Abstract:

Objective To establish and evaluate a novel colorimetric method (sodium 3,3'-{1-((phenylamino) carbonyl)-3,4-tetrazolium}-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT)/1-methoxy-5-methylphenazinium methylsulfate (1-methoxy-PMS) assay, XTT/mPMS assay) for the rapid anti-mycobacteria drug susceptibility testing.Methods The properties of the XTT/mPMS and XTT/2-methyl-1,4-NQ (mNQ) colorimetric assay, including optimal reagent concentrations, linear range, reaction time, stability, cytotoxicity and drug interference, were assessed. The performance of XTT/mPMS for the rapid drug susceptibility testing of clinical isolated Mycobacteria strains was evaluated taking solid culture method as the gold standard, and the minimum inhibitory concentration (MIC) was determined. The results were compared with the resazurin assay.Results The optimal concentrations of XTT and the electron mediators in XTT/mPMS and XTT/mNQ systems were 0.2 mmol/L and 0.04 mmol/L, respectively. The chromogenic reaction time with Mycobacterium smegmatis was shorter than that with Mycobacterium bovis and Mycobacterium tuberculosis in both systems (XTT/mPMS: 60, 420 and 420 min; XTT/mNQ: 60, 420 and 420 min, respectively). Both systems showed low cytotoxicity to the three mycobacteria, and could prolong the time of reaching the state phase for 12 to 48 hours approximately. The XTT/mPMS system was stable when co-cultured with Middlebrook 7H9 liquid medium at 37 ℃ or when long-term stored at 37 ℃ for 8 d, 25 ℃ for 15 d and 4 ℃ for 30 d. Moreover, using the solid culture method as the gold standard, the specificity of the XTT/mPMS colorimetric method for the rapid drug susceptibility testing (rifampicin and isoniazid) of clinical isolated Mycobacteria strains were 100.0% (17/17) and 100.0% (13/13) and the sensitivity were 95.7% (22/23) and 92.6% (25/27), respectively, which were consistent with the resazurin assay (specificity: 100.0% (17/17) and 100.0% (13/13); sensitivity: 100.0% (23/23) and 92.6% (25/27)).Conclusion We found that the XTT/mPMS chromogenic system is stable, low-toxic and inexpensive, and is well suited for the rapid drug susceptibility testing of Mycobacterium.

Key words: Mycobacterium, Mycobacterium tuberculosis, Drug resistance, bacterial, Microbial sensitivity tests, Evaluation studies