Permeability of TB drugs through the mycolic acid monolayer: a tale of two force fields.

Tuberculosis (TB) treatment becomes challenging due to the unique cell wall structure of Mycobacterium tuberculosis (M. tb). Among various components of the M.tb cell wall, mycolic acid (MA) is of particular interest because it is speculated to exhibit extremely low permeability for most of the drug molecules, thus helping M.tb to survive against medical treatment. However, no quantitative assessment of the thermodynamic barrier encountered by various well-known TB drugs in the mycolic acid monolayer has been performed so far using computational tools. On this premise, our present work aims to probe the permeability of some first and second line TB drugs, namely ethambutol, ethionamide, and isoniazid, through the modelled mycolic acid monolayer, using molecular dynamics (MD) simulation with two sets of force field (FF) parameters, namely GROMOS 54A7-ATB (GROMOS) and CHARMM36 (CHARMM) FFs. Our findings indicate that both FFs provide consistent results in terms of the mode of drug-monolayer interactions but significantly differ in the drug permeability through the monolayer. The mycolic acid monolayer generally exhibited a higher free energy barrier of crossing with CHARMM FF, while with GROMOS FF, better stability of drug molecules on the monolayer surface was observed, which can be attributed to the greater electrostatic potential at the monolayer-water interface, found for the later. Although both the FF parameters predicted the highest resistance against ethambutol (permeability values of 8.40 × 10-34 cm s-1 and 9.61 × 10-31 cm s-1 for the CHARMM FF and the GROMOS FF, respectively), results obtained using GROMOS were found to be consistent with the water solubility of drugs, suggesting it to be a slightly better FF for modelling drug-mycolic acid interactions. Therefore, this study enhances our understanding of TB drug permeability and highlights the potential of the GROMOS FF in simulating drug-mycolic acid interactions.

Natural deep eutectic solvent for the simultaneous derivatization and microextraction of isoniazid from human plasma.

BACKGROUND: Personalized medicine is a rapidly revolving field that offers new opportunities for tailoring disease treatment to individual patients. The main idea behind this approach is to carefully select safe and effective medications and treatment plant based on each patient's unique pharmacokinetic profile. Isoniazid is a first-line anti-tuberculosis drug that has interindividual variability in its metabolic processing, leading to significant differences in plasma concentrations among patients receiving equivalent doses. This variability necessitates the creation of individualized treatment regimens as part of personalized medicine to achieve more effective therapy. RESULTS: In this work, a deep eutectic solvent-based liquid-liquid microextraction approach for the separation and determination of isoniazid in human plasma by high-performance liquid chromatography with UV-Vis detection was developed for the first time. A new natural deep eutectic solvent based on thymol as a hydrogen bond donor and 4-methoxybenzaldehyde as a hydrogen bond acceptor was proposed as the extraction solvent. The developed microextraction procedure assumed two simultaneous processes during the mixing of the sample and extraction solvent: the derivatization of the polar analyte in the presence of 4-methoxybenzaldehyde (component of the natural deep eutectic solvent) with the formation of a hydrophobic Schiff base (1); mass transfer of the Schiff base from the sample phase to the extraction solvent phase (2). Under optimal conditions, the limits of detection and quantification were 20 and 60 µg L-1, respectively. The RSD value was <10 %, the extraction recovery was 95 %. SIGNIFICANCE: In this work, the possibility of isoniazid derivatization in the natural deep eutectic solvent phase with the formation of the Schiff base was presented for the first time. The approach provided the separation and preconcentration of polar isoniazid without the use of expensive derivatization agents and solid-phase extraction cartridges. The formation of the Schiff base was confirmed by mass spectrometry.

Factors associated with uptake of isoniazid preventive therapy among children living with HIV in Mwanza region, Tanzania: a cross-sectional study.

