested by Chrysops during its blood meal.
- 236. • Theycast offtheirsheaths, penetrate the stomach wall and reach thoracic muscles where they develop into infective larvae. • Development in Chrysops is completed in about lOdays. Pathogenicity and Clinical Features The pathogenesis of loiasis depends on the migratory habit ofthe adult worm. • Their wanderings through subcutaneous tissues set up temporary foci of inflammation, which appear as swellings, of up to 3 cm in size, usually seen on the extremities. These are the Calabar swellings or fugitive swellings, because they disappear in a few days, only to reappear elsewhere. • Ocular manifestations occurwhen the worm reaches the subconjunctival tissues during its wanderings. l h e ocular lesions include granulomata in the bulbar conjunctiva, painless edema of the eyelids and proptosis. • Complications like nephropathy, encephalopathy and cardiomyopathy can occur but are rare. Laboratory Diagnosis Diagnosis rests on the appearance of fugitive swelling in persons exposed to infection in endemic area. • Definitive diagnosis requires the detection ofmicrofilaria in peripheral blood or the isolation of the adult worm from the eye. • Microfilariae may be shown in peripheral blood collected during the day. • The adult worm can be demonstrated by removal from the skin or conjunctiva or from a subcutaneous biopsy specimen from a site ofswelling. • High eosinophil count is common. Treatment Diethylcarbamazine (8-10 mg/ kg per day for 21 days) is effective against both the adult and the microfilarial forms of l. Loa, but requires multiple courses. It has to be used with caution as severe adverse reactions may develop following the sudden death oflarge numbers of microfilariae. • Simultaneous administration of corticosteroids minimizes such reaction. • lvermectin or albendazole although not approved by Food and Drug Administration (FDA) for this purpose, is effective in reducing microfilarial loads. lvermectin is contraindicated in patients with heavy microfilaremia (>5,000 microfilaria/mL). • Treatment by surgical removal of the adult worms is rarely done. Filarial Worms KEY POINTS OF LOA LOA • Loa loa is also known as African eyeworm and causes loiasis. • Vectors: Day-biting flies (Chrysops). • Microfilaria is sheathed and nuclei extend up to tail tip. • Microfilaria appears during the day (diurnal periodic). • Clinical features: Subcutaneousswellings (Calabar swellings), ocular granuloma, edema of eyelid and proptosis. • Diagnosis: Demonstration of adult worm from s kin and conjunctiva. Demonstration of microfilaria in peripheral blood during day. High eosinophil count • Treatment: Diethylcarbamazine with simultaneous administration of corticosteroid of other drugs which may be used. lvermectin or albendazole. Onchocerca Volvulus History and Distribution Onchocerca volvulus, the "convoluted filaria'; or the "blinding filaria" producing onchocerciasis or "river blindness" was first described by Leuckart in 1893. • Itaffects about40 million people, mainly in tropicalAfrica, but also in Central and South America. A small focus of infection exists in Yemen and South Arabia. • Onchocerciasis is the second major cause of blindness in the world. Habitat The adult worms are seen in nodules in subcutaneous connective tissue ofinfected persons. Morphology Adult worm: The adult worms are whitish, opalescent, with transverse striations on the cuticle (Fig. 10). Fig. 10: Onchocerca volvulus
- 237. Paniker'sTextbook of MedicalParasitology • the posterior end is curved, hence the name Onchocerca, which means "curved tail''. • the male worm measures about 30 mm in length and 0.15 mm in thickness and the female measures 50 cm by 0.4mm. Microfilaria: The microfilariae are unsheathed and nonperiodic. • They measure about 300 by 0.8 µm. • The microfilaria is found typically in the skin and subcutaneous lymphatics in the vicinity of parent worms. • They may also be found in the conjunctiva and rarely in peripheral blood. Life Cycle Life cycle is completed in two hosts: l. Definitive host: Humans are the only definitive host. 2. Intermediate hosts: Day-biting female black flies of th e genus Simulium (black flies). The vector Simulium species breed in "fast-flowing rivers"; and therefore, the disease is most common along the course ofrivers. Hence, the name "river blindness''. • The female black flies are "pool feeders" and suck in blood and tissue fluids. Microfilariae from the skin and lymphatics are ingested and develop within the vector, becoming the infective third-stage larvae, which migrate to its mouth parts. • The extrinsic incubation period is about 6 days. Infection is transmitted when an infected Simulium bites a person. • The prepatent period in man is 3- 15 months. • The adultworm lives in the human host for about 15 years and the microfilariae for about 1year. Pathogenicity and Clinical Features Pathogenesisdepends onthe host's allergicand inflammato1 y reactions to the adult worm and microfilariae. • The infective larvae deposited in the skin by the bite ofthe vector develop at thesite to adult worms. Adult worms are seen singly, in pairs, or in tangled masses in subcutaneous tissues. They may occur in the subcutaneous nodules or free in the tissues. • The subcutaneous nodule or onchocercoma is a circumscribed, firm, nontender tumor, formed as a result of fibroblastic reaction aroun d the worms. 1 odules vary in size from a few mm to about 10 cm. 111ey tend to occur over anatomical sites where the bones are superficial, such as the scalp, scapulae, ribs, elbows, iliac crest, sacrum and knees. the nodules are painless and cause no trouble except for their unsightly appearance • Microfilariae cause lesions in tl1e skin and eyes. The skin lesion is a dermatitis with pruritus, pigmentation, atrophy and fibrosis. In an immuno- logically hyperactive form of onchodennatitis called as Sowdah, the affected skin darkens as a result of intense inflammation, which occurs as result of clearing ofmicrofilariae from blood. Ocular manifestations range from photophobia to gradual blurring of vision, progressing to total blindness. Lesions may develop in all parts ofthe eye. The most common early finding is conjunctivitis with photophobia. Other ocular lesions include punctale or sclerosing keratitis, iridocyclitis, secondary glaucoma, choroidoretinitis and optic atrophy. Laboratory Diagnosis Microscopy: The microfilariae may be demonstrated by examination ofskinsnip from theareaofmaximalmicrofilariaJ density such as iliac crest or trapezius region, which is placed on a slide in water or saline. the specimen is best collected around midday. this method is specific and most accurate. • Microfilariae may also be shown in aspirated material from subcutaneous nodules. • ln patients with ocular manifestations, microfilariae may be found in conjunctival biopsies. • Adultworms can be detected in the biopsy material of the subcutaneous nodule. Serology: Serologicaltests are useful for the diagnosis ofcases in which microfiJariae are not demonstrated in the skin. • Enzyme-linked immunosorbent assay is more sensitive than skin snip tests. The test detects antibodies against specific onchocercal antigen. • A rapid card test using antigen 0Vl 6 to detect IgG4 in serum has been evaluated. Molecular diagnosis: Polymerase chain reaction from skin snipsis donein specialized laboratories and is highlysensitive and specific. Prophylaxis In 1974, World Health Organization (WHO) launched a control program inWestAfrica usingaerial larvicide for vector control and treatment of patients with ivermectin. This is believed to have prevented blindness in millions ofchildren. Treatment • Chemotherapy with ivermectin is the main stay of treatment. Ivermectin is given orally in a single dose of 150 µg/kg either yearly or semiannually. ln areas of Africa coendemic for 0. volvutus and Loa Loa, however, ivermectin is contraindicated because of severe post- trea011enl encephalopathy seen in patients. • Diethylcarbamazine and suramin have also been used. DEC destroys microfilariae, but usually causes an intense reaction (Mazzotti reaction) consisting of pruritus, rash,
- 238. lymphadenopathy, fever, hypotensionand occasionally, eye damage. • A 6 week course of doxycycline is macrofilariastatic, rendering the female worm sterile as it targets the Wolbachia endosymbiont offilarial parasites. • Surgical excision is recommended when nodules arc located on the head due to the proximity of the worm to the eyes. KEY POINTS OF ONCHOCERCA VOLVULUS • Onchocerca volvulus, produces onchocerciasis or "river blindness". • The adult worm is white with transverse striation on the cuticle. The posterior end is curved. • Microfilaria is unsheathed, tail-tip free of nuclei and nonperiodic. • Definitive host: Humans. • Intermediate host: Female black flies (Simulium). • Clinical features: Subcutaneous nodule formation (onchocercoma). Ocular manifestations-sclerosing keratitis, secondary glaucoma. optic atrophy, chorioretinitis. It is the second major cause of blindness in world. • Diagnosis: Demonstration of microfilaria from skin snips and aspirated material form subcutaneous nodules. Demonstration of lgG4 antibody and PCR. • Treatment: lvermectin is the drug of choice except in areas coendemic for 0. volvulus and L. loa. Mansonella Streptocerca Also known as Acanthocheilonema, Dipetalonema, or Tetrapetalonema streptocerca, this worm is seen only in West Africa. • 1he adult worms live in the dermis, just under the skin surface. • The unsheathed microfiliariae are found in the skin. • Culicoides species are the vectors. Chimpanzees may act as reservoir hosts. Infection may cause dermatitis with pruritus and hypopigmented macuJes. • Diagnosis is made by demonstration of the microfilariae in skin clippings. • Ivermectin (single dose of 150 µg/ kg) is effective in treating streptocerciasis. • SEROUS CAVITY FILARIASIS Mansonella Ozzardi Mansonella ozzardi is a New World filaria seen only in Central and South America and the West Indies. • the adult worms are found in the peritoneal and pleural cavities ofhumans. Filarial Worms • The nonperiodic unsheathed microfilariae are found in the blood. • Culicoides species are the vectors. • Infection does not cause any illness. • Diagnosis is made by demonstrating microfilariae in blood. • lvermectin (single dose 6 mg) is effective in treatment. Mansonel/a Perstans Also known as Acanthocheilonema, Dipetalonema, or Tetrapetalonema perslans, this worm isextensivelydistributed in tropical Africa and coastal South America. • The adult worms live in the body cavities of humans, mainly in peritoneum, less often in pleura, and rarely in pericardium. • The microfilariae arc unsheathed and subperiodic. • Vectors are Culicoides species. African primates have been reported to act as reservoir hosts. Infection is generally asymptomatic, though ii has been claimed that it causes transient abdominal pain, rashes, angioedema and malaise. Diagnosis is by demonstration of the microfilariac in peripheral blood or serosal effusion. • Doxycycline (200 mg twice a day for 6 weeks) targeting the Wolbachia endosymbiont in M. perstans is the first effective treatment. Zoonotic Filariasis Filariae naturally parasitic in domestic and wild animals may rarely cause accidental infection in man through the bite of their vectors. • In such zoonotic filariasis, the infective larvae develop into adults, but do not mature to produce microfilariae. The worm dies and the inflammatory reaction around the dead worm usually causes clinical manifestations. Brugia Pahangi A parasite of dogs and cats in Malay ia may infect man and cause lymphangitis and lymphadenitis. Dirofilaria lmmitis The dog "heartworm" is a common parasite of dogs, widely distributed in the tropics and subtropics. When humans get infected, the worm lodges in the right heart or branches of the pulmonary artery. l he dead worm becomes an embolus blocking a small branch of the pulmonary artery, producing a pulmonary infarct. The healed infarct may appear as a "coin lesion" on chest radiography and can be mistaken for malignancy.
- 239. Paniker's Textbook ofMedicalParasitology Dirofilaria Repens A natural parasite of dogs, it may sometimes infect humans, causing subcutaneous and subconjunctival nodules. Many Dirojilaria species may form nodules in human conjunctiva and are collectively calied Dirofilarla conjunctivae. REVIEW QUESTIONS 1. Name the species of filarial worms that infect humans and describe briefly the life cycle and laboratory diagnosis of Wuchereria bancrofti. 2. Short notes on: a. Microfilariae b. Periodicity of microfilariae c. Pathogenesis of lymphatic filariasis d. Tropical pulmonary eosinophilia e. Filariasis f. Preventive measures in filariasis g. Brugia malayi h. Loaloa i. Onchocerca volvulus 3. Differentiate between: a. Occult and classical filariasis b. Micron/aria bancrofti and Micron/aria malayi MULTIPLE CHOICE QUESTIONS 1. All are true regarding filariasis except a. Man is an intermediate host b. Caused by Wuchereria bancrofti c. Involves lymphatic system d. DEC is used in treatment 2. All of the following are true about Brugia malayi except a. The intermediate host in India is Mansonia mosquito b. The tail tip is free from nuclei c. Nuclei are blurred, so counting is difficult d. Adult worm is found in the lymphatic system 3. Hydrocele and edema in foot occurs in a. Wuchereria bancrofti b. Brugia malayi c. Brugia timori d. Onchocerca volvulus 4. In which stage of filariasis are microfilaria seen in peripheral blood a. Tropical eosinophilia b. Early adenolymphangitis stage c. Late adenolymphangitis stage d. Elephantiasis 5. Diurnal periodicity is seen in larvae of a. Brugia malayi b. Wuchereria bancrofti c. Loa loa d. Mansonella perstans 6. Which of the following microfilariae is unsheathed a. Mf. loa b. Mf. bancrofti c. Mf. malayi d. Mf. perstans 7. All of t he following parasites can be detected in urine sample except a. Wuchereria bancrofti b. Schistosoma haematobium c. Trichomonas vaginalis d. Giardia lamblia 8. Fugitive or calabar swelling is seen in infection with a. Onchocerca volvulus b. Loa Joa c. Wuchereria bancrofti d. Brugia timori 9. River blindness is the name given to disease caused by a. Loaloa b. Onchocerca volvulus c. Toxoplasma gondii d. Acanthamoeba culbertsoni 10. The filarial worm which can be seen in conjunctiva is a. Brugia malayi b. Loaloa c. Onchocerca volvulus d. None of the above Answer 1. a 2. b 8. b 9. b 3. a 10. b 4. b 5. C 6. d 7. d
- 240. CHAPTER 21 • COMMONNAME Guinea worm. • HISTORY AND DISTRIBUTION The guinea worm has been known from antiquity. It is believed to have been the "fiery serpent"in the Bible, which tormented the Israelites on the banks ofthe Red Sea. • The technique of extracting the worm by twisting it on a stick, still practiced by patients in endemic areas is said to have been devised by Moses. The picture of the "serpent worm" on a stick may have given rise to the physician's symbol ofcaduceus. • Galen named the disease dracontiasis, (Greek draco- dragon or serpent). Avicenna called it the Medina worm as it was prevalent there. llence, the name Dracunculus medinensis (Dracunculus being the diminutive ofDraco). • The worm was present in tropical Africa, the Middle East in Arabia, Iraq, Iran, and in Pakistan and India. ln India, it was seen in the dry areas in Rajasthan, Gujarat, Madhya Pradesh, Andhra Pradesh, Maharashtra, Tamil Nadu and Karnataka (fig. 1). About 50 million people were estimated to be infected with the worm. Fig. 1: Geographical distribution of Dracunculus medinensis infection (before its eradication) • The infection has been eradicated from India and all of Southeast Asia region by 2000. • The disease still remains endemic in 13 African countries including Sudan (highest incidence), Niger, etc. • HABITAT The adult females of D. medinensis are usually found in the subcutaneous tissue of the legs, arms and back in man. • MORPHOLOGY Adult Worm The adult female is a long, cylindrical worm with smooth milky-while cuticle resembling a long piece of white twine. It has a blunt anterior end and a tapering recurved tail (Fig. 2). It measures about a meter (60-120 cm) in length and 1-2 mm in thickness. • The body of the gravid female is virtually filled with the branches of an enormous uterus, containing some 3 million embryos. • The female worm is viviparous (Box 1). • The male worm, which is rarely seen, is much smaller than female being 10-40 mm long and 0.4 mm thick. Female worm survives for about a year, whereas life span ofmale worm is not more than 6 months. Larva The larva measures 500-750 µmin length and 15-25 µmin breadth. • It has a broad anterior end and a slender filiform tail which extends fora third ofthe entire body length (Fig. 3). • The cuticle shows prominent striations. • The larva swims about with a coiling and uncoiling motion.
