GENERAL PROPERTIES AND CLASSIFICATION OF FUNGI

 Mycology is the study of fungi, which are eukaryotic organisms.

 However, unlike animals, most fungi are nonmotile and possess a rigid cell wall.
Unlike plants, fungi are nonphotosynthetic.
 Fungi have an essential rigid cell wall that determines their shape and
protects them from osmotic and environmental stress. Cell walls are composed
largely of carbohydrate layers, long chains of polysaccharides as well as
glycoproteins and lipids. Some sugar polymers are found in the cell walls of many
fungi, such as chitin, glucans, and mannans, polymers of mannose.
 Fungal infections are mycoses .
 Most pathogenic fungi are exogenous, their natural habitats being water, soil,
and organic debris.
 The mycoses with the highest incidence—candidiasis and dermatophytosis—
are caused by fungi that are part of the normal human microbiota and highly
adapted to survival on the human host.
 Mycoses may be classified as superficial, cutaneous, subcutaneous, or systemic
and opportunistic mycoses.

Morphology

 Fungi grow in two basic forms, as yeasts and moulds.

 Yeasts are single cells, usually spherical to ellipsoid in shape and varying
in diameter from 3 to 15 μm. Most yeasts reproduce by budding. Some
species produce buds that characteristically fail to detach and become
elongated; continuation of the budding process then produces a chain of
elongated yeast cells called pseudohyphae. Yeast colonies are usually soft,
opaque, 1–3 mm in size, and cream-colored.
 Mould form occurs by the production of multicellular filamentous colonies.
These colonies consist of branching cylindric tubules called hyphae, varying
in diameter from 2 to 10 μm. The mass of intertwined hyphae that
accumulates during active growth is a mycelium. Some hyphae are divided
into cells by cross-walls or septa, which typically form at regular intervals
during hyphal growth. Vegetative or substrate hyphae penetrate the supporting
medium, anchor the colony, and absorb nutrients. In contrast, aerial hyphae
project above the surface of the mycelium and usually bear the reproductive
structures of the mold.
 Some species of fungi are dimorphic and capable of growth as a yeast or mould
depending on environmental conditions.
Staining characters
 Lactophenol cotton blue, Gram stain, India ink and nigrosin are commonly used.

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 Lactophenol cotton blue – fungal structures are stained blue
 Gram stain – gram positive
 India ink or nigrosin – negative staining is particularly useful for demonstrating
capsules which unstained against a dark background.
 H & E stain, Periodic acid-Schiff stain – for demonstrating fungi in tissues

Reproduction

 Fungi reproduce by means of spores.

 Fungal spores are of two types, the sexual spores and the asexual spores.

Sexual spores

 Zygospore – when the tips of the approximating hyphae come together and their
contents fuse, developing a large thick-walled body.
 Ascospore – following primary nuclear fission, sexual spores usually 4 to 8 in
number are formed within a sac-like structure or ascus.
 Oospore – developed by the fertilization of the contents of a special female
structure on the mycelium by the nucleus of a male structure developed nearby.
This spore contains an oosphere.
 Basidiospore – four spores developed from the ends of club-shaped structures
called basidium.

Asexual spores

 The medical fungi produce two major types of asexual spores, conidia,
which are produced by most pathogenic fungi, and sporangiospores.
 In some fungi, vegetative cells may transform into conidia (eg, arthroconidia,
chlamydospores). In others, conidia are produced by a conidiogenous cell, such
as a phialide, which itself may be attached to a specialized hypha called a
conidiophore.
 Sporangiospores result from mitotic replication and spore production within a
sac-like structure called a sporangium, which is supported by a sporangiophore..

Growth and isolation of fungi

 Most fungi occur in nature and grow readily on simple sources of nitrogen and
carbohydrate.
 Sabouraud’s agar, which contains glucose and modified peptone (pH 7.0),
has been used because it does not readily support the growth of bacteria.
 To culture medical fungi from non-sterile specimens, antibacterial antibiotics
(eg, gentamicin, chloramphenicol) and cycloheximide are added to the media
to inhibit bacteria and saprobic moulds, respectively.

Classification
Superficial mycoses --- Pityriasis Versicolor, Tinea Nigra and Piedra

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Cutaneous mycoses --- Dermatophytosis
Subcutaneous mycoses --- Mycetoma
Systemic mycoses ---Histoplasmosis
Opportunistic mycoses --- Candidiasis, Cryptococcosis, Aspergillosis, Penicilliosis

Classification based on morphology

1. Mold: Hyphal or mycelial colony or form of growth
2. Yeasts: Unicellular, spherical to ellipsoid fungal cells that usually reproduce by
budding.
3. Yeasts like fungi
4. Dimorphic fungi

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 Figure (1.1) Fungal cell

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Figure (1.2) Fungal cell morphology

Figure (1.3) Examples of asexual spores

Figure (2.1) Pityriasis versicolor & Lesion Figure (2.2) Tinea nigra

Fig (2.3) Trichophyton Fig (2.4) Microsporum Fig (2.5) Epidermophyton

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Figure (4.1) Pseudohyphae & Figure (4.2) Chlamydospore (Red) and budding
yeast of Candida species by germ tube (black) of Candida albicans
Gram stain

Figure (4.3) Indian ink preparation of Figure (4.4) Life cycle of cryptococcosis
Cryptococcus neoformans

Figure (4.5) Aspergillus fumagatus Figure (4.6) Pneumocystis jirovecii

(Lactophenol cotton blue stain) in bronchoalveola

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 GENERAL PARASITOLOGY

Parasite A living organism which receives nourishment and shelter from another
organism where it lives
Host An organism which harbours the parasite

Association of living things which live together

An association which is formed between animals of different species may be divided
into three categories:

Symbiosis: An association in which both are so dependent upon each other that one
cannot live without the help of the other

Commensalism: An association in which the parasite only is deriving benefit without

causing injury to its host.

