Skip to 0 minutes and 10 secondsVANESSA YARDLEY: The Direct Agglutination Test, or DAT, is used for the clinical diagnosis of a suspected case of visceral leishmaniasis, also known as kala-azar. This video shows how a DAT is carried out in practice. The methods in each laboratory may differ slightly. This video will demonstrate best practice for performing the test. The test is straightforward but requires good controls and careful execution. After a short introduction, we will gather the materials needed and take you through each step of the test before moving on to interpreting the results.

Skip to 0 minutes and 47 secondsWhat is the principle of the DAT? A Direct Agglutination Test reveals whether a patient has been infected with leishmania. It does so by detecting whether antibodies against the parasite are present in a blood or serum sample taken from a patient with suspected VL. The patient sample, also referred to as the test sample, is serially diluted in a microtiter plate. If the patient carries leishmania antibodies, they will bind to the leishmania antigen provided by the Coomassie blue stained leishmania promastigotes within the test, and can therefore be detected. The binding of antigen and antibody is typically referred to as agglutination and is a basic property of immunology.

Skip to 1 minute and 33 secondsAs the DAT reagents are labelled with Coomassie blue dye, agglutination during the test becomes easy to detect by the naked eye. In the reaction well, the agglutination can look like a cloud, a dot with frayed edges, or an enlarged dot. If no anti-leishmania antibodies are present, the blue-coloured leishmania promastigotes will settle in the bottom of the well and form a small blue dot. The patient sample tested in a DAT can either be a drop of serum or blood, or a blood spot dried onto philtre paper. Please note that this video only demonstrates a DAT using serum. Samples will need more preparation time if dried blood spots are being used.

Skip to 2 minutes and 18 secondsThe advantage of using these is that they can be stored for several days or more and are more field friendly. To prepare the dried blood spot, a blood sample is taken and put onto absorbent Whatman philtre paper, which is then left to dry. Once dried, the sample can either be stored or used for the DAT. The blood sample is cut, or more usually punched out, using a five-millimetre biopsy puncher, and the paper circle placed into the well of the DAT microtiter plate. The sample can then be eluted to check for the presence of leishmania antibodies. Safety is important. It is crucial to remember that you will be handling human samples and hazardous chemicals.

Skip to 3 minutes and 1 secondWear protective clothing-- namely, a lab coat and disposable gloves. All samples and materials must be disposed of safely in appropriate waste containers. To start the DAT, make sure all the materials and reagents you need are to hand. You will need a 96-well microtiter plate with a v-shaped bottom to perform the testing. You will also need a variety of pipettes and appropriate tips. A timer is also useful, as well as markers and paper to record the results. You will need the DAT antigen, which may be freeze-dried in serum or whole blood. You will also need physiological saline and 2-mercaptoethanol. The latter reagent must be used in a safety cabinet or fume hood and treated as hazardous.

Skip to 3 minutes and 49 secondsYou will also need control samples, a negative control sample that you know does not contain leishmania antibodies, and a positive control sample that has already been validated, such as a pooled sample of serum from patients who have tested positive for VL. To start the test, some of these reagents will first need to be prepared. First, you will need to prepare the assay diluent. Do this by mixing 50 millilitres of saline with 390 microliters of 2-mercaptoethanol. Next, prepare the antigen and controls. Leishmania antigen that is either freeze-dried in serum or whole blood will need to be reconstituted.

Skip to 4 minutes and 30 secondsHere, we use the Leish-KIT antigen, which required removing the DAT antigen from the fridge, reconstituting in 5 millilitres of sterile saline, and mixing well by rotation for one hour before use. Do not shake this, as it will destroy the antigen. Freeze-dried serum controls will also need reconstitution. Use a new set of control sera every time a new set of DAT tests is performed. Reconstitute each bottle that contains 2 microliters of freeze-dried serum with 100 millilitres of saline and mix gently. Once all the reagents are ready, you should be able to start filling in the microtiter plates to allow the binding reaction to occur.

Skip to 5 minutes and 16 secondsBefore you start, make sure that you have labelled the plate with the samples and that you have a list of the samples with the positions on the microtiter plate noted. Remember that the volumes of the reagents added into the wells may be different depending on the samples you are using. Here we are testing three patient serum samples as an example. If you are using dried blood spot samples, adjust accordingly. When filling the microtiter plate, make sure to change tips to avoid cross-contamination. Add 98 microliters of the assay diluent to the unused wells of the first column. Then add 50 microliters of the assay diluent to all the other wells, namely, columns 2 to 12, using the multipette.

Skip to 6 minutes and 3 secondsSecondly, add 2 microliters of each of the patient's serum samples to the first column-- A1 in this instance, and B1 and C1. Here, add positive to D1 and negative to E1.

Skip to 6 minutes and 20 secondsFor the sero-dilution, adjust the multichannel pipette to 50 microliters and firmly fix the tips to avoid pipetting errors. Mix the contents of the first column by pipetting gently up and down three to five times, avoiding bubbles. Then transfer 50 microliters from the first column to the second. Mix gently again, and transfer 50 microliters to the third column. Continue this until and including column 11. Once you have finished mixing column 11, expel the 50 microliters. The last column, column 12, is a blank control, which means it should not contain any test sample. Once the sero-dilution is complete, the antigen can be added.

Skip to 7 minutes and 9 secondsAdd 50 microliters of antigen preparation to each well in reverse order, i.e., from column 12 to 1, blank to highest titre. This will help reduce cross-contamination.

Skip to 7 minutes and 26 secondsNow, cover the plate, gently rotate by hand, and leave the plate to incubate for a minimum of 16 hours at ambient temperature. After incubation, carefully place the plate on a white surface or on top of a sheet of white paper. Check the control samples first. You should be able to see differences. The first column of the positive control sample, the most concentrated sample, should be clear. Looking along the road to the lower concentrations of the sample from column 1 to 12, a distinct blue dot should progressively appear. This is the sediment of unbound leishmania promastigotes. The results of the DAT are interpreted according to titers, and in relation to the dilutions of the patient samples.

Skip to 8 minutes and 13 secondsThe titer is expressed as the last dilution that still shows a difference in an agglutination, compared with the blank wells or the negative control. There are three classifications of results. If the titer is found at 1 to 100, 1 to 200, or 1 to 400 dilution, then the result is negative. If the titer is at 1 to 800, 1 to 1,600, or 1 to 3,200, it is considered borderline. And if the titer is above 1 in 3,200, the sample is positive for leishmania antibody. In this video, the positive and negative controls have worked as expected, and the blank wells have also worked-- i.e., there is no agglutination.

Skip to 8 minutes and 57 secondsIn the test sample wells, the first patient's serum tested in the first row looks negative, whereas samples from patients in rows 2 and 3 look positive.

Skip to 9 minutes and 10 secondsKeep in mind that DAT is a serological test and only detects antibodies, not the parasite. The test can give a positive result a long time after the patient has been successfully treated for VL and discharged. Therefore, the DAT cannot differentiate between a new VL infection and relapse. During an epidemic season, patients may present with VL symptoms but have a negative DAT result. This is because the antibody level may not have risen sufficiently enough to react with the test antigen. In these instances, it is prudent for the suspected VL patient to return for repeat DAT a week or two later. Patients with borderline DAT titers should either be retested or have a parasitological assessment.

Skip to 10 minutes and 0 secondsBefore reporting, it is good practice that a second person also reads the DAT plate and confirms the results. When finished, do not forget to dispose of the materials in a proper and safe way.

DAT demonstration video

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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This video is from the free online course:

Control and Elimination of Visceral Leishmaniasis

London School of Hygiene & Tropical Medicine