Development and accuracy evaluation of a new loop-mediated isothermal amplification assay targeting the HSP70 gene for the diagnosis of cutaneous leishmaniasis.

Cutaneous leishmaniasis (CL) is a global public health problem caused by species on the genus Leishmania and is the most prevalent clinical form of leishmaniasis. The aim of this study was to develop a new LAMP assay for Leishmania sp. based on HSP70 gene and evaluate it clinically for molecular diagnosis of CL. The study was carried out in the following stages: i) design of primers based on HSP70 gene of Leishmania sp.; ii) evaluation of detection limit and analytical specificity; iii) estimation of the accuracy of LAMP-Leish/HSP70 assay for diagnosing CL. A total of 100 skin biopsy samples from patients, comprising 60 CL cases and 40 non-cases, were analyzed in this study. One LAMP assay using HSP70 gene as molecular target were standardized, and the observed detection limit was 100fg of L. braziliensis purified DNA. The LAMP-Leish/HSP70 assay was specific for Leishmania spp. The LAMP-Leish/HSP70 assay showed an accuracy of 92%, and positivity rates were not affected by lesion onset time or parasite load. This novel LAMP assay targeting the HSP70 gene of Leishmania sp. has the potential to be a useful tool to integrate into routine diagnosis for suspected cases of CL.

Impact assessment of different DNA extraction methods for non-invasive molecular diagnosis of tegumentary leishmaniasis.

The aim of this study was to evaluate two methods of nucleic acid extraction (spin-column-based method - commercial kit and direct boil - DB) from swab sampling compared to biopsy sampling for the diagnosis of tegumentary leishmaniasis (TL), (cutaneous - CL and mucocutaneous - MCL forms). The impact of these nucleic acid extraction protocols on different types of PCR and LAMP techniques were compared regarding nucleic acid quality, molecular assays accuracy, indirect quantitation, and costs. The evaluated patients were 57 TL cases (36 CL and 21 MCL) and 34 non-cases. Swab samples extracted by the DB method showed a higher DNA degradation rate and worse DNA quality in comparison to the commercial kit. Molecular tests performed on biopsy samples showed identical or higher performance in all analysis, as compared to their own performance on swab samples for TL (CL and MCL). However, only the SSU rRNA TaqMan™ RT-PCR test showed a significant difference between the performance of biopsy and swab samples extracted by commercial kit. The kDNA-cPCR coupled with swab extracted by commercial kit showed the highest accuracy (95.6%) for TL diagnosis. The sensitivity of the LAMP-RT 18S method in swab samples extracted with a commercial kit (82.5%) was close to that found in biopsy samples (86%) for TL diagnosis. The DB extraction method presented the lowest cost. The use of swab as a minimally-invasive sampling method, associated with an efficient nucleic acid extraction protocol, may represent a low-cost alternative for the diagnosis of CL and MCL.