Rapid identification of mycobacteria and drug-resistant Mycobacterium tuberculosis by use of a single multiplex PCR and DNA sequencing

Abstract

Tuberculosis (TB) remains a significant global health problem for which rapid diagnosis is critical to both treatment and control. This report describes a multiplex PCR method, the Mycobacterial IDentification and Drug Resistance Screen (MID-DRS) assay, which allows identification of members of the Mycobacterium tuberculosis complex (MTBC) and the simultaneous amplification of targets for sequencing-based drug resistance screening of rifampin-resistant (rifampin(r)), isoniazid(r), and pyrazinamide(r) TB. Additionally, the same multiplex reaction amplifies a specific 16S rRNA gene target for rapid identification of M. avium complex (MAC) and a region of the heat shock protein 65 gene (hsp65) for further DNA sequencing-based confirmation or identification of other mycobacterial species. Comparison of preliminary results generated with MID-DRS versus culture-based methods for a total of 188 bacterial isolates demonstrated MID-DRS sensitivity and specificity as 100% and 96.8% for MTBC identification; 100% and 98.3% for MAC identification; 97.4% and 98.7% for rifampin(r) TB identification; 60.6% and 100% for isoniazid(r) TB identification; and 75.0% and 98.1% for pyrazinamide(r) TB identification. The performance of the MID-DRS was also tested on acid-fast-bacterium (AFB)-positive clinical specimens, resulting in sensitivity and specificity of 100% and 78.6% for detection of MTBC and 100% and 97.8% for detection of MAC. In conclusion, use of the MID-DRS reduces the time necessary for initial identification and drug resistance screening of TB specimens to as little as 2 days. Since all targets needed for completing the assay are included in a single PCR amplification step, assay costs, preparation time, and risks due to user errors are also reduced.

Fig 1

Proposed MID-DRS testing algorithm. Multiplex PCR is performed using AFB smear-positive processed sputum samples or cultures positive for AFB. Amplification of IS6110 and RD9 prompts preliminary identification of M. tuberculosis and sequencing to screen for mutations associated with RIF^r, INH^r, and PZA^r. Lack of RD9 with IS6110 amplification or lack of IS6110 with RD9 amplification prompts hsp65 sequencing for confirmation of MTBC. All IS6110-negative specimens are evaluated with hsp65 sequencing for confirmation of an (i) MTBC-negative result, (ii) M. avium result, or (iii) NTM identification. rDNA, rRNA gene.

Fig 2

Amplification products produced with the MID-DRS assay. Results of agarose gel electrophoresis of PCR amplicons produced from the individual and multiplexed primer pairs in the MID-DRS assay using genomic DNA are shown. Lane 1, a Hyperladder II standard; lane 2, rpoB; lane 3, pncA; lane 4, katG; lane 5, hsp65; lane 6, RD9; lane 7, 16S rRNA gene MAC; lane 8, IS6110; lane 9, multiplex PCR (7-plex) of all above-listed targets using TB DNA; lane 10, multiplex PCR (7-plex) of all above-listed targets using MAC DNA.

Fig 3

Limit of detection of MID-DRS multiplex PCR. The analytical sensitivity of the MID-DRS multiplex PCR was determined by comparison of resulting PCR amplification products to numbers of CFU produced with serial dilutions of either M. tuberculosis H37Rv ATCC 27294 (A) or M. avium ATCC 25291 (B). Cell counts were obtained by the direct plate count method.

Fig 4

Specificity of primers for the 16S rRNA gene of MAC. Strains shown represent a subset of all strains used for in silico primer selection. Light gray areas represent the locations of forward and reverse primers with respect to the conventional base pair numbering of the 16S rRNA gene. Dark gray sections represent sites where changes were made to the forward primer for the design of the secondary forward primer used in PCRs. av., avium; paratuberc., paratuberculosis; intracell., intracellulare.