Optimizing design of research to evaluate antibiotic stewardship interventions: consensus recommendations of a multinational working group.

BACKGROUND: Antimicrobial stewardship interventions and programmes aim to ensure effective treatment while minimizing antimicrobial-associated harms including resistance. Practice in this vital area is undermined by the poor quality of research addressing both what specific antimicrobial use interventions are effective and how antimicrobial use improvement strategies can be implemented into practice. In 2016 we established a working party to identify the key design features that limit translation of existing research into practice and then to make recommendations for how future studies in this field should be optimally designed. The first part of this work has been published as a systematic review. Here we present the working group's final recommendations. METHODS: An international working group for design of antimicrobial stewardship intervention evaluations was convened in response to the fourth call for leading expert network proposals by the Joint Programming Initiative on Antimicrobial Resistance (JPIAMR). The group comprised clinical and academic specialists in antimicrobial stewardship and clinical trial design from six European countries. Group members completed a structured questionnaire to establish the scope of work and key issues to develop ahead of a first face-to-face meeting that (a) identified the need for a comprehensive systematic review of study designs in the literature and (b) prioritized key areas where research design considerations restrict translation of findings into practice. The working group's initial outputs were reviewed by independent advisors and additional expertise was sought in specific clinical areas. At a second face-to-face meeting the working group developed a theoretical framework and specific recommendations to support optimal study design. These were finalized by the working group co-ordinators and agreed by all working group members. RESULTS: We propose a theoretical framework in which consideration of the intervention rationale the intervention setting, intervention features and the intervention aims inform selection and prioritization of outcome measures, whether the research sets out to determine superiority or non-inferiority of the intervention measured by its primary outcome(s), the most appropriate study design (e.g. experimental or quasi- experimental) and the detailed design features. We make 18 specific recommendation in three domains: outcomes, objectives and study design. CONCLUSIONS: Researchers, funders and practitioners will be able to draw on our recommendations to most efficiently evaluate antimicrobial stewardship interventions.

Oral amoxicillin and amoxicillin-clavulanic acid: properties, indications and usage.

BACKGROUND: Amoxicillin has been in use since the 1970s; it is the most widely used penicillin both alone and in combination with the ß-lactamase clavulanic acid. OBJECTIVES: In this narrative review, we re-examine the properties of oral amoxicillin and clavulanic acid and provide guidance on their use, with emphasis on the preferred use of amoxicillin alone. SOURCES: Published medical literature (MEDLINE database via Pubmed). CONTENT: While amoxicillin and clavulanic acid have similar half-lives, clavulanic acid is more protein bound and even less heat stable than amoxicillin, with primarily hepatic metabolism. It is also more strongly associated with gastrointestinal side effects, including Clostridium difficile infection, and, thus, in oral combination formulations, limits the maximum daily dose of amoxicillin that can be given. The first ratio for an amoxicillin-clavulanic acid combination was set at 4:1 due to clavulanic acid's high affinity for ß-lactamases; ratios of 2:1, 7:1, 14:1 and 16:1 are currently available in various regions. Comparative effectiveness data for the different ratios are scarce. Amoxicillin-clavulanic acid is often used as empiric therapy for many of the World Health Organization's Priority Infectious Syndromes in adults and children, leading to extensive consumption, when some of these syndromes could be handled with a delayed antibiotic prescription approach or amoxicillin alone. IMPLICATIONS: Using available epidemiological and pharmacokinetic data, we provide guidance on indications for amoxicillin versus amoxicillin-clavulanic acid and on optimal oral administration, including choice of combination ratio. More data are needed, particularly on heat stability, pharmacodynamic effects and emergence of resistance in 'real-world' clinical settings.

The quality of studies evaluating antimicrobial stewardship interventions: a systematic review.

