Comparison of PCR-based diagnoses for visceral leishmaniasis in Bangladesh.

The diagnosis of visceral leishmaniasis (VL) is performed using multiple methods encompassing parasitological, serological and nucleic acid-based diagnostic tools, each method with its own unique advantages and disadvantages. Conventional parasitological methods are risky for the patient and require skilled personnel to collect specimens from spleen or bone marrow, and hence they are not generally available in impoverished areas. Polymerase chain reaction (PCR) has been validated as an excellent alternative to microscopy in terms of sensitivity and specificity. Here, we evaluate four different PCR assays targeting ITS1, ITS2, mini-exon and small subunit-rRNA (SSUrRNA) using DNA extracted from peripheral blood buffy coat in order to avoid more invasive processes. A total of 61 VL patients and 75 non-VL infected control individuals were enrolled. The VL patients were confirmed to be positive for Leishmania amastigotes in splenic smears by microscopy. Sensitivities of the PCR targeting ITS1, ITS2, SSUrRNA and mini-exon were 96.7%, 91.8%, 88.5% and 34.4%, respectively, while the specificity was 98.7% for all methods. Nested PCR for ITS1 resulted in 100% sensitivity. The efficacy of each PCR was evaluated with various Leishmania amastigote parasite loads in each spleen smear, graded from 1+ to 5+. The PCR targeting ITS1 showed 100% sensitivity for the detection of Leishmania donovani in all samples from grades ≥3, ≥4, and ≥5, respectively. The restriction fragment length polymorphism observed in ITS1 amplicons digested by HaeIII classified the parasite into L. donovani complex. The ITS1 PCR was found to be equal to conventional, but very invasive and risky parasitological diagnoses and superior to other PCR based methods in sensitivity and examination of genetic heterogeneity. We recommend the PCR targeting ITS1 using peripheral blood buffy coat DNA as an alternate, less invasive diagnostic choice for the confirmation of L. donovani infection.

Diagnostic accuracy of loop-mediated isothermal amplification (LAMP) for detection of Leishmania DNA in buffy coat from visceral leishmaniasis patients.

BACKGROUND: Visceral leishmaniasis (VL) remains as one of the most neglected tropical diseases with over 60% of the world's total VL cases occurring in the Indian subcontinent. Due to the invasive risky procedure and technical expertise required in the classical parasitological diagnosis, the goal of the VL experts has been to develop noninvasive procedure(s) applicable in the field settings. Several serological and molecular biological approaches have been developed over the last decades, but only a few are applicable in field settings that can be performed with relative ease. Recently, loop-mediated isothermal amplification (LAMP) has emerged as a novel nucleic acid amplification method for diagnosis of VL. In this study, we have evaluated the LAMP assay using buffy coat DNA samples from VL patients in Bangladesh and compared its performance with leishmania nested PCR (Ln-PCR), an established molecular method with very high diagnostic indices. METHODS: Seventy five (75) parasitologically confirmed VL patients by spleen smear microcopy and 101 controls (endemic healthy controls -25, non-endemic healthy control-26, Tuberculosis-25 and other diseases-25) were enrolled in this study. LAMP assay was carried out using a set of four primers targeting L. donovani kinetoplast minicircle DNA under isothermal (62 °C) conditions in a heat block. For Ln-PCR, we used primers targeting the parasite's small-subunit rRNA region. RESULTS: LAMP assay was found to be positive in 68 of 75 confirmed VL cases, and revealed its diagnostic sensitivity of 90.7% (95.84-81.14, 95% CI), whereas all controls were negative by LAMP assay, indicating a specificity of 100% (100-95.43, 95% CI). The Ln-PCR yielded a sensitivity of 96% (98.96-87.97, 95% CI) and a specificity of 100% (100-95.43, 95% CI). CONCLUSION: High diagnostic sensitivity and excellent specificity were observed in this first report of LAMP diagnostic evaluation from Bangladesh. Considering its many fold advantages over conventional PCR and potential to be used as a simple and rapid test in the VL endemic areas of the Indian subcontinent, our findings are encouraging, but further evaluation of LAMP is needed.