ß-Lactam pharmacodynamics in Gram-negative bloodstream infections in the critically ill.

OBJECTIVES: To determine the ß-lactam exposure associated with positive clinical outcomes for Gram-negative blood stream infection (BSI) in critically ill patients. PATIENTS AND METHODS: Pooled data of critically ill patients with mono-microbial Gram-negative BSI treated with ß-lactams were collected from two databases. Free minimum concentrations (fCmin) of aztreonam, cefepime, ceftazidime, ceftriaxone, piperacillin (co-administered with tazobactam) and meropenem were interpreted in relation to the measured MIC for targeted bacteria (fCmin/MIC). A positive clinical outcome was defined as completion of the treatment course or de-escalation, without other change of antibiotic therapy, and with no additional antibiotics commenced within 48 h of cessation. Drug exposure breakpoints associated with positive clinical outcome were determined by classification and regression tree (CART) analysis. RESULTS: Data from 98 patients were included. Meropenem (46.9%) and piperacillin/tazobactam (36.7%) were the most commonly prescribed antibiotics. The most common pathogens were Escherichia coli (28.6%), Pseudomonas aeruginosa (19.4%) and Klebsiella pneumoniae (13.3%). In all patients, 87.8% and 71.4% achieved fCmin/MIC ≥1 and fCmin/MIC >5, respectively. Seventy-eight patients (79.6%) achieved positive clinical outcome. Two drug exposure breakpoints were identified: fCmin/MIC >1.3 for all ß-lactams (predicted difference in positive outcome 84.5% versus 15.5%, P < 0.05) and fCmin/MIC >4.95 for meropenem, aztreonam or ceftriaxone (predicted difference in positive outcome 97.7% versus 2.3%, P < 0.05). CONCLUSIONS: A ß-lactam fCmin/MIC >1.3 was a significant predictor of a positive clinical outcome in critically ill patients with Gram-negative BSI and could be considered an antibiotic dosing target.

Therapeutic drug monitoring of ß-lactam antibiotics in the critically ill: direct measurement of unbound drug concentrations to achieve appropriate drug exposures.

Objectives: To describe the achievement of unbound ß-lactam antibiotic concentration targets in a therapeutic drug monitoring (TDM) programme in critically ill patients, and the factors associated with failure to achieve a target concentration. Patients and methods: Plasma samples and clinical data were obtained for analysis from a single centre prospectively. Unbound concentrations of ceftriaxone, cefazolin, meropenem, ampicillin, benzylpenicillin, flucloxacillin and piperacillin were directly measured using ultracentrifugation. Factors associated with the achievement of pharmacokinetic/pharmacodynamic (PK/PD) targets or negative clinical outcomes were evaluated with binomial logistic regression. Results: TDM data from 330 patients, and 369 infection episodes, were included. The range of doses administered was 99.4% ± 45.1% relative to a standard daily dose. Dose increases were indicated in 33.1% and 63.4% of cases to achieve PK/PD targets of 100% fT>MIC and 100% fT>4×MIC, respectively. Dose reduction was indicated in 17.3% of cases for an upper PK/PD threshold of 100% fT>10×MIC. Higher protein bound ß-lactams (ceftriaxone and benzylpenicillin) had better therapeutic target attainment (P < 0.01), but were prone to excessive dosing. Augmented renal clearance (calculated CLCR >130 mL/min) increased the odds of failure to achieve 100% fT>MIC and 100% fT>4×MIC (OR 2.47 and 3.05, respectively; P < 0.01). Conclusions: Measuring unbound concentrations of ß-lactams as part of a routine TDM programme is feasible and demonstrates that a large number of critically ill patients do not achieve predefined PK/PD targets. The clinical significance of this finding is unknown due to the lack of correlation between PK/PD findings and clinical outcomes.

Laboratory medicine best practice guideline: vitamins a, e and the carotenoids in blood.