BACKGROUND: Antimicrobial stewardship aims to optimize antibiotic use and minimize selection of antimicrobial resistance. The methodological quality of published studies in this field is unknown. AIMS: Our objective was to perform a comprehensive systematic review of antimicrobial stewardship research design and identify features which limit validity and translation of research findings into clinical practice. SOURCES: The following online database was searched: PubMed. STUDY ELIGIBILITY CRITERIA: Studies published between January 1950 and January 2017, evaluating any antimicrobial stewardship intervention in the community or hospital setting, without restriction on study design or outcome. CONTENT: We extracted data on pre-specified design quality features and factors that may influence design choices including (1) clinical setting, (2) age group studied, (3) when the study was conducted, (4) geographical region, and (5) financial support received. The initial search yielded 17 382 articles; 1008 were selected for full-text screening, of which 825 were included. Most studies (675/825, 82%) were non-experimental; 104 (15%) used interrupted time series analysis, 41 (6%) used external controls, and 19 (3%) used both. Studies in the community setting fulfilled a median of five out of 10 quality features (IQR 3-7) and 3 (IQR 2-4) in the hospital setting. Community setting studies (25%, 205/825) were significantly more likely to use randomization (OR 5.9; 95% CI 3.8-9.2), external controls (OR 5.6; 95% CI 3.6-8.5), and multiple centres (OR 10.5; 95% CI 7.1-15.7). From all studies, only 48% (398/825) reported clinical and 23% (190/825) reported microbiological outcomes. Quality did not improve over time. IMPLICATIONS: Overall quality of antimicrobial stewardship studies is low and has not improved over time. Most studies do not report clinical and microbiological outcome data. Studies conducted in the community setting were associated with better quality. These limitations should inform the design of future stewardship evaluations so that a robust evidence base can be built to guide clinical practice.

The relationship between Gram-negative colonization and bloodstream infections in neonates: a systematic review and meta-analysis.

OBJECTIVES: Neonates admitted to neonatal intensive care units (NICU) are at significant risk of developing bloodstream infections (BSIs). Gram-negative bacteria (GNB) both colonize and infect, but the association between these entities is unclear. By conducting a systematic literature review, we aimed to explore the impact of factors on the association between GN colonization and GN-BSI at both baby-level and unit-level. METHODS: We searched Medline, Embase, and Cochrane Library. Observational cohort studies published after 2000 up to June 2016 reporting data on the total number of neonates (0-28 days) colonized with GNB assessed by rectal/skin swab culture and the total number of neonates with GN-BSI (same bacteria) were included. Studies were excluded if data on skin/rectal colonization, neonates, and GNB could not be identified separately. Meta-analyses along with multivariate meta-regression with a random-effect model were performed to investigate factors associated with the GN colonization and GN-BSI at baby-level and unit-level. RESULTS: Twenty-seven studies fulfilled our inclusion criteria, 15 for the baby-level and 12 for the unit-level analysis. Study heterogeneity was high, with suboptimal overall quality of reporting assessed by the STROBE-NI statement (44.8% of items adequately reported). In 1984 colonized neonates, 157 (7.9%) developed GN-BSI compared with 85 of 3583 (2.4%) non-colonized neonates. Considerable heterogeneity was observed across studies. Four factors were included in the meta-regression model: gross domestic product (GDP), pathogen, outbreak, and frequency of screening. There was no statistically significant impact of these factors on GN colonization and GN-BSI in baby-level. We were unable to perform the multivariate meta-regression because of insufficient reported data for unit-level. CONCLUSIONS: Study limitations include the small number and the high heterogeneity of the included studies. While this report shows a correlation between colonization and BSI risk, these data currently do not support routine screening for GNB. Analysis of large cohorts of colonized neonates with clinical outcomes is still needed to define the major determinants leading from colonization to infection.

Risk in the "Red Zone": Outcomes for Children Admitted to Ebola Holding Units in Sierra Leone Without Ebola Virus Disease.

We collected data on 1054 children admitted to Ebola Holding Units in Sierra Leone and describe outcomes of 697/1054 children testing negative for Ebola virus disease (EVD) and accompanying caregivers. Case-fatality was 9%; 3/630 (0.5%) children discharged testing negative were readmitted EVD-positive. Nosocomial EVD transmission risk may be lower than feared.

