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Objectives To evaluate a testing algorithm for the rapid identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that includes the use of PCR-based targeted single nucleotide polymorphism (SNP) detection assays preceded by a multiplex PCR sensitive to S-Gene Target Failure (SGTF). Methods PCR SNP assays targeting SARS-CoV-2 S-gene mutations ΔH69–V70, L452R, E484K, N501Y, H655Y and P681R using melting curve analysis were performed on 567 samples in which SARS-CoV-2 viral RNA was detected by a multiplex PCR. Viral whole-genome sequencing (WGS) was performed to confirm the presence of SNPs and to identify the Pangolin lineage. Additionally, 1133 SARS-CoV-2 positive samples with SGTF were further assessed by WGS to determine the presence of ΔH69–V70. Results The N501Y-specific assay (n = 567) had an overall percentage agreement (OPA) of 98.5%. The ΔH69-V70-specific (n = 178) and E484K-specific (n = 401) assays had OPA of 96.6% and 99.7%, respectively. Assessment of H655Y (n = 139) yielded a 100.0% concordance when applied in the proposed algorithm. The L452R-specific (n = 67) and P681R-specific (n = 62) assays had an OPA of 98.2% and 98.1%, respectively. The proposed algorithm identified six variants of concern/interest (VOC/VOI)—Alpha (n = 149), Beta (n = 65), Gamma (n = 86), Delta (n = 49), Eta (n = 6), Kappa (n = 6)—and 205 non-VOC/VOI strains—including the variants under monitoring B.1.214.2 (n = 43) and B.1.1.318 (n = 18) and Epsilon (n = 1). An excellent concordance was observed for the identification of all SARS-CoV-2 lineages evaluated. Conclusions We present a flexible testing algorithm for the rapid detection of current and emerging SARS-CoV-2 VOC/VOIs, which can be easily adapted based on the local endemicity of specific variants.

Objectives: This study determined associations between respiratory viruses and subsequent illness course in primary care adult patients presenting with acute cough and/or suspected lower respiratory tract infection. Methods: A prospective European primary care study recruited adults with symptoms of lower respiratory tract infection between November 2007 and April 2010. Real-time in-house polymerase chain reaction (PCR) was performed to test for six common respiratory viruses. In this secondary analysis, symptom severity (scored 1 = no problem, 2 = mild, 3 = moderate, 4 = severe) and symptom duration were compared between groups with different viral aetiologies using regression and Cox proportional hazard models, respectively. Additionally, associations between baseline viral load (cycle threshold (Ct) value) and illness course were assessed. Results: The PCR tested positive for a common respiratory virus in 1354 of the 2957 (45.8%) included patients. The overall mean symptom score at presentation was 2.09 (95% confidence interval (CI) 2.07-2.11) and the median duration until resolution of moderately bad or severe symptoms was 8.70 days (interquartile range 4.50-11.00). Patients with influenza virus, human metapneumovirus (hMPV), respiratory syncytial virus (RSV), coronavirus (CoV) or rhinovirus had a significantly higher symptom score than patients with no virus isolated (0.07-0.25 points or 2.3-8.3% higher symptom score). Time to symptom resolution was longer in RSV infections (adjusted hazard ratio (AHR) 0.80, 95% CI 0.65-0.96) and hMPV infections (AHR 0.77, 95% CI 0.62-0.94) than in infections with no virus isolated. Overall, baseline viral load was associated with symptom severity (difference 0.11, 95% CI 0.06-0.16 per 10 cycles decrease in Ct value), but not with symptom duration. Conclusions: In healthy, working adults from the general community presenting at the general practitioner with acute cough and/or suspected lower respiratory tract infection other than influenza impose an illness burden comparable to influenza. Hence, the public health focus for viral respiratory tract infections should be broadened. (C) 2020 Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases.