Despite apparent method similarities between laboratories there appear to be confounding factors inhibiting uniform reporting and standardisation of vitamin assays. The Australasian Association of Clinical Biochemists (AACB) Vitamins Working Party, in conjunction with The Royal College of Pathologists of Australasia Quality Assurance Programs, has formulated a guideline to improve performance, reproducibility and accuracy of fat-soluble vitamin results. The aim of the guideline is to identify critical pre-analytical, analytical and post-analytical components of the analysis of vitamins A, E and carotenoids in blood to promote best practice and harmonisation. This best practice guideline has been developed with reference to the Centers for Disease Control and Prevention (CDC) "Laboratory Medicine Best Practices: Developing an Evidence-Based Review and Evaluation Process". The CDC document cites an evaluation framework for generating best practice recommendations that are specific to laboratory medicine. These 50 recommendations proposed herein, were generated from a comprehensive literature search and the extensive combined experience of the AACB Vitamins Working Party members. They were formulated based on comparison between an impact assessment rating and strength of evidence and were classified as either: (1) strongly recommend, (2) recommend, (3) no recommendation for or against, or (4) recommend against. These best practice recommendations represent the consensus views, in association with peer reviewed evidence of the AACB Vitamins Working Party, towards best practice for the collection, analysis and interpretation of vitamins A, E and carotenoids in blood.

Protein binding of ß-lactam antibiotics in critically ill patients: can we successfully predict unbound concentrations?

The use of therapeutic drug monitoring (TDM) to optimize beta-lactam dosing in critically ill patients is growing in popularity, although there are limited data describing the potential impact of altered protein binding on achievement of target concentrations. The aim of this study was to compare the measured unbound concentration to the unbound concentration predicted from published protein binding values for seven beta-lactams using data from blood samples obtained from critically ill patients. From 161 eligible patients, we obtained 228 and 220 plasma samples at the midpoint of the dosing interval and trough, respectively, for ceftriaxone, cefazolin, meropenem, piperacillin, ampicillin, benzylpenicillin, and flucloxacillin. The total and unbound beta-lactam concentrations were measured using validated methods. Variabilities in both unbound and total concentrations were marked for all antibiotics, with significant differences being present between measured and predicted unbound concentrations for ceftriaxone and for flucloxacillin at the mid-dosing interval (P < 0.05). The predictive performance for calculating unbound concentrations using published protein binding values was poor, with bias for overprediction of unbound concentrations for ceftriaxone (83.3%), flucloxacillin (56.8%), and benzylpenicillin (25%) and underprediction for meropenem (12.1%). Linear correlations between the measured total and unbound concentrations were observed for all beta-lactams (R(2) = 0.81 to 1.00; P < 0.05) except ceftriaxone and flucloxacillin. The percent protein binding of flucloxacillin and the plasma albumin concentration were also found to be linearly correlated (R(2) = 0.776; P < 0.01). In conclusion, significant differences between measured and predicted unbound drug concentrations were found only for the highly protein-bound beta-lactams ceftriaxone and flucloxacillin. However, direct measurement of unbound drug in research and clinical practice is suggested for selected beta-lactams.

Vitamin B1 and B6 method harmonization: comparison of performance between laboratories enrolled in the RCPA Quality Assurance Program.

OBJECTIVES: The RCPA Quality Assurance Program (RCPA QAP) offers monthly proficiency testing for vitamins A, B1, B6, ß-carotene, C and E to laboratories worldwide. A review of the results submitted for the whole blood vitamin B1/B6 sub-program revealed a wide dispersion. Here we describe the results of a methodology survey for vitamins B1 and B6. DESIGN AND METHODS: A questionnaire was sent to thirteen laboratories. Eleven laboratories were returning QAP results for vitamin B1 (thiamine diphosphate) and five were returning results for vitamin B6 (pyridoxal-5-phosphate). RESULTS: All nine respondents provided a clinical service for vitamins B1 and B6. HPLC with fluorescence detection was the most common method principle. For vitamin B1, six respondents used a commercial assay whilst three used in-house methods; whole blood was the matrix for all. For vitamin B6, five respondents used commercial assays and four used in-house assays. The choice of matrix for vitamin B6 varied with three respondents using whole blood and five using plasma for analysis. Sample preparation incorporated protein precipitation and derivatization steps. An internal standard was employed in sample preparation by only one survey respondent. CONCLUSIONS: The immediate result of this survey was the incorporation of plasma vitamin B6 into the RCPA QAP vitamin program. The absence of an internal standard in current vitamin B1 and B6 assays is a likely contributor to the wide dispersion of results seen in this program. We recommend kit manufacturers and laboratories investigate the inclusion of internal standards to correct the variability that may occur during processing.