Patterns and trends of pediatric bloodstream infections: a 7-year surveillance study.

We characterize the epidemiology of pediatric bloodstream infections (BSIs) in Switzerland. We analyzed pathogen distribution and resistance patterns in monomicrobial and polymicrobial BSIs in children from 2008 to 2014 using data from the Swiss antibiotic resistance centre (ANRESIS). A confirmatory statistical analysis was performed comparing pathogens and resistance across 20 acute care hospitals. We identified 3,067 bacteremia episodes, of which 1,823 (59 %) were considered true BSI episodes. Overall, S. aureus (16.5 %, 300) was the most frequent pathogen, followed by E. coli (15.1 %, 276), coagulase-negative staphylococci (CoNS, 12.9 %, 235), S. pneumoniae (11.1 %, 202) and non-E. coli Enterobacteriaceae (8.7 %, 159). S. aureus and E. coli showed similar frequencies in all of the variables analyzed (e.g., hospital acquisition, hospital type, medical specialty). The proportion of these microorganisms did not change over time, resistance rates remained low (4.3 % methicillin resistance in S. aureus; 7.3 % third-/fourth-generation cephalosporin resistance in E. coli), and no significant resistance trends were observed. We observed a 50 % increase of CoNS BSIs from 2008 (9.8 %, 27) to 2014 (15.2 %, 46, p value for trend = 0.03). S. pneumoniae decreased from 17.5 % (48) to 6.6 % (20) during that timeframe (p for trend = 0.007). S. aureus and E. coli remained the most significant pathogens among pediatric BSIs in Switzerland, exhibiting low resistance rates. CoNS accounted for a greater proportion of BSIs over time. The decrease in bacteremic pneumococcal infections can likely be attributed to the introduction of the 13-valent conjugate vaccine in 2011.

Surveillance of Gram-negative bacteria: impact of variation in current European laboratory reporting practice on apparent multidrug resistance prevalence in paediatric bloodstream isolates.

This study evaluates whether estimated multidrug resistance (MDR) levels are dependent on the design of the surveillance system when using routine microbiological data. We used antimicrobial resistance data from the Antibiotic Resistance and Prescribing in European Children (ARPEC) project. The MDR status of bloodstream isolates of Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa was defined using European Centre for Disease Prevention and Control (ECDC)-endorsed standardised algorithms (non-susceptible to at least one agent in three or more antibiotic classes). Assessment of MDR status was based on specified combinations of antibiotic classes reportable as part of routine surveillance activities. The agreement between MDR status and resistance to specific pathogen-antibiotic class combinations (PACCs) was assessed. Based on all available antibiotic susceptibility testing, the proportion of MDR isolates was 31% for E. coli, 30% for K. pneumoniae and 28% for P. aeruginosa isolates. These proportions fell to 9, 14 and 25%, respectively, when based only on classes collected by current ECDC surveillance methods. Resistance percentages for specific PACCs were lower compared with MDR percentages, except for P. aeruginosa. Accordingly, MDR detection based on these had low sensitivity for E. coli (2-41%) and K. pneumoniae (21-85%). Estimates of MDR percentages for Gram-negative bacteria are strongly influenced by the antibiotic classes reported. When a complete set of results requested by the algorithm is not available, inclusion of classes frequently tested as part of routine clinical care greatly improves the detection of MDR. Resistance to individual PACCs should not be considered reflective of MDR percentages in Enterobacteriaceae.

Outbreak investigation for toxigenic Corynebacterium diphtheriae wound infections in refugees from Northeast Africa and Syria in Switzerland and Germany by whole genome sequencing.

