Insufficient β-lactam concentrations in the early phase of severe sepsis and septic shock

This was a prospective, multicenter, observational study performed in four Departments of Intensive Care in Belgium (at the St-Luc Hospital, Erasme Hospital, and UZ-VUB in Brussels and St Pierre Hospital in Ottignies). The study protocol was approved by the university ethics committees of the different hospitals. Before enrolment, written consent was obtained from the patient or their nearest relative.

All patients admitted to one of the four ICUs between January 2005 and July 2006 were considered for inclusion. Inclusion criteria were a diagnosis of severe sepsis or septic shock [18], either at admission or during the ICU stay, and treatment with a broad-spectrum β-lactam antibiotic (ceftazidime, cefepime, piperacillin-tazobactam, or meropenem). Patients meeting one of the following criteria were excluded: age less than 18 years or more than 85 years; pregnancy or lactation; previous administration of any of the investigated antibiotics; chronic renal failure requiring dialysis; or allergy to any of the investigated antibiotics. The study period was limited to the first 24 hours of antibiotherapy.

Administration of the four β-lactams was made according to local guidelines. These drugs are generally used in the participating centers to treat hospital- or ICU-acquired infections or in the case of community-acquired infection when a more-resistant pathogen may be involved (recent hospitalization or antibiotic therapy, previous colonization by more resistant strain). Piperacillin-tazobactam was preferred as first-line therapy in cases of proven or suspected intra-abdominal infections. Ceftazidime and cefepime was used as first-line therapy in other cases. Meropenem was used as second-line therapy (i.e. failure of piperacillin-tazobactam or cephalosporins) or in case of suspected or previous colonization by extended spectrum beta-lactamase Gram-negative bacteria.

In all study patients, demographics, pre-existing chronic diseases, admission diagnosis and biological data were collected in institutional databases. The severity of illness of each patient was characterized using the Acute Physiology and Chronic Health Evaluation (APACHE) II [19] and sequential organ failure assessment (SOFA) [20] scores determined on the first day of antibiotic treatment. Creatinine clearance (CrCl) was calculated using a standard formula [21]. Treatment of patients with catecholamines, mechanical ventilation, hemofiltration or hemodialysis was recorded, as was length of ICU and hospital stay, overall mortality, and cause of death. Hemodynamic data were collected at baseline, and 8 and 24 hours after the start of the protocol.

All the patients included in the study received a first dose of 2 g ceftazidime or cefepime, 4 g/0.5 g piperacillin-tazobactam, or 1 g meropenem. The usual daily doses of these antibiotics and dose adjustments for renal function are presented in Additional file 1. All the patients also received amikacin and the two antibiotics were administered simultaneously over 30 minutes using an infusion pump. Blood samples of 5 mL were collected without anticoagulant immediately before the infusion (0 hour) and 1, 1.5, 4.5, and 6 or 8 hours (depending on the frequency of administration of the β-lactam) thereafter; these blood-draw time points were chosen as they belong to the elimination phase of all four antibiotics. The exact sampling time was recorded by the nursing or medical staff. Blood samples were centrifuged at 4000 g for 10 minutes after blood clotting. To allow for possible drug instability at room temperature, serum samples were stored at -80°C until analysis.

