Abstract
This study correlates Minimum Inhibitory Concentrations (MICs) of rifampicin (RIF) and isoniazid (INH) with GenoType MTBDRplus assay results for drug-resistant Mycobacterium tuberculosis (MTB) clinical isolates. MICs of RIF and INH were established for 84 and 90 isolates, respectively, testing six concentrations of each drug. Genotypic resistance to each drug was determined by GenoType MTBDRplus assay with 50 representative mutations confirmed by pyrosequencing, with mutations in the rpoB gene associated with RIF-resistance and mutations in the katG and/or inhA genes associated with INH-resistance. Based upon the correlation of MICs with specific genetic profiles, relative resistance levels were established for each isolate. Results indicate that MTB phenotypic resistance, currently based upon the testing of isolate susceptibility to a single drug concentration, may be more accurately profiled via quantitative MICs, and therefore the correlation of molecular diagnostic results with specific MICs may allow for more optimal treatment of infections.
Keywords: Drug susceptibility testing, multidrug-resistant tuberculosis, level of resistance, mutations
1. Introduction
The increase in prevalence of drug-resistant tuberculosis (DR-TB), including multi- and extensively drug-resistant tuberculosis (M/XDR-TB) (WHO, 2010), and the recent emergence of “totally drug-resistant TB” (Udwadia et al., 2012; Velayati et al., 2009) poses a significant threat to global TB control efforts (WHO, 2010; Shah et al., 2011; Gandhi et al., 2010; Jonsson et al., 2012; WHO, 2011).
Mycobacterium tuberculosis (MTB), the main agent of TB, is known to have heterogeneous phenotypic drug resistance profiles. TB phenotypic resistance can range from low- and moderate-resistance to drugs, resulting in infections that might still be effectively treated with increased dosing of the relevant TB medications, to high-level resistance, as seen in infections that cannot be treated with the drugs with which the isolate is resistant to (Springer et al., 2009). As such, estimating the Minimum Inhibitory Concentrations (MICs) of drugs against an individual patient’s MTB infection can be an effective, if tedious, clinical diagnostic method (Heifets et al., 2009). However, in low-resource clinical settings with facility and cost limitations, these methods are beyond basic laboratory diagnostic capabilities.
Molecular diagnostics, including line probe assays (WHO, 2008a), such as the Hain GenoType MTBDRplus assay, and pyrosequencing assays, such as those utilizing the Qiagen PyroMark platform (Lin et al., 2014), have the potential to significantly improve MDR diagnosis and to facilitate real-time treatment decisions. However, these assays represent only indirect measures of phenotypic resistance, and the relationship between mutations in the rpoB and katG, inhA, and ahpC genes and rifampicin (RIF) - and isoniazid (INH)-MICs is not clearly defined. Additional data is necessary to fully exploit and interpret molecular diagnostics for the clinical evaluation of RIF and INH drug resistance in TB patients.
For RIF, mutations within 81bp region of the rpoB gene, encoding the B subunit of a DNA-dependent RNA polymerase, are responsible for conferring RIF resistance. Canonical mutations in this gene, including 516Val, 526Asp/Tyr and 531Leu, are well documented, whereas amino acid changes at codons 511, 515, 516, 518, 521, 522, and 533 have not been thoroughly evaluated for their association with phenotypic RIF-resistance. Notably, high-level RIF-resistance is reported more frequently than lower levels of resistance, supporting the analysis of these other, potentially RIF-resistance associated mutations for diagnostic purposes (Sirgel et al., 2013).
For INH, the accumulation of mutations in the katG gene and inhA promoter leads to development of resistance. Mutations within katG prevent the activation of the gene’s respective prodrug, resulting in high-level INH resistance. Mutations in the inhA promoter gene are known to increase the level of protein expression, and are generally correlated with low-level INH resistance. (Santos et al., 2012).
In this study, we determined the MICs of RIF and INH for MTB clinical isolates and categorized the isolates by phenotypic levels of resistance based on MIC values. Ultimately, we correlated these phenotypic resistance levels with GenoType MTBDRplus assay results, with 50 representative mutations confirmed by pyrosequencing.