Toxigenic Corynebacterium diphtheriae is an important and potentially fatal threat to patients and public health. During the current dramatic influx of refugees into Europe, our objective was to use whole genome sequencing for the characterization of a suspected outbreak of C. diphtheriae wound infections among refugees. After conventional culture, we identified C. diphtheriae using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and investigated toxigenicity by PCR. Whole genome sequencing was performed on a MiSeq Illumina with >70×coverage, 2×250 bp read length, and mapping against a reference genome. Twenty cases of cutaneous C. diphtheriae in refugees from East African countries and Syria identified between April and August 2015 were included. Patients presented with wound infections shortly after arrival in Switzerland and Germany. Toxin production was detected in 9/20 (45%) isolates. Whole genome sequencing-based typing revealed relatedness between isolates using neighbour-joining algorithms. We detected three separate clusters among epidemiologically related refugees. Although the isolates within a cluster showed strong relatedness, isolates differed by >50 nucleotide polymorphisms. Toxigenic C. diphtheriae associated wound infections are currently observed more frequently in Europe, due to refugees travelling under poor hygienic conditions. Close genetic relatedness of C. diphtheriae isolates from 20 refugees with wound infections indicates likely transmission between patients. However, the diversity within each cluster and phylogenetic time-tree analysis suggest that transmissions happened several months ago, most likely outside Europe. Whole genome sequencing offers the potential to describe outbreaks at very high resolution and is a helpful tool in infection tracking and identification of transmission routes.

Most international guidelines on prevention of healthcare-associated infection lack comprehensive recommendations for neonates and children.

International infection prevention and control (IPC) guidelines provide standardized recommendations for healthcare-associated infection (HCAI) prevention in adults, but often lack specific information about neonates and children. We reviewed ten international IPC/HCAI guidelines to identify paediatric-specific recommendations for HCAI prevention. Hand hygiene, bloodstream infection, ventilator-associated pneumonia, environmental control and outbreak management were frequently reported with recommendations applicable to children and newborns, but documents on catheter-associated urinary tract infection and surgical site infection were lacking.

Variation in paediatric hospital antibiotic guidelines in Europe.

OBJECTIVE: To assess the availability and source of guidelines for common infections in European paediatric hospitals and determine their content and characteristics. DESIGN: Participating hospitals completed an online questionnaire on the availability and characteristics of antibiotic prescribing guidelines and on empirical antibiotic treatment including duration of therapy for 5 common infection syndromes: respiratory tract, urinary tract, skin and soft tissue, osteoarticular and sepsis in neonates and children. RESULTS: 84 hospitals from 19 European countries participated in the survey of which 74 confirmed the existence of guidelines. Complete guidelines (existing guidelines for all requested infection syndromes) were reported by 20% of hospitals and the majority (71%) used a range of different sources. Guidelines most commonly available were those for urinary tract infection (UTI) (74%), neonatal sepsis (71%) and sepsis in children (65%). Penicillin and amoxicillin were the antibiotics most commonly recommended for respiratory tract infections (RTIs) (up to 76%), cephalosporin for UTI (up to 50%) and for skin and soft tissue infection (SSTI) and bone infection (20% and 30%, respectively). Antistaphylococcal penicillins were recommended for SSTIs and bone infections in 43% and 36%, respectively. Recommendations for neonatal sepsis included 20 different antibiotic combinations. Duration of therapy guidelines was mostly available for RTI and UTI (82%). A third of hospitals with guidelines for sepsis provided recommendations for length of therapy. CONCLUSIONS: Comprehensive antibiotic guideline recommendations are generally lacking from European paediatric hospitals. We documented multiple antibiotics and combinations for most infections. Considerable improvement in the quality of guidelines and their evidence base is required, linking empirical therapy to resistance rates.

Systemic antifungal prescribing in neonates and children: outcomes from the Antibiotic Resistance and Prescribing in European Children (ARPEC) Study.

