Oral Fosfomycin Efficacy with Variable Urinary Exposures following Single and Multiple Doses against Enterobacterales: the Importance of Heteroresistance for Growth Outcome.

Oral fosfomycin trometamol is licensed as a single oral dose for the treatment of uncomplicated urinary tract infections, with activity against multidrug-resistant uropathogens. The impact of interindividual variability in urinary concentrations on antimicrobial efficacy, and any benefit of giving multiple doses, is uncertain. We therefore performed pharmacodynamic profiling of oral fosfomycin, using a dynamic bladder infection in vitro model, to assess high and low urinary exposures following a single oral dose and three repeat doses given every 72 h, 48 h, and 24 h against 16 clinical isolates with various MICs of fosfomycin (8 Escherichia coli, 4 Enterobacter cloacae, and 4 Klebsiella pneumoniae isolates). Baseline fosfomycin high-level-resistant (HLR) subpopulations were detected prior to drug exposure in half of the isolates (2 E. coli, 2 E. cloacae, and 4 K. pneumoniae isolates; proportion, 1 × 10-5 to 5 × 10-4% of the total population). Fosfomycin exposures were accurately reproduced compared to mathematical modeling (linear regression slope, 1.1; R2, 0.99), with a bias of 3.8% ± 5.7%. All 5/5 isolates with MICs of ≤1 µg/ml had no HLR and were killed, whereas 8/11 isolates with higher MICs regrew regardless of exposure to high or low urinary concentrations. A disk diffusion zone of <24 mm was a better predictor for baseline HLR and regrowth. Administering 3 doses with average exposures provided very limited additional kill. These results suggest that baseline heteroresistance is important for treatment response, while increased drug exposure and administering multiple doses may not be better than standard single-dose fosfomycin therapy.

Oral Fosfomycin Treatment for Enterococcal Urinary Tract Infections in a Dynamic In Vitro Model.

There are limited treatment options for enterococcal urinary tract infections, especially vancomycin-resistant Enterococcus (VRE). Oral fosfomycin is a potential option, although limited data are available guiding dosing and susceptibility. We undertook pharmacodynamic profiling of fosfomycin against E. faecalis and E. faecium isolates using a dynamic in vitro bladder infection model. Eighty-four isolates underwent fosfomycin agar dilution susceptibility testing (E. faecalis MIC50/90 32/64 µg/ml; E. faecium MIC50/90 64/128 µg/ml). Sixteen isolates (including E. faecalis ATCC 29212 and E. faecium ATCC 35667) were chosen to reflect the MIC range and tested in the bladder infection model with synthetic human urine (SHU). Under drug-free conditions, E. faecium demonstrated greater growth restriction in SHU compared to E. faecalis (E. faecium maximal growth 5.8 ± 0.6 log10 CFU/ml; E. faecalis 8.0 ± 1.0 log10 CFU/ml). Isolates were exposed to high and low fosfomycin urinary concentrations after a single dose, and after two doses given over two days with low urinary concentration exposure. Simulated concentrations closely matched the target (bias 2.3%). E. faecalis isolates required greater fosfomycin exposure for 3 log10 kill from the starting inoculum compared with E. faecium The ƒAUC0-72/MIC and ƒ%T > MIC0-72 for E. faecalis were 672 and 70%, compared to 216 and 51% for E. faecium, respectively. There was no rise in fosfomycin MIC postexposure. Two doses of fosfomycin with low urinary concentrations resulted in equivalent growth inhibition to a single dose with high urinary concentrations. With this urinary exposure, fosfomycin was effective in promoting suppression of regrowth (>3 log10 kill) in the majority of isolates.

Efficacy of single and multiple oral doses of fosfomycin against Pseudomonas aeruginosa urinary tract infections in a dynamic in vitro bladder infection model.

