[Study on the method for rapid detection of Mycobacterium tuberculosis by phage amplified biologically assay].

OBJECTIVE: To set up a method for rapid detection of Mycobacterium tuberculosis (MTB) by phage amplified biologically (PhaB) assay and to investigate the optimal test condition. METHODS: Various test conditions were compared in order to observe the influence on detective results after MTB was infected by Mycobacteriophage. The test condition established was used for detection of sputum samples, and the results were compared with BIOTEC Lab test. RESULTS: The bacterial concentration of MTB in 200 - 500/ml was detected by PhaB assay at 1 x 10(9) plaque forming unite (PFU)/ml of Mycobacteriophage, 37 degrees C for 60 min. The optimal concentration of virucidal for inactivation of Mycobacteriophage was 100 mmol/ml for 5 min at room temperature. The bacteriolytic plaque was clear at the concentration of 1 x 10(8)/ml indicator cells. Bacterium inactivated by heat can not be infected by Mycobacteriophage. Positive result was observed for control strains of H(37)Rv, H(37)Ra and M. bovis while negative result was obtained for 7 strains of non-Mycobacterium and 16 control strains of non-Tuberculosis Mycobacteria (NTM). The 4 strains of NTM (M. fortuitum, M. intrcellulare, M. aurum, M. phlei) showed positive reaction at higher concentrations (> 1 x 10(5)/ml). The repetition test showed that the differentiation coefficient in batch and inner was all under 15%. There was a significantly difference (P < 0.01) in positive rate between two digestion-decontamination procedure with N-acetyl-cysteine-NaOH liquefacient (94%) and NaOH liquefacient (62%). The positive rate of the samples cultured one day (65%) was significantly higher than that of the samples without preculture (40%). The results for detection of clinical samples by two reagents, ours and BIOTEC Lab, were nearly the same. CONCLUSION: Because its rapidity, simplicity, and sensitivity, PhaB assay can be used for rapid detection of MTB, but the condition of test is very important.

[Application of rapid detection for rifampin resistance in clinical isolates of Mycobacterium tuberculosis by phage amplified biologically assay].

OBJECTIVE: To set up a rapid detection method for rifampin susceptibility with phage amplified biologically (PhaB) assay and to evaluates its value in the detection of rifampin resistance in clinical isolates of Mycobacterium tuberculosis. METHODS: The assay was established to detect rifampin resistance in 524 clinical isolates of Mycobacterium tuberculosis, and the result was compared to that of the absolute concentration method. The minimum inhibitory concentration (MIC) was detected by BACTEC MGIT 960 method for the discrepant isolates. RESULTS: Rifampin susceptibility results were available for 524 strains of Mycobacterium tuberculosis. A total of 223 strains were found to be rifampin resistant and 301 strains were rifampin susceptible detected by PhaB assay, but 211 and 313 strains were respectively found to be rifampin resistant and susceptible by conventional methods. There were 198 and 288 rifampin resistant and susceptible strains both detected by the two methods. The drug susceptibility of 35 strains was the same in 38 discrepant isolates by the PhaB assay and absolute concentration method. The sensitivity, specificity, positive and negative predictive values as well as the overall accuracy for the PhaB assay was 93.8%, 92.0%, 88.8%, 95.7% and 92.7% respectively if the judgment standard was adopted by conventional methods. CONCLUSION: The result of PhaB assay was available within 2 days. This method, which is simple and does not need special equipment, can be used for rapid screening for rifampin resistance from Mycobacterium tuberculosis.