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.

Field Validation of SYBR Green- and TaqMan-Based Real-Time PCR Using Biopsy and Swab Samples To Diagnose American Tegumentary Leishmaniasis in an Area Where Leishmania (Viannia) braziliensis Is Endemic

“…We evaluated primer and probe specificity for Leishmania (V.) braziliensis using NCBI BLAST. Two primer pairs were ultimately selected: MP1L/MP3H (for SYBR Green qPCR) (34) and L. braziliensis kDNA3 (for TaqMan qPCR) (33). For the L. braziliensis kDNA3 primer pair, we opted to use the described probe to enhance specificity.…”

“…Primer pair choices were based on previously described sequences that amplify fragments of the Leishmania (V.) braziliensis minicircle kDNA (5,(32)(33)(34)(35)(36). We evaluated primer and probe specificity for Leishmania (V.) braziliensis using NCBI BLAST.…”

“…For the L. braziliensis kDNA3 primer pair, we opted to use the described probe to enhance specificity. Standard curves were generated using Leishmania (V.) braziliensis (MHOM/BR/1994/M15176) DNA from 6 ϫ 10 5 to 6 ϫ 10 Ϫ5 parasite DNA equivalents/reaction for MP1L/MP3H and from 2.4 ϫ 10 5 to 2.4 ϫ 10 Ϫ5 parasite DNA equivalents/reaction for L. braziliensis kDNA3, as described elsewhere (33,34).…”

“…TaqMan qPCR was performed with primers targeting sequences of the kDNA of Leishmania (Viannia) and a hydrolysis probe specific for Leishmania (V.) braziliensis, namely, primers 5=-TGCTATAAAATCGTAC CACCCGACA-3= and 5=-GAACGGGGTTTCTGTATGCCATTT-3=, and probe FAM-TTGCAGAACGCCCCTACCCA GAGGC-TAMRA (FAM, 6-carboxyfluorescein; TAMRA, 6-carboxytetramethylrhodamine), resulting in an amplified product of 83 bp (33). The reaction was performed in a final volume of 20 l, consisting of 1ϫ TaqMan Universal master mix (Life Technologies, Carlsbad, CA, USA), 400 nM each primer, 250 nM probe, 2 l of ultrapure water, and 20 ng of DNA template.…”

Detection and Differentiation of Leishmania spp. in Clinical Specimens by Use of a SYBR Green-Based Real-Time PCR Assay

“…The advantages of real-time quantitative PCR (qPCR) approaches in comparison with conventional PCR assays include the potential for increased sensitivity and specificity and for a decreased risk of contamination of the laboratory environment (14,(18)(19)(20)(21)(22)(23)(24)(25)(26). The efficiency of real-time platforms for Leishmania species identification has been discussed (24,(27)(28)(29)(30). Although real-time PCR approaches have the potential for identifying different Leishmania spp.…”