Rapid detection of Mycobacterium tuberculosis and pyrazinamide susceptibility related to pncA mutations in sputum specimens through an integrated gene-to-protein function approach.

Abstract

Testing the pyrazinamide (PZA) susceptibility of Mycobacterium tuberculosis isolates is challenging. In a previous paper, we described the development of a rapid colorimetric test for the PZA susceptibility of M. tuberculosis by a PCR-based in vitro-synthesized-pyrazinamidase (PZase) assay. Here, we present an integrated approach to detect M. tuberculosis and PZA susceptibility directly from sputum specimens. M. tuberculosis was detected first, using a novel long-fragment quantitative real-time PCR (LF-qPCR), which amplified a fragment containing the whole pncA gene. Then, the positive amplicons were sequenced to find mutations in the pncA gene. For new mutations not found in the Tuberculosis Drug Resistance Mutation Database (www.tbdreamdb.com), the in vitro PZase assay was used to test the PZA resistance. This approach could detect M. tuberculosis within 3 h with a detection limit of 7.8 copies/reaction and report the PZA susceptibility within 2 days. In an initial testing of 213 sputum specimens, the LF-qPCR found 53 positive samples with 92% sensitivity and 97% specificity compared to the culture test for M. tuberculosis detection. DNA sequencing of the LF-qPCR amplicons revealed that 49 samples were PZA susceptible and 1 was PZA resistant. In the remaining 3 samples, with new pncA mutations, the in vitro PZase assay found that 1 was PZA susceptible and 2 were PZA resistant. This integrated approach provides a rapid, efficient, and relatively low-cost solution for detecting M. tuberculosis and PZA susceptibility without culture.

• Authors: Heng Li, Jun Chen, Man Zhou, Xuelei Geng, Junping Yu, Weihua Wang, Xian-En Zhang, Hongping Wei
• Journal: Journal of clinical microbiology (J Clin Microbiol) Vol. 52 Issue 1 Pg. 260-7 (Jan 2014) ISSN: 1098-660X [Electronic] United States
• PMID: 24226918 (Publication Type: Evaluation Study, Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Antitubercular Agents
• Pyrazinamide
• Amidohydrolases
• PncA protein, Mycobacterium tuberculosis

Topics

• Adult
• Amidohydrolases (genetics, metabolism)
• Antitubercular Agents (pharmacology)
• Humans
• Microbial Sensitivity Tests (methods)
• Molecular Diagnostic Techniques (methods)
• Mutation, Missense
• Mycobacterium tuberculosis (drug effects, genetics, isolation & purification)
• Pyrazinamide (pharmacology)
• Real-Time Polymerase Chain Reaction (methods)
• Sensitivity and Specificity
• Sequence Analysis, DNA
• Sputum (microbiology)
• Time Factors
• Tuberculosis, Pulmonary (diagnosis, microbiology)