[Comparison of P-nitrobenzoic acid and hydroxylamine hydrochloride for the primary screening of mycobacterial species].

OBJECTIVES: To examine the screening methods for mycobacteria recommended by the China Anti-tuberculosis Association, in order to increase laboratory diagnostic accuracy for mycobacterial screening. METHODS: Using P-nitrobenzoic acid (PNB 0.5 g/L) as the control group, and hydroxylamine hydrochloride (HA, in 125, 150 and 175 mg/L concentrations) as the study group, laboratory preserved strains of H37Rv M.tuberculosis, and standard and clinically isolated strains of M.nontuberculosis (NTM) from Guangzhou Chest Hospital were tested for both PNB and HA sensitivity. Differences between groups were analyzed by χ(2) test. RESULTS: Among the 2529 MTB strains, the resistance rate to PNB was 3.0% (76/2529), to HA was 12.2% (308/2529), 4.8% (121/2529), and 0.9% (23/2529), respectively, corresponding to the aforementioned 3 different concentrations of HA. Among the 1766 NTM strains, the sensitive rate to PNB was 8.3% (147/1766), to HA was 0.1% (2/1766), 0.5% (9/1766), and 0.9% (16/1766), respectively, corresponding to the aforementioned 3 different concentrations of HA. There was significant difference (χ(2) = 5.44-83.50, P < 0.05). CONCLUSION: HA at 175 mg/L concentration was the optimal condition for laboratory tuberculosis preliminary screening.

[A study on the drug resistance to p-nitrobenzoic acid in Mycobacterium tuberculosis].

OBJECTIVE: To investigate the resistance of Mycobacterium tuberculosis (MTB) to p-nitrobenzoic acid (PNB), in order to provide the scientific basis for reliable identification of laboratory strains of mycobacterium. METHODS: Strains of mycobacterium grown in PNB media were identified with additional traditional biochemical tests, according to the standard protocols of laboratory diagnostics for tuberculosis by the Chinese Antituberculosis Association. For mycobacteria grown in the PNB media but highly suspected as MTB by traditional biochemical tests, MPB64 monoclonal antibody was used for the differentiation between MTB and non-tuberculosis mycobacteria group (NTM). Minimal inhibitory concentrations (MIC) of PNB was further determined for culture-confirmed MTB, 10 strains of clinically isolated MTB (control), H(37)Rv standard strain, mycobacterium avium standard strain, and 5 strains of clinically isolated NTM. RESULTS: A total of 1114 strains of MTB were confirmed, among which 58 PNB manifested resistance. The rate of resistance was 5.21% (58/1114), with an MIC ranging for 1.0 - 1.5 g/L. The MICs of control MTB and H(37)Rv standard strain were 0.25 - 0.5 g/L and 0.25 g/L, respectively. Both mycobacterium avium standard strain and clinically isolated NTM showed an MIC of > 2.0 g/L. Differences between groups were statistically significant (t = 4.87, 5.09, 6.68, respectively, P < 0.01). CONCLUSION: In order to avoid laboratory misdiagnosis, for primary screening with NTB with PNB culture, the presence of MTB characteristics, including cream-colored broccoli-like colony morphologies, as well as clinical response to first-line anti-tuberculosis medications, despite PNB tolerance, warrants further investigations of traditional biochemical tests, differentiation with MPB64 monoclonal antibodies, or simply by the use of temperature manipulation or drug-sensitivity test results.

[Analysis of the changes in bacterial types and drug sensitivity profiles of mycobacterial strains in Guangzhou over the last twelve years].

OBJECTIVE: To improve evidence-based care in the management of tuberculosis, we retrospectively analyzed the bacterial types and drug sensitivity test results of mycobacteria in Guangzhou over the past twelve years (from July 1998 to March 2010). METHODS: Over these twelve years, a total of 14 095 mycobacterial strains isolated from different samples were subjected to type identification and drug sensitivity tests according to the Standard Protocols of Laboratory Diagnostics for Tuberculosis by the Chinese Antituberculosis Association. Chi-square test was performed for statistical analyses for comparisons between groups. RESULTS: Of 14 095 strains of mycobacteria isolated, 10 844 strains (76.84%) were MTB, and 3251 strains (23.16%) were non-tuberculosis mycobacteria (NTM). Compared with the result of the fourth national survey of tuberculosis epidemiology, which showed 11.1% of NTM, the one of our study was significantly different (χ(2) = 69.79, P < 0.001). Drug sensitivity tests of MTB showed tolerance rates of 28.99% (2729/9413), 21.75% (2047/9413), 17.45% (1643/9413) and 11.53% (1085/9413) against isoniazid, rifampin, streptomycin and ethambutol, respectively. CONCLUSION: An increasing trend was observed in MTB drug tolerance against streptomycin, rifampin and isoniazid, whereas more and more NTM strains were isolated in recent years. These findings are worthy of note for clinicians.

[Study on species identification of Mycobacteria by gas chromatography analysis of whole-cell fatty acid].

OBJECTIVE: To evaluate the accuracy and applicability of species identification method of Mycobacteria by applying gas chromatography analysis of whole-cell fatty acid. METHODS: Species identification of 14 reference strains and 727 clinical isolates of Mycobacteria were performed by using MIDI Sherlock Microbial Identification System (MIS)4.0 based on gas chromatography analysis of whole-cell fatty acid, and the results were compared with those of conventional species identification method. RESULTS: (1) By using the conventional method, all 14 reference strains were identified correctly. Except for Mycobacterium.vaccae, the result obtained by MIS were identical to that of conventional method. (2) Among 625 clinical isolates identified as Mycobacterium tuberculosis (MTB) and Mycobacterium bovis, 45 strains were mistakenly identified as Nontuberculous Mycobacteria (NTM) by MIS. Its accuracy was 93%. For 102 NTM strains, results of group identification showed no difference between the two methods. However, 7 results of species identification were not consistent, the accuracy being 93%. (3) MIS mainly mistook MTB for Mycobacterium gastri, Mycobacterium trivial and Mycobacterium smegmatis, and Mycobacterium scrofulaceum for Mycobacterium gordonae among NTM. CONCLUSIONS: The results of species identification of Mycobacteria by applying gas chromatography analysis of whole-cell fatty acid are in accordance with those of conventional method for the majority of the strains, and MIS can identify Mycobacteria to species level by a single experiment. It can be considered as a good method for species identification of Mycobacteria in terms of its accuracy and applicability.