title: Rifampicin resistance mutations in the 81 bp RRDR of rpoB gene in Mycobacterium tuberculosis clinical isolates using Xpert® MTB/RIF in Kampala, Uganda: a retrospective study.
abstract: BACKGROUND
  Introduction of Xpert® MTB/RIF assay has revolutionalised the diagnosis of tuberculosis (TB) by simultaneously detecting the bacteria and resistance to rifampicin (rif), a surrogate marker for multi-drug resistant TB (MDR-TB) as well as one of the principal first-line anti-tuberculosis drugs. In general, rpoB mutations can be found in 96.1% of rif-resistant Mycobacterium tuberculosis (MTB) strains worldwide and these mutations usually are located in a region at the 507-533rd amino acid residuals (81 bp) in the MTB rpoB gene, which is referred to as Rifampicin-resistance-determining region (RRDR). In this study, we determined the frequency of MDR-TB in Kampala using Xpert® MTB/RIF in comparison with the agar proportion method using Middlebrook 7H11and further determined the frequency of probes for different rpoB gene mutations using Xpert® MTB/RIF assay in the 81 bp RRDR.
  
  
  METHODS
  A total of 1501 specimens received at Mycobacteriology laboratory, Makerere University for Xpert testing between May 2011 and May 2014 were analysed by Xpert® MTB/RIF assay. Specimens that were positive for both MTB and rifampicin resistance were further subjected to a complete first line anti-mycobacterial drug susceptibility testing using Middlebrook 7H11 agar proportion method (APM).
  
  
  RESULTS
  Xpert® MTB/RIF assay detected 313 MTB positive specimens and out of which 12 specimens had both MTB and rifampicin- resistance conferred by four different rpoB gene mutations in the 81 bp-RRDR of MTB, further one (1/12), specimen was found to be rifampicin mono-resistant on APM while the 11 were found to be MDR-TB. Probes associated with the observed rif- resistance were as follows: E (7/12), B (3/12), A (1/12), D (1/12) and no rif-resistance was associated with probe C. No specimen yielded rif-resistance associated with more than one probe failure (mutation combinations). Probe D was associated with rifampicin mono-resistant.
  
  
  CONCLUSIONS
  MDR-TB was at 3.5% in the studied population. Mutations associated with Probe E (58%) also known as codons 531and 533 are the commonest rpoB gene mutation identified by Xpert® MTB/RIF assay in this setting and mutations identified by probe E of the assay, turned out to be MDR-TB strains by agar proportion method antimicrobial susceptibility testing. No mutation was detected in the codon 522.
authors.affiliation: Department of Medical Microbiology, School of Biomedical Sciences, College of Health Sciences, Makerere University, P,O Box 7072, Kampala, Uganda. gmboowa@yahoo.com.
authors.forename: Gerald
authors.initials: G
authors.lastname: Mboowa
authors.forename: Carolyn
authors.initials: C
authors.lastname: Namaganda
authors.forename: Willy
authors.initials: W
authors.lastname: Ssengooba
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases; D024881:Drug Resistance, Bacterial; D006801:Humans; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification*; D011933:Reagent Kits, Diagnostic; D012189:Retrospective Studies; D012293:Rifampin / Q000494:pharmacology*; D018088:Tuberculosis, Multidrug-Resistant / Q000175:diagnosis / Q000453:epidemiology / Q000382:microbiology*; D014454:Uganda / Q000453:epidemiology
chemical_list: D001426:Bacterial Proteins; D011933:Reagent Kits, Diagnostic; C505805:rpoB protein, Mycobacterium tuberculosis; D012321:DNA-Directed RNA Polymerases; D012293:Rifampin