Clinical assessment of momelotinib drug-drug interactions via CYP3A metabolism and transporters.

Momelotinib-approved for treatment of myelofibrosis in adults with anemia-and its major active metabolite, M21, were assessed as drug-drug interaction (DDI) victims with a strong cytochrome P450 (CYP) 3A4 inhibitor (multiple-dose ritonavir), an organic anion transporting polypeptide (OATP) 1B1/1B3 inhibitor (single-dose rifampin), and a strong CYP3A4 inducer (multiple-dose rifampin). Momelotinib DDI perpetrator potential (multiple-dose) was evaluated with CYP3A4 and breast cancer resistance protein (BCRP) substrates (midazolam and rosuvastatin, respectively). DDI was assessed from changes in maximum plasma concentration (Cmax), area under the concentration-time curve (AUC), time to reach Cmax, and half-life. The increase in momelotinib (23% Cmax, 14% AUC) or M21 (30% Cmax, 24% AUC) exposure with ritonavir coadministration was not clinically relevant. A moderate increase in momelotinib (40% Cmax, 57% AUC) and minimal change in M21 was observed with single-dose rifampin. A moderate decrease in momelotinib (29% Cmax, 46% AUC) and increase in M21 (31% Cmax, 15% AUC) were observed with multiple-dose rifampin compared with single-dose rifampin. Due to potentially counteracting effects of OATP1B1/1B3 inhibition and CYP3A4 induction, multiple-dose rifampin did not significantly change momelotinib pharmacokinetics compared with momelotinib alone (Cmax no change, 15% AUC decrease). Momelotinib did not alter the pharmacokinetics of midazolam (8% Cmax, 16% AUC decreases) or 1'-hydroxymidazolam (14% Cmax, 16% AUC decreases) but increased rosuvastatin Cmax by 220% and AUC by 170%. Safety findings were mild in this short-term study in healthy volunteers. This analysis suggests that momelotinib interactions with OATP1B1/1B3 inhibitors and BCRP substrates may warrant monitoring for adverse reactions or dose adjustments.

Comprehensive assessment of invalid and indeterminate results in Truenat MTB-RIF testing across sites under the national TB elimination program of India.

Introduction: Truenat MTB-RIF assay (Truenat), a nucleic acid amplification test (NAAT), is a real-time polymerase chain reaction (RT-PCR) chip-based assay that can detect Mycobacterium tuberculosis (Mtb) and rifampicin (RIF) drug resistance using portable, battery-operated devices. The National TB Elimination Program (NTEP) in India introduced this novel tool at the district and subdistrict level in 2020. This study aimed to assess the level and causes of inconclusive results (invalid results, errors, and indeterminate results) in MTB and RIF testing at NTEP sites and the root causes of these in the programmatic setting. Methods: Truenat testing data from 1,690 functional Truenat sites under the NTEP from April to June 2021 were analyzed to assess the rates of errors, invalid MTB results, and indeterminate RIF results. Following this analysis, 12 Truenat sites were selected based on site performance in Truenat testing, diversity of climatic conditions, and geographical terrain. These sites were visited to assess the root causes of their high and low rates of inconclusive results using a structured checklist. Results: A total of 327,649 Truenat tests performed for MTB and RIF testing were analyzed. The rate of invalid MTB results was 5.2% [95% confidence interval (CI): 5.11-5.26; n = 16,998] and the rate of errors was 2.5% (95% CI: 2.46-2.57; n = 8,240) in Truenat MTB chip testing. For Mtb-positive samples tested using the Truenat RIF chip for detection of RIF resistance (n = 40,926), the rate of indeterminate results was 15.3% (95% CI: 14.97-15.67; n = 6,267) and the rate of errors was 1.6% (95% CI: 1.53-1.78; n = 675). There was a 40.1% retesting gap for Mtb testing and a 78.2% gap for inconclusive RR results. Among the inconclusive results retested, 27.9% (95% CI: 27.23-28.66; n = 4,222) were Mtb-positive, and 9.2% (95% CI: 7.84-10.76; n = 139) were detected as RR. Conclusion: The main causes affecting Truenat testing performance include suboptimal adherence to standard operating procedures (SOPs), inadequate training, improper storage of testing kits, inadequate sputum quality, lack of quality control, and delays in the rectification of machine issues. Root cause analysis identified that strengthening of training, external quality control, and supervision could improve the rate of inconclusive results. Ensuring hands-on training of technicians for Truenat testing and retesting of samples with inconclusive results are major recommendations while planning for Truenat scale-up. The recommendations from the study were consolidated into technical guidance documents and videos and disseminated to laboratory staff working at the tiered network of TB laboratories under the NTEP in order to improve Truenat MTB-RIF testing performance.

