1. Laboratory Diagnosis of
Toxoplasmosis

2. Outline
Disease Burden of Toxoplasmosis in
India
 The Test Formats available


Antibody Detection
 Detection of Toxoplasma- Microscopy, Animal, Culture
 Detection of Parasite DNA

Application in Clinical Situations

Screening in Pregnancy
Diagnosis in the Neonatal, pre natal stage
Diagnosis in immunocompromised host- Cerebral
Toxoplasmosis
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3. The Disease Burden

In India, the national seroprevalence
has been found to be 24.3%.(Dhumne et
al,2007. J Parasitol;93)


Lowest in the northern parts of India,
and highest in the south.
A high seroprevalence of 57% has been
reported from Almora district (Singh and
Nautiyal 1991. IJMR;93)

A recent study from Chandigarh: 15.33%
pregnant women were
seropositive.(Khurana et al 2010. IJMM;28).

4. The Disease Burden
Study from Hyderabad showed a
seropositivity (IgG) rate of 27% in
women with BOH, and IgM in 11.5%.
 Study from our centre revealed 9.3%
IgM positivity rate in BOH cases.


5. The Test Formats:1. Antibody

Remains the test method of choice.
1.
Use of Particulate Antigens:
Trophozoites, IgG
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2.
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Dye Test
IFAT
Agglutination, Differential Agglutination.
Use of Soluble Antigens: Cytosol
antigen.
ELISA
Avidity Test
Western Blot

6. 1. Antibody Detection
a.

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
Sabin Feldman Dye Test : IgG
Described in 1948 (Science, 1948:108)
Cytotoxic effect of antibodies in the
presence of complements.
End point is the dilution at which 50%
of the tachyzoites are dead.
Live tachyzoites necessary
Easy to read, Highly sensitive (2 IU/ml)
Detects very early antibodies.

7. 1. Antibody Detection
b. IFAT:
 Formalin treated tachyzoites used
 Can detect IgG, IgM
 Difficult to interpret
 BFP in autoimmune diseases.

8. 1. Antibody Detection
c. Agglutination Test: IgG
 Formalin treated antigen
 Highly sensitive if 2ME treatment is
done.
Differential Agglutination Test :
 Formalin treated antigen(HS) and
methanol treated antigen (AC) used
with a single sample
 To rule out recent infection.

9. 1. Antibody Detection
 AC
antigen specific for membrane,
strong antibody response in early
infection, wanes after 6-12 mts.
 Sensitivity: HS: 2 IU/ml, AC: 50IU/ml
 HS/AC≥ 4 : Infection has occurred
more than 6 months before.
 Antigens difficult to prepare, not
available commercially.

10. 1. Antibody Detection
Use of Soluble antigen: Cytosol
antigens enriched with membrane
antigen( P30, SAG 1) to enhance
sensitivity.
a. ELISA
 Extensively used
 IgG, IgM, IgA, IgE.
 IgG indirect ELISA: Sensitivity 10300 IU/ml.

11. 1. Antibody Detection
b. IgG Avidity Test
 Increasing humoral response, ↑avidity of IgG
 Early stage of infection: Weak avidity; late
stage: Strong avidity
 Two parallel ELISA: Untreated serum; serum
treated with urea/guanidine/thiocyanate
(Dissociates Ag-Ab complex of weak avidity)
 High avidity index→ Infection in remote past
 Caveat: Not always true, ↑in avidity may be
slow

12. 1. Antibody Detection
c. Western Blot Test
 Two samples tested in parallel:
Blood/CSF; Blood/aqueous humor;
Maternal/neonatal blood.
 Additional bands in the second
sample denotes organ/neonatal
infection

13. IgG and IgM Western blots of
serum from a mother (m)
and her newborn (b). Sera
were drawn from mother and
baby when the baby was 2
days of age.
Toxoplasmawas isolated from
the placental tissue.
Remington et al 2004.
JCM:42.

14. 2. Detection of Toxoplasma
a.


Microscopy:
Smears stained by Giemsa, FAT,
Biopsy material
Presence of foci of tacyzoites with or
without cysts suggest active disease.

15. 2. Detection of Toxoplasma
b. Animal Inoculation
 Body fluids, trypsinized tissue
 Intraperitoneal inoculation in 6-8 mice
 Seroconversion occurs after 7-10
days
 Peritoneal lavage examination shows
the tachyzoites.

16. 2. Detection of Toxoplasma
c. Cell Culture:
 Human embryo fibroblast (MRC5) or
monocyte lines (THP 1).
 4-5 days.

17. 3. PCR

Diagnosis of congenital, ocular, cerebral
toxoplasmosis.
 The most common use of PCR is for
prenatal diagnosis of the congenital
infection using amniotic fluid.
 Most laboratories use the 35-foldrepeated B1 gene
 Some laboratories in Europe are
switching to the AF146527 sequence, a
DNA fragment that is repeated 200- to
300-fold in the T. gondii genome.

18. 3. PCR
An evaluation of three targets (18S
ribosomal DNA, B1, and AF146527) in
parallel.
 Differences in the sensitivity and
specificity of these three PCR
techniques were not found to be
statistically significant ( Filisetti et al 2003.

JCM;41).

19. Diagnosis in Pregnancy
Active infection normally occur once
in life.
 Acquired immunity is life long
 The risk is only from an infection
caught for the first time during
pregnancy, or 2-3 months before
conception.
 Mothers who tested positive before
the pregnancy do not need
monitoring/screening.


20. Diagnosis in Pregnancy

Within 2–3 months before conception
- 1% or below risk of transmission but
a high risk of miscarriage
• Severity varies with age of fetus
• More severe manifestation early in
pregnancy, Less frequent transmission
• More frequent transmission later in
pregnancy, Less severe manifestation

21. Risk of Toxoplasma gondii congenital infection (transmission) and development of clinical
signs in offspring before age 3 years, according to gestational age at maternal
seroconversion.
Goldstein E J C et al. Clin Infect Dis. 2008;47:554-566
© 2008 by the Infectious Diseases Society of America

22. Diagnosis in Pregnancy
In most centres one sample is tested
for IgG and/or IgM
IgM

◦ Determine recent infection or in the distant past.
◦ Significant potential of misinterpretation of +ve
result, should be confirmed by other tests.
◦ IgM antibodies can persist for months to more
than one year.
◦ Persistence of these IgM antibodies does not
appear to have any clinical relevance .

23. Interpretation of results of serological tests for toxoplasmosis performed at clinical
(nonreference) laboratories.
Goldstein E J C et al. Clin Infect Dis. 2008;47:554-566
© 2008 by the Infectious Diseases Society of America

24. Neonatal/Prenatal Diagnosis
Diagnosis in neonatal period : WBT
with paired sample
 IgM and IgA detection may be used:
Chance of false negative because of
small amounts produced.
 Declining IgG level may be used to
rule out congenital infection.
 PCR of amniotic fluid+
Ultrasonogram done after 18 weeks.


25. Diagnosis in
Immunocompromised Patients
Peripheral blood, BAL, Bone marrow
may be used to demonstrate and/or
isolate the parasite.
 PCR with the above samples.
 If IgG in blood is positive, risk is high
in HIV positive patients with CD4+
count<200/cumm, and IgG >150IU/ml
OR presence of 22,25, or 69KD bands
in WBT.


26. Conclusion
Disease burden is high in India as
shown by seroprevalence studies.
 There is no regulation for antenatal
screening
 The rural population is highly
vulnerable but totally neglected
regarding testing for this important
pathogen .
 Toxoplasmosis remains a neglected
disease in India.
