Use of the recombinant K39 dipstick test and the direct agglutination test in a setting endemic for visceral leishmaniasis in Nepal

We evaluated the field use of two serologic tests for visceral leishmaniasis (VL), the direct agglutination test (DAT) and rK39 dipstick test, in the context of a case-control study. Most VL cases in Nepal are currently diagnosed on clinical grounds and with relatively non-specific tests such as the formol-gel test. Among 14 newly diagnosed VL patients with bone-marrow slides confirmed positive in two independent laboratories, the sensitivity of both tests was 100%. Among 113 controls with no personal or household history of VL, the specificity of the rK39 was 100% while that of the DAT was 93%. The rK39 was less expensive than DAT, and has the advantages of ease of use and obtaining results within minutes. The wider use of the rK39 dipstick test could improve the specificity of VL diagnosis in Nepal.     

Evaluation of a new recombinant K39 rapid diagnostic test for Sudanese visceral leishmaniasis

A new rK39 rapid diagnostic dipstick test (DiaMed-IT-Leish) was compared with aspiration and a direct agglutination test (DAT) for diagnosis of visceral leishmaniasis (VL) in 201 parasitologically confirmed cases, 133 endemic controls, and in 356 clinical suspects in disease-endemic and -epidemic areas in Sudan. The sensitivity of the rK39 test in parasitologically confirmed VL cases was 90%, whereas the specificity in disease-endemic controls was 99%. The sensitivity of the DAT was 98%. In clinically suspected cases, the sensitivity of the rK39 test was 81% and the specificity was 97%. When compared with the diagnostic protocol based on the DAT and aspiration used by Médecins sans Frontières in epidemic situations, the positive predictive value was 98%, and the negative predictive value was 71%. This rK39 rapid diagnostic test is suitable for screening as well as diagnosis of VL. Further diagnostic work-up of dipstick-negative patients with clinically suspected VL is important. The ease and convenience of the dipstick test will allow decentralization and improved access to care in disease-endemic areas in Sudan.     

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.     

Positive response to sodium antimony gluconate administration in visceral leishmaniasis seropositive patients.

In a prospective study conducted in Mymensingh district of Bangladesh, 1, 273 patients were assessed for the presence of visceral leishmaniasis (VL). Sodium antimony gluconate (SAG) was successfully administered to 715 patients with parasitologically confirmed infection. In the remaining 558, although there was clinical indication of VL, Leishmania donovani parasites could not be demonstrated. Administration of SAG in this group was on the grounds of the prevailing symptoms, exclusion of malaria and a positive direct agglutination test (DAT). Significant improvements in the clinical and hematological parameters were observed in 547 (98%) of the unconfirmed VL cases. On the basis of the parasitological findings or positive response to specific anti-Leishmania chemotherapy, the sensitivity and specificity of the DAT were 99.6% and 97.7% respectively. The results supported the reliability of DAT for diagnosis of VL at levels below that of parasitological detection.     

Direct agglutination test with urine samples for the diagnosis of visceral leishmaniasis

A new direct agglutination test (DAT) for use with urine samples for the diagnosis of visceral leishmaniasis (VL) has been developed and compared with the conventional DAT with serum samples and our previously reported enzyme-linked immunosorbent assay (ELISA) with urine samples (urine ELISA). The new DAT, in which anti-human IgG was used as enhancing antibody, was tested with urine samples from 75 VL patients and 225 non-VL patients and healthy people. The sensitivity of the new DAT (90.7%), was almost the same as that of the conventional DAT (91.0%) and the urine ELISA (93.3%). The specificity of the new DAT (96.4%) was nearly identical with that of the urine ELISA (97.3%). A urine-based DAT has several advantages over the conventional DAT: sample collection is non-invasive and it can process larger numbers of samples with smaller amounts of antigen.     

Latent class analysis of diagnostic tests for visceral leishmaniasis in Brazil

Objective To estimate the sensitivities and specificities of different diagnostic tests for visceral leishmaniasis (VL) using latent class analysis (LCA). Methods This study was performed using data from a prospective study conducted in four Brazilian states from May 2004 to May 2007. Five diagnostic tests for VL were evaluated in 285 VL cases and 119 non‐cases: microscopy, indirect fluorescence antibody test (IFAT), enzyme‐linked immunosorbent assay using recombinant K39 antigen (rK39‐ELISA), direct agglutination test (DAT) and the rK39 rapid test. Results Microscopy showed sensitivity of 77.0% (CI: 71.5–81.5) and specificity of 99.0% (CI: 94.0–99.7). The IFAT and the DAT showed similar sensitivities, 88.3% (CI: 84.0–92.0) and 88.5% (CI: 84.1–92.0), respectively, but the DAT had a higher specificity (95.4%, CI: 89.2–98.1) than did the IFAT (83.0%, CI: 75.0–88.2). The rK39‐ELISA and the rK39 rapid test showed sensitivities of 99.0% (CI: 96.3–99.6) and 94.0% (CI: 90.1–96.3), and specificities of 82.5% (CI: 75.0–88.3) and 100% (CI: 97.0–100.0%), respectively. Conclusions Considering the lack of an adequate reference standard, LCA proved to be a useful tool in validating diagnostic methods for VL. The DAT and the rK39 rapid test showed better performance. Thus, clinically suspected cases of VL in a Brazilian endemic area could be treated based on the positivity of one of these tests.     