Global burden of disease due to rifampicin-resistant tuberculosis: a mathematical modeling analysis.

In 2020, almost half a million individuals developed rifampicin-resistant tuberculosis (RR-TB). We estimated the global burden of RR-TB over the lifetime of affected individuals. We synthesized data on incidence, case detection, and treatment outcomes in 192 countries (99.99% of global tuberculosis). Using a mathematical model, we projected disability-adjusted life years (DALYs) over the lifetime for individuals developing tuberculosis in 2020 stratified by country, age, sex, HIV, and rifampicin resistance. Here we show that incident RR-TB in 2020 was responsible for an estimated 6.9 (95% uncertainty interval: 5.5, 8.5) million DALYs, 44% (31, 54) of which accrued among TB survivors. We estimated an average of 17 (14, 21) DALYs per person developing RR-TB, 34% (12, 56) greater than for rifampicin-susceptible tuberculosis. RR-TB burden per 100,000 was highest in former Soviet Union countries and southern African countries. While RR-TB causes substantial short-term morbidity and mortality, nearly half of the overall disease burden of RR-TB accrues among tuberculosis survivors. The substantial long-term health impacts among those surviving RR-TB disease suggest the need for improved post-treatment care and further justify increased health expenditures to prevent RR-TB transmission.

Highly Sensitive Detection of Complicated Mutations of Drug Resistance in Mycobacterium tuberculosis Using a Simple, Accurate, Rapid, and Low-Cost Tailored-Design Competitive Wild-Type Blocking Assay.

Establishing simple, rapid, and highly sensitive molecular assays is crucial for timely diagnosis and effective treatment of drug-resistant tuberculosis. However, current genotypic drug susceptibility testing (DST) still encounters enormous challenges including lower sensitivity than phenotypic DST and insufficient accuracy. Herein, a simple, low-cost, multiplex real-time polymerase chain reaction-based assay is established to achieve highly sensitive detection of low-abundant mutants through competitive wild-type blocking (COWTB). Analytical performance of the COWTB assay can achieve 1% or even 0.1% mutants under background of 10 000 wild-type genomes/test. Furthermore, clinical practice feasibility is evaluated to identify resistance to rifampicin (RIF), isoniazid (INH), and streptomycin (SM) on 92 actual clinical samples, its sensitivity is 93.8% for RIF and 100% for INH and SM, and specificity is 100% each for RIF, INH, and SM when using DNA sequencing as the reference standard. In comparison, the sensitivity of reverse dot blotting assay commonly used in clinics is 93.8%, 90.0%, and 84.6%, and the specificity is 96.1%, 98.6%, and 100% for RIF, INH, and SM, respectively. Importantly, the COWTB assay can also be applicable for other drug-resistant mutations and pave a promising detection strategy to fill the gap between phenotypic and genotypic DST for detecting low-abundant drug-resistant M. tuberculosis.

Clinical assessment of gepotidacin (GSK2140944) as a victim and perpetrator of drug-drug interactions via CYP3A metabolism and transporters.

Gepotidacin is a novel triazaacenaphthylene antibiotic in phase III development. Based on nonclinical in vitro characterization of gepotidacin metabolism, two phase I studies were conducted in healthy participants to investigate clinical drug-drug interactions (DDIs). We assessed gepotidacin as a DDI victim with a potent cytochrome P450 (CYP) 3A4/P-glycoprotein (P-gp) inhibitor (itraconazole), potent CYP3A4 inducer (rifampicin), and nonspecific organic cation transporter (OCT)/multidrug and toxic extrusion transporter (MATE) renal transport inhibitor (cimetidine) via single doses of gepotidacin before and after co-administration with multiple doses of the modulator drugs. Gepotidacin DDI perpetrator potential for P-gp inhibition (digoxin) and CYP3A4 inhibition (midazolam) was evaluated via single doses of the two-drug cocktail without and with gepotidacin. The DDI magnitudes were interpreted based on area under the concentration-time curve (AUC). A weak DDI (AUC increase 48%-50%) was observed for gepotidacin co-administered with itraconazole. A clinically significant decrease in gepotidacin plasma AUC (52%) was observed with rifampicin coadministration, indicating a moderate DDI. There was no DDI for gepotidacin with cimetidine; a unique biomarker approach showed increased serum creatinine (24%), decreased renal clearance of creatinine (21%), and N1-methylnicotinamide (39%), which confirmed extensive MATE inhibition and partial OCT2 inhibition. Gepotidacin was not a P-gp DDI perpetrator, although the maximum plasma concentration of digoxin increased (53%) and is potentially clinically relevant given its narrow therapeutic index. Gepotidacin demonstrated weak CYP3A4 inhibition with midazolam (<2-fold AUC increase). There were no new safety-risk profile findings. These results will inform the safe and efficacious clinical use of gepotidacin when co-administered with other drugs.