BACKGROUND: Tuberculosis (TB) is a leading cause of death among children living with HIV (CLHIV). Isoniazid preventive therapy (IPT) reduces the incidence of TB by 70% and mortality by 50% among CLHIV. However, in most developing countries including Tanzania, the uptake of IPT is suboptimal, below the 90% WHO-global uptake target. We assessed the factors associated with IPT uptake among CLHIV in Mwanza region, Tanzania. METHODS: This was a multicenter facility-based cross-sectional study among CLHIV aged 1 to 10 years in seven districts of Mwanza region, Tanzania from 1st November 2021 to 20th January 2022. Data were collected using a structured interview-administered questionnaire including information on children and caregivers' demographics, caregivers' health related information and children's clinical information. Our outcome variable was uptake of IPT, defined as initiation on IPT either during the time of the study or within past three years before this study We conducted modified Poisson regression to assess the association between IPT uptake and selected exposures in Stata version 15.0. RESULTS: A total of 415 CLHIV were enrolled, the median age of the children was 7 years (Interquartile range: 5-8). The uptake of IPT was 91% (n = 377). The majority of children's caregivers were HIV positive (86%, n = 387) and were aware about IPT (63.6%, n = 264). Factors associated with IPT uptake included; having an employed caregiver [Adjusted Prevalence Ratio (aPR): 1.06 95% Confidence Interval (CI): 1.00-1.13] and attending the ART clinic every month [aPR: 1.00; 95% CI: 0.87-1.00] . CONCLUSIONS: The uptake of IPT uptake among CLHIV in Mwanza, Tanzania exceeds the global WHO-target of ≥ 90%. Monthly ART clinic visits could be essential in promoting IPT uptake among CLHIV.

Factors associated with incomplete tuberculosis preventive treatment: a retrospective analysis of six-years programmatic data in Cambodia.

Tuberculosis (TB) preventive treatment (TPT) effectively prevents the progression from TB infection to TB disease. This study explores factors associated with TPT non-completion in Cambodia using 6-years programmatic data (2018-2023) retrieved from the TB Management Information System (TB-MIS). Out of 14,262 individuals with latent TB infection (LTBI) initiated with TPT, 299 (2.1%) did not complete the treatment. Individuals aged between 15-24 and 25-34 years old were more likely to not complete the treatment compared to those aged < 5 years old, with aOR = 1.7, p = 0.034 and aOR = 2.1, p = 0.003, respectively. Individuals initiated with 3-month daily Rifampicin and Isoniazid (3RH) or with 6-month daily Isoniazid (6H) were more likely to not complete the treatment compared to those initiated with 3-month weekly Isoniazid and Rifapentine (3HP), with aOR = 2.6, p < 0.001 and aOR = 7, p < 0.001, respectively. Those who began TPT at referral hospitals were nearly twice as likely to not complete the treatment compared to those who started the treatment at health centers (aOR = 1.95, p = 0.003). To improve TPT completion, strengthen the treatment follow-up among those aged between 15 and 34 years old and initiated TPT at referral hospitals should be prioritized. The national TB program should consider 3HP the first choice of treatment.

A public health development intervention between acceptability and feasibility: descriptive-interpretive discussion of an example.

Introduction: the National Tuberculosis Control Program (NTP) in Cameroon participated between 2016 and 2018 in a multi-country operational study of the Union against Tuberculosis and Lung Disease (The UNION) aiming at demonstrating the efficiency and feasibility of systematic tuberculosis preventive treatment (TPT) with 3 months of an isoniazid/rifampicin (3RH) combination in under-five child contacts of bacilliferous TB patients. Cameroon was one of the participating countries of the study. Despite the promising results communicated following this study, the coverage of TPT with 3RH in Cameroon remains low. We explored the intervention under aspects of acceptability and perceived feasibility. Methods: key participants and stakeholders in this descriptive interpretative study in Cameroon were interviewed in five focus groups or individually (31 individuals). The Focus Group Discussion (FGD) and interview transcripts were analysed for different components of acceptability using a theoretical framework and the results discussed confronting them with the main objective of the study, i.e. demonstrating feasibility. Results: the children's parents expressed overall positive feelings about and acceptance of the intervention, emphasizing the unexpected empathy shown by the health staff. The involved field staff, too, showed unreserved acceptance. On the other hand, managers at the intermediate and central levels showed scepticism as to the process of initiation of the study as well as to its feasibility in the given context, neglecting aspects of resources necessary for a scaling-up and of prioritisation. Conclusion: the adoption of a public health strategy, also internationally recognized as an effective and efficient intervention, requires more than the demonstration of its acceptability or feasibility during the term of a showcase project introduced by an external development partner. Adoption is conditioned by adoption and circumspect planning involving at each stage the stakeholders on all levels of the program.

