Recommendations for enterovirus diagnostics and characterisation within and beyond Europe.

Enteroviruses (EV) can cause severe neurological and respiratory infections, and occasionally lead to devastating outbreaks as previously demonstrated with EV-A71 and EV-D68 in Europe. However, these infections are still often underdiagnosed and EV typing data is not currently collected at European level. In order to improve EV diagnostics, collate data on severe EV infections and monitor the circulation of EV types, we have established European non-polio enterovirus network (ENPEN). First task of this cross-border network has been to ensure prompt and adequate diagnosis of these infections in Europe, and hence we present recommendations for non-polio EV detection and typing based on the consensus view of this multidisciplinary team including experts from over 20 European countries. We recommend that respiratory and stool samples in addition to cerebrospinal fluid (CSF) and blood samples are submitted for EV testing from patients with suspected neurological infections. This is vital since viruses like EV-D68 are rarely detectable in CSF or stool samples. Furthermore, reverse transcriptase PCR (RT-PCR) targeting the 5'noncoding regions (5'NCR) should be used for diagnosis of EVs due to their sensitivity, specificity and short turnaround time. Sequencing of the VP1 capsid protein gene is recommended for EV typing; EV typing cannot be based on the 5'NCR sequences due to frequent recombination events and should not rely on virus isolation. Effective and standardized laboratory diagnostics and characterisation of circulating virus strains are the first step towards effective and continuous surveillance activities, which in turn will be used to provide better estimation on EV disease burden.

Population pharmacokinetics of continuous infusion of piperacillin in critically ill patients.

Dosing recommendations for continuous infusion of piperacillin, a broad-spectrum beta-lactam antibiotic, are mainly guided by outputs from population pharmacokinetic models constructed with intermittent infusion data. However, the probability of target attainment in patients receiving piperacillin by continuous infusion may be overestimated when drug clearance estimates from population pharmacokinetic models based on intermittent infusion data are used, especially when higher doses (e.g. 16 g/24 h or more) are simulated. Therefore, the purpose of this study was to describe the population pharmacokinetics of piperacillin when infused continuously in critically ill patients. For this analysis, 270 plasma samples from 110 critically ill patients receiving piperacillin were available for population pharmacokinetic model building. A one-compartment model with linear clearance best described the concentration-time data. The mean ± standard deviation parameter estimates were 8.38 ± 9.91 L/h for drug clearance and 25.54 ± 3.65 L for volume of distribution. Creatinine clearance improved the model fit and was supported for inclusion as a covariate. In critically ill patients with renal clearance higher than 90 mL/min/1.73 m2, a high-dose continuous infusion of 24 g/24 h is insufficient to achieve adequate exposure (pharmacokinetic/pharmacodynamic target of 100% fT>4 x MIC) against susceptible Pseudomonas aerginosa isolates (MIC ≤16 mg/L). These findings suggest that merely increasing the dose of piperacillin, even with continuous infusion, may not always result in adequate piperacillin exposure. This should be confirmed by evaluating piperacillin target attainment rates in critically ill patients exhibiting high renal clearance.

Development and validation of an LC tandem MS assay for the quantification of ß-lactam antibiotics in the sputum of cystic fibrosis patients.

OBJECTIVES: Antibiotic therapy is of vital importance for the control of infectious exacerbations in cystic fibrosis (CF) patients. However, very little is known regarding the fraction of systemically administered antibiotics reaching the lower respiratory tract secretions. We developed and validated a method to measure the concentrations of piperacillin, ceftazidime, meropenem and aztreonam in CF sputum, and present the validation data. METHODS: Ultra-performance LC coupled to tandem MS was used. A single sample can be measured in 2.5 min with multiple reaction monitoring in positive electrospray ionization mode. Deuterated internal standards were used and a concentration range of 0.7-160 mg/L was covered. The method was validated according to the EMA guideline on analytical method validation. RESULTS: The boundaries within which a reliable measurement in CF sputum can be performed were determined. A few constraints are linked to the instability of the antibiotics in sputum. Piperacillin showed limited stability at room temperature and during freeze-thaw cycles. Autosampler instability was observed after 15 h for aztreonam at low concentrations. CONCLUSIONS: The method allows a reliable measurement of the selected antibiotics, if precautions are taken regarding the limited stability of piperacillin at room temperature. Due to freeze-thaw instability, piperacillin should always be analysed on the day of sampling. Quick review of the analytical data and reanalysis are needed as low concentrations of aztreonam are not stable in the autosampler.

