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Concept: Pyrazinamide

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Routine full characterization of Mycobacterium tuberculosis (TB) is culture-based, taking many weeks. Whole-genome sequencing (WGS) can generate antibiotic susceptibility profiles to inform treatment, augmented with strain information for global surveillance; such data could be transformative if provided at or near point of care.We demonstrate a low-cost DNA extraction method for TB WGS direct from patient samples. We initially evaluated the method using the Illumina MiSeq sequencer (40 smear-positive respiratory samples, obtained after routine clinical testing, and 27 matched liquid cultures). M. tuberculosis was identified in all 39 samples from which DNA was successfully extracted. Sufficient data for antibiotic susceptibility prediction was obtained from 24 (62%) samples; all results were concordant with reference laboratory phenotypes. Phylogenetic placement was concordant between direct and cultured samples. Using an Illumina MiSeq/MiniSeq the workflow from patient sample to results can be completed in 44/16 hours at a reagent cost of £96/£198 per sample.We then employed a non-specific PCR-based library preparation method for sequencing on an Oxford Nanopore Technologies MinION sequencer. We applied this to cultured Mycobacterium bovis BCG strain (BCG), and to combined culture-negative sputum DNA and BCG DNA. For flowcell version R9.4, the estimated turnaround time from patient to identification of BCG, detection of pyrazinamide resistance, and phylogenetic placement was 7.5 hours, with full susceptibility results 5 hours later. Antibiotic susceptibility predictions were fully concordant. A critical advantage of the MinION is the ability to continue sequencing until sufficient coverage is obtained, providing a potential solution to the problem of variable amounts of M. tuberculosis in direct samples.

Concepts: DNA, Molecular biology, Tuberculosis, Full genome sequencing, Mycobacterium, Mycobacterium tuberculosis, Mycobacterium bovis, Pyrazinamide

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We report the high-pressure response of three forms (α, δ, and γ) of pyrazinamide (C5H5N3O, PZA) by in situ Raman spectroscopy and synchrotron X-ray diffraction techniques with a pressure of about 14 GPa. These different forms are characterized by various intermolecular bonding schemes. High-pressure experimental results show that the γ phase undergoes phase transition to the β phase at a pressure of about 4 GPa, whereas the other two forms retain their original structures at a high pressure. We propose that the stabilities of the α and δ forms upon compression are due to the special dimer connection that these forms possess. On the other hand, the γ form, which does not have this connection, prefers to transform to the closely related β form when pressure is applied. The detailed mechanism of the phase transition together with the stability of the three polymorphs is discussed by taking molecular stacking into account.

Concepts: Spectroscopy, Diffraction, X-ray, Chemical bonding, Raman spectroscopy, X-ray crystallography, Tuberculosis treatment, Pyrazinamide