Diagnostic accuracy of the Xpert® MTB/XDR assay for detection of Isoniazid and second-line antituberculosis drugs resistance at central TB reference laboratory in Tanzania.

INTRODUCTION: Early diagnosis of tuberculosis (TB) and universal access to drug-susceptibility testing (DST) are critical elements of the WHO End TB Strategy. Current rapid tests (e.g., Xpert® MTB/RIF and Ultra-assays) can detect rifampicin resistance-conferring mutations, but cannot detect resistance to Isoniazid and second-line anti-TB agents. Although Line Probe Assay is capable of detecting resistance to second-line anti-TB agents, it requires sophisticated laboratory infrastructure and advanced skills which are often not readily available in settings replete with TB. A rapid test capable of detecting Isoniazid and second-line anti-TB drug resistance is highly needed. METHODS: We conducted a diagnostic accuracy study to evaluate a new automated Xpert MTB/XDR 10-colour assay for rapid detection of Isoniazid and second-line drugs, including ethionamide, fluoroquinolones, and injectable drugs (Amikacin, Kanamycin, and Capreomycin). Positive Xpert MTB/RIF respiratory specimens were prospectively collected through routine diagnosis and surveillance of drug resistance at the Central TB Reference Laboratory in Tanzania. Specimens were tested by both Xpert XDR assay and LPA against culture-based phenotypic DST as the reference standard. FINDINGS: We analysed specimens from 151 TB patients with a mean age (SD) of 36.2 (12.7) years. The majority (n = 109, 72.2%) were males. The sensitivity for Xpert MTB/XDR was 93.5% (95% CI, 87.4-96.7); for Isoniazid, 96.6 (95% CI, 92.1-98.6); for Fluoroquinolone, 98.7% (95% Cl 94.8-99.7); for Amikacin, 96.6%; and (95% CI 92.1-98.6) for Ethionamide. Ethionamide had the lowest specificity of 50% and the highest was 100% for Fluoroquinolone. The diagnostic performance was generally comparable to that of LPA with slight variations between the two assays. The non-determinate rate (i.e., invalid M. tuberculosis complex detection) of Xpert MTB/XDR was 2·96%. CONCLUSION: The Xpert MTB/XDR demonstrated high sensitivity and specificity for detecting resistance to Isoniazid, Fluoroquinolones, and injectable agents. This assay can be used in clinical settings to facilitate rapid diagnosis of mono-isoniazid and extensively drug-resistant TB.

Synthesis and characterisation of antimicrobial metal-organic frameworks as multi-drug carriers.

Antibiotic resistance is a significant global concern, necessitating the development of either new antibiotics or advanced delivery methods. With this in mind, we report on the synthesis and characterisation of a new family of Metal-Organic Frameworks (MOFs), OnG6 MOFs, designed to act as multi-drug carriers for bacterial infection treatment. OnG6 is based on the pro-drug 4,4'-azodisalicylic acid (AZDH4), which in vivo produces two equivalents of para-aminosalicylic acid (ASA), a crucial drug for M. tuberculosis treatment. X-ray and computational studies revealed that OnG6 MOFs are mesoporous MOFs with etb topology and an [M2(AZD)] formula (M = Zn, OnG6-Zn; Mg, OnG6-Mg; Cu, OnG6-Cu; and Co, OnG6-Co), featuring 1-dimensional channel type pores of 25 Å diameter. OnG6 MOFs are the first reported MOFs bearing the ligand AZDH4, joining the family of mesoporous MOFs arranged in a honeycomb pattern. They absorb isoniazid (INH) and ciprofloxacin (CIPRO) with the former being a specific antibiotic for M. tuberculosis, and the latter being a broader-spectrum antibiotic. The stability of the MOFs and their capacity for antibiotic uptake depend on the nature of the metal ion, with OnG6-Mg demonstrating the highest drug absorption. The antimicrobial activity of these species was assessed against S. aureus and E. coli, revealing that the carriers containing CIPRO displayed optimal efficacy.