- 241. Paniker'sTextbook ofMedical P arasitology Fig.2: Adult worm of Dracuncu/us medinensis Box 1: Viviparous nematodes • Dracunculusmedinensis • Trichinella spiralis • Wuchereria bancrofti • Brugia malayi • Brugia timori Ovoviviparous nematodes • Strongyloidesstercoralis. • LIFE CYCLE D. medinensis passes its life cycle in two hosts: l. Definitive host: Man 2. Intermediate host: Cyclops, in which embryos undergo developmental changes. There is no animal reservoir (Table l ). Infective Form 1hird-stage larva present in the hemocele of infected Cyclops. • Mode oftransmission: Humans get infected by drinking unfiltered water containing infected Cyclops. • Incubation period: About 1year. • The adult worm, which is viviparous discharges larvae, which are ingested by thefreshwater crustacean.Cyclops, the intermediate host. Development of Adult Worm in Man When water containing infected Cyclops is swallowed by man, the Cyclops is killed by the gastric acidity and the guinea worm larvae present in its hemocele are released. • The larvae penetrate the wall of the duodenum and reach the retroperitoneal and subcutaneous connective tissues. Fig. 3: Larva of Dracunculus medinensis Table 1: Parasites requiring one intermediate host to complete their life cycle Intermediate host Man Pig Cow Snail Cyclops Sandfly Tsetse fly Chrysops Mosquito Tick Triatomine bug Flea Parasite • Plasmodium species • Echinococcusgranu/osus • Echinococcus multilocularis • Taenia multiceps • Taenia solium • Taenia saginata asiatica • Sarcocystissuihominis • Trichinella spiralis • Taenia saginata • Sarcocystishominis Schistosoma species Dracunculusmedinensis Leishmania species T rypanosoma species Loa/oa • Wuchereria bancrofti • Brugia spp. • Mansonella spp. Babesia species Trypanosoma cruzi • Hymenolepisnano • Hymenolepisdiminuta • Dipylidium caninum • Here, the larvae develop into male and female adults in about 3-4 months and mate. • After mating, the male worms die in the tissues and sometimes become calcified.
- 242. • In another6 months time, the fertilized female worm grows in size, matures, and migrates within the connective tissues throughout the body, to finally reach a site where it is likely to come into contact with water. • The most common site involved is the leg, but other sites such as arms, shoulder, breast, buttocks, or genitalia may also be affected. • At this site, it secretes a toxin that causes a blister formation, which eventually ruptures, discharging a milky-white fluid containing numerous LI stage larvae. • this process continues for 2-3 weeks, till aUthe larvae are released. Development of Larvae in Cyclops The larvae swim about in water, where they survive for about a week. • They are swallowed by the freshwater copepod Cyclops, which is the intermediate host (Fig. 4). • The larvae penetrate the gut wall ofthe Cyclops and enter its body cavity, where they molt twice. • In about 2-4 weeks, they develop into the infective third- stage larvae (L3). • The entire life cycle takes about a year, so that all the infected persons develop the blisters and present with clinical man ifestations at about the same time of the year (Fig. 4). • PATHOGENICITY AND CLINICAL FEATURES D. medinensiscauses dracunculiasis or dracunculosis. • Infection induces no illness till the gravid female worm comes to lie under the skin, readyto discharge its embryos. • The body fluid of the adult worm is toxic and leads to blisterformation. • A few hours before the development of the blister, there may be constitutional symptoms such as nausea, vomiting, intense pruritus and urticaria! rash. DracunculusMedinensls • The blister develops initially as a reddish papule with a vesicular center and surrounding induration. The most common sites for blister formation are the feet between the metatarsal bones or on the ankles. • The fluid in the blister is a sterile yellowish liquid with polymorphs, eosinophils and mononuclear cells. • The local discomfort diminishes with the rupture of the blister and release ofthe embryos. • Seconda.ry bacterial infection is frequent. Sometimes, it may lead to tetanus. • Sometimes, the worm travels to unusual sites such as the pericardium, the spinal canal, or the eyes, with serious effects. • Dracunculiasis lasts usually for 1-3 months. • LABORATORY DIAGNOSIS • Detection of adult worm: Diagnosis is evident when the tip of the worm projects from the base of the ulcer. Calcified worms can be seen by radiography. • Detection oflarva: By bathing the ulcer with water, the worm can be induced to release the embryos (LI larvae), which can be examined under the microscope. • Skin test: An intradermal test with guinea worm antigen elicits positive response. Serological test: Enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (lFA) are frequemJy used to detected antibodies to D. medinensis (Flow chart 1). • TREATMENT • Antihistaminics and steroids are ofhelp in the initialstage ofallergic reaction. • Metronidazole, niridazole and thiabendazole are useful in treatment. Flow chart 1: Laboratory diagnosis of Dracunculus medinensis Laboratory diagnosis ~ + + + + t Detection of adult Detection of X-ray Skin test Serological test Blood test worm larva Calcified worms Guinea worm to detect antibodies: reveals From the base Under the can be seen in antigen injected · ELISA eosinophilia of ulcer microscope radiography intradermally elicits • IFA positive response Abbreviations: ELISA, enzyme-linked immunosorbent assay; IFA, immunofluorescence assay
- 243. Paniker'sTextbook ofMedical Parasitology Cyclopsare digested in stomach and L3 larvae released Man (Definitive host) Cyclops containing L3 stage larva Larvae reach the retroperitoneal and subcutaneous connective tissues, and mature into adult worms Man (Definitive host) Adult worm in the subcutaneous tissue Gravid female in subcutaneous tunnel ready to discharge larvae on contact with water Adult female discharging larvae in Water water (L1 stage) Cyclops (Intermediate host) Larva penetrate the gut wall of Cyclops (intermediate host) and enter the body cavity Fig. 4: Life cycle of Dracuncu/us medinensis Motile L1 stage larva in water
- 244. Fig. 5: Ancienttechnique of removing adult worm from blister • For removal of the worm, the best method is the ancient technique of patiently twisting it around a stick. It may take 15-20 days to extract the whole worm but if care is taken not to snap the worm, this method is safe and effective (Fig. 5). • Surgical removal of the worm under anesthesia is another m ethod of treatment. • PROPHYLAXIS • Provision of protected piped water supply is the best method of prevention or else boiling or fil tering water through a cloth and then consuming water. • Destroying Cyclops in water by chemical treatment wilh Abate (temephos). • Not allowing infected persons to bathe or wade in sources ofdrinking water. Note: Because of its simple life cycle, localized distribution, and the absence of animal reservoirs, guinea worm infection was eradicable. Measuresto eliminate theinfection have been successful. Global eradication of the infection is imminent. 1 KEY POINTS OF DRACUNCULUS MEDINENSIS • Guinea worm infection has been eradicated from India. • Adult females are found in subcutaneous tissue of man. • Female worm is viviparous releasing thousands of motile first- stage larvae into the water. • Definitive host: Humans. Dracunculus Medinensis - • Intermediate host: Cyclops, in which larvae undergo development changes to become third-stage larvae. • Infective form to humans: Cyclops containing L3 larvae. • Clinical features: Pruritus, urticaria! rash, blister formation in skin and cellulitis. • Diagnosis: Detection of adult worm and larval form in ulcer. Demonstration of dead worm by X-ray. Serology-ELISA and IFA. • Treatment: Antihistaminics and steroids in initial stage. Metronidazole and niridazole are useful. Surgical removal of the worm. REVIEW QUESTIONS 1. List viviparous nematodesand describe briefly the lifecycle and laboratory diagnosisof Dracunculus medinensis. 2. Short notes on: a. Pathogenicity and clinical features of dracunculosis b. Tissue nematodes c. Prophylaxisofguinea worm infection MULTIPLE CHOICE QUESTIONS 1. Which ofthe following parasite does not enter into the body by skin penetration a. Dracunculus b. Necator americanus c. Ancylostoma duodenale d. Strongyloides 2. Definitive host for Guinea worm is a. Man b. Cyclops c. Snail d. Cyclops and man 3. Guinea worm is a. Enterobius b. Trichuris c. Dracunculus d. Taenia solium 4. Cyclops is the source ofinfection in a. Dracunculus b. Spirometra c. Both d. None Answer 1. a 2. a 3. C 4. C
- 245. CHAPTER 22 • ANG/OSTRONGYLUSCANTONENSIS Common Name Rat lungvvorm. History and Distribution Angiostrongylus cantonensis causes eosinophilic meningo- encephalitis (cerebral angiostrongyliasis) in humans. • This condition was first reported from Taiwan in 1945. • Since then, hundreds of cases have occurred in Taiwan, Thailand, Indonesia and the Pacific islands. • Human infection has also been recorded in lndia, Egypt, Cuba and the United States ofAmerica (USA). Habitat The adult worm is present in the branches of pulmonary artery in rats. Morphology • It is about 20 mm long and 0.3 mm thick. • Eggs of Angiostrongylus resemble those of hookworms. Life Cycle Natural host: Rats. Intermediate hosts:Molluscs, slugs and snails. Jnfectiveform:Third-stage larvae. . . . The eggs hatch in the lungs and the larvae which migrate up the trachea are swallowed and expelled in the feces. The larvae infect molluscs, slugs and snails, which are the intermediate hosts. Crabs, freshwater prawns and frogs have also been fow1d to be naturally infected (Box 1). The larva undergoes two molts. In about 2 weeks, the infective third-stage larvae develop, which can survive in the body of the intermediate host for about a year. Rats become infected when they eat the molluscs. Box 1: Nematodes with crabs and crayfishes as source of infection • Angiosrrongy/us canronensis • Paragonimuswestermani • In the rat, the larvae penetrate the gut wall to enter the venules and are carried in circulation to the brain, where they develop into young adults in about a month. • These penetrate the cerebral venules and reach the pulmonary artery, where they lodge, mature, and start laying eggs. • Human infection is acquired by eating infected molluscs and other intermediate hosts containing the third-stage larvae. Infection may also occur through raw vegetables or water contaminated with the larvae. • The larvae penetrate the gut and are carried to the brain, but they are unable to develop further. • They die and induce an inflammatory reaction in the brain and meninges to produce meningoencephalitis. • The incubation period is about 2-3 weeks. Clinical Features Patients present with intense headache, fever, neck stiffness, convulsions and various degrees ofpareses. • The worm may also cause ocular complications. • Infection docs not seem to confer immunity, as second attacks have been recorded. • Fatality is rare. Diagnosis Peripheral eosinophilia and high cerebrospinal fluid (CSF) eosinophilia (up to 90%) are constant features. • Larvae and adult worms may be seen in CSF (Table 1). Treatment Most cases recover spontaneously, only some develop residual pareses.
- 246. Table 1: Parasitesfound in cerebrospinal fluid Protozoa Helminths • Trypanosoma bruceispp. Angiasrrongylus cantonensis • Naegleria fowleri • Acanthamoeba spp. • Anthelmintic treatment is not recommended, as the disease is due to dead larvae. • The drugs mayeven enhance the illness due to destruction ofmore larvae. Nole: Angiostrongylus costaricensis, inhabiting the mesenteric arteries of wild rodents in Costa Rica in Central America, may cause human infections. The disease presents as inflammation ofthe lower bowels and is known as abdominal angiostrongyliasis. • CAPILLARIA PHILIPPINENSIS C. phillippinensis is a small nematode, about 3-4 mm long. It belongs to the superfamilyTrichuroidea. History and Distribution It has been responsible for several fatal cases of diarrheal illness in the Philippines from 1963. • It has also been reported from Thailand, Japan, Iran and Egypt. Habitat The adult worn inhabits the small intestine particularly the jejunum. Life Cycle Definitive host: Birds (fish-eating birds) Intermediate host:Fish. • Its life cycle has not been worked out. • Human infection is believed to occur by eating infected fish, which are the intermediate hosts harboring the infective larvae. • Autoinfection is stated to be responsible for the high degree ofinfection in man. Clinical Features lhe clinical disease consists of malabsorption syndrome with severe diarrhea, borborygmi and abdominal pain. Seriou cases may be fatal in 2 weeks to 2 months. Miscellaneous Nematodes Diagnosis Diagnosis is made by detection of the eggs, larvae and adults in stools. The eggs resemble those of Trichuris trichiura, but are smaller. Treatment Mebendazole is useful in treatment. ote: C. hepatica is a common parasite of rats, which may occasionally infect man causing hepatitis that may be fatal. • GNATHOSTOMA SPINIGERUM History and Distribution Gnathostoma spi11igerum, originally described from gastric tumors of a tiger, parasitizes dogs, tigers, lions, cats and their wild relative . Gnathostomiasis is a zoonotic infection ofman. • lluman infections have been reported from 7hailand and other countries in the Far East. Cases ofhuman infection with G. spinigerum and a related species G. hispidum have also been reported from India. Morphology It is a small spirurid nematode. The female (25-55 mm) is longer than the male ( I0-25 mm). • The eggs are oval, brown, unsegmented bearing a transparent knob-like thickening at one end {Fig. 1). Life Cycle Definitive host: Dog, cat and other carnivorous animals First intermediate lwst: Cyclops Second intermediate host: Freshwater fish and frog Fig. 1: Adult worm and egg of Gnathostoma splnigerum
- 247. - Paniker'sTextbook ofMedical Parasitology Table 2: Helminths causing central nervous system (CNS) infection Cestodes Trematodes Nematodes • Taenia solium • Schistosomajaponicum • Trichinella spiralis • Taenia mult,ceps • Paragonimus westermani • Angiostrongyluscantonensis • Spirometra spp. • Echinococcus granulosus • Echinococcus multi/ocularis Paratenic host: Birds and humans. Adult worm resides in the tumors or granulomatous lesions of the sromach wall of cat and dog. Eggs are laid in the tumors. • They pass into gastric lumen by means ofan aperture and are discharged in feces into water, where they hatch into first-stage larva. • L1 larvae are ingested by Cyclops (first intermediate host) in which the second-stage larvae develop. • Cyclops is eaten by fishes, frogs and snakes, in which the third-stage larvae develop (L3). • When the third-stage larvae are eaten by cats, dogs, or other suitable hosts, the larvae develop into adults inside their body. • When other hosts that are not suited to be a definitive host (reptiles, buds or mammals) get infected, the larva does not undergo any further development and such a host is paratenic. • Humans get infected by eating undercooked fish containing third-stage larvae, but further development of the worm cannot proceed normally in paratenic host. The larvae migrate in the tissues of infected persons, causing indurated nodules or abscesses and creeping eruplion (larva migrans) (Table 2). Clinical Features The migration of larvae in the tissues of the infected persons leads to indurated nodules or abscesses and creeping eruption. • When the nodules are superficial, they can be incised and the larvae can be removed. thhe wandering larvae may reach the brain oreyescausing severe damage. Diagnosis An intradermal test using the larval or adult antigens has been described. • The lesion can be biopsied and the presence of typical larva confirms the diagnosis. • Toxocara canis • Toxocara coli • Gnathostoma spinigerum • Strongyloides stercoralis Table 3: Parasites with fishes as the source of infection Freshwaterfish • Gnathostoma spinigerum • Capillaria philippinensis • Clonorchis s/nensis • Heterophyes heterophyes • Metagonimus yokogawai • D1phyllobothrium /arum Treatment Marine fish Anisakis simplex • Incision of the lesion and removal of larva. Albendazole, mebendazole in high closes has also been recommended. • ANISAKIASIS Anisakis species are nematode parasites ofmarine mammals like dolphins, seals and whales. Anisakiasis is common in Japan and other places like etherland and USA where fresh or undcrtreatcd fish is a popular food (Table 3). Life Cycle Dejinitive host: Dolphin, seals and whales Intermediate host: Sea fishes • 1he eggs are passed in seawater, hatch and infect marine crustacea (krill). Marine fish eats the infccrcd krill and the infective larvae remain in the fish's viscera and flesh. • When humans consume uncooked or improperly preserved fish containing the infective larvae, they penetrate the gut wall at the level of the throat, stomach, or intestine, leading to local inflammation and granuloma formation.
