C-210. Development and Implementation of a Real-Time PCR Assay for Rapid Identification of Mycobacterium tuberculosis Complex DNA from Clinical Samples in New York State

T. A. Halse, P. Cunningham, W. J. Wolfgang, N. Dumas, K. Musser;
New York State Dept. of Hlth., Albany, NY.

Real-time PCR (rtPCR) has become a standard method for rapid detection in the diagnostic microbiological laboratory. Our laboratory has developed a rtPCR protocol for the rapid detection of the Mycobacterium tuberculosis (Mtb) complex from clinical specimens. The assay targets the multi copy IS6110 insertion element, and the protocol includes an assessment of inhibition and a simple pre-processing step utilizing the BD Geneohm™ Lysis kit. The analytical sensitivity of the assay is 0.05 colony-forming units per PCR reaction and the specificity was determined to be 100% in the 84 Mycobacterium spp. and other respiratory organisms tested. This rtPCR is inexpensive, requires minimal hands-on time, and results are available within 3 hours of specimen decontamination. Historically, the Gen-Probe Amplified Mycobacterium Tuberculosis Direct (MTD) Test has been used by our laboratory in conjunction with culture to rapidly detect Mtb complex rRNA in acid-fast bacilli (AFB) smear positive and negative concentrated sediments prepared from respiratory specimens. A study was conducted to evaluate this rtPCR and MTD with conventional culture identification methods on 320 clinical specimens received by our laboratory. Of these, 99 specimens were culture positive for MTb complex and 81 were culture positive for Mycobacterium other than tuberculosis (MOTT). Applying culture as the gold standard, sensitivity, specificity, and positive and negative predictive values of the rtPCR were similar to the MTD test (98, 96.4, 92.3, 99.1% and 97.9, 95.4, 90.3, 99.0%, respectively). However, the rtPCR yielded fewer inconclusive results. As a result of this study, the Mycobacteriology laboratory of the NYS Department of Health has found rtPCR to be a good alternative to MTD testing for rapid reporting of respiratory specimens. The sensitivity, specificity, and ease of use of this method permit rapid acquisition of reliable results that aid in the initial management of suspect tuberculosis patients prior to obtaining results from cultured material.