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.     

Performance of Latex Agglutination Test (KAtex) In Diagnosis of Visceral Leishmaniasis in Iran

Background: Visceral leishmaniasis (VL) is an endemic disease in some parts of Iran. Many techniques have been used for diagnosis of VL, among which the urine based la-tex agglutination test (KAtex) is a promising one. Objective: To compare three diag-nostic tests of VL including KAtex, ELISA and Direct Agglutination Test (DAT) in VL patients and healthy controls in the south west of Iran. Methods: Serum (n = 29) and urine samples (n = 31) were collected from parasitologically confirmed VL patients. Control samples were obtained from healthy individuals (n = 61) and also from patients with infectious diseases other than VL. The collected serum samples were tested by DAT and ELISA using crude antigen from promastigotes of Leishmania infantum and the urine samples were tested by KAtex. Results: Sensitivity and specificity of KAtex for diagnosis of VL was found to be 83.9% and 100%, respectively. Sensitivities of DAT and ELISA were 93.1% and 86.2% and their specificities were 100% and 90.5%, respectively. Conclusion: KAtex yielded a satisfactory sensitivity and specificity in di-agnosis of VL in Iran and can be recommended as a rapid, field applicable and reliable test for diagnosis of VL in this region.     

A comparative study of the effectiveness of diagnostic tests for visceral leishmaniasis

We compared the validity of pancytopenia, the formol-gel test (FGT), the indirect fluorescence antibody test (IFAT), the direct agglutination test (DAT), and the rK39 dipstick test as diagnostic criteria for visceral leishmaniasis (VL) in Nepal. Between September 2000 and January 2002, 310 clinical suspects had a bone marrow aspirate, and if negative, a spleen aspirate smear examined for Leishmania donovani. Sensitivity and specificity of all tests were determined compared with parasitology and by latent class analysis (LCA). Compared with parasitology, the sensitivities of the other tests were as follows: pancytopenia = 16.3% (95% confidence interval [CI] = 11.3-22.5%), FGT = 39.9% (95% CI = 32.7-47.4%), IFAT = 28.4% (95% CI = 22.0-35.5%), DAT = 95.1% (95% CI = 90.8-97.7%), and the rK39 dipstick test = 87.4% (95% CI = 81.7-91.9%). Sensitivity estimates obtained by LCA were similar, but specificity estimates were substantially higher (DAT = 93.7% versus 77.8%; rK39 dipstick test = 93.1% versus 77.0%). The DAT or the rK39 dipstick test can replace parasitology as the basis of a decision to treat VL in Nepalese peripheral health services.     

Evaluation of direct agglutination test (DAT) as an immunodiagnostic tool for diagnosis of visceral leishmaniasis in Nepal

Before field application of the direct agglutination test (DAT) for leishmaniasis, it was assessed as a diagnostic tool. Fifteen confirmed visceral leishmaniasis cases (bone marrow aspiration positive), 120 tuberculosis, 58 leprosy, 15 malaria, 26 intestinal parasitic infection cases, 24 endemic healthy controls from adjacent to the study area, and 18 controls from Kathmandu (who had never visited the VL endemic areas) were tested for anti-leishmanial antibody agglutination titers. Two of the tuberculosis cases were positive for anti-leishmanial agglutinating antibodies at 1:800. All the visceral leishmaniasis confirmed cases were reactive to anti-leishmanial antibody at > or = 1:3,200. Other specimens were negative for serology. The sensitivity of the direct agglutination test was 100% and the specificity was 99.2%. The direct agglutination test had positive and negative predictive values of 100% and 99.2% respectively. The direct agglutination test has been found to be simple, rapid, reliable, economic, safe and adaptable to micro-techniques using microtiter plates. It is specific and sensitive. The direct agglutination test is simple enough for it to be performed in a field laboratory.     