- 248. Clinical Features infection withthe larva ofanisakis is known as anjsakJasls or herring worm disease. • Local inflammation and granuloma formation is present at the level of throat, stomach, or intestine, depending on the level ofpenetration ofgut wall. • The illness varies according to the site involved, such as throat irritation or acute gastric or bowelsymptoms. • No case has been reported from India. Treatment Endoscopic surgical treatment of gastric and intestinal anisaldasis is the method ofchoice. Prophylaxis Proper cooking ofsea fish. REVIEW QUESTIONS 1. Short notes on: a. Anisakiasis b. Gnathostomaspinigerum c. Angiostrongylus cantonensis d. Paratenic host Miscellaneous Nematodes - MULTIPLE CHOICE QUESTIONS 1. Rat lung worm is the common name of a. Paragonimus westermani b. Toxocara canis c. Angiostrongylus cantonensis d. Mansonella streptocerca 2. Paratenic host for Angiostrongylus cantonensis is a. Rat b. Man c. Frog d. Camel 3. All of the following parasites are found in CSF except a. Naegleria b. Acanthamoeba c. Angiostrongylus d. Trypanosoma 4. Definitive host for Capillaria philippinensis is a. Man b. Rat c. Birds d. Fish Answer 1. c 2. b 3. d 4. C
- 249. CHAPTER 23 Diagnostic Methodsin Parasitology • INTRODUCTION Laboratory procedures play an important role in the diagnosis of parasitic infections, both for confirmation of clinical suspicion and for identifying unsuspected infections. The principles of laboratory diagnosis are the same as in bacterial and viral infections, but the relative importance of the different methods varies greatly. • While isolation of the infecting agent and detection of specific antibodies are the major methods in bacteriology and virology, they are of much less importance in parasitology than morphological identification of the parasite by microscopy. • Compared to bacteria and viruses, parasites are very large and possess distinctive shape and structure, which enables their specific diagnosis on morphological grounds. • Due to tl1eir complex antigenic structure and extensive cross-reactions, serological diagnosis is of limited value in parasitic infections. • Although many pathogenic parasites can be grown in laboratory cultures, this method is not suitable for routine diagnosis because ofitsrelative insensitivity and the delay involved. • Morphological diagnosis ofparasites consists oftwo steps: (1) detection ofthe parasite or its parts in clinical samples and (2) its identification. l. Detection depends on collection of the appropriate samples and their examination by suitable techniques. 2. Identification requires adequate skill and expertise in recognizing the parasite in its various stages and its differentiation from morphologically similar artifacts. A description of the common diagnostic techniques in parasitology is given here. • EXAMINATION OF STOOL Collection of Fresh Stool Specimen • All stool specimens should be collected in a suitable, clean, wide mouthed container like a plastic container with a light-fitting lid,waxed cardboard box, or match box. • All fresh specimens should be handled carefully because each specimen represents a potential source of infectious material. • The specimen should not be contaminated with water, urine, or disinfectants. Liquid stools should be examined or preserved within 30 minutes of passage. Soft stools should be examined or preserved within 1 hour of passage and formed stool should be examined or preserved within 24 hours of passage. • Normally passed stools are preferable, although samples obtained after purgative (sodium sulfate) or high saline enema may also be used. • Examination offresh specimens is necessary for observing motility ofprotozoan parasites. • Stool should be examined for its consistency, color, odor and presence of blood or mucus. • T n some instances, parasites may be seen on gross inspection, as in the case of roundworm, pinwonn , or tapeworm proglottids. Microscopic Examination • 111e microscope should be equipped with a micrometer eyepiece, as it is often essential to measure the size of parasites. For example, the differentiation between cysts of the pathogenic Entamoeba histolytica and the nonpathogenic E. hartmanni is based entirely on their sizes.
- 250. • Microscopy shouldalso include contributory findings such as the presence of Charcot-Leyden crystals and cellular exudates such as pus cells, red blood cells (RBCs) and macrophages. • For detection of parasites, ii is best to employ a combination of methods, as different m ethods serve different purposes. • The methods include examination of: (i) wet mounts, (ii) thicksmears, and (iii) permanent-stained preparations. • Various concentration methods can be used to increase the sensitivity of microscopic examination. • If there is a delay in examination, use of preservatives such as formalin, sodium acetate and polyvinyl alcohol is recommended. Wet Mounts • Unstainedwet.film:The unstained wetfilm isthe standard preparation and is made by emulsifying a small quantity of stool in a drop of (0.85%) saline placed on a slide and applying a coverslip (22 mm x 22 mm) on top, avoiding air bubbles. A proper preparation should be just dense enough for newspaper print to be read through it. If the feces con tains mucus, it is advisable to prepare films using the mucus part.1he entire field under coverslip should be systematically examined with low-power objective (l0X) under lowlight intensity. Any suspicious object may then be examined with the high-power objective. • Wet saline mounts: Wet saline mounts are particularly useful for detecting live motile trophozoites of E. histolytica, Balantidium coli and Giardia lamblia. Eggs of helminths are also readily seen. Rhabditiform larvae of Strongyloides stercoralis are detected in freshly passed stool. • Eosin staining: Eosin 1% aqueous solution, can be used for staining wet films. Eosinstains everything exceptliving protoplasm. Trophozoites and cysts ofprotozoa, as well as helminth larvae and thin-walled eggs stand out as pearly- white objects against a pink background and can be easily detected. Chromatoid bodies and nuclei of amebic cysts can be seen prominently. Eosin also indicates the viability ofcysts; live cysts are unstained and dead ones are stained pink. • Iodine staining: Iodine staining orwet mounts is another standard method of examination. Either Lugol's iodine diluted (5 g iodine, 10 g potassium iodide and 100 mL of distilled water) or Dobell and O'Connor iodine solution (1 g iodine, 2 g potassium iodide and 50 m l of distilled water) are used. Iodine helps to confirm the identiry of cysts, as it prominently stains the glycogen vacuoles and nuclei. Protozoan cyst stained with iodine show yellow- gold cytoplasm, brown glycogen material and pale refractile nuclei. Diagnostic Methods in Parasitology - Thick Smears These are not useful for routine examination, but are valuable in surveys for intestinal helminth eggs. The method described by Kato and Miura in 1954 is known as the Kato thick smear technique. • About 50 mg stool is taken on a slide and covered with a special wettable cellophane coverslip soaked in glycerin containing aqueous malachite green. • The preparation is left for about a n hour at room temperature, during which the glycerin clears the stool, enabling the helminth eggs to be seen distinctly under low-power magnification. • This method is, however not useful for diagnosis of protozoa or helminth larvae. PermanentStainedSmears Permanent stained smears are examined normally under oil immersion {lO0X} objective. • Confirmation of the intestin al p rotozoan, both trophozoites and cysts, is the primary purpose of this technique. • Helminthic eggs and larvae take up too much stain and usually cannot be identified. • Permanent sm ear can be prepared with both fresh and polyvinyl alcohol preserved stool specimen. • The two methods commonly used are: (1) the iron- hematoxylin stain and (2) Wheatley's trichrome stain. The iron-hematm.. xylinis the older method, but is more difficult. 1. .lron-hematoxylin stain Procedure: • Fecal smear on a slide is fixed in Schaudinn's solution for 15 minutes and is immersed successively for 2-5 minutes in 70% alcohol, 70% alcohol containing a trace of iodine, and then 50% alcohol for 2-5 minutes. • It is washed in water for 5- l O minutes and immersed in 2% aqueous ferric ammonium sulfate solution for 5- 15 minutes. • It is again washed in water for 3-5 minutes and stained with 0.5% aqueous hematoxylin for 5- 15 m inutes. • It is washed for 2-5 minutes and differentiated in saturated aqueous solution of picric acid for 10- 15 minutes. • It is then washed for 10- 15 minutes and dehydrated by passing through increasing strengths of alcohol, cleared in toluene or xylol and mounted. 2. Trlchrome stain (Wheatley's method) • The trichrome technique of Wheatley for stool specimens is a modification of Gomori's original staining procedure for tissue.
- 251. - Paniker'sTextbook ofMedical Parasitology Box 1: Reagents of trichrome stain • Chromotrope 2R: 0.6 g • Light green SF: 9.3 g • Phosphotungstic acid: 0.7 g • Acetic acid (glacial): 1.0 ml • Distilled water: 100 ml. • lt is a quicker and simpler method, which produces uniformly well-stained smears of the intestinal protozoa, human cells, yeast cells and artifact material in about 45 minutes or less. Procedure: • 1he smear is fixed in Schaudinn's solution and taken successively through alcohol, as earlier. • Trichrome stain (chromotrope 2R, light green SF, phosphotungstic acid in glacial acetic acid and distilled waler) is then applied for 5-10 minutes, differentiated in acid-alcohol dehydrated, cleared and mounted (Box l). Modified trichrome stainfor microsporidia: - This staining method is based on the fact that stain penetration of the microsporidial spore is very difficult, thus more dye is used in d1e chromotrope 2R than that routinely used to prepare Wheatley's modification of Lrichrome method and the staining time is much longer (90 m inutes). Other staining techniques are used for special purpose. For example, modified acid-fast or Giemsa stain is employed for detection of oocysts of Cryptosporidium and fsospora. ModifiedZiehl-Neelsen (acid-fast) stain (hot method): Oocysts of Cryptosporidium and Isospora in fecal specimens may be difficult to detect, without special staining. Modified acid-fast stains are recommended to demonstrate these organisms. Application of heat to the carbolfuchsin assists in the staining and the use of a milder decolorizer (5% sulfuric acid) allows the organisms lo retain more of their pink-red color. Kinyoun's acid-fast stain (cold method): Cryptosporidium and Isospora have been recognized as causes of severe diarrhea in immunocompromised hosts but can also cause diarrhea in immunocompetent hosts. Kinyoun's acid-fast stains are recommended to demonstrate these organisms. - Unlike the Ziehl-Neelsen modified acid-fast stain, Kinyoun's stain does not require the heating of reagents for staining (Box 2). Procedure: - Smear 1-2 drops of specimen on the slide and allow it to air dry. Box 2:Reagents of Kinyoun's acid-fast stain • 50% ethanol (add 50 ml of absolute ethanol and 50 ml ofdistilled water). • Kinyoun's carbolfuchsin: - Solution A: Dissolve 4 g of basic fuchsin in 20 ml of 95% ethanol. - Solution 8: Dissolve 8 g of phenol crystals in 100 ml ofdistilled water. - Mix solution A and B, and store at room temperature. 1% sulfuric acid. • Alkaline methylene blue. • Dissolve 0.3 g of methylene blue in 30 ml of 95% ethanol, and add 100 ml of dilute (O.O1%) potassium hydroxide. Fix with absolute methanol for l minute. Flood the slide with Kinyoun's carbolfuchsin and stain it for 5 minutes. Rinse the slide briefly(3-5 seconds) with 50%elhanol. Rinse the slide thoroughlywith water. Oecolorize by using 1% sulfuric acid for 2 minutes or until no more color runs from the slide. - Rinse the slide with water (it may take less than 2 minutes; do not destain too much) and drain. Counterstain with methylene blue for l minute. Rinse the slide with water and air dry. - Examine with the low or high dry objective. To see internal morphology, use the oil objective (1 00X). • Auramine O stainfor coccidia: Coccidia are acid-fast organisms and also stain well with phenolized auramine 0 . The size and typical appearance of Cryptosporidium, Cyclospora and /sospora oocysts enable auramine 0 -stained slides to be examined at low-power under the l0X objective. The entire sample area can usually be examined in less than 30 seconds. The low cost of the reagenrs, the simple staining protocol and the rapid microscopic exam ination also make this staining method suitable for screening unconcentrated stool specimens. Concentrated sediment from fresh or nonpolyvinyl alcohol- preserved stool may also be used. Concentration Methods When the parasites are scanty in stools, routine microscopic examination may fail to detect them. It is then necessary to selectively concentrate the protozoan cysts and helminth eggs and larvae. Concentration may be done using fresh or preserved feces. Several concenn·ation techniques have been described. They can be classified as Lhe floatation or sedimentation methods. I j
- 252. Infloatation method, thefeces are suspended in a solution of high specific gravity, so that parasitic eggs and cysts float up and get concentrated at the surface. • In sedimentation method, the feces are suspended in a solution with low specific gravity, so that the eggs and cysts get sedimented at the bottom, either spontaneously or by centrifugation. Floatation Methods • Saturated saltsolution technique Procedure: - A simple and popular method is salt floatation using a saturated solution of sodium chloride, having a specific gravity of 1.2. About 2 ml ofthe salt solution is taken in a flat bottomed tube (or penicillin bottle) and 1 g of feces is emulsified in it. - 1he container is then filled completely to the brim with the salt solution. - A slide is placed on the container, so that it is in contact with the surface of the solution without any intervening air bubbles. After standing for 20-30 minutes, the slide is removed, without jerking, reversed to bring the wet surface on lop, and examined under the microscope. A coverslip need not to be applied, if examination is done immediately. Any delay in exam ination may cause salt crystals to develop, interfering with clarity ofvision. This simple method is quite useful for detecting the eggs of the common nematodes such as roundworm, hookworms and whipworm, but is not applicable for eggs of tapeworms, unfertilized egg of Ascaris lumbricoides, eggs oftrematodes and protozoan cysts. • Zinc sulfate centrifugalfloatation Procedure: - Make a fine suspension ofabout 1 g of feces in 10 mL of water and strain through gauze L o remove coarse particles. - Collect the liquid in a small test tube and centrifuge for 1 minute at 2,500 revolutions per minute. Pour off the supernatant, add water, resuspend, and centrifugein the same manner, repeating the process, till the supernatant is clear. Pour off the clear supernatant, add a small quantity ofzinc sulfate solution (specific gravity 1.18- 1.2) and resuspend the sediment well. Add zinc sulfate solutio n to a little below the brim and centrifuge at 2,500 revolution per minute for 1 minute (Fig. lA). - Take samples carefully from the surface, using a wire loop, transfer to slide and examine under the microscope (Fig. 18). Adrop ofdilute iodine helps to bring out the protozoan cysts in a better way. Diagnostic Methods in Parasitology This technique is useful for protozoan cysts and eggs of nematodes and small tapeworms, but it does not detect unfertilized roundworm eggs, nematode larvae, and eggs of most trematodes and large tapeworms. Sugarfloatation technique: Sheather's sugar floatation technique is recommended for the detection of cryptosporidia infection. Sedimentation Methods m B Formal-ether sedimentation technique Formol-ether concentration method has been the most widely used sedimentation method (Fig. IC). Procedure: Emulsify 1-2 g feces in 10 mL of water and let large particles sediment. Take the supernatant and spin at 2,500 revolutions per minute for 2-3 minutes. Discard the upernatant. Add 10% formol-saline, mix well and let it stand for l Ominutes. Add 3 mL ether and shake well. Spin at 2,500 revolutions per minute for 2-3 minutes. Four layers Zinc sulfate Sediment / 0 7 Ethyl acetate Debris/fat Formalin Sediment Figs 1A to C: (A) Zinc sulfate floatation concentration technique; (B) Method used to remove surface film in the zinc sulfate floatation concentration procedure: and (C) Formol-ether sedimentation technique
