Evaluation of preservation methods and simulated multiple infection on the fidelity of real-time PCR detection of Leishmania DNA

ABSTRACT

To detail the evaluation of a real-time polymerase chain reaction [PCR]-based approach to Leishmania detection. Also to test the fidelity of PCR diagnostics in a series of experiments mimicking infection by two species of Leishmania. Leishmania major [a causal agent of cutaneous leishmaniasis] infected Phlebotomus papatasi sandflies were generated to test the effect of four preservation methods on the fidelity of real-time PCR detection of Leishmania DNA. There was no effect of preservation methods on the sensitivity or specificity of two different assays. The ability of these assays to correctly diagnose cases of multiple infection was tested with artificial double infections created by combining DNA from pairs of Leishmania species [L, major, L. tropica and L, panamensis]. One assay failed to properly diagnose certain double infections but overall the PCR methodologies were robust. These findings provide important reassurances for subsequent real world investigations of Leishmania in vector, reservoir and human populations