2. Materials and Methods
2.1 Setting
The study was performed at the Mycobacteriology Laboratory of P. D. Hinduja National Hospital (PDHNH) and Medical Research Centre (MRC), a tertiary care hospital in Mumbai, India that has a referral bias towards nonresponders.
2.2 Ethical approval
The study was approved by the Institutional Review Board (IRB) of Hinduja Hospital. Written consent was waived as the study was carried out on 120 consecutively archived MTB isolates for which GenoType MTBDRplus assay, as well as MGIT 960 drug susceptibility testing (DST) for RIF and INH, had been previously performed.
2.3 Phenotypic MGIT Drug Susceptibility Testing
All isolates were subjected to conventional phenotypic DST for RIF and INH by MGIT 960 (Becton Dickinson Diagnostic System, Sparks, MD) with Epicenter software, using critical concentrations recommended by the World Health Organization (WHO, 2008b). MGIT DST quality control was ensured daily using a known (genotypically confirmed) resistant isolate as well as the H37Ra pansusceptible strain.
Preparation of Drugs
A stock solution of RIF (Becton Dickinson Diagnostic System, Sparks, MD Catalogue 245126) was prepared by unfiltered dissolution of the drug in dimethyl sulfoxide (DMSO) and further dilution with distilled water. INH (Becton Dickinson Diagnostic System, Sparks, MD and catalogue 245126) was prepared by dissolution in distilled water, filtration, and then further dilution with distilled water. Both drugs were stored at −80°C for up to 6 months. Preparation of the inoculum, inoculation and incubation was performed as per MGIT 960 manufacturer instructions (Becton Dickinson Diagnostic System, Sparks, MD) (Siddiqi et al., 2012).
2.3.1 MIC using MGIT 960
MICs were performed via MGIT 960 using a macrodilution method where different concentrations of the drugs were prepared and injected into different MGIT tubes. The subsequent inoculation and incubation was performed as for phenotypic DST determination.
Selection of drug concentrations for MICs
MIC testing levels were selected in order that the seven drug concentrations would define the phenotypical resistance levels of the majority of mutations. The WHO has established a critical concentration of 1μg/ml for RIF and 0.1μg/ml for INH (WHO, 2008b). For RIF and INH, concentrations above and below the critical concentration were tested as follows: For estimation of RIF MICs we used two concentrations below the critical concentration (0.5 and 0.25μg/ml), and four concentrations above the critical concentration (2.0, 4.0, 10.0 and 20.0μg/ml). For estimation of INH MICs we used two concentrations below the critical concentration (0.05 and 0.025μg/ml), and four concentrations above the critical concentration (0.5, 1.0, 3.0 and 10.0μg/ml).
2.4 Genotypic Methods
GenoType MTBDRplus assay
120 consecutive archived isolates were selected for which both GenoType MTBDRplus assay and DST (at the WHO approved critical concentration) had been performed previously. The GenoType MTBDRplus assay (Hain Lifescience, Nehren, Germany) procedure was comprised of 3 steps. First, a GenoLyse kit was used for DNA extraction from decontaminated patient samples/bacterial cultures. Multiplex polymerase chain reaction (PCR) amplification with biotinylated primers was then performed in a thermal cycler (Eppendorf) under the following cycling conditions: denaturation 95°C/15 min, initial denaturation 95°C/30 sec, annealing 58°C/2 min (10 cycles), denaturation 95°C/25 sec, annealing 53°C/40 sec, extension 70°C/40 sec (30 cycles), final extension 70°C/8 min. Finally, reverse hybridization was performed as per manufacturer instructions.
A strict, unidirectional workflow was adhered to for all experiments. Quality control was ensured by testing a pansusceptible MTB strain, H37Ra, along with a strain confirmed to carry resistance-associated mutations, during each MGIT 960 run. In order to confirm that no cross contamination occurred, a negative control was also run with each batch (Ajbani et al., 2012).