- 253. Paniker'sTextbookofMedical Parasitology will form-(1)a top layer ofether, (2) a plug of debris at the interface, (3) the formalin-saline layer and (4) the sediment at the bottom (Fig. IC). - Carefully detach the debris from the sides of the tube and discard the top three layers. Suspend the sediment in a few drops of flujd and examine wet mount and iodine preparation. As ether is inflammable an d explosive, its use can be hazardous. Ethyl acetate can be conveniently used in its place, with equallygood results. The method is useful for all helminth eggs and protozoan cysts. • Baermann concentration method Procedure: Another method of examination of stool specimen suspected of having small numbers of Strongyloides larvae is the use of a modified Baermann apparatus (Fig. 2). The Baermann technique, which involves using a funnel apparatus, relies on the principle that active larvae migrate from a fresh fecal specim en that has been placed on a wire mesh with several layers of gauze, which are in contact with tap water. Larvae migrate through the gauze into the water and settle to the bottom of the funnel, where tl1ey can be collected and examjned. - Besides being used for patient's stool specimens, this technjque can be used to examine soil specimens for the presence oflarvae. Egg Counting Methods A semiquantitative assessment of the worm burden can be made by estimating the number of eggs passed in stools.This is done by eggcounts and by relating the counts to the number ofworms present by assuming the number ofeggs passed per worm per day. However, these are at best approximations and only a rough indication ofworm burden can be obtained.Egg counts help to classify helminth infections as heavy, moderate, or light. Egg counts can be done by different methods. • The standard wet mount gives rough indication of the number of eggs. Ordinarily, 1-2 mg of feces is used for preparing a wet film, and if all the eggs in the film are counted. The number of eggs per gram of feces can be assessed. • The modified Kato thick smeartechnique using50 mg of stoolcleared byglycerin-soaked cellophane coverslip can be used for egg counting. • McMaster's egg counting chamber can also be used. In this method, eggs in 20 mg of stool are concentrated by salt floatation on the squared grid on the roof of the chamber, which can be coun ted. Soil or fecal material Gauze Wire screen Water---i,--,,i..- --41k'I Rubber tubing _ ___ Clamp - --e::::::::11• Container _ _,.___ Fig. 2: Baermann concentration method Box 3: Hatching test for schistosoma eggs This test is used to demonstrate the viability of the miracidia within the schistosome eggs recovered from the urine or stool. Fecal or urine specimens must be processed without any preservative.The specimens are placed in 10 volumes of dechlorinated or spring water. Living miracidia may be released by hatching within few hours.The specimens are examined microscopically for presence of miracidia, which indicates active infection. ln Stoll's dilution technique, 4 g of feces is mixed thoroughly with 56 ml of N/10 sodium hydroxide using beads in a rubber stoppered glass tube. Using a pipetle, exactly 0.075 mL of the sample is transferred to a slide, cover glassis applied,and all the eggs presentare counted. The number multiplied by 200 gives the number of eggs per gram of feces. this figure requires to be corrected for the consistency of feces, by multiplying by 1 for hard formed feces, by 2 for mushy formed feces, by 3 for loose stools and by4 for liquid stools. Watery stools are unfit for counting. Special techniques have been described for particular purposes as for example, Bell's dilution-filtration count for schistosome eggs (Box 3). Scotch tape method:This is a simple and effective metl1od for detection of eggs and female worms of Enterobius vermicularis and occasionally eggs of Taenia solium, T. saginata and Schistosoma mansoni. In this method, a piece of transparent adhesive tape is pressed firmly
- 254. Et Use apiece or clear cellophane B tape approximately 4 inches long Press the sticky side or the tape against the skin across the anal opening Diagnostic Methods in Parasitology m Hold the tape between thumbs and forefingers with sticky side facing upward m Place the sticky side of the tape down against the surface or a clean glass slide Figs 3A to D: Method for collection of a cellophane (scotch) tape preparation for pinworm diagnosis. This method dispenses with the tongue depressor, requiring only tape and a glass microscope slide. The tape must be pressed deep into the anal crack against perianal skin, and the adhesive surface of the tape is spread on a glass slide (Figs 3A to D). The slide is then placed undermicroscope and observed for parasitic eggs. A drop of toluene or xylol may be placed between the tape and the slide to clear the preparation. The specimen should be collected for 3 consecutive days at night or early in the morning. Fecal Culture Fecal culture is not used for routine diagnosis, but for species identification, for example in differentiation between Ancylostoma and Necator. Harada-Mori Filter Paper Strip Culture 1he test detects light infection with hookworm, S. stercoralis, Trichostrongylus spp, as well as to facilitate species identification ofhelminths. The Harada-Mori culture method uses strips of filter paper on which feces is smeared in the middle third. The paper strips are kept in conical centrifuge tubes with water at the bottom, in which the strips dip (Fig. 4). The tubes are kept at room temperature in the dark for 7-10 days, during which time the larvae develop and fall into the water at the bottom, from which they can be collected. Also, caution must be exercised in handling the paper strip itself, since infective Strongyloides larvae may migrate upwards, as well as downwards on the paper strip. Fig. 4: Harada-Mori tube method and petri dish culture method AgarPlate Culture for Strongyloides Agar plate cultures are used to recover larvae of S. stercoralis and appear to be more sensitive. Approximately, 2 g fecal specimens are inoculated onto agar plates. Then the plates are sealed with tape to prevent accidental infection and placed in room temperature for 2 days. In positive cases, larvae will crawl over the agar, making visible tracks over it. For further confirmation of larvae, the plates are examined microscopically.
- 255. Paniker'sTextbook of MedicalParasitology Charcoal Culture Charcoal culrures are simple and efficient. oftened feces is mixed with 5-10 parts of moistened charcoal granules and packed into a suitable container and kept covered. In 7-10 days, the larvae hatch out and come to the surface. They can be collected by applying a pad of soft cotton cloth on the surface for half an hour. The cloth is removed and kept upside down on a sedimentation flask filled to the brim with warm water. The larvae fall to the bottom of the flask, while the charcoal particles remain on the cloth. • EXAMINATION OF BLOOD Next to feces, the largest number of parasites are found in blood. Blood examination i the routine diagnostic method in malaria, filariasis, African trypanosomiasis and babesiosis. It is sometimes positive in Chagas di ease and rarely, in kala- azar and toxoplasmosis. Blood examination is done in the following ways. Examination for Malarial Parasites 1he standard diagnostic method in malaria is the examination ofstained blood fiJms- boLh thin and thick smears. Collection of Blood For demonstration of malarial parasites, blood should be collected not during the peak of fever, but optimally several hours after it. Bouts of fever follow the synchronous rupture of large number of parasitized erythrocytes, releasing their membrane shreds and contents. the emerging merozoites parasitize other erythrocytes and initiate a fresh cycle of erythrocylicschiwgony. The timing is particularlyimportant in P. Jalciparum infections, as here the late stages ofschizogony are not seen in peripheral circuJarion. • In practice, the rule is to take a blood smear when a uspected malaria patient is first seen and then again subsequently afrer a bout offever. Smears should invariably be collected before starting antimalarial treatment. Thin smear: • A thin smear is prepared from finger prick or in infants from heel prick blood or ethylene diaminetetra-acetic acid (EDTA) anticoagulated venous blood can also be used, provided blood films are made within 30 minutes. • A small drop (10-15 µL ) is spread on a clean grease-free slide with a spreader to give a uniform smear, ideally a single celJ thick smear. The margins of the smear should be well short of the sides of the slide, and the rail should end a little beyond the center ofits length. • The thin smear displays blood cells and parasites clearly. Its only disadvantage is that only a small volume of blood can be surveyed, so that a light infection could be missed. • If the smear are prepared from anticoaguJated blood, which is more than an hour old, the morphology of both parasites and infected RBCs may not be typical. • After drying, the smear is stained with Giemsa or Leishman stain. • For Giemsa stain, the smear is fixed in methanol for 3-5 minutes. After drying, Giemsa stain, diluted 1 drop in l mL of buffered water, pH 7-7.2, is applied for 30-45 minutes. The slide is then flushed gently with tap water, dried and examined under the oil immersion objective. The cytoplasm of malarial parasites is stained blueand the chromatin dot is stained red. • For Leishman's sta in, prior fixation is not necessary as the stain is an alcoholic solution, which fixes as it stains. Leishman stain isapplied for 1-2 minutes and diluted with rwice its volume ofbuffered water, pH 7-7.2 and is kept for 10-15 minutes. the smear is then dried and examined. lleporting of thin blood.films: In malignant tertian malaria, only the ring stage and gamctocyte are seen in peripheral smear, while in benign tertian malaria, all stages of schizogony and gametocytes can be seen. Thin smear examination enables the appreciation of changes in the erythrocytes, such as enlargement, alteration of shape, fimbriation, red cells stippling (Schuffner's dots) as seen with P. vivax, and irregular stippling (Maurer'sclefts), as seen in mature P.Jalciparum trophozoites. Any marked increase in white cell numbers and if indicated perform a differential white cell count. Parasitized erythrocytes are seen most often in the upper and lower margins ofthe tail ofthe smear. Count the percentage of parasitized red cells, when there is high falciparum malaria parasitemia (+++ or more para ites seen in the thick film) to monitor a patient's response to treatment. • A minimum of 100 fields should be examined before a negative report is given. Thick smear: • Thick smears have the advantage that a larger quantity of blood can be tested. Increased volume of blood present on thick film mayallowthe malaria parasite to be detected even with low para itemia. Compared with a thin film, a thick film is about 30 times more sensitive and can detect about 20 parasites/ µL ofblood. • The disadvantages are that the red cells arc lysed and the morphology of the para ices is di toned, so that species identification becomes difficult.
- 256. • A bigdrop or blood (20-30 µL) from finger or heel prick is collected on a clean grease-free slide and spread with the corner of another clean slide to form a uniformly thick smear of about 1 cm2 • The thickness of the smear should be such that the hands ofa wristwatch can be seen through it, but not the figures on the dial. • The smear is dried in a horizontal position, kept covered from dust. • Thick smears have to be dehemoglobinized before staining. • They can be stained with Giemsa or l eishman's stains as described earlier. Wright's stain and )SB stain (so called because it was devised by Jaswant Singh and Bhattacharjee, in 1944) are very useful for staining large numbers ofthick films as in malaria surveys. Wright's stain consists of two solutions: l. Solution A contains methylene blue and azure B in phosphate buffer. 2. solution B contains eosin in phosphate buffer. The film is immersed in solution Afor 5 seconds, washed in tap water, immersed in solution B for 5 seconds, washed, dried and examined. Staining times may need adjustment. Ifthe smear is too blue, stain longer in solution B;iftoo pink, in solution A. Jaswant Singh and Bhattacharj ee stain also consists of two solutions: l. The fi rst contains methylene blue, potassium dichromate, sulfuric acid, potassium hydroxide and water. 2. The second solution is aqueous eosin. For staining, the smear is immersed in solution 1 for 10 seconds, washed for 2 seconds in acidulated water pH 6.2- 6.6, stained in solution 2 for 1 second, washed in acidulated water, immersed again in solution l and washed. Reporting ofthick bloodfilms: • Select an area that is well-stained and not too thick. • Examine for malaria parasites and malaria pigment under oil immersion objective (l00X). • Examine at least I00 high-power microscope fields for parasites. • Reporttheapproximatenumberofparasites(trophozoites, schizonls and gametocytes) and also whether malaria pigment is present in white cells or not. • The plus sign scheme that can be used to repon parasite numbers are described in Box 4. Box 4: Plus sign scheme for reporting parasite numbers • 1- 10 per 100 high-power fields: + • 11-10 per 100 high-power fields: ++ • 1-10 in every high-power field:+++ • More than 10 in every high-power field: ++++. Diagnostic Methodsin Parasitology Combined thick and thin blood.films: • Combined thick and thin smears can be taken on the same slide. Draw a thick line with a glass-marking pencil on a slide, dividing it into two'un equal parts. The thick smear is made on the smaller part and the thin smear drawn on the larger. • Thick smear is first dehemoglobinized and the two are then stained together. An easy method is to add undiluted Leishman stain over the thin smear, and then the diluted stain flooded over to the thick smear also. • Do not allow the methanol to contact the thick film when fixing the thin film. • The stained thin smear is examined first. If the thin smear is negative, the thick smear should be searched for parasites. When a slide is positive for malarial parasites, the report should indicate the species, the developm ental stages found and the density of parasites in the smear. Examination for Microfilaria Microfilariae may be detected in peripheral blood, both in unstained mounts and in stained smear (Table I and Box 5). Wet Mount • Two or three drops ofblood are collected on a clean glass slide and mixed with two drops of water to lyse the red cells. The preparation is covered with a coverslip and sealed. The preparation is examined under the low-power microscope for the motile microfilariae, which can be seen wriggling about, swirling the blood cells in their neighborhood. Table 1: Parasites found in peripheral blood film Protozoa Nematodes • Plasmodium spp. • Wuchereria bancrofti • Babes,a spp. • Brugia spp. • Leishmania spp. • Loaloa • Trypanosoma spp. • Mansonella ozzardi Box 5: Time of collection In case of nocturnal periodic microfilariae, blood should be collected between 10 PM and 2 AM. In subperiodic nocturnal infection, the time of collection of blood should be between 8 PM and 10 PM and for subperiodic diurnal infection the time of collection should be ideally between 2 PM and 6 PM.
