Optimizing the Design and Analysis of Clinical Trials for Antibacterials Against Multidrug-resistant Organisms: A White Paper From COMBACTE's STAT-Net.

Innovations are urgently required for clinical development of antibacterials against multidrug-resistant organisms. Therefore, a European, public-private working group (STAT-Net; part of Combatting Bacterial Resistance in Europe [COMBACTE]), has reviewed and tested several innovative trials designs and analytical methods for randomized clinical trials, which has resulted in 8 recommendations. The first 3 focus on pharmacokinetic and pharmacodynamic modeling, emphasizing the pertinence of population-based pharmacokinetic models, regulatory procedures for the reassessment of old antibiotics, and rigorous quality improvement. Recommendations 4 and 5 address the need for more sensitive primary end points through the use of rank-based or time-dependent composite end points. Recommendation 6 relates to the applicability of hierarchical nested-trial designs, and the last 2 recommendations propose the incorporation of historical or concomitant trial data through Bayesian methods and/or platform trials. Although not all of these recommendations are directly applicable, they provide a solid, evidence-based approach to develop new, and established, antibacterials and address this public health challenge.

Development of a Novel Collagen Wound Model To Simulate the Activity and Distribution of Antimicrobials in Soft Tissue during Diabetic Foot Infection.

Diabetes has major implications for public health, with diabetic foot ulcers (DFUs) being responsible for significant morbidity and mortality. A key factor in the development of nonhealing ulcers is infection, which often leads to the development of biofilm, gangrene, and amputation. A novel approach to treating DFUs is the local release of antibiotics from calcium sulfate beads. We have developed a novel model system to study and compare the release and efficacy of antibiotics released locally, using collagen as a substrate for biofilm growth and incorporating serum to mimic the biochemical complexity of the wound environment. We found that our soft-tissue model supports the growth of a robust Pseudomonas aeruginosa biofilm, and that this was completely eradicated by the introduction of calcium sulfate beads loaded with tobramycin or gentamicin. The model also enabled us to measure the concentration of these antibiotics at different distances from the beads and in simulated wound fluid bathing the collagen matrix. We additionally found that a multidrug-resistant Staphylococcus aureus biofilm, nonsusceptible to antibiotics, nonetheless showed an almost 1-log drop in viable counts when exposed to calcium sulfate beads combined with antibiotics. Together, these data suggest that locally applied antibiotics combined with calcium sulfate provide surprising efficacy in diabetic foot infections and offer an effective alternative approach to infection management. Our study additionally establishes our new system as a biochemically and histologically relevant model that may be used to study the effectiveness of a range of therapies locally or systemically for infected DFUs.

Amikacin use and therapeutic drug monitoring in adults: do dose regimens and drug exposures affect either outcome or adverse events? A systematic review.

OBJECTIVES: The objectives of this study were to identify the amikacin dosage regimens and drug concentrations consistent with good outcomes and to determine the drug exposures related to nephrotoxicity and ototoxicity. METHODS: A literature review was conducted in Medline, EMBASE and the Cochrane Central Register of Controlled Trials. Full journal articles reporting randomized controlled trials, controlled clinical trials, interrupted time series trials, and controlled before and after studies involving amikacin therapeutic drug monitoring (TDM) and dose adjustment were considered for inclusion. RESULTS: Seventeen studies for inclusion were identified, comprising 1677 participants. Amikacin doses ranged from 11 to 15 mg/kg/day with 13 studies using 15 mg/kg/day. Studies were generally designed to compare different aminoglycosides rather than to assess concentration-effect relationships. Only 11 papers presented data on target concentrations, rate of clinical cure and toxicity. Target peak concentrations ranged from 15 to 40 mg/L and target troughs were typically <10 or <5 mg/L. It was not clear whether these targets were achieved. Measured peaks averaged 28 mg/L for twice-daily dosing and 40-45 mg/L for once-daily dosing; troughs averaged 5 and 1-2 mg/L, respectively. Fifteen of the included studies reported rates of nephrotoxicity; auditory and vestibular toxicities were reported in 12 and 8 studies. CONCLUSIONS: This systematic review found little published evidence to support an optimal dosage regimen or TDM targets for amikacin therapy. The use of alternative approaches, such as consensus opinion and a review of current practice, will be required to develop guidelines to maximize therapeutic outcomes and minimize toxicity with amikacin.

The role of microbial biofilms in prosthetic joint infections.

