ObjectivesThe increasing prevalence of antimicrobial resistance (AMR) in Escherichia coli, notably those producing AmpC, ESBL and carbapenemases among food-producing animals, poses a significant public health threat, especially in low- and middle-income countries. With support from the Fleming Fund and in alignment with the WHO, FAO and WOAH tripartite Tricycle project, the EQASIA Matrix External Quality Assessment (EQA) scheme aimed at evaluating laboratories’ proficiency from different One Health (OH) sectors in detecting resistant E. coli from food-like matrices in South and South-East Asia.MethodsBetween 2021 and 2024, four Matrix EQA rounds were implemented across OH sectors. Each round involved four lyophilized simulated meat samples spiked with well-characterized E. coli strains expressing β-lactam resistance phenotypes. Participating laboratories performed selective isolation, species identification, antimicrobial susceptibility testing (AST) and phenotypic classification using routine methods. Results were submitted via a secure online platform and evaluated against expected outcomes, using CLSI-based interpretive criteria and a standardized error categorization system.ResultsTwenty-one laboratories from 10 countries participated across the four rounds. Accuracy in E. coli identification declined from 75% in 2021 to 23.1% in 2024. Only one laboratory achieved full identification accuracy in any round. AST performance showed inter-laboratory variation, with deviation rates ranging from 0.5% to 41%. Misclassification of resistance phenotypes, particularly AmpC and combined ESBL + AmpC, was common. Quality control testing improved modestly over time but remained incomplete.ConclusionsThe Matrix EQA revealed critical insights into regional laboratory capacity and highlighted persistent technical gaps in detecting complex resistance mechanisms from food matrices. With the conclusion of the Fleming Fund, maintaining these gains will require sustained national investment, EQA integration into AMR strategies and continued regional coordination to support Tricycle-aligned surveillance.

ObjectivesTo characterize the population pharmacokinetics of unbound cloxacillin in patients undergoing total arthroplasty of the hip (THA) or knee (TKA), and to explore alternative dosing regimens for cloxacillin prophylaxis.MethodsPlasma concentrations of total and unbound cloxacillin from 200 patients undergoing primary elective THA (n & 95) or TKA (n & 105) were analysed. Intravenous cloxacillin doses of 2 g were administered pre-surgery, and repeated after 2 and 6 hours. Samples (n & 496) were analysed using HPLC-MS/MS. Non-linear mixed-effects modelling was performed to develop a population pharmacokinetic model describing unbound cloxacillin exposure. Using this model, alternative prophylaxis regimens were explored with Monte Carlo simulations.ResultsA two-compartment model with non-linear protein binding adequately described the data. Estimated glomerular filtration rate (eGFR) and body weight (kg) were significant covariates on unbound cloxacillin clearance. In 13% of patients sampled at the end of surgery (n & 25/187), unbound cloxacillin <2 mg/L was observed. A model-predicted 18–22% (n & 36–43/200) of patients failed to sustain plasma levels ≥2 mg/L throughout the two-hour dosing interval with the current regimen. In contrast, a continuous 1 g/h infusion after a 1 g loading dose would ensure target attainment in >99% of patients, according to the model predictions.ConclusionsFor many THA and TKA patients, the current cloxacillin prophylaxis regimen may fail to provide adequate target site concentrations during the entire surgical procedure. Transitioning to a prolonged infusion protocol could increase attainment of PK/PD targets without exceeding the currently recommended total perioperative dose amounts.

