Co-resistance to isoniazid and second-line anti-tuberculosis drugs in isoniazid-resistant tuberculosis at a tertiary care hospital in Thailand

ABSTRACT Isoniazid-resistant tuberculosis (Hr-TB) is an important drug-resistant tuberculosis (TB). In addition to rifampicin, resistance to other medications for Hr-TB can impact the course of treatment; however, there are currently limited data in the literature. In this study, the drug susceptibility profiles of Hr-TB treatment and resistance-conferring mutations were investigated for Hr-TB clinical isolates from Thailand. Phenotypic drug susceptibility testing (pDST) and genotypic drug susceptibility testing (gDST) were retrospectively and prospectively investigated using the Mycobacterium Growth Indicator Tube (MGIT), the broth microdilution (BMD) method, and whole-genome sequencing (WGS)-based gDST. The prevalence of Hr-TB cases was 11.2% among patients with TB. Most Hr-TB cases (89.5%) were newly diagnosed patients with TB. In the pDST analysis, approximately 55.6% (60/108) of the tested Hr-TB clinical isolates exhibited high-level isoniazid resistance. In addition, the Hr-TB clinical isolates presented co-resistance to ethambutol (3/161, 1.9%), levofloxacin (2/96, 2.1%), and pyrazinamide (24/118, 20.3%). In 56 Hr-TB clinical isolates, WGS-based gDST predicted resistance to isoniazid [katG S315T (48.2%) and fabG1 c-15t (26.8%)], rifampicin [rpoB L430P and rpoB L452P (5.4%)], and fluoroquinolones [gyrA D94G (1.8%)], but no mutation for ethambutol was detected. The categorical agreement for the detection of resistance to isoniazid, rifampicin, ethambutol, and levofloxacin between WGS-based gDST and the MGIT or the BMD method ranged from 80.4% to 98.2% or 82.1% to 100%, respectively. pDST and gDST demonstrated a low co-resistance rate between isoniazid and second-line TB drugs in Hr-TB clinical isolates. IMPORTANCE The prevalence of isoniazid-resistant tuberculosis (Hr-TB) is the highest among other types of drug-resistant tuberculosis. Currently, the World Health Organization (WHO) guidelines recommend the treatment of Hr-TB with rifampicin, ethambutol, pyrazinamide, and levofloxacin for 6 months. The susceptibility profiles of Hr-TB clinical isolates, especially when they are co-resistant to second-line drugs, are critical in the selection of the appropriate treatment regimen to prevent treatment failure. This study highlights the susceptibility profiles of the WHO-recommended treatment regimen in Hr-TB clinical isolates from a tertiary care hospital in Thailand and the concordance and importance of using the phenotypic drug susceptibility testing or genotypic drug susceptibility testing for accurate and comprehensive interpretation of results.