Prosthetic joint infection (PJI) still remains a significant problem. In line with the forecasted rise in joint replacement procedures, the number of cases of PJI is also anticipated to rise. The formation of biofilm by causative pathogens is central to the occurrence and the recalcitrance of PJI. The subject of microbial biofilms is receiving increasing attention, probably as a result of the wide acknowledgement of the ubiquity of biofilms in the natural, industrial, and clinical contexts, as well as the notorious difficulty in eradicating them. In this review, we discuss the pertinent issues surrounding PJI and the challenges posed by biofilms regarding diagnosis and treatment. In addition, we discuss novel strategies of prevention and treatment of biofilm-related PJI.

The TARGET cohort study protocol: a prospective primary care cohort study to derive and validate a clinical prediction rule to improve the targeting of antibiotics in children with respiratory tract illnesses.

BACKGROUND: Children with respiratory tract infections are the single most frequent patient group to make use of primary care health care resources. The use of antibiotics remains highly prevalent in young children, but can lead to antimicrobial resistance as well as reinforcing the idea that parents should re-consult for similar symptoms. One of the main drivers of indiscriminate antimicrobial use is the lack of evidence for, and therefore uncertainty regarding, which children are at risk of poor outcome. This paper describes the protocol for the TARGET cohort study, which aims to derive and validate a clinical prediction rule to identify children presenting to primary care with respiratory tract infections who are at risk of hospitalisation. METHODS/DESIGN: The TARGET cohort study is a large, multicentre prospective observational study aiming to recruit 8,300 children aged ≥3 months and <16 years presenting to primary care with a cough and respiratory tract infection symptoms from 4 study centres (Bristol, London, Oxford and Southampton). Following informed consent, symptoms, signs and demographics will be measured. In around a quarter of children from the Bristol centre, a single sweep, dual bacterial-viral throat swab will be taken and parents asked to complete a symptom diary until the child is completely well or for 28 days, whichever is sooner. A review of medical notes including clinical history, re-consultation and hospitalisations will be undertaken. Multivariable logistic regression will be used to identify the independent clinical predictors of hospitalisation as well as the prognostic significance of upper respiratory tract microbes. The clinical prediction rule will be internally validated using various methods including bootstrapping. DISCUSSION: The clinical prediction rule for hospitalisation has the potential to help identify a small group of children for hospitalisation and a much larger group where hospitalisation is very unlikely and antibiotic prescribing would be less warranted. This study will also be the largest natural history study to date of children presenting to primary care with acute cough and respiratory tract infections, and will provide important information on symptom duration, re-consultations and the microbiology of the upper respiratory tract.

Exploring the Pharmacokinetics of Phenoxymethylpenicillin (Penicillin-V) in Adults: A Healthy Volunteer Study.

This healthy volunteer study aimed to explore phenoxymethylpenicillin (penicillin-V) pharmacokinetics (PK) to support the planning of large dosing studies in adults. Volunteers were dosed with penicillin-V at steady state. Total and unbound penicillin-V serum concentrations were determined, and a base population PK model was fitted to the data.

Susceptibility of monomicrobial or polymicrobial biofilms derived from infected diabetic foot ulcers to topical or systemic antibiotics in vitro.

Diabetic foot ulcers can become chronic and non-healing despite systemic antibiotic treatment. The penetration of systematically-administered antibiotics to the site of infection is uncertain, as is the effectiveness of such levels against polymicrobial biofilms. We have developed an in vitro model to study the effectiveness of different treatments for infected diabetic foot ulcers in a wound-like environment and compared the activity of systemic levels of antibiotics with that for topically applied antibiotics released from calcium sulfate beads. This is the first study that has harvested bacteria from diabetic foot infections and recreated similar polymicrobial biofilms to those present in vivo for individual subjects. After treatment with levels of gentamicin attained in serum after systemic administration (higher than corresponding tissues concentrations) we measured a 0-2 log reduction in bacterial viability of P. aeruginosa, S. aureus or a polymicrobial biofilm. Conversely, addition of gentamicin loaded calcium sulfate beads resulted in 5-9 log reductions in P. aeruginosa, S aureus and polymicrobial biofilms derived from three subjects. We conclude that systemically administered antibiotics are likely to be inadequate for successfully treating these infections, especially given the vastly increased concentrations required to inhibit cells in a biofilm, and that topical antibiotics provide a more effective alternative.

Assay of ertapenem in human serum by high-performance liquid chromatography.