OBJECTIVES: We used a dynamic bladder infection in vitro model with synthetic human urine (SHU) to examine fosfomycin exposures to effectively kill, or prevent emergence of resistance, among Pseudomonas aeruginosa isolates. METHODS: Dynamic urinary fosfomycin concentrations after 3 g oral fosfomycin were simulated, comparing single and multiple (daily for 7 days) doses. Pharmacodynamic response of 16 P. aeruginosa (MIC range 1 to >1024 mg/L) were examined. Baseline disc diffusion susceptibility, broth microdilution MIC and detection of heteroresistance were assessed. Pathogen kill and emergence of resistance over 72 h following a single dose, and over 216 h following daily dosing for 7 days, were investigated. The fAUC0-24/MIC associated with stasis and 1, 2 and 3 log10 kill were determined. RESULTS: Pre-exposure high-level resistant (HLR) subpopulations were detected in 11/16 isolates after drug-free incubation in the bladder infection model. Five of 16 isolates had >2 log10 kill after single dose, reducing to 2/16 after seven doses. Post-exposure HLR amplification occurred in 8/16 isolates following a single dose and in 11/16 isolates after seven doses. Baseline MIC ≥8 mg/L with an HLR subpopulation predicted post-exposure emergence of resistance following the multiple doses. A PK/PD target of fAUC0-24/MIC >5000 was associated with 3 log10 kill at 72 h and 7 day-stasis. CONCLUSIONS: Simulated treatment of P. aeruginosa urinary tract infections with oral fosfomycin was ineffective, despite exposure to high urinary concentrations and repeated daily doses for 7 days. Emergence of resistance was observed in the majority of isolates and worsened following prolonged therapy. Detection of a baseline resistant subpopulation predicted treatment failure.

Impact of bacterial species and baseline resistance on fosfomycin efficacy in urinary tract infections.

OBJECTIVES: To assess the antibacterial effects of a single 3 g oral fosfomycin dose on Escherichia coli and Klebsiella pneumoniae clinical isolates within a dynamic bladder infection model. METHODS: An in vitro model simulating dynamic urinary fosfomycin concentrations was used. Target fosfomycin exposure (Cmax = 1984 mg/L and Tmax = 7.5 h) was validated by LC-MS/MS. Pharmacodynamic responses of 24 E. coli and 20 K. pneumoniae clinical isolates were examined (fosfomycin MIC ≤0.25-128 mg/L). Mutant prevention concentration (MPC), fosfomycin heteroresistance, fosfomycin resistance genes and fosA expression were examined. Pathogen kill and emergence of high-level resistance (HLR; MIC >1024 mg/L) were quantified. RESULTS: Following fosfomycin exposure, 20 of 24 E. coli exhibited reductions in bacterial counts below the lower limit of quantification without regrowth, despite baseline fosfomycin MICs up to 128 mg/L. Four E. coli regrew (MIC = 4-32 mg/L) with HLR population replacement. At baseline, these isolates had detectable HLR subpopulations and MPC >1024 mg/L. All E. coli isolates were fosA negative. In contrast, 17 of 20 K. pneumoniae regrew post exposure, 6 with emergence of HLR (proportion = 0.01%-100%). The three isolates without regrowth did not have a detectable HLR subpopulation after dynamic drug-free incubation. All K. pneumoniae had MPC >1024 mg/L and were fosA positive. WGS analysis and fosA expression failed to predict fosfomycin efficacy. CONCLUSIONS: E. coli and K. pneumoniae isolates demonstrate discrepant responses to a single fosfomycin dose in a dynamic bladder infection in vitro model. Treatment failure against E. coli was related to an HLR subpopulation, not identified by standard MIC testing. Activity against K. pneumoniae appeared limited, regardless of MIC testing, due to universal baseline heteroresistance.

Optimizing dosing of nitrofurantoin from a PK/PD point of view: What do we need to know?

Nitrofurantoin is an old antibiotic and an important first-line oral antibiotic for the treatment of uncomplicated urinary tract infections. However despite its long term use for over 60 years, little information is available with respect to its dose justification and this may be the reason of highly variable recommended doses and dosing schedules. Furthermore, nitrofurantoin is not a uniform product -crystal sizes of nitrofurantoin, and therefore pharmacokinetic properties, differ significantly by product. Moreover, pharmacokinetic profiling of some products is even lacking, or difficult to interpret because of its unstable chemical properties. Pharmacokinetic and pharmacodynamic data is now slowly becoming available. This review provides an overview of nitrofurantoins antibacterial, pharmacokinetic and pharmacodynamic properties. This shows that a clear rationale of current dosing regimens is scanty.

