Bertrand Guignard

Detection of Live and Antibiotic-Killed Bacteria by Quantitative Real-Time PCR of Specific Fragments of rRNA

ABSTRACT Assessing bacterial viability by molecular markers might help accelerate the measurement of antibiotic-induced killing. This study investigated whether rRNA could be suitable for this purpose. Cultures of penicillin-susceptible and penicillin-tolerant (Tol1 mutant) Streptococcus gordonii were exposed to mechanistically different penicillin and levofloxacin. Bacterial survival was assessed by viable counts and compared to quantitative real-time PCR amplification of either the 16S rRNA genes or the 16S rRNA, following reverse transcription. Penicillin-susceptible S. gordonii lost ≥4 log10 CFU/ml of viability over 48 h of penicillin treatment. In comparison, the Tol1 mutant lost ≤1 log10 CFU/ml. Amplification of a 427-bp fragment of 16S rRNA genes yielded amplicons that increased proportionally to viable counts during bacterial growth but did not decrease during drug-induced killing. In contrast, the same 427-bp fragment amplified from 16S rRNA paralleled both bacterial growth and drug-induced killing. It also differentiated between penicillin-induced killing of the parent and the Tol1 mutant (≥4 log10 CFU/ml and ≤1 log10 CFU/ml, respectively) and detected killing by mechanistically unrelated levofloxacin. Since large fragments of polynucleotides might be degraded faster than smaller fragments, the experiments were repeated by amplifying a 119-bp region internal to the original 427-bp fragment. The amount of 119-bp amplicons increased proportionally to viability during growth but remained stable during drug treatment. Thus, 16S rRNA was a marker of antibiotic-induced killing, but the size of the amplified fragment was critical for differentiation between live and dead bacteria.

Drug-related problems identification in general internal medicine: The impact and role of the clinical pharmacist and pharmacologist

BACKGROUND Patients admitted to general internal medicine wards might receive a large number of drugs and be at risk for drug-related problems (DRPs) associated with increased morbidity and mortality. This study aimed to detect suboptimal drug use in internal medicine by a pharmacotherapy evaluation, to suggest treatment optimizations and to assess the acceptance and satisfaction of the prescribers. METHODS This was a 6-month prospective study conducted in two internal medicine wards. Physician rounds were attended by a pharmacist and a pharmacologist. An assessment grid was used to detect the DRPs in electronic prescriptions 24h in advance. One of the following interventions was selected, depending on the relevance and complexity of the DRPs: no intervention, verbal advice of treatment optimization, or written consultation. The acceptance rate and satisfaction of prescribers were measured. RESULTS In total, 145 patients were included, and 383 DRPs were identified (mean: 2.6 DRPs per patient). The most frequent DRPs were drug interactions (21%), untreated indications (18%), overdosages (16%) and drugs used without a valid indication (10%). The drugs or drug classes most frequently involved were tramadol, antidepressants, acenocoumarol, calcium-vitamin D, statins, aspirin, proton pump inhibitors and paracetamol. The following interventions were selected: no intervention (51%), verbal advice of treatment optimization (42%), and written consultation (7%). The acceptance rate of prescribers was 84% and their satisfaction was high. CONCLUSION Pharmacotherapy expertise during medical rounds was useful and well accepted by prescribers. Because of the modest allocation of pharmacists and pharmacologists in Swiss hospitals, complementary strategies would be required.

Impact of clinical decision support guidelines on therapeutic drug monitoring of gentamicin in newborns.

Background: Our institutions gentamicin dosing and therapeutic drug monitoring (TDM) practices for newborns were suspected to be very heterogeneous. Once-daily dosing (ODD) or extended-interval dosing (EID) and trough concentration measurement were recommended. Clinical decision support guidelines were developed and implemented as clinical decision support in the computerized prescriber order entry system. Impact on dosing, TDM practices, and blood sampling were evaluated. Methods: A 1-year retrospective historically controlled study before (April 2008–March 2009) and after the implementation of guidelines (January 2010–December 2010) for newborns (<30 days of life) receiving gentamicin. Blood concentrations (% of peak concentrations sampled, % of patients with zero or one concentration sampled, % of trough concentrations ⩽1 mg/L) and dose regimen (ODD/EID) were compared between groups. Factors potentially associated with gentamicin concentration were analyzed (multivariate analysis). Results: One hundred thirty-two (postguidelines) versus 102 (preguidelines) patients were included (median gestational age: 34.3 versus 35.8 weeks, P > 0.05). After implementation of the guidelines, an ODD/EID regimen was almost exclusively used (97.7% versus 61.6%, P < 0.001), the percentage of peak concentrations (0.9% versus 17.2%, P < 0.001) and the number of blood samples per patient (87.1% having 0 or 1 concentration measured versus 48.0, P < 0.001) sharply reduced. A significantly higher percentage of trough concentrations were ⩽1 mg/L (68.5% versus 33.0%, P < 0.001). The probability of a trough concentration ⩽1 mg/L increased with an ODD/EID regimen (odds ratio, 7.23; 95% confidence interval: 3.48–15.0, P < 0.001) and in the postguidelines group (odds ratio, 2.02; 95% confidence interval: 1.01–4.02, P = 0.045). Conclusions: Guideline implementation generated a sharp reduction in blood sampling. Clinical benefits of better gentamicin dosing and TDM practices were evident. Cost-effectiveness and clinical benefit of reduced blood sampling should be evaluated.