Field validity, reproducibility and feasibility of diagnostic tests for visceral leishmaniasis in rural Nepal

OBJECTIVES: To assess the field accuracy, reproducibility and feasibility of the formol gel test (FGT), the urine latex agglutination test (KAtex) and a rK39 antigen-based dipstick for the diagnosis of visceral leishmaniasis (VL) in rural Nepal. METHOD: Patients with clinical suspicion of VL were recruited at Rangeli District Hospital (DH), a 15-bed government hospital located in south-eastern Nepal. FGT, KAtex and rK39 dipstick tests were performed on site and later repeated at a reference kala-azar diagnostic laboratory to assess reproducibility. Diagnosis of VL was confirmed by either a positive bone marrow aspirate examination or a positive direct agglutination test (DAT titre > or = 1:3200) in patients who later responded to anti-leishmanial therapy. RESULTS: Of 155 patients initially recruited, 142 (85 with VL and 57 with another diagnosis) were included in the study. The sensitivity of the rK39 dipstick [89%; 95% confidence interval (CI): 81-94] was significantly higher than that of the KAtex (57%; 95% CI: 46-67) and the FGT (52%; 95% CI: 41-62). All three tests had a specificity of at least 90%. Agreement was higher for the rK39 dipstick (kappa = 0.87) than for the FGT (0.68) and the KAtex (0.43). All tests required < or = 20 min of actual work and < or = 40 min to obtain the results. CONCLUSION: The rK39 dipstick was easy to do, more accurate and reproducible than other rapid diagnostic tests for VL in a DH of rural Nepal. It should be integrated into the field diagnostic algorithm of VL in this region and mechanisms to secure its availability should be found.     

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.     

Pediatric visceral leishmaniasis in Albania.

OBJECTIVE: Visceral leishmaniasis (VL) in children is endemic in southern Europe but has not been previously reported from Albania. This prospective study reports the clinical and laboratory findings in 50 children with visceral leishmaniasis, the value of a direct agglutination test (DAT), and the result of treatment with meglumine antimonate. MATERIALS AND METHODS: Sera obtained from 50 children with VL confirmed by bone marrow examination, 40 household contacts, and 30 hospitalized children with other infections were examined using DAT. RESULTS: Clinical features included fever (100%), hepatosplenomegaly (100%), pallor (100%), weight loss (98%), vomiting (68%), diarrhea (32%), and bleeding disorders (8%). Laboratory findings were anemia (94%), neutropenia (85%), hypergammaglobulinemia (70%), and thrombocytopenia (22%). Thirty children who developed secondary bacterial infections had significantly lower hemoglobin and neutrophil counts (P<0.0001). Direct agglutination test had a sensitivity of 98%, a specificity of 100%, and a positive predictive value of 100%. One child with severe generalized bleeding died within 48 hours of admission before receiving treatment. CONCLUSION: The direct agglutination test was highly valuable in diagnosis of VL in this series. Meglumine antimonate was an effective therapeutic agent. Post-treatment bone marrow examination confirmed recovery in all patients. There were no relapses of VL during one-year follow up.     

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.     

Utility of lymph node aspiration in the diagnosis of visceral leishmaniasis in Sudan

We evaluated lymph node aspiration (LNA) as a simple diagnostic procedure for visceral leishmaniasis (VL). Lymph node aspiration was compared with the direct agglutination test (DAT) using a diagnostic titer > or = 1:6,400 in 7,880 suspected VL patients in eastern Sudan. Compared with DAT, LNA had a sensitivity of 65.1% (95% confidence interval = 63.5-66.6%). Parasite density in LNA correlated strongly with DAT titers (P < 0.0001), and low parasite density accounted for 78.1% of positive LNA results with DAT titers < 1:6,400 (n = 782). Risk factors predictive of a positive LNA result were an age of 1-29 years, male sex, a hemoglobin level < 10.0 g/dL, a DAT titer > or = 1:800, and a location with a higher prevalence of VL. Lymph node and splenic aspirations were similarly accurate as tests of cure after treatment of 50 VL patients in southern Sudan. Pre-treatment LNA results were negative in 20 cases of severe post kala-azar dermal leishmaniasis.     

Evaluation of rK-39 Strip Test Using Urine for Diagnosis of Visceral Leishmaniasis in an Endemic Region of India

The definitive diagnosis of visceral leishmaniasis (VL) requires invasive procedures for demonstration of parasites in tissue smear or culture. These procedures need expertise and laboratory supports and cannot be performed in the field. The aim of the present study was to evaluate the existing rK-39 immunochromatographic nitrocellulose strips test (ICT) with some modification in human urine for diagnosis of VL. The test was performed on both sera and urine samples on the same 786 subjects (365 confirmed VL and 421 control subjects). The sensitivity of the rK-39 ICT in serum was 100%, whereas the specificity was 93.8%, 100%, and 96.2% in healthy controls from endemic, non-endemic, and other infectious diseases, respectively. However, in urine samples, the test showed 96.1% sensitivity and 100% specificity. Considering sensitivity and feasibility of the test in the field, rK-39 ICT using urine samples can be an alternative to conventional invasive VL diagnosis.     

