ARTIKEL PENELITIAN

Toxoplasma gondii Identification in Mothers Blood

and Fetal Tissue with Nested PCR
Aloysius Suryawan1, Juliana A. Masengi-Rumopa 2, Josef S. B. Tuda3,
Theresia M. Rahardjo4, Mihoko Imada5
1Department

of Obstetrics and Gynecology,Maranatha Christian University / Immanuel

Hospital Bandung,2Department of Obstetrics and Gynecology, General Hospital Manado,
3Department of Parasitology, 4Department of Biology, Sam Ratulangi University, Manado,
Indonesia and, 5 JICA (Japanese International Cooperation Agency) Manado, Indonesia.

Abstrak
Toxoplasma gondii identification in mothers blood and fetal tissue with nested PCR
Objective: To examine the correlation between Toxoplasma gondii infection with spontaneous
abortion on pregnant women based on nested PCR result from mothers blood and fetal tissue.
Methods: A prospective clinical diagnostic study using nested PCR performed on 30
cases of pregnant women with spontaneous abortion fulfilling the inclusion criteria, latex
agglutination test (+) and exclusion criteria, latex agglutination test (-). Mothers blood and
fetal tissues samples, which gave positive result in serologic test, were analyzed with nested
PCR using18S-rDNA gene primers.
Results: Five of 30 mothers blood samples (16.7%) and 9 of 30 fetal tissue samples
(30%) gave positive PCR results. According to Fishers Exact test, PCR detected the presence
of Toxoplasma gondii in significant value (P < .001). Conclusion: There is a strong correlation
between Toxoplasma gondii infection with spontaneous abortion (P < 001).
Keywords: nested PCR Toxoplasma gondii infection spontaneous abortion

JKM.
Vol. 5, No. 2, Februari 2006

asymptomatic or silent infection,

will limit and cause difficulty in
the diagnosis process so the
abnormality or mortality of the
fetus will be increased.4,5,6,7
Current diagnosis of Toxoplasma gondii is based on parasite
isolation and serological assay.
Toxoplasma gondii can be isolated by
mice inoculation or tissue cul-ture
but this technique need longer time,
about 3-6 weeks. Serological test
can overcome this problem and
detect Toxoplasma gondii anti-body.
Positive serologic result was
determined by showing a seroconversion of immunoglobulin G antibodies for primary infection and
detection of specific immunoglobulin M. This method is also timeconsuming and complicated by the
presence of crossreactive antibodies, and influenced by immunology condition, especially in
immunosuppression or immunodeficiency patients.8,9,10,11,12,13
Based on this fact, PCR
becomes a very important diagnostic tool because it has a very
high sensitivity and specificity in
detecting Toxoplasma gondii infection, both in acute and chronic
stage. One of the PCR methods
which has very high sensitivity and
accuracy is a nested PCR using
primer which is very conserved
and species-specific14, like 18SrDNA primers used in this study.
The PCR is performed both on
mothers blood and aborted tissue
and the result can be performed in
a few hours.15-22

Toxoplasma gondii is an obligate intracellular protozoan parasite that can infect an extremely
wide host range, from birds to
mammals, including humans. This
parasite can survive in all nucleated cells, including blood cells in
acute stage, forms a specific
vacuole that protect the parasite
from host cell immune system. In
the chronic stage, the parasite can
form a cyst in the central nervous
system, skeletal muscle and eye
tissue and can exist for the lifetime
of its host. The cysts can rupture
and
release
highly
invasive
trophozoite, which may cause a
recurrent infection and potentially
fatal if the host is in a state of
immune deficiency.1,2,3
Toxoplasma gondii infection is
often asymptomatic in healthy individuals, but a primary Toxoplasma gondii infection in pregnant
women may cause a range of
abnormalities including abortion,
fetal death and congenital defect,
depending on gestation age when
infection occurs. The fetus is
infected by Toxoplasma gondii
through placental circulation. The
cyst can form in placental tissue
and fetal brain. When the fetus is
infected at first trimester, abortion
can occur. In Norway between 1992
to 1994, 10.9% women were
infected before pregnancy and
0.17% were infected during pregnancy. In Indonesia, toxoplasmosis
prevalence is 14% and it is still high
in pregnant women, about 5.5-84%.
The high score of toxo-plasmosis in
pregnant
women,
especially