burden (1).Drug-resistant TB (DR-TB) is an emerging global concern, posing challenges to TB treatment and infection control.Multidrug-resistant tuberculosis (MDR-TB) is a Mycobacterium tuberculosis (MTB) strain that is resistant to rifampicin (RIF) and isoniazid (INH), while isoniazid-resistant TB (Hr-TB) refers to the MTB strain that is susceptible to RIF but resistant to INH (1,2).INH is an active drug with bactericidal activity and is used to treat active TB and prevent latent TB reactivation (3).The prodrug INH requires activation by the mycobacterial catalase-peroxidase enzyme (KatG) to interact with enoyl-acyl carrier protein reductase (InhA) and inhibit mycolic acid synthesis in the cell wall.Mutations in the katG gene are commonly involved in resistance to INH, followed by the promoter region of the fabG1-inhA operon and the ahpC promoter (4,5).
Hr-TB can be diagnosed using conventional mycobacterial culture followed by phenotypic drug susceptibility testing (pDST), such as the agar proportion method, fluorescence-based liquid medium system, or the broth microdilution (BMD) method (6,7).However, these tests are time-consuming, which can delay the initiation of the appropriate treatment.Therefore, rapid molecular tests are important for the diagnosis of DR-TB.Furthermore, the resistance of MTB to INH can be classified as low-or highlevel resistance based on minimal inhibitory concentration (MIC) results when tested on liquid medium: low-level resistance as an MIC of INH greater than 0.1 mg/L to 0.4 mg/L and high-level resistance as an MIC greater than 0.4 mg/L (8).This information plays a critical role in the selection of the appropriate treatment regimen, such as the inclusion of high-dose INH or the exclusion of INH.The current treatment regimen for Hr-TB recommended by the WHO includes RIF, ethambutol (EMB), pyrazinamide (PZA), and levofloxacin (LFX) for a duration of 6 months (3).Therefore, accurate pDST or genotypic drug susceptibility testing (gDST) for INH and other drugs is a key factor in carefully selecting patients who can benefit from the Hr-TB regimen at the start of TB treatment.For gDST, several WHO-recommended molecular tests, namely, Xpert MTB/RIF, Xpert MTB/RIF Ultra, and Truenat MTB-RIF Dx (9), cannot simultaneously detect resistance to INH, RIF, and other anti-TB drugs in the same reaction.Whole-genome sequencing (WGS) is a reliable tool for predicting resistance to first-and second-line drugs of the M. tuberculosis complex (MTBC) (5,10).
Currently, the susceptibility profiles of Hr-TB clinical isolates circulating in Thailand are still limited, especially for drugs in the WHO-recommended treatment regimen.Therefore, this study aimed to address this research gap and evaluate the concordance of susceptibility results from the Mycobacterium Growth Indicator Tube (MGIT) and BMD methods and the WGS approach for the diagnosis of Hr-TB and resistance to the treatment regimen.

MTBC clinical isolates and patient populations
A retrospective study investigated Hr-TB clinical isolates recovered from clinical specimens at King Chulalongkorn Memorial Hospital (KCMH) from January 2017 to December 2021.A total of 183 Hr-TB clinical isolates were recovered from 144 patients with TB based on the pDST results for streptomycin (STM), INH, RIF, and EMB (SIRE) tested by the BD BACTEC Mycobacterium Growth Indicator Tube 960 system (MGIT; BD Biosciences, Franklin Lakes, NJ, USA) in a routine laboratory service without additional clinical specimens requested for this study.The MGIT results of 161 nonduplicate Hr-TB clinical isolates from 144 patients and characteristics of the patients with Hr-TB (age at the time of the first diagnosis of Hr-TB, sex, and treatment history) were recovered from medical records.This study was approved by the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand (COA.1297/2019).until use.The isolates were previously identified using line probe assays (LPAs) such as GenoType Mycobacterium CM VER 2.0 (MTBC level) and GenoType MTBC VER 1. X (MTB species level) (Hain Lifescience, Nehren, Germany) following the manufacturer's instructions.The frozen stock of the mycobacteria was subcultured in Lowenstein-Jen sen medium at 37°C for 3 to 4 weeks or until a visible colony was observed and was sufficient for prospective pDST and genomic DNA extraction for WGS analysis.

Phenotypic drug susceptibility tests
The MGIT 960 SIRE kit was previously tested with the following final drug concentra tions (mg/L): STM (1.0), INH (0.1), RIF (1.0), and EMB (5.0), following the manufacturer's instructions.A prospective pDST study was conducted for nonduplicate Hr-TB isolated from an individual patient based on distinguishable SIRE MGIT results or from different types of specimens in each disease episode.Some clinical isolates were excluded from prospective pDSTs due to bacterial contamination and no growth after subculture.Finally, for the MGIT system, the Hr-TB clinical isolates were prospectively tested with high-dose INH (0.4 mg/L) (n = 108), PZA (100.0 mg/L) (n = 118), and LFX (1.0 mg/L) (n = 96).Drug susceptibility testing (DST) for high-dose INH and PZA was performed using the MGIT 960 INH 0.4 and PZA kits (BD BACTEC), respectively, following the manufactur er's instructions, and the susceptibility test for LFX was performed as recommended by the WHO (6).

