Diagnóstico molecular da hanseníase com biomarcadores ML0024 e 85B pela PCR em tempo real

Abstract

Leprosy is a spectral disease caused by Mycobacterium leprae, whose clinical and laboratory diagnosis is performed by means of sputum smear techniques of low sensitivity and specificity, they do not detect the bacilli in paucibacillary (PB). In the present study were tested by qPCR molecular markers, in order to assist the diagnosis of disease, mainly in the form PB. The total DNA of skin lesion biopsy was taken from 185 treatment-naïve patients. The qPCR was performed using primer pairs for amplification of the sequence ML0024 and 85B of the genome of the bacillus. Both markers were highly specific. Detection of DNA of the bacillus by qPCR and qPCR ML0024 85B was 59.45% (110/185) and 57.29% (106/085), respectively, while the bacterial index (BI) smear and dermal skin lesion detected, respectively, the presence of the bacillus in 45.94% (85/185) and 44.86% (83/185) of cases. Among all cases negative for the IB dermal smear, the qPCR and qPCR ML0024 85B detected the presence of the bacillus DNA in 26.00% (26/100) and 23.00% (23/100), respectively. Among those negative for the IB skin lesions, the ML0024 qPCR was positive in 27.45% (28/102) and qPCR 85B in 23.52% (24/102). There was significant difference between positive molecular tests and two tests bacilloscopic (p <0.0001) for the clinical forms of T, and DT-DT +. The agreement between the results of qPCR and qPCR ML0024 85B was more than 90.00% and showed excellent repeatability. Among the tests and molecular tests bacilloscopic the concordance observed were higher than 80.00%. As for the quantitative analysis, we observed a positive correlation between the clinical forms and bacillary load. Molecular markers evaluated in this study also have the same number of copies in the genome, are specific, have virtually the same amplicon size and have similar behavior, although with significant differences in sensitivity. Therefore, this study corroborates with the Ridley- Jopling classification (1966) showing the presence of DNA of M. leprae in samples bacilloscopic whose tests were negative, correlating directly with the bacterial load of each clinical spectrum of disease, increasing certainty in the diagnosis of leprosy.