Laboratory Diagnosis of Toxoplasmosis

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

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).

The Disease BurdenStudy 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.

The Test Formats:1. Antibody Remains the test method of choice.

1. Use of Particulate Antigens: Trophozoites, IgG

Dye Test IFAT Agglutination, Differential Agglutination.

2. Use of Soluble Antigens: Cytosol antigen.

ELISA Avidity Test Western Blot

1. Antibody Detectiona. 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.

1. Antibody Detection

b. IFAT: Formalin treated tachyzoites

used Can detect IgG, IgM Difficult to interpret BFP in autoimmune diseases.

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.

1. Antibody DetectionAC 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.

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

10-300 IU/ml.

1. Antibody Detectionb. 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

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

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.

2. Detection of Toxoplasmaa. Microscopy: Smears stained by Giemsa, FAT,

Biopsy material Presence of foci of tacyzoites

with or without cysts suggest active disease.

2. Detection of Toxoplasmab. Animal Inoculation Body fluids, trypsinized tissue Intraperitoneal inoculation in 6-8

mice Seroconversion occurs after 7-10

days Peritoneal lavage examination

shows the tachyzoites.

2. Detection of Toxoplasmac. Cell Culture: Human embryo fibroblast (MRC5)

or monocyte lines (THP 1). 4-5 days.

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-fold-repeated 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.

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).

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.

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

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

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 .

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

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.

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.

ConclusionDisease 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.