Efficacy and tolerability of a 4-month ofloxacin-containing regimen compared to a 6-month regimen in the treatment of patients with superficial lymph node tuberculosis: a randomized trial.

BACKGROUND: Tuberculosis (TB) lymphadenitis is the most common form of extra-pulmonary TB, and the treatment duration is six months. This non-inferiority based randomized clinical trial in South India evaluated the efficacy and safety of a four-month ofloxacin containing regimen in tuberculosis lymphadenitis (TBL) patients. METHODS: New, adult, HIV-negative, microbiologically and or histopathologically confirmed superficial lymph node TB patients were randomized to either four-month oflaxacin containing test regimen [ofloxacin (O), isoniazid (H), rifampicin (R), pyrazinamide (Z) -2RHZO daily/ 2RHO thrice-weekly] or a six-month thrice-weekly control regimen (2HRZ, ethambutol/4RH). The treatment was directly observed. Clinical progress was monitored monthly during and up to 12 months post-treatment, and thereafter every three months up to 24 months. The primary outcome was determined by response at the end of treatment and TB recurrence during the 24 months post-treatment. RESULTS: Of the 302 patients randomized, 298 (98.7%) were eligible for modified intention-to-treat (ITT) analysis and 294 (97%) for per-protocol (PP) analysis. The TB recurrence-free favourable response in the PP analysis was 94.0% (95% CI: 90.1-97.8) and 94.5% (95% CI: 90.8-98.2) in the test and control regimen respectively, while in the ITT analysis, it was 92.7% and 93.2%. The TB recurrence-free favourable response in the test regimen was non-inferior to the control regimen 0.5% (95% CI: -4.8-5.9) in the PP analysis based on the 6% non-inferiority margin. Treatment was modified for drug toxicity in two patients in the test regimen, while one patient had a paradoxical reaction. CONCLUSION: The 4-month ofloxacin containing regimen was found to be non-inferior and as safe as the 6-month thrice-weekly control regimen.

Drug resistance patterns and treatment outcomes in DR-TB patients at a tertiary care centre in Mumbai.

BACKGROUND: Drug-resistant tuberculosis (DR-TB) is a major health problem and threatens Tuberculosis (TB) control and outcomes globally. India holds one-fourth of global DR-TB burden.1 AIMS: 1- To study drug resistance patterns and outcomes in DR-TB patients under National Tuberculosis Elimination Programme (NTEP) at a tertiary care-centre. 2- To correlate outcome of DR-TB with drug resistance patterns. METHODS: It is a retrospective study of 302 Drug Resistant Tuberculosis patients from Jan 2020 to May 2022. Common mutations of drug resistance, pyrazinamide resistance in DR-TB patients, correlation of High dose Moxifloxacin sensitivity by Line Probe Assay (LPA) and drug sensitivity test (DST), outcome of DR-TB patients with drug resistance patterns and correlation of outcome of DR-TB patients with their initial body-weight were studied. RESULTS: Kat G was the most common mutation in Isoniazid (96%) resistance for MDR TB as well as Isoniazid Mono-resistance TB (p = 0.001). 91% cases with MDR-TB were resistant to pyrazinamide. 51.2% cases had low dose Fluroquinolone resistance. 18.8% cases had low and high dose Fluroquinolone resistance. 8.5% cases had resistance to injectables. 21.7% of cases who were resistant to High dose Moxifloxacin on second line LPA were found to be sensitive on DST. Outcomes were not dependent on the LPA resistance patterns. Body-weight greater than 45 Kg at the time of initiation of treatment was associated with better outcomes (p = 0.007). CONCLUSION: DR-TB patients are resistant to pyrazinamide in nearly all cases; hence pyrazinamide is not suitable for initial replacement sequence. Second line resistance doesn't impact outcome in DR-TB patients.

