Avaliação da reação em cadeia da polimerase quantitativa na detecção de Mycobacterium leprae em casos de hanseníase de difícil diagnóstico

Abstract

Leprosy is an infectious disease caused by Mycobacterium leprae (M. leprae) that affects the skin and peripheral nerves and can cause physical disabilities which evolve to permanent physical deformities. The leprosy patient can be classified as paucibacillary and multibacillary and paucibacillary forms difficult to diagnose. New molecular methods of high sensitivity and specificity as RT-PCR qPCR, has contributed to leprosy diagnosis, especially in cases of difficult diagnosis. This study was developed in Fundação de Dermatologia Tropical e Venereologia Alfredo da Matta, Manaus – Amazonas, from March 2014 to August 2015, and we aimed to validate the qPCR technique in the detection of M. leprae in skin biopsies patients with clinical suspicion of leprosy and subjected to histopathological examination. The qPCR amplifications from a specific sequence of the 16S gene from M. leprae were performed on 180 samples, of which 82 were fresh biopsies and 98 paraffin biopsies. From Fresh biopsies were analyzed 65 samples, of which 9 are samples of patients with other dermatoses, 12 clinically inconclusive samples and 38 samples from patients with leprosy. Of these, 68.5% (26/38) were positive in molecular test for leprosy and 33,3% (3/9) were positive for other dermatoses. The sensitivity was 68.5%, specificity of 66.70%, with positive predictive value of 89.7% and a negative predictive value of 33.3%. Among the paraffin biopsies were analyzed 61 samples from patients with leprosy, which 73.8% were positive. In these samples, the molecular test had a sensitivity of 73.8%. Furthermore, the number of genomes was estimated in both groups biopsies and ranged from 8.53 (paraffin in a biopsy) to 2,060,230 (on a fresh biopsy). This data suggest that the qPCR confirm the difference between patients MB and PB both fresh biopsies as for paraffin biopsies and the amounts of genomes percentage and quantity was greater in MB patients in the fresh specimens group compared to the group of paraffin samples. Interestingly, the figures in the samples of PB patients even though they have fewer genomes, also has a higher percentage of positivity. Despite adjustments to improve the reaction efficiency, the results show that the test can be useful, particularly in inconclusive cases and difficult diagnosis, as in Paucibacillary forms when the number of genomes is high (greater than 50) reduces the risk of false positives.