title: 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.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Central Leprosy Teaching and Research Institute, Chengalpattu, Tamil Nadu, India. drbsekar@yahoo.com
authors.forename: Balaraman
authors.initials: B
authors.lastname: Sekar
authors.forename: Kamalanathan
authors.initials: K
authors.lastname: Arunagiri
authors.forename: Balan Nirmal
authors.initials: BN
authors.lastname: Kumar
authors.forename: Sujatha
authors.initials: S
authors.lastname: Narayanan
authors.forename: Kandhaswami
authors.initials: K
authors.lastname: Menaka
authors.forename: Puthenparambil Kuruvilla
authors.initials: PK
authors.lastname: Oommen
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D001706:Biopsy; D002991:Clofazimine / Q000494:pharmacology; D003430:Cross-Sectional Studies; D027081:DNA Gyrase / Q000235:genetics*; D003622:Dapsone / Q000494:pharmacology; D004094:Dihydropteroate Synthase / Q000235:genetics*; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D020022:Genetic Predisposition to Disease; D006801:Humans; D007917:Leprostatic Agents / Q000494:pharmacology*; D007918:Leprosy / Q000235:genetics*; D051379:Mice; D008826:Microbial Sensitivity Tests; D009154:Mutation; D009166:Mycobacterium leprae / Q000187:drug effects* / Q000235:genetics*; D015242:Ofloxacin / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D012293:Rifampin / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D007917:Leprostatic Agents; D003622:Dapsone; D015242:Ofloxacin; D002991:Clofazimine; D004094:Dihydropteroate Synthase; D027081:DNA Gyrase; D012293:Rifampin