The pharmacokinetics of nitrofurantoin in healthy female volunteers: a randomized crossover study.

BACKGROUND: Use of nitrofurantoin has increased significantly since its recent repositioning as a first-line agent for uncomplicated cystitis by multiple guidelines. However, current dosing regimens were developed in an era before robust pharmacokinetic testing and may not be optimal. Furthermore, formulations have been modified over the years. OBJECTIVES: To reassess the plasma and urinary pharmacokinetic profile of macrocrystalline nitrofurantoin in two commonly used dosing regimens. METHODS: In this open-label, randomized crossover pharmacokinetic trial, 12 healthy adult female volunteers were randomized to receive oral nitrofurantoin 100 mg q8h on days 1 and 2 and, after a washout period, 50 mg q6h on days 30 and 31, or the same dosing schemes in reversed order. Urine and blood were collected at steady state and analysed by UPLC. Pharmacokinetic analysis was performed by WinNonlin. RESULTS: Plasma peak concentrations were low (mean 0.33 mg/L, SD 0.08, and 0.69 mg/L, SD 0.35, after 50 and 100 mg, respectively) and dose dependent. The AUC0-24 was higher (6.49 versus 4.43 mg·h/L, P = 0.021) for the 100 mg q8h dosing regimen, but the dose-normalized AUC was similar for the two regimens. In contrast, urinary concentrations were dose independent: increasing the nitrofurantoin dose delayed the time to peak urinary concentration, while steady-state AUC0-24 values remained unchanged (943.49 and 855.95 mg·h/L at 50 mg q6h and 100 mg q8h, respectively). CONCLUSIONS: Plasma concentrations were relatively low and dose dependent. The dose-independent urinary concentrations suggest that excretion of nitrofurantoin into the urine is saturable. Pharmacodynamic studies are urgently required to determine the impact of these findings.

Review of the pharmacokinetic properties of nitrofurantoin and nitroxoline.

Nitrofurantoin and nitroxoline are oral antibiotics for the treatment or prophylaxis of acute urinary tract infections. New interest in both these drugs is increasing because of the emergence of resistance to other antibiotics, but knowledge of their pharmacokinetics (PK) is lacking since they were developed before the advent of standardized research for drug approval. The aims of this review were to (i) summarize the PK data reported in the literature and (ii) to identify PK knowledge gaps. The current body of PK knowledge of both drugs appears to be poor and mainly based on old studies. Nitrofurantoin PK values were obtained from studies using many variables, e.g. formulations, crystal sizes and analytical methods, resulting in high interindividual variability in PK parameters and no uniform PK profile. Clinical experience and PK data for nitroxoline are even more limited since the drug is registered in only Germany and a few (Eastern European) countries. Clinical studies in relevant patient populations are needed with commercially available nitrofurantoin and nitroxoline formulations at approved dosing regimens to more fully characterize their PK profiles, and to investigate the influence of patient characteristics on these profiles in order to optimize efficacy and avoid toxicity and emergence of resistance. Only with this updated knowledge and efficacy data from well-structured trials can both drugs maintain their antimicrobial activity against uropathogens.

Fosfomycin efficacy and emergence of resistance among Enterobacteriaceae in an in vitro dynamic bladder infection model.

