Combined Real-Time PCR and rpoB Gene Pyrosequencing for Rapid Identification of Mycobacterium tuberculosis and Determination of Rifampin Resistance Directly in Clinical Specimens

• DOI: 10.1128/jcm.02149-09
• Publication Date: 2010-01-28T02:48:42Z
• Authors: Tanya A. Halse; Justine Edwards; Phyllis L. Cunnin...; William J. Wolfgang; Nellie B. Dumas; Vincent E. Escuyer; Kimberlee A. Musser
• Keywords: rpoB; Pyrosequencing; Mycobacterium tuberculosis complex; Gold standard (test); Nontuberculous Mycobacteria

Our laboratory has developed a rapid, sensitive, and specific molecular approach for detection in clinical specimens, within 48 h of receipt, both Mycobacterium tuberculosis complex (MTBC) DNA mutations the 81-bp core region rpoB gene that are associated with rifampin (RIF) resistance. This approach, which combines an initial real-time PCR internal inhibition assessment pyrosequencing assay, was validated direct use specimens. To assess suitability respiratory, nonrespiratory, acid-fast bacillus (AFB)-positive AFB-negative we evaluated specimens received our between 11 October 2007 30 June 2009. With culture used as "gold standard," sensitivity, specificity, positive negative predictive values were determined 1,316 to be follows: respiratory 94.7%, 99.9%, 99.6%, 98.6%, respectively; nonrespiratory 88.5%, 100.0%, 96.9%, AFB-positive 97.7%, 75.4%, 98.0%, 98.4%, respectively. minimal this occurring 0.2% tests. The assay similar prospective study, 148 MTBC by tested. final results revealed testing 98.6% concordant conventional susceptibility RIF liquid displayed adequate sensitivity 96.6% Used together, these assays provide reliable aid management patients suspected prior availability cultured material, they also ability predict resistance tuberculosis-positive little from time specimen receipt.