Background: Neonatal sepsis is associated with high mortality, but there is limited clinical data on outcomes in hospital facilities in low and middle income countries (LMIC). The NeoOBS study aimed to determine the clinical features, pathogens and clinical outcomes of infants presenting with neonatal sepsis in hospitals globally. Methods: Infants

Background: The COVID-19 pandemic has resulted in many health systems worldwide facing an imbalance between patients’ need and available resources. No previous studies have developed explicit recommendations for selecting patients most likely to benefit from hospitalisation in settings with an unsolvable hospital-bed shortage. Methods: Multi-Criteria Decision Analysis (MCDA) was used to determine weights for criteria to prioritise COVID-19 patients for admission to hospital. The MCDA was applied in two main steps: specification of criteria for prioritising COVID-19 patients, and levels within each criterion; and determination of weights for the criteria based on experts’ knowledge and experience in managing COVID-19 patients, via an online survey. Criteria were selected based on available COVID-19 evidence with a focus on healthcare settings with limited resources. Findings: Eleven criteria for prioritising patients were specified, and 103 MDs caring for COVID-19 patients completed the survey to determine the criteria’s weights. The 11 criteria (and levels), in decreasing order of importance [mean weights, summing to 100%], are: PaO2 (>80, 70-80, and 96%, 92-96%, and 20 breaths/min) [9∙5%]; comorbidities (diabetes, respiratory/cardiovascular diseases, and onco-haematological diseases) [6.5%]; living with vulnerable people (people with comorbidities, pregnant women, and immunosuppressed patients) [6∙4%]; body mass index ( 40) [5∙6%]; duration of symptoms before hospital evaluation ( 7 days) [5∙4%]; CRP (normal / high based on local cut off) [5∙1%]; and age (18-50, 50-70, and >70) [3∙8%]. Interpretation: MCDA can be used for prioritising patients for hospitalisation when prioritisation is necessary and evidence for disease predictors is limited or unavailable. MCDA is likely to be especially useful in low- and middle-income countries where effective national contingency plans are difficult to establish. Funding Statement: This project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No 820755. This Joint Undertaking receives support from the European Union’s Horizon 2020 research and innovation programme and EFPIA and bioMerieux SA, Janssen Pharmaceutica NV, Accelerate Diagnostics S.L., Abbott, Bio-Rad Laboratories, BD Switzerland Sarl, and The Wellcome Trust Limited. Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: There were no humans participating in the research project. Briefly, in the study a list of variables/criteria W ere selected from the scientific literature and the Multi-Criteria Decision Analysis (based on PAPRIKA method) was applied in order to give a weight/score (total 100%) to each criterion. For this reason, the study was exempted from ethical review/clearance.

Objectives We quantified the impact of antibiotics prescribed in primary care for urinary tract infections (UTIs) on intestinal colonization by ciprofloxacin-resistant (CIP-RE) and extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-PE), while accounting for household clustering. Methods Prospective cohort study from January 2011 to August 2013 at primary care sites in Belgium, Poland and Switzerland. We recruited outpatients requiring antibiotics for suspected UTIs or asymptomatic bacteriuria (exposed patients), outpatients not requiring antibiotics (non-exposed patients), and one to three household contacts for each patient. Faecal samples were tested for CIP-RE, ESBL-PE, nitrofurantoin-resistant Enterobacteriaceae (NIT-RE) and any Enterobacteriaceae at baseline (S1), end of antibiotics (S2) and 28 days after S2 (S3). Results We included 300 households (205 exposed, 95 non-exposed) with 716 participants. Most exposed patients received nitrofurans (86; 42%) or fluoroquinolones (76; 37%). CIP-RE were identified in 16% (328/2033) of samples from 202 (28%) participants. Fluoroquinolone treatment caused transient suppression of Enterobacteriaceae (S2) and subsequent two-fold increase in CIP-RE prevalence at S3 (adjusted prevalence ratio (aPR) 2.0, 95% CI 1.2–3.4), with corresponding number-needed-to-harm of 12. Nitrofurans had no impact on CIP-RE (aPR 1.0, 95% CI 0.5–1.8) or NIT-RE. ESBL-PE were identified in 5% (107/2058) of samples from 71 (10%) participants, with colonization not associated with antibiotic exposure. Household exposure to CIP-RE or ESBL-PE was associated with increased individual risk of colonization: aPR 1.8 (95% CI 1.3–2.5) and 3.4 (95% CI 1.3–9.0), respectively. Conclusions These findings support avoidance of fluoroquinolones for first-line UTI therapy in primary care, and suggest potential for interventions that interrupt household circulation of resistant Enterobacteriaceae .

