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

Abstract

The parasite protozoan Leishmania, the causative agent of leishmaniasis, includes two subgenera of medical interest: Leishmania (Leishmania) and Leishmania (Viannia). Parasite species detection and characterization is crucial to choose treatment protocols and to monitor the disease evolution. Molecular approaches can speed up and simplify the diagnostic process. In particular, several molecular assays target the mitochondrial DNA minicircle network (kDNA) that characterizes the Leishmania genus. We previously proposed a qPCR assay targeting kDNA, followed by high resolution melt (HRM) analysis (qPCR-ML) to distinguish L. (L.) infantum and L. (L.) amazonensis from L. Viannia species. Successively, this assay has been integrated with other qPCR assays, to differentiate L. (L.) infantum, L. (L.) amazonensis and L. (L.) mexicana. In this work, we tested the applicability of our qPCR-ML assay on L. (L.) donovani, L. (L.) major, L. (L.) tropica and L. (L.) aethiopica, showing that the qPCR-ML assay can also amplify Old World species, different from L. (L.) infantum, with good quantification limits (1 × 10−4–1 × 10−6 ng/pcr tube). Moreover, we evaluated 11 L. (L.) infantum strains/isolates, evidencing the variability of the kDNA minicircle target molecules among the strains/isolates of the same species, and pointing out the possibility of quantification using different strains as reference. Taken together, these data account for the consideration of qPCR-ML as a quantitative pan-Leishmania assay.

Molecular Diagnosis of Leishmaniasis: Quantification of Parasite Load by a Real-Time PCR Assay with High Sensitivity

“…have 35 to 36 chromosomes in their nuclear genomes [ 35 , 36 ], and it is characterized by both mini and maxi circles kinetoplast DNA structures [ 37 ]. Various chromosomal and kinetoplast DNA sequences have been evaluated as targets for diagnostic systems and the extra-chromosomal circles showed always the best sensitivity and represent currently the most employed target with worldwide diffusion [ 38 , 39 ]. The use of high-copy 18S and minicircle kDNA sequences as a target usually allows amplification at the genus or subgenus level, due to their conserved sequences or to the heterogeneity of minicircle classes [ 21 ].…”

A novel leishmanial copper P-type ATPase plays a vital role in parasite infection and intracellular survival

“…as evident from Leishmania -specific kinetoplast DNA transcript level ( Fig. S4 A ) ( 39 ). The results suggested that host Cu uptake and utilization is downregulated during the early stages of infection.…”

Case report: Diagnosis of visceral leishmaniasis using metagenomic next-generation sequencing and bone marrow smear

“…However, the specificity is low, and false-positive results may occur (van Griensven and Diro, 2019;Scarpini et al, 2022). In recent years, some studies have found that qPCR analysis of mitochondrial DNA minicircle network (kDNA) based on the characteristics of Leishmania genus has high sensitivity and specificity, and can thus be useful for quantitative detection of pan-Leishmania (Ceccarelli et al, 2020;de Carvalho et al, 2022;Mota et al, 2022). However, this technique is expensive and has not been used for routine clinical testing.…”