Ertapenem is a new carbapenem with a broad spectrum of activity and an extended half-life, permitting once daily administration. Although high-performance liquid chromatography (HPLC) methods have been described for ertapenem, these are complex and involve column switching and thus this type of assay may not suitable for use in routine clinical microbiology laboratories. In this study we report a rapid, straightforward HPLC method for the detection of ertapenem in human serum.

Evidence of excessive concentrations of 5-flucytosine in children aged below 12 years: a 12-year review of serum concentrations from a UK clinical assay reference laboratory.

5-flucytosine (5-FC) is an antifungal drug used for the treatment of serious infections caused by Candida or Cryptococcus spp. In the UK, the recommended pre- and post-dose serum therapeutic ranges are 30-40 mg/L and 70-80 mg/L, respectively. A 12-year retrospective review of serum concentrations of 5-FC in three groups of children aged 1-30 days (n=167), 31-60 days (n=102) and 91 days to 12 years (n=122) was conducted. In these three age groups, 65.1%, 44.4% and 21.3% of pre-dose samples and 39.3%, 29.2% and 19.7% of post-dose samples were above the recommended ranges. Both the mean concentration and the percentage of concentrations above the recommended ranges were significantly higher in the youngest age group (1-30 days old), suggesting that the standard dose of 100 mg/kg daily may not be an appropriate dose in this age group.

Development and internal validation of a clinical rule to improve antibiotic use in children presenting to primary care with acute respiratory tract infection and cough: a prognostic cohort study.

BACKGROUND: Antimicrobial resistance is a serious threat to public health, with most antibiotics prescribed in primary care. General practitioners (GPs) report defensive antibiotic prescribing to mitigate perceived risk of future hospital admission in children with respiratory tract infections. We developed a clinical rule aimed to reduce clinical uncertainty by stratifying risk of future hospital admission. METHODS: 8394 children aged between 3 months and 16 years presenting with acute cough (for ≤28 days) and respiratory tract infection were recruited to a prognostic cohort study from 247 general practitioner practices in England. Exposure variables included demographic characteristics, parent-reported symptoms, and physical examination signs. The outcome was hospital admission for respiratory tract infection within 30 days, collected using a structured, blinded review of medical records. FINDINGS: 8394 (100%) children were included in the analysis, with 78 (0·9%, 95% CI 0·7%-1·2%) admitted to hospital: 15 (19%) were admitted on the day of recruitment (day 1), 33 (42%) on days 2-7; and 30 (39%) on days 8-30. Seven characteristics were independently associated (p<0·01) with hospital admission: age <2 years, current asthma, illness duration of 3 days or less, parent-reported moderate or severe vomiting in the previous 24 h, parent-reported severe fever in the previous 24 h or a body temperature of 37·8°C or more at presentation, clinician-reported intercostal or subcostal recession, and clinician-reported wheeze on auscultation. The area under the receiver operating characteristic (AUROC) curve for the coefficient-based clinical rule was 0·82 (95% CI 0·77-0·87, bootstrap validated 0·81). Assigning one point per characteristic, a points-based clinical rule consisting of short illness, temperature, age, recession, wheeze, asthma, and vomiting (mnemonic STARWAVe; AUROC 0·81, 0·76-0·85) distinguished three hospital admission risk strata: very low (0·3%, 0·2-0·4%) with 1 point or less, normal (1·5%, 1·0-1·9%) with 2 or 3 points, and high (11·8%, 7·3-16·2%) with 4 points or more. INTERPRETATION: Clinical characteristics can distinguish children at very low, normal, and high risk of future hospital admission for respiratory tract infection and could be used to reduce antibiotic prescriptions in primary care for children at very low risk. FUNDING: National Institute for Health Research (NIHR).

Clinical presentation and microbiological diagnosis in paediatric respiratory tract infection: a systematic review.