Parasitism: An association in which the parasite derives benefit and the host gets
nothing in return but always suffers some injury, however slight the injury
may be. The host, at the same time, offers some resistance to the injury
done by the parasite and there may be some adaptation (tolerance)
between parasite and the host

Classes of Parasites
Ecto-parasite (Ectozoa) : Lives outside on the surface of the body of the host
Endo-parasite (Endozoa) : Lives inside the body of the host, in the blood, tissues,
body cavities, digestive tract and other organs
Temporary parasite : Visits its host for a short period
Permanent parasite : Leads a parasitic life throughout the whole period of its
life
Facultative parasite : Lives a parasitic life when opportunity arises
Obligatory parasite : Cannot exist without a parasitic life
Occasional or Accidental parasite : Attack an unusual host
Wandering or Aberrant Parasite : Happens to reach a place where it cannot
live

Classes of Hosts
Definitive Host : Either harbours the adult stage of the parasite or where the
parasite utilizes the sexual method of reproduction

Intermediate Host : Harbour the larval stages of the parasite. In some cases larval
developments are completed in two different intermediate hosts

Paratenic Host (A carrier or transport host): A host where the parasite remains
viable without further development.

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 Sources of infection and portal of entry

Infective stage of the parasite may reach the human body in the following ways

1. By contamination of food or drink

(a) Cysts of Entamoeba histolytica and eggs of Ascaris Iumbricoides which
contaminate the food or drink.
(b) The infective form may remain in the flesh of some intermediate hosts, which are
taken as food, i) Beef containing the larval stage of Taenia saginata (Cysticercus
bovis), (ii) Pork containing the larval forms of Taenium soliurn (Cysticercus
cellulosae) and the larval forms of Trichinella spiralis; (iii) Fish containing the
plerocercoid larvae of Diphylobothrium latum and metacercarial forms of
Clonorcis sinesis; (iv) Crab or crayfish containing metacercarial forms of
Paragonimus westermani
(c) Sometimes the intermediate host harbouring the infective form may be taken up
as a whole, cyclops infected with the larval forms of Dracanculus medinensis are
ingested with water
(d) The infective forms may come out of its intermediate host and encyst in aquatic
plants, eaten as food by man, eg. metacercarial form of Fasiola buski and
F. hepatica.

2. By contamination of the skin or mucous membrane

(a) The filariform larvae of Ancylostoma duodenale, Necator americanus and
Strongyloides stercoralis which abound in damp soil, may penetrate the unbroken
skin of an individual walking over such places bare-footed.
(b) The cercarial form of Schistosoma haematobiurn, S. mansoni and S. japonicum in
infected water, may penetrate the akin of a person coming in contact with such
water.

3. By the agency of insect host

 An infected blood-sucking arthropod may introduce the organism directly into the
blood or into the skin or into the skin layers at the time of obtaining a blood-meal,
e.g., Plasmodia (malarial parasites) by Anopheline mosquitoes, Trypanosoma by
Glossina (tsetse flies), Leishmania by Phlebotomus (sandflies) and Wuchereria by
Culicine mosquitoes.
 In this group, the parasites undergo a biological development for a certain period
before becoming infective to man.

Laboratory diagnosis

 Depending on the nature of the parasitic infections, the following materials should be
collected for specific diagnosis:
1. Blood. In those parasitic infections, where the parasite itself, or in any stage of its
development, circulates in the blood stream, examination of blood film forms one of

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the main procedures for specific diagnosis. Examples are:
 In malaria, the parasites are found inside the erythrocytes (R.B.C.).
 In kala-azar, L. donovani are found inside the monocytes of blood.
 In African sleeping sickness and Chagas' disease, trypomastigotes are found in the
blood plasma.
 In Bancroftian and Malayan filariasis, microfilariae are found in the blood plasma.

2. Stool. Examination of the stool forms an important part in the diagnosis of intestinal
parasitic infections and also for those helminthic parasites which localize in the biliary
tract and discharge their eggs into the intestine.
 In protozoal infections, either trophozoites or cystic forms may be detected; the
former during the active phase and the latter during the chronic phase. Examples are
amoebiasis, giardiasis and balantidiasis.
 In the case of helminthic infections, either the adult worms or their eggs are found in
the stool. Examples are:
 Eggs are found in intestinal helminthiasis (ascariasis, hookworm infection,
trichuriasis, fasciolopsiasis, intestinal schistosomiasis, taeniasis,
diphyllobothriasis, hymenolepiasis and dipylidiasis) and also where the adult
worms inhabit the biliary tract (fascioliasis and clonorchiasis).
 In enterobiasis, eggs are rarely found in the stool because they are deposited
on the perianal skin and hence anal swabs are to be taken for the diagnosis.
 In strongyloidiasis, larvae, not eggs, are commonly present in freshly-passed
stool.
 Adult worms are found in ascariasis and after a vermifuge in hookworm
infection and enterobiasis. Segments of adult worms are found in taeniasis,
diphyllobothriasis and other intestinal tapeworm infections.

3. Urine. When the parasite localises in the urinary tract, examination of the urine will
be of help in establishing the parasitological diagnosis. Examples are:
 In vesical schistosomiasis, terminal-spined eggs of S. haematobium are found.
 In cases of chyluria causcd by W. bancrofti, microfilariae are found.

4. Sputum. Examination of the sputum is useful in the following:

 In cases where the habitat of the parasite is in the respiratory tract, as in
paragonimiisis, the eggs of P. westermani are found.
 In amoebic abscess of lung or in the case of amoebic liver abscess bursting into the
lung, the trophozoites of E. histolytica are detected.
 In case of rupture of hydatid cyst of the lung, scolices and hooklets of E.granulosus
obtained.