Background: Urinary tract infections (UTIs) are among the most common bacterial infections and a frequent indication for antibiotic use. Fosfomycin, an important oral antibiotic for outpatient UTIs, remains a viable option for MDR uropathogens. We aimed to perform pharmacodynamic profiling simulating urinary concentrations to assess the adequacy of the current dosing regimen. Methods: A dynamic in vitro bladder infection model was developed, replicating urinary fosfomycin concentrations after gastrointestinal absorption, systemic distribution and urinary elimination. Concentrations were measured by LC-MS/MS. Twenty-four Enterobacteriaceae strains (Escherichia coli, Klebsiella pneumoniae and Enterobacter cloacae; MIC range 0.25-64 mg/L) were examined. Pathogen kill and emergence of resistance was assessed over 72 h. Results: Observed in vitro fosfomycin concentrations accurately simulated urinary fosfomycin exposures (Tmax 3.8 ±âŸ0.5 h; Cmax 2630.1 ±âŸ245.7 mg/L; AUC0-24 33 932.5 ±âŸ1964.2 mg·h/L). Fifteen of 24 isolates regrew, with significant rises in fosfomycin MIC (total population MIC50 4 to 64 mg/L, MIC90 64 to > 1024 mg/L, P = 0.0039; resistant subpopulation MIC50 128 to > 1024 mg/L, MIC90 >1024 mg/L, P = 0.0020). E. coli and E. cloacae isolates were killed with pharmacokinetic/pharmacodynamic EI50 of fAUC0-24/MIC = 1922, fCmax/MIC = 149 and fTime>4×MIC = 44 h. In contrast, K. pneumoniae isolates were not reliably killed. Conclusions: Using dynamic in vitro simulations of urinary fosfomycin exposures, E. coli and E. cloacae isolates with MIC >16 mg/L, and all K. pneumoniae isolates, were not reliably killed. Emergence of resistance was significant. This challenges fosfomycin dosing and clinical breakpoints, and questions the utility of fosfomycin against K. pneumoniae. Further work on in vitro dose optimization is required.

Dried Blood Spots Combined With Ultra-High-Performance Liquid Chromatography-Mass Spectrometry for the Quantification of the Antipsychotics Risperidone, Aripiprazole, Pipamperone, and Their Major Metabolites.

BACKGROUND: Risperidone, aripiprazole, and pipamperone are antipsychotic drugs frequently prescribed for the treatment of comorbid behavioral problems in children with autism spectrum disorders. Therapeutic drug monitoring (TDM) could be useful to decrease side effects and to improve patient outcome. Dried blood spot (DBS) sample collection seems to be an attractive technique to develop TDM of these drugs in a pediatric population. The aim of this work was to develop and validate a DBS assay suitable for TDM and home sampling. METHODS: Risperidone, 9-OH risperidone, aripiprazole, dehydroaripiprazole, and pipamperone were extracted from DBS and analyzed by ultra-high-performance liquid chromatography-tandem mass spectrometry using a C18 reversed-phase column with a mobile phase consisting of ammonium acetate/formic acid in water or methanol. The suitability of DBS for TDM was assessed by studying the influence of specific parameters: extraction solution, EDTA carryover, hematocrit, punching location, spot volume, and hemolysis. The assay was validated with respect to conventional guidelines for bioanalytical methods. RESULTS: The method was linear, specific without any critical matrix effect, and with a mean recovery around 90%. Accuracy and imprecision were within the acceptance criteria in samples with hematocrit values from 30% to 45%. EDTA or hemolysis did not skew the results, and no punching carryover was observed. No significant influence of the spot volume or the punch location was observed. The antipsychotics were all stable in DBS stored 10 days at room temperature and 1 month at 4 or -80°C. The method was successfully applied to quantify the 3 antipsychotics and their metabolites in patient samples. CONCLUSIONS: A UHPLC-MS/MS method has been successfully validated for the simultaneous quantification of risperidone, 9-OH risperidone, aripiprazole, dehydroaripiprazole, and pipamperone in DBS. The assay provided good analytical performances for TDM and clinical research applications.

Identification and quantification of the antipsychotics risperidone, aripiprazole, pipamperone and their major metabolites in plasma using ultra-high performance liquid chromatography-mass spectrometry.