All antibiotic quantitative analyses were performed in a centralized reference laboratory (St Luc Hospital). Importantly, as the PK of piperacillin and tazobactam are highly correlated [22], we only measured piperacillin levels. Serum β-lactam concentrations were determined by high-performance liquid chromatography with diode array detection. The intravenous antibiotic formulations were reconstituted according to the manufacturers' recommendations and diluted in water in order to reach stock solution aliquots of 1 mg/mL, stored at -20°C. Before each assay, a fresh calibration curve was prepared from the stock solution and blank serum at the following concentrations: 0.75, 1, 2, 5, 10, 25, and 50 μg/mL for piperacillin-tazobactam; 5, 10, 25, 50, and 100 μg/mL for ceftazidime; 0.1, 0.25, 0.5, 1, 5, 10, 25, and 50 μg/mL for cefepime or meropenem. The calibration and liquid-liquid extraction procedures as well as chromatographic conditions have been described previously [23]. The validation of the four analytical methods was performed over a three-day period with five calibration curves per day (i.e., 15 serum samples per concentration level). All methods were validated according to the published acceptance criteria for specificity, linearity, accuracy, precision (intra-day (repeatability), inter-day (intermediate precision)) and sensitivity (limit of detection (LOD) and limit of quantification (LOQ)). Specificity was determined by the ability to identify the β-lactam from its characteristic retention time and ultraviolet spectrum, and the fine resolution of its chromatographic peak. The linearity of the calibration curve was demonstrated by a significant linear regression analysis, with a determination coefficient (r²) more than 0.99. Accuracy was expressed as the percent deviation of the mean observed concentration from the theoretical value, which should not exceed 15%, except at the LOQ (20%). Precision was acceptable if the intra- and inter-day coefficients of variation (CV) were 20% or less at the LOQ and 15% or less at all other concentrations. LOQ was defined as the lowest concentration of the calibration curve, which could be reliably differentiated from background noise with a signal-to-noise ratio of at least 10:1 and quantified with acceptable accuracy (80 to 120%) and precision (CV ≤ 20%); LOD was defined as the lowest concentration that could be detected and reliably differentiated from background noise with a signal-to-noise ratio of at least 3:1.

The carry-over effect was tested by injecting regular blank samples and ultrapure water into the high-performance liquid chromatography system after high concentration calibrators. Under the described chromatographic conditions, piperacillin-tazobactam, ceftazidime, cefepime, and meropenem were identified by sharp and well-resolved peaks. The linearity was statistically confirmed over the concentration range tested for each β-lactam and was associated with an r² of more than 0.999. The four analytical methods were accurate and precise. LOD and LOQ were 0.50 and 0.75 μg/mL, respectively, for piperacillin-tazobactam, 2.00 and 5.00 μg/mL for ceftazidime, and 0.07 and 0.10 μg/mL for cefepime and meropenem. Appropriate dilution was performed for clinical samples with concentrations above the upper analytic range (corresponding to the calibration curve).

The PK of the four antibiotics was individually assessed using WinNonlin Professional version 5.0.1. software (Pharsight Corporation, Mountain View, CA, USA). A one-compartment model with first-order elimination was selected to fit the data. Investigated PK parameters included maximal serum concentration (Cmax, calculated by extrapolation of the elimination phase at the end of the infusion) Vd, total clearance (CL), elimination half-live (t_1/2) and area under the serum concentration-time curve (AUC). Vd and CL were normalized to the body weight.

The threshold of MIC required for maximal β-lactam activity is still controversial. In this study, the adequacy of β-lactam therapy was assessed by calculating the time spent greater than four times the target MIC (T > 4 × MIC). For each drug, the optimal T > 4 × MIC was considered as: above 50% for piperacillin-tazobactam, above 70% for ceftazidime and cefepime, and above 40% for meropenem in Gram-negative bacterial infections [24]. As the dosage interval of β-lactams is prolonged in renal impairment [see Additional file 1], we calculated the time before the subsequent administration according to the adjustment of the drug regimen to CrCl for each patient. As there is a large variance in MIC values for different bacteria, we considered the MICs for problematic pathogens, such as P. aeruginosa, commonly isolated in ICU patients, as the empiric target threshold [25]. Sensitivity thresholds of MIC for this pathogen, as defined by European Committee on Antimicrobial Susceptibility Testing (EUCAST), are: 8 μg/mL or less (ceftazidime, cefepime), 16 μg/mL or less (piperacillin-tazobactam), and 2 μg/mL or less (meropenem) [see Additional file 1] [26]. We, therefore, classified each patient as having an 'adequate' or 'inadequate' PK profile according to the percentage of time during which serum drug concentrations remained more than four times the clinical breakpoint for P. aeruginosa (% T > 4 × MIC): 32 μg/mL or more (ceftazidime, cefepime), 64 μg/mL or more (piperacillin-tazobactam), and 8 μg/mL or more (meropenem). Finally, by simulation using the PK parameters of our population, we calculated the probability of achieving target T > 4 × MIC values for other MICs that can be found in ICU-isolated Gram-negative bacteria.