The appropriate use of systemic antifungals is vital in the prevention and treatment of invasive fungal infection (IFI) in immunosuppressed children and neonates. This multicenter observational study describes the inpatient prescribing practice of antifungal drugs for children and neonates and identifies factors associated with prescribing variability. A single-day point prevalence study of antimicrobial use in hospitalized neonates and children was performed between October and December 2012. The data were entered through a study-specific Web-based portal using a standardized data entry protocol. Data were recorded from 17,693 patients from 226 centers. A total of 136 centers recorded data from 1,092 children and 380 neonates receiving at least one antifungal agent. The most frequently prescribed systemic antifungals were fluconazole (n=355) and amphotericin B deoxycholate (n=195). The most common indications for antifungal administration in children were medical prophylaxis (n=325), empirical treatment of febrile neutropenia (n=122), and treatment of confirmed or suspected IFI (n=100 [14%]). The treatment of suspected IFI in low-birthweight neonates accounted for the majority of prescriptions in the neonatal units (n=103). An analysis of variance (ANOVA) demonstrated no significant effect of clinical indication (prophylaxis or treatment of systemic or localized infection) on the total daily dose (TDD). Fewer than one-half of the patients (n=371) received a TDD within the dosing range recommended in the current guidelines. Subtherapeutic doses were prescribed in 416 cases (47%). The predominance of fluconazole and high incidence of subtherapeutic doses in participating hospitals may contribute to suboptimal clinical outcomes and an increased predominance of resistant pathogenic fungi. A global consensus on antifungal dosing and coordinated stewardship programs are needed to promote the consistent and appropriate use of antifungal drugs in neonates and children.

Time-course of leptin levels in term and preterm human milk.

OBJECTIVE: To compare the time-course of breast milk leptin levels between term and preterm pregnancy. DESIGN: Open longitudinal prospective randomised study. METHODS: RIA of leptin levels in milk from 33 mothers (term pregnancy: n=24; preterm: n=9) at three postpartum intervals: 2-3 days, 4-5 days and 6 weeks (intervals A, B and C), combined with serum in 23 mothers (term: n=17; preterm: n=6) in interval A. Milk samples were sonicated before incubation. RESULTS: Interval A leptin levels were approximately tenfold higher in serum than in milk (term: 13.24+/-2.48 vs 1.34+/-0.14 ng/ml, P<0.0001; preterm: 4.46+/-1.05 vs 0.63+/-0.18 ng/ml, P<0.0005), and higher in term than in preterm serum (P=0.03). Milk levels were higher in the term vs preterm group in intervals A (P<0.01) and B (P<0.05). In the term group they declined significantly from interval A to interval B (P<0.05) but did not vary significantly in the preterm group. Serum levels correlated with maternal body mass index; milk levels showed only moderate correlation with maternal and infant weight or body mass index. CONCLUSION: The reasons for the presence and differential longitudinal expression of leptin in human milk after term and preterm pregnancy remain unknown. A hypothesis, requiring further study, is that persistently lower leptin levels in preterm milk act as a compensatory release of a brake on neonatal growth.

Cysteine substitutions in apolipoprotein A-I primary structure modulate paraoxonase activity.

Paraoxonase (PON) is transported primarily on apolipoprotein A-I (apoA-I) -containing high-density lipoprotein (HDL) and is thought to protect against early atherogenic events including low-density lipoprotein (LDL) oxidation and monocyte migration. It has been proposed that apoA-I may be necessary for PON's association with plasma HDL. On the basis of this, we examined the effect of apoA-I on PON's enzymatic activity and its ability to associate with HDL. Additionally, we examined whether changes in apoA-I primary structure (cysteine substitution mutations) could modulate these effects. Chinese hamster ovary cells stably transfected with human PON1A cDNA were incubated in the presence and absence of recombinant wild-type apoA-I (apoA-I(WT)) and specific Cys substitution mutations. Extracellular accumulation of PON activity in the presence of apoA-I(WT) was 0.095 +/- 0.013 unit/mg of cell protein (n = 7) compared to 0.034 +/- 0.010 unit/mg of cell protein in the absence of apoA-I (n = 7), a 2.79-fold increase in activity when apoA-I was incubated with the cells. Lipid-free apoA-I did not increase PON activity, while preformed nascent HDL increased PON activity only 30%, suggesting that maximal PON activity is lipid-dependent and requires coassembly of PON and apoA-I on nascent HDL. The cysteine mutations R10C, R27C, and R61C significantly increased (p < 0.01) PON activity 32.6% +/- 14.7%, 31.6% +/- 18.9%, and 27.4% +/- 20%, respectively, over that of wild type (WT). No changes in PON activity were observed with apoA-I cysteine substitution mutations in the C-terminal portion of the protein. The data suggest that, for optimal PON activity, coassembly of the enzyme onto nascent HDL is required and that the N-terminal region of apoA-I may be important in the assembly process.

