Usefulness of the direct agglutination test in the early detection of subclinical Leishmania donovani infection: a community-based study

The value of a direct agglutination test (DAT) in the detection of subclinical infections with Leishmania donovani has recently been investigated in the Indian state of Bihar, after the sensitivity and specificity of the test had been determined. When used to screen sera from 108 parasitologically confirmed cases of visceral leishmaniasis, 50 patients with active, non-leishmanial infection, and 641 healthy controls living close to, or distant from, an endemic area, the test was found to be 91.7% sensitive and 100% specific if a titre of 1:800 was used as the threshold for seropositivity.During a longitudinal clinical study in a rural, VL-endemic area of the Indian state of Bihar, the test was used, with 1:800 set as the threshold titre, to determine the baseline prevalence of infection with L. donovani among villagers who, though showing no symptoms of VL, had recently been febrile for at least 2 weeks. The 234 subjects of this study were either VL-case contacts [i.e. members of households in which there were active or cured VL cases (N=78)] or the members of control households with no cases or history of the disease (N=156). The results of DAT at the start of the study indicated that 49 (20.9%) of the subjects--29 (37.2%) of the VL-case contacts and 20 (12.8%) of the other subjects--were seropositive and therefore probably had subclinical infections with L. donovani. During the subsequent 9 months of follow-up, however, only eight of the subjects found seropositive at the start of the study--seven (24.1%) of the seropositive case contacts but only one (5.0%) of the other seropositives--developed symptomatic VL, all by month 6 of the follow-up. Compared with their neighbours, therefore, individuals who shared households with active or cured cases of VL appeared at greater risk not only of L. donovani infection (indicating focal transmission) but also of developing symptomatic disease once infected. Curiously, among the seropositive case contacts, those from the households that harboured active cases of VL at the baseline survey were less likely to develop symptomatic VL during the 9 months of follow-up than those from households that harboured only cured cases (18.8% v. 30.8%). The wide-spread use of DAT could allow the detection and early treatment of latent L. donovani infections and so contribute to the elimination of VL, at least as a public-health problem, from India.     

Leishmania donovani infection in Eastern Sudan: Comparing direct agglutination and rK39 rapid test for diagnosis-a retrospective study

Objective: To compare diagnostic accuracy and agreement between direct agglutination test and rK39 rapid tests for diagnosis of visceral leishmaniasis in an endemic area, the Doka area in Eastern Sudan.Methods: Stored sera of confirmed visceral leishmaniasis cases, unconfirmed visceral leishmaniasis-suspects and negative controls were tested by direct agglutination test and rK39 rapid test. The sera were collected from the Doka area in Eastern Sudan. Diagnostic accuracy of direct agglutination test and rK39 rapid test was assessed in terms of sensitivity, specificity, positive predictive value and negative predictive value, estimated at 95% confidence interval(CI). Agreement between the two tests was determined by the kappa(κ) value.Results: Taking lymph node aspiration of Leishmania as a gold standard, direct agglutination test showed 91.0% sensitivity, 99.3% specificity, resulting in a positive and negative predictive value of 99.3% and 91.0%, respectively. In contrast, the sensitivity of rK39 rapid test was 85.2% and specificity 98.6%, resulting in a positive and negative predictive value of 98.5% and 85.9%, respectively. Most(81.3%) of the confirmed visceral leishmaniasis sera revealed strong antibody titers(≥1:6 400). Some sera(n=5) that were positively tested with rK39 rapid test were negative in direct agglutination test(≤1:800); in contrast, direct agglutination test was positive in 12 confirmed visceral leishmaniasis sera that were negatively tested with rK39 rapid test. There was moderate to good agreement between direct agglutination test and rK39 rapid test for confirmed visceral leishmaniasis patients(κ=0.42, 95% CI=0.21-0.63) and control sera(κ=0.80, 95% CI=0.41-1.00).Conclusions: Both direct agglutination test and rK39 rapid test are satisfactory test systems for visceral leishmaniasis diagnosis in East Sudan. Their simplicity makes them ideal for first healthcare in rural areas. These data are relevant also for other East African endemic countries because of the geographical and overlapping distribution of the Leishmania parasite.     

