Sensitive Molecular Diagnostics for Cutaneous Leishmaniasis

Abstract

Background: Rapid diagnosis of cutaneous leishmaniasis (CL) and identification of Leishmania species is highly important for the disease management. In Israel, CL is caused mainly by Leishmania major and Leishmania tropica species.

Methods: We established an easy to handle point of care lesion-swabbing, combined with a highly sensitive multiplex real time PCR (multiplex qPCR) for accurate and rapid diagnosis of Leishmania species.

Results: Using three probes: one general for: Leishmania species, and two specific for L major, and L tropica, we screened 1783 clinical samples collected during two years. Leishmania species was found in 1086 individuals, 1008 L major, and 70 L tropica. Eight samples positive for Leishmania species only, were further tested using a second set of multiplex qPCR developed, and were found positive for Leishmania braziliensis and Leishmania infantum/donovani (2 and 6 samples, concomitantly).

Conclusions: Taken together, the test enabled diagnostics and better treatment of Leishmania infections from the Old World (1078 samples) and the New World (8 samples), and the subtyping of the dominant strains in the region, as well as in returning travelers'.

Keywords: Leishmania major; Leishmania species; qPCR; real-time PCR..

Figure 1.

Multiplex quantitative polymerase chain reaction (qPCR) of set I and set II for Leishmania diagnosis. (A) Representative multiplex qPCR amplification of set I, positive for Leishmania species (Yakima yellow-tagged probe), Leishmania major ([L major] 6-carboxyfluorescein-tagged probe), and Leishmania tropica ([L tropica] ROX-tagged probe). Upper left corner demonstrates multiplex qPCR amplification of set I that was negative for Leishmania and positive for the human gene PHP, which serves as internal control (IC) for sampling, extraction, and amplification of the specimen. The concentration of the IC primers and probe was limited, to prevent competition with the sets of Leishmania. (B) Representative multiplex qPCR of set II for Leishmania braziliensis ([L braziliensis] 6-carboxyfluorescein-tagged probe) and Leishmania infantum/donovani (Cy5-tagged probe), positive for L braziliensis. (C) A standard curves for L major and Leishmania species (L species) sets. Promastigotes were grown in culture and counted and deoxyriboneucleic acid was extracted. Serial dilutions were prepared and tested in triplicates by multiplex qPCR for both: L major (gray diamonds, and gray trend line) and L species (empty black squares, and black trend line). Standard deviations are depicted for each set. Shown is 1 experiment of 3. Cq, quantification cycle.

Figure 2.

Diagnosis of cutaneous leishmaniasis by in-laboratory multiplex quantitative polymerase chain reaction test. The bars represent the number of samples tested during July 2014 to August 2016. Depicted are (1) total number of patients tested and (2) the Leishmania species samples that were found to be positive (black bars) and negative (gray bar).

Figure 3.

Seasonality of Leishmania major infections in Israel Southern District. The graphs depicts the number of samples that were sent for diagnosis (open squares) and the number of positive samples for L major (black squares), in each month, during July 2014 to August 2016.