- 257. Paniker'sTextbook of MedicalParasitology • The examination may conveniently be deferred till next morning, asmicrofilariae retain theirviabilityand motility for 1 or 2 days at room temperature. • By using a simple counting chamber, microfilariae in the wet mount can be counted. Stained Smears • A thick smear is prepared as for malaria, dehemo- globinized, and stained with Leishman's, Giemsa, or Delafield's hematoxylin stains. • Stained smears have the advantage that the morphology ofmicrofilariae can be studied and species identification can be made. Thus, for differentiation between Mf bancrofti and Mf malayi stained smears are necessary. • Sometimes, microfilariae may be seen in thin smears also. • By using a measured quantity of blood for preparing smears, as for example with a 20 cubic mm pipette and counting the total number of microfilariae in the smear, microfilaria counts can be obtained. Multiplying the number of microfilariae in a 20 cubic mm smear by 50 gives the count per mL of blood. Concentration Methods These methods have been developed to recover low numbers ofmicrofilariae from blood and employ venous blood. • Sedimentation method: - In sedimentation method, the sample of blood is first lysed with acetic acid, saponin, or other lytic substance, or by freeze-thawing, and then centrifuged. - The sediment is stained and the microfilariae are counted. • Membranefiltration concentration: In membrane filtration method, a measured quantity (1-5 mL) of blood is collected into an anticoagulant solution and passed through membrane filters fixed on syringes with Swinney filter holder. Blood cells and proteins sticking on to the filter are washed away by repeatedly passing saline through it. The filter is removed, placed on a slide, stained with dilute Giemsa stain and examined under low-power microscope for microfilariae. Millipore and nucleopore membrane filters (5 ~un porosity) are available for this purpose; the latter being more sensitive, as it can screen larger volumes of blood. - Membrane filtration recovers most species of microfilariae; however, because of their small size, Mansonella perstans and M. ozzardi may not be recovered. Membranes with smaller pores (3 ~un) have been suggested to recover these two species. - The membrane filter method is much more sensitive than the finger prick method as the blood samples are taken during day, it also give reliable results even with nocturnal periodic microfilariae. However, the method has the disadvantages that venipunclure is necessary, membranes are costly, and microfilariae may not be in a satisfactory condition for detailed morphological study. The number of microfilariae counted divided by 10 gives the number ofmicrofilariae per mL ofblood. - This is the most sensitive method of detecting small numbers of microfilariae, but it is expensive for routine use. • Microhematocrit tube method: Capillary blood is collected in two heparinized capillary tubes or about 100 µL is first collected into EDTA anticoagulant, and then transferred to plain capillary tubes. - The blood is centrifuged in a microhematocrit centrifuge. - The huffy coat is examined microscopically for motile microfilariae. In areas where the species is known and Mansonella microfilariae are not found, tl1is is a rapid technique for detecting microfilariae. • Buffy coatbloodfilm: The buffy coat containing white blood cells (WBCs) and platelets obtained after centrifugation of whole anticoagulated blood and the layer of RBCs just below the buffy coat layer, can be used to prepared thick and thin blood films in suspected infections with filaria, Leishmania, Trypanosoma and malaria. TI1e sensitivity of this method is much higher than that ofroutine thick film. Diethylcarbamazine Provocation Test Oral administration of diethylcarbamazine (DEC; 100 mg or 2 mg/kg of body weight) brings about mobilization of microfilariae into peripheral blood. Blood collected 20-50 minutes after the drug is given, will show microfilariae so that blood collection can be done during day time. This is a great advantage for surveys. But the drug may cause febrile reactions, particularly in brugiasis. It cannot be used in areas endemic for onchocerciasis because of the danger of provoking severe reactions. • SPUTUM EXAMINATION Sputum is examined commonly for the demonstration ofeggs of Paragonimus westermani, and sometimes for detection oftrophozoites ofE. histolytica in amebic pulmonary abscess. Rarely, the larval stages of hookworm, A. lumbricoides, or
- 258. Box 6: Parasitesfound in sputum • Paragonimus wesrermani • Enramoebahis10/ytica (trophozoites in case of pulmonary abscess) • Pneumocysrisjirovecii • Rarely migrating larvae of Ascarislumbricoides • Rarely migrating larvae of Suongyloides stercorahs • Rarely migrating larvae ofAncylostoma duodenale • Rarely migrating larvae of Necatoramericanus. S. stercoraLis or the cestode hooklets may be seen in sputum samples (Box 6). • Concentrated stained prepa rations of induced sputum are commonly used to detect P. jirovecii and differentiate trophozoite a nd cyst forms from other possible causes of pne umonia, pa rticula rly in an acquired immunodeficiency syndrome (AJDS) patient. • orm ally, direct saline mount preparation is done for microscopy. • If the sputum is thick, equal volume of 3% N-aceryl cysteine or 3% sodium hydroxide is added to the sputum to liquefy the specimen and after centrifugation, the sedimenr is examined for microscopic examination under low (!OX) and high (40X) power magnifications. • In a Paragonim us spp. infection, the sputum may be viscous and tinged with brownish necks, which are clusters of eggs (iron.fili11gs) and may be streaked with blood. • URINE OR BODY FLUIDS EXAMINATION • Largevolume ofurine samples should be allowed to settle for 1- 2 hours. • About 50 mL of the bottom sediment of L he sample is taken for centrifugation. • The highly concentrated sediment after centrifugationis examined for direct wet mount microscopy. Mayshow eggs ofSchistosoma and Trichomonas vaginaLis. Mic rofilaria m ay be de tected from chylous urine in lymphatic filariasis. • TISSUE BIOPSY Tissue biopsies and fine-needle aspirations a rc taken from cutaneous ulcers of trypan osomiasis or leishmaniasis and from skin nodules of onchocerciasis and post-kala-azar de rma l leishmaniasis (PKDL). A skin snip can be obtained to diagnose subcutaneous filariasis or leishmaniasis by grasping with a forceps or elevating a portion ofskin with the tip ofneedle. Tip ofthe small cone of the skin is, then sliced with a sharp blade or razor. Diagnos tic Methods in Parasitology • Wet m ount prepararion of lymph node aspirate and chancre fluid are used as rapid methods for demonstration ofrrypanosomes. • Biopsies from liver,spleen,bone marrowand lymph nodes are taken in visceral leishmaniasis for demon tration of Leishman-Donovan (LD) bodies. • All biopsy tissues must be submitted to the laboratory without the addition of formalin fixative. If there is delay in transport or processing, the specimen should be placed in polyvinyl alcohol fixative. In soft specimens, a small partshould be scraped and examined as direct saline wet mount. • Impression smears can be made from freshly cut tissue specimens on a glass slide and examined after fixation with Schaudinn' solution. Trichrome or other stains can be used. • The residual partofthe biopsyspecimen maybe processed for histopathological examination. • Adult filarial worms can sometimes be found in section of biopsied lymph node. Cornealscrapings arc useful in diagnosisofacanlhamoeba keratitis. • MUSCLE BIOPSY Spiral larval form of Trichinella spiralis, larval form of T. solium (cyslicercus cellulosae) a nd am astigote o f Trypanosoma cruzi can be demonstrated in skeletal muscle biopsy. In trichinosis, muscle biopsy (gascrocnemius, deltoid and biceps) specimen must be examined by compressing the tissue between two slides and checkingthe preparation under low-power (lO X) objective. this me thod does not become positive until 2-3 weeks after the illness. • DUODENAL CAPSULE TECHNIQUE (ENTEROTEST) Enlerotest is a simple method ofsampling duodenal contents. • the device is composed of a length of nylon yarn-coiled inside a gelatin capsule. t he end of the yarn is affixed to the patient's face. • The capsule is then swallowed and the gelatin dissolves in the stomach. • The weighted string is carried into the duodenum by peristal is. • Bile-stained mucus is then retrieved after 3-4 hours and duodenal contents adherent to the yarn is scrapped off and exam ined under microscope as wet mount or a stained smear after preservation in formalin or polyvi nyl alcohol. • Usually 4-5 drops of material is obtained.
- 259. Paniker'sTextbook of MedicalParasitology • Enterotest is used for detecting trophozoites of Ciardia, larvae of Strongyloides, eggs of Liver flukes and oocysts of lsospora. • SIGMOIDOSCOPY MATERIAL Material obtained from sigmoidoscopy is useful in the diagnosis of E. histolytica that cannot be diagnosed by routine examination for at least 3 days. • Material from the intestinal mucosa should be aspirated or scraped and not to be collected by cotton swabs. • the material should be processed immediately. • 1n heavy infection of Trichuris, sigmoidoscopy may show white bodies of the worms hanging from the inflamed mucosa oflarge intestine. • UROGENITAL SPECIMEN The detection of T. uaginalis is usually based on wet preparation of vaginal and urethral discharges and prostatic specimens. Specimens should be collected in small volume of0.85% saline and should be sent immediately for detection of actively motile organisms, as the jerky movements of Trichomonas begin to dimjnish with time. • CULTURE METHODS Many parasites can now be grown in culture, but this has not become a routine diagnostic method in parasitic infections (Box 7). It is sometimes employed for accurate identification of the parasite species. It is more often employed for obtajning large yields of the parasite as a source of antigen, animal inoculation, drug-sensitivity testing, experimental or physiological studies and teaching purposes. Some of the culture methods used for different parasites are indicated here. Ameba E. histolylica and other intestinal amebae can be grown in diphasic or monophasic media, media containing other microorganisms, or axenic cultures. • Boeck and Drbohlau diphasic medium, the classical culture medium for ameba has been modified by various workers (Box 8). - The medium as used now, is basically an egg slant, with an overlay of sterile serum or liver extract in buffered saline. A loopful of sterile rice powder is added to the medium just before inoculation with fresh feces or its saline centrifugal sediment. - Cultures can be obtained from feces-containing cysts or trophozoites. Box 7: Parasites which can be cultured in the laboratory • Entamoeba histolytica • Giardia /amblia • Trichomonas vagina/is • Leishmania spp. • Trypanosoma spp. • Acanthamoeba spp. • Naegleria fowleri • Balanridium coli • Plasmodium spp. Box 8: Composition ofBoeck and Drbohlav medium (L ocke's solution) • Sodium chloride: 9 g • Potassium chloride:0.4 g • Calcium chloride: 0.2 g • Sodium bicarbonate:0.2 g • Glucose:2.5 g • Distilled water: 1000 ml • Egg: Four (clean and washed) Box 9: Composition of Balamuth's medium • Liverconcentrate powder: 1 part • Egg yolk medium: 9 part • Phosphatebuffer • Tribasic potassium phosphate: 212 g • Monobasic potassium: 136 g • Distilledwater - The cultures are incubated at37°C andsubcultured at 48-hour intervals. Arnebae can be demonstrated in the Liquid phase in unstained mounts or stained smears. • Balamuth's monophasic liquid medium is also used commonly for cultivation of amcbae and other intestinal protozoa.This is an eggyolk-Liverextract infusion medium (Box9). - Both protozoa and bacteria present in stools grow in the earlier media. - Bacterial growth can be reduced by addition of penicillin or other antibiotics that do not inhibit protozoa. Axenic cultures (pure cultures without bacter. ia or other microorganisms) were first developed by Diamond in 1961. Axenic cultivation has enabled precise antigenic and biochemical studies on amebae. 8. coli grows well in Balamuth's medium. G. lamblia had been established in association with Candida
- 260. and Saccharomyces, butaxenic cultures were developed in 1970. - T. uaginalis grows very well in several commercially available media such as trypticase serum media. - Naegleria and Acanthamoeba from cerebrospinal fluid (CSF) can be grown on agar plates heavily seeded with Escherichia coli. Leishmania and Trypanosomes • Nouy-MacNeal-Nicolle medium: The classical Nouy- MacNeal-Nicolle (NNN} medium first described in 1904 for cultivation of Leishmania, is equally satisfactory for trypanosomes also. This is a defibrinated rabbit blood agar medium (Box 10). Several modifications of this medium have been introduced. Two bottles ofculture are aseptically inoculated with 0.1 mL ofspecimen in each and incubated at 24°C for 4 weeks. The primary culture is examined every 4 days for promasrigotes in leishmaniasis and for epimastigote stages in trypanosomiasis for up to 30 days. • Schneider's insect tissue culture medium: It is recommended in vitro culture ofLeishmania. this medium is said to the more sensitive than NN medium (Box 11). Malaria Parasites • Cultivation of malaria parasites was first obtained byBass and Jones in 1912. A simple method of cultivation is as follows: About 10- 12 mLofdefibrinated or heparinized blood rich in ring forms of malaria parasite, mixed with 0.2 mL of50%dexLrose solution are incubated at 37°C in a sterile test tube in an upright position. The blood separates into the erythrocytes below, plasma above and the huffy coat in between. Malaria parasites grow in the erythrocyte layer immediately below the huffy coat. - Smears are collected from this layer at intervals, without tilting the tube. Box 10: Composition of Novy-MacNeal-Nicolle (NNN) medium • Bactoagar (Difeo): 1.4 g • Sodium chloride:0.6 g • Double distilled water: 90 ml • Defibrinated rabbit blood (10%):10 ml. Box 11 : Composition of Schneider's insect tissue culture medium • Schneider'sDrosophila tissue culture medium: 80 ml • Fetal calf serum: 20 ml • Antibiotic-antimycotic solution: 1.2 ml. Diagnostic Methods in Parasitology Segmented schizonts are usually observed after incubation for 24-36 hours. • The breakthrough in cultivation ofmalarial parasites came in 1976 when Trager and Jensen successfully maintained P. Jalciparum in continuous cul tu res in h uman erythrocytes using Roswell Park Me morial Institute (RPMI) 1640 medium. The cultures are incubated at 38°C with 10% human serum at pH 6.8-7.2 under an atmosphere with 7% carbon dioxide and 1-5% oxygen. A continuous flow system is used in which the mediwn flows slowly and continuously over the layer of erythrocytes. The method has been applied to various species ofPlasmodia. It has been employed for preparation of antigens, drug-sensitivitystudies, vaccine tests and manyother purposes. • ANIMAL INOCULATION Animal inoculation is not a routine diagnostic procedure in parasitic infections, but can be used in some instances because of its sensitivity. Toxoplasmosis: Animal inoculation can be used for isolating Toxoplasma gondii from infected persons. Lymph node or other biopsy materials are inoculated intrapcritoneally into im munosuppressed mice. Peritoneal fluid obtained 7-10 days later, may show the parasite in Giemsa-stained smears. However, serial passages may be necessary for its isolation. Brain smears may be examined for cysts after sacrificing the mice 3-4 weeks after inoculation. Seroconversion of the animal inoculation also inclicates a positive result. • Visceral leishmaniasis: Bone marrow, liver, spleen, or lymph node aspirates from kala-azar patients, injected intraperitoneally into hamsters is a very sensitive method for diagnosing visceral leishmaniasis. Even a single amastigote can establish the infection in the animal. Spleen smears taken 4-6 weeks later show Leishmania donovani (LO) bodies. • Trypanosomiasis: Blood from patients with trypano- somiasis can be injected intraperitoneally or into the tail vein of mice, rats and guinea pigs, etc. These animals are susceptible to infection by T. brucei rhodesiense. Parasitenlia can be demonstrated in 2 weeks. • XENODIAGNOSIS This method involves the diagnostic infection of a vector, in wh ich the parasite multiplies and can be demonstrated. In T. cruzi, diagnosis may be established by letting the vector reduviid bug feed on suspected patients. In 4-5 weeks, live flagellate forms can be seen in the feces ofthe bugs.