Toxoplasma gondii Identification in Mothers Bloodand Fetal Tissue with Nested PCR
Aloysius Suryawan, Juliana A. Masengi-Rumopa, Josef S. B. Tuda, Theresia M. Rahardjo, Mihoko Imada

produces a 290-bp DNA segment

from base 58 to 348. The first
primer
pair
used
are
5'CCATGCATGTCTAAGTATAA GC
and
5'-GTTACCCGTCACTG
CCAC. The second primer used are
5'-CTAAGTATAAGCTTTTATACG
GC and 5'-TGCCACGGTAGTCC
AATAC.
Preparation of DNA template for PCR. DNA templates were prepared from mothers blood
and fetal tissue. Fifty l of each
sample was putt into 2.5 ml a
Eppendorf tube and 100 l Triton
X-100 was added into the tube.
Then the tube was boiled for 5
minutes and stored at -20oC until
used. This technique was used in
Keio University Japan and has
proven to be successful in PCR
process.
Amplification protocol. The
reagents
used
for
PCR
amplification were given from Keio
University Japan. PCR mix-tures
were prepared for 50 l react-ion
volume as following: distilled
water 33.25 l, 10xPCR buffer 5 l,
2mM dNTPs 5l, 25 mM MgCl2 4l,
each primer 0.5l, Taq poly-merase
0.5l and DNA template 1.5 l.
DNA thermal cycler (Perkin-Elmer)
was programmed for 40 cycles of
amplification, both for first and
second round PCR. Parameters for
the first round PCR cycle consisted
of 5 min at 95oC (initial
denaturation), 30 sec at 94oC
(denaturation), 1 min at 64oC
(primer annealing), 2 min at 72oC
(polymerization) and 5 min at 72oC

Materials And Methods

Patients characteristic and
selection. (i) Mothers. All pregnant
women with spon-taneous abortion
within 20 weeks of gestation from
October 2002 to March 2003 were
identified and characterized by age,
gestational
age,
spontaneous
abortion history and parity. They
were offered serological test
(agglutination test) and suggested
to perform PCR examination both
on mothers blood and fetal tissue if
serological test gives positive result.
After the informed consent was
granted, 5 cc venous blood was
drawn and centrifuged to separate
its serum from blood, then the
serum was serologically tested.
Thirty preg-nant women with
abortion and whose gave positive
result in serologically test were
included in this study. The remains
of blood samples were preserved at
-20oC for subsequent DNA isolation
step. (ii) Fetal tissue. Five cc fetal
tissues from all serologic positive
mothers were taken and preserved
at -20oC for subsequent DNA
isolation step.22,23,24
Selection of Primer. Primer
used for this nested PCR will
amplify a DNA sequence from 18SrDNA gene. The primer was used
in Keio University, Japan and proven to give positive result in HIVpositive
individuals
with
encephalitis toxoplasmosis. The
first primer pair produces a 311-bp
DNA segment from base 48 to 359
and the second primer pair

JKM.
Vol. 5, No. 2, Februari 2006

only 1 positive case from fetal

tissue on women aged over 40.
(Table 2)
On gestational age, the
largest positive cases were found at
> 8-12 weeks pregnancy, with 4
cases (13.33%) from mothers blood
and 8 cases (26.67%) from fetal
tissue. Four positive cases from
mothers blood consisted of each 2
cases at 8-10 and 10-12 weeks
pregnancy, all of them showed
positives result in their fetal tissue
samples. Eight positive cases from
fetal tissue consisted of each 4
cases at 10-12 and 8-10 weeks
pregnancy. At gestational age
below 8 weeks, we found 1 positive
result (3.33%), both on mothers
blood and fetal tissue, but there are
no positive results at > 12-16 and >
16-20 weeks pregnancy. (Table 3)
According to spontaneous
abortion history, women with first
spontaneous abortion history gave
the largest positive results with 5
positive cases (16.67%) from mothers blood and 7 positive cases
(23.33%) from fetal tissue, 5 positive
results occurred both on mothers
blood and their fetal tissues. Each
followed by 1 posi-tive case from
fetal tissue for women with 2-3
times and more of 3 times
spontaneous
abortion
history.
(Table 4)
All data were analyzed with
Fischers Exact Test and showed
significant relationship (P < .001,
95% CI) between fetal tissue and
mothers blood examination by
nested PCR.