5. Biopsy material. It varies with different parasitic infections. For example :

 Spleen puncture in cases of kala-azar.
 Bone marrow puncture in cases of kala-azar and African trypanosomiasis.
 Lymph node puncture in cases of African sleeping sickness and filariasis

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6. Indirect evidences. Changes indicative of internal parasitic infection are:
 Eosinophilia often gives an indication of tissue invasion by a helminth
 Biochemical Alteration of the Blood, Hypergammaglobulinaemia in cases of kala-
azar, African trypanosomiasis, schistosomiasis and visceral larva migrans
 Serological tests
 Intradermal Reaction (Skin Test)

PROTOZOOLOGY

Phylum Protozoa
Protozoal parasite consists of the single “cell-like unit” which is morphologically and
functionally complete. The differences between protozoa and metazoan are as follows :

Protozoa Metazoa
Morphology Unicellular Multicellular
A single “cell-like unit” A number of cells, making
up a complex individual.
Physiology A single cell performs all the functions: Each special cell performs
reproduction, respiration, excretion etc. a particular function.

Morphology

The structure of a protozoal “cell” is composed of 1. a cytoplasmic body and

2. a nucleus
Cytoplasm
 It may be divisible into two portions:
a. Ectoplasm
The external hyaline portion; its function is protective, locomotive and sensory.
b. Endoplasm
The internal granular portion; its function is nutritive and reproductive.

Structures developed from ectoplasm

i. Organelles of locomotion:
a. Pseudopodia : Prolongation of temporary ectoplasmic process,
seen in Rhizopodea.
b. Flagella : Long delicate thread-like filaments, seen in
Zoomastigophorea.
c. Cilia : Fine needle-like filaments covering the entire surface
of the body, seen in Ciliatea.
Speed of locomotion
Amoeba - 0.2 to 3 micrometres (m) per second
Flagellates - 15 to 30 micrometres (m) per second
Ciliatea - 400 to 2,000 micrometres (m) per second
1 micrometre (m) = 0.001 millimetre (mm)

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ii. Contractile vacuoles
situated inside the endoplasm : excretory function

iii. Rudimentary digestive organ

e.g. cytostome. (cell mouth ) and cytopharynx, seen in Balantidium coli.

iv. Cyst-wall
A thickened resistant wall.
Seen in the cystic stage

Nucleus
 Controls the various functions and regulate reproduction. It is situated inside the
endoplasm and its structure helps in the differentiation of genera and species. Its
structure comprises of:
 Bounded externally by a well-defined nuclear membrane
 Made up of a network of linin, enclosing within it the nuclearsap
 Chromatin granules, lining the inner side of the nuclear membrane or appearing
as condensation on linin threads
 Karyosome (palstin) situated inside the nucleus either centrally or peripherally

Kinetoplast, micronucleus and macronucleus

 Most of the protozoa has one nucleus, but in Ciliatea two nuclei are present a small
micronucleus and a large macronucleus.
 Certain protozoa, such as trypanosomatid flagellates, show in addition to the nucleus,
a non-nuclear DNA-containing body called the kinetoplast, near which the flagellum
originates; there is a small body at the point of the origin of the flagellum known as
the basal body.

Encystment
 The protozoal parasite can be transformed from an active (trophozoite) to an inactive
stage, losing its power of motility and enclosing itself within a tough wall. The
protoplasmic body thus formed is known as a cyst. At this stage the parasite loses its
power of growth and multiplication.
 The cyst is the resistant stage of the parasite and is also infective to man.

Reproduction

 The protozoa parasite may exist in two stages : trophozoite and cyst, as in
intestinal flagellates and amoebae.
 The methods of reproduction among the parasitic protozoa are as follows :

1. Asexual multiplications
i. By simple binary fission :
Before division all the structures are duplicated. The individual parasite divides
either longitudinally or transversely into or more less equal parts.

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 ii. By multiple fission or schizogony :
In this process more than two individuals are produced, as in Plasmodia. The
nucleus of the parent cell at first undergoes repeated divisions which are then
surrounded by the cytoplasm. When the multiplication is completed, the parasitic
body or the schizont ruptures and liberated these daughter individuals which in
their turn repeat their life cycle.

2. Sexual reproduction
i. By conjugation :
In this process, a temporary union of two individuals occurs during which time
interchange of nuclear material takes place. Later on, the two individuals
separate, each being rejuvenated by the process, as in Ciliatea.

ii. By syngamy :
In this process, sexually differentiated cells, gametes, united permanently and a
complete fusion of the nuclear material takes place. The resulting product is then
known as a zygote, as in Plasmodia.

Life cycle

 A protozoal parasite may pass its life cycle in one or two hosts.
 Second host-not required. E.g. Rhizopodea, intestinal flagellates and Ciliatea
 Second host-required. E.g. Trypanosoma, Leishmania and Plasmodia

Classification of Protozoa

A. Systematic Classification

Protozoa

Subphylum Plasmodroma Ciliophora

Class Rhizopoda Mastigophora Sporozoa Ciliata

Order Amoebida Protomonadida Diplomanadida Coccidiida Heterotrichida

In intestine In blood & tissue

Genus Entamoeba Chilomastix Trypanosoma Giardia Plasmodium Balantidium

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 Spp E. histolytica C. mesnili T. brucei G .intesti- P. vivax B.coli
E. coli Trichomonas subgroup nails P. falciparum
E. gingivalis T.hominis T. cruzi P. malariae
Endolimax T. vaginalis T. rangeli P. ovale
E. nana Enteromonas Lesihmania Isospora
Iodamoeba E. hominis L.donovani I. hominis
I. butschlii Embadomonas L.tropica I. belli
Dientamoeba E. intestinalis L.brasiliensis Toxoplasma
D. fragilis T. gondii

B. Classification based on pathogenicity

The pathogenicity of the protozoal parasites are divided into two main groups:
i. Pathogenic
ii. Non-pathogenic.

