Dados do Trabalho

Título

PCR-ITS-1 followed by sequencing: definition of Leishmania species from smears of lesions of patients with cutaneous leishmaniasis residing in Santarém, Pará, Brazil

Introdução

Cutaneous leishmaniasis(CL) is an infectious disease caused by the protozoan Leishmania. For the diagnosis of the disease, parasitological tests are used, but they present variable sensitivity and specificity. The serodiagnosis has low sensitivity, in addition to cross-reactivity with other pathogens. For molecular diagnosis, PCR has high sensitivity and specificity and can be performed from several biological sample, such as smears stained on slides and previously examined by microscopy. As a target gene target for PCR, the ITS-1 region (internal transcriptional spacer1) of ribosomal RNA has an adequate number of polymorphisms for species-level distinction, in addition to conserved regions of diagnostic importance.

Objetivo(s)

Our objective was evaluate PCR-ITS-1 followed by sequencing to identify the Leishmania species from lesion smears from patients with suspected CL, residing in Santarém, Pará, Brazil.

Material e Métodos

Forty smears of lesion stained by Giemsa on slides, from 40 patients with suspected CL, were analyzed by microscopy (1000X) in the laboratory of the Núcleo Técnico de Vigilância em Saúde de Santarém (NTVS)/Pará. These smears were subjected to DNA extraction, followed by PCR-ITS1 and Sanger sequencing.

Resultados e Conclusão

Of the 40 smears examined microscopically, 30 were positive and 10 were negative. The 10 negatives were from patients who clinically did not correspond to the CL and 100% (10/10) of them were negative in the PCR. Of the 30 positives on microscopy, 83% (25/30) were positive on PCR-ITS-1. Nine samples so far were subjected Sanger sequencing and showed the following results after alignment: 3 with L. (V.) lainsoni, 1 L. (L.) amazonensis, 1 L. (V.) shawi, 1 L. (V.) guyanensis, 1 not identified, 1 aligned with L.(V.) braziliensis and L.(V.) shawi simultaneously and 1 aligned with L.(V.) panamensis and L.(V.) guyanensis simultaneously. Molecular investigation using ITS-1 as a target showed a specificity of 100% and a sensitivity of 83%, the last value can be explained by the storage time of the slides with smears or the quantity of parasites in the lesions. The sequencing of the ITS-1 region showed that there is a great diversity of Leishmania Viannia species in Santarém and L.(V.) braziliensis is not the most prevalent species in Santarém, Pará, Brazil.

Palavras-chave

Cutaneous leishmaniasis, PCR-ITS-1 diagnosis, PCR-ITS-1 sequencing, molecular diagnosis of smears.

Área

Eixo 06 | Protozooses