WHO comparative evaluation of serologic assays for Chagas disease Journal Club April 2, 2009

Chagas disease (American trypanosomiasis) caused by the parasite Trypanosoma cruzi, which is transmitted: –By the triatomine bug (kissing or reduviid bug) –Vertically –Blood transfusions –Organ transplants

Epidemiology Endemic in Mexico, Central and South America –8-11 million people are infected 45,000 fatalities may occur annually –Those living in rural areas at highest risk (triatomine bugs love mud walls and thatched roofs) In the US and Canada, infection is increasing as a result of the immigration of infected individuals from endemic areas

Transfusion relevence Acute, vector-borne infections are mostly mild –Chronic, asymptomatic infection results –acute infection in patients with compromised immune systems can be very serious It has been estimated that there may be as many as 100,000 legal immigrants in the US and Canada who are unknowingly infected The rate of seropositive blood donors in the US ranges from 1 in 5400 to 1 in 25,000 –Among high risk populations in Washington and California, the prevalence was 1 in 500, which 20% able to transmit infection –2 cases of TT-Chagas disease have been identified in Canada

Screening In 2006, the US FDA approved a serologic enzyme-linked immunosorbent assay (ELISA) for screening donations –Ortho-Clinical Diagnostics

Challenges Laboratory diagnosis of Chagas disease is challenging –Direct detection of the parasites is difficult Low parasitemia during chronic phase Hence, the laboratory diagnosis is based on serologic assays –i.e. looking for T. cruzi antibodies with a lysate of epimastigotes grown in liquid culture –There is no accepted/accessible gold standard New assays are validated using high-antibody-titre, consensus positive specimens (and thus their ability to detect low-antibody-titre cases is untested) –Sensitivity is overestimated

Purpose Evaluation of commercially available test kits for Chagas disease for use in blood bank screening is difficult –Lack of large and well-characterized panels Collaborate effort of Latin American blood centres and the WHO to establish a Chagas disease panel

Design 437 specimens (Chagas positive and Chagas negative) were provided in 2000 to the WHO Collaborating Centre in San Paulo –10 Latin American blood centres in 10 countries –Specimens were assigned a positive or negative status based on concordant results in at least three of the four confirmatory assays Between 2001 and 2005, this panel was used to evaluate 19 screening assays

Confirmatory Assays Indirect immunofluorescence IF Western blot WB Radioimmunoprecipitation assay RIPA –Only RIPA gave 100% agreement with the final serologic status of the specimens Recombinant immunoblot IB –RIBA

Indirect Immunofluorescence epimastigotes Donor’s antibodies against parasite Anti-human IgG- fluorescein conjugate

Trypomastigote antigens Anti-human IgG conjugate The conjugate develops colour, and the results interpreted by visual inspection for bands between kDa

RIPA Radiolabeled antigen fragments are combined with the specimen Specific antibodies, if present, will bind these antigen fragments The resulting antigen-antibody complexes are precipitated and separated by electrophoresis The pattern is detected using autoradiography (the exposure of sensitive X-ray film by the radioactive emissions of the bound, labeled antigens)

Recombinant immunoblot (RIBA) Recombinant parasite antigens are applied to paper strips Exposure to specimen allows anti-parasite antibodies, if present, to bind to the antigen Anti-human labelled conjugate allows visualization

Results 437 specimens –Positive 39% –Negative 61% –Inconclusive 2% (excluded from analysis) Since their true serologic status cannot be determined, there is no way to conclude how they might have affected the calculated sensitivities and specificities

Results Screening assays –Sensitivity and specificity varied considerably Sensitivity 80%-100% Specificity % EIAs performed better than other screening methods –Four EIAs had specificity and sensitivity in excess of 99%

Conclusions At least four of the commercially available EIAs are sufficiently sensitive and specific for use as a single-assay screen of blood donations The majority of commercially available indirect hemagglutinin assays should not be used Sensitivity and/or specificity was low Subject to reader bias False negatives reported even for high-titre specimesn (prozone effect) The RIPA is the gold standard But it is complex

Prozone effect

Evaluation What is the background/rationale for the investigation? –There was a clear need for establishing a Chagas panel –The report helps to raise awareness among clinicians/blood bankers about the challenges of evaluating new assays

Evaluation Study design –The setting was a blend of the small Latin American endemic countries with the resources of WHO over several years Variables –Variables and confounders (e.g. concurrent disease) were left undescribed –Diagnostic criteria (e.g. diagnostic test) varied from country to country

Evaluation Bias –Bias is a possible confounder, since there was no apparent randomization in the submission of samples –Centres may have submitted specimens that were “easy” (clearly positive or negative), or specimens that were challenging Submitting either a preponderance of easy or tough specimens could affect the sensitivity and specificity of the test results

Evaluation Study size –No particular study size was pre- determined

Evaluation Statistical methods were not described in detail –95% CI were calculated and provided

Evaluation Participants –The number of specimens received was reported –The number of specimens eventually excluded from the study also reported Main results –Sensitivities, specificities and 95% CI were reported for the 19 screening methods and the four confirmatory assays –The “gold standard” was the consensus result of the four confirmatory assays

Evaluation Results –Four EIA kits were appropriate to be used as single assay screens –RIPA is proposed as the gold standard Limitations & Generalization –The specimens provided by the blood services may differ from those that would be randomly encountered (especially in countries were Chagas is not endemic) –Questions of cost and availability for small blood services –Issues of convenience with RIPA technology