Background: Antivirals are infrequently prescribed in primary care for influenza-like-illness (ILI), mostly because of perceived ineffectiveness in real world primary care, and as individuals who will especially benefit have not been identified in independent trials. We aimed to determine whether adding antiviral treatment to usual primary care for patients with ILI reduces time to recovery overall and in key subgroups. Methods: We conducted an open-label, pragmatic, randomized controlled trial of adding oseltamivir to usual care in patients aged one year and older consulting with ILI in primary care. The primary endpoint was time to recovery (return to usual activities, with fever, head- and muscle-ache minor/absent), following a Bayesian piece-wise exponential model. Baseline nasopharyngeal swabs were analyzed after study completion. Findings: We ascertained the primary outcome for 3059 of the 3266 participants recruited in 15 European countries during three seasonal influenza seasons (2015-2018); 52.0% (1590/3059) had PCR-confirmed influenza infection. Time to recovery was shorter in those given oseltamivir by 1.02 days (95% Bayesian CI: 0.73-1.32). Hazard ratios of proportional benefit were similar across pre-specified subgroups; absolute benefit in time to recovery varied, with an estimated benefit of >2 days in older patients, more severe illness, comorbidity, or longer prior illness duration. The effect was independent of influenza status. Fewer antibiotics were prescribed in the oseltamivir arm (9.3% vs 13.2%, mean difference 4.0; 95% CI: 1.7-6.3), while nausea and/or vomiting was shorter in usual care patients (HR 0.92; 95% CI: 0.85-1.00). Interpretation: Primary care patients with ILI treated with oseltamivir recovered sooner than those managed by usual care alone, irrespective of influenza virus test results. Older, sicker, patients with comorbidities and longer prior illness duration showed greater absolute benefit. Trial Registration: The trial is registered with the ISRCTN Registry, number ISRCTN27908921. Funding Statement: CB reports receiving advisory board fees from Roche Molecular Systems and grant support from Roche Molecular Diagnostics; CB was supported by funding from an NIHR Protection Research Unit on Health Care Associated Infections and Antimicrobial Resistance, by the NIHR MedTech and In Vitro Diagnostics Co-Operative at Oxford NHS Foundation Trust, and by an NIHR Senior Investigator Award. TV is PI in a project that is funded by Innovative Medicines Initiative (IMI) of the European Union, in which Janssen Pharmaceutica is a partner and researcher in another IMI project in which Biocartis, Janssen, Biomerieux and Berry Consultants are partners. In addition he is co PI of a NIHR funded RCT, and PI in several studies funded by the Netherlands Organization of Health Research and Development. CL has received advisory boards fees from MSD and grant support from GILEAD, Global Bridges, Servier, Galenica-Olvos. Declaration of Interests: CB reports receiving advisory board fees from Roche Molecular Systems and grant support from Roche Molecular Diagnostics; CB was supported by funding from an NIHR Protection Research Unit on Health Care Associated Infections and Antimicrobial Resistance, by the NIHR MedTech and In Vitro Diagnostics Co-Operative at Oxford NHS Foundation Trust, and by an NIHR Senior Investigator Award. TV is PI in a project that is funded by Innovative Medicines Initiative (IMI) of the European Union, in which Janssen Pharmaceutica is a partner and researcher in another IMI project in which Biocartis, Janssen, Biomerieux and Berry Consultants are partners. In addition he is co PI of a NIHR funded RCT, and PI in several studies funded by the Netherlands Organization of Health Research and Development. CL has received advisory boards fees from MSD and grant support from GILEAD, Global Bridges, Servier, Galenica-Olvos. All other authors declare no conflict of interest. Ethics Approval Statement: All participating countries gained national research ethics committees and CTA approval as required. Ethics Ref: 15/SC/0138.