The antipsychotics risperidone, aripiprazole and pipamperone are frequently prescribed for the treatment in children with autism. The aim of this study was to validate an ultra-high performance liquid chromatography-mass spectrometry method for the quantification of these antipsychotics in plasma. An ultra-high performance liquid chromatography-mass spectrometry assay was developed for the determination of the drugs and metabolites. Gradient elution was performed on a reversed-phase column with a mobile phase consisting of ammonium acetate, formic acid in methanol or in Milli-Q ultrapure water at a flow rate of 0.5 mL/min. The method was validated according to the US Food and Drug Administration guidelines. The analytes were found to be stable enough after reconstitution and injection of only 5 µL improved the accuracy and precision in combination with the internal standard. Calibration curves of all five analytes were linear. All analytes were stable for at least 72 h in the autosampler and the high quality control of 9-OH-risperidone was stable for 48 h. The method allows quantification of all analytes. The advantage of this method is the combination of a minimal injection volume, a short run-time, an easy sample preparation method and the ability to quantify all analytes in one run. Copyright © 2015 John Wiley & Sons, Ltd.

Evaluation of pooled human urine and synthetic alternatives in a dynamic bladder infection in vitro model simulating oral fosfomycin therapy.

The impact of the bladder environment on fosfomycin activity and treatment response is uncertain. Standard laboratory media does not reflect the biomatrix of urine, where limited nutritional factors are important for growth and antimicrobial kill rates. We compared fosfomycin activity against Enterobacteriaceae in laboratory media, human urine and synthetic alternatives. Sixteen clinical isolates (8-Escherichia coli, 4-Enterobacter cloacae, 4-Klebsiella pneumoniae) were studied with broth microdilution (BMD) susceptibility, static time-kill assays and dynamic testing in a bladder infection model simulating a 3 g oral fosfomycin dose. Mueller-Hinton broth (MHB) with and without 25 mg/L glucose-6-phosphate (G6P), pooled midstream urine (MSU), pooled 24 h urine collection (24 U), artificial urine medium (AUM) and synthetic human urine (SHU) were compared. BMD susceptibility, bacterial growth and response to static fosfomycin concentrations in urine were best matched with SHU and were distinctly different when tested in MHB with G6P. Fosfomycin exposure in the bladder infection model was accurately reproduced (bias 4.7 ± 6.2%). Under all media conditions, 8 isolates (2-E. coli, 2-E. cloacae, 4-K. pneumoniae) re-grew and 4 isolates (4-E. coli) were killed. The remaining isolates (2-E. coli, 2-E. cloacae) re-grew variably in urine and synthetic media. Agar dilution MIC failed to predict re-growth, whereas BMD MIC in media without G6P performed better. Emergence of resistance was restricted in synthetic media. Overall, SHU provided the best substitute for urine for in vitro modelling of antimicrobial treatment of uropathogens, and these data have broader utility for improved preclinical testing of antimicrobials for urinary tract infections.

An audit of nitrofurantoin use in three Australian hospitals.

BACKGROUND: International guidelines have recommended the long-acting formulation of nitrofurantoin as first-line treatment for uncomplicated urinary tract infections (UTIs) since 2010. Australian guidelines have only recently listed nitrofurantoin as a first-line agent, but the long-acting formulation is not available. In the setting of increasing multidrug-resistance, the unavailability of the long-acting formulation of nitrofurantoin in Australia, and anecdotal perception of confusion regarding dosing, we audited nitrofurantoin use. METHODS: We performed a retrospective audit of nitrofurantoin use at Alfred Health. All patients dispensed nitrofurantoin from January 2016 to June 2018, as identified from pharmacy dispensing records, were eligible. We used a standardised case report form to extract data from medical records, including dosing regimen and indication. RESULTS: We included 150 patients with 151 nitrofurantoin prescriptions in the analysis, of whom 74% [111/150] were female. Nitrofurantoin was most commonly dispensed for the treatment of UTIs (68% [103/151] versus 32% [48/151] for UTI prophylaxis). For the treatment of uncomplicated UTIs, the most frequently used dose was 100 mg twice daily for five days. In male patients, the 100 mg twice daily for seven days was the most popular regimen. The prophylactic dose of 50 mg once daily was used in women but rarely in men. We did not find evidence of dose adjustment for renal impairment. CONCLUSION: While treatment duration was consistent with guidelines, the dosage and frequency used was often incorrect for the formulation and was not adjusted for renal function. Nitrofurantoin use is likely to increase, so clarification regarding optimal nitrofurantoin dosing regimens may be appropriate.

