Isoniazid resistance profile in rifampicin resistant Mycobacterium tuberculosis

Background: Muldrug-resistant tuberculosis (MDR-TB) is a global public health problem. Rifampicin (RIF) resistance has been used as a surrogate marker for MDR-TB but isoniazid (INH) resistance within RIF resistance cases is li�le known. This study aimed to determine the propor�on of INH resistance among RIF-resistant MTB. Methods: In this cross-seconal study, from March 2021 to February 2022, 53 RIF-resistant MTB isolates in sputum samples detected by Xpert-MTB RIF assay were enrolled. All samples were tested for muta�on in katG (codon 315) and inhA promoter (-5, -8, -15 and -16) genes to detect INH resistance by real-me PCR. Sta�s�cal analysis was done using IBM SPSS (version 26). Results: Out of 53 RIF-resistant samples, 15.1% were sensi�ve to INH, and the rest had concomitant resistance to INH. The propor�on of newly diagnosed and previously treated cases was nearly equal, and most of the


INTRODUCTION
Tuberculosis (TB) ranks as the 13th most significant contributor to global mortality and stands as the second most fatal infectious disease, surpassed only by COVID-19. 1 Bangladesh holds the seventh position on a global scale and is placed fourteenth among countries with a high burden of multidrug-resistant TB (MDR-TB). 2R-TB is defined as mycobacterium tuberculosis (MTB) resistant to at least isoniazid (INH) and rifampicin (RIF).It has emerged as a major pitfall of global TB control programs, requiring longer treatment, costly therapies, and higher treatment failure and mortality rates. 3During 2019, it was approximated that 3.3% of newly reported TB cases and 18% of cases with prior treatment history exhibited MDR-TB or resistance to rifampicin (RR-TB). 2 Prevalence of any resistance to INH, regardless of rifampicin resistance status, is 10.7% among new TB patients and 27.2% among previously treated TB patients. 4In Bangladesh, resistance to INH in previously treated cases is 49.9% and 10.8% in new patients. 5,6RIF and INH resistance often occur concurrently as in MDR-TB strains but such resistance arises independently from each other and can occur without resistance to the other as well. 3 the molecular assay, more than 95% of RIF resistance is associated with a mutation in 81 base pair rifampin resistance determining region (RRDR) of the bacterial RNA polymerase β subunit (rpoB) gene (codons 507-533), with the most frequent mutation in codon 531.
0 On the other hand, inhA mutation is associated with low-level resistance, and higher doses may overcome this condition and translate into efficacy. 11In 6.8% INH-resistant isolates, other less common may be responsible. 12In such situations, INH resistance may potentially be caused by the upregulation of INH inactivators or efflux pumps. 13fective management of MDR-TB begins with early diagnosis of the cases. 14DNA probes are used in molecular approaches such as GeneXpert MTB/RIF to identify mutations linked to RIF resistance in the rpoB gene. 15 4 Increasing rates of RIF monoresistance cases may be responsible for this change. 14Prevalence of RIF monoresistance was 0.2% and 0.4% in newly diagnosed and previously treated TB patients, respectively, in Bangladesh. 25In 2020, this prevalence increased to 0.3% and 0.7% accordingly. 5Similarly, the increasing rate of RIF monoresistance in India (22%), has also raised questions regarding the presence of INH coresistance in RIF-resistant isolates. 20So, Xpert MTB/ RIF assay, which is recommended as a first-line test for detection of MDR TB, considering RIF resistance as a 'surrogate marker', may not detect considerable number of INH susceptible, RIF resistant isolates and may need to be complemented by other DST methods. 14e proportion of previously treated and newly diagnosed tuberculosis patients had a small difference (52.8% versus 47.2%) in this study.However, the prevalence of RIF-resistant TB is higher among previously treated patients than newly diagnosed cases (18% compared to 3.8%) globally. 1Though newly diagnosed patients are also at risk of RIF-resistant TB  due to either spontaneous mutations or transmission of drug-resistant strains, different findings in this study may occur due to a smaller number of samples, which may not represent the population. 26 cases to be INH resistant. 29An underlying factor contributing to this observation might stem from the prolonged previous TB therapy in cases requiring retreatment, potentially elevating the susceptibility to drug resistance.The likelihood of having drug-resistant tuberculosis was found to be directly related to the total time (measured in months) of prior anti-tuberculosis treatment. 30 the present study, the predominant (53.3%) mutation responsible for INH-resistance was in katG (codon 315), followed by mutation in both katG and inhA gene (37.8%) and mutation in inhA gene alone (8.9%).