Summary Background Antibacterial resistant infections are rising continuously, resulting in increased morbidity and mortality worldwide. With no new antibiotic classes entering the market and the possibility of returning to the pre-antibiotic era, the Joint Programming Initiative on Antimicrobial Resistance (JPIAMR) was established to address this problem. We aimed to quantify the scale and scope of publicly funded antibacterial resistance research across JPIAMR countries and at the European Union (EU) level to identify gaps and future opportunities. Methods We did a systematic observational analysis examining antibacterial resistance research funding. Databases of funding organisations across 19 countries and at EU level were systematically searched for publicly funded antibacterial resistance research from Jan 1, 2007, to Dec 31, 2013. We categorised studies on the basis of the JPIAMR strategic research agenda's six priority topics (therapeutics, diagnostics, surveillance, transmission, environment, and interventions) and did an observational analysis. Only research funded by public funding bodies was collected and no private organisations were contacted for their investments. Projects in basic, applied, and clinical research, including epidemiological, public health, and veterinary research and trials were identified using keyword searches by organisations, and inclusion criteria were based on the JPIAMR strategic research agenda's six priority topics, using project titles and abstracts as filters. Findings We identified 1243 antibacterial resistance research projects, with a total public investment of €1·3 billion across 19 countries and at EU level, including public investment in the Innovative Medicines Initiative. Of the total amount invested in antibacterial resistance research across the time period, €646·6 million (49·5%) was invested at the national level and €659·2 million (50·5%) at the EU level. When projects were classified under the six priority topics we found that 763 (63%) of 1208 projects funded at national level were within the area of therapeutics, versus 185 (15%) in transmission, 131 (11%) in diagnostics, 53 (4%) in interventions, and only 37 (3%) in environment and 39 (3%) in surveillance. Interpretation This was the first systematic analysis of research funding of antibacterial resistance of this scale and scope, which relied on the availability and accuracy of data from organisations included. Large variation was seen between countries both in terms of number of projects and associated investment and across the six priority topics. To determine the future direction of JPIAMR countries a clear picture of the funding landscape across Europe and Canada is needed. Countries should work together to increase the effect of research funding by strengthening national and international coordination and collaborations, harmonising research activities, and collectively pooling resources to fund multidisciplinary projects. The JPIAMR have developed a publicly available database to document the antibacterial resistance research collected and can be used as a baseline to analyse funding from 2014 onwards. Funding JPIAMR and the European Commission.

Background aims Dendritic cell (DC)-based immunotherapy has shown potential to counteract autoimmunity in multiple sclerosis (MS). Methods We compared the phenotype and T-cell stimulatory capacity of in vitro generated monocyte-derived DC from MS patients with those from healthy controls. Results Except for an increase in the number of C-C chemokine receptor 7expressing DC from MS patients, no major differences were found between groups in the expression of maturation-associated membrane markers or in the in vitro capacity to stimulate autologous T cells. Conclusions Our observations may pave the way for the development of patient-tailored DC-based vaccination strategies to treat MS.

Clin Microbiol Infect 2012; 18: E308E313 Abstract Antimicrobial resistance is a serious threat and compromises the management of infectious disease. This has particular significance in relation to infections of the respiratory tract, which are the lead cause of antibiotic prescribing. Education is fundamental to the correct use of antibiotics. A novel open access curriculum has been developed in the context of a European Union funded research project Genomics to combat Resistance against Antibiotics in Community-acquired lower respiratory tract infections in Europe (GRACE http://www.grace-lrti.org). The curriculum was developed in modular format and populated with clinical and scientific topics relevant to community-acquired lower respiratory tract infections. This curriculum informed the content of a series of postgraduate courses and workshops and permitted the creation of an open access e-Learning portal. A total of 153 presentations matching the topics within the curriculum together with slide material and handouts and 104 webcasts are available through the GRACE e-Learning portal, which is fully searchable using a mindmap to navigate the contents. Metrics of access provided a means for assessing usage. The GRACE project has permitted the development of a unique on-line open access curriculum that comprehensively addresses the issues relevant to community-acquired lower respiratory tract infections and has provided a resource not only for personal learning, but also to support independent teaching activities such as lectures, workshops, seminars and course work.