Protozoal parasites pathogenic to man

Subphylum Genus Species Habitat Pathogenic effects

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Sarcomastigophora
Superclass Sarcodina –
Entamoeba E. histolytica Large intestine Dysentery

Superclass Mastigophora
Intestinal Flagellate Giardia G. intestinalis Small intestine Diarrhoea

Genital Flagellate Trichomonas T. vaginalis Vagina Vaginitis

Blood and tissue Trypanosoma T. brucei Blood, Lymph node, African trypanosomiasis
C.N.S (Sleeping sickness)
T. cruzi Heart, South Amecrican trypanosomiasis
Nervous system (Chagas’ disease)

Leishmania L. donovani R.E. System Kala-azar and Dermal

leishmanoid
L. tropica Skin Oriental sore
L. brasiliensis Oro-nasal Espundia
mucous membrane

Sporozoa
Class Telosporea Plasmodium P. vivax R.B.C Benign tertian malaria
P. falciparum R.B.C Malignant tertian malaria
and pernicious malaria
P. malariae R.B.C Quartan malaria
P. ovale R.B.C Ovale tertian malaria

Isospora I. hominis Ep.cells of intes Diarrhoea

I. belli Ep.cells of intes Diarrhoea

Toxoplasma T. gondii R.E system Encephalomyelitis

Parenchyma cell choroidoretinitis, etc.

Cyclospora C. cayentanensis Intestine Cyclosporiasis

Cryptosporidium C. parvum Large intestine Cryptosporidiosis

Ciliophora
Class Ciliatea Balantidium B. coli large intestine Dysentery

Microspora
Enterocytozoon E. bieneusi Epi cells of intestine Microsporidiasis
Encephalitozoon E. intestinalis Epi cells of intestine Microsporidiasis

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 HELMINTHOLOGY

 The helminthic parasites are multicellular, bilaterally symmetrical animals having

three germ layers (triploblastic metazoan). The helminths of importance to human
being are divided into two main groups showing the following characteristic.

HELMINTHS

Phylum Platyhelminthes Phylum-Nemathelminthes

(Class Cestoidea and Trematoda) (Class Nematoda)
Flattened, leaf-like or tape-like and segmented Elongated, cylindrical,
unsegmented bodies
Mostly hermaphrodite (monoecious) Sexes separate (diecious)
Alimentary canal incomplete or entirely lacking Alimentary canal complete
Body cavity absent Body cavity present

Table 1.1 Differences Between Cestodes, Trematodes and Nematodes

CESTODE TREMATODE NEMATODE

Shape: Tape-like, Leaf-like, Elongated, cylindrical
segmented unsegmented unsegmented
Sexes: Not separate, Not separate Separate (diecious)
i.e, (monoecious),except
hermaphrodite Schistosomes which
are diecious
“Head” end. Suckers, often Suckers, no hooks No suckers, no hooks
with hooks. well-developed buccal
capsule in some spps.
Alimentary Absent Present but Present and complete;
canal incomplete; no anus anus present
Body cavity Absent Absent Present

Class : Cestoidea

General characters of cestodes

1. The majority of cestodes are long, segmented and tape-like, hence called
tapeworms. They are flattened dorsoventrally.
2. Sizes vary from a few millimeters to several metres.
3. Adult worms are found in the intestinal canal of man and animal.
4. “Head” is provided with suckers (slit-like or cup-like) and sometimes with hooks
which serve as organs of attachment.
5. There are there regions in an adult worm ;

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 i. a “head” (scolex),
ii. a “neck” and
iii. a strobila (a body or trunk) consisting of a series of segments
(proglottides).
6. Sexes are not separate, i.e. each individual worm is a hermaphrodites.
7. Body cavity is absent.
8. Alimentary canal is entirely absent.
9. Excretory and nervous systems are present.
10. Reproductive system is highly developed and complete in each segment.
According to the maturity of reproductive organs, three types of segments of the
strobila can be recognized from the front backwards.
Immature : Male and female organs not differentiated,
Mature : Male and female organs have become differentiated (male organs
appear first).
Gravid : Uteri are filled with eggs (other organs atrophied or disappeared).

Classification of cestodes infecting man according to habitat

I. Pseudophyllidean cestodes
Possessing false or slit-like grooves (bothria)
1. Adult worms in intestine
Genus - Diphyllobothrium : D. latum (fish tapeworm)
2. Larval stages (Plerocercoid) in man
i. Sparganum mansoni
ii. Sparganum proliferum
II. Cyclophyllidean cestodes
Possessing cup-like and round suckers (acetabula)
1. Adult worms in intestine
i. Genus Taenia : T. saginata (beef tapeworm)
T. solium (pork tapeworm)
ii. Genus Hymenolepis : H. nana (dwarf tapeworm)
H. diminuta (rat tapeworm)
iii. Genus Dipylidium : D. caninum (double pored dog tapeworm)
2. Larval stages in man
i. Genus Echinococcus : Hydatid cyst of E. granulosus (dog tapeworm)
E. multilocularis
ii. Genus Taenia : Cysticercus cellulosae of T. solium
iii. Genus Multiceps : Coenurus cerebralis of M.multiceps and
Coenurus glomeratus of M. glomeratus

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 CLASS : Nematoda
General characters of nematodes

1. The nematodes are unsegmented worms without any appendage. They are
elongated and cylindrical or filiform in appearance: both ends are often pointed.
2. The sizes show a great variation, the smallest (T. spiralis and S. stercoralis).
3. The body is covered with a rough cuticle.
4. The worm possesses a body cavity in which the various organs, such as the
digestive and genital systems, float. Excretory and nervous systems are
rudimentary.
5. The alimentary canal is complete, consisting of an oral aperture, mouth cavity,
oesophagus, intestine and a subterminal anus. The mouth cavity, when present,
may have teeth or cutting plates: in other cases where the mouth cavity is absent,
the oral aperture is directly continuous with the oesophagus.
6. The nematodes of man are all diecious helminthes i.e. the sexis are separate: The
male is smaller than the female and its posterior end is curved or coiled ventrally.
Modes of infection of nematodes

1. By ingestion of
a. Embryonated eggs contaminating food and drinks,
eg. A. lumbricoides, E. vermicularis, T. trichiura.
b. Growing embryos in intermediate host (infected cyclops)
eg. D. medinensis.
c. Encysted embryos in infected pig’s flesh.eg. T. spiralis.
2. By penetration of the skin : - The filariform larvae boring through the skin :
eg. A. duodenale. N. americanus, S. stercoralis
3. By blood-sucking insects, W. bancrofti.
4. By inhalation of infected dust containing embryonated eggs.
eg. A. lumbricoides, E. vermicularis.

