CURRENT APPROACHES FOR CONTROL OF ISONIAZID-RESISTANT TUBERCULOSIS

Isoniazid (H; INH) is an important first-line drug for the treatment of active tuberculosis (TB) and latent TB infection because of its potent early bactericidal activity against Мycobacterium tuberculosis . Currently, TB resistant to INH, alone or in combination with other drugs, is the most common type of drug-resistant TB. Epidemiology of INH-resistant TB, the molecular mechanisms of drug resistance, current methods for diagnosis and therapeutic regimens of this TB form are presented. Studies in the


INTRODUCTION
Tuberculosis (TB), an ancient communicable disease caused by Mycobacterium tuberculosis (MTB), is one of the top 10 causes of death worldwide. Until the Coronavirus disease 2019 (COVID-19) pandemic, TB was the leading cause of death from a single infectious agent. Based on World Health Organization (WHO) data, in 2020, an estimated 9.9 million people fell ill with TB worldwide, equivalent to 127 cases per 100,000.. Among all incident cases of TB, 8% were people living with Human immunodeficiency virus (HIV). Globally in 2020, there were an estimated 1.3 million deaths among HIV-negative individuals and an additional 214,000 deaths among HIV-positive individuals (1). Drug-resistant TB (DR-TB) continues to be a public health threat. In MTB, drug resistance develops through spontaneous genetic mutations. The development of acquired drug resistance usually occurs when there is a large bacterial population, such as in pulmonary cavities (2) or when an inadequate drug combination or dosages of the first-line drugs (FLDs) has been prescribed (3,4). Rarely, malabsorption of anti-TB drugs may account for acquired resistance (5). WHO currently uses five categories to classify cases with DR-TB: -Multidrug-resistant TB (MDR-TB) -with resistance to both isoniazid (H; INH) and rifampicin (R; RIF)the two most effective FLDs; -Rifampicin-resistant TB (RR-TB); -Resistant to INH and sensitive to RIF TB (Hr-TB); -Pre-extensively drug-resistant TB (pre-XDR-TB) -MDR/RR-TB plus resistance to any fluoroquinolone (FQ) -a class of second-line anti-TB drugs (SLDs); -Extensively drug-resistant TB (XDR-TB) -MDR/ RR-TB plus resistance to any FQ, plus to at least one of the drugs bedaquiline (Bdq) and linezolid (Lzd) (1).  (11), i.e. the prevalence of INH-resistant TB in the country seems to be higher than MDR-TB rates.

MOLECULAR-GENETIC MECHANISMS OF DEVELOPMENT OF ISONIAZID-RESISTANT TUBERCULOSIS
MTB has the ability for spontaneous, slow but steady mutations, leading to the development of drug resistance. This natural phenomenon is genetically determined and varies for the different drugs. Drug intake increases the likelihood for its appearance. Isoniazid exerts its effects only in metabolically active mycobacterial cells. The probability for development of spontaneous resistance to INH is 1 in every 10 6 cell divisions (12). Drug resistance is the result of the selection of mycobacteria with mutations among the bacterial population due to destruction of the susceptible bacteria by anti-TB drugs. The problem is exacerbated if the patient is treated with monotherapy or with combination of FLDs in suboptimal concentrationsmost bacteria die, but those with mutations survive, multiply and dominate the bacterial population (13). Table 1 presents the main MTB genes, affected by specific molecular mechanisms (mutations) leading to development of resistance to the anti-TB drugs (14).
Resistance to INH is usually due to a mutation in katG or inhA, and more seldom -in other genes, such as ahpC32 (15,16). In order to be effective against TB, INH must be activated by catalase-peroxidase, an enzyme regulated by katG gene,. Muta tions in katG, most commonly at Ser315Thr (Ser → Thr), result in insufficient activation of the drug and are associated with a high level of resistance -Minimum inhibitory concentration (MIC) > 1 μg/ml (17,18). The inhA gene encodes an enoyl ACP reductase involved in fatty acid synthesis in MTB. As these fatty acids are targeted by the active derivative of INH, mutations in inhA or its pro moter region block INH binding and result in low-level INH resistance (MIC <1 μg/ml). The most frequent mutation in the promoter region is at position 15C/T (Cys → Thr) (19,20). Isolates with an inhA mutation are also typically resistant to ethionamide (Eto) and prothionamide (Pto) -SLDs for treatment of DR-TB (21). According to most of the studies, risk factors for developing INH resistance include a history of TB, and origin from regions with a higher TB prevalence (Asia, Pacific Islans, etc.) (22).