Comparative evaluation of parasitology and serological tests in the diagnosis of visceral leishmaniasis in India: a phase III diagnostic accuracy study

In this phase III trial for diagnostics for visceral leishmaniasis (VL) in India, we compared parasitological diagnosis with several serological tests: direct agglutination test (freeze dried; DAT-FD), rK-39 strip test, rK-26 strip test and a latex agglutination test for antigen detection in urine (KAtex) in 452 subjects from the endemic regions of Bihar, India. The subjects were segregated into four categories: 230 confirmed patients, 52 probable cases, 70 non-cases and 100 healthy endemic controls. The first two groups were used for estimating sensitivity, the latter two for specificity. Sensitivity of DAT-FD was 98.9%, rK-39: 98.9%, KAtex: 67.0% and rK-26: 21.3%. Sensitivity of DAT-FD on blood taken on filter paper (DAT-FDF) was 99.3%, which was comparable with that using serum. Specificity of serological tests was comparable and high (DAT-FD and DAT-FDF: 94%, rK-39 strip test: 97%, KAtex: 99% and rK-26 strip test: 100%). The classical 'gold standard' parasitological demonstration in splenic smear performed poorly as it missed 18.4% of cases that benefited from VL treatment. Reproducibility of the serological tests between field and central laboratories was excellent (kappa = 1.0, 0.99, 0.96 and 0.94 respectively for microscopy, DAT-FD, rK-39 strip test and rK-26 strip test). A high degree of agreement was observed between DAT-FD and rK-39 strip test (kappa = 0.986). Although DAT-FD and rK-39 strip test were highly sensitive with excellent specificity, the ease of use of the latter makes it most suitable for the diagnosis of VL in the field conditions.     

Diagnosing visceral leishmaniasis with the recombinant K39 strip test: experience from the Sudan

We compared a strip test employing recombinant K39 (rK39) antigen and protein A/colloidal gold as read-out agents with the rK39 ELISA for IgM and IgG antibodies and the direct agglutination test (DAT) using 55 sera from patients with parasitologically confirmed visceral leishmaniasis (VL). The rK39 strip test was positive in 37/55 (67%), the DAT in 50/55 (91%) at > or = 1 : 1600 cut-off value and in 47/55 (85%) at > or = 1 : 6400 cut-off value. The rK39-ELISA gave positive IgG results for all sera; those who had a positive strip test had significantly higher IgG levels than those with a negative strip test (31.1 (SD=3.6) and 17.7 U/ml (SD=9.8), respectively, P < 0.0001). A total of 31/55 (56%) sera showed a positive IgM result; of these 27 (49%) had a positive strip test. We tested 115 apparently cured VL patients with the strip test during follow-up; 68 were also tested with DAT. In the strip test, 25-43% of patients had a positive result at time points 3, 6, 9 and 12 months after treatment; for DAT (cut-off > or = 1 : 1600) these results were 67-83%. In neither test did a significant decrease in positivity rates occur over time (P=0.37 for the strip test, P=0.17 for the DAT). No correlation (P=0.33) was found between a positive strip test and a positive DAT result (cut-off > or = 1: 1600), indicating that the strip test and DAT are complementary rather than interchangeable. Of 61 endemic controls two (3%) had a positive strip test result; both had a positive leishmanin skin test. The rK39 strip test has the ideal format for use in the field, but its sensitivity is limited; like DAT, but to a lesser extent, it remains positive after treatment.     

Latent class analysis permits unbiased estimates of the validity of DAT for the diagnosis of visceral leishmaniasis

BACKGROUND: Substantial uncertainty surrounds the specificity of the Direct Agglutination Test (DAT) for visceral leishmaniasis (VL) in clinical suspects, since no good gold standard exists for unequivocally identifying diseased subjects. We explored the Latent Class Analysis (LCA) modelling technique to circumvent this problem. PATIENTS AND METHODS: Data on 149 clinical suspects recruited in 1993-96 during a multicentre study in Sudan were re-examined. Clinical data, lymph node and bone marrow aspirate and DAT results were available. IFAT was performed in 1997 on stored filter paper blood of 80 individuals. Classical Validity Analysis (CVA) in a 2 x 2 contingency table with parasitology as a gold standard was compared with the parameter estimates produced by the best fitting LCA model. RESULTS: The sensitivity estimates of DAT produced by CVA (98% (89%-100%)) were almost exactly reproduced by LCA. The specificity estimates by LCA were substantially higher than those obtained in CVA. Specificity of DAT depended, however, on whether the subject was treated for VL before. In subjects without prior treatment, CVA estimated DAT specificity at 68% (56%-79%), whereas LCA estimated it at 85% (63%-100%). CONCLUSION: LCA modelling proved a useful tool, as it gave consistent estimates of test characteristics and allowed for control of confounding factors and interaction effects. Since VL is a life-threatening disease for which expensive but effective and safe treatment exists, a clinical suspect in an endemic area should be treated on the basis of a positive DAT result.     

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.