- 261. Paniker'sTextbook ofMedical Parasitology •IMMUNOLOGICAL DIAGNOSIS Serology Several serological tests have been developed for detection of antibodies to parasites using antigens from cultured parasites or from natural or experimental infections in animals or humans. In some cases, antigens are obtained from related parasites or even sometimes from bacteria. Advances in cultivation of parasites have made parasitic antigens more readily available. Cloning ofparasitic antigens promises to be a new source. In some instances, diagnosis is attempted by serological demonstration of parasitic antigens in blood, tissues, or secretions ofsuspected patients. Virtually, all types of serological reactions have been used. However, serodiagnosis in parasitic infections has only limited value due to various factors: • Parasites are complex antigenically and exhibit wide range of cross-reactions, so that serological tests are not sufficiently specific. • Another difficulty is in distinguishing between past and current infections. This has been solved partly by looking for immunoglobulin M (IgM) antibody, as in amebiasis and toxoplasmosis. • In general, indirect hemagglutination (IHA), enzyme- linked immunosorbent assay (ELISA) and counter- immunoelectrophoresis (CIEP) are m ost sensitive; indirect immunofluorescence (IF), direct agglutination test (DAT) and complement fixation test (CFT) are moderately sensitive; and simple precipitation in gel and coated particle agglutination tests are least sensitive. Serology has not been very useful in the diagnosis of individual cases, but has been valuable as a screening method in epidemiological surveys. However, in some infections where parasites are seldom demonstrable in patients, for example in toxoplasmosis and hydatidosis, serology is of great help. Listed here are some of the applications ofserology. Amebiasis Serology is of no value in the diagnosis of acute amebic dysentery or luminal amebiasis. But in invasive amebiasis, particularly in liver abscess, serology is very useful. • Indirect hemagglu.tination is mos! widely employed. Titers of 1:256 or more are significant in cases of amebic liver abscess and have prognosticvalue. • Tech Lab E. histolytica test was able to detect galactose lectin (GalNAc) antigen in almost all patients of amebic liver abscess. Giardiasis Enzyme-Linked immunosorbent assay and indirect immu.nofluorescence (IIF) test have been developed for detection of Giardia. • Commercially available ELISA (ProSpec T / Giardia) kit detects Giardia-speciflc antigen 65 (GSA 65). The sensitivity ofthe test is 95% and specificity is 100%, when compared to conventional microscopy. Trypanosomiasis Serological rests used to detect trypanosomiasis are IHA, indirectfluorescent antibody (/FA} and ELISA. • Specific antibodies are detected by these tests in the serum within 2-3 weeks infection. • Specific antibodies can be demonstrated by !FA and ELISA in CSF. Leishmaniasis Indirect hemagglu.tinalion, CIEP and DOT-ELISA are usually positive in kala-azar. • Complement tesl using Witebsky, Klingenstein and Kuhn (WKK} antigen from the acid-fast Kedrowsky bacillus are relatively less sensitive. • Indirect fluorescent antibody rest is positive very early in the disease, even before the appearance ofsymptoms and becomes negative within 6 months ofcure. • rK39 micro ELISA test is a qualitative immuno- chromatographic assay for detection of antibodies to Leishmania. Malaria Indirect immunofluorescence, ELISA and IHA are sensitive and specific, but are not useful for diagnosis of acute malaria because antibodies persist for some years after cure. • A negative test may, however help to exclude malaria. • Serological tests are useful in epidemiological surveys for malaria. • Molecular assays such as antigen capture for detection of hislidine-rich protein II (HRP-2) and Plasmodium lactate dehydrogenase (pLDH} have been applied for developing rapid dipstick tests (e.g. ParaSight-F in malignant tertian malaria). Toxoplasmosis Serological tests offer the most useful diagnostic method in roxoplasmosis. • The original Sabin-Feldman dye test, though veryspecific and sensitive, is no longer in use. IIF IHA and CFT were
- 262. other useful tests.The dye test remains positive for life, while CFT becomes negative soon after active infection. • At present, ELISA is routinely used in Toxoplasma serology. It is very informative, as it provides titers of IgM and IgG antibodies separately for better interpretation of the results. Cryptosporidiosis Indirectfluorescent antibody and ELISA usingpurified oocysts as antigens have been used to detect circulating antibodies specific to Cryptosporidium parvum. Intestinal Helminths Antibodies can be demonstrated in most intestinal helminthiases, but extensive cross-reactions lim it their use in diagnosis. Trichinosis Serology is very useful in diagnosis of trichinosis. Bentonite flocculation slide tests and CFT become positive 3-4 weeks after infection. • Indirect immunofluorescence becomes positive even earlier. • Enzyme-linked immunosorbent assay is also available. Demonstration ofseroconversion is diagnostic. Toxocariasis lligh titers in serological tests are obtained in visceral larva migrans, but specificity is low due to cross-reactions wilh intestinal nematode antigens. Filariasis Indirect lzemagglutination and bentonite flocculation tests with antigen from Dirofllaria immitis gives positive reaction in patients, andhigh titers in tropical pulmonaryeosinophilia. But cross-reactions arc frequent. lmmunochromatographic card test (JCT) is a new and rapid filarial antigen test that detects soluble Wuchereria bancrofti antigens in the serum ofinfected humans. Echinococcosis Several serological tests have been developed using hydatid fluid or scolex antigens from hydatid cysts in sheep. IHA, JJF, CIEPand ELISA are very sensitive. Cross-reactions occur with cysticercosis. Diagnostic Methods in Parasitology • SKIN TESTS lntradermal tests have been used in manyparasitic infections. Theyare sensitive and persist for manyyears, sometimes even for life. But specificity is relatively low. • Casoni's test: This test had been used widely in the diagnosis ofhydatid disease since its original description in 1911. The antigen is sterile hydatid fluid drawn from hydatid cysts from cattle, sheep, pig, or humans, filtered and tested for sterility. Intradermal injection of 0.2 mL of the antigen induces a wheal and flare reaction within 20 minutes in positive cases. A saline control is used. False-positive tests are seen in schistosomiasis and some other conditions. Casoni's test is now largely replaced by serological tests. • l eishmanin (Montenegro) test:lhis test is used to measure delayed hypersensitivity. Leishmania test is sensitive and relatively specific. The antigen is obtained from cultured Leishmania and consists of killed promastigotes in phenol saline. lntradermal injection of 0.1 mL induces a papule of 5 mm or more in diameter in 48-72 hours. This delayed hypersensitivity test is positive in cutaneous leishmaniasis and negative in diffuse cutaneous and visceral leishmaniasis. • Fairley's lest: This skin test is group-specific and gives positive results in all schistosomiasis. The intradermal allergic test uses antigen infected snails, cercariae, eggs and adult schistosomes from experimentally infected laboratory animals. Skin test in Bancroftian filariasis: Intradermal injection of filarial antigens (extracts of microfilariae, adult worms and third-stage larvae ofBrugia malayi, or the dog filaria, Dirofilariaimmitis) induce an immediate hypersensitivity reaction, but the diagnostic value of the skin test is very limited due to the high rate of false-positive and negative reactions. • MOLECULAR METHODS ucleic acid-based diagnostic tests are mainly available in specialized or reference centers. Nucleic acid probes and amplification techniques such as polymerase chain reaction (PCR) and multiplex PCR, western blot and deoxyribonucleic acid (D A) hybridization techniques are increasingly used to detect parasites in specimens ofblood, stool, or tissue from patients. • These test are usefulfor detecting subspecies or stain level identification which is important for epidemiological studies and are also used to detect parasitic drug resistance. For example, specific 17 kDa and 27 kDa
- 263. Paniker'sTextbook of MedicalParasitology sporozoiteantigens are employed for seroepidemiological studies in cryptosporidiosis using western blot technique. • Deoxyribonucleic acid probe is a highly sensitive method for the diagnosis of malaria. It can detect even less than 10 parasite/ µL ofblood. • B, geneofT. gondiican be detected byPCRoftheamniotic fluid in case of congenital toxoplasmosis. PCR have been developed for detecL ion of filarial DNA from patients blood. If parasite cannot be identified by microscopy, amplification of babesial 18S ribonucleic acid (RNA) by PCR is recommended. • Drug resistances in malaria are detected now by PCR techniques. PCR is increasingly used now for species specification and for detection of drug resistance in malaria. Chloroquine resistance in P.falciparum has been attributed to mutation in the Plasmodium Jalciparum chloroquine resistance transporter (PfCHT), a transporter gene in the parasite. Poirit mutation in another gene Plasmodium falciparum multidrug resistance protein 1 (PfMDHl) has also been implicated in determining resistance in vitro. Pyrimethamine and sulfadoxine resistances are associated with point mutations in dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) genes respectively. Mutation in PfATPase gene is associated with reduced susceptibility to artemisinin derivatives. REVIEW QUESTIONS 1. Enumerate the various methods employed for examination of stools and describe in detail the concentration methodsof stool examination. 2. Describe various skin tests used for diagnosis in many parasitic infections. 3. Write short notes on: a. Scotch tape method b. Blood examination for malarial parasite c. Blood examination for microfilaria d. Enterotest e. Casoni's test f. Floatation method ofstool examination MULTIPLE CHOICE QUESTIONS 1. time of collection of blood is important in a. Microfilaria b. Trypanosoma spp. c. Leishmania spp. d. Babesia spp. 2. Modified acid-fast stain is used for the diagnosis of a. Entamoeba histolytica b. Toxoplasma gondii c. Cryptosporidium parvum d. Leishmania donovani 3. Sputum examination is commonly done for detecting the eggs of a. Strongyloides stercoralis b. Entamoeba histolytica c. Paragonimus westermani d. Ascaris lumbricoides 4. larval forms of which parasite can be found in muscle biopsy a. Ascaris lumbricoides b. Taenia so/ium c. Trichuris trichiura d. Ancylostoma duodenale Answer 1. a 2. C 3. C 4. b
- 264. INDEX Page numbers followedby b refer to box,frefer to figure.Jcrefer to flow chartand I refer to table A Abscess, splenic 19 Acanthamoeba 12, l3, IS, 26, 28,291 ,231, 233, 244 culbertsoni, life cycle of 29f keralicis 29, 30 Acanthocheilonema 223 Acanthopodia 28 Accidental host 2, 121 Acephalocysts 133 Acetabulum 14 l Acid-fast parasitic organisms 105b stain IOOJ, 236 Acidosis, metabolic 79 Acquired immunodeficiencysyndrome 5, 13, 29, 93, 104, 184 243 Adenolymphangitis, acute 214 Adenophorea 166 Adoral cilia 107 Adult Trichuris rrichiura worms l76f Adult worm 112, 144, 151, 154, 156, 160, 170, 175, 180, 181, 198, 203 African trypanosomiasis 42,46 Agar plate culture 239 Albendazole 128, 135 Alimentary canal, amebae of 13 Alphonse laveran 67 Amastigote 42, 48, 48f, 53, 54f Ameba 244 classification of 151 drug sensitivity of 23 Amebapores 18 Amebiasis 20fc,246 cutaneous 19, 21 genitourinary 19, 21 hepatic 20 lesions of 22f metastatic 2I pulmonary 19, 21 Amebic antigen detection 23 appendicitis 19 colitis 24 cysteine proteases 18 dysentery 13, L S, 19, 20 encephalitis, granulomacous 26, 29 granuloma 30 hepatitis 19, 20 keratitis 26, 29 lectin 18 liver abscess 13, 15, 19, 20f, 21, 2lfc, 24 meningoencephalitis 13, 26-29 ulcer 18, 19f flask-shaped 19/ Amebida 12 Ameboflagellate 27 Ameboma 19, 30 Amebostomes 27 American rrypanosomiasis 47 American visceral leishmaniasis 56 Amoeboflagellate 39 Amphotericin B 28, 61 Ampulla ofVater 155 Ancylostoma 6, 140, 165, 189 brazilie11se 165, 167 caninum 167 ceyla11iwm 193 duodenale 3, 7, 165, 176, 180, 187-189, 192,194,207,229, 243 adult worm of 188/ egg of 189] life cycle of 190/ Anemia 46, 57 causesof 56b, 78b, 192b dimorphic 192 severe 56, 87 Angiostrongyliasis, abdominal 231 Angiosrrongylus cantonensis 167, 230-233 Animal inoculation 8, 47, 50, 59, 94, 245 Anisakiasis 232, 233 Anisakis simplex 167, 232 Anodic antigen, circulating 147 Anopheles barbirostris 2LO, 219 Anthroponotic urban type 62 Anthropozoonoses 2 Antiamebic drugs 24 I Antibody demonstration of 217 detection 7, 23, 35, 51, 60, 95, 128, 147 Antigen 7 detectio11 7, 35, 47, 51, 59, 95, 128, 135, 146 tests, rapid 83 Anti-oocyst antibody 100 Apansporoblastina 12 Aphasmidia 165 Apicomplexa, phylum 66, 661 Appendicitis 178 Artcmisinin-based combination therapy 84 Ascariasis, ectopic 205 Ascaris 6, 8, 140, 207 eggs, types of 202f fertilized egg of 202/ lumbricoirles 3, 7,112,165, 167, 176,180, 189, 194, 199-201, 20 If, 204, 206f c, 207, 243,248 life cycle of 204/ pneumonia 203 suum 167 unfertilized egg of 202/ Ascites 57 Aspirates, splenic 58 Aspiration 135, 135b biopsies 59 Atovaquone 88 Autoimmune hemolysis 56 Axoneme 41, 42, 53 Azithromycin 88 B Babesin 4, 12, 66 bovis 86 microti 13, 86, 86/ Babesiosis 87,871 Bachman intradermal test 174 Bacillary dysentery 20, 201 Bacterial infection, secondary 227 Baermann concenrration method 238, 238/ Balamuth's medium 23 composition of 244b Balamuth's monophasic liquid medium 244 Balamuthia 26 mnndrillaris 15, 30 Balantidiasis I09 Balantidium 12, 109 coli 3, 7, 11, 13, 14, 39, 107, 107f, 109, 150,244 life cycle of 108/ Bancroftian filariasis 213, 247 Basal body 10 Basophilic stippling 73 Baylisascaris 207 procyonis 167 Bell's dilution-filtration count 238 Bentonite flocculation tests 247 Benznidazole 51 Bile duct carcinoma 145 staining 123 Bilharziasis 143 Biliary cirrhosis 156 obstruction, acute 205 passage 152 tract 142, 154
