Nasal PCR assay for the detection of Mycobacterium leprae pra gene to study subclinical infection in a community.

Leprosy is a chronic infectious disease caused by Mycobacterium leprae. Identification of Mycobacterium leprae is difficult in part due to the inability of the leprosy bacillus to grow in vitro. A number of diagnostic methods for leprosy diagnosis have been proposed. Both serological tests and molecular probes have shown certain potential for detection and identification of Mycobacterium leprae in patients. In this study, we have investigated whether Mycobacterium leprae DNA from the nasal secretion of healthy household contacts and the non contacts could be detected through PCR amplification as a method to study the sub clinical infection in a community. A total of 200 samples, 100 each from contacts and non contacts representing all age groups and sex were included in this study. The M. leprae specific primer (proline-rich region) of pra gene was selected and PCR was performed using extracted DNA from the sample. A total of 13 samples were found to be positive for nasal PCR for pra gene among the male and female contacts out of which 7% were males and 6% were females. Even though several diagnostic tools are available to detect the cases of leprosy, they lack the specificity and sensitivity. PCR technology has demonstrated the improved diagnostic accuracy for epidemiological studies and requires minimal time. Although nasal PCR studies have been reported from many countries it is not usually recommended due to the high percentage of negative results in the contact.

Detección de mutaciones en los genes folp1, rpoB y gyrA de M. Leprae por secuenciación directa PCR-una técnica rápida para el cribaje de resistencias farmacológicas en la lepra / No disponible

Introducción: La técnica convencional para evaluar la susceptibilidad antimicrobiana de la multiterapia (MDT) recomendado por la OMS frente al Mycobacterium leprae en la almohadilla plantar del ratón (MFP) es laboriosa y larga (aproximadamente entre 6 y 12 meses). Actualmente se han definido dianas moleculares para distintos medicamentos para tratar la lepra. Se han estandarizado diversas técnicas moleculares para la rápida detección de estas resistencias. Este estudio ha comparado dichos métodos moleculares con la técnica MFP para determinar la susceptibilidad del M. leprae frente a los anti-microbianos. Métodos: Participaron en este estudio 40 pacientes con índice bacteriológico (IB) positivo, de entre ellos había 25 con característica clara de recaída, 11 nuevos casos y 4 incumplidores del tratamiento. Se obtuvo una biopsia cutánea de todos los casos que fue procesado para MFP y métodos moleculares. Se diseña primero PCR para amplificar fragmentos DNA de 388 bp del gen folP1 para la resistencia a la dapsona, de 305 bp del gen rpoB para la resistencia a la rifampicina y de 342 bp del gen gyrA para la resistencia a ofloxacino, seguida por la secuenciación DNA. Resultados: Solamente se obtuvo un crecimiento significativo con la técnica MPF en 28 de las 40 biopsias procesadas (70%). De entre ellas, 10 muestras resultaron ser dapsona resistentes, una muestra presentó resitencia frente a la dapsona, rifampicina y clofazimina. Se obtuvo amplificación de genes en 40 (100%) de las muestras. De entre los amplificados folP1 secuenciados, 6 cepas presentan mutaciones en posición 53 o 55 de los aminoácidos. Las cepas que resultaron muy resistentes con crecimiento dos log con la técnica MFP y/o crecimiento mediante pasaje presentaban mutaciones en el gen folP1. No se detectaron mutaciones en los amplificados rpoB y gyrA. No e detectaron pues resistencia frente a la rifampicina en el DNA aislado de las biopsias. Conclusión: Se puede aplicar la técnica PCR-secuenciación directa por su sensibilidad y rapidez para sustituir a la más laboriosa MFP para la detección de resistencia frente a dapsona, rifampicina y ofloxacino (AU)

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Detection of mutations in folp1, rpoB and gyrA genes of M. leprae by PCR- direct sequencing--a rapid tool for screening drug resistance in leprosy.

