Dados do Trabalho

Título

PCR on scarification blades for the diagnosis of cutaneous leishmaniasis

Introdução

Cutaneous leishmaniasis (CL) is a zoonotic disease caused by the protozoan Leishmania spp. through the bite of infected sandflies. CL diagnosis is based on direct examination of a scarification sample (EDE) or by apposition of biopsy (EDB). If the result is negative, Polymerase Chain Reaction (PCR) is performed on a biopsy sample from the lesion. The decentralization of diagnosis and sample collection for PCR is a major challenge due to the lack of trained physicians to perform this invasive procedure. In addition, parasitemia tends to decrease with disease progression, leading to difficulties in identifying the parasite and, consequently, to a misdiagnosis, increasing the need to take biopsy samples.

Objetivo(s)

In order to minimize this problem, a technique for extracting DNA from the parasites directly from the scarification glass slide to perform PCR (S-PCR) was standardized.

Material e Métodos

Fifty slides from the diagnostic routine of the Parasitic Diseases Service (SDP/Funed) with different parasitemias were selected. Twelve positive slides for other parasites, including Trypanosoma cruzi (Chagas disease), Plasmodium falciparum and P. vivax (malaria) and Toxoplasma gondii (toxoplasmosis), were also selected for negative control.The samples from the slides were scraped using a lysis buffer and then DNA extraction and PCR were performed.

Resultados e Conclusão

The S-PCR technique achieved 75% (3/4) positivity on slides with 1-9 amastigotes, detecting 100% of slides with adequate diagnostic quality even with only one amastigote on the slide, 100% of slides with more than 10 amastigotes and 38% positivity even on negative slides by direct examination, from suspected patients. There were no cross-reactions in samples containing the other tested parasites.This study was a proof of concept that successfully validated the proposed technique as a tool to aid the diagnosis of CL, clarifying unresolved cases with direct examination and reducing the need for biopsy.

Palavras-chave

Cutaneous leishmaniasis. Diagnosis. Polymerase Chain Reaction. Direct examination.