ObjectivesTo evaluate the penetration of olorofim, a novel antifungal agent into the tissues of the rat.Materials and methodsQuantitative whole-body autoradiography was used to assess the distribution and tissue penetration in male and female rats following a single 2-hour IV infusion of [14C]-olorofim.ResultsRadioactivity was rapidly and extensively distributed throughout many tissues in both sexes with maximal concentrations observed soon after start of the infusion. Thereafter radioactivity declined and at 336 h (final timepoint), concentrations of radioactivity in most tissues were below the limit of quantification. The pattern of radioactive distribution was similar at all timepoints with concentrations greatest in liver, kidney cortex, adrenal cortex, abdominal and brown fat; the lowest concentrations were observed in the whole eye and at the bone surface (with levels similar to plasma in bone marrow and uveal tract/retina). Tissue:plasma radioactivity concentration ratios for most tissues were above unity during the period 2.25 to 72 h post-start of infusion. Radioactivity was detected in tissues frequently reported as sites of systemic fungal infections such as the lung, brain, kidney, bone and eye. Pharmacokinetic analyses showed that at early timepoints >75% of circulating radiolabelled material was intact olorofim confirming that olorofim is present in these key tissues at levels similar to or exceeding those seen in plasma.ConclusionsOlorofim distributes widely into rat tissues including those frequently infected by fungi, indicating its potential to treat systemic fungal infections caused by susceptible species, a finding in agreement with recent clinical reporting from a Phase 2 trial.

BackgroundOlorofim is an antifungal agent with a novel mechanism of action against Aspergillus spp., rare moulds and dimorphic fungi. Preclinical studies reported unidirectional antagonism between olorofim and mould-active azoles (e.g. azoles other than fluconazole) against Aspergillus fumigatus in which the azole reduces the antifungal effect of olorofim.MethodsTo assess the potential clinical impact of this preclinical finding, we compared outcomes of olorofim with and without concomitant azoles from a Phase 2b salvage study in patients with invasive fungal diseases with few or no treatment options.ResultsAnalyses are limited by the study size but outcomes were similar for olorofim with and without an azole in combination (whether mould-active or not) at Days 42 (the primary study endpoint) and 84 for both Mycoses Study Group–European Organization for Research and Treatment of Cancer (MSG-EORTC) responses and for all-cause mortality in patients with aspergillosis (77 not receiving an azole, 24 receiving an azole in combination), in patients with coccidioidomycosis (11 not receiving an azole, 30 receiving an azole in combination) and in patients with other fungal infections (50 not receiving an azole, 10 receiving an azole in combination). In addition, the rate of drug-induced liver injury was not increased in patients receiving combination with an azole.ConclusionsAlthough limited by small numbers of patients for individual fungi, this analysis did not demonstrate a pattern of reduced clinical efficacy or tolerability of olorofim when given in combination with either the mould-active azoles or with fluconazole.Trial registration number. NCT03583164.

Background and objectivesOritavancin is a lipoglycopeptide with sustained bactericidal activity against Gram-positive bacteria due to its prolonged half-life. This Systematic Review aimed to extrapolate, from in vitro/in vivo or clinically study, the most relevant PK/PD target to inform therapeutic drug monitoring-guided oritavancin dose optimization in clinical practice.Materials and methodsFollowing the PRISMA 2020 Statement and adopting the PICO strategy, a comprehensive search was conducted in PubMed, Scopus and Cochrane databases up to September 2025.ResultsOf 186 articles screened, 52 were considered eligible for full-text assessment. Nine studies were included and proceeded with data extraction and synthesis steps. In vitro studies showed a marked concentration-dependent bactericidal activity at fCmax > 4–16 mg/L against different bacterial strains, further confirmed by in vivo animal models (fCmax/MIC > 6 to 14). However, the only identified in-human daily repeated doses study supported the findings of an exposure–response relationship with %fT > MIC as predictive of microbiological and clinical success.ConclusionsThe peculiar pharmacokinetics profile of oritavancin results in a borderline collinearity between the two PK/PD indices fCmax/MIC and %fT > MIC in relation to microbiological and clinical success rates. On the basis of available in vitro/in vivo data supporting concentration-dependent killing activity, a single 1200 mg oritavancin dose should be adequate for most infections. In special patient populations, or when multidose oritavancin regimens are adopted for long-term antibiotic treatment, therapeutic drug monitoring supported by expert clinical pharmacological advice may be valuable to optimize the initial and next-dose strategy (1200 mg or 800 mg) and to define the timing of re-administration.