Isoniazid resistance in Rifampicin sensitive pulmonary tuberculosis in children and adolescents.

BACKGROUND: Isoniazid (INH) and Rifampicin (RIF) are two crucial drugs used in antitubercular therapy. INH is known for its potent bactericidal effects and has a relatively higher prevalence of resistance compared to RIF. However, RIF resistance has been the subject of more extensive research. On the other hand, Ethambutol (EMB) and Streptomycin (STR) resistance have not been thoroughly studied, particularly in the context of children and adolescents. To address this knowledge gap, a study was designed to investigate the resistance patterns of INH, EMB, and STR in RIF-sensitive pulmonary tuberculosis (PTB) cases among children and adolescents. METHODS: Seventy-five newly diagnosed RIF sensitive PTB cases up to 18 years of age were enrolled. Retreatment cases were excluded. Sputum/gastric aspirate sample of these patients were sent for culture in Mycobacterium Growth Indicator Tube (MGIT) followed by drug susceptibility testing and Line Probe Assay. RESULTS: INH, EMB and STR resistance among RIF sensitive PTB cases was found to be 5.7%, 0% and 0.7% respectively. RIF resistance detected by CBNAAT was found to be 8.4%. CONCLUSION: Detection of INH resistance is as important as detecting RIF resistance as prevalence of INH resistance in RIF sensitive PTB among children and adolescents up to 18 years is around 6%.

Study of adverse drug reactions during the treatment of drug resistant tuberculosis.

BACKGROUND: Pharmacovigilance entails monitoring of patients for timely detection of ADR and reporting them so that more information about drug safety can be obtained. This may help in the future for dose modification or alteration of regimen. In NTEP, ADSm (Active Drug Safety monitoring) is part of pharmacovigilance. In this study we shall be studying ADRs to Anti TB drugs in DRTB. METHODOLOGY: This study is observational, retrospective and record based, of patients admitted from 2021 to 2023 in the DOTS ward of Respiratory Medicine Department of a tertiary care hospital in Goa. Data such as age, sex, regimen, date of AKT initiation and adverse effects documented has been noted and compiled. RESULTS: ADRs have been tabulated in the form of tables. Statistical analysis is done to find out the commonest ADR, time when they are likely to occur, which age and gender are most likely affected and if there are any other associated risk factors for ADRs. CONCLUSION: This study will enable in future to better monitor patients with regard to particular adverse drug reaction, patient safety and if needed to alter the regimen as early as possible.

In-silico transcriptome analysis of antibiotic-treated Mycobacterium tuberculosis identifies novel antibiotic resistance factors.

The emergence of drug resistant Mycobacterium tuberculosis strains increases the burden on the treatment of tuberculosis. In this study, through in-silico transcriptome analysis of drug-treated M. tuberculosis samples, novel drug targets for the treatment of drug resistance in tuberculosis were identified. Gene expression datasets of tuberculosis patients samples treated with different antibiotics (Isoniazid, Rifampicin, Pyrazinamide, Bedaquiline and Linezolid) were considered in this study. DESeq2 was used to identify the differentially regulated genes. Novel genes which were up-regulated during antibiotic treatment were identified which could be antibiotic resistance factors. Further, to understand the resistance mechanism of the novel genes, we performed gene ontology and gene network analysis for the differentially up-regulated genes. Thus, the in-silico transcriptome analysis paves way for a deeper understanding of the antibiotic resistance in M. tuberculosis.

Mutational analysis in drug resistant Mycobacterium tuberculosis in Western Uttar Pradesh.