A method for determining the free (unbound) concentration of ten beta-lactam antibiotics in human plasma using high performance liquid chromatography with ultraviolet detection.

With the clinical imperative to further research in the area of optimising antibiotic dosing in the intensive care setting, a simple high performance liquid chromatography method was developed and validated for routinely determining the free (unbound) concentration of ten beta-lactam antibiotics in 200 µL of human plasma. Antibiotics determined include three cephalosporins (ceftriaxone, cephazolin and cephalotin); two carbapenems (meropenem and ertapenem); and five penicillins (ampicillin, piperacillin, benzylpenicillin, flucloxacillin and dicloxacillin). There was a single common sample preparation method involving ultracentrifugation and stabilisation. Chromatography was performed on a Waters XBridge C18 column with, depending on analytes, one of four acetonitrile-phosphate buffered mobile phases. Peaks of interest were detected via ultraviolet absorbance at 210, 260 and 304 nm. The method has been validated and used in a pathology laboratory for therapeutic drug monitoring in critically ill patients. The significant variability in the level of protein binding that is common with antibiotics traditionally considered to have high protein binding (e.g. ceftriaxone, cephazolin, ertapenem, flucloxacillin and dicloxacillin) suggests that this assay should be preferred for measuring the pharmacologically active concentration of beta-lactam antibiotics in a therapeutic drug monitoring programme.

Therapeutic drug monitoring of beta-lactam antibiotics in burns patients--a one-year prospective study.

BACKGROUND: Beta-lactams are first-line antibiotics for the management of superficial infections due to burn injury. There is sparse data available on therapeutic drug monitoring (TDM) in patients with burns in a ward setting. This study was conducted to evaluate the utility of a beta-lactam TDM program in a cohort of burn injury patients in a ward environment. METHODS: Steady-state blood samples were collected immediately before a scheduled dose. The therapeutic concentration targets assessed were (1) free antibiotic concentrations exceeding the minimum inhibitory concentration (MIC; fT > MIC) and (2) free concentrations ≥4× MIC of the known or suspected pathogen (fT > 4× MIC). The duration of therapy was also assessed. RESULTS: A total of 50 patients were included for TDM over a 12-month period. The mean (±SD) age was 49 ± 16 years. The mean percent total body surface area burn was 17 ± 13%. The mean serum creatinine concentration was 86 ± 20 µmole/L. Sixty percent of the patients did not achieve fT > MIC, and only 18% achieved the higher target of fT > 4× MIC. Although all the patients achieved a positive clinical outcome, the duration of antibiotic treatment was shorter in patients who achieved fT > MIC compared with those who did not (4.2 ± 1.1 versus 5.3 ± 2.3 days; P = 0.03). CONCLUSIONS: We found TDM to be a reliable intervention for burn injury patients in a ward environment. This study supports pharmacokinetic data that burns patients may be at risk of subtherapeutic dosing, which may prolong the duration of antibiotic therapy.

Subtherapeutic initial ß-lactam concentrations in select critically ill patients: association between augmented renal clearance and low trough drug concentrations.

