We have applied a loop-mediated isothermal amplification (LAMP) technique to detect Leishmania donovani DNA. The LAMP technique detected 1 fg of L. donovani DNA, which was 10-fold more sensitive than a conventional polymerase chain reaction (PCR). All nested PCR–positive blood samples from visceral leishmaniasis patients were positive with the LAMP technique, and DNA samples from L. infantum, L. major, L. mexicana, L. tropica, L. braziliensis, Plasmodium falciparum, and healthy humans were negative with the LAMP technique. The advantages of the LAMP method are its shorter reaction time, a lack of requirement of sophisticated equipment, and visual judgment of positivity based on the turbidity of reaction mixture. Our LAMP technique can be a better alternative to a conventional PCR, especially under field conditions.

We developed an enzyme-linked immunosorbent assay (ELISA) that detects filaria-specific immunoglobulin G4 antibodies in unconcentrated urine. The ELISA was positive in 87 of 91 (95.6%) urine samples collected from people with Wuchereria bancrofti microfilariae, antigen, or both. Of 298 urine samples collected in Thailand, Lao People's Democratic Republic, and Japan, where no human filariasis is known, 295 (99.0%) were negative by ELISA. Various intestinal nematode and fluke infections did not interfere with the ELISA. Urine samples with sodium azide could be kept at 37 degrees C for 4 weeks, and the time of urine collection did not influence ELISA results. This ELISA can be used to identify endemic foci of filariasis.

Visceral leishmaniasis (VL), caused by an intracellular parasite Leishmania donovani in the Indian subcontinent, is considered to be anthroponotic. The role of domestic animals in its transmission is still unclear. Although cattle are the preferred blood host for Phlebotomus argentipes, the sandfly vector of VL in the Indian subcontinent, very little information is available for their role in the disease transmission. In this study, we examined domestic cattle for serological and molecular evidence of Leishmania infection in a VL-endemic area in Bangladesh. Blood samples from 138 domestic cattle were collected from houses with active or recently-treated VL and post-kala-azar dermal leishmaniasis patients. The presence of anti-leishmanial antibodies in serum was investigated using enzyme-linked immunosorbent assay (ELISA) and then with direct agglutination tests (DAT). Nested PCR (Ln PCR) was performed to amplify the ssu-rRNA gene using the DNA extracted from Buffy coat. Recently-developed molecular assay loop-mediated isothermal amplification (LAMP) was also performed for further sensitive detection of parasite DNA. In this study, 9.4% (n = 13) of the cattle were found to be positive by ELISA. Of the 13 ELISA-positive cattle, only four (30.8%) were positive in DAT. Parasite DNA was not detected in either of the molecular assays (Ln PCR and LAMP). The study confirmed the presence of antibodies against Leishmania parasite in cattle. However, the absence of Leishmania DNA in the cattle indicates clearly that the cattle do not play a role as reservoir host. Similar study needs to be undertaken in the Indian subcontinent to determine the role of other domestic animals on which sandflies feed.

Diagnosis of visceral leishmaniasis (VL) by demonstration of parasites in tissue smears obtained from bone marrow, spleen or lymph nodes is risky, painful, and difficult. The rK-39 strip test is widely used for the diagnosis of VL using blood/serum samples in endemic countries. The aim of the study was to evaluate the rK-39 strip test using urine sample as a non-invasive means for the diagnosis of VL. The rk-39 strip test was performed using urine from 100 suspected VL cases along with 25 disease control (malarial febrile cases) and 50 healthy control (from endemic and non-endemic areas). All the VL suspected cases were positive with the rK-39 strip test using serum. The sensitivity and specificity of the rK-39 strip test using urine samples was 95% and 93.3%, respectively, compared to serum based rK-39 test. The findings suggest that the urine based rK-39 test could be a practical and efficient tool for the diagnosis of VL patients in rural areas, particularly where resources are limited.

We reported a highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) that detects immunoglobulin G (IgG) in urine using rKRP42 antigen for the diagnosis of visceral leishmaniasis (VL). The ELISA was applied to study chronological change in antibody titers in five study areas in Rajshahi district, Bangladesh. A total of 585 subjects without a past VL history were examined at least three times in the 30-month follow-up period; of these subjects, 137 (23.4%) subjects became ELISA-positive at least one time during the study. Among the positive cases, 40 (29.2%) subjects developed clinical VL, and 31 (77.5%) of these subjects showed IgG titers of ≥ 1,000 U more than one time in the study period. Considering only the first ELISA results, 22 subjects with IgG titers of ≥ 1,000 U could be found, and 21 (95.5%) of these subjects turned out to be clinical cases. The high urinary IgG titers (≥ 1,000 U) will help predict possible clinical VL cases and thus, identify an outbreak in its earlier stage.