Rapid colorimetric testing for pyrazinamide susceptibility of M. tuberculosis by a PCR-based in-vitro synthesized pyrazinamidase method.

Abstract

Pyrazinamide (PZA) is an important first-line anti-tuberculosis drug. But PZA susceptibility test is challenging because PZA activity is optimal only in an acid environment that inhibits the growth of M. tuberculosis. For current phenotypic methods, inconsistent results between different labs have been reported. Direct sequencing of pncA gene is being considered as an accurate predictor for PZA susceptibility, but this approach needs expensive sequencers and a mutation database to report the results. An in-vitro synthesized Pyrazinamidase (PZase) assay was developed based on PCR amplification of pncA gene and an in vitro wheat germ system to express the pncA gene into PZase. The activity of the synthesized PZase was used as an indicator for PZA susceptibility. Fifty-one clinical isolates were tested along with pncA sequencing and the BACTEC MGIT 960 methods. The in-vitro synthesized PZase assay was able to detect PZA susceptibility of M. tuberculosis within 24 h through observing the color difference either by a spectrometer or naked eyes. This method showed agreements of 100% (33/33) and 88% (14/16) with the pncA sequencing method, and agreements of 96% (27/28) and 65% (15/23) with the BACTEC MGIT 960 method, for susceptible and resistant strains, respectively. The novel in-vitro synthesized PZase assay has significant advantages over current methods, such as its fast speed, simplicity, no need for expensive equipment, and the potentials of being a direct test, predicting resistance level and easy reading results by naked eyes. After confirmation by more clinical tests, this method may provide a radical change to the current PZA susceptibility assays.

• Authors: Man Zhou, Xuelei Geng, Jun Chen, Xude Wang, Dianbing Wang, Jiaoyu Deng, Zhiping Zhang, Weihua Wang, Xian-En Zhang, Hongping Wei
• Journal: PloS one (PLoS One) Vol. 6 Issue 11 Pg. e27654 ( 2011) ISSN: 1932-6203 [Electronic] United States
• PMID: 22102918 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Antitubercular Agents
• DNA, Bacterial
• Pyrazinamide
• Amidohydrolases
• pyrazinamide deamidase

Topics

• Amidohydrolases (metabolism)
• Antitubercular Agents (pharmacology)
• Colorimetry
• DNA, Bacterial (genetics)
• Drug Resistance, Microbial
• Humans
• Microbial Sensitivity Tests
• Mutation (genetics)
• Mycobacterium tuberculosis (genetics, isolation & purification)
• Polymerase Chain Reaction
• Pyrazinamide (pharmacology)
• Tuberculosis (drug therapy, microbiology)