- 265. Paniker'sTextbook ofMedical Parasitology Binaryfission 11, J6, 41 Binucleate cyst 16f, 25f Biopsy 217 BiLhionol 153 Blackwater fever 79 Bladder carcinoma 145 containing seeds 142 worm ll7, 123 Blastocyslis hominis 10 I, 10 If Blastomeres 188 Blepharoplast 42, 53 Blinding filaria 221 Blister formation 227 Blood 13, 142 collection of 240 examination 6, 135 fluke 141-143 incubation infectivity test 47 loss 178 picture 87 smear 82b transfusion malaria 801 urea nitrogen 88 Boeckand Drbohlav diphasic medium 244, 244b, Bone marrow 56 aspirate 58 macrophage of 13 Bothriocephalus anemia 120 Bradyzoites 9 l, 93, 102 Brain 21, 104,232 parenchyma l28f Bronchi 161 Brugia malayi 4, 7,165,208,210,218, 219f, 224,226 Brugia pahangi 167, 223 Brugia patei 167 Brugia Limori 165,208,210, 219,226 Buffy coat blood film 242 Bunostomum phlebotomum 167 C Cachexia 57 Calabar swellings 219, 22 l Calcofluor white staining 29 Candidate vaccine 61 Capil/aria a eropltila 16 1 Capillaria philippinensis 4, 165, 180, 231, 232 Card agglutination trypanosomiasis test 46, 47 Cardiac implantable electronic device 133 Cartwheel appearance 16 Casoni's inrradermal 1es1 134 Casoni's test 247 Cat liver fluke 156 Cathodic antigen, circulating 147 Caudal papillae l66 Cecum 18 Cellular exuda1 es 20,235 Cellulose acetate membrane precipitation test 23 Central nervous system 13, 46, 129, 150, 171 infection 232t Centrilobular necrosis 77 Cercarial dermatitis 148 Cerebral amebiasis 19 angiostrongyliasis 230 malaria 79 paragonimiasis 161 Cerebrospinal fluid 6, 27-29, 45-47, 128, 230,231,245 Cestodes 4, 112, l l 5 classification of 115, l 16t living 122b Chagas disease 13, 42, 47 acute 49, 50 chronic 50 Chagas radioimmune precipitation assay 5 1 Chagoma 50 Chancre painless 45 trypanosomal 45 Charcoal culture 240 Charcot-Leyden crystals 19, 20, 22, 22f, 235 Chemoprophylaxis 84, 85 Chiclero's ulcer 53, 63 Chilomastix 12 mesnili 32, 38 egg of 38f Chinese liver fluke 154 Chocolate brown sputum 21 Cholangiocarcinoma 156 Cholangitis 156 Chopra's antimony test 60 Chromatoid bodies 10, 16 Chrysops 220, 221 Chylous urine 215/ Cilia 11 Ciliophora 11, 12 Cloaca 164 Clonorchis 113, 141,207 sinensis 4, 7, 143, 145, 151, 154, 172, 194, 201,232 egg of 154f life cycle of 155 / Coccidia 12, 66, 90 Coenurns 117, 129 Colon 13, 18 Complement fixation test 7, 46, 47, 58, 133, 135,216,246 Complete blood count 205 Congestive cardiac failure 87 Conjunctiva 165 Conjunctiva! biopsies 222 Conjunctiva] epithelium 104 Coproantigen, detection of 23 Copulatory spicules 200 Coracidium 118, 120 Cornea 29, 104 Corneal stroma 104 Counter-currem immunoelectrophoresis 23 Craig's medium 22 Creeping myiasis 167 Crescentic tachyzoites 90f Crustacea 232 Cruzin 51 Cryptosporidiosis 247 Cryptosporidium 12, 14, 66, 100, 236 paruum 3, 4, 7, 13, 10, 97, 99, 100, 105, 199,204,248 life cycle of 99f oocysrs of 98f, 100 / Clenodactylusgundi 90 Cu/ex quinquefasciatus 2 10, 213 Culicoides 2!0, 223 Cutaneous leishmaniasis, diffuse 53, 62 cyclophyllidean 117, 1171 tapeworms 122 cyclops 118, 232 Cyclospora 66, 101,236 cayetanensis 3, 7,39,99, 100,105 Cylindrical esophagus 180 Cyst 14, 16, 17, 26, 27, 29, 30, 39, 107, 107 f fluid 134 mature 96 / uninucleate 16f cysteine-pep1one-liver-maltose 37 cysticerci in muscles 124} cysticercosis 126, 128, 140 Cysticercus 117, 123 bouis 123, 124f cellulosae 123, 124f cysts of I28 f Cytoadherence 74 Cytolysis 18 Cytopharynx 107 Cytoplasm 10, 16 Cytopyge 107 Cytos10me 107 D Deoxyribonucleic acid 8, 21, 35, 47, 58, 83, 127, 133,217,247 Dermatitis 184 Diamond's axenic medium 23 Diarrhea 13, 34b, 97 bloody 1501 1 Oichlorodiphenyltrichloroethane 85, 218 Dicrocoelium dendriticum 150, 153 Dienlamoeba 12, 39 fragilis 32, 39 trophozoite of 39{
- 266. Died1ylcarbamazine 168, 215,217, 222 medicated salts 218 provocation test 216, 242 Dihydrofolate reductase 84, 248 Dihydropteroate synthase 84,248 Dipetalonema 223 Diphyllobothrium 113, 115 latum 4, 7, 112,116,117, 118f. L 22, 151, 172,232 life cycle of l 19f Dipylidium 113, 115 caninum 7, 116, 139, 139f. 226 Direct agglutination test 51, 58, 60,246 Direct fluorescent meL hod 105 Dirofilaria 167 conjunctivae 224 immitis 161,167,223 repens 224 Disseminated intravascular coagulaLion 87 Distomata 141 Doxycycline 218 Dracunculiasis 227 Dracunculus medinensis 4, 164, 165, 225, 226, 227fc, 229 adult worm of 226/ infection 225 .f larva of 226 .f life cycle of 228/ Dumdum fever 52, 53 Duodenal aspirates 97, 184, 205 Duodenal capsule technique 243 Duodenwn 156 Dysentery 13 E East Africa n trypanosomiasis 45,451 Echinococcosis 247 £chinococcus 8, I15, 117 granulosus 2, 4, 46, 116, 117, 129, 130.f. 133/ c, 140, 16 1,204,226,232 life cycle of 131/ multilocularis 2, 116, 136, 226, 232 Echinostoma 113, 156, 159 Echinostomatoidea 141 Ectocyst 132 Ect0parasite 1 Ectopic infection 146, 167 Ectoplasm Io Edema 46, 57 painless 221 Elephantiasis 210,214, 215f Embryophore, inner 123 Encephalitis 13 granulomatous 29 £ncephalitowon 12, 104 inleslinalis 105 Encephalopamy, diffuse symmeLric 79 Encysted larvae 165 Endemic foci 160 Endocyst 132 Endodyogeny 11 Endogony 91 Endolimax nana 15, 25, 26 .f Endoparasite I Endoplasm 10 Endoscopy 5 1 Endospore 105 Entamoeba 6, 12 coli LS, 24, 25/ gingivalis 15, 25 hartmanni 15, 25 trophozoite of 25f hislolytica 3, 6, 7, 10, 13, 15, 16.f. 18/J, 21 /c, 23.f. 99, 105, 109, 150, 199, 204,234,243,244, 248 life cycle of 17!, 17/c polecki 15 Enteric cycle 92 Enterobius vermicularis 3, 4, 6, 7, 39, 165, 175, 176, 189, 195, 196, 196); 198/ c, 199,207 adult worm of 195/ life cycle of 197/ Enterocyte 105 J;'nterocylozoon bieneusi 105 Enteromonadina 12 Enteromonas 12 hominis 32, 38, 40 cyst of 38/ Enterotest 35 Enzyme-linked immunosorbent assay 7, 21,23,35, 46,47,51,58,83,94, 95, 127, 133, 147, 168, 173, 185,205, 206,2 16,217,227,246,247 Eosinophil count 215 Eosinophilia 128, 178 peripheral 185 Eosinophils 5( Epilepsy, focal 133 Epimastigotes 42, 43, 45, 48, 48) Erythematous patches 57/ Erythrocyte mature 73 sedimentation rate 46 sequestration 79 surface antigens, ring-infected 85 Erythrocyticschizogony 68, 69, 76); 240 Escherichia coli 29 Esophagus, double bulb 181 Espundia 63 Ethylene diamineterra-acetic acid 240 Eucoccidia 12 Eurytrema pancreaticum 154 ExcystaLion 17 Exilagellating male gametocytes 71 Exoenteric cycle 93 Exoerythrocytic schizogony 68 schizont 69 stage 69 Extrinsic incubation period 45, 55 Eyes 232 F Fairley's lCSt 147, 247 Index Falciparum malaria, complications of 79b Falcon assay screening test 147 Fasciola 113, 141, 167 gigantica 7, 151 hepatica 4, 7, 143, 150, 15 1, 151.f. 153, 194,201 egg of 151/ life cycle of 152f Fascioliasis 153 Fasciolidae 141 Fasciolopsis 113, 141 buski 4, 7, 143, 151, 153, 156, 157 !, 201 egg of 157( life cycle of 158/ Fast-flowing rivers 222 Fat malabsorption 34 Ferrissia tenuis 145 Fever 20 high-grade 56 Fibrin degradation products 84 Filarial antigen, circulating 2 17 Filarial worm 208 classification of 208t Filariasis 208, 247 lymphatic 210 subcutaneous 210,219 Filariform 183 larva 181, 181/. 184, 188, 19lt, 213 third-stage 188 Flagella 13 Flagellates 32, 321 zoological classification of 41 Flagellwn 4 1, 42 Floatation method 237 Flukes 141 Fluorescent antibody direct 37 indirect 83, 205, 206 Fluorescent staining 100 Formogcl test 60 Formol-ether sedimentation technique 237, 237f Fragilis 39 Free-livingsoil cycle 182 Frenkel, skin test of 95 Fulminant amebic colitis 19 Furcocercous cercaria 145 Fusiform worms 195
- 267. Paniker'sTextbook of MedicalParasitology G Gametocytes 68, 71 Gametogony 11, 71, 73, 90, 97 Gastric washings 205 Gastrodiscoides 113, 141 hominis 7,151, 153, 156, 159, l59 f Gastrointestinal tract J42 Gastrophilus 167 Gelatin capsule 243 Gelminths 112 Genital flagellates 32 Geohelminths 207b Giant intestinal fluke 156 Giardia 6, 12, 13 lamblia 3, 5-7, 13, 14, 32, 33/, 35 .f. 99, 109, 199,244 life cycle of 34f Giardiasis 246 Giardia-specific antigen 35, 65 Giemsa stain 46, 59 /, 9lf, 240, 241 Glisson's capsule 153 Glucose-6-phosphate dehydrogenase 78, 79 deficiency 80 Glycogen mass of 16 vacuole, large 16 Glycophorin 69 Glycoproteins 18 Glycosylphosphatidylinositol 56, 74 Gnathostoma spinigerum 167, 231, 232 egg of 23lf Gnalhostomiasis 166 Golgi 67 cycle 67 Gomori methenamine silver 94 Gram's stain I 05 Granules, column of 211 Granuloma formation 214 Ground glass appearance 16 Guinea worm 165 Gymnamebia 12 Gynecophoric canal 142 H Harada-Mori filter paper strip culture 239 Harada-Mori tube method 192, 239 f IIartmannella culbertsoni 28 llearr 13 Ileidenhain's hematoxylin magnification 25f llelminths L, 111, U lt, 113 zoological classification of 113 Hemagglutination, indirect 7, 83, 127, 133, 205,206 l lemonagellates 13, 14, 32, 41 stages of 421 Hemoglobin 79, 83 nature of 80 Hemoglobinuric nephrosis 79 Hemoptysis 161 Hemorrhage 56 Hemosporina 12 Ilemozoin pigment 69, 77 Ilcpatic lobe, right 134f Hermaphrodites 112, 116 Hermaphroditic flukes 143, 150 Hermaphroditic trematode, morphology of l42f Herring worm disease 233 Heterophyes 113, 141, 156 heterophyes 7, 15L, 158, 232 l leterophyidae J4l 1lexacanth 117 embryo 118, 123, 130 oncosphere 136 Histidine rich protein 7, 74, 83 Jlookworm 187 diagnosis of l93fc filariform larva of .l 80t infection 187b, 190, 192/J Host-parasite relationships 2, 3Jc Human African trypanosomiasis 4511, 47 treatment of 47/ Human acquire infection 93 hookworm 166 immunodeficiencyvirus 10, 24, 36, 57, 105b infection 230 large intestine 159 leukocyte antigen 80 malaria 66 parasites 69t nematode 167 trematode 167 Humoral immunity 8 1 Hydatid cyst 130, 131, 131 /, 132}; 134}, 136 fate of 133 disease, malignant 136b fluid 132 sand 132 Hydrocelc 214 Hymenolepiasis 139 Ilymenolepis 113, 115 diminuta 7, 116,139,226 nana 3, 4, 7,112,116, 122, 136, 139, 189, 199,226 adult worm of l37f egg of 137 f life cycle of 138 J llypergammaglobulinemia 60 Hyperinfection 184 Hypnozoites 69, 71, 81 reactivation of 81 Ilypochromic microcytic anemia 192 Hypoglycemia 79 Iatrogenic transmission 4 Iliac crest 58 Immature cyst 96f Immunity 5, 24, 58, 80 lmmunochromatographic card test 58,216, 2 17,247 lmmunofluorescence assay 227 indirect 35 Immunoglobulin E 198, 215 M 5, 80,246 Indian visceral leishmaniasis 56 Indirect fluorescent antibody 23, 216, 217, 246 test 94, 95 Indirect hemagglutination 21, 23, 46, 47, 51, 216, 217,246,247 assay 23 Indirect immunofluorescence 47, 51,246, 247 Infective rhabditiform 201 larva 176 lnflammatory reaction 5 Innate immunity 80 lntercellular adhesion molecule 74 Interferon gamma 74 Intestinal amebiasis 18, 19, 19 /, 21, 24 chronic 19b sequelae of 19b bilharziasis 148 biopsy 97 entamoeba 261 flagellates 13, 32 flukes 141,142, 156,1 76 helminths 247 human nematodes 165 invasion, stage of 173 sarcocystosis l 02 taeniasis 126, 128 Intestine 13 large 13,107, 142,165, 175, 175b small 13, 32b, 122b, 142, 165, 180, 180h, 200 lntradermal allergic tests .156 skin test 147 test 51 Intravenous pyelogram 134, 147 Iodamoeba 12, 26 butschlii 15, 25, 26 Iodine staining 235 Iodophilic body 26 Iodoquinol 24 Iron-hematoxylin stain 235 lsoenzym e study 47
