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Comparison between microsatellites and Ml MntH gene as targets to identify Mycobacterium leprae by PCR in leprosy.

Abstract

BACKGROUND: The Polymerase Chain Reaction (PCR) technique has been frequently used in the molecular diagnosis of leprosy.

OBJECTIVES: To compare the results of PCR with four pairs of Mycobacterium leprae specific primers as well as to compare these results to multibacillary (MB) and paucibacillary (PB) leprosy according to the WHO operational classification.

METHOD: 28 DNA samples, collected from the frozen skin biopsies and biopsy imprints on filter paper of 23 patients (14 MB and PB 9), were examined for PCR using primers which amplify 131, 151 and 168bp of specific microsatellite regions and a 336 fragment of the Ml MntH (ML2098) gene.

RESULTS: M.leprae bacillus could be detected in 22 (78.6%) of the 28 samples. 9 (45%) of the 20 biopsy samples and 6 (75%) of the 8 imprints were positive to TTC. 7 (35.5%) skin biopsy specimens and 5 (62.5%) imprints were positive to AGT, and 11 (55%) biopsies and 4 (50%) were positive to AGT. 11 (55%) skin biopsies and 4 (50%) imprints were positive to AT. 8(38%) skin biopsies and 5 (62.5%) imprints were positive to the Ml MntH gene. In the MB group, the microsatellites detected the bacillus in 78.5% of the samples, and the Ml MntH gene in 57.1% of the samples, independent of the clinical material. In the PB group 55.5% of samples were positive to the microsatellite primers, while 22.2% were positive to the Ml MntH gene.

CONCLUSIONS: These results show that both the specific regions of microsatellites, as well as the Ml MntH gene fragment can be useful tools for detecting the M. leprae DNA by PCR in frozen skin biopsy samples and filter paper biopsy imprints.

Translated Abstract
FUNDAMENTOS: PCR tem sido frequentemente utilizada no diagnóstico molecular da hanseníase. OBJETIVOS: comparar os resultados da PCR com 4 pares de primers específicos para Mycobacterium leprae, bem como os resultados da PCR à classificação operacional, segundo a OMS, de multibacilar (MB) e paucibacilar (PB) da hanseníase. MÉTODO: Vinte e oito amostras de DNA, extraído de biópsias congeladas de pele e de imprint de biópsias em papel de filtro de 23 pacientes (14 MB e 9 PB), foram utilizadas na PCR com primers que amplificam 131pb, 151pb e 168pb de regiões de microssatélites, e um fragmento de 336pb do gene Ml MntH (ML2098) do bacilo. RESULTADOS: O bacilo pôde ser detectado em 22 (78,6%) das 28 amostras. Nove (45%) das 20 amostras de biópsia e 6 (75%) das 8 amostras de imprints foram positivas para TTC. Sete (35,5%) amostras de biópsias e 5 (62,5%) imprints foram positivos para AGT, e 11 (55%) biópsias e 4 (50%) imprints foram positivos para AT. Oito (38%) amostras de biópsias e 5 (62,5%) imprints foram positivos para o gene Ml MntH. Dentre o grupo MB, os microssatélites detectaram o bacilo em 78,5% das amostras, e o gene Ml MntH, em 57,1% das amostras, independentemente do material clínico. No grupo PB, 55,5% das amostras foram positivas para os microssatélites, enquanto que 22,2% o foram para o gene Ml MntH. CONCLUSÕES: Estes resultados mostram que, tanto as regiões específicas de microssatélites quanto o gene Ml MntH, podem representar ferramentas úteis na detecção do Ml MntH por PCR em amostras de biópsias e imprint de biópsias. Palavras-chave: Hanseníase; Mycobacterium leprae; Reação em cadeia da polimerase; Repetições de microssatélites

More information

Type
Journal Article
Author
Cruz AF
Furini RB
Roselino AMF