Detecção molecular de Mycobacterium leprae: investigação da resistência à poliquimioterapia em indivíduos classificados como casos e com suspeição de hanseníase no Leste de Minas Gerais

Abstract

Leprosy is a chronic infectious disease caused by Mycobacterium leprae, which can affect peripheral nerves and lead to physical disabilities when diagnosis is delayed. Although clinical assessment and bacilloscopy remain essential for diagnosis, molecular detection may improve bacillus identification in cases of low bacillary load. In addition, resistance to multidrug therapy challenges disease control. In this context, this study aimed to perform molecular detection of Mycobacterium leprae and investigate resistance to multidrug therapy in individuals classified as leprosy cases and individuals suspected of having leprosy in eastern Minas Gerais, Brazil.This observational study used samples and information collected from 2017 to 2021 at the Center for Reference in Endemic Diseases and Special Programs in Governador Valadares. Initially, we evaluated 411 samples by quantitative polymerase chain reaction (qPCR), targeting the repetitive element RLEP of the Mycobacterium leprae genome. We considered samples with a cycle threshold (Ct) value below 37 positive. We classified individuals as cases when a leprosy diagnosis was recorded in the Brazilian Notifiable Diseases Information System (SINAN) and as suspected cases when they had molecular positivity but no previous notification. We characterized individuals with molecular positivity by sociodemographic variables and spatial distribution. Individuals suspected of having leprosy were clinically followed up, when possible, to assess diagnostic progression.For the molecular investigation of drug resistance, we selected samples with Ct values below 26 and subjected them to amplification and sequencing of the folP1, rpoB, and gyrA genes, which are associated with resistance to dapsone, rifampicin, and fluoroquinolones, respectively. We compared Ct values between groups using the Mann–Whitney U test.Of the 411 initial samples, 278 were RLEP-positive, including 158 from cases and 120 from individuals suspected of having leprosy. In the sociodemographic analysis, we found no statistically significant associations between qPCR positivity and sex, age group, or race/ethnicity. Positive individuals came from 31 municipalities, 13 of which had at least one positive sample, indicating heterogeneous territorial distribution.Among individuals suspected of having leprosy who resided in Governador Valadares, 29 underwent clinical follow-up; of these, 12 (41.4%) were subsequently diagnosed with leprosy. The interval between molecular detection and clinical confirmation averaged six years.For the comparison of Ct values, we evaluated 258 samples with available results, including 138 from the Case group and 120 from the group suspected of having leprosy. No statistically significant difference was observed between the groups (p = 0.121). However, among the 54 samples selected for sequencing, including 39 from cases and 15 from individuals suspected of having leprosy, Ct values differed significantly (p < 0.001).Skin smear samples were a suitable source of DNA for sequencing, with satisfactory results for 87.0% of samples for the rpoB and gyrA genes and 81.5% for folP1. We identified no drugresistance mutations in the genes evaluated. The findings indicate that RLEP qPCR, when integrated with clinical assessment, territorial analysis, and longitudinal follow-up, may help identify individuals who require systematic surveillance. The differences in Ct values among the samples selected for sequencing highlight the usefulness of quantitative analysis in selecting material with a greater relative amount of amplifiable DNA. The absence of resistance-associated mutations should be interpreted in light of the limited number of samples and does not rule out the circulation of resistant variants. Overall, the results reinforce the potential of molecular detection and sequencing as complementary tools to conventional strategies for leprosy diagnosis, surveillance, and investigation of drug resistance.