- 268. Isospora 12, 66,236 belli 3, 7, 13, 96, 105 oocysts of 96/ Itching papules 165 lvermectin 218,222 J Jaundice, obstructive 136 JejunaJ biopsies 184 Jejunum 129, 156, 187 K Kala-azar 13, 52, 53, 55, 56, 56b, 57 /. 58 / c Karyosome 10 Katayama disease 150 fever 148, 150 Kato d1ick smear technique 235 modified 238 Kato-Katz smear tech nique 192 Kawamoto technique 83 Keratitis 13, 29 stromal 104 Keratoconjunctiviris 104 Kidney 21 Kinetoplast I0, 41 , 42, 53 Kinetoplastida 12, 13 Kinyoun's acid-fast stain 97,236 reagents of 236b Kinyoun's carbol fuchsin 236 Knott's concentration technique 216 Kupffer cells 56 L Lactophenol cotton blue 135 Lancet fluke I53 Larva 165,171, 225 currens 166, 184 detection of 227 development of 227 infective stage of 166 migrans 165, 232 cutaneous 167, 167t, 168, 1681 third-stage I9lt Latex agglutination test 23, 95 Laverania 66 Leishman's stain 240, 241 Leishman-Donovani body 54/. 59 / Leishmania 4, 12, 13, 41 , 52, 245 aethiopica 53, 61 braziliensis 13 complex 53 classification of 531 donovani 7, 13, 52, 53, 59 /. 248 life cycle of 55/ morphology of 54/ transmission of 561 infantum 53 major 53, 61 mexican a complex 53 peruviana 53 tropica 13, 53, 61 complex 61 Leishmaniasis 246 cutaneous 13, 52, 53 mucocutaneous 13, 52, 53, 63 Leishmanin skin test 60,247 Lepromatous leprosy 184 Leukopenia 56, 57 Lieberkuhn, crypts of 18 Lipophosphoglycan 23, 55 Liposomal amphotericin-B 61 Liver 56, 131 !, 151 abscesses 21 biopsy 23 fluke 141,142, 150 rot 151 Loaloa 7, 165,167,208, 2 10,219,221, 222, 224, 226,241 Lobopodia 27 Lobosea 12 Locke's solution 244b Locomotion 112 Loeffler's syndrome 166, 203, 204b Lugol's iodine 235 Lumbricoides 200 Lumen-dwelling flagellates 32 Luminal amebicides 24 Lung 150, 160 flukes 141, 142, 160 right lower lobe of 13'l Lutzomyia 63 jlaviscutellata 53 longipalpis 53 olmeca 53 umbratilis 53 Lymph node 13 aspirates 59 peripheral 56 Lymphadenitis 214 Lymphadenopathy 45, 57, 93 Lymphangiovarix 214 Lymphangitis 2 14 Lympheclema 214 l.ymphoreticular malignancies 184 Lymphorrhagia 214 M Machado-Guerreiro test 51 Macrogamete 92, 98 Macrogametocyte 7l, 73 Macules, hypopigmented 223 Malaria 13, 66, 78/J, 83/J, 871 congenital 80 Control Programs 86 global distribution of 67/ initiative, roll-back 86 merozoite-incluced 80 organs in 78/ Index parasite 14, 66, 70!, 73/. 74!, 82b, 82 !, 240, 245 culture of 82 pigment 69, 69b septicemic 79 tertian malignant 79 vaccine 85 Malarial parasite drug resistance of 85 types of 71 Malnutrition, severe 184 Mansonella ozzardi 165, 208, 210,223, 241 perstans 165, 208, 210, 223, 224, 242 strepLocerca 208, 210, 223, 233 Mass d1erapy 217 Mastigophora 12, 13 Mastigote 41 Maurer's clefts 73 Mazzotti reaction 222 McMaster's egg counting 238 Melarsoprol 47 Membrane filtration concentration 242 Meningoencephalitis 230 Merogony 69, 105 Merozoites 68 Mesoendemic 67 Metacyclic trypomastigotes 43, 49 Metacyst 18 Metacystic trophozoites 18 Metagonimus 141 yokogawai 7, 143, 156, 159, 172, 232 Metazoa 10 Metriphonate 147 Metronidazole 24 Meyers Kouwenaar syndrome 215 Microabscesses 148 Microconcentration technique 82 Microfilaria 208,211,219,241, 248 bancrofti 211, 220 J morphology of 21V demonstration of 215 malayi 220 f Microgamete 92, 98 Microhematocrit rube method 242 Microspora 11, 12, 14, 66, 104 Microsporidia 3, 48, I05, I06 classification of 1041 infective stage of 105 Microsporidium 104, 105 Microsporum 12 Migrating larva 190, 203 Mild flu 93 Monocytosis 46 Montenegro test 60, 247
- 269. Paniker'sTextbook of MedicalParasitology Mosquito-borne malaria 80t Motile bacteria 20 Motile nophozoites 20 Mucus plug 175 Multiceps multiceps . 129 Multilocular hydatid 136 Multiple fission 11 Murine strain 139 Muscle 104, 171 biopsy 172, 173/J, 243 invasion, stage of 173 Muscular cysticercosis 126 Myocarditis 46, 46b Myositis 104 N Naegleria 12, 15,291,233 fowleri I , 13, 15, 26, 231, 244 life cycle of 28/ Napier's aldehyde test 60 ational Rural Ilealth Mission 86 National Vector borne Disease Control Programme 86 Necator 165 americanus 3, 7, 165-167, 176,180,187, 189,192,204,207,229,243 Nelson's medium 23 Nematodes 111-113, 164 classification or 1651 zoological classification of 166t Nematohelminthes 11 l Neoplasia 5 Nerves 13 Neural larva migrans 168,207 Neurocysticercosis l26 Neutropenia 56 Nifurtimox 51 Nitazoxanide LOO Nocturnal enuresis 197 Noncalcified hydatid cyst 134/ Nonspecific serum tests 60 Normocytic normochromic anemia 60 Nosema bombycis 104 ovy-Macneal-NicolJe medium 245, 245b ucleic acid amplificaLion test 37 Nuclepore filtration 216 Nucleus LO, 16, 41, 42 0 Ocular cysticercosis 127, 128 Ocular toxoplasmosis 94 Onchocerca volvulus 165, 208, 210, 221, 22 1 1; 223 Onchocercoma 222 Onchodermatilis 222 Oncosphere 117, 118 Oocysr 71, 90, 92, I05 mature 96/ spherical 98 thin-walled 98 Ookinete 71 Operculate snails 154 Operculum 118 Ophthalmic larva migrans 168,207 Opisthorchioidea 141 Opislhorchis I 13, 14 1 felineus 143, 151 viverrini 143, 145, 151 Opportunistic infections 105b Oral flagellates 32 Ovarian lobe, accessory 122 Oxyuris vermicularis 195 p Packed cell volume 79, 83, 84 Pancreatic duct 154 PancreaLitis 205 Panstrongylus megistus 48 Parabasal body 42, 53 Paragonimiasis, abdominal 161 Paragonimus 113, 14 1 westermani 4, 143, 151, 160, 160f. I61, 163,204,230,232,233,242,243, 248 egg of 161/ life cycle of 162/ Paramphistomatidae 141 Parasite I, 2/J, 3t, 7b, 115, 201/J, 204b aberrant l accidental l detection of 205 escape mechanisms 6t exhibitingantigenic variations 5b P test 83 facultative l free-living l infectious 199b lactate dehydrogenase 83 life cycle of 3 quantification of 82/J types of 2/c Parasitic diseases 7t Parasitology l Paratenic host 2 Paromomycin 24, 61, 100 Pelvic plexuses 144 venous plexuses 145 Pentamidine 47 Peribronchial granulomatous lesions 161 Pericardia! amebiasis 19 Pericyst I31 Peridomestic cycle 48 Periodic acid-Schiffstain 91, 105 Peripheral blood 71 , 82/ Peristome 107 Petridish culture meL hod 239/ Phasmid 166 Phlebotomus argenlipes 53 ariasi 53 longipes 53 orientalis 53 papatasi 53 pedifer 53 perniciosus 53 sergenti 53 Pinworm 165 Piroplasmia 12 Pistia plant, removal of 218 Plagiorchioidea 141 Planoconvex egg 196/ Plasmodium l, 11, 12, 66 falciparum 5, 7, 66, 67, 69, 73, 74/, 77, 78, 82/, 83, 88, 89 chloroquine resistance transporter 84,248 erythrocyte membrane protein-I 74, 79 histidine-rich protein 83 lactate dehydrogenase 7 multidrug resistance protein 84, 248 lactate clehydrogenase 246 malariae 66, 67, 69, 75,, 77, 78, 89 stages 76/ ovale 66,67,69, 75, 77, 78,89 uivax 7, 66, 67, 69, 7 l, 72f. 73f. 77, 78, 88, 89 life cycle of 68/ Plastic envelope medium 37 Platyhelminthes 111, 115 Pleistophora 104 Plerocercoid larva 118 Pneumocystis jirovecii 5, 243 pneumonia 94 PneumoniLis 204b Polar tubule 105 Polymerase chain reaction 8, 35, 83, 84, 87, 127,133,216,217,247 Portal hypertension 148, 156 Post-kala-azar dermal leishmaniasis 13, 52, 57, 57t, 243 treatment of 57 Praziquantel 128, 147, 148, 158 Precysr 16 Pre-erythrocytic schizogony 68, 691 Primaquine 84, 85 Procercoid larva 118, 120 Proglonids 116, 127 Promastigote 42, 53, 54/ Protein 34 merozoite surface 85 Protozoa I, 410,, 32b 1
- 270. classification of 11,I2t transmitted 37b Protozoan parasites 34h Protozoology 1 Pruritus ani 196 Pseudocele 164 Pseudocyst 91 Pseudophyllidean 117, 1171 tapeworms l J7 Pseudopodia I0, 13, 15, J6 Pseudotumoral growth granulomatous 19 Pulmonary capillaries 130 Pyknotic bodies 22 Pyriform 129, 159 Pyrimethamine 95 Q Quadrinucleate ameba 18 Quadrinucleate cyst 26J; 33( mature 16 Quantitative buffy coat 82, 83 Quartan malaria 75 Quinine 84 R Rat fleas 138 Rat tapeworm 139 Rectal biopsy 146, 148 Red blood cell 13, 19, 20, 39, 67, 68, 74, 78, 00,82,235 splenic sequestration of 56 Renal transplantation80 Respiratory distress syndrome, acute 87 Reticulocyces 72 Reliculoendothelialsystem 13 Retortamonadida 12 Retortamonas 12 intestinalis 32, 38 Retroinfection 196 Rhabditiform larva 181, 18 1{. 183, 188, 203 Rhizopoda 12 Rhodnius prolixus48 Ribonucleic acid 82, 248 River blindness 222 RK39 tesr 60 Robinson's medium 23 Rodent feces 138 Romana's sign 50 Roundworm egg 202t s Sabin.Feldman dye test 95, 246 Salivarygland 68 Salpingitis, chronic 197 Sandy patches 145 Snrcocystis 12, 66, I02 homl11is 32, J72, 226 oocyst of 102( suihominis 172, 226 Sarcocystosis, muscular 102 Sarcodina 12, 13 Sarcomastigophora 11, 12, 15 Saturated sail solution 176h technique 237 Schaudinn's solution 236 Schistosoma 141, 144/ eggs 238b hematobium 5, 7, 143·145, 147/c, 149, 207,217 egg of 144/ intercalatum 150 japonicum 143, 149, 232 mansoni 143, 1< 17, 149 mekongi 150 Schistosomatidae 141 Schistosomes 143, 143/J, Iso morphology of 143/ Schistosomiasis 145 acuce 145 chronic 145 Schizogony 11, 68, 73, 90, 97, 98 Schneider's drosophila tissue culture medium 59 Schneider's insect tissue culture medium 245, 245h Schuffner's docs 72 Scotch tape method 198, l 98/ ; 238 Segmemina 157 Serological tests 7, 205 Serous cavity filariasis 210, 223 Serpent worm 225 erurn glutamic pyruvate transaminase 88 Sheep liver fluke 150 Sigmoidorectal region 18, 144 Sigmoido copy 178 Silkworm disease J04 Skeletal muscle 104, 171 Skin 13 snip 243 test 7, 8h, 60, 62, 63,217,227,247 transmission 4 Sleepingsickness 13, 42 Slender thread•like worms 208 Smooth curves 216 Somatic cells 211 Somatic human nematodes 165 outh American trypanosorniasis 42 Sparganosis 120, 121, 166 Sparganum ll8 larva 120 Spherical nucleus 38 Spirometra 115, 120 erinacei 116 life cycle of 121/ theileri 116 Spirurid nematode 231 Index - Spleen 21, 56, 78 Spoliative effects 203 Sporoblasts 96 porocyst 96, 142, 153, 154 Sporogony 68, 71, 90, 104, 105 Sporozoa 12, 661 Sporozoites 68, 71 StalJion's disease 43 Scercoraria 42 StoU's dilution technique 238 Strawberry mucosa 37 String test 35 Strobila 116, 11 7 Strongyloides 165,229,239 stercora/is 4, 105, 164,165, 167, 180, 184/ c, 185, 199,204,207,226,232, 243, 248 egg or 181/ larvae of 181/ life cycle or 182); 183/ c Strongyloidiasis 185 Sugar floatation technique 237 Suihominis 32 Sulfadiazine 95 Suppurative inflammation, acute 29 Swimmer's itch 145 Sylvatic zoonosis 48 Syngamy 11 Syscemic lupus erythematosus 29 T Tachyzoites 91, 93 Taenia 115 antigen, detection or 127 eggof 124/ multiceps 129, 226, 232 saginata 4, 7, 116, 122, 123t, 140, 172, 176,201,226 asiatica 122, 129, 226 life cycle of 125/ solium 2·4, 7, 112, 116, ll7, l 22, l 22/ , 1231 , 140, 199, 201, 207, 226, 229, 232 adult worm or 122/ eggof 176 life cycle of 125, 126/ Tapeworm 115, llS J; 122, 129, Tetracyclines 218 Tetrapetalonema perstans 223 streplocerca 223 Thromhocytopenia 56, 57, 60 Tick•borne disease 86 Tinidazole 24 Tiny knob 154 Tissue 6 amebicides 24 biopsy 243 - - - ~...~- - - -!~ii.i=l.lo,oiii•
- 271. B !aniker'sTextbookofMedical Parasitology cyst90, 91, 93 hypoxia 77 necrosis 18 Toxic megacolon 19 Toxocara canis 167, 206, 232, 233 adult worms of 206/ Toxocara cati 167,206,232 Toxocariasis 247 Toxoplasma 11, 12, 14, 66, 90, 94 encephalitis 95, 96 gondii 1, 2, 4, 5, 10, 14, 46, 48, 90, 90{. 91 /, 93, 94/c,96, I 05, l 72, 248 lire cycle of 92 f inrection 93, 94 pneumonia 94 Toxoplasmosis 94, 245, 246 acquired 93 acute 93 congenital 93, 95 Trachipleistophora I04 Transfusion malaria 80 Transovarian transmission 87 Transverse binary fission I08 Trau ma 5 Traveler' diarrhea 99/, Trematodes 4, 112, 141, 143/, classification of 141 zoological classification of 14 l/ Triatoma inrestans 48 Trichina worm 170 Trichinel/a 140, 164 cyst 171 Spira/is 4, 7, 46, 165, 170, 172, 173/< , 174, 180, 199,207, 226,232,243 adult worms or 170/ life cycle of 172 /, l73t Trichinosis 247 Trichomonadida 12 Trichomonas 12, 13, 36, 39 hominis 32, 36 /, 38 tenax 32, 36 /. 37, 204 vagina/is 3, 6, 7, 13, 36, 36 ); 95, 109, 217, 244 Trichostomatina 12 Trichostrongyliasis 193 Trichostrongylus orienlalis 7 Trichrome stain 235 modified LOS, 236 reagents of 236b Trichuris 175,229 lrichiura 3, 7, l09, J50, 165, 175, J76, 178, 178 f c,189, 196,20l,207 egg of 176/ life cycle or L 77] Triclabenda1ole 153 Tripartite 187 Trop-bio test 2 I7 Trophozoite 11, 14, 16, 16/; 27/ , 29, 33,33{, 38/, 39, 86/; 90, 91, 91{. 107, 107/ extracellular 90{ Tropical pulmonary eosinophilia 215, 2 17 Trypa11osoma 12, 13, 41, 233, 241, 244, 248 brucei 4, 13, 32, 42,231 brucei 43 lire cycle or 44 / gambiense 5, 42, 50 rhodesiense 5, 43, 45, 46, 50 cruzi 4, 13, 32, 43, 46-48, 48{. 50, SO J, 51, 93,226 life cycle or 49f equiperdum 43 evansi 43 gambiense 43, 43/ lewisi 43 infections 95 rangeli 43, 51 rhodestense43f. 46[ Trypanosomatidae 41 Trypanosomatina 12 Trypanosomes 42, 5 1 classification or 42 Trypanosomiasis 43{. 46/< , 245, 246 Trypomastigote 42, 48, 48 ] Tsetse fly 45 Tubercles 147 Tubulina 12 Typhus-like examhema 94 u Uncinaria stenoceplzala 167 United Nations Children's Fund 86 United ations Development Programme 86 Upper respiratory tract 29 Urethra 13 Urethritis 13 Urinary bladder 144 carcinoma 145 Urine 6, 7/J V Vaccination 5 Vacuole 53 Vagina 13 Vaginal sphincter, prominent 122 Vaginitis 13 Vascular cell adhesion molecule- I 74 Vector mosquito, eradication of 2 18 Vector transmission 4 Vermicules 87 Vertebrate host 44 Visceral larva migrans 167, 1671, 168, 1681, 206,207 Visceral leishmaniasis 52, 53, 54/, 61 h, 245 Viviparous nematodes 226/J w Water plants, ingestion of 159 Watsonius watsoni 153, 156, 159 West African trypanosomiasis 43, 451 Western blot 100 Wet saline mounts 235 Wheatley's lrichrome stain 235 Whip-like flagella 32 Whipworm 165, 175, 1761 White blood cell 29, 83, 242 Winterbonom's sign 45 Wolbachia 208,223 Wright's tain 241 · wuchereria 164 y bancrofti 4, 7, 165, 199,208, 2 10, 211{. 212,213, 2 16/,, 2 17,218,224,226, 241 adult worm or 21J/ life cycle of 212/ Young erythrocytes 72 Young trophowites 69 z Ziehl-Neelsen stain 100( modified 97, 98, 236 Ziemann's stippling 75 Zinc sulfate noatation concentration technique 237/ Zooa11throponoses 2 Zoomastigophorea 12 Zoonoses 2, 8 Zoonotic filariasis 223 Zoophilic nematode 167 Zygocotylidae 141 Zygote 7 1