Out
Similar distributions were observed in laboratory-based surveillance in Pakistan, where any katG mutation was present in 76.1% and inhA mutation was present in 7.6% in RIF-resistant isolates, but the proportion of double mutation found was 3.1%. 31Globally, with a wide variation, katG mutations tend to be more frequent (42 -95% of isolates), while inhA mutations occur in 6-43% of isolates; around 10% of M. tuberculosis isolates have both mutations. 11Though the findings of this study are consistent with these ranges regarding the katG and inhA mutation, the proportion of isolates conferring double mutations was much higher.This phenomenon could arise due to the considerable variation in the prevalence of mutations in the katG and inhA genes across different geographical regions. 32Moreover, isoniazid-resistant M. tuberculosis isolates with katG gene (codon-315) mutation are found to be strongly associated with high-level drug resistance due to extensive loss of enzymatic activity. 33In these conditions, isoniazid will not be effective even when administered in higher dose, resulting in high-level INH resistance. 34

Conclusion
A considerable proportion of samples were susceptible to INH among RIF resistance isolates.Most of the isoniazid-resistant isolates were associated with mutation in katG followed by mutation in inhA.
KatG encodes catalase/ peroxidase enzymes, which causes activation of INH, that ultimately disrupts the mycolic acid biosynthesis by inhibiting inhA, which is the NADH-dependent enoyl-ACP reductase enzyme encoded by inhA gene. 8A mutation in the inhA gene leads to the overexpression of the target this tends to elevate the minimal inhibitory concentration (MIC) of INH. 9 KatG mutation, particularly at codon 315, results in high-level INH resistance, while some katG mutations that retain catalage-peroxidase activity may result in low-level INH resistance.
treatment history, previous treatment regimens, and treatment adherence) was meticulously recorded in a predefined data sheet.Patients who have never been treated for TB or have taken anti-TB drugs for less than one month were defined as new TB patients, whereas patients who had received treatment for more than one month in the past were included as previously treated patients.After collection, the samples were decontaminated using 2% NaOH-NALC solution and phosphate buffer saline following the Mycobacteriology laboratory manual, WHO, 2014.23DNA was extracted from the decontaminated sample using GenoType MTBDRplus 2.0 Genolyse kit following user's manual and stored at -20ºC.Real-time Quantitative PCR (qPCR) was carried out according to the manufacturer's recommendation in thus extracted DNAs, using a qPCR kit (TRUPCR Rif/INH MTB Drug Resistant Detection Kit, India) in Applied Biosystem 7300 Real-time PCR system.For each sample, a reaction mixture was made where 20µl of PCR master mix was added to 10µl of DNA sample.Then, the plate was sealed and loaded into the instrument.The plate was read at the end of the sixth thermal cycle, and fluorescence was detected.(Fluorescein amidites-FAM for katG and Victoria-VIC for inhA), Mutation caused the katG or inhA probe in the assay to drop out completely, resulting in no detectable cycle threshold due to no amplification.In such cases, where either one of the probes or both the probes that did not show amplification had point mutations in katG gene (codon 315) and/or inhA promoter gene (-5, -8, -15 and -16).Samples that had point mutation in katG gene (codon 315) and/or inhA promoter gene (-5, -8, -15 and -16) by real-time PCR were considered as INH resistant isolates.Statistical analysis Statistical analysis was done using Statistical Package for Social Sciences (SPSS) Version 26.Data were normally distributed.Categorical data were expressed in frequency and percentage.The statistical significance was assessed using chi-squared test.

FIGRE 2 (
FIGRE 2 (A) History of tuberculosis treatment among INH resistant and INH sensitive cases by real-time PCR (B) Mutation profile of INH resistant cases by real-time PCR.
WHO has recommended for short-course MDR-TB treatment regimen containing high-dose INH in MDR-TB patients since 2016, which is also included in the National Guideline and Operational Manual for Drug-resistant TB of Bangladesh in 2020. 6Results of this study suggest that patients should be evaluated for isoniazid sensitivity before starting standardized shortcourse treatment regimen for MDR-TB, containing high dose INH to avoid dose-related toxicity.One limitation of this study was that the sample size was relatively low.However, the strength of the study lies in the fact that, samples had been collected from the screening and referral centres for tuberculosis.Additionally, for INH resistance, the two most common responsible gene mutations (katG and inhA) were detected.Moreover, though several studies demonstrated RIF and INH-monoresistance status, no other recent study has revealed INH resistance in RIFresistant cases in our country.

TABLE 1 Distribution of RIF-resistant cases according to the treat- ment history RIF resistant cases Number of cases (%)
of 53 RIF-resistant cases, 15.1% of isolates were INH sensitive, and 84.9% had concomitant resistance to 6NH by Real-time PCR (FIGURE1).A total of 28 (52.8%)patients had a previous history of receiving treatment and 25 (47.2%)patients were newly diagnosed cases.Among previously treated cases, 24 (85.7%)patients received Category-I treatment and 4 (14.3%)cases were treated with an MDR-TB treatment regimen.Most previously treated cases (92.9%) received treatment regularly, while 7.1% did not (TABLE1).Among INH-resistant cases, 55.6% were previously treated, whereas 62.5% of INH-sensitive cases were newly diagnosed (FIGURE2).DISCUSSIONBangladesh is one of the countries with the highest global MDR-TB burden.6Detection of RIF resistance is Shareef N et al.Bangabandhu Sheikh Mujib Medical University Journal 2023; https://doi.org/10.3329/bsmmuj.v16i3.64496Isoniazid resistance in rifampicin resistant tuberculosis cases 162 considered a marker for diagnosis of MDR-TB, without directly testing for INH.Despite being a major arm of anti-TB chemotherapy, INH susceptibility remains unevaluated.14 This study focuses on INH resistance status in RIF-resistant cases.isolates is increasing.In South Africa, one of the highest drug-resistant TB burden countries, a retrospective data analysis revealed RIF-resistant INH-sensitive cases increased from 15.3% in 2011 to 21.4% in 2014.