Quantitative Cytochrome P450 3A4 Induction Risk Assessment Using Human Hepatocytes Complemented with Pregnane X Receptor-Activating Profiles.

Reliable in vitro to in vivo translation of cytochrome P450 (CYP) 3A4 induction potential is essential to support risk mitigation for compounds during pharmaceutical discovery and development. In this study, a linear correlation of CYP3A4 mRNA induction potential in human hepatocytes with the respective pregnane-X receptor (PXR) activation in a reporter gene assay using DPX2 cells was successfully demonstrated for 13 clinically used drugs. Based on this correlation, using rifampicin as a positive control, the magnitude of CYP3A4 mRNA induction for 71 internal compounds at several concentrations up to 10 µM (n = 90) was predicted within 2-fold error for 64% of cases with only a few false positives (19%). Furthermore, the in vivo area under the curve reduction of probe CYP substrates was reasonably predicted for eight marketed drugs (carbamazepine, dexamethasone, enzalutamide, nevirapine, phenobarbital, phenytoin, rifampicin, and rufinamide) using the static net effect model using both the PXR activation and CYP3A4 mRNA induction data. The liver exit concentrations were used for the model in place of the inlet concentrations to avoid false positive predictions and the concentration achieving twofold induction (F2) was used to compensate for the lack of full induction kinetics due to cytotoxicity and solubility limitations in vitro. These findings can complement the currently available induction risk mitigation strategy and potentially influence the drug interaction modeling work conducted at clinical stages. SIGNIFICANCE STATEMENT: The established correlation of CYP3A4 mRNA in human hepatocytes to PXR activation provides a clear cut-off to identify a compound showing an in vitro induction risk, complementing current regulatory guidance. Also, the demonstrated in vitro-in vivo translation of induction data strongly supports a clinical development program although limitations remain for drug candidates showing complex disposition pathways, such as involvement of auto-inhibition/induction, active transport and high protein binding.

Mutations compensating for the fitness cost of rifampicin resistance in Escherichia coli exert pleiotropic effect on RNA polymerase catalysis.

The spread of drug-resistant bacteria represents one of the most significant medical problems of our time. Bacterial fitness loss associated with drug resistance can be counteracted by acquisition of secondary mutations, thereby enhancing the virulence of such bacteria. Antibiotic rifampicin (Rif) targets cellular RNA polymerase (RNAP). It is potent broad spectrum drug used for treatment of bacterial infections. We have investigated the compensatory mechanism of the secondary mutations alleviating Rif resistance (Rifr) on biochemical, structural and fitness indices. We find that substitutions in RNAP genes compensating for the growth defect caused by ßQ513P and ßT563P Rifr mutations significantly enhanced bacterial relative growth rate. By assaying RNAP purified from these strains, we show that compensatory mutations directly stimulated basal transcriptional machinery (2-9-fold) significantly improving promoter clearance step of the transcription pathway as well as elongation rate. Molecular modeling suggests that compensatory mutations affect transcript retention, substrate loading, and nucleotidyl transfer catalysis. Strikingly, one of the identified compensatory substitutions represents mutation conferring rifampicin resistance on its own. This finding reveals an evolutionary process that creates more virulent species by simultaneously improving the fitness and augmenting bacterial drug resistance.

Assessment of persistent antimicrobial and anti-biofilm activity of p-HEMA hydrogel loaded with rifampicin and cefixime.

Catheter-associated urinary tract infections (CAUTIs) are nosocomial infections causing more than one million hospital cases annually. The progress of CAUTIs leads to severe health complications. Infections result in blockage of the medical device due to biofilm formation, which necessitates the replacement of the device. The objective of this study is to improve urological biomaterials to minimize microbial growth and reduce the incidence of CAUTIs. Challenges from mixed biofilm are crucial and need to be addressed in the development of new coating materials. Herein, an investigation highlighted the reduction of mixed biofilm overgrowth and attachment tendency on poly-2-hydroxyethyl methacrylate (p-HEMA) surface by loading the hydrogel with rifampicin (RIF), cefixime trihydrate (CFX), and combined ratios of RIF and CFX. Mixed biofilm-formation ability in (3:1) RIF: CFX-loading p-HEMA (F6) surface showed best tendency to resist form biofilm. Persistent antimicrobial activity increased in p-HEMA loaded with combined ratios of RIF and CFX surface compared to p-HEMA alone, antimicrobial activity lasted for 8 days. All fabricated films exhibited %cell viability higher than 75% on HEK 293 cells. The addition of RIF and CFX may improve the duration of urological device employment before replacement.

