The use of fluorescent fragment length analysis (PCR-FFL) in the direct diagnosis and identification of cutaneous Leishmania species

Abstract

Leishmaniasis is a disease caused by different species belonging to the genus Leishmania. It presents different epidemiological and clinical features and requires the development of rapid, sensitive techniques to improve specific diagnosis. In this study, we compared the traditional technique of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) with PCR-fluorescent fragment length analysis (PCR-FFL). Fluorescently tagged primers, designed in the rRNA fragment ITS-1 and 7SL region, were used to amplify fragments, which were later digested and whose sizes were accurately determined using an automated DNA sequencer. We validated the technique using 19 Leishmania strains from five cutaneous Leishmania species before testing 36 clinical samples: 23 skin biopsies and 13 skin scrapings/lesion exudates on filter paper. In real diagnostic, PCR-FFL has proved to be quick, accurate, and more sensitive (83.3% testing the ITS-1 fragment and 94.4% testing the 7SL) than PCR-RFLP analysis (75% and 80.6%). Filter papers improved the specific diagnosis in both techniques using non-invasive samples. Copyright © 2013 by The American Society of Tropical Medicine and Hygiene.