Automated liquid culture system misses isoniazid heteroresistance in Mycobacterium tuberculosis isolates with mutations in the promoter region of the inhA gene

Abstract

Heteroresistance in Mycobacterium tuberculosis isolates remains the major challenge for phenotypic drug susceptibility testing (DST) methods to detect drug resistance. The aim of this study was to investigate the abilities of phenotypic DST methods to identify the isoniazid (INH) heteroresistance in M. tuberculosis. We found that the broth dilution method was able to detect INH resistance if 0.5 % resistant bacteria with mutations in the katG and oxyR-ahpC regions were present, while the detection limit ranged from 1 to 10 % for the INH-resistant strains harboring inhA mutations, which was associated with the different mutant types. Additionally, MGIT DST was able to find the recommended 1 % INH resistance due to katG mutations. In contrast, MGIT DST detected resistance in suspensions with 20 % resistant bacteria with inhA mutations. Statistical analysis revealed that the ability of the broth dilution method to detect heteroresistance was better than that of the MGIT DST (p = 0.004). When we further pairwise compared the two methods for detecting heteroresistance according to different mutant loci, the broth dilution method found more heteroresistance due to inhA mutations than MGIT DST (p = 0.001), while the differences for katG and oxyR-ahpC mutations were both not statistically significant (p > 0.05). In conclusion, our findings demonstrate that MGIT DST fails to detect INH heteroresistance in M. tuberculosis isolates with mutations in the promoter region of inhA. In addition, the broth dilution method is more sensitive than MGIT DST in finding INH heteroresistance, indicating that this method may serve as an alternative method to detect the heteroresistance of M. tuberculosis.

Rapid diagnosis of MDR and XDR tuberculosis with the MeltPro TB assay in China

“…Indirect drug susceptibility of the culture-positive isolates identified as MTB was detected by the Bactec MGIT 960 automated system according to the manufacturer’s instructions. The critical concentrations were 1.0 μg/mL for rifampicin (RIF), 0.1 μg/ml for isoniazid (INH), 2 μg/mL for ofloxacin(OFLX), 0.5 μg/mL for amikacin (AMK), and 1.0 μg/mL for kanamycin (KAN) 27 32 33 . The clinical TB laboratories enrolled in this study have passed the proficiency testing for conventional DST organized by the National TB Reference Laboratory of the China CDC.…”

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“…The range in the abilities for drug-resistant TB detection among these studies may be explained by the differences in the molecular characteristics of predominant drug-resistant MTB strains that exist across different regions. In addition, heteroresistance is considered an important mechanism in the emergence of drug resistance 21,22. However, the HAIN test could not detect minor drug-resistant populations from tubercle bacilli.…”

Agreement of Middle brook 7H10 with Lowenstein Jensen and accuracy of the Sensititre MYCOTB plate using either method as a reference standard for Mycobacterium tuberculosis first line drug susceptibility testing

“…The sub-culturing of isolates for MB7H10 and MYCTB plate DST may have caused changes in the sample strain population from the original sample used for DST in presence of hetero-resistance [ 18 ]. Studies have indicated hero-resistance to affect DST results from the sub-cultured strains [ 19 , 20 ], however, this is expected to be minimal given the low prevalence of hetero-resistance. This is one of the few studies which have compared LJ and MB7H10 DST with MYCOTB plate and our findings are vital for future studies and recommendations.…”