Background A new or re-emerging infectious disease pandemic ranks among the highest priorities for civic contingency planning. Despite advances in preclinical and clinical research methods, patient-centered clinical research is not effectively embedded in outbreak responses to inform clinical management of patients and public health responses. Prefunded clinical research networks offer a solution but require well-specified processes for rapid response. We aimed to define a model for how a prefunded clinical research network, Platform for European Preparedness Against (Re-)emerging Epidemics (PREPARE), could rapidly respond at the earliest stages of a new or re-emerging infectious disease outbreak. Methods To develop and test the operational framework, we consulted with multiple stakeholders and facilitated tabletop simulation exercises in Lisbon (May 3, 2017), Brussels (Oct 3, 2017), and Berlin (May 14, 2018). Delegates simulated clinical research planning and delivery, based on PREPARE's operational capabilities, in response to infectious disease outbreak scenarios. Meeting outcomes informed iteration of an operational framework. Findings We developed an operational framework to detail how PREPARE is triggered, and escalates and de-escalates a response. On receipt of a valid trigger signalling an infectious disease outbreak, a rapidly convened expert committee determines the scale and scope of a proportionate clinical research response in one of three outbreak response modes (preparation, mobilisation, response). Modes are defined by the risk to the health and security of European citizens and the need for clinical research contribution. An operational team develops and delivers bespoke outbreak-relevant research plans through research-active clinical research networks across Europe. Simulation exercises have rehearsed the consortium's response to a hypothetical influenza pandemic and estimate it would take up to 3 weeks for existing sites to be open to patient recruitment. A tiered approach and amendment of existing protocols, contracts, and study materials enable a rapid response. However, ethical, regulatory, administrative, laboratory, and logistic bottlenecks present an ongoing challenge to rapid research deployment. Pragmatic solutions have been mapped, including expedited regulatory review, generic contracts, and modified research enrolment procedures. Interpretation PREPARE's outbreak capability is an important step forward in delivering clinical research during an infectious disease pandemic. When properly aligned with public health activities, both patient outcomes and public health responses will benefit from sustained clinical networks. PREPARE's operational framework may be adapted for use by clinical trial networks operating in different global regions. Funding PREPARE is funded by the European Commission's FP7 programme (grant agreement 602525).

An external quality assessment (EQA) panel consisting of a total of 13 samples in broncho alveolar lavage (BAL) or transport medium was prepared to assess the proficiency of laboratories in the correct detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae by nucleic acid amplification techniques (NAATs) (6 samples containing various concentrations (4.9–490 inclusion forming units (IFU)/ml) of C. pneumoniae, 5 samples containing various concentrations (20–5000 color-changing units (CCU)/ml) of M. pneumoniae and 2 samples negative for both). Seventy-nine laboratories from 18 countries participated in this EQA study. Sixty-four datasets were returned for C. pneumoniae (n = 5 conventional commercial, n = 10 conventional in-house, n = 4 real-time commercial, n = 43 real-time in-house, and n = 2 SDA). Sixty-seven datasets were obtained for M. pneumoniae (n = 5 conventional commercial, n = 10 conventional in-house, n = 4 real-time commercial, n = 46 real-time in-house, and n = 2 strand displacement amplification (SDA)). For the total panels, correct results per sample varied between 95.3% and 100% for C. pneumoniae and between 53.7% and 95.5% for M. pneumoniae. In general, commercial conventional NAATs showed possible sensitivity issues when compared to conventional in-house NAATs for both organisms. On the other hand, real-time commercial NAATs scored better than real-time in-house assays in terms of sensitivity for both organisms. For C. pneumoniae and M. pneumoniae, 0.8% and 2.2% true false-positive results and 1.9% and 2.0% false positives were reported in the samples spiked with the other organism. Analysis of the data for C. pneumoniae showed that the concentrations used were easily detectable by the vast majority of participants. The percentage of correct qualitative results for M. pneumoniae demonstrated that the concentrations included in this panel proved challenging for a number of participants.

