Development of a rapid loop-mediated isothermal amplification assay for diagnosis and assessment of cure of Leishmania infection.

BACKGROUND: Leishmaniasis is a spectrum of diseases with great relevance to public health. Conventional diagnostic methods are time consuming, needing trained personnel. A robust, rapid and cost effective diagnostic test is warranted for on-time diagnosis and field application. METHODS: We have developed a loop mediated isothermal amplification (LAMP) assay with primers (n = 6) based on Leishmania donovani kDNA for detection of Leishmania infection, using a closed tube to prevent cross-contamination. The assay was used to detect Leishmania infection in biological samples obtained from patients of visceral leishmaniasis (VL), post kala-azar dermal leishmaniasis (PKDL) and cutaneous leishmaniasis (CL). RESULTS: The assay was positive for L. donovani, L. tropica and L. major parasites, with the highest sensitivity towards L. donovani (1 fg DNA). The high sensitivity of the assay for detection of L. donovani was reflected in its ability to detect parasite DNA within 30 min of amplification time with a threshold detection limit of ≥25 copies per reaction. The assay detected parasite in 64 of 66 VL blood samples (sensitivity, 96.9%; 95% CI: 89.6-99.2%), 15 of 15 VL bone marrow aspirate samples (sensitivity, 100%; 95% CI:79.6-100%), 65 of 67 PKDL tissue biopsy samples (sensitivity, 97%; 95% CI:89.7-99.2%). The assay was evaluated in a few cases of CL wherein it was found positive in 8 of 10 tissue biopsies (sensitivity, 80%; 95% CI: 49-94.3%). The assay was negative in all control blood (n = 76) and tissue biopsy (n = 24) samples (specificity, 100%; 95% CI: 96.3-100%). Further, the assay was evaluated for its utility in assessment of cure in treated VL and PKDL patients. The assay detected parasite DNA in 2 of 20VL blood samples and 2 of 21 PKDL tissue samples. Out of 4 cases that were positive for parasite DNA at post treatment stage, 2 patients (1VL and 1 PKDL) returned with relapse. CONCLUSIONS: The study demonstrated a Leishmania genus specific closed tube LAMP assay for reliable and rapid molecular diagnosis of VL and PKDL with potential for application in assessment of cure.

Cutaneous leishmaniasis caused by Leishmania tropica in Bikaner, India: parasite identification and characterization using molecular and immunologic tools.

Identification of new foci of cutaneous leishmaniasis (CL), along with reports of Leishmania donovani causing the disease, is an issue of concern. Clinico-epidemiologic analysis of 98 cases in the endemic regions of Rajasthan state, India, suggested the preponderance of infection in men (62.24%) compared with women (37.75%). Species characterization by internal transcribed spacer 1 (ITS1) polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP), kDNA-PCR, and immunofluorescence assay established L. tropica as the causative organism. When applied directly to 32 clinical samples, kDNA PCR had a sensitivity of 96.6%, whereas ITS1 PCR had a sensitivity of 82.75%, thus facilitating diagnosis and species identification. Either parasite culture or direct microscopy alone detected 48.2% and 65.5% of the positive samples, respectively, whereas culture and microscopy together improved overall sensitivity to 89.3% (25/28). Except for the kDNA PCR, all other assays were 100% specific. This study provides the first comprehensive molecular and immunologic studies of CL in India.