BACKGROUND: Antibiotic prescribing decisions for respiratory tract infection (RTI) in primary care could be improved if clinicians could target bacterial infections. However, there are currently no evidence-based diagnostic rules to identify microbial aetiology in children presenting with acute RTIs. AIM: To analyse evidence of associations between clinical symptoms or signs and detection of microbes from the upper respiratory tract (URT) of children with acute cough. DESIGN AND SETTING: Systematic review and meta-analysis. METHOD: A literature search identified articles reporting relationships between individual symptoms and/or signs, and microbes detected from URT samples. Associations between pathogens and symptoms or signs were summarised, and meta-analysis conducted where possible. RESULTS: There were 9984 articles identified, of which 28 met inclusion criteria. Studies identified 30 symptoms and 41 signs for 23 microbes, yielding 1704 potential associations, of which only 226 (13%) have presently been investigated. Of these, relevant statistical analyses were presented for 175 associations, of which 25% were significant. Meta-analysis demonstrated significant relationships between respiratory syncytial virus (RSV) detection and chest retractions (pooled odds ratio [OR] 1.9, 95% confidence interval [CI] = 1.6 to 2.3), wheeze (pooled OR 1.7, 95% CI = 1.5 to 2.0), and crepitations/crackles (pooled OR 1.7, 95% CI = 1.3 to 2.2). CONCLUSIONS: There was an absence of evidence for URT pathogens other than RSV. The meta-analysis identified clinical signs associated with RSV detection, suggesting clinical presentation may offer some, albeit poor, diagnostic value. Further research is urgently needed to establish the value of symptoms and signs in determining microbiological aetiology and improve targeting of antibiotics in primary care.

Determination of avilamycin A and B in pig faeces by solid phase extraction and reverse-phase HPLC assay.

A HPLC method is described for the simultaneous determination of avilamycin A and B in pig faeces, following extraction using acetonitrile and normal-phase solid phase extraction. The HPLC stationary phase was Kromosil 5 micro C-18 with a mobile phase of 48% acetonitrile and 52% 0.01N ammonium acetate buffer, pumped at a flow rate of 1 ml/min. Detection was by UV absorbance at 295 nm and an injection volume of 50 microl was used. Recovery from faeces was >98% and intra-assay precision (CV) was <9.0% for both compounds. The lowest limit of quantification was 0.9 mg/kg (avilamycin A) and 0.2 mg/kg (avilamycin B) with an accuracy of <15% error. No interference was seen from endogenous materials in pig faeces and commonly used veterinary antibiotics.

A reverse-phase HPLC assay for the simultaneous determination of enrofloxacin and ciprofloxacin in pig faeces.

A reverse-phase HPLC assay is described for the simultaneous assay of enrofloxacin (ENR) and ciprofloxacin (CPX) in pig faeces. Extraction used dichloromethane, 2-propanol and 0.3M ortho-phosphoric acid (1:5:4 v/v/v). Separation was achieved using a Spherisorb S5 C8 column, heated to 50 degrees C and a mobile phase of 0.16% ortho-phosphoric acid (adjusted to pH 3.0 with tetrabutylammonium hydroxide solution) with 20 ml acetonitrile per litre solution. The method used fluorescence detection (Ex 310 nm; Em 445 nm), a flow rate of 1 ml/min and a 20 microl injection volume. Retention times were approximately 6 min for ciprofloxacin and 10 min for enrofloxacin. The linearity range for both compounds was 0-20 mg/kg, lowest limit of quantification 0.3 mg/kg and recoveries were >92%.

Impact of soaking gentamicin-containing collagen implants on potential antimicrobial efficacy.

BACKGROUND: The purpose of this study is to evaluate how wetting of Collatamp (a gentamicin-containing collagen implant [GCCI]) impacts on the gentamicin content of the implant and whether this affects its potential antibacterial efficacy. METHODS: GCCI (Collatamp(®), EUSA Pharma [Europe], Oxford, United Kingdom) containing 130 mg gentamicin and 280 mg collagen (10 cm × 10 cm) were immersed in 300 mL normal saline for up to 6h. At set times after immersion the GCCI were removed, the saline diluted in normal human serum and the gentamicin content assayed by a validated immunoassay (Cedia, Microgenics Ltd, UK) to provide an estimate of the loss from each implant. The mean concentration data were then fitted to an exponential decay model (WinNonLin, Pharsight, US). RESULTS: After a very short immersion period there was significant loss of gentamicin from the implants with a mean loss of 6.7% at 2 s, increasing to 40.5% at 1 min and essentially total loss by 6 h of immersion. Loss of gentamicin followed a complex elution profile, with elution half-lives ranging from 50 s on initial immersion to 99 min late in the elution period. CONCLUSION: This study provides clear evidence that even a short period of dipping of Collatamp implants, and probably other GCCI, before insertion into the patient results in a significant loss of gentamicin which may be of clinical significance unless the period of soaking is very short. We therefore recommend that wetting of these implants before insertion is not undertaken.

The effect of a modified posterior approach on blood flow to the femoral head during hip resurfacing.

