Revealing the Interaction Mechanism between Mycobacterium tuberculosis GyrB and Novobiocin, SPR719 through Binding Thermodynamics and Dissociation Kinetics Analysis.

With the rapid emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb), various levels of resistance against existing anti-tuberculosis (TB) drugs have developed. Consequently, the identification of new anti-TB targets and drugs is critically urgent. DNA gyrase subunit B (GyrB) has been identified as a potential anti-TB target, with novobiocin and SPR719 proposed as inhibitors targeting GyrB. Therefore, elucidating the molecular interactions between GyrB and its inhibitors is crucial for the discovery and design of efficient GyrB inhibitors for combating multidrug-resistant TB. In this study, we revealed the detailed binding mechanisms and dissociation processes of the representative inhibitors, novobiocin and SPR719, with GyrB using classical molecular dynamics (MD) simulations, tau-random acceleration molecular dynamics (τ-RAMD) simulations, and steered molecular dynamics (SMD) simulations. Our simulation results demonstrate that both electrostatic and van der Waals interactions contribute favorably to the inhibitors' binding to GyrB, with Asn52, Asp79, Arg82, Lys108, Tyr114, and Arg141 being key residues for the inhibitors' attachment to GyrB. The τ-RAMD simulations indicate that the inhibitors primarily dissociate from the ATP channel. The SMD simulation results reveal that both inhibitors follow a similar dissociation mechanism, requiring the overcoming of hydrophobic interactions and hydrogen bonding interactions formed with the ATP active site. The binding and dissociation mechanisms of GyrB with inhibitors novobiocin and SPR719 obtained in our work will provide new insights for the development of promising GyrB inhibitors.

Tuberculosis treatment outcomes and associated factors among tuberculosis patients treated at healthcare facilities of Motta Town, Northwest Ethiopia: a five-year retrospective study.

Tuberculosis (TB) remains a significant public health concern, particularly in low-resource settings. The treatment outcome is a crucial indicator of the effectiveness of TB treatment programs. Assessing the current treatment outcome and its associated factors is essential for improving patient care and reducing the spread of TB. Therefore, this study aimed to assess TB treatment outcomes and their associated factors among TB patients who received treatment at public healthcare facilities in Motta Town, Northwest Ethiopia. A facility-based retrospective cross-sectional study design was employed in two TB treatment centers in Motta town from January 2017 to December 2021. The study participants were all patients diagnosed with TB who received treatment. A p-value of 0.05 with a 95% confidence interval (CI) was used to determine statistical significance. A total of 362 TB patients were included in the study. The overall treatment success rate was 88.4% (95% CI 85.1, 91.7). Male gender (AOR = 2.40, 95% CI 1.16, 4.98), normal nutritional status (AOR = 3.11, 95% CI 1.33, 7.25), HIV negative status (AOR = 3.35, 95% CI 1.31, 8.60), and non-presumptive drug resistance to TB (AOR = 3.72, 95% CI 1.74, 7.98) were significantly associated with successful TB treatment outcomes (p < 0.05). In the current study, nine out of ten study participants had successful TB treatment outcome rates. Male gender, normal nutritional status, non-presumed drug resistance to TB, and HIV-negative status were significantly associated with successful TB treatment outcomes. By taking risk factors associated with poor treatment outcomes like those found in this study into account, patient management and treatment can be optimized. Sufficient TB control measures for populations are imperative and could significantly reduce the nation's total TB burden.

Design, synthesis and characterization of ethyl 3-benzoyl-7-morpholinoindolizine-1-carboxylate as anti-tubercular agents: In silico screening for possible target identification.