Dose optimization of piperacillin/tazobactam in critically ill children.

Objectives: To characterize the population pharmacokinetics of piperacillin and tazobactam in critically ill infants and children, in order to develop an evidence-based dosing regimen. Patients and methods: This pharmacokinetic study enrolled patients admitted to the paediatric ICU for whom intravenous piperacillin/tazobactam (8:1 ratio) was indicated (75 mg/kg every 6 h based on piperacillin). Piperacillin/tazobactam concentrations were measured by an LC-MS/MS method. Pharmacokinetic data were analysed using non-linear mixed effects modelling. Results: Piperacillin and tazobactam blood samples were collected from 47 patients (median age 2.83 years; range 2 months to 15 years). Piperacillin and tazobactam disposition was best described by a two-compartment model that included allometric scaling and a maturation function to account for the effect of growth and age. Mean clearance estimates for piperacillin and tazobactam were 4.00 and 3.01 L/h for a child of 14 kg. Monte Carlo simulations showed that an intermittent infusion of 75 mg/kg (based on piperacillin) every 4 h over 2 h, 100 mg/kg every 4 h given over 1 h or a loading dose of 75 mg/kg followed by a continuous infusion of 300 mg/kg/24 h were the minimal requirements to achieve the therapeutic targets for piperacillin (60% f T >MIC >16 mg/L). Conclusions: Standard intermittent dosing regimens do not ensure optimal piperacillin/tazobactam exposure in critically ill patients, thereby risking treatment failure. The use of a loading dose followed by a continuous infusion is recommended for treatment of severe infections in children >2 months of age.

Augmented renal clearance implies a need for increased amoxicillin-clavulanic acid dosing in critically ill children.

There is little data available to guide amoxicillin-clavulanic acid dosing in critically ill children. The primary objective of this study was to investigate the pharmacokinetics of both compounds in this pediatric subpopulation. Patients admitted to the pediatric intensive care unit (ICU) in whom intravenous amoxicillin-clavulanic acid was indicated (25 to 35 mg/kg of body weight every 6 h) were enrolled. Population pharmacokinetic analysis was conducted, and the clinical outcome was documented. A total of 325 and 151 blood samples were collected from 50 patients (median age, 2.58 years; age range, 1 month to 15 years) treated with amoxicillin and clavulanic acid, respectively. A three-compartment model for amoxicillin and a two-compartment model for clavulanic acid best described the data, in which allometric weight scaling and maturation functions were added a priori to scale for size and age. In addition, plasma cystatin C and concomitant treatment with vasopressors were identified to have a significant influence on amoxicillin clearance. The typical population values of clearance for amoxicillin and clavulanic acid were 17.97 liters/h/70 kg and 12.20 liters/h/70 kg, respectively. In 32% of the treated patients, amoxicillin-clavulanic acid therapy was stopped prematurely due to clinical failure, and the patient was switched to broader-spectrum antibiotic treatment. Monte Carlo simulations demonstrated that four-hourly dosing of 25 mg/kg was required to achieve the therapeutic target for both amoxicillin and clavulanic acid. For patients with augmented renal function, a 1-h infusion was preferable to bolus dosing. Current published dosing regimens result in subtherapeutic concentrations in the early period of sepsis due to augmented renal clearance, which risks clinical failure in critically ill children, and therefore need to be updated. (This study has been registered at Clinicaltrials.gov as an observational study [NCT02456974].).

Assays for therapeutic drug monitoring of ß-lactam antibiotics: A structured review.

In some patient groups, including critically ill patients, the pharmacokinetics of ß-lactam antibiotics may be profoundly disturbed due to pathophysiological changes in distribution and elimination. Therapeutic drug monitoring (TDM) is a strategy that may help to optimise dosing. The aim of this review was to identify and analyse the published literature on the methods used for ß-lactam quantification in TDM programmes. Sixteen reports described methods for the simultaneous determination of three or more ß-lactam antibiotics in plasma/serum. Measurement of these antibiotics, due to low frequency of usage relative to some other tests, is generally limited to in-house chromatographic methods coupled to ultraviolet or mass spectrometric detection. Although many published methods state they are fit for TDM, they are inconvenient because of intensive sample preparation and/or long run times. Ideally, methods used for routine TDM should have a short turnaround time (fast run-time and fast sample preparation), a low limit of quantification and a sufficiently high upper limit of quantification. The published assays included a median of 6 analytes [interquartile range (IQR) 4-10], with meropenem and piperacillin being the most frequently measured ß-lactam antibiotics. The median run time was 8 min (IQR 5.9-21.3 min). There is also a growing number of methods measuring free concentrations. An assay that measures antibiotics without any sample preparation would be the next step towards real-time monitoring; no such method is currently available.

