Differentiation of Leishmania (L.) infantum, Leishmania (L.) amazonensis and Leishmania (L.) mexicana Using Sequential qPCR Assays and High-Resolution Melt Analysis

Abstract

Leishmania protozoa are the etiological agents of visceral, cutaneous and mucocutaneous leishmaniasis. In specific geographical regions, such as Latin America, several Leishmania species are endemic and simultaneously present; therefore, a diagnostic method for species discrimination is warranted. In this attempt, many qPCR-based assays have been developed. Recently, we have shown that L. (L.) infantum and L. (L.) amazonensis can be distinguished through the comparison of the Cq values from two qPCR assays (qPCR-ML and qPCR-ama), designed to amplify kDNA minicircle subclasses more represented in L. (L.) infantum and L. (L.) amazonensis, respectively. This paper describes the application of this approach to L. (L.) mexicana and introduces a new qPCR-ITS1 assay followed by high-resolution melt (HRM) analysis to differentiate this species from L. (L.) amazonensis. We show that L. (L.) mexicana can be distinguished from L. (L.) infantum using the same approach we had previously validated for L. (L.) amazonensis. Moreover, it was also possible to reliably discriminate L. (L.) mexicana from L. (L.) amazonensis by using qPCR-ITS1 followed by an HRM analysis. Therefore, a diagnostic algorithm based on sequential qPCR assays coupled with HRM analysis was established to identify/differentiate L. (L.) infantum, L. (L.) amazonensis, L. (L.) mexicana and Viannia subgenus. These findings update and extend previous data published by our research group, providing an additional diagnostic tool in endemic areas with co-existing species.

Evaluation of a kDNA-Based qPCR Assay for the Detection and Quantification of Old World Leishmania Species

“…panamensis , L. ( V .) braziliensis ] and among these species, distinguishing between Leishmania and Viannia subgenera [ 6 , 8 ]. In this work, we show for the first time that the qPCR-ML assay can also amplify the Old World species different from L. ( L .)…”

“…Moreover, the use of FRET probes for species discrimination [ 31 ] could increase the cost compared to approaches based on intercalating dyes. The lack of species specificity of the qPCR-ML assay can be overcome using sequential PCR assays, as already demonstrated [ 8 ] or, in part, with the contextualization between the clinical presentation and the geographical origin of the patient.…”

“…amazonensis [ 7 ] and from L. ( L .) mexicana [ 8 ]. These approaches have also been applied to Brazilian clinical samples, allowing to distinguish different species causing infection in the New World [ 9 ].…”

Multiplex PCR Detection of Five Common Respiratory Pathogens from Bronchoalveolar Lavages Using High Resolution Melting Curve Analysis

“…One of these methods is high-resolution melting (HRM) analysis, which relies on a real-time PCR method in the presence of the double stranded DNA (dsDNA) intercalating uorescent dye and monitors changes in melting of dsDNA with increasing temperature [10][11][12][13][14]. HRM has been used for genotyping, detection of bacterial resistance genes, as well as for detection and differentiation of various pathogenic organisms such as bacteria, fungi, viruses and parasites [11,[15][16][17][18][19][20][21]. However, this method has not been applied directly on BAL samples to simultaneously distinguish several pathogens.…”