Pyrosequencing
Fifty representative isolates of the 120 MTB isolates utilized in this study were sequence-confirmed by pyrosequencing (PSQ). A total of 30 isolates had the rpoB gene region pyrosequenced to confirm the genotypic RIF-resistance, based upon the presence or absence of known resistance-associated rpoB mutations. PSQ results confirmed GenoType MTBDRplus assay findings, in that a PSQ-resistant result was marked by the absence of wildtype probes and the presence of the corresponding mutation probe (MUT1, MUT2A, MUT2B and/or MUT3), or, as in the case of 14 isolates, with absence of wildtype probes and the absence of the corresponding mutation probe in the GenoType assay. Similarly, 20 representative isolates had the relevant katG and inhA gene regions pyrosequenced to confirm GenoType INH-resistance results. PSQ data was collected for six isolates showing GenoType mutations in both the katG and inhA genes, six showing only katG mutations, and four showing only inhA mutations. Four isolates determined to be INH- and RIF-wildtype via the GenoType assay were also pyrosequenced.
PSQ consisted of PCR amplification followed by PSQ reaction on the the PyroMark Q96 ID system (Qiagen, Valencia, CA). The HotStarTaq kit and deoxynucleoside triphosphate (dNTP) mixtures (Qiagen, Valencia, CA) were used in the PCR master mix. The PCR mixture contained 2.5μl of DNA and 22.5μl of a PCR master mix, which included 1× supplied PCR buffer, 2.5mM MgCl2, 0.96mM dNTP mixture, 1× Q-solution, 0.5μM primer, and 1U of HotStarTaq. Amplification involved initial activation of the HotStarTaq at 95°C for 15 min, 50 cycles of amplification at 94°C for 15 s, 60°C for 30 s, and 72°C for 20 s, and final extension at 72°C for 5 min. PyroMark Q96 (5 by 96) reagents were used for PSQ with the sequence analysis mode of the PyroMark Q96 ID system utilized for result interpretation (Lin et al., 2014). The molecular marker IS6110 was utilized to confirm the presence of MTB. The targeted loci for detection of DR-TB were the RIF resistance-determining region (RRDR) of rpoB for RIF resistance and regions of the katG gene and inhA and ahpC promoters for INH resistance (Lin et al., 2014).
3. Results
3.1 Phenotypic Results
Phenotypic MGIT DST results found 18/120 isolates to be MDR (resistant to INH and RIF) but susceptible to fluoroquinolones (FQs) and to injectables (amikacin, kanamycin and capreomycin), while 53/120 were MDR with additional resistance to FQs, 9/120 were XDR with resistance to injectables and FQs, and 7/120 were INH-monoresistant. Three isolates were found to be susceptible to RIF and to injectables, but resistant to all other drugs tested. A total of 30/120 isolates were phenotypically susceptible to all drugs tested.
3.1.1 MIC results
We determined the MICs of RIF and INH for the MTB isolates and assessed their correlation with known genetic mutations. The genotypic and phenotypic findings of the 120 MTB isolates, with regards to RIF and INH, are summarized in Table 1. Resistance levels were established for the various mutations with respect to their MIC range. The correlation of specific mutations with phenotypic resistance levels is summarized in Tables 2 and 3.
Table 1.