A cost-benefit algorithm for rapid diagnosis of tuberculosis and rifampicin resistance detection during mass screening campaigns.

BACKGROUND: Active tuberculosis (TB) case finding is important as it helps detect pulmonary TB cases missed by the other active screening methods. It requires periodic mass screening in risk population groups such as prisoners and refugees. Unfortunately, in these risk population groups periodic mass screening can be challenging due to lengthy turnaround time (TAT), cost and implementation constraints. The aim of this study was to evaluate a diagnostic algorithm that can reduce the TAT and cost for TB and Rifampicin resistance (RR) detection. The algorithm involves testing with TB-LAMP followed by Xpert MTB/RIF for positive TB-LAMP cases to diagnose TB during mass campaigns in prisons and refugee camps. METHODS: The National Tuberculosis Control Program (NTCP) organized routine TB mass-screening campaigns in 34 prisons and 3 villages with refugees camps in Cameroon in 2019. TB LAMP was used for initial TB diagnosis and all TB-LAMP positive cases tested with the Xpert MTB/RIF assay to determine RR. TAT and cost benefits analysis of the combined use of TB-LAMP and Xpert MTB/RIF assays was determined and compared to the Xpert MTB/RIF assay when used only. RESULTS: A total of 4075 sputum samples were collected from TB presumptive, 3672 cases in 34 prisons and 403 samples in 3 villages. Of the 4,075 samples screened with TB-LAMP, 135 were TB positive (3.31%) and run on the Xpert MTB/RIF. Of the 135 positives cases, Xpert MTB/RIF revealed 3 were RR (2.22%). The use of TB-LAMP followed by testing with Xpert MTB/RIF for TB and RR detection reduced the TAT by 73.23% in prisons and 74.92% in villages. In addition to a reduced TAT, the two molecular tests used in synergy is cost benefit from year 2 onwards. CONCLUSION: This study demonstrates the advantages of a diagnostic algorithm based on an initial testing with TB-LAMP followed by testing with Xpert MTB/RIF for TB diagnosis. This approach improved early and rapid TB detection with an added advantage of providing RR status. The proposed algorithm is effective and less costly from the second year of implementation and should be used by TB control programs.

Assessment of epidemiological and genetic characteristics and clinical outcomes of resistance to bedaquiline in patients treated for rifampicin-resistant tuberculosis: a cross-sectional and longitudinal study.

BACKGROUND: Bedaquiline improves outcomes of patients with rifampicin-resistant and multidrug-resistant (MDR) tuberculosis; however, emerging resistance threatens this success. We did a cross-sectional and longitudinal analysis evaluating the epidemiology, genetic basis, and treatment outcomes associated with bedaquiline resistance, using data from South Africa (2015-19). METHODS: Patients with drug-resistant tuberculosis starting bedaquiline-based treatment had surveillance samples submitted at baseline, month 2, and month 6, along with demographic information. Culture-positive baseline and post-baseline isolates had phenotypic resistance determined. Eligible patients were aged 12 years or older with a positive culture sample at baseline or, if the sample was invalid or negative, a sample within 30 days of the baseline sample submitted for bedaquiline drug susceptibility testing. For the longitudinal study, the first surveillance sample had to be phenotypically susceptible to bedaquiline for inclusion. Whole-genome sequencing was done on bedaquiline-resistant isolates and a subset of bedaquiline-susceptible isolates. The National Institute for Communicable Diseases tuberculosis reference laboratory, and national tuberculosis surveillance databases were matched to the Electronic Drug-Resistant Tuberculosis Register. We assessed baseline resistance prevalence, mutations, transmission, cumulative resistance incidence, and odds ratios (ORs) associating risk factors for resistance with patient outcomes. FINDINGS: Between Jan 1, 2015, and July 31, 2019, 8041 patients had surveillance samples submitted, of whom 2023 were included in the cross-sectional analysis and 695 in the longitudinal analysis. Baseline bedaquiline resistance prevalence was 3·8% (76 of 2023 patients; 95% CI 2·9-4·6), and it was associated with previous exposure to bedaquiline or clofazimine (OR 7·1, 95% CI 2·3-21·9) and with rifampicin-resistant or MDR tuberculosis with additional resistance to either fluoroquinolones or injectable drugs (pre-extensively-drug resistant [XDR] tuberculosis: 4·2, 1·7-10·5) or to both (XDR tuberculosis: 4·8, 2·0-11·7). Rv0678 mutations were the sole genetic basis of phenotypic resistance. Baseline resistance could be attributed to previous bedaquiline or clofazimine exposure in four (5·3%) of 76 patients and to primary transmission in six (7·9%). Odds of successful treatment outcomes were lower in patients with baseline bedaquiline resistance (0·5, 0·3-1). Resistance during treatment developed in 16 (2·3%) of 695 patients, at a median of 90 days (IQR 62-195), with 12 of these 16 having pre-XDR or XDR. INTERPRETATION: Bedaquiline resistance was associated with poorer treatment outcomes. Rapid assessment of bedaquiline resistance, especially when patients were previously exposed to bedaquiline or clofazimine, should be prioritised at baseline or if patients remain culture-positive after 2 months of treatment. Preventing resistance by use of novel combination therapies, current treatment optimisation, and patient support is essential. FUNDING: National Institute for Communicable Diseases of South Africa.

