Leishmania (Viannia) species identification on clinical samples from cutaneous leishmaniasis patients in Peru: assessment of a molecular stepwise approach

Abstract

We present an algorithm based on three PCR assays for Leishmania (Viannia) species identification and assessed its performance using 70 specimens from Peruvian patients. The succession of the assayed targets can be ordered according to species prevalence. Sequential progression through the algorithm reduced the number of samples here studied by approximately 30% after each step.

Fig 1

kDNA PCR band intensity categories used as references to allocate diagnosis of clinical specimens. PCR mixtures (lanes 1 to 8) contained 5 ng of human DNA plus different amounts (5 ng down to 1 fg) of Leishmania DNA. PCR product sizes correspond to 70 bp for Leishmania kDNA and 197 bp for human beta-globin gene (internal control). Abbreviations: w/o, without; M, molecular size ladder.

Fig 2

Reduction of numbers of clinical specimens through the algorithm steps. PCR steps are shown with black shading. Subsequent steps are indicated with arrows, while identification endpoints in the flow chart are indicated within gray-shaded boxes. The algorithm was evaluated using 70 specimens on filter paper lesion impressions, and the numbers processed in each step are indicated (n). mpi Lp and mpi Lb correspond to PCRs using reverse allele-specific primers for L. (V.) peruviana and L. (V.) braziliensis, respectively. *, two specimens that could not be typed with cpb PCR due to negative or weak cpb amplification but were identified with hsp70 PCR-restriction fragment length polymorphism (PCR-RFLP).