Serological markers for leishmania donovani infection in Nepal: Agreement between direct agglutination test and rK39 ELISA

Visceral leishmaniasis (VL) is an important vector-borne disease caused by Leishmania donovani in the Indian subcontinent. The actual incidence and role of asymptomatic infections in the region are not wellknown. We used the direct agglutination test (DAT) and the rK39 ELISA as L. donovani infection markers in 10 VL endemic villages in Nepal. DAT titre distribution showed two subgroups in the population (infected and non-infected individuals), while rK39 did not. The agreement between both tests was moderate (j = 0.53; 95% CI 0.49–0.57). More research is needed to develop validated markers for Leishmania infection.     

Rapid detection of human Leishmania infantum infection: a comparative field study using the fast agglutination screening test and the direct agglutination test

This study aimed to evaluate the performance of a fast agglutination screening test (FAST) for serodiagnosis of human Leishmania infantum infection in Iran. FAST is based on the direct agglutination test (DAT) but combines with a higher parasite concentration and is performed with only one serum dilution. The validity of FAST for the detection of L. infantum infection in the field was compared with the direct agglutination test on 110 confirmed or patients suspected of infection with leishmaniasis, 177 healthy individuals and 41 patients with other infectious diseases who were from northwestern and southern parts of Iran. In this study, we found a 1:1600 cut-off point empirically by seeking the best correlation (90.8) between sera confirmed with visceral leishmaniasis and healthy control sera. A sensitivity of 95.4% (95% CI, 91.4-99.4) and specificity of 88.5% (95% CI, 84.2-92.8) were found with 1:1600 as a cut-off titer when DAT-confirmed cases were compared with the control groups. A good degree of agreement was found between FAST and DAT (90.8%) by Kappa analysis. FAST requires 2?h for reading the results versus the 12-18?h needed for DAT. As FAST is simple, rapid, sensitive and non-invasive and does not require a higher volume of antigens or much expertise, it can be used for screening and serodiagnosis of human L.?infantum infection.     

Usefulness of the rK39-immunochromatographic test, direct agglutination test, and leishmanin skin test for detecting asymptomatic Leishmania infection in children in a new visceral leishmaniasis focus in Amhara State, Ethiopia

In areas where visceral leishmaniasis is anthroponotic, asymptomatically infected patients may play a role in transmission. Additionally, the number of asymptomatic patients in a disease-endemic area will also provide information on transmission dynamics. Libo Kemkem and Fogera districts (Amhara State, Ethiopia) are now considered newly established areas to which visceral leishmaniasis is endemic. In selected villages in these districts, we conducted a study to assess the usefulness of different approaches to estimate the asymptomatic infection rate. Of 605 participants, the rK39 immunochromatographic test was able to detect asymptomatic infection in 1.5% (9 of 605), direct agglutination test in 5.3% (32 of 605), and leishmanin skin test in 5.6% (33 of 589); the combined use of serologic methods and leishmanin skin test enabled detecting asymptomatic infection in 10.1% (61 of 605). We conclude that the best option to detect asymptomatic infection in this new visceral leishmaniasis-endemic focus is the combined use of the direct agglutination test and the leishmanin skin test.     

Prospective evaluation and comparison of the direct agglutination test and an rK39-antigen-based dipstick test for the diagnosis of suspected kala-azar in Nepal

The diagnosis of visceral leishmaniasis (kala-azar) remains difficult in rural endemic areas and practical and reliable tests are badly needed. Two serological tests, the Direct Agglutination Test (DAT) and an rK39-antigen-based dipstick test, were compared to parasitological diagnosis in a group of 184 patients presenting at a tertiary care centre in south-eastern Nepal with a history of fever > or = 14 days and splenomegaly; 139 patients had a parasitologically proven kala-azar and 45 patients had a negative parasitological work-up. The rK39 dipstick showed a sensitivity of 97% and a specificity of 71%. The DAT was up to 99% sensitive with a low cut-off titre (1:400) but its specificity did not exceed 82% even with a high cut-off titre (1:51 200). Both tests could be used for screening suspect patients in endemic areas. However, their use as confirmatory tests should be restricted to situations where the proportion of kala-azar among clinical suspect patients is high. The rK39 dipstick is cheaper and easier to use than the DAT and could be used widely provided that both its performance and production remain stable.     