MIC results of RIF and INH:
| Mutations | GenoType MTBDRplusassay | Total no. of isolates | No. of isolates | MIC range of INH | MIC range of RIF | ||
|---|---|---|---|---|---|---|---|
| katG with inhA | rpoB | katG with inhA | rpoB | n=24 | |||
| Ser315Thr1 C15T | Ser531Leu | MUT1 MUT1 | MUT3 | 18 | 18 | >10 | >20 |
| Ser315Thr1 T8C | Ser531Leu | MUT1 MUT3A | MUT3 | 1 | 1 | 10 | >20 |
| Ser315Thr1 T8C | Ser531Leu | MUT1 MUT3A | MUT3 | 1 | 1 | >10 | >20 |
| Ser315Thr2 T8C | Ser531Leu | MUT2 MUT3A | MUT3 | 1 | 1 | 10 | >20 |
| Ser315Thr1 C15T | Asp516Tyr | MUT1 MUT1 | Absence of WT3/4 | 1 | 1 | >10 | 4 |
| Ser315Thr1 C15T | His526Asp | MUT1 MUT1 | MUT2B | 1 | 1 | >10 | >20 |
| Ser315Thr1 T8C | His526Asp | MUT1 MUT3A | MUT2B | 1 | 1 | 10 | >20 |
| katG | rpoB | katG | rpoB | n=50 | |||
| Ser315Thr1 | His526Cys | MUT1 | Absence of WT7 | 1 | 1 | 10 | 2 |
| Ser315Thr1 | His526Asn | MUT1 | Absence of WT7 | 3 | 2 | 10 | 0.25 |
| 1 | 10 | 0.5 | |||||
| Ser315Thr1 | Leu533Pro | MUT1 | Absence of WT8 | 4 | 1 | 10 | 2 |
| 2 | 10 | 4 | |||||
| 1 | >10 | 4 | |||||
| Ser315Thr1 | His526Tyr | MUT1 | MUT2A | 2 | 2 | 3.0 | >20 |
| Ser315Thr1 | Asp516Val | MUT1 | MUT1 | 3 | 3 | 10 | 10 |
| Ser315Thr1 | Asp516Tyr | MUT1 | Absence of WT3,4 | 2 | 1 | 10 | 10 |
| 1 | 10 | 0.25 | |||||
| Ser315Thr1 | Asp516Phe | MUT1 | Absence of WT 3/4 | 1 | 1 | 10 | 10 |
| Ser315Thr1 | Asp516Ala | MUT1 | Absence of WT 3+8 | 1 | 1 | 3.0 | 10 |
| Ser315Thr1 | Ser531Leu | MUT1 | MUT3 | 25 | 7 | 3.0 | >20 |
| 16 | 10 | >20 | |||||
| 2 | >10 | >20 | |||||
| Ser315Thr2 | Ser531Leu | MUT2 | MUT3 | 2 | 2 | 10 | >20 |
| Ser315Thr1 | His526Asp | MUT1 | MUT2B | 5 | 2 | >10 | >20 |
| 3 | 10 | >20 | |||||
| Ser315Thr1 | His526Arg | MUT1 | Absence of WT7 | 1 | 1 | 10 | >20 |
| inhA | rpoB | inhA | rpoB | n=16 | |||
| C15T | WT | MUT1 | WT | 6 | 6 | 0.5 | 0.25 |
| C15T | Ser531Leu | MUT1 | MUT3 | 9 | 6 | 0.5 | >20 |
| 3 | 1 | >20 | |||||
| C15T | His526Tyr | MUT1 | MUT2A | 1 | 1 | 1 | >20 |
| WT | WT | 30 | 25 | 0.1 | 0.25 | ||
| 5 | 0.05 | 0.25 | |||||
Table 2.
MIC of RIF and correlation of mutations with level of resistances:
| Mutations/ Level of resistance | Asp516Ala | Asp516Phe | Asp516Tyr | Asp516Val | His526Cys | His526Asn | His526Tyr | His526Asp | His526Arg | Ser531Leu | Leu533Pro |
|---|---|---|---|---|---|---|---|---|---|---|---|
| ≤4 (Low) | 2 | 1 | 3 | 4 | |||||||
| 10 (Moderate) | 1 | 1 | 1 | 3 | |||||||
| >20 (High) | 3 | 7 | 1 | 57 |
Table 3.