Key to the diagnosis of intestinal nematodes

Intestinal Nematodes

Larvae in Stool Eggs in Stool Eggs in Perianal Skin

(S. stercoralis)

Coloured Colourless Colourless

(Bile stained) (Not bile-stained) (Not bile-stained)
1. A. lumbricoides 1. Hookworms E. vermicularis
2. T. trichiura A. duodenale
N. americanus
E. vermicularis
(rarely)

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 Classification of nematodes according to habitat of adult worms

1. Intestinal
Small Intestine Only : Ascaris lumbricodies (Common round worm)
Ancylostoma duodenale (The Old world hookworm)
Necator americanus (American hook-worm)
Strongyloides stercoralis
Trichinella spiralis
Caecum and Vermiform Appendix :
Enterobius vermicularis (Thread worm or pin-worm)
Trichuris trichiura (Whipworm)

2. Somatic (inside the tissues and organs)

Lymphatic system : Wuchereria bancrofti
Brugia malayi
Subcutaneous tissues : Loa loa (African eye worm)
Onchocerca volvulus
Dracunculus medinensis (Guinea worm)
Lungs : Strongylodies stercoralis

Mesentery : Dipetalonema-perstans
Mansonella ozzardi
Conjunctiva : Loa loa
Class : Trematoda

General characters of trematodes

 Flat, leaf like, unsegmented worm (flukes)

 Male, female organs are present (except schistosomes)
 Distomata (oral and ventral suckers)
 Oviparous, operculated eggs (except schistosomes)
 Body cavity - absent
 Alimentary canal - incomplete (no anus)

Anatomical classification

Group Species
Blood flukes - Schistosomes (S. haematobium, S. mansoni, S. japonicum)
Liver flukes - Fasciola hepatica, Clonorchis sinensis
Intestinal flukes - Fasciolopsis buski, Heterophyes heterophyes, Metagonimus
yokogawai
Lung fluke - Paragoniums westermani

Department of Microbiology, UMMG 18

 GENERAL PROPERTIES OF VIRUSES
 Viruses are the small infectious agents (ranging from about 20 nm to 300 nm
in diameter) and contain only one kind of nucleic acid (RNA or DNA) as their
genome.
 The nucleic acid is encased in a protein shell, which may be surrounded by a
lipid-containing membrane.
 The entire infectious unit is termed a virion.
 Viruses are inert in the extracellular environment; they replicate only in living
cells, being parasites at the genetic level.
 The viral nucleic acid contains information necessary for programming the
infected host cell to synthesize virus-specific macromolecules required for the
production of viral progeny.
 During the replicative cycle, numerous copies of viral nucleic acid and coat proteins
are produced.
 The coat proteins assemble together to form the capsid, which encases and
stabilizes the viral nucleic acid against the extracellular environment and facilitates
the attachment and penetration by the virus upon contact with new susceptible cells.

Terms and definitions in virology

 Capsid: The protein shell, or coat that encloses the nucleic acid genome.
 Capsomeres: Morphologic units seen in the electron microscope on the
surface of icosahedral virus particles.
 Defective virus: A virus particle that is functionally deficient in some aspect of
replication.
 Envelope: A lipid-containing membrane that surrounds some virus particles. It is
acquired during viral maturation by a budding process through a cellular membrane.
Virus-encoded glycoproteins are exposed on the surface of the envelope. These
projections are called peplomers.
 Nucleocapsid: The protein–nucleic acid complex representing the packaged form
of the viral genome
 Virion: The complete virus particle. In some instances (e.g. papillomaviruses,
picornaviruses), the virion is identical with the nucleocapsid. In more complex virions
(herpesviruses, orthomyxoviruses), this includes the nucleocapsid plus
a surrounding envelope.

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 Classification of viruses
 The following properties have been used as a basis for the classification of viruses.
 Genomic sequence data are advancing taxonomic criteria (e.g. gene order) and may
provide the basis for the identification of new virus families.
 Virion morphology, including size, shape, type of symmetry, presence or
absence of peplomers, and presence or absence of membranes.
 Virus genome properties, including type of nucleic acid (DNA or RNA), size
of genome in kilobases (kb) or kilobase pairs (kbp), strandedness (single or
double)
 Genome organization and replication
 Virus protein properties
 Antigenic properties.
 Physicochemical properties of the virion
 Biologic properties, including natural host range, mode of transmission, vector
relationships, pathogenicity, tissue tropisms, and pathology.

Universal system of virus taxonomy

 A system has been established in which viruses are separated into major
groupings—called families—on the basis of virion morphology, genome structure,
and strategies of replication.
 Within each family, subdivisions called genera are usually based on biological,
genomic, physicochemical, or serologic differences.
 Genus names carry the suffix -virus. In several families (Herpesviridae,
Paramyxoviridae, Parvoviridae, Poxviridae, Reoviridae, Retroviridae), a larger
grouping called subfamilies has been defined, reflecting the complexity of
relationships among member viruses.
 Virus orders may be used to group virus families that share common
characteristics. For example, order Mononegavirales encompasses the Bornaviridae,
Filoviridae, Paramyxoviridae, and Rhabdoviridae families.
 By 2000, the International Committee on Taxonomy of Viruses had organized
more than 4000 animal and plant viruses into 56 families and 233 genera, with
hundreds of viruses still unassigned. Of these, 24 families contained viruses that
infect humans and animals.

