Polymorphism-specific PCR enhances the diagnostic performance of American tegumentary leishmaniasis and allows the rapid identification of Leishmania species from Argentina

Jorge D Marco,Paola A Barroso,Yoshihisa Hashiguchi,Ayako Tomatani,Tatsuyuki Mimori,María C Mora,Luis A Parada,Taketoshi Taniguchi,Julio R Nasser,S Pamela Cajal,Fabricio M Locatelli,Masataka Korenaga,Miguel A Basombrío

doi:10.1186/1471-2334-12-191

Abstract

BackgroundThe diagnosis of the leishmaniases poses enormous challenges in Argentina. The Polymorphism-Specific PCR (PS-PCR) designed and validated in our laboratories has been proven effective for typifying the Leishmania genus from cultured material. Here we evaluated the performance of this method in the diagnosis of American tegumentary leishmaniasis (ATL) and the rapid identification of Leishmania spp. directly from clinical specimens.MethodsA total of 63 patients from northwestern Argentina, with cutaneous or mucocutaneous lesions, underwent an ATL diagnosis protocol which included clinical examination, Leishmanin skin test, and microscopic examination of dermal smears. In addition, we performed PS-PCR on DNA directly extracted from the specimens scraped from the lesions.ResultsOut of the 63 patients, 44 were classified as ATL cases and 19 as non-ATL cases. The diagnostic sensitivity of the microscopic analysis of dermal smears and PS-PCR individually were 70.5% and 81%, respectively. When performing both tests in parallel, this parameter increased significantly to 97.6% (p = 0.0018). The specificities, on the other hand, were 100%, 84.2%, and 83.3% for the combination, respectively (p > 0.05). Using the PS-PCR analysis we successfully identified the Leishmania spp. in 31 out of the 44 ATL cases. Twenty-eight (90.3%) cases were caused by L. (V.) braziliensis, two (6.5%) by L. (V.) guyanensis, and one (3.2%) by L. (V.) panamensis.ConclusionsThe efficacy of the ATL diagnosis was significantly improved by combining the dermal smear examination with a PS-PCR analysis. Our strategy allowed us to reach the diagnosis of ATL with high accuracy regarding the species of the etiological agent in 70.5% of the cases. Moreover, we diagnosed two cases of the disseminated cutaneous form caused by L. (V.) braziliensis and a cutaneous case due to L. (V.) panamensis infection, both findings reported for the first time in Argentina.

Highlights

The diagnosis of the leishmaniases poses enormous challenges in Argentina
In the north of Salta province, where 53.1% of all cases reported in the Argentina occur, some institutions enforce applying the Montenegro skin test (MST) together with microscopic examination of smears to diagnose American tegumentary leishmaniasis (ATL)
In the present study we evaluated the performance of a modified PolymorphismSpecific PCR (PS-PCR) approach applied directly on clinical samples

Summary

Introduction

The diagnosis of the leishmaniases poses enormous challenges in Argentina. The PolymorphismSpecific PCR (PS-PCR) designed and validated in our laboratories has been proven effective for typifying the Leishmania genus from cultured material. We evaluated the performance of this method in the diagnosis of American tegumentary leishmaniasis (ATL) and the rapid identification of Leishmania spp. directly from clinical specimens. The leishmaniases are a group of neglected tropical diseases caused by flagellated parasites of the genus Leishmania. American tegumentary leishmaniasis (ATL) is endemic in Argentina with an estimated incidence of 8.76 cases/ year/106 inhabitants, calculated from 1984 to 2005 [2]. These data might not be accurate since the laboratory resources for confirming ATL are not always available, and local physicians have to report suspected cases merely based on clinical evidence without any diagnostic test. MST detects past infections or previous contact with the parasite, it is a useful tool for screening or as a complementary test for the diagnosis of an active disease [4]

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