A Rapid Drug Resistance Genotyping Workflow for Mycobacterium tuberculosis, Using Targeted Isothermal Amplification and Nanopore Sequencing.

Phenotypic drug susceptibility testing (DST) for tuberculosis (TB) requires weeks to yield results. Although molecular tests rapidly detect drug resistance-associated mutations (DRMs), they are not scalable to cover the full genome and the many DRMs that can predict resistance. Whole-genome sequencing (WGS) methods are scalable, but if conducted directly on sputum, typically require a target enrichment step, such as nucleic acid amplification. We developed a targeted isothermal amplification-nanopore sequencing workflow for rapid prediction of drug resistance of TB isolates. We used recombinase polymerase amplification (RPA) to perform targeted isothermal amplification (37°C for 90 min) of three regions within the Mycobacterium tuberculosis genome, followed by nanopore sequencing on the MinION. We tested 29 mycobacterial genomic DNA extracts from patients with drug-resistant (DR) TB and compared our results to those of WGS by Illumina and phenotypic DST to evaluate the accuracy of prediction of resistance to rifampin and isoniazid. Amplification by RPA showed fidelity equivalent to that of high-fidelity PCR (100% concordance). Nanopore sequencing generated DRM predictions identical to those of WGS, with considerably faster sequencing run times of minutes rather than days. The sensitivity and specificity of rifampin resistance prediction for our workflow were 96.3% (95% confidence interval [CI], 81.0 to 99.9%) and 100.0% (95% CI, 15.8 to 100.0%), respectively. For isoniazid resistance prediction, the sensitivity and specificity were 100.0% (95% CI, 86.3 to 100.0%) and 100.0% (95% CI, 39.8 to 100.0%), respectively. The workflow consumable costs per sample are less than £100. Our rapid and low-cost drug resistance genotyping workflow provides accurate prediction of rifampin and isoniazid resistance, making it appropriate for use in resource-limited settings. IMPORTANCE Current methods for diagnosing drug-resistant tuberculosis are time consuming, resulting in delays in patients receiving treatment and in transmission onwards. They also require a high level of laboratory infrastructure, which is often only available at centralized facilities, resulting in further delays to diagnosis and additional barriers to deployment in resource-limited settings. This article describes a new workflow that can diagnose drug-resistant TB in a shorter time, with less equipment, and for a lower price than current methods. The amount of TB DNA is first increased without the need for bulky and costly thermocycling equipment. The DNA is then read using a portable sequencer called a MinION, which indicates whether there are tell-tale changes in the DNA that indicate whether the TB strain is drug resistant. Our workflow could play an important role in the future in the fight against the public health challenge that is TB drug resistance.

Analysing the fitness cost of antibiotic resistance to identify targets for combination antimicrobials.

