Genotyping of Mycobacterium leprae present on Ziehl-Neelsen-stained microscopic slides and in skin biopsy samples from leprosy patients in different geographic regions of Brazil

We analysed 16 variable number tandem repeats (VNTR) and three single-nucleotide polymorphisms (SNP) in Mycobacterium leprae present on 115 Ziehl-Neelsen (Z-N)-stained slides and in 51 skin biopsy samples derived from leprosy patients from Ceará (n = 23), Pernambuco (n = 41), Rio de Janeiro (n = 22) and Rondônia (RO) (n = 78). All skin biopsies yielded SNP-based genotypes, while 48 of the samples (94.1%) yielded complete VNTR genotypes. We evaluated two procedures for extracting M. leprae DNA from Z-N-stained slides: the first including Chelex and the other combining proteinase and sodium dodecyl sulfate. Of the 76 samples processed using the first procedure, 30.2% were positive for 16 or 15 VNTRs, whereas of the 39 samples processed using the second procedure, 28.2% yielded genotypes defined by at least 10 VNTRs. Combined VNTR and SNP analysis revealed large variability in genotypes, but a high prevalence of SNP genotype 4 in the Northeast Region of Brazil. Our observation of two samples from RO with an identical genotype and seven groups with similar genotypes, including four derived from residents of the same state or region, suggest a tendency to form groups according to the origin of the isolates. This study demonstrates the existence of geographically related M. leprae genotypes and that Z-N-stained slides are an alternative source for M. leprae genotyping.

Estudio de resistencia a la rifampicina y la dapsona en tres pacientes con recurrencia de lepra / Study of rifampin and dapsone resistance in three patients with recurring leprosy

OBJETIVO: Detectar la presencia de cepas de Mycobacterium leprae resistentes a la rifampicina y la dapsona en tres pacientes con recurrencia de lepra y sospecha clínica de resistencia antimicrobiana, mediante la aplicación de técnicas moleculares. MÉTODOS: Se realizó un estudio descriptivo retrospectivo en tres pacientes multibacilares del Sanatorio de Agua de Dios, Cundinamarca, Colombia, que habían presentado recidivas de lepra documentadas por su historia clínica, baciloscopia y biopsia. Se obtuvieron biopsias de lesiones cutáneas que se procesaron para la extracción y purificación del ADN bacilar. Se amplificaron regiones de los genes rpoB y folP1 asociadas con la resistencia antimicrobiana, mediante la reacción en cadena de la polimerasa "touch-down" y se secuenciaron los productos amplificados mediante el método de Sanger. RESULTADOS: Se detectó una mutación puntual en el nucleótido 1367 del gen rpoB en dos de las muestras estudiadas. No se encontró la mutación estudiada en el gen folP1 en ninguno de los tres pacientes. CONCLUSIONES: La mutación identificada demostró la presencia de bacilos de M. leprae resistentes a la rifampicina en dos de los tres pacientes estudiados con recurrencia de la enfermedad. No se detectó la mutación indicadora de resistencia a la dapsona en ninguno de los tres pacientes.

OBJECTIVE: To detect the presence of rifampin- and dapsone-resistant strains of Mycobacterium leprae in three patients with recurring leprosy and clinically-suspected antimicrobial resistance through molecular techniques. METHODS: A retrospective, descriptive study was conducted of three multibacillary patients at the "Agua de Dios" Sanitarium in Cundinamarca, Colombia, that presented leprosy relapses that were documented by medical history, bacilloscopy, and biopsy. Biopsies were taken of the skin lesions and the bacteria were subject to DNA extraction and purification. Regions of the rpoB and folP1 genes associated with antimicrobial resistance were amplified and subjected to touch-down polymerase chain reaction and the amplified products were sequenced using the Sanger method. RESULTS: A punctual mutation was identified in nucleotide 1367 of the rpoB gene in two of the samples studied. This mutation was not found in the folP1 gene of any of the three patients. CONCLUSIONS: The mutation identified showed strains of rifampin-resistant M. leprae in two of the three patients with recurring leprosy. Mutations that indicate dapsone-resistance were not detected in any of the three patients.