Population pharmacokinetics and dosing simulations of cefepime in septic shock patients receiving continuous renal replacement therapy.

The aim of this study was to describe the population pharmacokinetics of cefepime in septic shock patients requiring continuous renal replacement therapy and to determine whether current or alternative dosing regimens can achieve PK/PD targets. In this observational PK study, 62 samples from 13 patients were analysed using non-linear mixed-effects modelling. Different dosing regimens were evaluated using Monte Carlo simulations with ultrafiltration flow rates (UFRs) of 1000, 1500 and 2000 mL/h. The probability of target attainment was calculated against a conservative (60% T(>MIC)) and a higher PK/PD target (100% T(>MIC)) against an MIC of 8 mg/L, the clinical susceptibility breakpoint for Pseudomonas aeruginosa. A one-compartment model with between-subject variability (BSV) on clearance and volume of distribution (V(d)) described the data adequately. UFR was supported as a covariate on both parameters. Typical values for clearance and V(d) were 4.4L/h (BSV 37%) and 40.9L (BSV 20%), respectively. Dosing simulations showed failure to achieve both a conservative and a higher PK/PD target using a dose of 1g q12h for patients treated with a high UFR (≥1500 mL/h). The dose of 2g q8h or 1g q6h leads to optimal target attainment for high UFR. One gram q8h is optimal for low UFR (≤1000 mL/h). We found important variability in PK parameters. Dosing simulations show that a dose of 2g q8h or 1g q6h is needed to ensure rapid achievement of adequate levels if the UFR is ≥1500 mL/h and 1g q8h for low UFR (≤1000 mL/h).

A Simulation Study Reveals Lack of Pharmacokinetic/Pharmacodynamic Target Attainment in De-escalated Antibiotic Therapy in Critically Ill Patients.

De-escalation of empirical antibiotic therapy is often included in antimicrobial stewardship programs in critically ill patients, but differences in target attainment when antibiotics are switched are rarely considered. The primary objective of this study was to compare the fractional target attainments of contemporary dosing of empirical broad-spectrum ß-lactam antibiotics and narrower-spectrum antibiotics for a number pathogens for which de-escalation may be considered. The secondary objective was to determine whether alternative dosing strategies improve target attainment. We performed a simulation study using published population pharmacokinetic (PK) studies in critically ill patients for a number of broad-spectrum ß-lactam antibiotics and narrower-spectrum antibiotics. Simulations were undertaken using a data set obtained from critically ill patients with sepsis without absolute renal failure (n = 49). The probability of target attainment of antibiotic therapy for different microorganisms for which de-escalation was applied was analyzed. EUCAST MIC distribution data were used to calculate fractional target attainment. The probability that therapeutic exposure will be achieved was lower for the narrower-spectrum antibiotics with conventional dosing than for the broad-spectrum alternatives and could drastically be improved with higher dosages and different modes of administrations. For a selection of microorganisms, the probability that therapeutic exposure will be achieved was overall lower for the narrower-spectrum antibiotics using conventional dosing than for the broad-spectrum antibiotics.

Subtleties in practical application of prolonged infusion of ß-lactam antibiotics.

Prolonged infusion (PI) of ß-lactam antibiotics is increasingly used in order to optimise antibiotic exposure in critically ill patients. Physicians are often not aware of a number of subtleties that may jeopardise the treatment. In this clinically based paper, we stress pragmatic issues, such as the importance of a loading dose before PI, and discuss a number of important practicalities that are mandatory to benefit from the pharmacokinetic advantages of prolonged ß-lactam antibiotic administration.

Comparison of different equations to assess glomerular filtration in critically ill patients.

