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DAT demonstration video

Video demonstrating how to perform a direct agglutination test for the diagnosis of visceral leishmaniasis.

Direct Agglutination Test (DAT)

The DAT antigen consists of freeze-dried L. donovani promastigotes that are stained blue. Suspended in solution, the antigen is mixed with patient serum. If anti-Leishmania antibodies are present, a visible antibody-antigen agglutination reaction occurs. By preparing different dilutions of antigen, the quantity of antibody can be estimated.

The specificity and sensitivity of DAT varies per region where the test is used and cut-off values should be validated for every endemic area and regularly re-evaluated. A high cut-off will yield no false positives, but may compromise sensitivity, whereas a low cut-off titre may detect subclinical infection and will have a low specificity. The cut-off titre depends on the context – the positive predictive value will be higher in case of a high prevalence of KA, so outbreaks and timing in relation to the transmission season must be taken into account.

DAT results are negative (low titers), positive (high titers) or borderline (intermediate titers). In the borderline group, 50% of patients are predicted to have positive parasitology, so these patients must always be further tested. In some cases it may take repeated serological and/or parasitological testing before diagnosis can be confirmed. In early disease, parasite load and antibody titres may still be too low to be detected by microscopy and rK39 rapid tests respectively, and in such cases a quantitative DAT test can give earlier confirmation of disease. An increasing DAT titre (≥2 wells) between two DAT tests 5-7 days apart is adequate confirmation, even if the titres are still below the positive threshold. DAT can only be performed by trained lab technicians in an established lab, and causes a delay in diagnosis as the results take over 18 hours to obtain. The test can be relatively expensive (about twice as expensive as rK39 rapid tests).

Notes

  1. If facilities to validate a positive control are not available, you are recommended to use manufactured negative controls. A reliable confirmatory test uses the PCR technique and respective equipment, which requires careful execution and can be expensive and less field friendly. A manufactured negative control is a reliable source for a truly negative sample.
  2. DISCLAIMER: LSHTM and partners collaborating in this video can take no legal responsibility for the use of any information contained. Users implementing the techniques shown do so at their own risk and must take responsibility for local verification/ validation of these techniques before they are used for the diagnosis of patients.
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Control and Elimination of Visceral Leishmaniasis

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