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.
Highlights
The emergence of drug-resistant strains is one of the main causes for the current spread of Tuberculosis (TB) and it is clinically important to do drug susceptibility testing for better control of TB [1,2]
In order for PCR products to cover possible mutations on the whole pncA gene, PCR primers were designed from the upstream and the downstream regions of pncA gene on M. tuberculosis genome (Fig. 1A and 1B)
The in-vitro PZase method showed very good agreements (100% and 88% for susceptible and resistant strains, respectively) in results with the pncA sequencing method, which demonstrated that this method could be used to replace the pncA sequencing method for accurate prediction of PZA susceptibility
Summary
The emergence of drug-resistant strains is one of the main causes for the current spread of Tuberculosis (TB) and it is clinically important to do drug susceptibility testing for better control of TB [1,2]. The radiometric assay (BACTEC 460TB) using pH 6.0 and PZA concentration of 100ug/mL was considered as most reliable and the currently recommended assay (Clinical Laboratory Standard Institute, M24-A2). Even for this reference method, inconsistent results between different labs have been reported [10,11]. Previous studies found that the radiometric assay can lead to inconclusive results up to 3.5% of the strains [12], falsely resistant results for at least 0.8% of the strains [13], and falsely susceptible results for some strains [14]
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