PIM-Check: development of an international prescription-screening checklist designed by a Delphi method for internal medicine patients

Objectives Potentially inappropriate medication (PIM) occurs frequently and is a well-known risk factor for adverse drug events, but its incidence is underestimated in internal medicine. The objective of this study was to develop an electronic prescription-screening checklist to assist residents and young healthcare professionals in PIM detection. Design Five-step study involving selection of medical domains, literature review and 17 semistructured interviews, a two-round Delphi survey, a forward/back-translation process and an electronic tool development. Setting 22 University and general hospitals from Canada, Belgium, France and Switzerland. Participants 40 physicians and 25 clinical pharmacists were involved in the study. Agreement with the checklist statements and their usefulness for healthcare professional training were evaluated using two 6-point Likert scales (ranging from 0 to 5). Primary and secondary outcome measures Agreement and usefulness ratings were defined as: >65% of the experts giving the statement a rating of 4 or 5, during the first Delphi-round and >75% during the second. Results 166 statements were generated during the first two steps. Mean agreement and usefulness ratings were 4.32/5 (95% CI 4.28 to 4.36) and 4.11/5 (4.07 to 4.15), respectively, during the first Delphi-round and 4.53/5 (4.51 to 4.56) and 4.36/5 (4.33 to 4.39) during the second (p<0.001). The final checklist includes 160 statements in 17 medical domains and 56 pathologies. An algorithm of approximately 31 000 lines was developed including comorbidities and medications variables to create the electronic tool. Conclusion PIM-Check is the first electronic prescription-screening checklist designed to detect PIM in internal medicine. It is intended to help young healthcare professionals in their clinical practice to detect PIM, to reduce medication errors and to improve patient safety.

Prevention of potentially inappropriate medication in internal medicine patients: A prospective study using the electronic application PIM-Check

Potentially inappropriate medication (PIM) is a risk factor for drug‐related problems (DRPs) and an important inpatient safety issue. PIM‐Check is a screening tool designed to detect PIM in internal medicine patients.

Muropeptide signature of inhibitors of protein synthesis correlates with β-lactam synergism against methicillin-resistant Staphylococcus aureus.

Quinupristin/dalfopristin (Q/D) and β-lactams interact positively against methicillin-resistant Staphylococcus aureus (MRSA). The effect extends to other inhibitors of protein synthesis, but not to inhibitors of polynucleotide synthesis or assembly, or to Q/D plus non-β-lactam cell wall inhibitors. Moreover, electron microscopy studies have correlated this effect with a thickened cell wall. In this study, we sought to determine whether inhibitors of protein synthesis might produce a specific peptidoglycan muropeptide signature that would correlate with their positive β-lactam interaction. The muropeptides of six S. aureus isolates (three methicillin-susceptible and three MRSA) were analysed using high-performance liquid chromatography and mass spectrometry. Exposure to 0.25× the minimum inhibitory concentration of inhibitors of protein synthesis consistently produced three main alterations irrespective of methicillin resistance: (i) an increase in peak 12 (a cyclic dimer of glycine-containing disaccharide-tetrapeptide); (ii) an increase in poorly resolved late-eluting materials; and (iii) a decrease in peak 1 (a disaccharide-pentapeptide). Eventually, the rate of autolysis was also decreased, supporting the structural alteration of the peptidoglycan. Other drug classes did not produce these anomalies. An increase in peak 12 was also observed in staphylococci treated with fosfomycin, which decreases expression of the native penicillin-binding protein (PBP) 2 and 4. Parallel blockage of normal PBPs with β-lactams abolished the anomalies, indicating that they resulted from altered function of native PBPs. This underlines the potential of inhibiting both protein synthesis and transpeptidation simultaneously and suggests that such a drug combination strategy might be efficaciously exploited.