Evaluation of two rK39 dipstick tests, direct agglutination test, and indirect fluorescent antibody test for diagnosis of visceral leishmaniasis in a new epidemic site in highland Ethiopia

We assessed the performance characteristics of two rK39 immunochromatographic tests, a direct agglutination test (DAT), and an indirect immunofluorescent antibody test (IFAT) in the site of a new epidemic of visceral leishmaniasis (VL) in northwestern Ethiopia. The study population was composed of 179 patients with suspected VL and 67 controls. The sensitivities of Kalazar Detect^®, DiaMed-IT Leish^®, DAT, and IFAT in 35 polymerase chain reaction–confirmed VL cases were 94.3%, 91.4%, 91.4%, and 100%, respectively, and the specificities were 98.5%, 94%, 98.5%, and 98.5%, respectively. In a Bayesian latent class analysis of all 246 specimens, the estimated sensitivities were 90.5%, 89%, 88.8%, and 96% for Kalazar Detect^®, DiaMed-IT Leish^®, DAT, and IFAT, respectively; DAT showed the highest estimated specificity (97.4%). Both rK39 immunochromatographic tests perform as well as DAT, and are suitable for VL diagnosis in first-level health centers in this area of Ethiopia.     

Comparison of an rK39 dipstick rapid test with direct agglutination test and splenic aspiration for the diagnosis of kala-azar in Sudan

We compared an rK39 dipstick rapid test (Amrad ICT, Australia) with a direct agglutination test (DAT) and splenic aspirate for the diagnosis of kala-azar in 77 patients. The study was carried out under field conditions in an endemic area of north-east Sudan. The sensitivity of the rK39 test compared with splenic aspiration was 92% (46/50), the specificity 59% (16/27), and the positive predictive value 81% (46/57). Compared with the diagnostic protocol used by Médecins sans Frontières, the sensitivity of the rK39 test was 93% (50/54), the specificity 70% (16/23), and the positive predictive value 88% (50/57). Compared with splenic aspirates, the sensitivity of a DAT with a titre > or =1:400 was 100% (50/50), but its specificity only 55% (15/27) and the positive predictive value was 80% (50/62). Using a DAT titre > or =1:6400, the sensitivity was 84% (42/50), the specificity 85% (23/27) and the positive predictive value 91% (42/46). All four patients with DAT titre > or =1:6400 but negative splenic aspirate were also rK39 positive; we consider these are probably 'true' cases of kala-azar, i.e. false negative aspirates, rather than false DAT and rK39 seropositives. There were no false negative DATs (DAT titre < or =1:400 and aspirate positive), but there were four false negative rK39 tests (rK39 negative and aspirate positive). The rK39 dipstick is a good screening test for kala-azar; but further development is required before it can replace the DAT as a diagnostic test in endemic areas of the Sudan.     

Evaluation of the direct agglutination test based on freeze-dried Leishmania donovani promastigotes for the serodiagnosis of visceral leishmaniasis in Sudanese patients

The direct agglutination test (DAT) based on freeze-dried (FD) Leishmania donovani antigen was evaluated for the serodiagnosis of kala-azar in a rural setting in eastern Sudan. The performance of the FD-DAT was compared with standard liquid antigen (LQ) by testing serum samples and blood samples collected on filter paper of microscopically and PCR-confirmed VL patients, apparently healthy endemic controls and patients with other relevant infectious diseases for the region. In the present study, the FD-DAT had a sensitivity of 96.8% and a specificity of 96.2%. The LQ-DAT had a sensitivity of 91.0% and a specificity of 96.6%. A high degree of agreement (97.3%; r-value 0.94) was observed between the FD-DAT and the LQ-DAT, as well as between the FD-DAT performed on serum samples and corresponding blood samples collected on filter paper (agreement 97.8%; r-value 0.79). The FD-DAT is very suitable as diagnostic test for kala-azar in remote rural conditions as it is sensitive, specific and stable. The antigen is affordable, reproducible and available, which contributes to the sustainability of the DAT as a diagnostic test for VL.     