BACKGROUND: Multi-drug resistant tuberculosis (MDR-TB) results in treatment failure and poor clinical outcomes. This study was carried out with the aim to determine the pattern of drug resistance against Mycobacterium tuberculosis towards first line ATT (anti-tubercular treatment) in sputum smear-positive patients using Line Probe Assay (LPA). METHODS: A cross sectional prospective study was carried out in a tertiary care Hospital of Meerut. A total of 898 sputum samples (on spot and early morning) collected from 449 suspected pulmonary tuberculosis patients as per RNTCP guidelines were screened by microscopy. Decontamination was done by N-acetyl-l-cysteine and sodium hydroxide. Then smear positive samples were subjected to 1st line drug susceptibility testing (DST) using LPA GenoType® MTBDRplus (HAIN Life Science) assay, a molecular method which allows rapid detection of Rifampicin (Rif) and Isoniazid (INH) resistance. RESULTS: The overall burden of MDR TB in this geographical area was 7.9 %. Mono-resistance with Rif alone was around 2.8 %. However, the mono-resistance with INH (inhA gene) and INH (katG gene) was 2.8 % and 1.1 % respectively. Drug resistance of Rif was due to mutations in rpoB gene while resistances to INH were more commonly due to mutation in inhA gene followed by katG gene. TB was more commonly seen in the age group of 30-59 years (43.8 %) and predominantly in males. CONCLUSION: Tuberculosis positivity rate is high in Western Uttar Pradesh. Burden of MDR TB in Western Uttar Pradesh was similar to National data. Line probe assay can be used as a primary method to diagnose multi drug resistant TB as done in present study which can help in earlier initiation of correct therapy.

Isoniazid monoresistant tuberculosis treatment outcomes in Puducherry, South India - A mixed methods study.

SETTING: On a programmatic basis, a new regimen was introduced in the National Tuberculosis Elimination Programme for isoniazid monoresistant tuberculosis in a few states in India. OBJECTIVE: To describe the clinical attributes and treatment outcomes of isoniazid mono-resistant tuberculosis patients on the new 6-month levofloxacin-containing regimen in Puducherry, India. DESIGN: The study is designed as a convergent parallel mixed-methods study: a retrospective cohort study and a descriptive qualitative study. A total of 180 Hr-TB patient health records were reviewed, and in-depth interviews with 35 participants were conducted. (20 Hr-TB patients and 15 HCWs). RESULTS: Of the total 180 Hr-TB patients included, we documented unfavourable outcomes in 26.1% of cases, and the KatG gene mutation was the most common mutation observed (63.9%). A significant risk of unfavourable outcomes was associated with low adherence and positive sputum at the third-month culture report. In interviewing the stakeholders, major challenges observed were the increased pill burden, delay in diagnosis, shortage of drugs, and lack of staff. CONCLUSION: Hr-TB patients have difficulty in adhering to the 6-month levofloxacin regimen, with the need for rigorous early 3-month follow-up and assessment.

Pyrazinamide resistance due to pncA gene mutation and its association with treatment outcome among tuberculosis patients of South India- A longitudinal observational study.

INTRODUCTION: Mycobacterium tuberculosis has been extensively studied for mutations leading to drug resistance. Pyrazinamide is a drug acting on the semi-dormant bacteria that is responsible for relapse of tuberculosis. This drug helped reduce the treatment duration of tuberculosis from nine to six months. However, this drug is not being screened for resistance along with Rifampicin and Isoniazid. AIMS AND OBJECTIVES: This study aimed to estimate the proportion of pncA gene mutation among tuberculosis patients and its association between treatment outcomes, clinical characteristics, and phenotypic drug resistance. METHOD: ology: A total of 154 samples included 73 drug-resistant and 81 drug-susceptible isolates. The isolates were subjected to DNA extraction and amplification using conventional PCR. The PCR product was sequenced by the Sanger sequencing method, and phenotypic drug susceptibility testing was done using the broth dilution method. The association of this gene with the treatment outcome was done by following up with the patients till the end of the regimen. RESULTS: None of the drug susceptible tuberculosis patients showed significant non-synonymous mutations. Among the drug-resistant TB patients, seven unique significant mutations out of 73 isolates (9.6%) were distributed among Isoniazid-resistant tuberculosis and Multi-Drug Resistant Tuberculosis isolates. No association was found between the mutations and the clinical characteristics of the subjects harboring these isolates. CONCLUSION: This study estimated seven unique mutations in drug-resistant tuberculosis and none in drug-sensitive tuberculosis. Isolates harboring was not significantly associated with the participant's treatment outcome and other clinical characteristics. The pyrazinamide resistance testing by the phenotypic and genotypic methods was found to be in concordance.

