Padronização e avaliação de um sistema de PCR quantitativo para diagnóstico molecular da hanseníase

Abstract

Leprosy persists as a public health problem in the world despite efforts to eliminate it. The number of people infected with Mycobacterium leprae who develop the disease contributes to Brazil being one of the countries with highest rates of new cases of leprosy in the world. It is well known that individuals who live close to leprosy patients are at higher risk of developing the disease. This is caused by an obligate intracellular pathogen, Mycobacterium leprae, which is not cultivable in vitro; such a feature hinders the classical diagnostic strategies that are based on bacteriological and / or histopathological tests. Early diagnosis of leprosy in endemic areas requires effective testing to identify individuals at risk of developing the disease prior to clinical manifestation. In order to support clinical diagnosis, molecular methods with high sensitivity and specificity, such as quantitative polymerase chain reaction (qPCR), have been used in the diagnosis of patients. The overall objective of the present study was to develop and standardize a qPCR multiplex system that is efficient for the diagnosis of leprosy. The kit consists of national reagents (manufactured by the Institute of Molecular Biology of Paraná - FIOCRUZ), developed in the form of a multiplex system containing a molecular target as a quality control. The developed multiplex system was able to detect the presence of the pathogen DNA (16S rRNA target) and the patient (18S2 target) in the same reaction. The efficiency of the established protocol was high (rho = 0.99) for the two analyzed targets and the validation parameters were satisfactory, presenting good levels of sensitivity (0.65), specificity (1) and accuracy (0.82). The findings of this study demonstrate the efficacy and importance of qPCR as a tool for early and complementary diagnosis within a laboratory routine. Such support for clinical evaluation could lead to the correct initiation of chemoprophylactic treatment, which would contribute to the interruption of the leprosy transmission chain.