RESUMO
To determine the best molecular method for diagnosing leprosy, two sets of Mycobacterium leprae-specific primers were compared. Fresh biopsies and slit skin smear samples were obtained from 67 leprosy patients and examined by touchdown (TD) PCR using primers amplifying either a 129-bp fragment of the RLEP repetitive sequence or a 360-bp fragment of the 18-kDa protein gene of M. leprae. Seventeen of 30 (56.7%) biopsy specimens and four of 37 (10.8%) slit skin smear specimens were positive using the primer for the 18-kDa protein gene, whereas 24 of 30 (80%) biopsy and 27 of 37 (73%) slit skin smear samples showed detectable PCR products in the RLEP repetitive sequence. Twenty-one of 31 cases (67.7%) with a bacterial index of zero were PCR positive for the primer RLEP repetitive sequence. These results demonstrate that detection of M. leprae using PCR with primers to a RLEP sequence is more sensitive and specific than PCR with the 18-kDa protein gene primers and also slit smears with acid fast staining. PCR of RLEP repetitive sequences is therefore a useful means of detecting M. leprae DNA even when it is present at very low levels.
Assuntos
Proteínas de Bactérias/genética , Hanseníase/diagnóstico , Mycobacterium leprae/genética , Reação em Cadeia da Polimerase/métodos , Anticorpos Antibacterianos/sangue , Primers do DNA , Humanos , Hanseníase/microbiologia , Mycobacterium leprae/isolamento & purificação , Sequências Repetitivas de Ácido Nucleico , Pele/microbiologiaRESUMO
To determine the best molecular method for diagnosing leprosy, two sets of Mycobacterium leprae-specific primers were compared. Fresh biopsies and slit skin smear samples were obtained from 67 leprosy patients and examined by touchdown (TD) PCR using primers amplifying either a 129-bp fragment of the RLEP repetitive sequence or a 360-bp fragment of the 18-kDa protein gene of M. leprae. Seventeen of 30 (56.7 per cent) biopsy specimens and four of 37 (10.8 per cent) slit skin smear specimens were positive using the primer for the 18-kDa protein gene, whereas 24 of 30 (80 per cent) biopsy and 27 of 37 (73 per cent) slit skin smear samples showed detectable PCR products in the RLEP repetitive sequence. Twenty-one of 31 cases (67.7 per cent) with a bacterial index of zero were PCR positive for the primer RLEP repetitive sequence. These results demonstrate that detection of M. leprae using PCR with primers to a RLEP sequence is more sensitive and specific than PCR with the 18-kDa protein gene primers and also slit smears with acid fast staining. PCR of RLEP repetitive sequences is therefore a useful means of detecting M. leprae DNA even when it is present at very low levels.
Assuntos
Humanos , Anticorpos Antibacterianos/sangue , Hanseníase/diagnóstico , Hanseníase/microbiologia , Mycobacterium leprae/genética , Mycobacterium leprae/isolamento & purificação , Pele/microbiologia , Primers do DNA , Proteínas de Bactérias/genética , Reação em Cadeia da Polimerase/métodos , Sequências Repetitivas de Ácido NucleicoRESUMO
Although there is no genetic diversity in isolates of Mycobacterium leprae, the variance of tandem repeats in the rpoT gene was recently demonstrated. We have typed clinical isolates of M. leprae in Korea using difference of the tandem repeats in the rpoT gene. Among 69 patients, 65 Korean isolates (94.2%) demonstrated four copies of the 6 bp tandem repeat (GACATC) in the rpoT gene, and incidences of three copies were found in only two Koreans and two foreigners (2.9%, respectively).
Assuntos
DNA Bacteriano/análise , Hanseníase/epidemiologia , Mycobacterium leprae/classificação , Mycobacterium leprae/genética , Sequência de Bases , Feminino , Genótipo , Humanos , Incidência , Coreia (Geográfico)/epidemiologia , Hanseníase/genética , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Probabilidade , Sensibilidade e Especificidade , Sequências de Repetição em TandemAssuntos
Dapsona/uso terapêutico , Hansenostáticos/uso terapêutico , Hanseníase/tratamento farmacológico , Mutação/genética , Mycobacterium leprae/genética , Adulto , Idoso , Animais , Di-Hidropteroato Sintase/genética , Resistência Microbiana a Medicamentos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium leprae/efeitos dos fármacos , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Análise de Sequência de DNARESUMO
Toll-like receptor 2 (TLR2) is critical in the immune response to mycobacterial infections and the mutations in the TLR2 have been shown to confer the susceptibility to severe infection with mycobacteria. To define this, we screened the intracellular domain of TLR2 in 131 subjects. Groups of 45 lepromatous and 41 tuberculoid leprosy (TT) patients and 45 controls were investigated. Ten subjects among the lepromatous leprosy (LL) patients had a band variant detected by single-stranded conformational polymorphism. DNA sequencing detected a C to T substitution at nucleotide 2029 from the start codon of the TLR2. The mutation would substitute Arg to Trp at amino acid residue 677, one of the conserved regions of TLR2. In our results, the mutation was involved in only LL, not TT and control. Thus, we suggest that the mutation in the intracellular domain of TLR2 has a role in susceptibility to LL.