Safety and pharmacokinetics-pharmacodynamics of a shorter tuberculosis treatment with high-dose pyrazinamide and rifampicin: a study protocol of a phase II clinical trial (HighShort-RP).

INTRODUCTION: Increased dosing of rifampicin and pyrazinamide seems a viable strategy to shorten treatment and prevent relapse of drug-susceptible tuberculosis (TB), but safety and efficacy remains to be confirmed. This clinical trial aims to explore safety and pharmacokinetics-pharmacodynamics of a high-dose pyrazinamide-rifampicin regimen. METHODS AND ANALYSIS: Adult patients with pulmonary TB admitted to six hospitals in Sweden and subjected to receive first-line treatment are included. Patients are randomised (1:3) to either 6-month standardised TB treatment or a 4-month regimen based on high-dose pyrazinamide (40 mg/kg) and rifampicin (35 mg/kg) along with standard doses of isoniazid and ethambutol. Plasma samples for measurement of drug exposure determined by liquid chromatography tandem-mass spectrometry are obtained at 0, 1, 2, 4, 6, 8, 12 and 24 hours, at day 1 and 14. Maximal drug concentration (Cmax) and area under the concentration-time curve (AUC0-24h) are estimated by non-compartmental analysis. Conditions for early model-informed precision dosing of high-dose pyrazinamide-rifampicin are pharmacometrically explored. Adverse drug effects are monitored throughout the study and graded according to Common Terminology Criteria for Adverse Events V.5.0. Early bactericidal activity is assessed by time to positivity in BACTEC MGIT 960 of induced sputum collected at day 0, 5, 8, 15 and week 8. Minimum inhibitory concentrations of first-line drugs are determined using broth microdilution. Disease severity is assessed with X-ray grading and a validated clinical scoring tool (TBscore II). Clinical outcome is registered according to WHO definitions (2020) in addition to occurrence of relapse after end of treatment. Primary endpoint is pyrazinamide AUC0-24h and main secondary endpoint is safety. ETHICS AND DISSEMINATION: The study is approved by the Swedish Ethical Review Authority and the Swedish Medical Products Agency. Informed written consent is collected before study enrolment. The study results will be submitted to a peer-reviewed journal. TRIAL REGISTRATION NUMBER: NCT04694586.

A novel mycobacterial growth inhibition assay employing live-cell imaging of virulent M. tuberculosis and monitoring of host cell viability.

Our aim was to develop a Mycobacterium tuberculosis (Mtb) growth inhibition assay (MGIA) as a summary estimate of host immune control of virulent Mtb. Mycobacterial growth inhibition (MGI) using previously frozen human PBMCs infected with H37Rv was assessed by live-cell imaging (Incucyte©) complemented by imaging flow cytometry analysis of phagocytosis. MGI measured as relative fluorescence units (RFU) was calibrated to time to positive culture (TTP) in BACTEC 960 MGIT. At a MOI (multiplicity of infection) of 5, there was a wide range of MGI of blood donors (1.1*106-2.7*106 RFU, n = 14). Intra- and inter-assay variability were at most 17.5 and 20.7 CV%. Cell viability at day 5 was 57 and 62% monitored by the LDH and Draq7 assays respectively. There was a strong correlation between a readout for Mtb growth using CFU counts or TTP compared to RFU (r2≥0.96). Our MGIA enabling live-cell imaging and monitoring of cell viability was able to detect a wide range of Mtb growth inhibition by PBMCs and was calibrated to several readout options for bacterial growth. This MGIA may be valuable as a surrogate marker of host immunity in a personalized medicine approach.