Serial Quantitative PCR Assay for Detection, Species Discrimination, and Quantification of Leishmania spp. in Human Samples

Abstract

The Leishmania species cause a variety of human disease syndromes. Methods for diagnosis and species differentiation are insensitive and many require invasive sampling. Although quantitative PCR (qPCR) methods are reported for leishmania detection, no systematic method to quantify parasites and determine the species in clinical specimens is established. We developed a serial qPCR strategy to identify and rapidly differentiate Leishmania species and quantify parasites in clinical or environmental specimens. SYBR green qPCR is mainly employed, with corresponding TaqMan assays for validation. The screening primers recognize kinetoplast minicircle DNA of all Leishmania species. Species identification employs further qPCR set(s) individualized for geographic regions, combining species-discriminating probes with melt curve analysis. The assay was sufficient to detect Leishmania parasites, make species determinations, and quantify Leishmania spp. in sera, cutaneous biopsy specimens, or cultured isolates from subjects from Bangladesh or Brazil with different forms of leishmaniasis. The multicopy kinetoplast DNA (kDNA) probes were the most sensitive and useful for quantification based on promastigote standard curves. To test their validity for quantification, kDNA copy numbers were compared between Leishmania species, isolates, and life stages using qPCR. Maxicircle and minicircle copy numbers differed up to 6-fold between Leishmania species, but the differences were smaller between strains of the same species. Amastigote and promastigote leishmania life stages retained similar numbers of kDNA maxi- or minicircles. Thus, serial qPCR is useful for leishmania detection and species determination and for absolute quantification when compared to a standard curve from the same Leishmania species.