PURPOSE: To evaluate equations for estimation of glomerular filtration rate (GFR) and measured urinary creatinine clearance, compared to measured GFR in critically ill patients. METHODS: GFR was measured using inulin clearance. Multiple blood samples were collected per patient for determination of serum creatinine, cystatin C and inulin. GFR was estimated by the use of the following estimation equations (eGFR): four commonly used creatinine-based equations [Cockcroft-Gault, Modification of Diet in Renal Disease (both the short and long formula) and Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI)], five cystatin C based estimation equations (Hoek, Larsson, Filler, Le Bricon, CKD-EPIcys) and one equation combining cystatin C and serum creatinine (CKD-EPIcr-cys). In addition we measured urinary creatinine clearance. Bias, precision and accuracy of all estimates were compared to those of the inulin clearance. RESULTS: Data were collected from 83 patients, of whom 68 were considered evaluable. The median age was 58 years [interquartile range (IQR) 39-68]. The median inulin clearance was 80 mL/min/1.73 m(2) (IQR 31-114). Equations based on creatinine had much bias and poor precision and accuracy. Measured urinary creatinine clearances overestimated GFR. Equations based on cystatin C were free of bias, but also had limited precision and accuracy. CONCLUSIONS: In this cohort of patients, estimates of GFR had low accuracy and precision. Cystatin C based formulas, especially CKD-EPIcr-cys, showed limited bias; however, the accuracy and precision of these estimates were still insufficient. Measured urinary creatinine clearance overestimates GFR, but may provide a cheap alternative, when this is taken into account.

Measuring unbound versus total vancomycin concentrations in serum and plasma: methodological issues and relevance.

BACKGROUND: Studies on the unbound fraction (fu) of vancomycin report highly variable results. Great controversy also exists about the correlation between unbound and total vancomycin concentrations. As differences in (pre-)analytic techniques may explain these findings, we investigated the impact of the procedure used to isolate unbound vancomycin in serum/plasma on fu and the correlation between total and unbound concentrations. METHODS: Patient samples (n = 39) were analyzed for total and unbound vancomycin concentrations after ultrafiltration (UF, Centrifree at 4°C and 37°C) or equilibrium dialysis (ED, using a Fast Micro-Equilibrium Dialyzer at 37°C) on an Architect i2000SR. To investigate correlations with potential binding proteins, total protein, albumin, alpha-1-acid glycoprotein, and IgA concentrations were also measured. RESULTS: The median fu after ED was 72.5% [interquartile range (IQR), 68.7%-75.0%]. Ultrafiltration at 4°C and 37°C resulted in a median fu of 51.6% (IQR, 48.6%-54.8%) and 75.2% (IQR, 69.3%-78.6%), respectively, with no significant difference between unbound vancomycin concentrations after ED and UF at 37°C (P = 0.13). Unbound concentrations obtained through ED and UF correlated linearly (4°C: r = 0.9457; 37°C: r = 0.9478; both P < 0.0001). Linear mixed-model regression showed that total vancomycin as such was the predominant determinant for the unbound concentration, allowing a reliable prediction (mean bias ± SD, 5.0% ± 7.6%). The studied protein concentrations were of no added value in predicting the unbound concentration. CONCLUSIONS: Vancomycin fu after UF at 4°C was on average 30.6% lower than that after UF at 37°C, demonstrating the importance of temperature during UF. Ultrafiltration at 37°C resulted in unbound vancomycin concentrations equivalent with ED. As the unbound concentration could be reliably predicted based on total vancomycin concentrations as such, measurement of unbound vancomycin concentrations has little added value over measurements of total vancomycin concentrations.

Ultrafast quantification of ß-lactam antibiotics in human plasma using UPLC-MS/MS.

There is an increasing interest in monitoring plasma concentrations of ß-lactam antibiotics. The objective of this work was to develop and validate a fast ultra-performance liquid chromatographic method with tandem mass spectrometric detection (UPLC-MS/MS) for simultaneous quantification of amoxicillin, cefuroxime, ceftazidime, meropenem and piperacillin with minimal turn around time. Sample clean-up included protein precipitation with acetonitrile containing 5 deuterated internal standards, and subsequent dilution of the supernatant with water after centrifugation. Runtime was only 2.5 min. Chromatographic separation was performed on a Waters Acquity UPLC system using a BEH C18 column (1.7 µm, 100 mm × 2.1 mm) applying a binary gradient elution of water and methanol both containing 0.1% formic acid and 2 mmol/L ammonium acetate on a Water TQD instrument in MRM mode. All compounds were detected in electrospray positive ion mode and could be quantified between 1 and 100 mg/L for amoxicillin and cefuroxime, between 0.5 and 80 mg/L for meropenem and ceftazidime, and between 1 and 150 mg/L for piperacillin. The method was validated in terms of precision, accuracy, linearity, matrix effect and recovery and has been compared to a previously published UPLC-MS/MS method.