DNA - containing viruses

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 Parvoviruses, Polyomaviruses, Papillomaviruses, Adenoviruses, Hepadnaviruses,
Herpesviruses, Poxviruses

RNA - containing viruses

 Picornaviruses, Astroviruses, Caliciviruses, Hepesviruses, Reoviruses, Arboviruses

and rodent-Borne Viruses, Togaviruses, Flaviviruses, Arenaviruses,
Coronaviruses,Retroviruses, Orthomyxoviruses, Paramyxoviruses, rhabdoviruses,
Filoviruses

Viroids

 Viroids are small infectious agents that cause diseases of plants.

 Viroids are agents that do not fit the definition of classic viruses.
 They are nucleic acid molecules without a protein coat. Plant viroids are single-
stranded, covalently closed circular RNA molecules.
 Viroids replicate by an entirely novel mechanism.
 Viroid RNA does not encode any protein products.
 To date, viroids have been detected only in plants; none have been demonstrated to
exist in animals or humans.

Prions

 Prions are infectious particles composed solely of protein with no detectable nucleic
acid. Prions do not appear to be viruses.
 They are highly resistant to inactivation by heat, formaldehyde, and ultraviolet light
that inactivate viruses.
 The prion protein is encoded by a single cellular gene.
 Prion diseases, called “transmissible spongiform encephalopathies,” include
scrapie in sheep, mad cow disease in cattle, and kuru and Creutzfeldt-Jakob
disease in humans.

Principles of virus structures

 Viruses come in many shapes and sizes. Structural information is necessary for virus
classification and for establishing structure–function relationships of viral proteins.
Viral architecture can be grouped into three types based on the arrangement of
morphologic subunits:
(1) Cubic symmetry (e.g. adenoviruses),
(2) Helical symmetry (e.g. orthomyxoviruses)

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 (3) Complex structures (e.g. poxviruses).

A. Cubic symmetry

 All cubic symmetry observed with animal viruses is of the icosahedral pattern.
 Icosahedron has 20 faces.
 There are exactly 60 identical subunits on the surface of an icosahedron.
 Most viruses that have icosahedral symmetry do not have an icosahedral shape
- rather, the physical appearance of the particle is spherical.
 Both DNA and RNA viral groups exhibit examples of cubic symmetry.

B. Helical symmetry

 In cases of helical symmetry, protein subunits are bound in a periodic way to the viral
nucleic acid, winding it into a helix.
 The filamentous viral nucleic acid - protein complex (nucleocapsid) is then coiled
inside a lipid - containing envelope.
 All known examples of animal viruses with helical symmetry contain RNA genomes
and, with the exception of rhabdoviruses, have flexible nucleocapsids that are wound
into a ball inside envelopes.

C. Complex structures

 Some virus particles do not exhibit simple cubic or helical symmetry but are more
complicated in structure. For example, poxviruses are brick shaped, with ridges on
the external surface and a core and lateral bodies inside.

Measuring the sizes of viruses

 Direct observation in the electron microscope is the most widely used method for
estimating particle size.
 Another method that can be used is sedimentation in the ultracentrifuge. The
relationship between the size and shape of a particle and its rate of sedimentation
permits determination of particle size.
 Viruses range in diameter from about 20 nm to 300 nm.
 Bacterial viruses (bacteriophages) vary in size (10 - 100 nm).

Chemical composition of viruses

Viral protein

 The structural proteins of viruses have several important functions.

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 Their major purpose is to facilitate transfer of the viral nucleic acid from one host cell
to another.
 They serve to protect the viral genome against inactivation by nucleases, participate
in the attachment of the virus particle to a susceptible cell, and provide the structural
symmetry of the virus particle.
 The proteins determine the antigenic characteristics of the virus. The host’s protective
immune response is directed against antigenic determinants of proteins or
glycoproteins exposed on the surface of the virus particle.
 Some surface proteins may also exhibit specific activities (e.g. influenza virus
hemagglutinin agglutinates red blood cells).
 Some viruses carry enzymes (which are proteins) inside the virions.
 The enzymes are essential for the initiation of the viral replicative cycle when the
virion enters a host cell.
 Examples include an RNA polymerase carried by viruses with negative -sense
RNA genomes (e.g. orthomyxoviruses, rhabdoviruses) that is needed to copy the
first mRNAs, and reverse transcriptase, an enzyme in retroviruses that makes a DNA
copy of the viral RNA, an essential step in replication and transformation.

Viral nucleic acid

 Viruses contain a single kind of nucleic acid - either DNA or RNA - that encodes
the genetic information necessary for replication of the virus.
 The genome may be single or double stranded, circular or linear, and segmented or
non-segmented. The type of nucleic acid, its strandedness, and its size are major
characteristics used for classifying viruses into families.
 Viral RNAs exist in several forms. The RNA may be a single linear molecule (e.g.
picornaviruses). For other viruses (e.g. orthomyxoviruses), the genome consists of
several segments of RNA that may be loosely associated within the virion.

Viral lipid envelopes

 A number of different viruses contain lipid envelopes as part of their structure.

 The lipid is acquired when the viral nucleocapsid buds through a cellular membrane
in the course of maturation.
 Budding occurs only at sites where virus - specific proteins have been inserted into
the host cell membrane.
 Lipid - containing viruses are sensitive to treatment with ether and other organic
solvents indicating that disruption or loss of lipid results in loss of infectivity.
 Nonlipid - containing viruses are generally resistant to ether.

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Viral glycoproteins

 Viral envelopes contain glycoproteins. In contrast to the lipids in viral membranes,

which are derived from the host cell, the envelope glycoproteins are virus encoded.
 The surface glycoproteins of an enveloped virus attach the virus particle to a
target cell by interacting with a cellular receptor. They are also often involved in
the membrane fusion step of infection.
 As a result of their position at the outer surface of the virion, they are frequently
involved in the interaction of the virus particle with neutralizing antibody.

Cultivation of viruses

Many viruses can be grown in

 Cell cultures
 Fertile eggs
 Growth of virus in animals is still used for the primary isolation of certain viruses and
for studies of the pathogenesis of viral diseases and of viral oncogenesis.

