Dados do Trabalho

Título

APPLICATION OF qPCR AND CONVENTIONAL PCR FOR THE DETECTION OF Leishmania (Viannia) braziliensis DNA IN OCULAR CONJUNCTIVA AND ORAL MUCOSA SWAB SAMPLES OF WILD AND SYNANTHROPIC RODENTS

Introdução

Leishmania (Viannia) braziliensis is the etiological agent for cutaneous leishmaniasis (CL). The disease affects the skin and mucous membranes with lesions of varied clinical spectrum. In the Northeast of Brazil, including in Pernambuco, L. (V.) braziliensis is the main circulating species. The presence of deforested areas favors the colonization of vectors and reservoirs such as wild and synanthropic rodents, which allow the maintenance and circulation of the protozoan in the region endemic. The diagnosis of the disease is performed through epidemiological, clinical and laboratory investigations. The conventional laboratorial techniques and collect sample methods have limitations such as variable sensitivity, low reproducibility, and invasive and painful way of material collection. In this context, molecular tests associated with the swab, a non-invasive collection method, provide a new easy, fast and more sensitive diagnostic tool.

Objetivo(s)

The aim of the study is to evaluate the sensitivity of molecular techniques (Conventional PCR and qPCR) in detecting the kDNA target in ocular conjunctival and oral mucosa swab samples.

Material e Métodos

The study area was Engenho Raiz de Dentro and Engenho Refrigério, municipalities of Amaraji-PE, Brazil, both endemic for CL. From 142 samples, 71 paired samples of swab ocular conjunctival and oral secretion of wild and synanthropic rodents were provided by the Leishmaniasis Reference Service of the Institute Aggeu Magalhães IAM/FIOCRUZ-PE. All samples were submitted to DNA extraction followed by Conventional PCR and an electrophoresis, which amplifies 750pb up to 10pg of DNA, and by qPCR, based on the SYBR Green system. The qPCR system amplifies the fragment of 138 bp specific for Leishmania braziliensis. On both molecular tests, the amplification target was kDNA.

Resultados e Conclusão

The qPCR results, 13.4% (CI=8 to 20%) of the samples were positive for Leishmania (V.) braziliensis, being statistically significant (p < 0.0001) when compared to conventional PCR, in which 100% of the samples were negative. Statistically, qPCR showed greater sensitivity, capable of detecting low parasite load (fentograms) in a non-invasive collection method. The use qPCR system, in association with the swab collection method, can be a valuable device in the management and diagnosis of reservoir animals in endemic areas of the disease.

Palavras-chave

qPCR. Conventional PCR. Swab. Rodents.