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BACKGROUND: Intensified antibiotic treatment might improve the outcome of tuberculous meningitis. We assessed pharmacokinetics, safety, and survival benefit of several treatment regimens containing high-dose rifampicin and moxifloxacin in patients with tuberculous meningitis in a hospital setting. METHODS: In an open-label, phase 2 trial with a factorial design in one hospital in Indonesia, patients (aged >14 years) with tuberculous meningitis were randomly assigned to receive, according to a computer-generated schedule, first rifampicin standard dose (450 mg, about 10 mg/kg) orally or high dose (600 mg, about 13 mg/kg) intravenously, and second oral moxifloxacin 400 mg, moxifloxacin 800 mg, or ethambutol 750 mg once daily. All patients were given standard-dose isoniazid, pyrazinamide, and adjunctive corticosteroids. After 14 days of treatment all patients continued with standard treatment for tuberculosis. Endpoints included pharmacokinetic analyses of the blood and cerebrospinal fluid, adverse events attributable to tuberculosis treatment, and survival. Analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT01158755. FINDINGS: 60 patients were randomly assigned to receive rifampicin standard dose (12 no moxifloxacin, ten moxifloxacin 400 mg, and nine moxifloxacin 800 mg) and high dose (ten no moxifloxacin, nine moxifloxacin 400 mg, and ten moxifloxacin 800 mg). A 33% higher dose of rifampicin, intravenously, led to a three times higher geometric mean area under the time-concentration curve up to 6 h after dose (AUC(0-6); 78·7 mg.h/L [95% CI 71·0-87·3] vs 26·0 mg.h/L [19·0-35·6]), maximum plasma concentrations (C(max); 22·1 mg/L [19·9-24·6] vs 6·3 mg/L [4·9-8·3]), and concentrations in cerebrospinal fluid (0·60 mg/L [0·46-0·78] vs 0·21 mg/L [0·16-0·27]). Doubling the dose of moxifloxacin resulted in a proportional increase in plasma AUC(0-6) (31·5 mg.h/L [24·1-41·1] vs 15·1 mg.h/L [12·8-17·7]), C(max) (7·4 mg/L [5·6-9·6] vs 3·9 mg/L [3·2-4·8]), and drug concentrations in the cerebrospinal fluid (2·43 mg/L [1·81-3·27] vs 1·52 mg/L [1·28-1·82]). Intensified treatment did not result in increased toxicity. 6 month mortality was substantially lower in patients given high-dose rifampicin intravenously (ten [35%] vs 20 [65%]), which could not be explained by HIV status or severity of disease at the time of presentation (adjusted HR 0·42; 95% CI 0·20-0·91; p=0·03). INTERPRETATION: These data suggest that treatment containing a higher dose of rifampicin and standard-dose or high-dose moxifloxacin during the first 2 weeks is safe in patients with tuberculous meningitis, and that high-dose intravenous rifampicin could be associated with a survival benefit in patients with severe disease. FUNDING: Royal Dutch Academy of Arts and Sciences, Netherlands Foundation for Scientific Research, and Padjadjaran University, Bandung, Indonesia.

Concepts: Tuberculosis, Cerebrospinal fluid, Meningitis, Tuberculous meningitis, Lumbar puncture, Tuberculosis treatment, Pyrazinamide, Rifampicin

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Standard tuberculosis (TB) treatment includes an initial regimen containing drugs that are both rapidly bactericidal (isoniazid) and sterilizing (rifampin and pyrazinamide), and ethambutol to help prevent the emergence of drug resistance. Antagonism between isoniazid and pyrazinamide has been demonstrated in a TB treatment mouse model. Because isoniazid’s bactericidal activity is greatest during the initial two treatment days, we hypothesized that removing isoniazid after the second day would increase the effectiveness of the standard regimen. To test this hypothesis, we developed a mouse model to measure the early bactericidal activity (EBA) of drug regimens designed to analyze the essentiality of both isoniazid and pyrazinamide during the first 14 d of therapy. Our results clearly indicate that discontinuation of isoniazid after the second day of treatment increases the EBA of standard therapy in the mouse model, whereas omitting pyrazinamide during the first 14 d was detrimental. Substitution of moxifloxacin for isoniazid on day 3 did not increase the EBA compared with only removing isoniazid after day 2. Our data show that a mouse model can be used to analyze the EBA of TB drugs, and our findings support pursuing clinical trials to evaluate the possible benefit of removing isoniazid after the first 2 treatment days.

Concepts: Pharmacology, Tuberculosis, Murinae, Tuberculosis treatment, Pyrazinamide, Rifampicin, Isoniazid, Ethambutol

