Evaluation of a commercial real-time PCR assay targeting Leishmania spp. with microscopically positive skin biopsies obtained from Nigerian patients

Abstract

Background: Although real-time polymerase chain reaction (PCR) assays for leishmaniasis are considered as highly sensitive, their standardization is still ongoing. In the study presented here, the sensitivity of a commercial real-time PCR assay targeting Leishmania spp. was evaluated with Nigerian skin biopsies from patients with microscopic diagnosis of cutaneous leishmaniasis (CL). Methods: The samples were subjected to two subsequently applied types of nucleic acid extraction and afterwards characterized by two in-house real-time PCR assays targeting kinetoplast DNA (desoxyribonucleic acid) and the small subunit rRNA (ribosomal ribonucleic acid) gene of Leishmania spp. as well as by Sanger sequence analysis of the leishmanial hsp70 gene. Subsequently, the VIASURE Leishmania real-time PCR detection kit (CerTest Biotec, S.L., San Mateo de Gállego, Zaragoza, Spain) was applied with the DNA eluates. Results: From 50 bioptic samples of Nigerian patients with CL, interpretable sequences could be obtained from 30 (60%), showing best matching with Leishmania major in all instances. The VIASURE assay confirmed the abundance of leishmanial DNA in all samples after both DNA extraction schemes, showing slightly better sensitivity compared to the reference PCR assays. Conclusion: The VIASURE assay reliably detects L. major in microscopically positive bioptic samples from Nigerian CL patients.