The deep branch of the medial femoral circumflex artery is vulnerable to injury during posterior approaches to the hip. We modified the posterior approach during hip resurfacing in 10 patients by dividing the short external rotators 2 cm from their insertion into the femur. The cefuroxime concentrations in bone samples from the femoral heads were compared with results for the extended posterolateral approach from previous published work. There was no difference between the concentration of cefuroxime in bone when using the modified posterior approach (mean 5.6 mg/kg; CI 3.6 - 7.8) compared to the extended posterolateral approach (mean 5.6 mg/kg; CI 3.5 - 7.8; p=0.95). The similarity in femoral head perfusion between approaches suggests that the blood supply may be impaired further by capsulotomy and capsulectomy rather than by damage to the deep branch of the MFCA alone.

Concentration and bioavailability of ciprofloxacin and teicoplanin in the cornea.

PURPOSE: To investigate the concentration and bioavailability of ciprofloxacin and teicoplanin in the cornea. METHODS: A biological assay was developed with corneal tissue used as a carrier for the antimicrobial. Concentration and biological activity were determined with a chemical assay and zone of inhibition (ZOI) around corneal samples with epithelial and endothelial surfaces in contact with the indicator organism. Patients undergoing penetrating keratoplasty received ciprofloxacin 0.3% or teicoplanin 1%. RESULTS: There were good correlations between antimicrobial concentration and ZOI, when either filter paper or corneal discs were used (R(2) > 92%). Of 33 patients, the mean (median) concentration of ciprofloxacin in the cornea was 1.37 mg/L (0.46 mg/L) and 1.89 mg/L (1.44 mg/L; bioassay) in the epithelial and endothelial orientations, respectively, and 14.87 mg/L (7.41) in the cornea and 0.51 mg/L (0.42) in the aqueous (chemical assay). For teicoplanin, the mean (median) concentration in the cornea was 9.58 mg/L (0 mg/L) in the epithelial and 4.78 mg/L (0 mg/L) in the endothelial orientations (bioassay). In the chemical assay, teicoplanin could not be detected in the cornea or aqueous at the lower limit of detection of 3.6 mg/L. CONCLUSIONS: The ZOI produced by corneal tissue provides a potential bioassay of antimicrobial activity and concentration. Although in contrast to teicoplanin ciprofloxacin shows good corneal penetration, with high endothelial-to-epithelial levels, only approximately 10% of measured levels in a chemical assay are available, according to a bioassay. Teicoplanin shows relatively poor corneal penetration through intact epithelium. These methods may be useful in evaluating the biological activity across the cornea of antimicrobials introduced into ophthalmic practice to deal with changing bacterial resistance.

Gentamicin concentrations in diagnostic aspirates from 25 patients with hip and knee arthroplasties.

BACKGROUND: There is little information on long-term release of antibiotics from impregnated bone cement. PATIENTS AND METHODS: We assayed joint fluids obtained for diagnostic purposes from 25 patients for the presence of gentamicin. All patients had presented with failing or painful joints up to 20 years following primary hip or knee arthroplasty, using gentamicin-impregnated cement. RESULTS: Gentamicin was detected in the joint fluids from 9 of 15 patients with knee prostheses and 4 of 10 patients with hip prostheses. The concentrations ranged from 0.06 mg/L to 0.85 mg/L with no significant differences in concentration between the patients with hip or knee prostheses, or the type of prosthesis. We found no relationship between the gentamicin concentration and the time after primary arthroplasty. INTERPRETATION: Although most concentrations were below the levels required to inhibit susceptible pathogens, we conclude that gentamicin release around failing implants may lead to false negative cultures in some patients and provide selective pressure for the emergence of resistance where infection is present in others.

Determination by HPLC of chlortetracycline in pig faeces.

An HPLC assay used to determine chlortetracycline (CTC) in pig faeces is reported. Prodigy ODS3 (4.6 x 150 mm) was used for the stationary phase, whereas the mobile phase comprised oxalic acid, sodium oxalate and sodium decane sulfonate (66%)--each of 4 mM, and 34% acetonitrile. The mobile phase was pumped at a flow rate of 1 mL/min. Detection of CTC was by ultraviolet absorbance at 370 nm, and a 20 micro L injection volume was used. Recovery from faeces was >90%, and coefficients of variability between runs were <10%. The lowest limit of quantification was 3.5 mg/kg, with an accuracy of <7% error. There was no interference from endogenous materials in the pig faeces, or commonly used antibiotics, and the method is suitable for use in drug disposition studies.