To date, the number of immunotherapy vaccines in clinical trials increases exponentially. To evaluate the efficacy of these clinical vaccination trials, we fervently need cellular immunomonitoring tools. In this study, we present a newly developed short-time assay which allows direct ex vivo analysis of multi-epitope antigen-specific T cell immune responses. This novel method is based on mRNA electroporation of isolated peripheral blood mononuclear cells (PBMC). Fresh and cryopreserved PBMC of both healthy volunteers as well as of allogeneic stem cell transplanted patients enrolled in a cytomegalovirus (CMV) dendritic cell vaccination trial were electroporated with CMV pp65-encoding mRNA. Using a direct IFN-γ EliSPOT and intracellular cytokine flow cytometry we detected significantly higher numbers of CMV pp65-specific IFN-γ-secreting T cells as compared to the assay with non-treated PBMC and as compared to PBMC electroporated with mRNA coding for an irrelevant protein. Compared to conventional methods to evaluate T cell-mediated immune responses, this method is time-saving and less labor-intensive because it obviates the need for in vitro cultured antigen-presenting cells and because an overnight incubation is sufficient for activation of T cells. Moreover, the use of CMV pp65-encoding mRNA will broaden the immune response because it covers every epitope with potential relevance. This is a major advantage compared to the recognition of a single epitope covered by a HLA-restricted peptides. In summary, we developed a highly efficient mRNA electroporation protocol for fresh and cryopreserved PBMC. This novel method is a rapid and elegant tool and will be convenient for monitoring the cellular immune status of patients in a clinical vaccination setting.

The worldwide increase in resistance to antimicrobial drugs has made reducing the unnecessary use of antibiotics a public health priority. There have been campaigns in many countries to educate the public about appropriate use of antibiotics in outpatients. By use of a comprehensive search strategy and structured interviews, we were able to identify and review the characteristics and outcomes of 22 campaigns done at a national or regional level in high-income countries between 1990 and 2007. The intensity of the campaigns varied widely, from simple internet to expensive mass-media campaigns. All but one campaign targeted the public and physicians simultaneously. Most campaigns that were formally evaluated seemed to reduce antibiotic use. The effect on resistance to antimicrobial drugs cannot be assessed accurately at present. Although the most effective interventions and potential adverse outcomes remain unclear, public campaigns can probably contribute to more careful use of antibiotics in outpatients, at least in high-prescribing countries.

Healthcare-associated infections (HAIs) have been a hot topic for several decades. An understanding of HAIs should be based on an understanding of the organisms that cause infection and determine prevention. Although some improvements in control in hospitals have been recorded, the community setting is now implicated, and the role of microbiology in diagnosis, detection of carriers and strain typing of organisms is evident. As healthcare systems vary widely, prevention strategies must be designed accordingly. Hand hygiene, however, remains applicable in all settings, and the WHO is strongly promoting alcohol-based hand rubs to interrupt transmission. Some countries are only beginning to develop standards, whereas compliance is obligatory in others. Economics and cost factors are common to all countries, and litigation is increasingly a factor in some.

The widespread use of immunosuppressive therapy and antimicrobial agents as well as novel probiotics without a long history of safe use has increased requirements for safety assessment of probiotic lactobacilli. In this study 44 faecal, 52 blood and 15 probiotic isolates (including 3 dairy strains) of Lactobacillus were assayed for their adhesion properties to extracellular matrix proteins and mucus, hemolysis, ability to avoid the induction of respiratory burst in peripheral blood mononucleocytes (PMN) and resistance to human serum. Among tested strains adhesion to collagen, fibrinogen and mucus was isolate-specific and no statistically significant differences were obtained between faecal, blood and probiotic isolates. However, blood isolates showed a trend for higher adhesion to mucus than probiotic strains (P=0.07). Probiotic strains induced lower respiratory burst in PMN when compared to the blood isolates (P

The objectives of the study were to determine the sensitivity of Nucleic Acid Sequence-Based Amplification (NASBA) for the detection of rhinovirus (RV) serotype 15 in water and in spiked respiratory specimens in the presence of a newly developed internal control (IC). The sensitivity of NASBA on RNA was 10 molecules per reaction. The sensitivity of RV NASBA on RV-15 in water and in respiratory specimens was equal to that in tissue culture. Addition of 10(4) molecules of rhinovirus internal control (RV IC) did not affect the sensitivity. Viral RNA should be extracted from clinical specimens as rapidly as possible after collection, since loss of detectable RNA occurs after 2 h. NASBA allows a sensitive detection of RV RNA in spiked respiratory specimens in the presence of an internal control.

