O papel da biologia molecular no diagnóstico, epidemiologia molecular e perfil de sensibilidade de cepas de M. leprae em região endêmica da Amazônia Brasileira

Abstract

Leprosy is a chronic disease, disabling and difficult to diagnose in all its clinical manifestations. The aims of this study were to identify the laboratory marker that presents greater sensitivity and specificity for diagnosis, to genetically understand the strains of M. leprae circulating in the Pará State and to evaluate the region drug resistance reality. A multidisciplinary team evaluated 833 individuals using different strategies at URE Dr. Marcello Candia, and in 14 municipalities in Pará. All individuals were clinically evaluated, and biological samples were collected for comparative analysis of the results of slit skin smear microscopy, molecular detection of the bacillus by qPCR RLEP in ear lobes, IgM Anti-PGL-I antibodies titration, and histopathological lesion skin biopsy, lesion qPCR RLEP, and M. leprae whole genome sequence. 351 cases were clinically diagnosed, divided into groups: individuals with classic and non-classic clinical manifestations, asymptomatic cases and 482 healthy contacts. The comparative analysis of the results demonstrated that ear lobe RLEP detection presented greater sensitivity, specificity and agreement with the clinical diagnosis (72.5, 70.4 and Kappa = 0.42 respectively), followed by skin biopsy (sensitivity= 65.8%), Anti-PGL-I serology with 61.2% (52.2 specificity), slit smears skin (41.7%) and histopathology (25.0%). The RLEP association to serology, showed an increased in laboratorial correlation with the clinic diagnosis (Kappa= 0.55). The circulant streains evaluation, we detected that the most frequent profile was profile 4N (52/66- 78.8%), followed by 4P subtype (4/66- 6.1%), 3I (9/66 -13, 6%), and 1D (1/66- 1.5%). In the drug resistance analysis, we obtained 3/101 (3%) mutation in folP1 gene, conferring dapsone resistance. 1/40 (2.5%) gyrB mutations, conferring quinolones resistance. The gyrB resistant strain also had mutation in folP1, and in the fadD9, ribD, pks4 and nth genes, considered a hypermutant strain. Our findings showed that qPCR RLEP molecular test associated with Anti-PGL-I serology were a good tool for leprosy laboratorial diagnosis, and that type 4 strains, originating in Africa, are the most frequent type in the Amazon. And we find drug-resistant strains, and a hypermuted strain circling in the region. The strains were resistant to the current polychemotherapy regimen and the alternative drug regimen.