(extended polymerization). Parameters for the second round PCR

consisted of 5 min at 95oC (initial
denaturation), 30 sec at 94oC (denaturation), 1 min at 60oC (primer
annealing), 2 min at 72oC (polymerization) and 5 min at 72oC
(extended polymerization). An
aliquot of the reaction mixture was
electrophoresis analyzed by 2%
agarose gel stained by 2 l
ethidium bromide and visualized
under UV light. A UV Camera was
used to take its photograph using
a 559 Polaroid film.
Result
Thirty pregnant women
with spontaneous abortion within
20 weeks of gestation, characterized by age, gestational age and
spontaneous abortion history, who
gave positive result in serological
test were submitted to nested PCR.
Nested PCR showed 5 positive
results from mothers blood and 9
positive results from fetal tissue.
(Figure 1, 2 and 3)
Based on PCR result, women at age 20-29 had the largest
positive cases with 2 cases (6.67%)
from mothers blood and 3 cases
(10%) from fetal tissue, including a
positive case both on mothers
blood and its fetal tissue. Women at
age below 20 showed 4 positive
cases, each 2 cases from mothers
blood and their fetal tissue (6.67%).
In women aged 30-39, there were
also 4 positive cases, 1 case (3.33%)
from mothers blood and 3 cases
(10%) from fetal tissue. There was

Toxoplasma gondii Identification in Mothers Bloodand Fetal Tissue with Nested PCR
Aloysius Suryawan, Juliana A. Masengi-Rumopa, Josef S. B. Tuda, Theresia M. Rahardjo, Mihoko Imada

obtained in women at age below 20

with 4 positive results, each 2
positive results (6.67%) from mothers blood and fetal tissue
(samples 26 and 30). In women
aged 30-39, there were also 4
positive results, 1 positive result
(3.33%) from mothers blood (sample 24) and 3 positive results (10%)
from fetal tissue (sample 11, 15 and
24). In older women over 40, only 1
positive result (3.33%) was found
from fetal tissue (sample 12).
According to this, we can see that
the highest Toxoplasma gondii
infection occurs especially in active
reproductive women, aged between 16 to 39 and the incidence of
infection decreases with age
increase. It gives a perception that
there is a lower toxoplasmosis
incidence in older patients (in this
study older than 40) because older
patients have a bigger chance to get
a contact with Toxoplasma gondii
which gives a higher anti-body
(IgG) protection to recurrent
infection. This assumption still
need further study with larger
subject sample with ELISA assay as
a quantitative screening tool. ELISA
can give more accurate result than
agglutination test be-cause it can
describe IgG antibody level, which
gives protection to recurrent
infection.25,26
According to gestational
age, the largest positive result was
found in gestational age > 8-12
weeks, with 4 positive cases
(13.33%) from mothers blood and 8
positive cases (26.67%) from fetal
tissue. Four positive cases from

Discussion
This study has some advantages. First, it describes the characterization of women with spontaneous abortion, which were
infected by Toxoplasma gondii and
the prevalence of toxoplasmosis at
Malalayang
General
Hospital
(RSUP) Manado, North Sulawesi,
Indonesia. Second, it shows the
significant advantage of nested
PCR to detect Toxoplasma gondii in
mothers blood and fetal tissue and
estimate the stage of Toxoplasma
gondii, whether it is in acute or
chronic form. Third, the study also
shows the correlation between fetal
tissue and mothers blood PCR
result. Beside some ad-vantages
above, there is also a possibility
that
patients
with
positive
serological result had no full
protection because they still had a
chance to get toxoplasmosis.
Based on the result, we can
describe the pattern of Toxoplasma
gondii infection in women with
spontaneous abortion at Manado,
who had a serological positive
result, according to age, gestational
age, spontaneous abortion history
and parity.
The result showed that from
30 pregnant women with spontaneous abortion, women at age 2029 had the higher risk for toxoplasmosis with 2 positive result
(6.67%) from mothers blood
(samples 5 and 21) and 3 positive
result (10%) from fetal tissue
(samples 5, 20 and 21). The lower
incidence of toxoplasmosis was