A thorough search for the development of innovative drugs to treat tuberculosis, especially considering the urgent need to address developing drug resistance, we report here a synthetic series of ethyl 3-benzoyl-7-morpholinoindolizine-1-carboxylate analogues (5a-o) as potent anti-tubercular agents. These morpholino-indolizines were synthesized by reacting 4-morpholino pyridinium salts, with various electron-deficient acetylenes to afford the ethyl 3-benzoyl-7-morpholinoindolizine-1-carboxylate analogues (5a-o). All synthesized intermediate and final compounds are characterized by spectroscopic methods such as 1H NMR, 13C NMR and HRMS and further examined for their anti-tubercular activity against the M. tuberculosis H37Rv strain (ATCC 27294-American type cell culture). All the compounds screened for anti-tubercular activity in the range of 6.25-50 µM against the H37Rv strain of Mycobacterium tuberculosis. Compound 5g showed prominent activity with MIC99 2.55 µg/mL whereas compounds 5d and 5j showed activity with MIC99 18.91 µg/mL and 25.07 µg/mL, respectively. In silico analysis of these compounds revealed drug-likeness. Additionally, the molecular target identification for Malate synthase (PDB 5CBB) is attained by computational approach. The compound 5g with a MIC99 value of 2.55 µg/mL against M. tuberculosis H37Rv emerged as the most promising anti-TB drug and in silico investigations suggest Malate synthase (5CBB) might be the compound's possible target.

IL-10 and TGF-ß1 may weaken the efficacy of preoperative anti-tuberculosis therapy in older patients with spinal tuberculosis.

Spinal tuberculosis is a common extrapulmonary type that is often secondary to pulmonary or systemic infections. Mycobacterium tuberculosis infection often leads to the balance of immune control and bacterial persistence. In this study, 64 patients were enrolled and the clinicopathological and immunological characteristics of different age groups were analyzed. Anatomically, spinal tuberculosis in each group mostly occurred in the thoracic and lumbar vertebrae. Imaging before preoperative anti-tuberculosis therapy showed that the proportion of abscesses in the older group was significantly lower than that in the younger and middle-aged groups. However, pathological examination of surgical specimens showed that the proportion of abscesses in the older group was significantly higher than that in the other groups, and there was no difference in the granulomatous inflammation, caseous necrosis, inflammatory necrosis, acute inflammation, exudation, granulation tissue formation, and fibrous tissue hyperplasia. B cell number was significantly lower in the middle-aged and older groups compared to the younger group, while the number of T cells, CD4+ T cells, CD8+ T cells, macrophages, lymphocytes, plasma cells, and NK cells did not differ. Meaningfully, we found that the proportion of IL-10 high expression and TGF-ß1 positive in the older group was significantly higher than that in the younger group. TNF-α, CD66b, IFN-γ, and IL-6 expressions were not different among the three groups. In conclusion, there are some differences in imaging, pathological, and immune features of spinal tuberculosis in different age groups. The high expression of IL-10 and TGF-ß1 in older patients may weaken their anti-tuberculosis immunity and treatment effectiveness.

Patterns and profiles of drug resistance-conferring mutations in Mycobacterium tuberculosis genotypes isolated from tuberculosis-suspected attendees of spiritual holy water sites in Northwest Ethiopia.

Purpose: This study examined the patterns and frequency of genetic changes responsible for resistance to first-line (rifampicin and isoniazid), fluoroquinolones, and second-line injectable drugs in drug-resistant Mycobacterium tuberculosis (MTB) isolated from culture-positive pulmonary tuberculosis (PTB) symptomatic attendees of spiritual holy water sites (HWSs) in the Amhara region. Patients and methods: From June 2019 to March 2020, a cross-sectional study was carried out. A total of 122 culture-positive MTB isolates from PTB-suspected attendees of HWSs in the Amhara region were evaluated for their drug resistance profiles, and characterized gene mutations conferring resistance to rifampicin (RIF), isoniazid (INH), fluoroquinolones (FLQs), and second-line injectable drugs (SLIDs) using GenoType®MTBDRplus VER2.0 and GenoType®MTBDRsl VER2.0. Drug-resistant MTB isolates were Spoligotyped following the manufacturer's protocol. Results: Genetic changes (mutations) responsible for resistance to RIF, INH, and FLQs were identified in 15/122 (12.3%), 20/122 (16.4%), and 5/20 (25%) of MTB isolates, respectively. In RIF-resistant, rpoB/Ser531Lue (n = 12, 80%) was most frequent followed by His526Tyr (6.7%). Amongst INH-resistant isolates, katG/Ser315Thr1 (n = 19, 95%) was the most frequent. Of 15 MDR-TB, the majority (n = 12, 80%) isolates had mutations at both rpoB/Ser531Leu and katG/Ser315Thr1. All 20 INH and/or RIF-resistant isolates were tested with the MTBDRsl VER 2.0, yielding 5 FLQs-resistant isolates with gene mutations at rpoB/Ser531Lue, katG/Ser315Thr1, and gyrA/Asp94Ala genes. Of 20 Spoligotyped drug-resistant MTB isolates, the majority (n = 11, 55%) and 6 (30%) were SIT149/T3-ETH and SIT21/CAS1-Kili sublineages, respectively; and they were any INH-resistant (mono-hetero/multi-). Of 15 RIF-resistant (RR/MDR-TB) isolates, 7 were SIT149/T3-ETH, while 6 were SIT21/CAS1-Kili sublineages. FLQ resistance was detected in four SIT21/CAS1-Kili lineages. Conclusion: In the current study, the most common gene mutations responsible for resistance to INH, RIF, and FLQs were identified. SIT149/T3-ETH and SIT21/CAS1-Kili constitute the majority of drug-resistant TB (DR-TB) isolates. To further understand the complete spectrum of genetic changes/mutations and related genotypes, a sequencing technology is warranted.