PCR and direct agglutination as Leishmania infection markers among healthy Nepalese subjects living in areas endemic for Kala-Azar

Objective  To compare a PCR assay and direct agglutination test (DAT) for the detection of potential markers of Leishmania infection in 231 healthy subjects living in a kala-azar endemic focus of Nepal.
Methods  The sample was composed of 184 (80%) persons without any known history of KA and not living in the same house as known kala-azar cases (HNK), 24 (10%) Healthy Household Contacts (HHC) and 23 (10%) past kala-azar cases which had been successfully treated (HPK).
Results  PCR and DAT positivity scores were, respectively: HNK, 17.6% and 5.6%; HHC, 12.5% and 20.8%; HPK, 26.1% and 95.7%. The ratio PCR-positives/DAT-positives was significantly higher in HNK (ratio = 3.1) than in HHC (ratio = 0.6, P  = 0.036) and in HPK (ratio = 0.2, P  = 0.012). The ratio PCR-positives/DAT-positives did not significantly differ between HHC (ratio = 0.6) and HPK (ratio = 0.2, P  = 0.473). The positive agreement index between PCR and DAT in HNK was 5%; in HHC, 0%; in HPK, 43%.
Conclusions  Our study highlights the specific character of PCR and DAT for the exploration of Leishmania asymptomatic infections. PCR is probably more informative for very recent infections among HNK, while DAT provides more information among HHC and HPK, a feature likely related to the power of serology to track less recent infections.     

Comparison of microscopy and culture in the detection of Leishmania donovani from splenic aspirates

Three culture media were compared with Giemsa-stained smears for the detection of Leishmania in splenic aspirates from Kenyan patients with visceral leishmaniasis. Ninety-nine splenic aspirates obtained from 26 patients at various times before, during, and after treatment were cultured in Schneider's Drosophila medium and RPMI medium 1640 (each supplemented with 20% fetal bovine serum) and McConnell's modification of Senekje's medium overlayed with 0.9% saline. From 13 splenic aspirates obtained before treatment, amastigotes were identified microscopically in all and promastigotes were cultured in 12. During and after treatment, Schneider's medium was the most sensitive method for detecting parasites, followed by microscopic examination of stained smears which was more sensitive than either of the other two media tested. Results indicate that, for initial diagnosis, both culture and direct microscopy of aspirates should be employed.     

A comparative study of fecal occult blood tests for early detection of gastrointestinal pathology

The question of what the most accurate and efficient fecal occult blood testing method is for the early detection of pathological gastrointestinal tract bleeding continues to be intensely debated. In this prospective study, the following five uniquely different slide tests were investigated in 120 patients who underwent gastrointestinal tract investigation: (1) a combination monoclonal antibody guaiac test (Monohaem); (2) an immunologic assay, enzyme-linked immunosorbent assay, with (3) a highly sensitive guaiac test (Fecatwin S/Feca enzyme immunoassay), (4) a popular guaiac test (Coloscreen III) (comparable with Hemoccult II), and (5) Coloscreen III/VPI (ie, with vegetable peroxidase) inhibitor. Computerized data show efficiency values for detection of fecal occult blood by Coloscreen III-Fecatwin S-Monohaem combined, 93%; Coloscreen III-Monohaem combined, 91%; Monohaem, 87%; Coloscreen III/VPI, 82%; Coloscreen III, 79 percent; enzyme-linked immunosorbent assay, 77%; and Fecatwin S, 68%. Results of sensitivity, specificity, false-positive and false-negative test results, tests' predictive value, simplicity, and costs of tests in this clinically based study suggests that the concomitant use of the monoclonal, monospecific test for human hemoglobin and an appropriately sensitive guaiac test is a potentially valuable approach to mass screening and early detection of occult bleeding gastrointestinal tract pathology, including colorectal cancer.     

Preparation and evaluation of a glycerol-preserved direct agglutination antigen for long-term preservation: a comparative study of the detection of anti-Leishmania infantum antibodies in human and dog

