Fast Identification and Quantification of Uropathogenic E. coli through Cluster Analysis.

Rapid diagnostic tools to detect, identify, and enumerate bacteria are key to maintaining effective antibiotic stewardship and avoiding the unnecessary prescription of broad-spectrum agents. In this study, a 15 min agglutination assay is developed that relies on the use of mannose-functionalized polymeric microspheres in combination with cluster analysis. This allows for the identification and enumeration of laboratory (BW25113), clinical isolate (NCTC 12241), and uropathogenic Escherichia coli strains (NCTC 9001, NCTC 13958, J96, and CFT073) at clinically relevant concentrations in tryptic soy broth (103-108 CFU/mL) and in urine (105-108 CFU/mL). This fast, simple, and efficient assay offers a step forward toward efficient point-of-care diagnostics for common urinary tract infections.

Exploring the Pharmacokinetics of Phenoxymethylpenicillin (Penicillin-V) in Adults: A Healthy Volunteer Study.

This healthy volunteer study aimed to explore phenoxymethylpenicillin (penicillin-V) pharmacokinetics (PK) to support the planning of large dosing studies in adults. Volunteers were dosed with penicillin-V at steady state. Total and unbound penicillin-V serum concentrations were determined, and a base population PK model was fitted to the data.

Challenges in the bioanalysis of tetracyclines: Epimerisation and chelation with metals.

Tetracyclines (TCs) are important broad spectrum antibiotics which are active against gram-positive and gram-negative bacteria. TCs readily form epimers, especially under weakly acidic conditions. The epimers are reported to have different antibacterial and toxicological properties and pose a significant challenge for selective bioanalysis, being isobaric with the parent drug and possessing very similar physicochemical properties. During the development, validation and use of bioanalytical methods for minocycline in plasma, urine and renal dialysate there were two unexpected findings. The first was that the analyte and the internal standard, tetracycline, were found to be unexpectedly stable and resistant towards epimerisation in the presence of the deproteinising agent, trichloroacetic acid (TCA). The second was that keeping minocycline spiked dialysate in a freezer led to significant losses which were worse for low concentrations at lower storage temperatures. Investigations into the stability of tetracycline, minocycline, omadacycline and tigecycline in aqueous acidic solutions, under typical analytical conditions, revealed that TCA acts as a stabiliser with respect to both epimerisation and other degradation pathways for these TCs. This gives the rarely used TCA a significant advantage over the commonly used deproteinising agents such as acetonitrile when analysing TCs. Studies of the recoveries of tetracycline and tigecycline from frozen renal dialysis buffer demonstrated similar losses to those for minocycline. These were assigned to deposition of insoluble Mg2+ or Ca2+ complexes on freezing, as pre-storage treatment of the samples by incubation in EDTA coated tubes at room temperature prevented the losses. Minocycline was stable in renal dialysis buffer samples when frozen, for up to ca. 3 months, when this treatment was employed. The TCs were analysed using LC-MS/MS based methods developed in-house using the assay that was originally developed for minocycline in plasma, urine and dialysate as a template.

Development, validation and application of a novel HPLC-MS/MS method for the measurement of minocycline in human plasma and urine.

New treatments are urgently required to treat infections caused by multi-drug resistant Acinetobacter baumanni,. To address this need, a new formulation of Minocin®, (minocycline for injection) has been developed that allows for higher doses of minocycline to be administered. Phase 1 clinical trials were conducted in healthy volunteers to assess the safety and pharmacokinetics (PK) of this new formulation at higher doses. In order to generate PK data, novel, selective and simple HPLC-MS/MS based assays were developed and validated for the determination of minocycline (MC) in human plasma and urine. The respective working ranges were 0.05 to 30 mg/L and 0.1 to 30 mg/L. Removal of endogenous proteins with trichloroacetic acid was used as a simple means of extracting MC from the samples. An analogue, tetracycline was used as the internal standard (IS). Chromatographic separation, including that of MC from its 4-epimer (4-EMC), was achieved on a Waters XBridge BEH C18 column (50 x 4.6 mm ID, 5 µm) with gradient elution. The mobile phases comprised water containing 5 mM ammonium formate at a pH of 2.5, and methanol containing 5 mM ammonium formate. The internal standard (IS) was tetracycline, a structural analogue of minocycline. The methods were fully validated and met regulatory acceptance criteria for intra-run and inter-run accuracy and precision, carryover, dilution integrity and matrix effects. Mean extraction recoveries ranged between 64.3% and 84.6% for MC and 64.3% for the IS. There was no significant ion suppression or enhancement for MC or the IS. The validated assays were successfully applied to 1423 plasma and 689 urine samples from a Phase 1 clinical study. There was no evidence of instability, or significant interconversion between MC and 4-EMC, in stored clinical samples, spiked plasma and urine samples, or their extracts, under various test conditions.