Successful High-Dosage Monotherapy of Tigecycline in a Multidrug-Resistant Klebsiella pneumoniae Pneumonia-Septicemia Model in Rats.

Background: Recent scientific reports on the use of high dose tigecycline monotherapy as a "drug of last resort" warrant further research into the use of this regimen for the treatment of severe multidrug-resistant, Gram-negative bacterial infections. In the current study, the therapeutic efficacy of tigecycline monotherapy was investigated and compared to meropenem monotherapy in a newly developed rat model of fatal lobar pneumonia-septicemia. Methods: A Klebsiella pneumoniae producing extended-spectrum ß-lactamase (ESBL) and an isogenic variant producing K. pneumoniae carbapenemase (KPC) were used in the study. Both strains were tested for their in vitro antibiotic susceptibility and used to induce pneumonia-septicemia in rats, which was characterized using disease progression parameters. Therapy with tigecycline or meropenem was initiated at the moment that rats suffered from progressive infection and was administered 12-hourly over 10 days. The pharmacokinetics of meropenem were determined in infected rats. Results: In rats with ESBL pneumonia-septicemia, the minimum dosage of meropenem achieving survival of all rats was 25 mg/kg/day. However, in rats with KPC pneumonia-septicemia, this meropenem dosage was unsuccessful. In contrast, all rats with KPC pneumonia-septicemia were successfully cured by administration of high-dose tigecycline monotherapy of 25 mg/kg/day (i.e., the minimum tigecycline dosage achieving 100% survival of rats with ESBL pneumonia-septicemia in a previous study). Conclusions: The current study supports recent literature recommending high-dose tigecycline as a last resort regimen for the treatment of severe multidrug-resistant bacterial infections. The use of ESBL- and KPC-producing K. pneumoniae strains in the current rat model of pneumonia-septicemia enables further investigation, helping provide supporting data for follow-up clinical trials in patients suffering from severe multidrug-resistant bacterial respiratory infections.

Development and validation of a fast and sensitive UHPLC-DAD assay for the quantification of nitrofurantoin in plasma and urine.

Nitrofurantoin is an antimicrobial drug that has been used in the treatment of lower urinary tract infections for more than 50 years. Despite its long use, surprisingly little is known of the pharmacokinetics of nitrofurantoin, whereas this is essential to optimize patient treatment. We developed a novel analytical method for the quantification of nitrofurantoin in plasma and urine using ultra-high performance liquid chromatography and diode array detection to allow pharmacokinetic studies in these two matrices. The sample preparation method consisted of protein precipitation for plasma and liquid-liquid extraction for urine. 100 µL was needed for the sample preparation. Furazolidone was used as internal standard. Gradient chromatographic separation was performed on a HSS-T3 column. UV detection was performed at a wavelength of 369 nm. The analysis time was 5 min. The method was successfully validated according to the FDA-guidelines (2018). Linearity was confirmed over a concentration range from 50 to 1250 µg/L in plasma and from 4 to 200 mg/L in urine (r2 > 0.95). Validation results of five QC concentrations for plasma and urine, respectively, are for within-day accuracy <± 13% in both matrices, for between-day accuracy <± 7% and <± 9%, for within-day precision <10% and <4% and for between-day precision <10% and <5%. Plasma samples are stable for seven days at 4 °C, and for 2 years at -20 °C and-80 °C. Urine samples are stable for at least seven days at 4 °C and at room temperature and for 2 years at -20 °C andat -80 °C, except from the lower concentrated samples, which are only stable at -80 °C. All samples were kept from daylight using amber colored glassware. The presented method meets all validation requirements and was successfully used in a clinical study where the pharmacokinetics of nitrofurantoin were investigated in healthy volunteers. The easy sample preparation method and the short analysis time make this method suitable for use during routine clinical practice to study the pharmacokinetics of nitrofurantoin.