 There are three basic types of cell cultures. Primary cultures are made by dispersing
cells (usually with trypsin) from freshly removed host tissues. In general, they are
unable to grow for more than a few passages in culture.
 Diploid cell lines are secondary cultures that have undergone a change that allows
their limited culture (up to 50 passages) but that retain their normal chromosome
pattern.
 Continuous cell lines are cultures capable of more prolonged - perhaps indefinite -
growth that have been derived from diploid cell lines or from malignant tissues. They
invariably have altered and irregular numbers of chromosomes.

A. Detection of virus - infected cells

 Multiplication of a virus can be monitored in a variety of ways:
 Development of cytopathic effects (i.e. morphologic changes in the cells). Types of
virus - induced cytopathic effects include cell lysis or necrosis, inclusion formation,
giant cell formation, and cytoplasmic vacuolization
 Appearance of a virus - encoded protein, such as the hemagglutinin of influenza
virus. Specific antisera can be used to detect the synthesis of viral proteins in infected
cells.
 Detection of virus - specific nucleic acid. Molecular - based assays such as
polymerase chain reaction provide rapid, sensitive, and specific methods of detection.
 Adsorption of erythrocytes to infected cells, called hemadsorption, caused by the
presence of virus - encoded hemagglutinin (parainfluenza, influenza) in cellular
membranes.

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 Viral growth in an embryonated chick egg may result in death of the embryo (e.g.
encephalitis viruses), production of pocks or plaques on the chorioallantoic
membrane (e.g. herpes, smallpox, vaccinia), or development of hemagglutinins in the
embryonic fluids or tissues (e.g. influenza).

B. Inclusion body formation

 In the course of viral multiplication within cells, virus specific structures called
inclusion bodies may be produced.
 They become far larger than the individual virus particle and often have an affinity for
acid dyes (e.g. eosin).
 They may be situated in the nucleus (herpesvirus), in the cytoplasm (poxvirus), or in
both (measles virus).
 In many viral infections, the inclusion bodies are the site of development of the
virions (the viral factories).
 The presence of inclusion bodies may be of considerable diagnostic aid.
 The intracytoplasmic inclusion in nerve cells - the Negri body - is pathognomonic for
rabies.
Reaction to physical and chemical agents

Heat and cold

 There is great variability in the heat stability of different viruses.
 Icosahedral viruses tend to be stable.
 Enveloped viruses are much more heat labile.
 Viral infectivity is generally destroyed by heating at 50 - 60.C for 30 minutes, although
there are some notable exceptions (e.g. hepatitis B virus, polyomaviruses).
 Viruses can be preserved by storage at subfreezing temperatures, and some may
withstand lyophilization.

Stabilization of viruses by salts

 Viruses are preferentially stabilized by certain salts.
 The stability of viruses is important in the preparation of vaccines.
 With the addition of salts for stabilization of the virus, potency can be maintained for
weeks at ambient temperatures even in the high temperatures of the tropics.

PH
 Viruses are usually stable between pH values of 5.0 and 9.0.
 Some viruses (e.g. enteroviruses) are resistant to acidic conditions.
 All viruses are destroyed by alkaline conditions.

Radiation
 Ultraviolet, x - ray, and high-energy particles inactivate viruses.

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Ether susceptibility
 Enveloped viruses are sensitive to ether.
 Ether susceptibility can be used to distinguish viruses that possess an envelope
from those that do not.

Detergents
 Nonionic and anionic detergents solubilize lipid constituents of viral membranes.

Formaldehyde
 Formaldehyde destroys viral infectivity by reacting with nucleic acid.
 Formaldehyde has minimal adverse effects on the antigenicity of proteins and
therefore has been used frequently in the production of inactivated viral vaccines

Photodynamic inactivation
 Viruses are penetrable to a varying degree by vital dyes such as toluidine blue,
neutral red and proflavine.
 These dyes bind to the viral nucleic acid, and the virus then becomes susceptible
to inactivation by visible light.

Antibiotics and other antibacterial agents

 Antibacterial antibiotics and sulfonamides have no effect on viruses.
 Larger concentrations of chlorine are required to destroy viruses than to kill bacteria,
especially in the presence of extraneous proteins.

Viral replication

 A variety of different viral strategies have evolved for accomplishing multiplication in

parasitized host cells.

(1) Attachment, penetration and uncoating

 The first step in viral infection is attachment, interaction of a virion with a specific
receptor site on the surface of a cell.
 Receptor molecules differ for different viruses but are generally glycoproteins.

(2) Expression of viral genomes and synthesis of viral components

 The synthetic phase of the viral replicative cycle ensues after uncoating of the viral
genome.
 The essential theme in viral replication is that specific mRNAs must be transcribed
from the viral nucleic acid for successful expression and duplication of genetic
information.
 After this is accomplished, viruses use cell components to translate the mRNA.

(3) Morphogenesis and release

 Newly synthesized viral genomes and capsid polypeptides assemble together to form
progeny viruses.

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 In general, nonenveloped viruses accumulate in infected cells, and the cells
eventually lyse and release the virus particles.
 Enveloped viruses mature by a budding process. Virus specific envelope
glycoproteins are inserted into cellular membranes; viral nucleocapsids then bud
through the membrane at these modified sites and in so doing acquire an envelope.
 Budding frequently occurs at the plasma membrane but may involve other
membranes in the cell.
 Excess amounts of viral components may accumulate and be involved in the
formation of inclusion bodies in the cell.
 As a result of the profound deleterious effects of viral replication, cellular cytopathic
effects eventually develop and the cell dies.

