Combined real-time pcr and rpob gene pyrosequencing for rapid identification of mycobacterium tuberculosis and determination of rifampin resistance directly in clinical specimens

Abstract

Our Lab. Has Developed A Rapid, Sensitive, and Specific Molec. Approach for Detection in Clin. Specimens, Within 48 H of Receipt, of Both Mycobacterium TB Complex (MTBC) DNA and Mutations Within the 81-Bp Core Reg. of the Rpob Gene That Are Assoc. with Rifampin (RIF) Resist. This Approach, Which Combines an Initial Real-time PCR with Int. Inhibition Assess. and A Pyrosequencing Assay, Was Validated for Direct Use with Clin. Specimens. to Assess the Suitability of Real-time PCR for Use with Resp., Nonrespiratory, Acid-fast Bacillus (AFB)-positive and AFB-negatlve Specimens, we Evaluated Specimens Received in Our Lab. between 11 October 2007 and 30 June 2009. with Culture Used as the "Gold Std.," the Sensitivity, Specificity, and Positive and Negative Predictive Values Were Determined for 1,316 Specimens to be as Follows: for Resp. Specimens, 94.7%, 99.9%, 99.6%, and 98.6%, Respectively; for Nonrespiratory Specimens, 88.5%, 100.0%, 100.0%, and 96.9%, Respectively; for AFB-positive Specimens, 99.6%, 100.0%, 100.0%, and 97.7%, Respectively; and for AFB-negative Specimens, 75.4%, 99.9%, 98.0%, and 98.4%, Respectively. PCR Inhibition Was Determined to be Minimal in This Assay, Occurring in 0.2% of Tests. the Rpob Gene Pyrosequencing Assay Was Evaluated in A Similar Prospective Stud., in Which 148 Clin. Specimens Positive for MTBC DNA by Real-time PCR Were Tested. the Final Results Revealed That the Results of Direct Test. of Clin. Specimens by the Pyrosequencing Assay Were 98.6% Concordant with the Results of Conventional Test. for Susceptibility to RIF in Liquid Culture and That Our Assay Displayed Adequate Sensitivity for 96.6% of the Clin. Specimens Tested. Used Together, These Assays Provide Reliable Results That Aid with the Initial Mgmt. of Patients with Suspected TB Prior to the Availability of the Results for Cultured Mat., and They Also Provide the Ability to Predict RIF Resist. in Mycobacterium TB-positive Specimens in as Little as 48 H from the Time of Clin. Specimen Receipt. Copyright 2010, Amer. Soc. for Microbiol. All Rights Reserved.