Urinary antibacterial activity of fosfomycin and nitrofurantoin at registered dosages in healthy volunteers.

Given emerging uropathogen resistance to more recent antibiotics, old antibiotics used for uncomplicated urinary tract infection (UTI) warrant re-examination. In this study, the urinary antibacterial activities of fosfomycin and nitrofurantoin were investigated by determining the urinary inhibitory titre and urinary bactericidal titre against uropathogens in urine samples from female volunteers following administration of single-dose fosfomycin (3 g) or nitrofurantoin (50 mg q6h or 100 mg q8h). Urine samples were collected over 48 h (fosfomycin) or 6 or 8 h (nitrofurantoin), with drug levels quantified with every void. Fosfomycin concentrations ranged from <0.75 mg/L [lower limit of quantification (LLOQ)] to 5729.9 mg/L and nitrofurantoin concentrations ranged from <4 mg/L (LLOQ) to 176.3 mg/L (50 mg q6h) or 209.4 mg/L (100 mg q8h). There was discrepancy in the response to fosfomycin between Escherichia coli and Klebsiella pneumoniae, with fosfomycin displaying strong bactericidal activity for 48 h against E. coli but moderate bactericidal activity for 18 h against K. pneumoniae. This effect was not related to the strain's baseline minimum inhibitory concentration but rather to the presence of a resistant subpopulation. Maximum titres of nitrofurantoin were obtained during the first 2 h, but no antibacterial effect was found in most samples regardless of the dose. In the rare samples in which antibacterial activity was detectable, titres were comparable for both species tested. These findings confirm doubts regarding fosfomycin administration in UTIs caused by K. pneumoniae and reveal a discrepancy between nitrofurantoin's measurable ex vivo activity and its clinical effect over multiple dosing intervals.

[A body packer with cannabis]. / Een bodypacker met cannabis.

BACKGROUND: In recent years, numerous reports have been published on body packers, i.e. people who use their own body to transport drugs. Most body packers swallow small packages of cocaine or heroin. CASE DESCRIPTION: We describe the case of a patient who, two days after ingestion of packages containing cannabis, was admitted for dizziness and drowsiness. Urinalysis was positive for cannabis and negative for other drugs. The deterioration in clinical status possibly indicated a burst package. The packages were surgically removed and the patient made a full recovery. CONCLUSION: Some body packers transport other drugs than cocaine or heroin, such as cannabis. In body packers who ingest cannabis, depressed consciousness is a main symptom of intoxication. They should be admitted and monitored for symptoms of intoxication or paralytic ileus. Laboratory tests provide qualitative support when interpreting clinical symptoms. Surgical intervention is indicated in cases of bowel obstruction or when intoxication due to package leakage is suspected.

A fast and sensitive LC-MS/MS method for the quantification of fosfomycin in human urine and plasma using one sample preparation method and HILIC chromatography.

Fosfomycin is an old antibiotic that is increasingly prescribed because of emergence of the antibiotic resistance and the growing incidence of multi-drug resistant infections. Surprisingly, little is known about its pharmacokinetics (PK) and the pharmacodynamics (PD). Quantification of fosfomycin in both urine and plasma provides insight into the PK/PD characteristics of fosfomycin, which is crucial for the optimization of the therapy and the prevention of the emergence of resistance. An analytical method is therefore needed for the quantification of fosfomycin in both urine and plasma. A fast and sensitive tandem mass spectrometry method in combination with HILIC chromatography for the quantification of fosfomycin with a universal sample preparation method for urine and plasma was developed and validated according to FDA guidelines. The universal sample preparation method only requires 100µL of a sample, the addition of the internal standard fosfomycin-13C3 benzylamine and an ultrafiltration step. The method is applicable for the concentration range of 0.75-375mg/L (R2 of 0.9998 in both matrices) encompassing the clinically relevant concentration range based on the susceptibility of possible (uro)pathogens in the clinical setting. The validation results for urine and plasma for all QC levels, were <2.1% and <3.2% for accuracy, <1.5% and <1.7% for within day precision and <5.0% and <3.8% for between day precision, respectively. No matrix effects were encountered and the total recovery in urine and plasma was high (102.5% and 99.4%). Prepared samples were stable at 4°C and 15°C for at least 72h and stored samples at -80°C were stable for at least 6 months. Selectivity and sensitivity were confirmed and no carry-over was observed. The method was successfully applied in two pharmacokinetic studies in healthy volunteers and patients respectively.