The clinical profile and outcomes of drug resistant tuberculosis in Central Province of Zambia.

BACKGROUND: The emergence of Drug Resistant Tuberculosis (DR-TB) is one of the main public health and economic problems facing the world today. DR-TB affects mostly those in economically productive years and prevents them from being part of the workforce needed for economic growth. The aim of this study was to determine the Clinical Profile and Outcomes of DR-TB in Central Province of Zambia. METHODS: This was a retrospective cross sectional study that involved a review of records of patients with confirmed DR-TB who were managed at Kabwe Central Hospital's Multi-Drug Resistant TB (MDR-TB) Ward from the year 2017 to 2021. 183 patients were managed during this period and all were recruited in the study. Data was collected from DR-TB registers and patient files and then entered in SPSS version 22 where all statistical analyses were performed. RESULTS: The study revealed that the prevalence of DR-TB among registered TB patients in Central Province was 1.4%. Majority of those affected were adults between the ages of 26 and 45 years (63.9%). The study also found that more than half of the patients were from Kabwe District (60.7%). Other districts with significant number of cases included Kapiri Mposhi 19 (10.4%), Chibombo 12 (6.6%), Chisamba 10 (5.5%), Mumbwa 7 (3.8%) and Mkushi 7 (3.8%). Furthermore, the analysis established that most of the patients had RR-TB (89.6%). 9.3% had MDR-TB, 0.5% had IR-TB and 0.5% had XDR-TB. RR-TB was present in 93.8% of new cases and 88.9% of relapse cases. MDR-TB was present in 6.2% of new cases and 10% of relapse cases. With regard to outcomes of DR-TB, the investigation revealed that 16.9% of the patients had been declared cured, 45.9% had completed treatment, 6% were lost to follow up and 21.3% had died. Risk factors for mortality on multivariate analysis included age 36-45 years (adjusted odds ratio [aOR] 0.253, 95% CI [0.70-0.908] p = 0.035) and male gender (aOR 0.261, 95% CI [0.107-0.638] p = 0.003). CONCLUSION: The research has shown beyond doubt that the burden of DR-TB in Central Province is high. The study recommends putting measures in place that will help improve surveillance, early detection, early initiation of treatment and proper follow up of patients.

Correlation between serum isoniazid concentration and therapeutic response in patients of pulmonary tuberculosis in Central India: A prospective observational study.