Modes of transmission of viruses

Viruses may be transmitted in the following ways:
 By droplet or aerosol infection (e.g. influenza, measles, smallpox);
 By sexual contact (e.g. papillomavirus, hepatitis B, herpes simplex type 2, human
immunodeficiency virus);
 By hand - mouth, hand - eye, or mouth - mouth contact (e.g. herpes simplex,
rhinovirus, Epstein - Barr virus);
 By exchange of contaminated blood (e.g. hepatitis B, human immunodeficiency
virus).
 By the fecal - oral route (e.g. enteroviruses, rotaviruses, infectious hepatitis A)
 By formites (e.g. Norwalk virus, rhinovirus).
 By means of an arthropod vector (e.g. arboviruses, togaviruses, flaviviruses, and
bunyaviruses).

Transmission from animal to animal, with humans an accidental host.

 By bite (rabies)
 By droplet or aerosol infection from rodent-contaminated quarters
(e.g. arenaviruses, hantaviruses).

Pathogenesis of viral diseases

A. Entry and primary replication

 For host infection to occur, a virus must first attach to and enter cells of one of
the body surfaces - skin, respiratory tract, gastrointestinal tract, urogenital tract, or
conjunctiva.
 Most viruses enter their hosts through the mucosa of the respiratory or
gastrointestinal tract.
 Some viruses are introduced directly into the bloodstream by needles (hepatitis B,
human immunodeficiency virus (HIV), by blood transfusions, or by insect vectors
(arboviruses).

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 Viruses usually replicate at the primary site of entry.
 Some, such as influenza viruses (respiratory infections) and noroviruses
(gastrointestinal infections), produce disease at the portal of entry.


B. Viral spread and cell tropism

 Many viruses produce disease at sites distant from their point of entry (eg,
enteroviruses, which enter through the gastrointestinal tract but may produce
central nervous system [CNS] disease).
 The most common route of spread is via the bloodstream or lymphatics.
 The presence of virus in the blood is called viraemia which is usually short.
 In some instances, neuronal spread is involved (rabies virus and herpes simplex
virus)

C. Cell injury and clinical illness

 Destruction of virus-infected cells in the target tissues and physiologic alterations

produced in the host by the tissue injury are partly responsible for the
development of disease.

D. Recovery from infection

 The host either succumbs or recovers from viral infection.

 Recovery mechanisms include both innate and adaptive immune responses.
Interferon (IFN) and other cytokines, humoral and cell-mediated immunity, and
possibly other host defense factors are involved.
 In acute infections, recovery is associated with viral clearance.

E. Virus shedding

 Shedding usually occurs from the body surfaces involved in viral entry.
 Shedding occurs at different stages of disease depending on the particular agent
involved.
 In some viral infections, such as rabies, humans represent dead-end infections, and
shedding does not occur.

Viral persistence
Chronic and latent virus

 Chronic infections (also called persistent infections) are those in which replicating
virus can be continuously detected, often at low levels; mild or no clinical symptoms
may be evident.

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 Infants infected with hepatitis B virus frequently become persistently infected (chronic
carriers); most carriers are asymptomatic.
 In chronic infections with RNA viruses, the viral population often undergoes many
genetic and antigenic changes.
 Latent infections are those in which the virus persists in an occult (hidden or
cryptic) form most of the time when no new virus is produced. There will be
intermittent fare - ups of clinical disease; infectious virus can be recovered during fare
- ups.
 Herpesviruses and Chickenpox virus (varicella-zoster) produce latent infections.
 Persistent viral infections are associated with certain types of cancers in humans as
well as with progressive degenerative diseases of the CNS of humans.

Prevention and treatment of viral infections

Antiviral chemotherapy

 Because viruses are obligate intracellular parasites, antiviral agents must be capable
of selectively inhibiting viral functions without damaging the host, making the
development of such drugs very difficult.
 Antivirals can be used to treat established infections when vaccines would not be
effective.

A. Nucleoside and nucleotide Analogs

 Nucleoside analogs inhibit nucleic acid replication by inhibition of polymerases

essential for nucleic acid replication. In addition, some analogs can be
incorporated into the nucleic acid and block further synthesis or alter its
function.
 The use of combinations of antiviral drugs can delay the emergence of resistant
variants (e.g. “triple drug” therapy used to treat HIV infections).
 Examples of nucleoside analogs include acyclovir (acycloguanosine), lamivudine
(3TC), ribavirin, vidarabine (adenine arabinoside), and zidovudine (azidothymidine;
AZT).
 Nucleotide analogs differ from nucleoside analogs in having an attached
phosphate group. Their ability to persist in cells for long periods of time increases
their potency. (e.g. Cidofovir)

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B. Reverse transcriptase inhibitors

 Nevirapine was the first member of the class of nonnucleoside reverse transcriptase
inhibitors.
 It acts by binding directly to reverse transcriptase and disrupting the enzyme’s
catalytic site.
 Resistant mutants emerge rapidly.

C. Protease inhibitors

 Saquinavir was the first protease inhibitor to be approved for treatment of HIV
infection
 Such drugs inhibit the viral protease that is required at the late stage of the replicative
cycle.
 Protease inhibitors include indinavir and ritonavir and others.

D. Other types of antiviral agents

 Fuzeon is a large peptide that blocks the virus and cellular membrane fusion step
involved in entry of HIV - 1 into cells.
 The synthetic amines amantadine and rimantadine specifically inhibit influenza A
viruses by blocking viral uncoating.
 Foscarnet (phosphonoformic acid) inhibits HSV replication.
 Methisazone is of historical interest as an inhibitor of poxviruses. It was the first
antiviral agent to be described and contributed to the campaign to eradicate
smallpox.

E. Interferons

 Interferons are host - coded proteins that are members of the large cytokine family
and that inhibit viral replication.
 They are produced very quickly (within hours) in response to viral infection or other
inducers and are one of the body’s first responders in the defense against viral
infection.
 IFN was the first cytokine to be recognized.
 IFNs are central to the innate antiviral immune response.
 There are multiple species of IFNs that fall into three general groups, designated IFN
- α, IFN - β, and IFN - γ. Both IFN - α and IFN - β are considered type I or viral IFNs;
IFN - γ is type II or immune IFN.
 IFN - α and IFN - β are resistant to low PH.

F. Viral vaccines

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