Simultaneous determination of nine ß-lactam antibiotics in human plasma by an ultrafast hydrophilic-interaction chromatography-tandem mass spectrometry.

Contemporary ß-lactam antibiotic dosing is debatable in severely ill patients, since the occurrence of pathophysiological changes in critical illness can result in great inter-individual variability. Therapeutic drug monitoring (TDM) is a commonly used dosing strategy to optimize exposure and thereby minimize toxicity and maximize the efficacy. Currently, TDM of ß-lactam antibiotics is rarely performed, due to poor availability in clinical practice. We describe an ultrafast Hydrophilic-Interaction Chromatography (HILIC) based UPLC-MS/MS method for the determination of amoxicillin, benzylpenicillin, cefotaxime, cefuroxime, ceftazidime, flucloxacillin, imipenem, meropenem and piperacillin in human plasma. This method involves simple sample preparation steps and was comprehensively validated according to standard FDA guidelines. For all analytes, mean accuracy and precision values were within the acceptance value. The lower and upper limits of quantification were found to be sufficient to cover the therapeutic range for all antibiotics. Finally, the method was successfully applied in a large pharmacokinetic study performed in the intensive care setting, and the feasibility of the analytical procedure was demonstrated in routine clinical practice. To the best of our knowledge, we report here the first HILIC-based UPLC-MS/MS assay for the determination of ß-lactam antibiotics in human plasma. This simple, sensitive and ultrafast assay requires small-volume samples and can easily be implemented in clinical laboratories to promote the TDM of ß-lactam antibiotics.

Multicentre open-label randomised controlled trial to compare colistin alone with colistin plus meropenem for the treatment of severe infections caused by carbapenem-resistant Gram-negative infections (AIDA): a study protocol.

INTRODUCTION: The emergence of antibiotic-resistant bacteria has driven renewed interest in older antibacterials, including colistin. Previous studies have shown that colistin is less effective and more toxic than modern antibiotics. In vitro synergy studies and clinical observational studies suggest a benefit of combining colistin with a carbapenem. A randomised controlled study is necessary for clarification. METHODS AND ANALYSIS: This is a multicentre, investigator-initiated, open-label, randomised controlled superiority 1:1 study comparing colistin monotherapy with colistin-meropenem combination therapy for infections caused by carbapenem-resistant Gram-negative bacteria. The study is being conducted in 6 centres in 3 countries (Italy, Greece and Israel). We include patients with hospital-associated and ventilator-associated pneumonia, bloodstream infections and urosepsis. The primary outcome is treatment success at day 14, defined as survival, haemodynamic stability, stable or improved respiratory status for patients with pneumonia, microbiological cure for patients with bacteraemia and stability or improvement of the Sequential Organ Failure Assessment (SOFA) score. Secondary outcomes include 14-day and 28-day mortality as well as other clinical end points and safety outcomes. A sample size of 360 patients was calculated on the basis of an absolute improvement in clinical success of 15% with combination therapy. Outcomes will be assessed by intention to treat. Serum colistin samples are obtained from all patients to obtain population pharmacokinetic models. Microbiological sampling includes weekly surveillance samples with analysis of resistance mechanisms and synergy. An observational trial is evaluating patients who met eligibility requirements but were not randomised in order to assess generalisability of findings. ETHICS AND DISSEMINATION: The study was approved by ethics committees at each centre and informed consent will be obtained for all patients. The trial is being performed under the auspices of an independent data and safety monitoring committee and is included in a broad dissemination strategy regarding revival of old antibiotics. TRIAL REGISTRATION NUMBER: NCT01732250 and 2012-004819-31; Pre-results.