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

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.

Multilocus sequence typing approach for a broader range of species of Leishmania genus: describing parasite diversity in Argentina.

Leishmaniasis is a vector-borne protozoan infection affecting over 350 million people around the world. In Argentina cutaneous leishmaniasis is endemic in nine provinces and visceral leishmaniasis is spreading from autochthonous transmission foci in seven provinces. However, there is limited information about the diversity of the parasite in this country. Implementation of molecular strategies for parasite typing, particularly multilocus sequence typing (MLST), represents an improved approach for genetic variability and population dynamics analyses. We selected six loci as candidates implemented in reference strains and Argentinean isolates. Phylogenetic analysis showed high correlation with taxonomic classification of the parasite. Autochthonous Leishmania (Viannia) braziliensis showed higher genetic diversity than L. (Leishmania) infantum but low support was obtained for intra-L. braziliensis complex variants suggesting the need of new loci that contribute to phylogenetic resolution for an improved MLST or nested-MLST scheme. This study represents the first characterization of genetic variability of Leishmania spp. in Argentina.

The isolation and molecular characterization of Leishmania spp. from patients with American tegumentary leishmaniasis in northwest Argentina.

American tegumentary leishmaniasis (ATL) is a group of zoonotic diseases caused by kinetoplastid flagellates of the genus Leishmania. A total of 66 patients diagnosed as positive ATL cases from northwest Argentina were included in this study. Leishmania stocks were isolated in vitro and analyzed over promastigote cultures sown on FTA through nested PCR and sequence of cytochrome b (cyt b). The molecular analysis resulted in the incrimination of L. (Viannia) braziliensis as the predominant species in the studied area, identifying two genotypes of L. (V.) braziliensis, 24 cases of Ab-1 cyt b and 41 cases of Ab-2 cyt b. One L. (V.) guyanensis strain was obtained from a traveler from the Brazilian Amazon. The prevalence of different genotypes was in agreement with previous studies, suggesting the necessity for new systems to study the genetic diversity in more detail. Most of the cases typified in this study were registered in the area of Zenta Valley (Orán, Hipólito Yrigoyen, and Pichanal cities), pointing a link between genotype and geographical origin of the sample. Sex and age distribution of the patients indicate that the transmission was predominantly associated with rural areas or rural activities, although the results might not exclude the possibility of peri-urban transmission. This work represents, so far, the largest isolation and molecular characterization of ATL cases in Argentina.