BACKGROUND: Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis is one of the top ten causes of death worldwide. Isoniazid (INH) is an important component of anti-tuberculosis therapy (ATT). Low isoniazid levels can serve as a risk factor for the development of treatment failure, relapse of disease and acquired secondary resistance. Hence, serum level of isoniazid becomes a critical factor in determining the treatment outcome of patients on ATT. This study aimed to evaluate the correlation between serum isoniazid concentration and therapeutic response in patients of pulmonary tuberculosis in Central India. METHODS: This was a prospective single cohort observational study conducted at a tertiary care hospital. Therapeutic response in newly diagnosed patients of pulmonary TB was determined based the microbiological, clinical and radiological parameters. Serum INH levels were estimated based on a spectrophotometric method using nano-spectrophotometer. RESULTS: In this study, patients had a significant improvement in treatment outcome as evident by a significant decrease in the TB score I at end of IP (p = 0.001) and a significant decline in the Timika score at end of CP (p = 0.001). Although all patients converted to sputum negative at end of CP, 20% remained positive at end of IP. Lower INH levels were seen in 13.3% of the study population. Higher INH levels were observed in sputum converters, patients with low TB score I and low Timika score, although no statistically significant difference was noted (p > 0.05). CONCLUSION: In this study, we could not find any statistically significant association between serum INH levels and therapeutic outcome of the patients. Further studies on a larger population could provide better understanding about the prevalence of low serum isoniazid levels among the Indian population and establish its relationship with therapeutic outcome. Also, the usage of a comparatively less expensive spectrophotometric method of analysis makes this feasible in almost every district hospital without the need of high-performance liquid chromatography which is costlier and needs more expertise.

Evaluation of clinical profiles, imaging findings and antituberculosis treatment outcome in granulomatous mastitis: An Indian scenario.

BACKGROUND: The management of choice for granulomatous mastitis (GM) has yet to be determined but few studies have demonstrated that anti-tubercular treatment (ATT) could be an effective alternative therapeutic option. Hence, the objective of the current study is to determine the clinical feature, radiological imaging findings, and histopathological examination results exhibited by GM and tuberculosis (TB)-proven GM as well as to evaluate the ATT clinical outcome in GM patients. METHODS: The study was performed on 68 GM patients who were referred to the department of pulmonology by the breast clinic (from January 2018 to August 2021). Study populations were categorized into two groups GM and TB-proven GM patients and all were prescribed with standard ATT regimen and were continuously followed up. SPSS version 25 was employed for statistical assessment. RESULTS: Our study showed that 6 patients from GM and 4 patients from the TB-proven GM group got relapsed. For patients who displayed partial remission, ATT treatment was started after assessing the side effects potential. 14.6% (n = 6) and 7.4% (n = 2) patients who initially demonstrated partial remission were also completely cured. ATT treatment curable rate was determined to be 90% (n = 37) and 81.5% (n = 22) for GM and TB-proven GM patients correspondingly. Therefore, the current study demonstrated nil significant differences between groups. CONCLUSION: The current study warrants that ATT therapy could be an effective and better treatment of choice for GM patients irrespective of their clinical condition.

Cost-consequence analysis of ambulatory clinic- and home-based multidrug-resistant tuberculosis management models in Eswatini.

BACKGROUND: We compared the cost-consequence of a home-based multidrug-resistant tuberculosis (MDR-TB) model of care, based on task-shifting of directly observed therapy (DOT) and MDR-TB injection administration to lay health workers, to a routine clinic-based strategy within an established national TB programme in Eswatini. METHODS: Data on costs and effects of the two ambulatory models of MDR-TB care was collected using documentary data and interviews in the Lubombo and Shiselweni regions of Eswatini. Health system, patient and caregiver costs were assessed in 2014 in US$ using standard methods. Cost-consequence was calculated as the cost per patient successfully treated. RESULTS: In the clinic-based and home-based models of care, respectively, a total of 96 and 106 MDR-TB patients were enrolled in 2014, with treatment success rates of 67.8% and 82.1%. Health system costs per patient treated were slightly lower in the home-based strategy (US$19 598) compared to the clinic-based model (US$20 007). The largest costs in both models were for inpatient care, administration of DOT and injectable treatment, and drugs. Costs incurred by patients and caregivers were considerably higher in the clinic-based model of care due to the higher direct travel costs to the nearest clinic to receive DOT and injections daily. In total, MDR patients in the clinic-based strategy incurred average costs of US$670 compared to US$275 for MDR-TB patients in the home-based model. MDR-TB patients in the home-based programme, where DOT and injections was provided in their homes, only incurred out-of-pocket travel expenses for monthly outpatient treatment monitoring visits averaging US$100. The cost per successfully treated patient was US$31 106 and US$24 157 in the clinic-based and home-based models of care, respectively. The analysis showed that, in addition to the health benefits, direct and indirect costs for patients and their caregivers were lower in the home-based care model. CONCLUSION: The home-based strategy used less resources and generated substantial health and economic benefits, particularly for patients and their caregivers, and decision makers can consider this approach as an alternative to expand and optimise MDR-TB control in resource-limited settings. Further research to understand the appropriate mix of treatment support components that are most important for optimal clinical and public health outcomes in the ambulatory home-based model of MDR-TB care is necessary.

