title: Molecular typing and differentiation of Mycobacterium tuberculosis clinical isolates using Double Repetitive Element PCR and Duplex PCR.
abstract: BACKGROUND
  To date, the advancements in polymerase chain reaction (PCR) assures accurate, fast identification and mycobacterial speciation in clinical settings, which promotes a better tuberculosis (TB) treatment regimen.
  
  
  METHODS
  In this study, a total of 78 clinically suspected cases of TB were processed for the detection of Mycobacterial infections by standard Ziehl Neelsen (ZN) staining, conventional Lowenstein-Jensen (LJ) and BACTEC MGIT-960™ liquid culture. Strain typing was performed by using Double Repetitive Element PCR (DRE-PCR) and Duplex PCR (DPCR) to differentiate Mycobacterium tuberculosis complex (MTB) from non-tuberculous mycobacteria (NTM), respectively.
  
  
  RESULTS
  Of 78 clinical isolates, 25 (32%) were drug-susceptible, and 53 (68%) were resistant to at least one drug. The BACTEC MGIT-960™ showed the highest (88.5%) positivity rate, compared with conventional LJ (82%) and ZN smear (61.5%). The mean time detection and drug susceptibility for MTB was 28 and 40days in LJ culture, and 10 and 13 days in BACTEC MGIT-960™ culture. Using DPCR, Mycobacterium avium infection was identified in HIV-positive (2.56%) and MTB in HIV-negative patients (97.4%), and the DRE-PCR system divulged 15 unique genotype patterns, and an institutional-based epidemiology database was created.
  
  
  CONCLUSIONS
  The combination of an in-house DRE-DPCR system could possibly identify and differentiate MTB from other mycobacterial species in a single reaction. In addition, restriction polymorphism analysis and DNA sequencing of NTM could assist in species identification directly from clinical isolates.
authors.affiliation: Department of Microbiology, Intermediate Reference Laboratory, Government Hospital for Chest Diseases, Gorimedu, Puducherry 605006, India. Electronic address: katkin14@gmail.com.
authors.forename: Kathirvel
authors.initials: K
authors.lastname: Maruthai
authors.affiliation: Department of Microbiology, Intermediate Reference Laboratory, Government Hospital for Chest Diseases, Gorimedu, Puducherry 605006, India; Department of Biotechnology, Sathyabama University, Chennai 600119, Tamilnadu, India. Electronic address: thirumuruganphd@gmail.com.
authors.forename: Thirumurugan
authors.initials: T
authors.lastname: Ravibalan
authors.affiliation: Department of Microbiology, Intermediate Reference Laboratory, Government Hospital for Chest Diseases, Gorimedu, Puducherry 605006, India. Electronic address: k.vallayyachari@gmail.com.
authors.forename: Kommoju
authors.initials: K
authors.lastname: Vallayyachari
authors.affiliation: Department of Microbiology, Intermediate Reference Laboratory, Government Hospital for Chest Diseases, Gorimedu, Puducherry 605006, India. Electronic address: surenmicro39@gmail.com.
authors.forename: Surendar
authors.initials: S
authors.lastname: Kesavan
authors.affiliation: Department of Biotechnology, Sathyabama University, Chennai 600119, Tamilnadu, India. Electronic address: antonysamrot@gmail.com.
authors.forename: Antony V
authors.initials: AV
authors.lastname: Samrot
authors.affiliation: Department of Microbiology, Intermediate Reference Laboratory, Government Hospital for Chest Diseases, Gorimedu, Puducherry 605006, India. Electronic address: stdcirlpdy@gmail.com.
authors.forename: Muthuraj
authors.initials: M
authors.lastname: Muthaiah
mesh_terms: D060085:Coinfection / Q000382:microbiology; D004269:DNA, Bacterial / Q000032:analysis; D024881:Drug Resistance, Bacterial; D005260:Female; D015658:HIV Infections / Q000453:epidemiology / Q000382:microbiology; D006801:Humans; D008297:Male; D058889:Molecular Typing / Q000379:methods; D009169:Mycobacterium tuberculosis / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012091:Repetitive Sequences, Nucleic Acid; D013183:Sputum / Q000382:microbiology; D014376:Tuberculosis / Q000382:microbiology*
chemical_list: D004269:DNA, Bacterial
keywords: Double Repetitive Element PCR; Drug resistant tuberculosis; Duplex PCR; Genotyping; Mycobacterium tuberculosis; Nontuberculous mycobacteria