Mutations in the rifampicin (Rif)-binding site of RNA polymerase (RNAP) confer antibiotic resistance and often have global effects on transcription that compromise fitness and stress tolerance of resistant mutants. We suggested that the non-essential genome, through its impact on the bacterial transcription cycle, may represent an untapped source of targets for combination antimicrobial therapies. Using transposon sequencing, we carried out a genome-wide analysis of fitness cost in a clinically common rpoB H526Y mutant. We find that genes whose products enable increased transcription elongation rates compound the fitness costs of resistance whereas genes whose products function in cell wall synthesis and division mitigate it. We validate our findings by showing that the cell wall synthesis and division defects of rpoB H526Y result from an increased transcription elongation rate that is further exacerbated by the activity of the uracil salvage pathway and unresponsiveness of the mutant RNAP to the alarmone ppGpp. We applied our findings to identify drugs that inhibit more readily rpoB H526Y and other RifR alleles from the same phenotypic class. Thus, genome-wide analysis of fitness cost of antibiotic-resistant mutants should expedite the discovery of new combination therapies and delineate cellular pathways that underlie the molecular mechanisms of cost.

Expression Dysregulation as a Mediator of Fitness Costs in Antibiotic Resistance.

Antimicrobial resistance (AMR) poses a threat to global health and the economy. Rifampicin-resistant Mycobacterium tuberculosis accounts for a third of the global AMR burden. Gaining the upper hand on AMR requires a deeper understanding of the physiology of resistance. AMR often results in a fitness cost in the absence of drug. Identifying the molecular mechanisms underpinning this cost could help strengthen future treatment regimens. Here, we used a collection of M. tuberculosis strains that provide an evolutionary and phylogenetic snapshot of rifampicin resistance and subjected them to genome-wide transcriptomic and proteomic profiling to identify key perturbations of normal physiology. We found that the clinically most common rifampicin resistance-conferring mutation, RpoB Ser450Leu, imparts considerable gene expression changes, many of which are mitigated by the compensatory mutation in RpoC Leu516Pro. However, our data also provide evidence for pervasive epistasis-the same resistance mutation imposed a different fitness cost and functionally distinct changes to gene expression in genetically unrelated clinical strains. Finally, we report a likely posttranscriptional modulation of gene expression that is shared in most of the tested strains carrying RpoB Ser450Leu, resulting in an increased abundance of proteins involved in central carbon metabolism. These changes contribute to a more general trend in which the disruption of the composition of the proteome correlates with the fitness cost of the RpoB Ser450Leu mutation in different strains.

Assessment of the GenoType MTBDRsl VER 2.0 compared to the phenotypic drug susceptibility testing and whole genome sequencing for the rapid detection of resistance to fluoroquinolone and second-line injectable drugs among rifampicin-resistant Mycobacterium tuberculosis isolates.

Molecular techniques have considerable advantages for rapid detection, a reduction of infectiousness, prevention of further resistance development and surveillance of drug-resistant TB. MTBDRsl VER 2.0 was used to detect resistance to second-line anti-tuberculosis drugs on 35 rifampicin-resistant M. tuberculosis (RR-MTB) isolates compared to the minimum inhibitory concentrations (MICs) and whole genome sequencing (WGS). The MTBDRsl VER 2.0 (Hain Life Science, Nehren, Germany) and WGS (San Diego, CA, USA) were performed for tracing mutations in resistant-related genes involved in resistance to fluoroquinolone (FLQ) and second-line injectable drugs. The broth microdilution method using 7H9 Middlebrook media supplemented with OADC was used to determine the MICs. The MTBDRsl VER 2.0 correctly detected 5/6 (83.3%) of FLQ-resistant strains. The MUT1 A1401G (seven strains) and MUT2 G1484T (one strain) mutations in rrs gene were detected in eight AMK/KAN/CAP-resistant strains. Four low-level KAN-resistant strains with the G-10A/C-12T (three strains) and eis C-14T (one strain) mutations in eis gene was diagnosed using MTBDRsl VER 2.0. Five errors were found in detecting resistance to kanamycin and capreomycin compared to the phenotypic drug susceptibility testing and WGS. Failling wild-type bands without improved mutant bands did not indicate a reliable resistance. WGS could efficiently resolve the discrepancies of the results. MTBDRsl showed better performance in detecting XDR strains than pre-XDR.

Performance Assessment of Xpert MTB/RIF Assay for Detecting Pulmonary Tuberculosis and Rifampin Resistance in a Tertiary Care Hospital in Korea.

