Quantificação por PCR em tempo real de Mycobacterium leprae em amostras de secreção nasal e biópsias de pele em hansenianos

Abstract

Mycobacterium leprae, the etiologic agent of leprosy, is not cultivable in axenic medium. The quantification of bacilli performed by skin bacilloscopy and histopathology is useful for the clinical classification of leprosy and treatment monitoring. However, this methodology yields low results regards to sensitivity and specificity. On the other way, the real-time quantitative PCR (qPCR) is a sensitive and specific assay that allows quantification of a varied of samples and also can be used for differential diagnosis of many pathogens. To this date, no studies have evaluated the sensitivity and specificity of qPCR for the diagnosis of leprosy from nasal secretion samples. Therefore, this study aimed at detecting and quantifying DNA from M. leprae by qPCR from nasal secretion and biopsy samples from leprosy cases, correlating with clinical and bacteriological index. We analyzed 61 samples of nasal secretion and 19 biopsy specimens of skin lesion (paired) from confirmed leprosy cases seen at the National Reference Center for Sanitary Dermatology Dona Libânia. All samples were subjected to DNA extraction followed by amplification by nested PCR of a region of 238 bp which targets a RLEP2 repetitive sequence. The samples were subjected to quantification of 16S rRNA region of the genome of M. leprae by qPCR, which specificity was verified by amplicon melting temperature (Tm = 79.5 ° C). The method was able to detect amounts over to 20 fg of M. leprae DNA, equivalent to four bacilli units. Nasal secretion assay was able to confirm the diagnosis in 89.7% of the multibacillary cases (MB) and 73.3% of the paucibacillary cases (PB). In addition, MB patient biopsies sensitivity was 100%. The number of bacilli detected in nasal secretion samples from leprosy patients ranged from 1.39 x 10³ to 8.02 x 105 bacilli, while detection in MB patient biopsy ranged from 1,87 x 103 to 1,50 x 106. The real-time PCR is more sensitive than conventional PCR and can be used as a complementary tool for the clinical and histopathological diagnosis of leprosy.