A large study of 1730 patients with presumed MDR-TB from Lima, Peru, Baku, Azerbaijan, South Africa (Cape Town and Durban) and India (Mumbai) was designed and supervised by the Foundation for Innovative New Diagnostics (FIND), Geneva, Switzerland to test an automated method of detecting rifampin (RIF) resistance. Support also came from the National Institutes of Health (NIH), Cepheid, and the Bill and Melinda Gates Foundation and was a multicenter endeavor at the various sites mentioned above from July 2008 to March 2009.
Three sputum samples, one direct and two indirect, processed with N-acetyl-L-cysteine and sodium hydroxide (NaOH) from each patient, were tested for MTB/RIF (rifampin resistant MTB) on the Xpert® (Cepheid) automated molecular system. The system uses a hemi-nested real-time polymerase chain reaction (PCR) assay to amplify a MTB sequence of the rpoB gene, followed by probes with molecular beacons for mutations within the rifampin-resistance-determining region. Results were available in two hours. Overall sensitivity of the test was 97.6% (culture positive patients), 99.8% (smear and culture-positive patients), and 90.2% for smear and culture-negative patients. Specificity was 99.2% for a single specimen, direct MTB/RIF test, 98.6% for two, and 98.1% for three MTB/Rif tests. Also detected were a group of MDR-TB patients (receiving treatment), who were resistant to both isoniazid (INH) and RIF.
Both safety and simplicity to operate the Xpert® MTB/RIF system (Cepheid) are positive aspects of the study, when procedures are carried out in reference centers by trained personnel. The system does not require use of a biosafety cabinet, but employs a simple, mainly hands-off step to process sputum by liquefaction and inactivation of viable TB bacilli. No infectious aerosols are generated and results are available within two hours. Simplicity is seen in the short technician training required (2-3 days compared to 2 weeks for Ziehl-Neelson microscopy) and limited manual time on the machine (15 minutes). Although application to laboratories outside these centers may not be cost effective at present, the FIND is working to lower the costs for low-income areas and eventual replacement of the expensive culture and susceptibility testing systems presently used.
The World Health Organization (WHO) recommended use of the Xpert® MTB/RIF automated molecular test for MTB resistance to RIF in 2010 and continues to monitor this technology in 21 countries under the STOP TB program. Other methods to detect RIF and INH resistance include INNO-LiPA Rif.TB (Immunogenetics, Ghent, Belgium) and GenoType® MTBDRplus (Hain LifeScience, Nehren, Germany), which are approved by the WHO for smear- positive clinical specimens and culture. The sensitivity range for RIF detection is 98% to 99% and for INH is 84% to 89%, but the methods are not yet approved by the FDA.