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

Abstract

Leishmaniasis in humans is caused by Leishmania spp. in the subgenera Leishmania and Viannia. Species identification often has clinical relevance. Until recently, our laboratory relied on conventional PCR amplification of the internal transcribed spacer 2 (ITS2) region (ITS2-PCR) followed by sequencing analysis of the PCR product to differentiate Leishmania spp. Here we describe a novel real-time quantitative PCR (qPCR) approach based on the SYBR green technology (LSG-qPCR), which uses genus-specific primers that target the ITS1 region and amplify DNA from at least 10 Leishmania spp., followed by analysis of the melting temperature (T m ) of the amplicons on qPCR platforms (the Mx3000P qPCR system [Stratagene-Agilent] and the 7500 real-time PCR system [ABI Life Technologies]). We initially evaluated the assay by testing reference Leishmania isolates and comparing the results with those from the conventional ITS2-PCR approach. Then we compared the results from the real-time and conventional molecular approaches for clinical specimens from 1,051 patients submitted to the reference laboratory of the Centers for Disease Control and Prevention for Leishmania diagnostic testing. Specimens from 477 patients tested positive for Leishmania spp. with the LSG-qPCR assay, specimens from 465 of these 477 patients also tested positive with the conventional ITS2-PCR approach, and specimens from 10 of these 465 patients had positive results because of retesting prompted by LSG-qPCR positivity. On the basis of the T m values of the LSG-qPCR amplicons from reference and clinical specimens, we were able to differentiate four groups of Leishmania parasites: the Viannia subgenus in aggregate; the Leishmania

Comparison and clinical validation of qPCR assays targeting Leishmania 18S rDNA and HSP70 genes in patients with American Tegumentary Leishmaniasis

“…This fact can lead to a lack of specificity, increasing the risk of false-positive diagnosis. Other studies were performed based on the development and comparison of qPCR methodologies for the molecular diagnosis of leishmaniasis analyzing a significant set of clinical samples, but the majority of them, despite the use of high copy number and conserved targets [42], focus on the diagnostic of canine visceral leishmaniasis [45][46][47]. Few studies quantify parasite load of Leishmania using HSP70 or 18S rDNA targets with a proper analytical validation.…”

Real-time PCR applications for diagnosis of leishmaniasis

“…β-actin or beta-2-microglobulin), not only to monitor PCR inhibition (i.e. false-negative results), but also to normalize the parasite load to the amount of host cells [ 33 , 34 , 48 , 58 ]. Although some clinical samples may contain a small amount of parasite DNA, the use of high copy number sequence as targets (e.g.…”

Semi-Quantitative, Duplexed qPCR Assay for the Detection of Leishmania spp. Using Bisulphite Conversion Technology

“…This may also leave the laboratory open to contamination risk during these post-PCR methods. Quantitative PCR is a closed-tube system, where one step is required between DNA addition and result, and results may be read in real-time [25]. It achieves sensitivities and specificities of up to 100% [26,27].…”