BACKGROUND: ß-Lactams are routinely used as empirical therapy in critical illness, with extended concentrations above the minimum inhibitory concentration (MIC) of the infecting organism required for effective treatment. Changes in renal function in this setting can significantly impact the probability of achieving such targets. METHODS: Analysis was made of trough plasma drug concentrations obtained via therapeutic drug monitoring, compared with renal function, in critically ill patients receiving empirical ß-lactam therapy. Drug concentrations were measured by means of high-performance liquid chromatography and corrected for protein binding. Therapeutic levels were defined as greater than or equal to MIC and greater than or equal to four times MIC (maximum bacterial eradication), respectively. Renal function was assessed by means of an 8-h creatinine clearance (CLCR). RESULTS: Fifty-two concurrent trough concentrations and CLCR measures were used in analysis. Piperacillin was the most frequent ß-lactam prescribed (48%), whereas empirical cover and Staphylococcus species were the most common indications for therapy (62%). Most patients were mechanically ventilated on the day of study (85%), although only 25% were receiving vasopressors. In only 58% (n = 30) was the trough drug concentration greater than or equal to MIC, falling to 31% (n = 16) when using four times MIC as the target. CLCR values ≥ 130 mL/min/1.73 m2 were associated with trough concentrations less than MIC in 82% (P < .001) and less than four times MIC in 72% (P < .001). CLCR remained a significant predictor of subtherapeutic concentrations in multivariate analysis. CONCLUSION: Elevated CLCR appears to be an important predictor of subtherapeutic ß-lactam concentrations and suggests an important role in identifying such patients in the ICU.

Free cortisol method comparison: ultrafiltation, equilibrium dialysis, tracer dilution, tandem mass spectrometry and calculated free cortisol.

BACKGROUND: Free cortisol (FC) can be calculated from measurements of total cortisol and binding proteins or measured after mechanical separation of unbound and bound fractions by equilibrium dialysis or ultrafiltration. FC can then be measured indirectly by 3H-cortisol dilution or directly by immunologic or tandem mass spectrometry assays. METHODS: We compared FC measured with ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC MS/MS) with 3H-cortisol dilution in ultrafiltrates and dialysates and also with calculated FC (Coolens equation). An adult FC reference interval was established. RESULTS: The UHPLC MS/MS and 3H-cortisol dilution methods were non-linearly related (Cusum linearity test p<0.001) but well correlated (R2=0.984). FC calculated with Coolens equation agreed with the UHPLC MS/MS method. Impurity of 3H-cortisol and non-specific adsorption were excluded as causes on non-linearity. Ultrafiltration was linearly related to equilibrium dialysis, simpler to perform and more repeatable. A gender non-specific FC reference interval of 2.1-19.1 nmol/L was established. CONCLUSIONS: In view of the non-linearity between measuring techniques and the variability of reported reference ranges, care should be exercised in adopting a reference range. The ultrafiltration UHPLC MS/MS method we described is robust and suitable for use in a routine laboratory.

Measurement of cortisol, cortisone, prednisolone, dexamethasone and 11-deoxycortisol with ultra high performance liquid chromatography-tandem mass spectrometry: Application for plasma, plasma ultrafiltrate, urine and saliva in a routine laboratory.

We describe an ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC MS/MS) method suitable for a routine laboratory to determine endogenous and exogenous glucocorticoids in plasma, plasma ultrafiltrate, urine and saliva in a single analytical run. After addition of a multi-analyte internal standard, a standardised sample preparation procedure with solid phase extraction followed, before injecting into a tandem mass spectrometer with positive mode electron spray ionisation and multiple reactant monitoring acquisition. The chromatography time was 3min. The limit of quantitation for cortisol and cortisone in plasma was 3.75nmol/L and linearity extended to 2000nmol/L. The limit of quantitation for cortisol in plasma ultrafiltrate and saliva was 0.6nmol/L. The limit of quantitation for 11-deoxycortisol and prednisolone was 5nmol/L and for dexamethasone 1nmol/L. The intra-assay CV was <5% and the inter-assay CV <10% for all analytes in all matrices. Comparison with an immuno-assay (IA) plasma cortisol method resulted in a regression equation of UHPLC=0.79×IA+31.12 with R(2)=0.960 (p<0.0001). Comparison with a high performance liquid chromatography (HPLC) cortisol method yielded a regression equation of UHPLC=1.06×HPLC+9.82, R(2)=0.992 (p<0.0001). The simultaneous measurement of endogenous and exogenous glucocorticoids contributed to patient care in cases with dexamethasone and metyrapone dynamic tests and unsuspected therapeutic glucocorticoid use.