Application of the Hollow-Fiber Infection Model to Personalized Precision Dosing of Isoniazid in a Clinical Setting.

BACKGROUND: The hollow-fiber infection model (HFIM) is a valuable tool for evaluating pharmacokinetics/pharmacodynamics relationships and determining the optimal antibiotic dose in monotherapy or combination therapy, but the application for personalized precision medicine in tuberculosis treatment remains limited. This study aimed to evaluate the efficacy of adjusted antibiotic doses for a tuberculosis patient using HFIM. METHODS: Model-based Bayesian forecasting was utilized to assess the proposed reduction of the isoniazid dose from 300 mg daily to 150 mg daily in a patient with an ultra-slow-acetylation phenotype. The efficacy of the adjusted 150-mg dose was evaluated in a time-to-kill assay performed using the bacterial isolate Mycobacterium tuberculosis (Mtb) H37Ra in a HFIM that mimicked the individual pharmacokinetic profile of the patient. RESULTS: The isoniazid concentration observed in the HFIM adequately reflected the target drug exposures simulated by the model. After 7 days of repeated dose administration, isoniazid killed 4 log10 Mtb CFU/mL in the treatment arm, while the control arm without isoniazid increased 1.6 log10 CFU/mL. CONCLUSION: Our results provide an example of the utility of the HFIM for predicting the efficacy of specific recommended doses of anti-tuberculosis drugs in real clinical setting.

[Annual progress of immunotherapy for tuberculosis in 2023].

As a chronic infectious disease, tuberculosis (TB) is closely related to immune regulation and immune effect. Immunotherapy which can improve the curative effect of tuberculosis and control the spread of tuberculosis, is one of the important means for the comprehensive treatment of tuberculosis. From October 2022 to September 2023, research on the immunotherapy of tuberculosis at home and abroad continues to increase, providing new opportunities for the treatment of multidrug-resistant and extensively drug-resistant tuberculosis. Host-targeted therapy and therapeutic vaccines are new directions for research into TB adjuvant therapy.

[Application and optimization of CRISPRi to the biology of Mycobacterium tuberculosis].

Tuberculosis, caused by infection with Mycobacterium tuberculosis (MTB), remains a global public health challenge. Multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) strains make tuberculosis more difficult to control. New tools to study the biology of MTB can identify novel targets for drug discovery. Recently, the Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) combined with next-generation sequencing has provided many novel insights into the physiology and genetics of MTB. This review summarizes the application and optimization of CRISPRi in MTB biology.

GenoMycAnalyzer: a web-based tool for species and drug resistance prediction for Mycobacterium genomes.

BACKGROUND: Drug-resistant tuberculosis (TB) is a major threat to global public health. Whole-genome sequencing (WGS) is a useful tool for species identification and drug resistance prediction, and many clinical laboratories are transitioning to WGS as a routine diagnostic tool. However, user-friendly and high-confidence automated bioinformatics tools are needed to rapidly identify M. tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM), detect drug resistance, and further guide treatment options. RESULTS: We developed GenoMycAnalyzer, a web-based software that integrates functions for identifying MTBC and NTM species, lineage and spoligotype prediction, variant calling, annotation, drug-resistance determination, and data visualization. The accuracy of GenoMycAnalyzer for genotypic drug susceptibility testing (gDST) was evaluated using 5,473 MTBC isolates that underwent phenotypic DST (pDST). The GenoMycAnalyzer database was built to predict the gDST for 15 antituberculosis drugs using the World Health Organization mutational catalogue. Compared to pDST, the sensitivity of drug susceptibilities by the GenoMycAnalyzer for first-line drugs ranged from 95.9% for rifampicin (95% CI 94.8-96.7%) to 79.6% for pyrazinamide (95% CI 76.9-82.2%), whereas those for second-line drugs ranged from 98.2% for levofloxacin (95% CI 90.1-100.0%) to 74.9% for capreomycin (95% CI 69.3-80.0%). Notably, the integration of large deletions of the four resistance-conferring genes increased gDST sensitivity. The specificity of drug susceptibilities by the GenoMycAnalyzer ranged from 98.7% for amikacin (95% CI 97.8-99.3%) to 79.5% for ethionamide (95% CI 76.4-82.3%). The incorporated Kraken2 software identified 1,284 mycobacterial species with an accuracy of 98.8%. GenoMycAnalyzer also perfectly predicted lineages for 1,935 MTBC and spoligotypes for 54 MTBC. CONCLUSIONS: GenoMycAnalyzer offers both web-based and graphical user interfaces, which can help biologists with limited access to high-performance computing systems or limited bioinformatics skills. By streamlining the interpretation of WGS data, the GenoMycAnalyzer has the potential to significantly impact TB management and contribute to global efforts to combat this infectious disease. GenoMycAnalyzer is available at http://www.mycochase.org .