ObjectiveTo prepare and evaluate a glycerol-preserved antigen from an Iranian strain of Leishmania infantum (L. infantum) for use in glycerol-preserved direct agglutination tests (GP-DAT) as an alternative to freeze dried direct agglutination tests (FD-DAT) that use freeze-dried antigen.MethodsGlycerol-preserved DAT antigen was prepared and stored at different temperatures. We tested antigen stored at 4 °C, 22-37 °C and 50 °C over a period of 365 days. Seven hundred twenty-nine serum samples were collected from different geographical zones of Iran from 2007-2009, and 80 of these samples were pooled to produce sera. Each pooled serum contained 10 sera. All positive and negative pooled sera were separately tested for anti-L. infantum antibodies with GP-DAT, FD-DAT and formaldehyde-fixed direct agglutination test (FF-DAT) antigens; tests were performed on both human and dog sera over a period of 12 months.ResultsThere was strong agreement between the results obtained using GP-DAT and FD-DAT antigens stored at 22–37 °C for 12 months for both human (100%) and dog (100%) pooled sera. The direct agglutination test results were highly reproducible (weighted kappa: GP=0.833, FD=0.979 and FF=0.917).ConclusionsBecause GP-DAT antigen is highly stable over a range of temperatures and is easy to transport in the field, this type of antigen may be particularly useful in areas with endemic visceral leishmaniasis.     

Evaluation of direct agglutination test, rk39 Test, and ELISA for the diagnosis of visceral leishmaniasis

The study reports an evaluation of the direct agglutination test (DAT) with use of promastigote/amastigote antigen, rk39 strip test, and ELISA for diagnosis of visceral leishmaniasis (VL). Out of 94 clinically suspected VL patients, 16 (17%) were seropositive by all the techniques; in addition, 6 were positive in rk39 strip test and ELISA. On retrospective analysis, out of 16 positive by all the techniques, 11 (69%) had demonstrable Leishmania donovani (LD) bodies in their bone marrow samples, while in 5 bone marrow was not examined. Out of 6 that were positive by ELISA and rk39 strip test, 2 had myelofibrosis and 4 had chronic myeloid leukemia. On the basis of bone marrow aspirate positivity, the sensitivity and specificity of DAT were 100% while those of rk39 strip test and ELISA were 100% and 87%, respectively. The study suggests that DAT appears to be the best technique for the serodiagnosis of VL.     

Detection of urinary antigens and their seroreactivity with serum of patients in Leishmania donovani infection

ObjectiveTo detect leishmanial antigens in pre and post treated urine of visceral leishmaniasis (VL) patients.MethodsUrine and serum sample from three VL patients were collected. Ammonium sulphate precipitation and purification of urine sample was done for proteins isolation. SDS PAGE of proteins was done followed by western blotting, with the patient's pre and post treatment serum.ResultsEight proteins of molecular weights 17 kDa, 25 kDa, 28 kDa, 42 kDa, 47 kDa, 54 kDa, 60 kDa and 85 kDa were detected in the urine of VL patients before treatment. After treatment with miltefosine, none of the above proteins was detected in urine samples. The western blot analysis with pre treatment serum confirmed the antigenicity of four urinary proteins of molecular weights 25 kDa, 28 kDa, 54 kDa and 60 kDa. The seropositivity with 25 kDa and 28 kDa antigens was negative with serum obtained after the completion of treatment.ConclusionsIn the context to unavailability of a prognostic tool, urinary leishmanial antigens may offer a better choice and may also be useful as immunoprophylactic candidates.     

The sensitivity and specificity of Leishmania chagasi recombinant K39 antigen in the diagnosis of American visceral leishmaniasis and in differentiating active from subclinical infection

The sensitivity and specificity of a Leishmania chagasi recombinant K39 (rK39)-based enzyme-linked immunosorbent assay (ELISA) for visceral leishmaniasis (VL) was assessed in Natal, Brazil. Anti-rK39 antibodies were detected in 93.3% of patients with parasitologically confirmed VL (n = 120) and in 33 others with clinically diagnosed disease. Anti-rK39 antibodies decreased significantly following treatment. The presence of antibodies was inversely correlated with development of a positive leishmanin skin test result. Anti-rK39 antibodies were detected in only 2.9% of asymptomatic subjects with a positive skin test result (n = 168). They were not detected in healthy controls (n = 30) or in persons with Chagas' disease (n = 13) or active tuberculosis (n = 31). Antibodies were found in only one of 13 patients with cutaneous leishmaniasis. In contrast, an ELISA using total L. chagasi promastigote antigen was sensitive, but not specific. The results indicate that the rK39-based ELISA is a sensitive and specific diagnostic test for symptomatic VL and can differentiate progressive from self-resolving infection.