JKM.
Vol. 5, No. 2, Februari 2006

positive cases (23.33%) from fetal

tissue (sample 5, 11, 20, 21, 24, 26
and 40). We found only 1 positive
case from fetal tissue (sample 15)
from mother with more than 3 times (5 times) spontaneous abortion history. Some previous studies
using serology method suggested
that it still need some further studies with larger sample in toxoplasmosis patients with recurrent
abortion.12,25
In this study, there are 5
positive cases on mothers blood
(16,67%) and 9 positive cases on
fetal tissue (30%). In fact, those 5
positive cases from mothers blood
also give positive result in their
fetal tissue samples and the
remaining 4 positive cases only
occurred on fetal tissue. There are
some possibilities that could cause
this result as following:
1. Late phase of acute infection
(early phase of chronic infection)
In this phase, the antibody already increased, reached the
peak state and started to going
down. The higher level of antibody will give a positive result
in serology test. The positive
result in mothers blood shows
that the parasite still is present
in circulation, while in trophozoite or bradyzoite form. Trophozoite is the acute form of
Toxoplasma gondii while bradyzoite is the chronic from of
the parasite and evolve after the
parasite penetrates the white
blood cell and forms a
protective specific vacuole. The

mothers blood consisted of sample 21 and 30 (8-10 weeks) and 24

and 26 (10-12 weeks), while 8
positive cases from fetal tissue
consisted of sample 11, 12, 24 and
26 (10-12 weeks) and sample 15, 20,
21 and 30 (8-10 weeks). We only
found 1 positive case (3.33%) in
gestational age < 8 weeks (sample
5), both on mothers blood and fetal
tissue and no positive result at
gestational age over 12 weeks. This
result shows that the highest
spontaneous abortion occurred at 812 weeks gestational age, which
becomes the most criti-cal period. It
support the suggest-ion made in
some previous studies, which used
serological
assay,
that
toxoplasmosis has a significant
correlation
with
spon-taneous
abortion in the first trimes-ter of
pregnancy and the rate of
spontaneous abortion will increase
if toxoplasmosis occurs at early
gestational age.27,28,29 The correlation is so strong because the overall positive result for mothers
blood were 5 cases (16.66%) and 9
cases for fetal tissue (30%). Further
study with larger sample is needed
to support the evidence based
medicine principle.
Beside age and gestational
age, spontaneous abortion history
and parity were some parameters
we used. In spontaneous abortion
history, the highest number of
positive cases was on women with
first spontaneous abortion history,
consisting of
5 positive cases
(16.67%) from mothers blood
(sample 5, 21, 24, 26 and 40) and 7

Toxoplasma gondii Identification in Mothers Bloodand Fetal Tissue with Nested PCR
Aloysius Suryawan, Juliana A. Masengi-Rumopa, Josef S. B. Tuda, Theresia M. Rahardjo, Mihoko Imada

ed that the parasite only presented

in cyst form and there are no
trophozoite and bradyzoite in
systemic circulation.
Nested PCR method used in
this study eliminated false positive
possibility because it has a better
sensitivity and specificity than an
ordinary PCR method. This study
has no
comparable stu-dies
because there were no pre-vious
published studies that used nested
PCR to detect Toxoplasma gondii in
blood and tissue. This study found
a quite higher positive result, 16.7%
positive result was found in
mothers blood and 30% in fetal
tissue. There is only one other
similar study titled Identifi-cation
of Toxoplasma gondii B1 gene with
PCR in aborted fetus preserv-ed in
formalin, conducted by M. Assmar
et al from Teheran, Iran, published
in Irn J Med Sci 2000; 25 (1&2): 5961. This study found a 20% positive
result in fetal tissue.23