UNLABELLED: Conventional Mouse foot-pad (MFP) assay for screening drug resistance in M. leprae is cumbersome and time-consuming (approximately 6 to 12 months). Molecular targets for different anti-leprosy drugs have been well defined. Molecular tools for rapid detection of drug resistance in M. leprae have been standardised. A study to compare molecular methods with MFP assay in determining the drug susceptibility of M. leprae was carried out. METHODS: Forty Bacteriological Index (BI) positive patients of leprosy with clinical features of relapse (25), new cases (11) and defaulters (4) were included in the study. A skin biopsy was done and the samples were processed using both MFP assay and Molecular method. PCR assays were carried out to amplify, 388 bp of folP1 gene for dapsone resistance, 305 bp of rpoB gene for rifampicin resistance and 342 bp of gyrA gene for ofloxacin resistance, followed by direct DNA sequencing. RESULTS: Significant growth in the MFP test was obtained in only 28 out of 40 biopsies processed (70%). Ten of these isolates were dapsone resistant; one isolate showed combined resistance against dapsone, rifampicin and clofazimine. Amplification for all three genes was successful in all the 40 (100%) samples. Among folP1 products sequenced, six isolates showed mutations at 53 (or) 55 amino acid positions. Those strains which showed high-level resistance with two log growth in MFP test, and/or showed growth in passage had mutations in folp1 gene. No mutation was detected in rpoB and gyrA products. Thus no molecular evidence of Rifampicin resistance was found in the DNA isolated from biopsies. CONCLUSION: Thus PCR-direct sequencing--the rapid and high sensitive molecular technique can be applied for detection of resistance against dapsone, rifampicin and ofloxacin in M. leprae, to over come the limitations of the conventional MFP assay.

Low frequency of moaA3 gene among the clinical isolates of Mycobacterium tuberculosis from Tamil Nadu and Pondicherry--south eastern coastal states of India.

BACKGROUND: Comparative genomic analysis of M. tuberculosis H37Rv and M. bovis BCG have shown that 16 RDs (Regions of Differences) are deleted in BCG and have shown six deletion regions in M. tuberculosis H37Rv. RD1, is present in M. tuberculosis but is absent in all M. bovis BCG sub-strains. A study from Kerala, a south-western coastal state of India aimed to find out differences in RD1 region showed for the first time the presence of moaA3 gene in majority of their clinical isolates, that was absent in type strain H37Rv. We attempted to find out such polymorphism between type strains and the clinical isolates within RD1, targeting moaA3 gene among the clinical isolates of Tamil Nadu & Pondicherry, south-eastern coastal states of India METHODS: One hundred and sixteen clinical isolates of M. tuberculosis were included in the study. PCR using RD1DLa and RD1DRa primers was carried out to amplify a 652 bp fragment, encoding for cfp10 and esat 6 proteins of RD1. A second PCR using primers designed from the surrounding regions of moaA3 gene was done to confirm the presence of the full Open Reading Frame (ORF) in clinical isolates. RESULTS: In M. tuberculosis H37Rv the expected 652 bp band was present. In BCG it was absent as expected, but a 386 bp fragment was amplified. Around 12/116 (10.3%) of our clinical isolates showed both 652 and 386 bp fragments. The additional 386 bp amplicon is a part of the moaA3 gene which codes for molybdopterin cofactor protein A in M. bovis. The second PCR amplified the flanking sequence of moaA3 and yielded the expected amplicon of 1254 bp in all those 10.3% of clinical isolates which had the 386 bp fragment. However the earlier study carried out in Kerala, reported the presence of moaA3 gene in majority (97%) of their clinical isolates. CONCLUSION: This finding showed that there was regional variation presenting polymorphism in moA3 gene, among the strains of M. tuberculosis and further strengthens the speculation of genetic differences among the strains of Kerala and Tamil Nadu & Pondicherry, the South Indian states.