Microneedle biosensors for real-time, minimally invasive drug monitoring of phenoxymethylpenicillin: a first-in-human evaluation in healthy volunteers.

BACKGROUND: Enhanced methods of drug monitoring are required to support the individualisation of antibiotic dosing. We report the first-in-human evaluation of real-time phenoxymethylpenicillin monitoring using a minimally invasive microneedle-based ß-lactam biosensor in healthy volunteers. METHODS: This first-in-human, proof-of-concept study was done at the National Institute of Health Research/Wellcome Trust Imperial Clinical Research Facility (Imperial College London, London, UK). The study was approved by London-Harrow Regional Ethics Committee. Volunteers were identified through emails sent to a healthy volunteer database from the Imperial College Clinical Research Facility. Volunteers, who had to be older than 18 years, were excluded if they had evidence of active infection, allergies to penicillin, were at high risk of skin infection, or presented with anaemia during screening. Participants wore a solid microneedle ß-lactam biosensor for up to 6 h while being dosed at steady state with oral phenoxymethylpenicillin (five 500 mg doses every 6 h). On arrival at the study centre, two microneedle sensors were applied to the participant's forearm. Blood samples (via cannula, at -30, 0, 10, 20, 30, 45, 60, 90, 120, 150, 180, 210, 240 min) and extracellular fluid (ECF; via microdialysis, every 15 min) pharmacokinetic (PK) samples were taken during one dosing interval. Phenoxymethylpenicillin concentration data obtained from the microneedles were calibrated using locally estimated scatter plot smoothing and compared with free-blood and microdialysis (gold standard) data. Phenoxymethylpenicillin PK for each method was evaluated using non-compartmental analysis. Area under the concentration-time curve (AUC), maximum concentration, and time to maximum concentration were compared. Bias and limits of agreement were investigated with Bland-Altman plots. Microneedle biosensor limits of detection were estimated. The study was registered with ClinicalTrials.gov, number NCT03847610. FINDINGS: Ten healthy volunteers participated in the study. Mean age was 42 years (SD 14). Seven (70%) were men. Microdialysis and microneedle results were similar for phenoxymethylpenicillin ECF maximum concentration (0·74 mg/L vs 0·64 mg/L; 95% CI -0·24 to 0·44; p=0·53), time to maximum concentration (1·18 h vs 1·10 h; -0·52 to 0·67; p=0·79), and AUC (1·54 mg × h/L vs 1·67 mg × h/L; -1·10 to 0·85; p=0·79). In total, 440 time points were compared with mean difference between measurements -0·16 mg/L (95% CI -1·30 to 0·82). Mean phenoxymethylpenicillin AUCs for free serum and microneedle PK were similar (1·77 mg × h/L [SD 0·59] vs 1·67 mg × h/L [1·00]; -0·77 to 0·97; p=0·81). Median coefficient of variation between sensors within individuals was 7% (IQR 4-17). Limit of detection for the microneedles was estimated at 0·17 mg/L. INTERPRETATION: This study is proof-of-concept of real-time, microneedle sensing of penicillin in vivo. Future work will explore microneedle use in patient populations, their role in data generation to inform dosing recommendations, and their incorporation into closed-loop control systems for automated drug delivery. FUNDING: National Institute for Health Research Imperial Biomedical Research Centre, Mérieux Foundation.