positive result on fetal tissue

shows that the parasite already
penetrated fetal tissue, in this
case placenta, and is followed
by cyste formation
causing
abortion to occur.30,31,32
2. Toxoplasmosis reinfection
Some literature mentioned that
Toxoplasma
gondii
infection
gives a total protection to a new
infection. Based on the re-sult
above, especially on 5 posi-tive
cases on mothers blood and
fetal tissue, there is a possibility
that the protection does work
but not totally. This gives a
chance for a new infect-ion to
occur.
The
previous
Toxoplasma gondii infection was
shown by the cyst form in
placenta that gives a positive
result on fetal tissue while the
new infection was shown by the
presence of the parasite in
blood circulation giving a
positive result on mothers
blood.3,12,23,24,27
3. Toxoplasmosis infection relapse
The parasite cyst in tissue can
rupture and release highly invasive trophozoite into blood
circulation. This situation develops in patients with decreasing immune
reaction. Cyst
already formed in tissue is
shown by positive result in fetal tissue and newly developing trophozoite in circulation
is shown by positive result in
mothers blood.2,3,35
The remaining 4 positive cases
occuring only in fetal tissue show-

Summary
Based on the result, we noticed that the incidence of Toxoplasma gondii infection in women
with spontaneous abortion is very
high at RSUP Manado. From 30
patients, there are 5 positive cases
from mothers blood (16,67%) and 9
positive cases (30%) from fetal
tissue that suggested a strong
correlation between abortion in
early pregnancy with toxoplasmosis, evidence by a very high
positive PCR result in fetal tissue at
early gestational rate.

JKM.
Vol. 5, No. 2, Februari 2006

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problem is the serological test used
to detect IgG antibody which was
not a quantitative method. Based
on those problems, further study
with more subjects and larger sample and more sensitive quantitative
method like ELISA combined with
PCR is needed. 12,36-38

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Acknowledgments
This research was supported by Keio University Japan and
JICA (Japan International Cooperation Agency).
We thank all the people
involved in this research especially
Maeda, MD from Keio University
Japan. We also thank the Parasitology Department, Medical Faculty,
Sam Ratulangi University Manado,
for research facilitation including
place, materials and equipment.

Toxoplasma gondii Identification in Mothers Bloodand Fetal Tissue with Nested PCR
Aloysius Suryawan, Juliana A. Masengi-Rumopa, Josef S. B. Tuda, Theresia M. Rahardjo, Mihoko Imada

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http://path.upmc.edu/divisions/neur
opath/bpath/cases/case50.html
accessed on January 15, 2003.
Wishnuwardhani
SD.
Penyakit
Menular. Dalam: Wiknjosastro H, ed.
Ilmu Kebidanan. Edisi 3. Yayasan Bina Pustaka Sarwono Prawirohardjo,
Jakarta. 1999; 38: 551-77.
Diagnosis and Follow-up of Toxoplasmosis Reactivation After Allogeneic
Stem Cell Transplantation Using Fluorescence Resonance Energy Transfer
Hybridization Probes. J of Clin
Microbiol, August 2000; 38(8): 2929-32.
Gandahusada S. Faktor-faktor Lingkungan yang Mempengaruhi Timbulnya Toksoplasmosis di Dalam Masyarakat. Bagian Parasitologi FKUI. Medika, Juni 1990; Nomor 6 Tahun 16:
485-9.

Toxoplasma gondii Identification in Mothers Bloodand Fetal Tissue with Nested PCR
Aloysius Suryawan, Juliana A. Masengi-Rumopa, Josef S. B. Tuda, Theresia M. Rahardjo, Mihoko Imada

APPENDIX
TABLE 1. Primers used for first and second round PCR.
Primer

Sequence

First primer

5'-CCATGCATGTCTAAGTATAAGC-3'
5'-GTTACCCGTCACTGCCAC-3'

Second primer

5'-CTAAGTATAAGCTTTTATACGGC-3'
5'-TGCCACGGTAGTCCAATAC-3'

TABLE 2. PCR result for women with spontaneous abortion characterized by age.
No

Age (y)

Latex

PCR

Agglutination

Mothers blood

Fetal tissue

(+)

(+)

(+)

< 20

13.33

6.67

6.67

20 29

13

43.33

6.67

10.00

30 39

26.67

3.33

10.00

> 40

16.67

0.00

3.33

Total

30

100.00

16.67

30.00

11

JKM.
Vol. 5, No. 2, Februari 2006

TABLE 3. PCR result for women characterized by gestational age.

