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

Abstract

Background: The 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.

Methods: A 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.

Results: Out 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.

Conclusions: The 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.

Figure 1

Disseminated cutaneousleishmaniasis. A patient with multiple ulcerated lesions of different degrees of evolution found on hands (A) and legs (B). Leishmania amastigotes were found in smears from the most recent lesions located on hands, face and the back of the patient. The lesions present in legs were the oldest. The Montenegro skin test was reactive, and L. (V.) braziliensis was incriminated as the causative agent by PS-PCR.

Figure 2

PCR-based diagnosis andLeishmaniaspecies assignation. PCR products from 12 samples from patients using V1-V2 primers for Viannia subgenus identification. Lanes 1, 4, 5, 8, 9 and 13 were positives, and lanes 2, 3, 10, 11, 12 and 14 were negatives. Lanes 6 and 7 are the negative and positive control, respectively (A). PCR using M1-M2 primers for L. mexicana and L. donovani complexes applied to five DNA samples. Lanes 1 to 5 were negatives. Lanes 6 and 7 negative and positive control, respectively (B). Species identification by PCR analysis with primers b1-b2 (b), g1-g2 (g) and p1-p2 (p) specific for L. (V.) braziliensis, guyanensis and panamensis, respectively (C). Patient 1: Lane b = positive, g and p = negatives. Patient 2: g = positive, b and p = negatives. Patient 3: p = positive, b and g = negatives. Lanes 4 and 5, negative and positive control, respectively. M: marker of molecular weight. Arrows indicate the expected location of the bands.