[Efficacy and safety of first-line anti-tuberculosis drugs combined with Linezolid for the treatment of pediatric tuberculous meningitis in real-word practice].

Objective: To evaluate the efficacy and safety of first-line anti-tuberculosis (TB) drugs combined with linezolid in treatment of children with tuberculous meningitis (TBM). Methods: A retrospective cohort study design was performed. Eight-nine Children diagnosed as TBM during January 1st 2016 and December 31st 2023 in Department of Infectious Disease, Children's Hospital of Chongqing Medical University were enrolled in the study. According to different treatment regimens, children were divided into a group of first-line anti-tuberculous drugs (isoniazid, rifampicin, pyrazinamide, ethambutol (HRZE)) and a group of HRZE and linezolid combination (HRZEL). The efficacy and safety of the 2 regimens were compared and the relationship between linezolid drug concentration and adverse reactions were analyzed. Comparisons between groups were performed using χ2 test and Mann-Whitney U test. Results: The 89 children with TBM included 53 males and 36 females with an onset age of 4.6 (1.4, 9.6) years. There were 27 cases in the HZREL group and 62 cases in the HRZE group. Before treatment, positive rate of interferon-gamma release assays (IGRA) in HRZEL group was lower than that in HRZE group (64% (16/25) vs.92% (55/60), χ2=9.82, P<0.05), but protein level of cerebrospinal fluid (CSF) was higher than that in HRZE group (1.2 (1.0, 2.0) vs.0.8 (0.4,1.4) g/L, Z=0.32, P<0.05). By the end of the intensive phase, there were no significant differences of rates of CSF improvement and etiology negativity between HRZEL group and HRZE group (both P>0.05).The 44 TBM children with high CSF protein (>1 g/L) included 25 males and 19 females with an onset age of 6.7 (3.0, 11.8) years. There were 21 cases in the HZREL group and 23 cases in the HRZE group accordingly. Before treatment, there were no significant differences of positive rate of IGRA test and CSF protein level between the 2 groups (62% (13/21) vs. 87% (20/23), 1.7 (1.1, 2.2) vs. 1.5 (1.2, 1.9) g/L, χ2=3.67, Z=0.23, both P>0.05). There were no significant differences in CSF indicators, etiology negativity or imaging remission between the two groups by the end of intensive phase (all P>0.05). Higher frequencies of granulocytopenia, gastrointestinal symptoms as well as withdrawal or change of drugs were found in HRZEL group when compared to those in HRZE group (44% (12/27) vs. 19% (12/62), 7% (2/27) vs. 0, 33% (9/27) vs. 3% (2/62), χ2=6.01, 4.70, 15.74, all P<0.05). Conclusions: The efficacy of HRZEL regimen is similar to conventional HRZE regimen in children with TBM, but with higher adverse effect. Prudentially evaluating the pros and cons of linezolid in the usage of drug-susceptible TB and carefully monitoring of linezolid associated adverse effects is suggested.

Effects of species of origin and mode of induction of microsomes on carbamazepine-induced cell toxicity.