No

Gestational
age (weeks)

Latex

PCR

Agglutination

Mothers blood

Fetal tissue

(+)

(+)

(+)

<8

6.67

3.33

3.33

> 8 12

21

70.00

13.33

26.67

> 12 16

23.33

0.00

0.00

> 16 20

0.00

0.00

0.00

Total

30

100.00

16.67

30.00

TABLE 4. PCR result for women characterized by spontaneous abortion history.

No

Spontaneous
abortion history

Latex

PCR

Agglutination

Mothers blood

Fetal tissue

(+)

(+)

(+)

01

27

90.00

16.67

23.33

23

6.67

0.00

3.33

>3

3.33

0.00

3.33

Total

30

100.00

16.67

30.00

12

Toxoplasma gondii Identification in Mothers Bloodand Fetal Tissue with Nested PCR
Aloysius Suryawan, Juliana A. Masengi-Rumopa, Josef S. B. Tuda, Theresia M. Rahardjo, Mihoko Imada

TABLE 5. Data analysis with Fischers Exact Test.

PCR of Mothers blood
Negative
PCR of Fetal
tissue

Negative

Positive

Total

Count

Total

Positive

21

21

% PCR JA

100,0%

100,0%

% PCR DI

84,0%

84,0%

% Total

70,0%

70,0%

Count

% PCR JA

44,4%

55,6%

100,0%

% PCR DI

16,0%

100,0%

30,0%

% Total

13,3%

16,7%

30,0%

25

30

% PCR JA

83,3%

16,7%

100,0%

% PCR DI

100,0%

100,0%

100,0%

% Total

83,3%

16,7%

100,0%

Count

13

JKM.
Vol. 5, No. 2, Februari 2006

TABLE 6. PCR and Serological Test Result from All Samples

Patient no.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
FT
MB
MS

Age (y)
29
20
25
32
27
35
20
40
18
46
35
41
31
23
32
28
29
24
21
23
25
16
39
35
40
19
30
40
20
16

Gestation age
(week)
8-10
10-12
14-16
10-12
7-8
10-12
10-12
12-14
8-10
12-14
12-14
10-12
8-10
8-10
8-10
8-10
8-10
8-10
8-10
8-10
8-10
10-12
6-8
10-12
12-14
10-12
10-12
14-16
12-14
8-10

Parity
P1A1
P0A1
P1A1
P1A1
P1A1
P2A1
P1A1
P3A1
P0A1
P2A2
P2A1
P4A5
P1A1
P1A1
PIA2
P1A1
P0A1
P1A1
P1A1
P0A1
P1A1
P0A1
P4A1
P4A1
P3A1
P0A1
P1A1
P2A1
P0A1
P0A1

: Fetal tissue
: Mothers blood
: Mother serum

14

Serologic Test
MS
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

PCR
FT
+
+
+
+
+
+
+
+
+

MB
+
+
+
+
+

Toxoplasma gondii Identification in Mothers Bloodand Fetal Tissue with Nested PCR
Aloysius Suryawan, Juliana A. Masengi-Rumopa, Josef S. B. Tuda, Theresia M. Rahardjo, Mihoko Imada

Figure 1 and 2. Positive result of nested PCR from fetal tissue.

Figure 1 (left) showed 4 positive result from sample 11, 12, 15 and 20 whenever figure
2 (right) showed 5 positive result from sample 5, 21, 24, 26 and 30.

Figure 3. Positive result of nested PCR from mothers blood.

Figure 3 showed 5 positive result from sample 5, 21, 24, 26 and 30.

15

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