WGS and data analysis
Genomic DNA extraction from culture materials was performed using the cetyltrimethy lammonium bromide-sodium chloride method (13).WGS was performed using the NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA) and a NovaSeq sequencer (Illumina Inc., San Diego, CA, USA) using a 2 × 150 paired-end configuration.NovaSeq Control Software, OLB, and GAPipeline-1.6(Illumina) were used for image analysis and base calling.
Trimmomatic V0.32 (14) and FastQC version 0.11.7 (15) were used to trim and check the quality of the FASTQ files (> Phred score 30).De novo assembly was performed with SPAdes ( 16) and quality-checked with CheckM (17) to validate the completeness (100%) and contamination (0%).Raw FASTQ sequencing data were analyzed using an online tool, TB-Profiler Version 4.4.0, to identify species and strain lineage and for drug susceptibility prediction (98% to 100% frequency of the variant) (18).Snippy software version 4.6.0(19) was used to identify single-nucleotide polymorphisms (SNPs) between the M. tuberculosis genome (GenBank accession number: NC_000962.3)and the generated core SNP alignment.Phylogenetic analysis of the 56 MTB isolates was performed using MEGA version 11 and a general time-reversible and gamma distribution model (chosen model based on data).The phylogenetic tree was built using 1,000 bootstrap repetitions.The phylogenetic tree was visualized with iTOL.

Statistical analysis
Data were analyzed using R version 4.2.1.The median and interquartile range (IQR) for age and categorical variables as percentages (%) were calculated using the rstatix package.Positive predictive value (PPV), negative predictive value, and 95% confidence interval (CI) were analyzed using the Yardstick version 1.1.0package.Graphs were created using the ggplot2 program, version 3.2.1 (20).

MTBC clinical isolates and patient populations
From a retrospective study, a total of 144 patients with TB were diagnosed with Hr-TB based on MGIT results, which showed the results of INH resistance and RIF susceptibility.Among the 144 patients with Hr-TB, 63 (43.8%) patients were women and 81 (56.3%) were men, with an age range of 2-88 years (median = 49, IQR = 32.5).Among these, a total of 143 Hr-TB cases with available medical records for review were classified as pulmonary TB (n = 97, 67.8%) and extrapulmonary TB (n = 46, 32.2%) based on history, clinical presentations, and imaging.From the medical record review, the prevalence of Hr-TB in our hospital setting was 11.2% during 2017-2021 (144 Hr-TB patients/1,282 total TB patients with MGIT results).Based on the treatment history, 89.5% (128/143) and 10.5% (15/143) were new and previously treated patients with TB, respectively.The treatment information of one patient was not available.A total of 161 nonduplicate Hr-TB isolates [MTB (151/161, 93.8%); MTBC (10/161, 6.2%)] from 144 Hr-TB patients were recovered from 128 (79.5%) pulmonary specimens (bronchoalveolar lavage fluid, endotracheal aspirate, and sputum) and 33 (20.5%) extrapulmonary specimens (lymph node, tissue biopsy, cerebrospinal fluid, urine, body fluid, gastric content, and pus).
The prediction of genotypic drug resistance using the TB-Profiler (98% to 100% frequency of the variant) revealed that the 56 MTB clinical isolates exhibited resistance    S2.

Fluoroquinolone
FLQ resistance was detected in one MTB isolate with a gyrA D94G mutation with an MIC value of 0.25 (S) mg/L for LFX and OFX (Fig. 1; Table S2).

