title: Evaluation of a biphasic media assay for pyrazinamide drug susceptibility testing of Mycobacterium tuberculosis.
abstract: OBJECTIVE
  Pyrazinamide is a key first-line tuberculosis drug. Reliable drug susceptibility testing (DST) data are of clinical importance, but in vitro testing is challenging since the activity of pyrazinamide is pH sensitive. The BACTEC MGIT 960 is considered the principal reference technique, but Wayne's test is an alternative, although it may be difficult to interpret. A further alternative is the use of a biphasic media assay (BMA). The objective of this work was to evaluate the BMA against the MGIT method and with screening of pncA gene mutations.
  
  
  METHODS
  Twenty strains were inoculated in tubes containing 2 mL of Löwenstein-Jensen (LJ) medium and 2 mL of semi-solid Kirchner medium with a critical concentration of 66 mg/L pyrazinamide at a pH of 5.2 or 5.5, incubated for 2 weeks and visually read. The results obtained were compared with MGIT 960 and DNA sequencing.
  
  
  RESULTS
  Results were obtained in duplicate for 19 strains. One strain failed to grow on two occasions and only one result was available. Reproducibility was 95%. Eleven of the 19 strains were susceptible to pyrazinamide, whereas 7 were resistant. One strain was susceptible initially and pyrazinamide resistant on repeat testing. At pH 5.5, two strains reported as susceptible at pH 5.2 gave resistant results.
  
  
  CONCLUSIONS
  The BMA might serve as a reliable low-cost DST alternative for pyrazinamide, particularly in laboratories using locally made solid media for DST. Its major drawback is the time to result. A reliable and affordable test method for the detection of pyrazinamide resistance is needed, especially in settings where multidrug-resistant tuberculosis is increasing. Proficiency testing should be routinely introduced wherever pyrazinamide DST is performed.
authors: PHE National Mycobacterium Reference Laboratory, London, UK Department of Infectious Diseases and Immunity, Faculty of Medicine, Imperial College, London, UK.
authors: X
authors: X
authors: Gonzalo
authors: PHE National Mycobacterium Reference Laboratory, London, UK Department of Infectious Diseases and Immunity, Faculty of Medicine, Imperial College, London, UK f.drobniewski@imperial.ac.uk.
authors: F
authors: F
authors: Drobniewski
authors: Department of Microbiology, Public Health Agency of Sweden, Solna, Sweden.
authors: S
authors: S
authors: Hoffner
authors: Department of Microbiology, Public Health Agency of Sweden, Solna, Sweden.
authors: J
authors: J
authors: Werngren
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical / Q000379:methods; D024881:Drug Resistance, Bacterial / Q000187:drug effects* / Q000235:genetics; D006801:Humans; D059021:Laboratory Proficiency Testing / Q000592:standards*; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification; D011718:Pyrazinamide / Q000494:pharmacology*
chemical_list: D000995:Antitubercular Agents; D011718:Pyrazinamide
keywords: TB; anti-tuberculosis drugs; pncA