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Bovine tuberculosis (BTB) is an endemic zoonosis in Morocco caused by Mycobacterium bovis, which infects many domestic animals and is transmitted to humans through consumption of raw milk or from contact with infected animals. The prevalence of BTB in Moroccan cattle is estimated at 18%, and 33% at the individual and the herd level respectively, but the human M. bovis burden needs further clarification. The current control strategy based on test and slaughter should be improved through local context adaptation taking into account a suitable compensation in order to reduce BTB prevalence in Morocco and decrease the disease burden in humans and animals. We established a simple compartmental deterministic mathematical model for BTB transmission in cattle and humans to provide a general understanding of BTB, in particular regarding transmission to humans. Differential equations were used to model the different pathways between the compartments for cattle and humans. Scenarios of test and slaughter were simulated to determine the effects of varying the proportion of tested animals (p) on the time to elimination of BTB (individual animal prevalence of less than one in a thousand) in cattle and humans. The time to freedom from disease ranged from 75 years for p = 20% to 12 years for p = 100%. For p > 60% the time to elimination was less than 20 years. The cumulated cost was largely stable: for p values higher than 40%, cost ranged from 1.47 to 1.60 billion euros with a time frame of 12 to 32 years to reach freedom from disease. The model simulations also suggest that using a 2mm cut off instead of a 4mm cut off in the Single Intradermal Comparative Cervical Tuberculin skin test (SICCT) would result in cheaper and quicker elimination programs. This analysis informs Moroccan bovine tuberculosis control policy regarding time frame, range of cost and levels of intervention. However, further research is needed to clarify the national human-bovine tuberculosis ratio in Morocco.

Concepts: Tuberculosis, Mycobacterium, Cattle, Mycobacterium bovis, Bacillus Calmette-Guérin, Tuberculosis treatment, Zoonosis, Pyrazinamide

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Accumulating evidence suggest that increasing doses of rifampicin may shorten tuberculosis treatment. The PanACEA HIGHRIF1 trial assessed safety, pharmacokinetics and anti-mycobacterial activity of rifampicin at doses up to 40 mg/kg. Eighty-three pulmonary tuberculosis patients received 10, 20, 25, 30, 35 or 40 mg/kg rifampicin daily over 2 weeks, supplemented with standard doses of isoniazid, pyrazinamide and ethambutol in the second week. This study aimed at characterizing rifampicin pharmacokinetics observed in HIGHRIF1 using non-linear mixed effects modeling. The final population pharmacokinetic model included an enzyme turn-over model accounting for time-dependent elimination due to auto-induction, concentration-dependent clearance and dose-dependent bioavailability. The relationship between clearance and concentration was characterized by a Michaelis-Menten relationship. The relationship between bioavailability and dose was described using an Emax relationship. The model will be key in determining exposure-response relationships for rifampicin and should be considered when designing future trials and when treating future patients with high dose rifampicin. This article is protected by copyright. All rights reserved.

Concepts: Pharmacology, Tuberculosis, Pharmacokinetics, Tuberculosis treatment, Pyrazinamide, Rifampicin, Isoniazid, Ethambutol

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Rapid diagnosis of drug-resistant tuberculosis is important in determining proper management. This letter reports on limitations of the Xpert MTB/RIF assay in determining rifampin resistance in tuberculosis isolates in Swaziland.

Concepts: Antibiotic resistance, Tuberculosis, Pyrazinamide, Rifampicin

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Pyrazinamide (PZA) is an important first-line drug in all existing and new tuberculosis (TB) treatment regimens. PZA-resistance in M. tuberculosis is increasing, especially among M/XDR cases. Noted issues with PZA Drug Susceptibility Testing (DST) have driven the search for alternative tests. This study provides a comprehensive assessment of PZA molecular diagnostics in M/XDR TB cases. A set of 296, mostly XDR, clinical M. tuberculosis isolates from four countries were subjected to DST for eight drugs, confirmatory Wayne’s assay, and whole-genome sequencing. Three genes implicated in PZA resistance, pncA, rpsA, and panD were investigated. Assuming all non-synonymous mutations cause resistance, we report 90% sensitivity and 65% specificity for a pncA-based molecular test. The addition of rpsA and panD potentially provides 2% increase in sensitivity. Molecular heterogeneity in pncA was associated with resistance and should be evaluated as a diagnostic tool. Mutations near the N-terminus and C-terminus of PZase were associated with East-Asian and Euro-American lineages, respectively. Finally, Euro-American isolates are most likely to have a wild-type PZase and escape molecular detection. Overall, the 8-10% resistance without markers may point to alternative mechanisms of resistance. Confirmatory mutagenesis may improve the disconcertingly low specificity but reduce sensitivity since not all mutations may cause resistance.

