Use of a Mycobacteriophage-Based Assay for Rapid Assessment of Susceptibilities of Mycobacterium tuberculosis Isolates to Isoniazid and Influence of Resistance Level on Assay Performance

Abstract

We standardized and assessed the performance of an in-house microtiter assay for determining the susceptibilities of Mycobacterium tuberculosis clinical isolates to isoniazid based on mycobacteriophage amplification technology. Seventy isolates (43 resistant and 27 sensitive according to the BACTEC 460 radiometric method and MIC determination) were studied. The isoniazid resistance molecular mechanism was previously determined by sequencing the entire katG gene and the mabA-inhA regulatory region. The sensitivity of the mycobacteriophage-based assay in detecting isoniazid resistance was 86.1%, the specificity achieved was 92.6%, and the overall accuracy was 88.6%. In order to assess the possible influence of resistance levels on the mycobacteriophage-based-assay sensitivity, the results were analyzed according to the isoniazid MICs. All the isolates exhibiting high-level resistance (MIC > 2 g/ml) were scored as resistant by the mycobacteriophagebased assay (100% concordance), and 95% showed mutations or deletions in the catalytic domain of the katG gene. In contrast, 26.1% of the low-level-resistance strains (MICs, 0.25 to 1 g/ml) were misclassified, and 66.7% had alterations in the mabA-inhA regulatory region. The mycobacteriophage-based assay could be used as a rapid method to detect the isoniazid susceptibility pattern, although data from those areas with high rates of low-level-resistance strains should be interpreted with caution. The features of the assay make it suitable for widespread application due to its low technical demand and cost.Despite intensive research done to improve tuberculosis (TB) diagnosis and drug susceptibility testing techniques, TB still remains one of the most threatening curable infectious diseases. Providing rapid diagnosis and antibiotic resistance detection systems is essential to prevent transmission, although these tools should be affordable in terms of cost and technical demand for those countries with limited resources and a high incidence of TB. In the vast majority of developing countries, conventional mycobacteriological techniques for culture isolation and antibiotic susceptibility testing are slow. More-rapid and newer methods such as the BACTEC 460 method (8) and the mycobacterium growth indicator tube method (22) are expensive and require sophisticated technology. There is an urgent need for accurate, simple, rapid, and inexpensive drug susceptibility testing methods adequate for widespread implementation.Several studies have reported promising results using mycobacteriophages for the rapid, simple, and inexpensive determination of drug susceptibilities (5, 6, 7, 23), especially for isoniazid (INH) and rifampin (RIF) resistance detection. These mycobacteriophage-based assays (MBAs) depend upon the ability of resistant mycobacteria to support phage replication after being exposed to drugs, while sensitive bacilli are not able to support phage infection. Extracellular phages are inactivated with a virucidal agent, whereas intracellular phages are protected and replicate...

Performance of the Genotype MTBDR Line Probe Assay for Detection of Resistance to Rifampin and Isoniazid in Strains of Mycobacterium tuberculosis with Low- and High-Level Resistance

“…1). These results confirm previous reports indicating that the replacement of Ser315 by Thr is the most frequent substitution encountered in INH-resistant strains of M. tuberculosis (1,6,10,19,24,26).…”

“…Among them, one strain with a high level of resistance had a Ϫ102 G-to-A mutation upstream from the inhA promoter. Several nucleotide substitutions in the inhA regulatory region have been previously reported from INH resistant strains, including positions Ϫ15 (C3T), Ϫ8 (T3C), Ϫ17 (G3T), and Ϫ39 (C3T) (1,6,7,26) but not the Ϫ102 G-to-A mutation found in the present study. The previously described mutations lead to the increased expression of the InhA protein, producing INH resistance via a titration mechanism (10).…”

GenoType MTBDR plus Assay for Molecular Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Strains and Clinical Samples

“…The emergence of strains resistant to the major anti-TB drugs speeds up the need for rapid methods for the identification of resistant M. tuberculosis strains in order to treat the disease effectively and, at the same time, prevent the spread of resistant strains (6,8,9,16). Multidrug-resistant (MDR) M. tuberculosis strains, which are resistant at least to rifampin (RIF) and isoniazid (INH), have emerged worldwide and seriously threaten TB control and prevention programs (30).…”

Pyrosequencing for Rapid Molecular Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Strains and Clinical Specimens

“…The low sensitivity in comparison with the sensitivity of detection of RIF resistance is due to the complex molecular basis of INH resistance and the existence of stillunidentified genes involved in the INH resistance mechanism (18,21). As has been previously described (10,16,25), the most common mutation involved in INH resistance is in katG, which has been related to high levels of INH resistance. In contrast, mutations causing low levels of INH resistance are not as clearly elucidated, as they are much more complex and involve different genes; however, a firm relationship has been found between mutations in the inhA regulatory region and low or intermediate levels of resistance (6,10).…”

“…As has been previously described (10,16,25), the most common mutation involved in INH resistance is in katG, which has been related to high levels of INH resistance. In contrast, mutations causing low levels of INH resistance are not as clearly elucidated, as they are much more complex and involve different genes; however, a firm relationship has been found between mutations in the inhA regulatory region and low or intermediate levels of resistance (6,10). Therefore, the combination of a susceptible result by the pyrosequencing assay but a pattern of resistance with the Bactec 460TB system for INH could reflect an infrequent mutation and could be related to low-level resistance.…”