Statistical analyses were performed using the SPSS 13.0 for Windows NT software package (SPSS Inc. 2004, Chicago, IL, USA). Descriptive statistics were computed for all study variables. A Kolmogorov-Smirnov test was used, and histograms and normal-quantile plots were examined to verify the normality of distribution of continuous variables. Discrete variables were expressed as counts (percentage) and continuous variables as means ± standard deviation or median (25th to 75th percentiles). Demographics and clinical differences between study groups were assessed using a chi-squared, Fisher's exact test, Student's t-test, or Mann-Whitney U test, as appropriate. The Pearson's (r) correlation coefficient was used to determine linear correlation as appropriate. Association between variables was tested by simple regression analysis and coefficient of determination (R²) in case of non-linear correlation. A P < 0.05 was considered as statistically significant.

We enrolled 80 patients (mean age 63 years, 64% male; Table 1) in the current study. Fifty-five (69%) of the patients were medical admissions and 55 had a hospital or ICU-acquired infection; 58 (72%) had septic shock. The median APACHE II score was 22 and the median SOFA score on admission was 8. Fifty-seven patients (71%) were treated with mechanical ventilation; 22 patients (27%) had acute renal failure. Overall ICU mortality was 38%, mostly due to sepsis. Most infections were respiratory or abdominal and were microbiologically documented in 56 patients (70%). Blood cultures were positive in 32 patients (40%). Forty (50%) cases of sepsis were secondary to Gram-negative bacilli, with 36 infections due to difficult-to-treat pathogens (P. aeruginosa (n = 18); Enterobacter species (n = 11); Citrobacter freundii, Hafnia alvei and Morganella morganii (n = 2 for each); Serratia marcescens (n = 1)).

Of the 80 patients, 16 were treated with meropenem, 18 with ceftazidime, 19 with cefepime, and 27 with piperacillin-tazobactam. The mean PK parameters for the four drugs are shown in Table 2. There was marked inter-individual variation in all PK parameters; Vd was increased for all four drugs when compared with healthy volunteers, with consequently a lower Cmax [see Additional file 1]. The median total CL was also reduced when compared with the median CL in healthy volunteers. The median percentage of T > 4 × MIC was 57% for meropenem, 45% for ceftazidime, 34% for cefepime, and 33% for piperacillin-tazobactam (Table 3). Thirteen patients had plasma concentrations less than four times the target MIC after only 90 minutes (ceftazidime = 1; cefepime = 1; piperacillin-tazobactam = 11). The number of patients who attained the target percentage T > 4 × MIC was 12 of 16 for meropenem (75%), 5 of 18 for ceftazidime (28%), 3 of 19 (16%) for cefepime, and 12 of 27 (44%) for piperacillin-tazobactam.

Drug regimens were adapted because of renal impairment in 41 patients (6 treated with meropenem, 9 with ceftazidime, 12 with cefepime, and 14 with piperacillin-tazobactam). The CrCl was similar among the four groups (piperacillin-tazobactam 56 (ranges: 13 to 164) mL/min; meropenem 64 (22 to 134) mL/min; ceftazidime 58 (15 to 145) mL/min; cefepime 40 (13 to 150) mL/min). In patients with renal dysfunction (CrCl <50 mL/min), 5 of 6 (83%) attained the target concentration for meropenem, 3 of 9 (33%) for ceftazidime, 2 of 12 (17%) for cefepime, and 10 of 14 (71%) for piperacillin-tazobactam. For piperacillin-tazobactam, but not for the other antibiotics, patients with renal dysfunction had a significantly higher probability of having adequate drug concentrations than patients with normal renal function (10 of 14 vs. 2 of 13, P = 0.03). Calculating the probability of target T > 4 × MIC attainment for several MICs, values more than 90% were obtained for ceftazidime and piperacillin-tazobactam with MIC of 2 μg/mL or less and for cefepime and meropenem with MIC of 1 μg/mL or less (Table 4).

No correlation was found between the T > 4 × MIC and any hemodynamic or clinical variable for any of the four drugs, including age, mechanical ventilation, APACHE II or SOFA score at admission, presence of shock, maximum dose of vasopressor agents or fluid balance. There was a significant correlation between CrCl at admission and CL for all drugs (data not shown).