DISCUSSION
This study investigated the phenotypic and genotypic susceptibility profiles of Hr-TB isolates at KCMH, Bangkok, Thailand, from 2017 to 2021 and compared the results of Hr-TB diagnostic methods.Hr-TB is the most prevalent DR-TB, with an estimated national prevalence of 9.7% and 21.3% among new and previously treated patients with TB, respectively (21).The current Hr-TB treatment regimen includes RIF, EMB, PZA, and LFX for 6 months (3).Susceptibility testing for INH, RIF, and other drugs is necessary to design an individual treatment regimen with other second-line TB drugs.In the present study, the prevalence of Hr-TB cases was 11.2% from 2017 to 2021 at KCMH, which is a tertiary care hospital; however, most of the cases (89.5%) were newly diagnosed patients with TB.Therefore, an investigation of the transmission of Hr-TB among the study population will be performed in subsequent studies.Furthermore, the low prevalence of previously treated Hr-TB cases in our study could be due to the underreported history of TB treatment in some patients.
SIRE MIGT was the initial pDST performed for MTBC isolates from patients suspec ted of TB in our hospital setting and categorically classified the MTBC as resistant or susceptible isolates.The BMD method can test up to 12 anti-TB agents simultane ously, and MTB resistance can be quantitatively evaluated as MIC values.However, the CLSI-established breakpoints are limited to RIF, INH, and EMB but not to other drugs due to their limited clinical relevance (7).Recently, the CRyPTIC endorsed ECOFFs/ECVs for various anti-TB drugs (12), which were used to interpret the BMD results in the present study.Our findings demonstrated a strong concordance between the results of the MGIT system and the BMD method in evaluating susceptibility to INH, RIF, EMB, and LFX.In addition, EMB and LFX still have active in vitro activity against MTB.
Currently, molecular assays play an important role in the diagnosis of DR-TB.Gene sequencing, specifically WGS, has been implemented for TB surveillance and predic tion of drug susceptibility in certain countries (22,23).WGS results are based on SNP-associated resistance identified using high-/good-performance databases such as the TB-Profiler (18).Therefore, WGS can provide information on mutations that current commercial gDST assays cannot detect.However, for second-line drugs, an additional pDST is necessary to detect resistance that may be missed by the WGS-based gDST due to the lack of sufficient mutation data to support the findings or standardize the bioinformatics pipeline (5,24).
The phylogenetic analysis of the Hr-TB isolates showed that the most common lineage was lineage 2 (East-Asian lineage), followed by lineage 1 (Indo-Oceanic lineage), and then lineage 4 (Euro-American lineage).Consistent with our results, a previous study has also shown that lineage 2 was the main lineage among DR-TB (MDR, pre-XDR, and XDR-TB) in Thailand (25).For high-level INH resistance, the MGIT results revealed that approximately half of the MTBC isolates (55.6%) were resistant to 0.4 INH.We further analyzed WGS-based gDST and pDST data and found that high-and low-level INH resistance was correlated with the katG S315T mutation and mutations in the promoter region of the inhA-fabG1 operon, respectively, which was consistent with the previous studies (5,8).Therefore, either pDST or gDST should be performed to avoid the use of INH in isolates with high-level INH resistance.
Furthermore, the categorical agreement between WGS-based gDST and pDST for detection of INH resistance was lower than that for other drugs because WGS-based gDST did not detect mutations associated with INH resistance in 11 Hr-TB isolates, suggesting that there may be new mutations that were not confirmed at the time as being associated with INH resistance due to insufficient evidence (5).This result may also be attributed to repeated subcultures of MTB in fresh medium, which might reduce genetic diversity within the MTB sample due to clone selection (26).Therefore, the discordance of the pDST/gDST results should be further analyzed in future studies.
Furthermore, WGS did not detect mutations associated with PZA resistance (5) in MGIT PZA-R MTB isolates.WGS analysis showed that missense mutations in clpC1, rv1258c, PPE35, and rv3236c are not associated with PZA resistance, and some mutations are not listed by the WHO (5).At present, MGIT is the only WHO-recommended PZA-pDST; however, this method is associated with a high rate of false-positive resistance due to improper preparation of the inoculum and pH of the culture medium (6,27,28).Currently, molecular PZA-DST is the most reliable method recommended by the WHO (6).Therefore, the resistance to PZA revealed using MGIT in our study may be a false-positive result.To confirm our findings, the PZA pDST was retested using the MGIT method, and all 15 PZA-resistant isolates revealed susceptible results.As a result, the initial PZA resistance was likely a false-positive result.However, if this resistance was a true positive, it could be conferred by other mutations (5) or other new mechanisms.In conclusion, clinicians should interpret PZA phenotypic susceptibility with caution, and molecular tests should be performed, in addition to the MGIT, to accurately detect resistance to PZA.
In addition, WGS-based gDST detected three RIF-R MTB isolates that were diagnosed as MDR-TB with rpoB L430P (n = 2) and rpoB L452P (n = 1) mutations, which have been previously demonstrated as rpoB low-level mutations and could not be detected using the MGIT method (23), consistent with the findings of this study.We also tested a newly proposed RIF critical concentration by the WHO (29), 0.5 mg/L, using the MGIT system.However, no resistance to RIF for these three MTB isolates was detected (unpublished results).Furthermore, the ETO, which shares resistance-associated mutations to INH, fabG1 c-15t (5), had discordant results between the BMD and WGS-based gDST (Tables S2 and S4).This discrepancy can be caused by the lack of sufficient reproducibility of this drug.Therefore, the pDST is not recommended for ETO by the WHO (6).A discordant result was also observed for FLQ, in which the WGS-based method detected a gyrA D94G mutation correlated with high-level MFX resistance in one isolate but was not detected by either pDST.The MICs of FLQ can vary in isolates with the same mutation (30).
The discordant results between the MGIT and BMD methods and between the pDST and gDST were observed for anti-TB drugs.These findings have been well described and recognized in previous studies for several anti-TB drugs and for both the pDST and gDST (24,31,32).The discrepancy can directly impact the decision of treatment regimen selection since most laboratories usually have only one validated pDST or gDST method for use.Laboratory personnel and clinicians should be aware that the DST can be discordant with other methods and communicate a possible explanation.The confirmation of DST results by a reference laboratory may be necessary for certain TB cases with complex resistance.Finally, the determination of the risk of drug resistance in patients from a history of prior TB treatment and compliance with anti-TB drugs as well as clinical manifestations are crucial and would assist in treatment regimen selection in this situation or when confirmation cannot be performed.
This study had a few limitations.First, no INH-susceptible MTBC isolate was evaluated for gDST and pDST.In the present study, only 56 Hr-TB clinical isolates were analyzed for broth microdilution and WGS.Moreover, different methods were not performed to reevaluate the discordance of the pDST/gDST results for some drugs.Nevertheless, to the best of our knowledge, this study presented the first susceptibility profiles of the WHO-recommended treatment regimen for Hr-TB isolates from patients in Thailand.Furthermore, the performance of the current pDST used in routine laboratory practices was evaluated and compared to that of the WGS-based gDST.

