RESUMO
Based on the discovery of three single nucleotide polymorphisms (SNPs) in Mycobacterium leprae, it has been previously reported that there are four major SNP types associated with different geographic regions around the world. Another typing system for global differentiation of M. leprae is the analysis of the variable number of short tandem repeats within the rpoT gene. To expand the analysis of geographic distribution of M. leprae, classified by SNP and rpoT gene polymorphisms, we studied 85 clinical isolates from Thai patients and compared the findings with those reported from Asian isolates. SNP genotyping by PCR amplification and sequencing revealed that all strains like those in Myanmar were SNP type 1 and 3, with the former being predominant, while in Japan, Korea, and Indonesia, the SNP type 3 was found to be more frequent. The pattern of M. leprae distribution in Thailand and Myanmar is quite similar, except that SNP type 2 was not found in Thailand. In addition, the 3-copy hexamer genotype in the rpoT gene is shared among the isolates from these two neighboring countries. On the basis of these two markers, we postulate that M. leprae in leprosy patients from Myanmar and Thailand has a common historical origin. Further differentiation among Thai isolates was possible by assessing copy numbers of the TTC sequence, a more polymorphic microsatellite locus.
Assuntos
Proteínas de Bactérias/genética , Hanseníase/transmissão , Mycobacterium leprae/genética , Polimorfismo de Nucleotídeo Único , Fator sigma/genética , Técnicas de Tipagem Bacteriana , Variações do Número de Cópias de DNA , DNA Bacteriano/genética , Genes Bacterianos , Loci Gênicos , Marcadores Genéticos , Genótipo , Humanos , Indonésia/epidemiologia , Japão/epidemiologia , Coreia (Geográfico)/epidemiologia , Hanseníase/epidemiologia , Hanseníase/microbiologia , Mycobacterium leprae/classificação , Mycobacterium leprae/isolamento & purificação , Reação em Cadeia da Polimerase , Tailândia/epidemiologiaRESUMO
Recently about 500 new cases of leprosy have been reported each year in Thailand. In addition to a steady rate of new case detection, Thailand is in Southeast Asia where leprosy is endemic in neighbouring countries; therefore, strain differentiation could be useful in tracing origins and routes of infection, and general leprosy surveillance. To identify suitable markers for differentiation of M. leprae strains in different global geographic regions and to determine the applicability of a systematic genotyping method for tracing leprosy transmission, variable nucleotide tandem repeats (VNTRs) of 14 loci were evaluated using DNA extracts from a total of 97 skin biopsies and slit skin smear samples. The alleles per locus ranged from 2-26 providing adequate strain differentiation. Microsatellite loci (GAA)21, (AT)17 are highly polymorphic followed by (GTA)9, (AC)8a, (AC)8b, and (AC)9. The minisatellites 6-7, 21-3 and 27-5 exhibited a limited number of alleles. The repeat of 23-3 showed no polymorphism. Overall, the strain types can be divided into two distinct Thai groups, according to the alleles at the (GGT)5 and 21-3 loci. However, there are no obvious geographical patterns of distribution of VNTR strain types. Closely matched VNTR profiles found in household members of two multi-case families suggested infection through a common source.
Assuntos
Hanseníase Multibacilar/microbiologia , Hanseníase Paucibacilar/microbiologia , Repetições Minissatélites , Mycobacterium leprae/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , DNA Bacteriano/química , DNA Bacteriano/genética , Feminino , Variação Genética , Humanos , Hanseníase Multibacilar/epidemiologia , Hanseníase Paucibacilar/epidemiologia , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular/métodos , Tailândia/epidemiologia , Adulto JovemRESUMO
Diagnosis of leprosy is usually based on clinical features and skin smear results including the number of skin lesions. Mycobacterium leprae is not cultivable and bacterial enumeration by microscopic examination is required for leprosy classification, choice in choosing and monitoring chemotherapy regimens, and diagnosis of relapse. However, detection and quantification using standard microscopy yields results of limited specificity and sensitivity. We describe an extremely sensitive and specific assay for the detection and quantification of M. leprae in skin biopsy specimens. Primers that amplified a specific 171-bp fragment of M. leprae 16S rRNA gene were chosen and specificity was verified by amplicon melting temperature. The method is sensitive enough to detect as low as 20 fg of M. leprae DNA, equivalent to four bacilli. The assay showed 100% concordance with clinical diagnosis in cases of multibacillary patients, and 50% of paucibacillary leprosy. The entire procedure of DNA extraction and PCR could be performed in c. 3 h. According to normalized quantitative real-time PCR, the patients in this study had bacilli numbers in the range of 1.07 x 10(2) -1.65 x 10(8) per 6-mm3 skin biopsy specimen. This simple real-time PCR assay is a facile tool with possible applications for rapid detection and simultaneous quantification of leprosy bacilli in clinical samples.
Assuntos
Hanseníase/diagnóstico , Mycobacterium leprae/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , RNA Ribossômico 16S/genética , Pele/microbiologia , Humanos , Mycobacterium leprae/genética , RNA Bacteriano/genética , Sensibilidade e Especificidade , Pele/patologiaRESUMO
Mycobacterium leprae isolates from Thai leprosy patients were typed for strain differentiation and analysis of leprosy transmission using the six base tandem repeat, GACATC, in rpoT gene and TTC repeat as genetic markers. M. leprae DNA was isolated from skin biopsies of new untreated leprosy patients living in remote areas or in suburban regions of Thailand where leprosy is in low prevalence. In M. leprae strains of 100 patients, TTC alleles exhibited variations in length with 10 to 30, 33 and 35 repeats, the most common alleles being 15, 16, 17 and 19 repeats. All isolates contained three copies of the six base repeat in rpoT gene. Application of TTC repeats in tracking leprosy transmission in two families with multi-cases identified a single (but different) strain of M. leprae in each family.
Assuntos
DNA Bacteriano/genética , Genes Bacterianos/genética , Hanseníase/microbiologia , Mycobacterium leprae/genética , Polimorfismo Genético/genética , Sequências de Repetição em Tandem/genética , Biópsia , Humanos , Hanseníase/transmissão , Mycobacterium leprae/classificação , Mycobacterium leprae/isolamento & purificação , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Pele , TailândiaRESUMO
An RNA-based assay is an additional molecular tool for leprosy diagnosis and determination of the viability of leprosy bacilli. To simplify RNA detection, a one-step reverse transcriptase PCR (RT-PCR) was established and evaluated. RNA and DNA could be isolated simultaneously. With the use of Mycobacterium leprae-specific primers targeting a 171-bp fragment of the M. leprae 16S RNA gene, RT-PCR resulted in detection of M. leprae in both slit skin smears and skin biopsy specimens. To enhance the positive signal, a digoxigenin-labeled DNA was developed, and successfully detected the amplified RT-PCR product. The method is sensitive, as it could detect one leprosy bacillus. When it was used directly on skin specimens collected from leprosy patients, 34 of 36 multibacillary (MB) and 13 of 24 paucibacillary (PB) cases showed positive results. The assay was also effective in monitoring bacterial clearance in leprosy patients during chemotherapy; after treatment with the multidrug therapy for 6 months, resulting in bacterial clearance, 16 of 36 MB patients and three of 24 PB patients tested were still positive for the 16S rRNA gene of M. leprae, suggesting the advisability of a more prolonged treatment course. This form of RT-PCR is of value in terms of simplicity and sensitivity in identifying M. leprae in routine skin specimens, especially when acid-fast bacilli are not discernable.