MIC of INH- Correlation of mutations with level of resistance:
| Mutations / Level of Resistance | katG with inhA | katG | inhA |
|---|---|---|---|
| Ser315Thr1withC15T Ser315Thr1withT8C, Ser315Thr2withT8C |
Ser315Thr1 Ser315Thr2 |
C15T | |
| 0.5–1.0 (Low) | 0 | 0 | 16 |
| 3.0–10.0 (Moderate) | 3 | 45 | 0 |
| >10.0 (High) | 21 | 5 | 0 |
3.2 Genotypic results
Mutations in the rpoB gene were observed in 84 MTB isolates; katG and inhA mutations were observed in 90 MTB isolates, with 24 of these isolates having mutations in both genes. Thirty-six isolates evaluated for RIF-resistance were wildtype, or lacking resistance-associated mutations in the rpoB RRDR, while 30 isolates tested for INH-resistance were wildtype for the associated gene regions of interest. 16.6% of isolates evaluated for RIF-resistance via GenoType assay showed the absence of wildtype probles, but also had an absence of the corresponding mutation probe. Agreement between phenotypic MGIT DST and genotypic MTBDRplus assay for INH-resistance was 100% and for RIF-resistance it was 95%. PSQ results of 30 MTB isolates evaluated for RIF-resistance and 20 isolates evaluated for INH-resistance are summarized in Tables 4 and 5.
Table 4.
PSQ results for rpoB (n=30):
| No. of isolates | GenoType MTBDRplus assay | PSQ |
|---|---|---|
| 1 | Absence of WT3 | 516GAC-GCC |
| 1 | Absence of WT3/4 | 516GAC-TTC |
| 3 | Absence of WT3/4/8 | 516GAC-TAC |
| 1 | Absence of WT7 | 526CAC-TGC |
| 1 | Absence of WT7 | 526CAC-CGC |
| 3 | Absence of WT7 | 526CAC-AAC |
| 3 | Absence of WT3/4 & presence of MUT1 | 516GAC-GTC |
| 3 | Absence of WT7 & presence of MUT2A | 526CAC-TAC |
| 3 | Absence of WT7/4 & presence of MUT2B | 526CAC-GAC |
| 3 | Absence of WT8 & presence of MUT3 | 531TGC-TTG |
| 4 | Absence of WT8 | 533CTG-CCG |
| 4 | WT | WT |
Table 5.
PSQ results for katG and inhA (n=20):
| No. of isolates | GenoType MTBDRplus assay | PSQ | ||
|---|---|---|---|---|
| katG | inhA | katG | inhA | |
| 4 | MUT1 | MUT1 | 315AGC-ACC | -15T |
| 1 | MUT1 | MUT3A | 315AGC-ACC | -8C |
| 1 | MUT2 | MUT3A | 315AGC-ACA | -8C |
| 4 | MUT1 | WT | 315AGC-ACC | WT |
| 2 | MUT2 | WT | 315AGC-ACA | WT |
| 4 | WT | MUT1 | WT | -15T |
| 4 | WT | WT | WT | WT |
4. Discussion
The various mutations our study identified within the katG, inhA and rpoB genes correlated with INH and RIF MICs, establishing specific drug-susceptibility levels for 120 MTB isolates (see Table 1). Clear differences were observed between the drug susceptibility levels of genotypically wildtype isolates and those harboring resistance-associated mutations. In total, mutations associated with RIF-resistance were found within the rpoB gene of 84 isolates and mutations associated INH-resistance were found within the katG and inhA genes of 90 of the 123 MTB isolates evaluated in this study.