In this study, we aimed to assess the performance of the Xpert MTB/RIF assay for the detection of pulmonary tuberculosis compared to the acid-fast bacilli (AFB) smear and culture analysis, and the incidence of rifampin resistance using the drug susceptibility test. The specimens referred for AFB smear and culture analysis and Xpert MTB/RIF assay from April 2015 to March 2018 were retrospectively reviewed. The sensitivity, specificity, and mean cycle threshold (Ct) values obtained in Xpert MTB/RIF assay and for rifampin resistance were analyzed. The results of Xpert MTB/RIF assay for pulmonary tuberculosis were evaluated based on the AFB smear grade. Among 3,840 specimens, 491 were positive in Xpert MTB/RIF assay and 626 in culture analysis. The sensitivity and specificity of Xpert MTB/RIF assay were 75.6% and 99.4%, respectively. The sensitivity of Xpert MTB/RIF assay for smear-positive/culture-positive specimens was 98.6% and that of smear-negative and -trace/culture-positive specimens was 63.1%. The positivity of Xpert MTB/RIF assay for culture-positive specimens was 89.9%, 98.6%, 95.7%, 100.0%, and 100.0% for the smear grades trace, 1+, 2+, 3+, 4+, respectively. The Ct values of 491 specimens significantly decreased as the AFB smear grade increased (P < 0.0001). The Ct values of smear-positive, -trace, and -negative specimens were 21.7 ± 4.2, 26.5 ± 3.9, and 27.4 ± 3.6, respectively. Rifampin resistance evaluated using Xpert MTB/RIF assay and culture analysis exhibited a correlation of 98.3%. The region covered by probe E was the most frequently mutated region (50.0%). Xpert MTB/RIF assay demonstrated reliable performance in detecting pulmonary tuberculosis from smear-positive and culture-positive specimens; however, further improvements are still required to detect smear-negative and culture-positive specimens.

Decreased classical monocytes and CD163 expression in TB patients: an indicator of drug resistance.

Tuberculosis (TB) is a disease instigated by Mycobacterium tuberculosis. Peripheral blood monocytes represent highly efficient effector cells of innate immunity against TB. Little is known about monocyte subsets and their potential involvement in the development of M. tuberculosis drug resistance in patients with TB. This study was conducted to investigate alterations in monocyte subsets, CD163 expression on monocytes, and its serum level in patients without and with rifampicin resistance TB (RR-TB) and healthy controls. A total of 164 patients with TB (84 without RR-TB and 80 patients with RR-TB) and 85 healthy controls were enrolled in this study. The percentages of various monocyte subsets and surface expression of CD163 on monocytes were quantitatively determined using flow cytometry. The serum level of CD163 was determined by commercially available ELISA kits. Decreased frequency of classical monocytes was detected in patients with RR-TB. Non-classical monocytes were decreased in patients without RR-TB; however, intermediate monocytes were raised in patients with RR-TB. The serum level of CD163 was decreased in patients of RR-TB that showsed a positive correlation with the frequency of CD14++CD16-CD163+ and CD14++CD16+CD163+ monocytes. It is concluded that decreased classical monocytes and sCD163 in patients with RR-TB could be an indicator of drug resistance.

Modelling the global burden of drug-resistant tuberculosis avertable by a post-exposure vaccine.

There have been notable advances in the development of vaccines against active tuberculosis (TB) disease for adults and adolescents. Using mathematical models, we seek to estimate the potential impact of a post-exposure TB vaccine, having 50% efficacy in reducing active disease, on global rifampicin-resistant (RR-) TB burden. In 30 countries that together accounted for 90% of global RR-TB incidence in 2018, a future TB vaccine could avert 10% (95% credible interval: 9.7-11%) of RR-TB cases and 7.3% (6.6-8.1%) of deaths over 2020-2035, with India, China, Indonesia, Pakistan, and the Russian Federation having the greatest contribution. This impact would increase to 14% (12-16%) and 31% (29-33%) respectively, when combined with improvements in RR-TB diagnosis and treatment relative to a scenario of no vaccine and no such improvements. A future TB vaccine could have important implications for the global control of RR-TB, especially if implemented alongside enhancements in management of drug resistance.

Quantitative Assessment of Levonorgestrel Binding Partner Interplay and Drug-Drug Interactions Using Physiologically Based Pharmacokinetic Modeling.

