Molecular Detection of Mutidrug-Resistant (MDR) Strains: Rifampin (RIF) Resistance

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The page below is a sample from the LabCE course Tracking Antibiotic-Resistant Tuberculosis. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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Molecular Detection of Mutidrug-Resistant (MDR) Strains: Rifampin (RIF) Resistance

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 used a hemi-nested real-time polymerase chain reaction (PCR) assay to amplify an 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% for culture-positive patients, 99.8% for 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 (two to three days contrasted to two weeks for Ziehl-Neelson microscopy) and limited manual time on the machine (15 minutes).
  • Application to laboratories outside these centers may not be cost-effective at present.
The World Health Organization (WHO) recommended the use of the Xpert® MTB/RIF automated molecular test for MTB resistance to RIF in 2010 and continues to monitor this technology in 28 countries. 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.