Antimicrobial susceptibility testing and sequencing of Mycobacterium tuberculosis clinical isolates
With the global rise in drug resistant tuberculosis (DR-TB), drug susceptibility testing (DST) is key to ending the disease. Universal access to a prompt and comprehensive DST is therefore a major component towards the End TB strategy. Currently, diagnosis of DR-TB still relies mainly on conventional DST, which distinguish resistant and susceptible strains based on critical concentration (CC). However, studies have shown that M. tuberculosis is not binary but diverse involving low, moderate and high levels of drug resistance. The CC could also change over time with more exposure to anti-TB drugs and for many of the anti-TB drugs the CC is near the wild type minimum inhibitory concentration (MIC). Consequently, phenotypic DST based on CC testing, may provide inaccurate results, possibly leading to suboptimal treatment regimens. This necessitates to continually revise and evaluate CCs. Thus, validation of quantitative methods determining MIC instead of CCs are needed to enable formulation of optimal regimens. In addition, determination of MIC facilitates monitoring trends of drugs resistance. Methods based on CC cannot detect subtle MIC changes until the mode shifts to the next category. The geometric mean MIC is a more sensitive marker for changes in MIC distributions. Shift in MIC population distributions may have important implications for treatment. While introduction of rapid molecular techniques has improved DR-TB case detection, a comprehensive catalogue of genetic markers of clinically relevant mutations does not exist and mutations associated with resistance to newer and repurposed drugs are yet to be identified. Literature suggests that different genetic polymorphisms are associated with distinct phenotypic resistance levels. However, the impact of those mutations on the MIC remains to be investigated. Previous studies on genetic association for drug resistance in M. tuberculosis mainly relied on phenotypes defined by DST performed at a single CC. MICs are more appropriate to assess the biological effects of genomic variation in understanding the mechanism of resistance. Thus, correlating specific mutations conferring drug resistance with specific MICs for given drug classes are essential to predict levels of resistance which can be used to guide clinical decision-making. This study aimed i) To validate the Sensititre MTCOTB broth microdilution (MYCOTB) method for first and second-line anti-TB drugs ii) To determine the association between specific rpoB mutations and the MIC of rifampin (RIF) and rifabutin (RFB) among clinical MDR-TB isolates and iii) To determine the association between different genetic polymorphisms and resistance at an MIC level and iv) To evaluate the trend of anti-TB MIC for M. tuberculosis clinical isolates over a three year period in order to observe a MIC creep, if any and investigate the role of mutations in MIC changes over time. The MYCOTB broth microdilution method was validated against the agar dilution method (ADM). Strains showing discordant results between the two methods after repeat testing, were resolved using the next generation sequencing. For this purpose, a collection of MDR-TB strains from a cross-sectional MDR-TB study were used. The MYCOTB plate is based on 12 lyophilized anti-TB drugs including first and second-line drugs. For ADM, 11-welled plates of Middlebrook 7H11 medium was used representing all drugs on MYCOTB except for CYC. The categorical, essential as well as sensitivity and specificity of MYCOTB were determined in comparison with ADM. The MYCOTB plate showed good overall performance with categorical agreement ranging from 88% to 98% for the drugs tested. The sensitivity of the plate ranged from 60-100%, with exception of para-aminosalicylic acid (PAS), which had 11%, while specificity ranged from 94% to 100%. Whole genome sequencing resolved 70% of the isolates in favour of the MYCOTB plate. Rifampicin resistance is often associated with the presence of mutations in the 81-bp RIF resistance determining region (RRDR) of the rpoB gene, but the effect of these rpoB mutations on RFB resistance is less well understood. Some rpoB mutations, detectable by rapid molecular diagnostics, confer resistance to RIF but not rifabutin (RFB), suggesting RFB may be effective for treatment of M. tuberculosis with these mutations. The current study investigated the association between rpoB mutations and MIC of RIF and RFB as well as the prevalence of RFB susceptible isolates among RIF resistant strains. MICs for first- and second-line drugs were determined using the MYCOTB method and the RRDR region of the rpoB gene was sequenced. Cross-resistance between RIF and RFB was found in 73% of the isolates. Mutations S531L, H526D and H526Y were associated with both RIF and RFB (p=0.0001), while, D516V and L533P mutations were found in RIF-resistant but RFB susceptible isolates (p=0.001). A total of 27% isolates were resistant to RIF but retained susceptibility to RFB. To determine the association of genetic polymorphism and resistance at MIC level, MICs for first- and second-line drugs were linked to the corresponding genetic mutations. The MICs were determined using the MYCOTB method and relevant genes were sequenced. The Kruskall Wallis static was used to determine the association between MICs and the different mutations. The katG mutations S315T, S315G and double peak S315T were significantly associated with high INH resistance (MIC: 2 to 4ug/ml; p=00001). However, katG mutations were not significantly associated with ETH MICs (p=0.832). The inhA mutations C-15T, T-8A and G-17T were significantly associated with high resistance to both INH (0.5 to 4 ug/ml, p=0.013) and ETH resistance (10 to 40 ug/ml, p=0.001). For MXF and OFX, gyrA mutations at codon 90 and 91 were associated with lower MIC compared to isolates at codon 94. Isolates with gyrA mutation at the codon 90 had lower MIC (OFX: 4 to 8 ug/ml; p=0.0001: MXF 1 ug/ml; p=0.0001) compared to isolates at codon 94 (OFX: 8 to 32 ug/ml; p=0.000, MXF: 1 to 8ug/ml; p=0.0001). The mutations A1401G, C492T, C492T_A1401G and A514C_A1401G were associated with high MICs for KAN (10 to 40ug/ml, P=0.0001) and AMI (16ug/ml, p=0.0001). Cross-resistance between AMI and KAN was found in 85% of resistant isolates. The embB mutation was not significantly associated with MIC ranges in this study. To investigate the MIC trend and possible MIC creep, the distributions of individual MICs were plotted against time to evaluate changes over the study period. Additionally, the MIC50, MIC90 and MIC range, modal MIC, geometric mean and median MIC were determined over the three-year period. The MIC trends over the three years and the significance of changes was assessed using paired T-test. A P-Value <0.05 was considered as significant. The study showed MIC creep for three drugs RFB (p=0.0001), MXF (p=0.0001) and OFX (p=0.0001), whereas EMB (p=0.0067) and INH (p=0.0218) showed decrease in MIC over time. All the other drugs; RIF, PAS, SM, KAN, CYC, AMI, and ETH showed stable MICs over three years. The study showed the MYCOTB assay is a good alternative to conventional DST methods; relatively rapid and provides quantitative data on susceptibility to first- and second-line drugs, thus facilitating therapeutic decision-making and therapeutic drug monitoring to optimize regimen efficacy. The 96-well microplate format without the need for equipment will allow its use in resource-limited settings. The study showed that up to 27% of MDR-TB patients may benefit from a treatment regime that include RFB as a substitute for RIF resistance. Different drug resistance mutations were associated with different MIC ranges; katG, inhA gyrA and rrs mutation were associated with high MICs of their respective drugs while mutations such as rpsL (for KAN and AMI) and embB were not significantly associated with MIC ranges. This information can help in guiding clinical decision-making. The study further showed a general increase in the proportion of resistant strains over the study period for 9 of the 11 drugs tested, with evidence of creep for three drugs (MOX, OFX and RFB). Thus the monitoring of MIC changes of drugs is important to prevent gradual loss of drug efficacy.