Conclusion
We demonstrated that the first-line regimen for Hr-TB still has active activity against the organism in vitro using the MGIT and BMD methods.The Hr-TB isolates had a low rate of EMB and LFX resistance, while the high PZA resistance rate was likely a false-positive result.In addition, the co-resistance rate to EMB and LFX was low.Moreover, the Hr-TB isolates were susceptible to other second-line TB drugs, such as BDQ, LZD, and CFZ, which can be used as substitutes in Hr-TB patients who cannot tolerate the adverse effects of the standard regimen.The findings of this study can help clinicians carefully Payungporn, Conceptualization, Supervision, Writing -review and editing | Suwatchar eeporn Rotcheewaphan, Conceptualization, Data curation, Funding acquisition, Writingoriginal draft, Writing -review and editing

a
The vertical bold line divides susceptible and resistant isolates based on the CLSI guidelines and CRyPTIC ECOFF/ECVs.

TABLE 1
Drug susceptibility patterns and co-resistance (%) of Hr-TB isolates using the MGIT method c 0.1 INH MGIT is the INH critical concentration of 0.1 mg/L.b 0.4 INH MGIT is the INH critical concentration of 0.4 mg/L.
a c No., number; R, resistant; S, susceptible.TABLE 2 Comparison of the results of the broth microdilution method (THAMYCO) and MGIT b