Among the 84 RIF-resistant isolates, 70 canonical mutations [Asp516Val (n=3), His526Tyr/Asp (n=10), and Ser531Leu (n=57)] and 14 disputed mutations [Asp516Tyr/Phe/Ala (n=5), His526Cys/Asn/Arg (n=5), and Leu533Pro (n=4)] were observed. We found the MIC of isolates with the Asp516Phe/Ala/Val mutation (5.9%, or 5/84 of RIF-resistant isolates) to be 10μg/ml. These isolates were defined as having a moderate level of resistance. This finding is in line with an earlier study reporting 85% (29/34) of isolates with Asp516Val mutations having a MIC range of 10–15μg/ml (Sirgel et al., 2013). Within our study, 11% (10/84) of isolates with His526Tyr/Asp mutations had MICs >20 μg/ml which is similar to a previously published study wherein 15% (3/19) of isolates with His526Tyr/Asp mutations had MICs >50μg/ml (Springer et al., 2008). RIF MICs >20μg/ml were confirmed for 67% (57/84) of isolates with Ser531Leu mutations in our study, a result comparable to another study’s report of 78% (15/19) of isolates with a Ser531Leu mutation having a MIC >20μg/ml (Springer et al., 2008). Isolates with Asp516Tyr mutations, and some with 526 mutations, showed discordant MGIT RIF MIC results. This is not a novel finding, as recent studies have shown that phenotypic RIF-resistance testing is not binary for all rpoB mutations, and MGIT 960 culture methods are known to incorrectly evaluate some RIF-resistance conferring mutations (Rigouts et al., 2013). Silent mutations, such as TTC514TTT and GGC507GGT, although possible sources of GenoType MTBDRplus wildtype probe absent and mutation probe absent results, were not encountered in the isolates evaluated in our study (Gracelin et al., 2014)
Out of 90 INH-resistant isolates, we observed the following mutations: Ser315Thr1/2 in the katG gene (n=50), C-15T within the inhA promoter (n=16), and katG with inhA (n=24) at Ser315Thr1/2 with C-15T or T-8C. Of the 90 isolates found to be resistant to INH at 0.1μg/ml, 14 were found to be susceptible to the drug at 0.5μg/ml (Table 1). 88% (14/16) of isolates with an inhA promoter mutation at C-15T, alone, showed low-level INH-resistance. This type of low-level resistance may potentially be overcome in TB treatment with increase in INH dosing, particularly in areas of low endemicity (Springer et al., 2008; Guo et al., 2006). In our study, 88% (21/24) of isolates having concurrent mutations in the katG and inhA gene regions evaluated i.e. Ser315Thr1 with C-15T or T-8C were determined to have MICs >10μg/ml. Finally, 100% (50/50) of isolates with the Ser315Thr1/2 mutation in katG were determined to have MICs of 3– ≥10μg/ml. Our findings, in this respect, confirm previously published results of MIC values associated with mutations at Ser315 (Springer et al., 2008).
Notably, both RIF and INH testing yielded low level MIC results in some instances, despite the presence of resistance-associated mutations in the RRDR of the rpoB gene, and the inhA promoter, respectively.
5. Conclusions
In order to effectively curb the rise and spread of MDR-TB throughout the globe, there is an urgent need to quickly and accurately determine clinical drug susceptiblity profiles of drug-resistant isolates. The correlation of genotypic test results, including line probe assays and pyrosequencing, with a specific range of MIC values for each drug, presents an invaluable tool for quickly guiding treatment decisions in clinical laboratories worldwide. Of particular interest, this study finds the majority of MTB isolates with the canonical rpoB mutations His526Asp/Tyr and Ser531Leu to have a high-level of resistance to RIF, whereas the Asp516Val mutation was associated with a moderate level of resistance to RIF, as seen via MIC testing. As each mutation within the rpoB gene does not confer the same level of phenotypic RIF-resistance, there is a need for additional MIC and genetic testing to confirm correlations between genotypic mutations and phenotypic resistance levels. We recommend additional clinical outcome studies to assess whether disputed mutations, with MICs around RIF-resistance critical concentration cutoffs, are associated with treatment failure. For example, the C-15T inhA mutation, found to correlate with low-level INH-resistance, is a point of clinical relevance, as isolates with this mutation might be treated with higher doses of INH. In conclusion, since effective MDR-TB treatment relies upon our knowledge of quantitative susceptibility testing results, the correlation of genotypic test results with phenotypic resistance levels is a critical component of TB diagnosis that should be further evaluated in order to prevent the spread of drug resistance and promote the optimum use of the few drugs available for TB treatment.
Highlights.
Mycobacterium tuberculosis resistance is heterogeneous i.e. involves Low, Moderate, High level of resistance.
MIC can predict this level of resistance.
Low level of resistance is a point of clinical relevance
Acknowledgments
We are thankful to National Health and Education Society, Hinduja Hospital and Medical Research Centre for funding this study. Antonino Catanzaro and Timothy C. Rodwell were funded by National Institute of Allergy and Infectious Diseases (NIAID) grant U01-AI082229 and grant R01AI111435-04.
Footnotes
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