Given the medical importance and challenges related to kinetoplastid diseases, a strategic roadmap is needed for the identification of high-quality leads and drug development candidates. Within the aim to deliver more compelling proof-of-concept read-outs, this part proposes a systematic flow-chart of laboratory experiments and decision criteria, focusing on African trypanosomiasis, Chagas disease and visceral and cutaneous leishmaniasis. Next to precision experimental design and reporting, an overview is provided of various complementary laboratory models reproducing kinetoplastid infection and disease. Technical aspects of conventional in vitro and in vivo approaches and, more recently, in silico methods are presented with reference to specific preclinical R&D stages from ‘hit finding’ to ‘profiling of a confirmed hit’, covering the expertise areas of medicinal chemistry, primary pharmacology, (eco)toxicology, pharmacokinetics and pharmaceutics (Figure 1).

BackgroundIntegrase mutations associated with dolutegravir resistance have been well characterized, but based on limited data from non-B subtypes.ObjectivesWe aim to identify potential integrase mutations not currently classified as integrase strand transfer inhibitor (INSTI) resistance mutations (DRMs) in individuals with viremia on dolutegravir-based regimens.MethodsWe included integrase sequences from DTG RESIST study sites in African countries. These were interpreted using Stanford HIVdb v9.8. We used a viral genome-wide association study-like approach restricted to the integrase region (INT-WAS) to identify mutations not classified as major or accessory INSTI DRMs but occurring more frequently in sequences carrying major INSTI DRMs than in those without major INSTI DRMs. We performed the same INT-WAS analysis with drug-naïve sequences from the Los Alamos HIV-1 database to test whether these identified mutations were enriched among sequences from individuals with viraemia whilst receiving DTG-based treatment.ResultsAmong 382 sequences, 104 (27.2%) showed at least intermediate dolutegravir resistance. Twelve integrase mutations not classified as major or accessory DRMs (S39R, L45I, I72L, L74I, V79I, I113V, S119R, K156N, I208M, T218M, A265V, and R284G) were significantly associated with predicted DTG resistance. Among them, V79I [adjusted odds ratio (aOR) 167.1, 95% credible interval (CrI) 17.9–2947.6] and I72L (aOR 65.6, 95% CrI 6.6–1273.7) were strongly associated. S39R, L45I, V79I, S119R, and K156N were linked to established INSTI resistance pathways, and I72L, L74I, V79I, K156N, I208M, and R284G were overrepresented in sequences from viraemic individuals on DTG-based treatment relative to drug-naïve sequences.ConclusionsWe identified several amino acid substitutions outside the established DRMs that are strongly associated with predicted dolutegravir resistance. Dolutegravir resistance evolution is complex and likely involves mutations not currently classified as DRMs.

BackgroundThe female genital tract (FGT) is a unique compartment with physiologically distinct properties complicating the extrapolation of drug efficacy; critical gaps remain in understanding regional variability within the FGT itself. We performed an in-depth investigation across endo- and ectocervical tissues on the utility of the cervical explant model to evaluate pre-exposure prophylaxis (PrEP) efficacy.MethodsUsing normal cervical tissues, we evaluated gene expression of relevant drug metabolizing enzymes and transporters (DMETs) via qRT-PCR and compared ecto- and endocervix. To determine differences in drug phosphorylation and to assess antiretroviral (ARV) efficacy, we incubated explants in tenofovir and emtricitabine then measured intracellular metabolites. Viral infectivity and dose–response with ARVs was measured using viral RNA and p24 following HIV-1JR-CSF challenge.ResultsABCC4 expression was 3-fold lower in ectocervical tissues compared with endocervical, whereas CYP3A5 was 2-fold higher. IL-6 was correlated with ABCB1 (r = 0.52, P = 0.01) and ABCG2 (r = 0.56, P =0.005). Dose-normalized phosphorylation did not differ between endo- and ectocervix (P > 0.5). Infectivity of explants was low (53%) but did not differ by compartment. Intracellular tenofovir diphosphate concentrations were associated with a decrease in ectocervical viral replication (r =0.39, P < 0.05). There was a strong relationship between the proportion of explants infected and emtricitabine dose (P =0.02) but no relationship between intracellular emtricitabine triphosphate and protection.ConclusionsWe identified differences in DMET expression and ARV metabolism between ecto- and endocervical tissues, as well as correlations between DMET and IL-6. Ectocervical explants demonstrated consistent viral infectivity and dose-dependent inhibition. The model is useful in determining tenofovir diphosphate targets.