Standardization and validation of in vitro drug metabolism is essential for pre-clinical drug development as well as for in vitro toxicity assays including the lymphocyte toxicity assay (LTA) and the in vitro platelet toxicity assay (iPTA). Use of isolated liver microsomes (MIC) in in vitro testing has been utilized for a long time; however, the effect of species of origin and induction agents on the metabolic capacities of MIC is not adequately evaluated. In this study we investigated the impact of species of origin and induction agent on the capacity of MICs to bioactivate carbamazepine (CBZ) using cytotoxicity as a gross endpoint to measure the levels of cytotoxic metabolites generated by each type of MICs. Jurkat E6.1 cell line was used and MICs from human, rat, mouse, minipig and rabbit origin as well as rat MICs that is either non-induced or induced by phenobarbitone (PHB), dexamethasone (DEXA), 3-methylcholanthrene (3MC), clofibrate (CLOF) and isoniazid (INH) were investigated. MICs from minipig and rat MICs induced with 3MC exhibited the highest capacity to produce cytotoxic metabolites of CBZ. These findings will help optimize and standardize in vitro toxicity assays and provide guidance to pre-clinical investigation of drugs.

EfpA is required for regrowth of Mycobacterium tuberculosis following isoniazid exposure.

Efflux of antibiotics is an important survival strategy in bacteria. Mycobacterium tuberculosis has approximately sixty efflux pumps, but little is known about the role of each pump or the substrates they efflux. The putative efflux pump, EfpA, is a member of the major facilitator superfamily and has been shown to be essential by saturation transposon mutagenesis studies. It has been implicated in the efflux of isoniazid (INH), which is a first-line drug used to treat tuberculosis (TB). This is supported by evidence from transcriptional profiling showing that efpA is induced in response to INH exposure. However, its roles in the physiology and adaptation of M. tuberculosis to antibiotics have yet to be determined. In this study, we describe the repression of efpA in M. tuberculosis, using CRISPR interference (CRISPRi) to knockdown the expression of this essential gene and the direct effect of this on the ability of M. tuberculosis to survive exposure to INH over a 45-day time course. We determined that wild-type levels of efpA were required for recovery of M. tuberculosis following INH exposure and that, after 45 days of INH exposure, only a few viable colonies were recoverable from efpA-repressed M. tuberculosis. We conclude that EfpA is required for recovery of M. tuberculosis following INH exposure, which could reduce the efficacy of INH in vivo, and that EfpA may have a role in the development of resistance during drug therapy.

Machine Learning Approach in Dosage Individualization of Isoniazid for Tuberculosis.

INTRODUCTION: Isoniazid is a first-line antituberculosis agent with high variability, which would profit from individualized dosing. Concentrations of isoniazid at 2 h (C2h), as an indicator of safety and efficacy, are important for optimizing therapy. OBJECTIVE: The objective of this study was to establish machine learning (ML) models to predict the C2h, that can be used for establishing an individualized dosing regimen in clinical practice. METHODS: Published population pharmacokinetic (PopPK) models for adults were searched based on PubMed and ultimately four reliable models were selected for simulating individual C2h datasets under different conditions (demographics, genotype, ethnicity, etc.). Machine learning models were trained on simulated C2h obtained from the four PopPK models. Five different algorithms were used for ML model building to predict C2h. Real-world data were used for predictive performance evaluations. Virtual trials were used to compare ML-optimized doses with PopPK model-optimized doses. RESULTS: Categorical boosting (CatBoost) exhibited the highest prediction ability. Target C2h can be predicted using the ML model combined with the dosing regimen and three covariates (N-acetyltransferase 2 [NAT2] genotypes, weight and race [Asians and Africans]). Real-world data validation results showed that the ML model can achieve an overall prediction accuracy of 93.4%. Using the final ML model, the mean absolute prediction error value decreased by 45.7% relative to the average of PopPK models. Using the ML-optimized dosing regimen, the probability of target attainment increased by 43.7% relative to the PopPK model-optimized dosing regimens. CONCLUSION: Machine learning models were developed with great predictive performance, which can be used to determine the individualized initial dose of isoniazid in adult patients.

Missed rifampicin and isoniazid resistance by commercial molecular assays.

Drug-resistant tuberculosis (TB) has poor outcomes unless resistance is detected early, ideally by commercially available molecular tests. We present a case of occult multidrug-resistant TB where both rifampicin and isoniazid resistance were missed by molecular testing and were only identified by phenotypic testing.