Levonorgestrel (LNG) is the active moiety in many hormonal contraceptive formulations. It is typically coformulated with ethinyl estradiol (EE) to decrease intermenstrual bleeding. Due to its widespread use and CYP3A4-mediated metabolism, there is concern regarding drug-drug interactions (DDIs), particularly a suboptimal LNG exposure when co-administered with CYP3A4 inducers, potentially leading to unintended pregnancies. The goal of this analysis was to determine the impact of DDIs on the systemic exposure of LNG. To this end, we developed and verified a physiologically-based pharmacokinetic (PBPK) model for LNG in PK-Sim (version 8.0) accounting for the impact of EE and body mass index (BMI) on LNG's binding to sex-hormone binding globulin. Model parameters were optimized following intravenous and oral administration of 0.09 mg LNG. The combined LNG-EE PBPK model was verified regarding CYP3A4-mediated interaction by comparing to published clinical DDI study data with carbamazepine, rifampicin, and efavirenz (CYP3A4 inducers). Once verified, the model was applied to predict systemic LNG exposure in normal BMI and obese women (BMI ≥ 30 kg/m2 ) with and without co-administration of itraconazole (competitive CYP3A4 inhibitor) and clarithromycin (mechanism-based CYP3A4 inhibitor). Total and free LNG exposures, when co-administered with EE, decreased 2-fold in the presence of rifampin, whereas they increased 1.5-fold in the presence of itraconazole. Although changes in total and unbound exposure were decreased in obese women compared with normal BMI women, the relative impact of DDIs on LNG exposure was similar between both groups.

Immortalization of human hepatocytes from biliary atresia with CDK4R24C, cyclin D1, and TERT for cytochrome P450 induction testing.

Hepatocytes are an important tool for in vitro toxicology testing. In addition to primary cultures, a limited number of immortalized cell lines have been developed. We here describe a new cell line, designated as HepaMN, which has been established from a liver associated with biliary atresia. Hepatocytes were isolated from a liver of 4-year-old girl with biliary atresia and immortalized by inoculation with CSII-CMV-TERT, CSII-CMV-Tet-Off, CSII-TRE-Tight-cyclin D1 and CSII-TRE-Tight-CDK4R24C (mutant CDK4: an INK4a-resistant form of CDK4) lentiviruses at the multiplicity of infection of 3 to 10. HepaMN cells exhibited morphological homogeneity, displaying hepatocyte-like phenotypes. Phenotypic studies in vivo and in vitro revealed that HepaMN cells showed polarized and functional hepatocyte features along with a canalicular cell phenotype under defined conditions, and constitutively expressed albumin and carbamoyl phosphate synthetase I in addition to epithelial markers. Since HepaMN cells are immortal and subcloned, kinetics and expression profiles were independent of population doublings. HepaMN cells showed increased CYP3A4 expression after exposure to rifampicin, implying that their close resemblance to normal human hepatocytes makes them suitable for research applications including drug metabolism studies.

Open-Label Assessment of the Effects of Itraconazole and Rifampicin on Balovaptan Pharmacokinetics in Healthy Volunteers.

INTRODUCTION: Balovaptan, an investigational vasopressin 1a receptor antagonist that has been evaluated for improvement of social communication and interaction, is primarily metabolized by cytochrome P450 3A4 (CYP3A4). METHODS: Two single-center, non-randomized, two-period, phase 1 studies assessed the effect of the strong CYP3A4 inhibitor itraconazole (study NCT03579719) or the strong CYP3A4 inducer rifampicin (study NCT03586726) at steady state on the pharmacokinetics (PK) of steady-state balovaptan in healthy volunteers. Participants received balovaptan (5 or 10 mg/day) alone for 10 days, or in combination with itraconazole (200 mg/day) for 15 days, or rifampicin (600 mg/day) for 10 days, following balovaptan washout and itraconazole/rifampicin pre-dosing. Geometric mean ratios (GMRs) and 90% confidence intervals (90% CIs) for the area under the concentration-time curve over the dosing interval (AUC) and maximum plasma concentration (Cmax) of balovaptan dosed with vs. without itraconazole/rifampicin were estimated from a mixed effects model. RESULTS: Both studies comprised 15-16 healthy male and female volunteers. Itraconazole 200 mg/day elevated steady-state exposure to 5 mg/day balovaptan approximately 4.5-5.5-fold (Day 15 GMR [90% CI], 4.46 [4.06-4.90] for Cmax and 5.57 [5.00-6.21] for AUC) and extended the time to steady state from ~ 5 days to ~ 13-14 days. Rifampicin 600 mg/day resulted in ~ 90% reductions in both the Cmax (Day 10 GMR [90% CI], 0.14 [0.12-0.15]) and AUC (0.07 [0.06-0.07]) of balovaptan 10 mg/day. Time to balovaptan steady state could not be determined with rifampicin. There were no clinically significant safety findings in either study. CONCLUSIONS: Strong modulators of CYP3A4 activity will significantly alter the PK of balovaptan, with the effect of CYP3A4 induction greater than that of inhibition. Caution should be taken when concomitantly dosing balovaptan with moderate or strong CYP3A4 inducers or strong CYP3A4 inhibitors. TRIAL REGISTRATION NUMBER: NCT03579719; NCT03586726.