CP-062 Subcutaneously implanted port-chamber central venous catheters: prevention and care of occlusion

Background Occlusion of subcutaneously implanted port-chamber central venous access devices (CVAD) is a commonly occurring problem in cancer patient care. A change of port-chamber catheter model in our institution was the opportunity to review nursing care techniques. Purpose To provide information on nursing care techniques for the prevention and management of thrombotic and non-thrombotic occlusions of subcutaneously implanted port-chamber central venous access devices, so as to contribute to the deliberations of an interdisciplinary working group charged with updating our institution’s best practices, and standardise them across adult and paediatric sectors. Materials and methods We carried out a structured literature review (Medline/Embase) and a manual search for non-indexed information sources up to February 2013. The keywords used were “central venous catheter”, “peripherally inserted central venous catheter” and “catheter occlusion”. Only publications presenting concrete facts on nursing care were included (drug volumes administered, exact durations of drug delivery, care techniques, written protocols). General recommendations were excluded. The criteria identified were: study methodologies, occlusion prevention techniques, definition and diagnosis, clearance techniques, effectiveness and safety. Results 26 publications were included: 14 studies (6 prospective, 8 retrospective), 9 review articles, 1 case study series, 1 survey and 1 reference book. Eleven publications concerned adult patients, 9 children and 4 both. Only 6 contained information with all the identified criteria. Fifteen only concerned occlusion prevention techniques, 14 concerned identification of blockages and 22 concerned blockage clearance techniques (17 thrombotic occlusion, 1 non-thrombotic lipid occlusion, 4 for both types). Highlighted points included: minimum 10 ml syringe volume, using NS (normal saline) for flushing and positive pressure filling (pulsed flux technique), thrombotic occlusion treatment using alteplase, the lack of validated, risk free treatment for non-thrombotic occlusion, and cost considerations. Conclusions Few studies of good methodological quality exist, with wide heterogeneity in types of catheter devices and occlusions evaluated. This renders comparison of preventive practices and occlusion treatment difficult. Literature review revealed a variety of useful insights for the interdisciplinary working group. The costs and risks of occlusion and the repeated use of alteplase call for good quality quantitative and qualitative prospective studies. No conflict of interest.

Effects of clinical decision support on the TDM of gentamicin and vancomycin in newborns

Background The dosing scheme for gentamicin and therapeutic drug monitoring (TDM) of gentamicin and vancomycin in newborns were evaluated as very heterogeneous in our institution. Once daily dosing (ODD) of gentamicin and trough levels measurement is recommended for most patients to ensure efficacy and reduce blood sampling. Guidelines were developed and implemented as a clinical decision support system. Purpose To evaluate dosing practices, blood sampling and therapeutic levels. Materials and methods Retrospective case– control study (01.2010-12.2010 and 04.2008 – 03.2009) in newborns (< 28 days of life) receiving either gentamicin or vancomycin before and after implementation of guidelines. Chart analysis criteria (mean +/-SD (Fishers exact, Wilcoxon rank sum tests)): % of ODD gentamicin dosing schemes, % of peak levels, mean number of levels sampled, % of therapeutic levels (trough level: gentamicin ≤1 mg/l; vancomycin: 5–10 mg/l). Results Gentamicin 132 (cases) versus 102 (controls) totalling 134 patients were included (mean gestational age: 33.8±5.4 versus 34.6±5.2 weeks, p>0.05). After guidelines had been implemented, an ODD scheme was used (97.7 vs 61.6%, p<0.001). Peak level measurement and mean number of levels were significantly reduced (0.9 vs 17.2% resp. 0.8±1.0 vs 1.7±1.4, p<0.001). A significantly higher % of trough levels were ≤1 mg/l (68.5 vs 33.0%, p<0.001). Vancomycin: 38 versus 37 patients included (mean gestational age: 29.1±3.8 vs 30.8±4.1 weeks, p>0.05). After guidelines had been implemented, peak level measurements were significantly reduced (0 vs 25.2%, p<0.001) and a trend to more patients with <2 levels sampled was noted (52.6% vs 29.7%, p=0.061). No differences were observed in the mean number of levels or in the % of therapeutic levels (2.7±3.4 vs 2.6±2.2, resp. 45.7 vs 57.1%, p>0.05). Trough levels > 15 mg/l were significantly more frequent (21.0% vs 5.2%, p=0.004) possibly due to an error in the guidelines. Conclusions Gentamicin dosing and TDM practices were improved after guidelines had been implemented. The effect of corrected guidelines on vancomycin TDM practice, the financial benefit of reduced blood sampling and clinical benefit should be evaluated in the future.