Concepts: Diagnosis, Type I and type II errors, Sensitivity and specificity, Tuberculosis, Mycobacterium, Mycobacterium tuberculosis, Pyrazinamide

4

Several infectious diseases of global importance - e.g. HIV, tuberculosis (TB) - require prolonged treatment with combination antimicrobial regimens, typically involving high-potency “core” agents coupled with additional “companion” drugs that protect against de novo emergence of mutations conferring resistance to the core agents. Often, the most effective (or least toxic) companion agents are re-used in sequential (first-line, second-line, etc…) regimens. We used a multi-strain model of M. tuberculosis transmission in Southeast Asia to investigate how this practice might facilitate the emergence of extensive drug resistance, i.e., resistance to multiple core agents. We calibrated this model to regional TB and drug resistance data using an Approximate Bayesian Computational approach. We reported the proportion of data-consistent simulations in which the prevalence of pre-extensively drug resistant (pre-XDR) TB - defined as resistance to both first-line and second-line core agents (rifampin and fluoroquinolones) - exceeded pre-defined acceptability thresholds (1-2 cases per 100,000 population by 2035). Using pyrazinamide (the most effective companion agent) in both first-line and second-line regimens increased the proportion of simulations exceeding the pre-XDR acceptability threshold seven-fold, compared to a scenario in which patients with pyrazinamide-resistant TB received an alternative drug. Model parameters related to emergence and transmission of pyrazinamide-resistant TB and resistance amplification were among those most strongly correlated with projected pre-XDR prevalence, indicating that pyrazinamide resistance acquired during first-line treatment subsequently promotes amplification to pre-XDR TB under pyrazinamide-containing second-line treatment. These findings suggest that appropriate use of companion drugs may be critical to preventing the emergence of strains resistant to multiple core agents.

Concepts: Pharmacology, Infectious disease, Mass drug administration, Antibiotic resistance, Tuberculosis, Tuberculosis treatment, Extensively drug-resistant tuberculosis, Pyrazinamide

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Rationale: A major priority in tuberculosis (TB) is to reduce effective treatment times and emergence of resistance. Recent studies in macrophages and zebrafish show that inhibition of mycobacterial efflux pumps with verapamil reduces the bacterial drug tolerance and may enhance drug efficacy. Objectives: Using mice, a mammalian model known to predict human treatment responses, and selecting conservative human bioequivalent doses, we tested verapamil as an adjunctive drug together with standard TB chemotherapy. As verapamil is a substrate for CYP3A4 which is induced by rifampin, we evaluated the PK/PD relationships of verapamil and rifampin co-administration in mice. Methods: Using doses that achieve human bioequivalent levels matched to those of standard verapamil but lower than those of extended release verapamil, we evaluated the activity of verapamil added to standard chemotherapy in both C3HeB/FeJ (which produce necrotic granulomas) and the wild type background C3H/HeJ mouse strains. Relapse rates were assessed after 16, 20 and 24 weeks of treatment in mice. Measurements and Main Results: We determined that a dose-adjustment of verapamil by 1.5 fold is required to compensate for concurrent use of rifampin during TB treatment. We found that standard TB chemotherapy plus verapamil accelerates bacterial clearance in C3HeB/FeJ mice with near sterilization and significantly lower relapse rates in just 4 months of treatment when compared with mice receiving standard therapy alone. Conclusions: These data demonstrate treatment shortening by verapamil adjunctive therapy in mice and strongly support further study of verapamil and other efflux pump inhibitors in human tuberculosis.

Concepts: Antibiotic resistance, Tuberculosis, Enzyme inhibitor, Efficacy, Inhibitor, Antibiotic, Leprosy, Pyrazinamide