Baculovirus-mediated expression and purification of human serum paraoxonase 1A.

Human paraoxonase 1 (hPON1) is a lipid-associated enzyme transported on HDL. There is considerable interest in hPON1 because of its putative antioxidative/antiatherogenic properties. We have created a recombinant baculovirus (BV) to generate hPON1A in large quantities for structure-function studies and here describe the method for production and isolation of the enzyme. A high level of recombinant hPON1 type A (rPON1A) was produced by Hi-5 insect cells (40 mg/l); a fraction ( approximately 10 mg/l) was secreted into the cell culture medium, but the majority ( approximately 30 mg/l) remained associated with the host insect cells. Cell-associated rPON1A was purified by detergent extraction (Tergitol NP-10) followed by three simple chromatography steps (DEAE-Sepharose, Sephacryl S-200, and concanavalin A). The purified enzyme bound to concanavalin A and was converted to a lower molecular mass by endoglycosidase H digestion, suggesting that rPON1A contained high-mannose N-glycan chains. There was a significant decrease in arylesterase activity (>99%) concomitant with enzymatic deglycosylation. rPON1A was dependent on Ca(2+) for arylesterase activity, exhibiting kinetic parameters similar to native hPON1A (K(m) = 3.8 +/- 2.1 vs. 3.7 +/- 2.0 mM and V(max) = 1,305 +/- 668 vs. 1,361 +/- 591 U/mg protein, rPON1A and hPON1A, respectively). Both rPON1A and hPON1A efficiently inhibited lipoxygenase-mediated peroxidation of phospholipid. In contrast to the arylesterase activity, which was sensitive to endoglycosidase H treatment, enzymatic deglycosylation did not inhibit the antioxidant activity of rPON1A. In conclusion, our BV-mediated PON1A expression system appears ideally suited for the production of relatively large quantities of rPON1A for structure-function studies.

Regulation and activity of the human ABCA1 gene in transgenic mice.

The ABCA1 transporter is one of the limiting steps in cellular cholesterol efflux. To study the expression and activity of the human ABCA1 gene in vivo we have examined mice containing two human BAC transgenes with different 5' ends. Mice containing a 255-kilobase (kb) BAC transgene, including 70 kb upstream of the previously defined exon 1, demonstrated a pattern of tissue-specific expression mimicking that of the endogenous mouse gene. Compared with macrophages from control mice, macrophages from these transgenics had increases in apoA-I cholesterol efflux heightened in response to increases in cell cholesterol content. The observed increase in macrophage apoA-I-mediated cholesterol efflux was not accompanied by alterations in plasma high density lipoprotein in the transgenics. Although mice containing a smaller 171-kb human BAC transgene, lacking the previously described exon 1 and ABCA1 promoter, did not express human ABCA1 in macrophages, they did express the human transgene in liver at levels comparable with those of the orthologous mouse gene. Analysis by 5' rapid amplification of cDNA ends of liver mRNA from these animals revealed a new ABCA1 exon 1 (exon 1A) and a previously unrecognized promoter. Analysis of human tissue revealed that exon 1A containing transcripts accounted for a high proportion of the ABCA1 mRNAs present in human liver. This analysis of ABCA1 transgenics showed that the expression of human ABCA1 transgenes can result in increased cholesterol efflux from macrophages, unaccompanied by changes in plasma high density lipoprotein, and identified a new ABCA1 promoter in humans.