DIAGNOSIS OF CUTANEOUS LEISHMANIASIS AND SPECIES DISCRIMINATION OF PARASITES BY PCR AND HYBRIDIZATION

The aim of this study was to assess the efficacy of PCR methodology in establishing the diagnosis of cutaneous leishmaniasis in patients fro m areas of endemicity in Venezuela. Biopsies from 233 patients with cu taneous ulcers suggestive of leishmaniasis were analyzed by PCR, emplo ying oligonucleotides directed against conserved regions of kinetoplas t DNA (kDNA), and the PCR products were then hybridized to nonradioact ively labeled, species-specific, cloned kDNA fragments. The ability of PCR to detect Leishmania cells was compared with those of the convent ional methodologies: skin testing with killed promastigotes (Montenegr o test), examination of Giemsa-stained biopsy smears, and in vitro cul ture of biopsy tissue. The PCR-hybridization technique detected the pr esence of Leishmania cells in 98% of patients clinically diagnosed as having leishmaniasis and also positive by the Montenegro skin test. In comparison, leishmania positivity was found in only 42% of cultures a nd 64% of biopsy smears. By hybridizing the PCR product to new kDNA pr obes specific for either Leishmania mexicana or Leishmania braziliensi s we found that both species are major causes of cutaneous leishmanias is in Venezuela, and the species identification was confirmed by restr iction enzyme analysis of kDNA from biopsy cultures. This work demonst rates that PCR coupled with hybridization is useful not only for the d iagnosis of cutaneous leishmaniasis but also for the taxonomic discrim ination essential for both epidemiology and therapy. This technique ca n be used to diagnose leishmaniasis in a country in which the disease is endemic and can perhaps be adapted for use in a rural clinic.