title: 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.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
authors.forename: Man
authors.initials: M
authors.lastname: Zhou
authors.forename: Xuelei
authors.initials: X
authors.lastname: Geng
authors.forename: Jun
authors.initials: J
authors.lastname: Chen
authors.forename: Xude
authors.initials: X
authors.lastname: Wang
authors.forename: Dianbing
authors.initials: D
authors.lastname: Wang
authors.forename: Jiaoyu
authors.initials: J
authors.lastname: Deng
authors.forename: Zhiping
authors.initials: Z
authors.lastname: Zhang
authors.forename: Weihua
authors.initials: W
authors.lastname: Wang
authors.forename: Xian-En
authors.initials: XE
authors.lastname: Zhang
authors.forename: Hongping
authors.initials: H
authors.lastname: Wei
mesh_terms: D000581:Amidohydrolases / Q000378:metabolism; D000995:Antitubercular Agents / Q000494:pharmacology*; D003124:Colorimetry; D004269:DNA, Bacterial / Q000235:genetics*; D004352:Drug Resistance, Microbial; D006801:Humans; D008826:Microbial Sensitivity Tests; D009154:Mutation / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D011718:Pyrazinamide / Q000494:pharmacology*; D014376:Tuberculosis / Q000188:drug therapy / Q000382:microbiology*
chemical_list: D000995:Antitubercular Agents; D004269:DNA, Bacterial; D011718:Pyrazinamide; D000581:Amidohydrolases; C022490:pyrazinamide deamidase
references.citation: Antimicrob Agents Chemother. 2008 May;52(5):1852-4
references.pmid: 18316515
references.citation: J Clin Microbiol. 2007 Feb;45(2):595-9
references.pmid: 17135430
references.citation: Nat Med. 1996 Jun;2(6):662-7
references.pmid: 8640557
references.citation: J Int Med Res. 2009 Sep-Oct;37(5):1430-5
references.pmid: 19930847
references.citation: Antimicrob Agents Chemother. 1983 Oct;24(4):600-1
references.pmid: 6418066
references.citation: Nucleic Acids Res. 2004 Jan 12;32(1):239-47
references.pmid: 14718549
references.citation: J Clin Microbiol. 2002 Feb;40(2):501-7
references.pmid: 11825963
references.citation: Res Microbiol. 1989 May-Jun;140(4-5):301-9
references.pmid: 2508202
references.citation: J Clin Microbiol. 2010 Jan;48(1):300-1
references.pmid: 19923479
references.citation: J Clin Microbiol. 2000 Apr;38(4):1498-501
references.pmid: 10747133
references.citation: J Korean Med Sci. 2001 Oct;16(5):537-43
references.pmid: 11641519
references.citation: Tuber Lung Dis. 1997;78(2):117-22
references.pmid: 9692180
references.citation: Antimicrob Agents Chemother. 2004 Aug;48(8):3209-10
references.pmid: 15273154
references.citation: J Med Microbiol. 2002 Jan;51(1):42-49
references.pmid: 11800471
references.citation: Int J Tuberc Lung Dis. 1999 Jul;3(7):564-81
references.pmid: 10423219
references.citation: Eur Respir J. 2011 Mar;37(3):690-711
references.pmid: 20693257
references.citation: JAMA. 1995 Mar 22-29;273(12):916-7
references.pmid: 7884943
references.citation: Chest. 2007 Feb;131(2):497-501
references.pmid: 17296653
references.citation: Front Biosci. 2004 Sep 01;9:2086-105
references.pmid: 15353272
references.citation: Arch Bronconeumol. 2010 May;46(5):255-74
references.pmid: 20444533
references.citation: J Med Microbiol. 2005 Dec;54(Pt 12):1127-1131
references.pmid: 16278424
references.citation: J Med Microbiol. 2002 Jan;51(1):11-12
references.pmid: 11800467
references.citation: J Clin Microbiol. 1996 Jan;34(1):84-6
references.pmid: 8748279
references.citation: PLoS One. 2011 Jan 24;6(1):e15785
references.pmid: 21283666
references.citation: J Clin Microbiol. 2004 Mar;42(3):1109-14
references.pmid: 15004061
references.citation: J Clin Microbiol. 1995 Sep;33(9):2468-70
references.pmid: 7494049
references.citation: Tuberculosis (Edinb). 2009 Mar;89(2):109-13
references.pmid: 19249243
references.citation: Antimicrob Agents Chemother. 2000 Mar;44(3):528-32
references.pmid: 10681313