A robust computational quest: Discovering potential hits to improve the treatment of pyrazinamide-resistant Mycobacterium tuberculosis.

The rise of pyrazinamide (PZA)-resistant strains of Mycobacterium tuberculosis (MTB) poses a major challenge to conventional tuberculosis (TB) treatments. PZA, a cornerstone of TB therapy, must be activated by the mycobacterial enzyme pyrazinamidase (PZase) to convert its active form, pyrazinoic acid, which targets the ribosomal protein S1. Resistance, often associated with mutations in the RpsA protein, complicates treatment and highlights a critical gap in the understanding of structural dynamics and mechanisms of resistance, particularly in the context of the G97D mutation. This study utilizes a novel integration of computational techniques, including multiscale biomolecular and molecular dynamics simulations, physicochemical and medicinal chemistry predictions, quantum computations and virtual screening from the ZINC and Chembridge databases, to elucidate the resistance mechanism and identify lead compounds that have the potential to improve treatment outcomes for PZA-resistant MTB, namely ZINC15913786, ZINC20735155, Chem10269711, Chem10279789 and Chem10295790. These computational methods offer a cost-effective, rapid alternative to traditional drug trials by bypassing the need for organic subjects while providing highly accurate insight into the binding sites and efficacy of new drug candidates. The need for rapid and appropriate drug development emphasizes the need for robust computational analysis to justify further validation through in vitro and in vivo experiments.

Efficacy, safety, and pharmacokinetics of isoniazid affected by NAT2 polymorphisms in patients with tuberculosis: A systematic review.

N-acetyltransferase 2 (NAT2) genetic polymorphisms might alter isoniazid metabolism leading to toxicity. We reviewed the impact of NAT2 genotype status on the pharmacokinetics, efficacy, and safety of isoniazid, a treatment for tuberculosis (TB). A systematic search for research articles published in Scopus, PubMed, and Embase until August 31, 2023, was conducted without filters or limits on the following search terms and Boolean operators: "isoniazid" AND "NAT2." Studies were selected if NAT2 phenotypes with pharmacokinetics or efficacy or safety of isoniazid in patients with TB were reported. Patient characteristics, NAT2 status, isoniazid pharmacokinetic parameters, early treatment failure, and the prevalence of drug-induced liver injury were extracted. If the data were given as a median, these values were standardized to the mean. Forty-one pharmacokinetics and 53 safety studies were included, but only one efficacy study was identified. The average maximum concentrations of isoniazid were expressed as supratherapeutic concentrations in adults (7.16 ± 4.85 µg/mL) and children (6.43 ± 3.87 µg/mL) in slow acetylators. The mean prevalence of drug-induced liver injury was 36.23 ± 19.84 in slow acetylators, which was significantly different from the intermediate (19.49 ± 18.20) and rapid (20.47 ± 20.68) acetylators. Subgroup analysis by continent showed that the highest mean drug-induced liver injury prevalence was in Asian slow acetylators (42.83 ± 27.61). The incidence of early treatment failure was decreased by genotype-guided isoniazid dosing in one study. Traditional weight-based dosing of isoniazid in most children and adults yielded therapeutic isoniazid levels (except for slow acetylators). Drug-induced liver injury was more commonly observed in slow acetylators. Genotype-guided dosing may prevent early treatment failure.