Beta-lactam antibiotic dosing during continuous renal replacement therapy: how can we optimize therapy?

Correct antibiotic treatment is of utmost importance to treat infections in critically ill patients, not only in terms of spectrum and timing but also in terms of dosing. However, this is a real challenge for the clinician because the pathophysiology (such as shock, augmented renal clearance, and multiple organ dysfunction) has a major impact on the pharmacokinetics of hydrophilic antibiotics. The presence of extra-corporal circuits, such as continuous renal replacement therapy, may further complicate this difficult exercise. Standard dosing may result in inadequate concentrations, but unadjusted dosing regimens may lead to toxicity. Recent studies confirm the variability in concentrations, and the wide variation in dialysis techniques used certainly contributes to these findings. Well-designed clinical studies are needed to provide the data from which robust dosing guidance can be developed. In the meantime, non-adjusted dosing in the first 1 to 2 days of antibiotic therapy during continuous renal replacement therapy followed by dose reduction later on seems to be a prudent approach.

Population pharmacokinetics and dosing simulations of cefuroxime in critically ill patients: non-standard dosing approaches are required to achieve therapeutic exposures.

OBJECTIVES: To investigate the population pharmacokinetics of cefuroxime in critically ill patients. METHODS: In this observational pharmacokinetic study, multiple blood samples were taken over one dosing interval of intravenous cefuroxime. Blood samples were analysed using a validated ultra HPLC tandem mass spectrometry technique. Population pharmacokinetic analysis and dosing simulations were performed using non-linear mixed-effects modelling. RESULTS: One hundred and sixty blood samples were collected from 20 patients. CL(CR) ranged between 10 and 304 mL/min. A two-compartment model with between-subject variability on CL, V of the central compartment and V of the peripheral compartment described the data adequately. Twenty-four hour urinary CL(CR) was supported as a descriptor of drug CL. The population model for CL was CL = θ(1) × CL(CR)/100, where θ(1) is the typical cefuroxime CL in the population, which is 9.0 L/h. The mean V was 22.5 L. Dosing simulations showed failure to achieve the pharmacokinetic/pharmacodynamic target of 65% fT(>MIC) for an MIC of 8 mg/L with standard dosing regimens for patients with CL(CR) ≥50 mL/min. CONCLUSIONS: Administration of standard doses by intermittent bolus is likely to result in underdosing for many critically ill patients. Continuous infusion of higher than normal doses after a loading dose is more likely to achieve pharmacokinetic/pharmacodynamic targets. However, even continuous infusion of high doses (up to 9 g per day) does not guarantee adequate levels for all patients with a CL(CR) of ≥300 mL/min if the MIC is 8 mg/L.

Does consistent piperacillin dosing result in consistent therapeutic concentrations in critically ill patients? A longitudinal study over an entire antibiotic course.

Piperacillin plasma concentrations are known to vary between critically ill patients. However, there are no comprehensive data on the variability of antibiotic concentrations within the same patient. The purpose of this study was to investigate the adequacy of dosing during an entire 7-day antibiotic course and to investigate the variability in antibiotic trough concentrations both between patients and within the same patient. Piperacillin trough concentrations were measured daily in critically ill patients with normal renal function. The drug assay was performed using UPLC-MS/MS. The pharmacokinetic/pharmacodynamic target was 100% fT>MIC of the Pseudomonas aeruginosa EUCAST breakpoint. Within- and between-patient variability were calculated as percent coefficient of variation (CV). Eleven patients treated for pneumonia were included in this nested prospective observational cohort study. The median (range) age was 67 (18-79) years, weight was 75 (57-90)kg and BMI was 23.5 (22.3-26.4). The median (range) creatinine clearance on Day 1 of antibiotic treatment was 102 (62-154)mL/min. Trough concentrations were variable, ranging from 4.9 mg/L to 98.0 mg/L. A median CV of 40% for within-patient variability and 57% for between-patient variability was found. Within-patient variability was inversely correlated with SOFA score (R = 0.65, P = 0.027) and APACHE II score on admission (R = 0.73, P = 0.009). In conclusion, piperacillin concentrations varied widely both between patients and within the same patient. Within-patient variability was inversely correlated with disease severity. Consistent dosing of piperacillin/tazobactam does not result in consistent piperacillin concentrations throughout the entire treatment period.