Whole Genome Sequencing (WGS)
-where the entire genetic code of MTB strains isolated from patients is described and compared to a reference set of genomes. The WGS has been introduced routinely in England, Germany and many EU/ EEA countries to guide clinical decision-making, earlier detection of resistance, and support of outbreak and epidemiological investigation. However, WGS requires sophisticated laboratory and bioinformatics infrastructure, and currently requires MTB isolation and DNA extraction before sequencing. Therefore the routine applicability of the method beyond research especially in low resource settings is uncertain. Nevertheless, the advances in the sequencing field may rapidly turn WGS to a TB diagnostic tool (26). Between January 2017 and December 2019 Bulgaria participated in the pilot study of the European Center for Disease Prevention and Control (ECDC), evaluating the systematic use of a WGSbased approach for MTB surveillance involving all European Union/European Economic Area (EU/ EEA) countries and highlighting the challenges to be considered for the future development of a WGS-based surveillance system (27). Yordanova et al. investigated MTB isolates from 36 TB cases from all over Bulgaria in a retrospective study for the period 2015-2016. All the cases were confirmed by BACTEC MGIT 960 with monoresistance to INH and additionally tested with GenoType MTBDRplus in the National Reference Laboratory of Tuberculosis (NRL TB) at the NCIPD. The authors found that only 25% of the tested MTB isolates with phenotypic INH mono-resistance had the S315T1 mutation in katG; all isolates were with MIC over 0.4 μg/ml. Resistance type C15T in the promoter region of inhA was detected in 22.22% of cases and only 1 of them showed MIC below 0.4 μg/ml. No mutations were detected in nearly half of the cases (n=19, 52.78%) and most of these isolates were with lower MIC values (n=12). The authors supposed that the resistance among the cases without mutations in katG or inhA can be explained with mutations in many other loci or genes (furA-katG, fabG1-inhA, ahpCoxyR intergenic region, efpA, fadE24, iniA, iniB, iniC, kasA, nat, ndh), which cannot be found using only GenoType MTBDRplus (28). At the end of 2021, WGS of all isolated DR-TB strains in Bulgaria started in the NRL TB at the NCIPD, which will expand our knowledge about the mutations of MTB in the country, including those corresponding with resistance to INH.

TREATMENT OF ISONIAZID-RESISTANT TUBERCULOSIS
The management of INH-resistant TB is important because the last systematic meta-analyses have shown that resistance to INH reduces the probability of treatment success and increases the risk of acquiring resistance to other impor tant FLDs such as RIF, thereby increasing the risk of MDR-TB. Moreover, INH-resistant TB generally requires longer treatment than drug-susceptible TB, increasing the burden of the disease (29,30). The recommended regimens for INH-resistant TB differ among countries and have differed over time. Table 2 summarizes the previous and current international guidelines for the treatment of INHresistant TB (22). There is a standard code for writing out anti-TB regimens. Each drug has an abbreviation (shown in the table). A DR-TB regimen consists of two phases, separated by a slash: the first is intensive phase and the second is prolonged phase. The number shown before each phase stands for the minimal required phase duration in months . The number in subscript (e.g., 3) after a letter is the number of drug doses per week. If there is no number in subscript, treatment is daily. Alternative drug(s) appears as a letter(s) in parentheses. The drugs in the higher groups are written first followed by the others in descending order of potency. Based on the most recent meta-analyses on the management of INH-resistant TB, in 2019 the WHO published the last key recommendations for Hr-TB treatmentwith RIF-EMB-PZA-Lfx for 6 months and no addition of SM or other injectable agents to the drug regimen. The WHO guidance emphasizes the importance of excluding resistance to RIF before starting the regimen for INH-resistant TB because of the risk for development of MDR-TB during the treatment course. The guidelines are based pri marily on individual patient data or observational studies con ducted in various settings. They indicate that addition of an FQ to RIF-EMB-PZA regimens compared to ≥6 months of RIF-EMB-PZA is leading to higher treatment success rate (aOR, 2.8; 95% CI, 1.1-7.3). The ad dition of an FQ to a 6-month RIF-EMB-PZA regimen tended to reduce the number of deaths (aOR, 0.4; 95% CI, 0.2-1.1) and the acquisition of RIF resistance (aOR, 0.10; 95% CI, 0.01-1.2). The main recommendations are to include Lfx rather than Mfx as a first choice because Lfx has a better safety profile than other FQs, and fewer drug interactions than Mfx; the plasma peak concentration of Lfx is not affected by the addi tion of RIF. Additionally, there are no contraindications for the use of Lfx with antiretroviral agents for the treatment of patients co-infected with HIV (31,32).

CONCLUSION
INH-resistant TB poses a significant challenge before public health systems. Many patients with Hr-TB would be missed by current diagnostic algorithms driven by RIF testing, thus receiving incomplete drug regimen. The development of new rapid molecular technologies is needed in order to ensure access to appropriate treatment and care. The WHO recommendations for Hr-TB treatment are based on observational studies, but not on randomized controlled trials, and are thus conditional and based on low certainty in the estimates of the effect. There fore, further work is needed to optimize the treatment and control of INH-resistant TB.