Abstract
Leishmaniasis is caused by the flagellated protozoan Leishmania, and is a neglected tropical disease (NTD), as defined by the World Health Organisation (WHO). Bisulphite conversion technology converts all genomic material to a simplified form during the lysis step of the nucleic acid extraction process, and increases the efficiency of multiplex quantitative polymerase chain reaction (qPCR) reactions. Through utilization of qPCR real-time probes, in conjunction with bisulphite conversion, a new duplex assay targeting the 18S rDNA gene region was designed to detect all Leishmania species. The assay was validated against previously extracted DNA, from seven quantitated DNA and cell standards for pan-Leishmania analytical sensitivity data, and 67 cutaneous clinical samples for cutaneous clinical sensitivity data. Specificity was evaluated by testing 76 negative clinical samples and 43 bacterial, viral, protozoan and fungal species. The assay was also trialed in a side-by-side experiment against a conventional PCR (cPCR), based on the Internal transcribed spacer region 1 (ITS1 region). Ninety-seven percent of specimens from patients that previously tested positive for Leishmania were positive for Leishmania spp. with the bisulphite conversion assay, and a limit of detection (LOD) of 10 copies per PCR was achieved, while the LOD of the ITS1 methodology was 10 cells/1000 genomic copies per PCR. This method of rapid, accurate and simple detection of Leishmania can lead to improved diagnosis, treatment and public health outcomes.
Highlights
Leishmaniasis is an infection caused by some species of Leishmania parasites that affect the skin, organs and mucosal regions of the body, leading to serious morbidity and possibly death
DNA was purified from cell-cultured promastigotes of the following species: L. donovani (MHOM/IN/80/DD8 supplied at 2.3 × cells/mL), L. braziliensis (MHOM/BR/75/M2903 supplied at 1.63 × cells/mL), L. tropica (MHOM/SU/74/K27 supplied at 1.03 × 107 cells/mL), L. amazonensis (MHOM/BR/73/M2269 supplied at 9.9 × 106 cells/mL), L. mexicana (MHOM/BZ/82/BEL21 supplied at 1.51 × 108 cells/mL) and L. major (MHOM/SU/73/5-ASKH supplied at 7.1 × 106 cells/mL), obtained from the American type culture collection (ATCC, Manassas, USA)
DNA converted from the panel of seven Leishmania quantitated standards (L. donovani, L. braziliensis, L. tropica, L. amazonensis, L. major, L. mexicana and L. infantum) were detected by the 18S rDNA assay (Table 2)
Summary
Leishmaniasis is an infection caused by some species of Leishmania parasites that affect the skin, organs and mucosal regions of the body, leading to serious morbidity and possibly death. It is classed as a Neglected Tropical Disease (NTD), affecting 12 million people worldwide, with a further 350 million people at risk of contracting the disease [1]. Many validated molecular Leishmania detection assays use conventional PCR (cPCR) for the detection of Leishmania infection [3,4,5]. Conventional PCR is a diagnostic method where DNA is amplified using a thermal cycler, amplicons are separated due to molecular weight by electrophoresis, and detected by stain (usually ethidium bromide or gel red) and UV light (via a transilluminator) [6]
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