This study evaluated retrospectively the efficacy of treatment with cefepime vs. a carbapenem, in combination with amikacin or ciprofloxacin, for seriously-ill patients infected with ESBL-producing Enterobacter aerogenes who were admitted to an intensive care unit. Forty-four episodes of infection were investigated in 43 patients: 21 treated with cefepime; 23 with a carbapenem. The two treatment groups did not differ statistically in terms of age, APACHE II scores, and infection sites, but the average duration of antibiotic exposure was significantly shorter in the cefepime group (8.5 days vs. 11.4 days; p 0.04). Clinical improvement was seen in 62% of patients receiving cefepime vs. 70% of patients receiving a carbapenem (p 0.59). Bacteriological eradication was achieved in 14% of patients receiving cefepime vs. 22% of patients receiving a carbapenem (p 0.76). The 30-day mortality rates related to infection were 33% in the cefepime group and 26% in the carbapenem group (p 0.44). Thus, outcome parameters did not differ significantly between the two groups. Nevertheless, a statistically significant increase in failure to eradicate ESBL-producing E. aerogenes was observed as the MICs of cefepime rose (p 0.017). Pulsed-field gel electrophoresis revealed three distinct clones, but one predominant clone harbouring the blaTEM-24 gene was associated with most (42/44) of the episodes of infection. It was concluded that cefepime may be an alternative agent for therapy of severe infections caused by TEM-24 ESBL-producing E. aerogenes, although further studies are required to confirm these observations.

Summary Background Resistance to antibiotics is a major public-health problem and antibiotic use is being increasingly recognised as the main selective pressure driving this resistance. Our aim was to assess outpatient use of antibiotics and the association with resistance. Methods We investigated outpatient antibiotic use in 26 countries in Europe that provided internationally comparable distribution or reimbursement data, between Jan 1, 1997, and Dec 31, 2002, by calculating the number of defined daily doses (DDD) per 1000 inhabitants per day, according to WHO anatomic therapeutic chemical classification and DDD measurement methodology. We assessed the ecological association between antibiotic use and antibiotic resistance rates using Spearman's correlation coefficients. Findings Prescription of antibiotics in primary care in Europe varied greatly; the highest rate was in France (32·2 DDD per 1000 inhabitants daily) and the lowest was in the Netherlands (10·0 DDD per 1000 inhabitants daily). We noted a shift from the old narrow-spectrum antibiotics to the new broad-spectrum antibiotics. We also recorded striking seasonal fluctuations with heightened winter peaks in countries with high yearly use of antibiotics. We showed higher rates of antibiotic resistance in high consuming countries, probably related to the higher consumption in southern and eastern Europe than in northern Europe. Interpretation These data might provide a useful method for assessing public-health strategies that aim to reduce antibiotic use and resistance levels.

Herpes simplex virus (HSV) is occasionally detected in the lower respiratory tract of patients in intensive care, but its clinical importance in such situations remains unclear. We did a prospective cohort study to define the prevalence, origin, risk factors, and clinical relevance of HSV in the respiratory tract of patients undergoing critical care.We tested 764 patients admitted to intensive care for the presence of HSV in the respiratory tract, and assessed statistical relations between this virus and clinical variables.HSV was detected by oropharyngeal swab in the upper respiratory tract of 169 (22%) of 764 patients, within 10 days of admission for 150 (89%) of these individuals. The virus was isolated in 58 (16%) of 361 patients whose lower respiratory tract was sampled. The presence of HSV in the throat was a risk factor for development of HSV infections in the lower respiratory tract (p0.001). HSV was isolated most frequently in patients with severe disease. HSV in the throat was associated with acute respiratory distress syndrome (p0.001) and with increased length of stay in intensive care (p0.001).Our data suggest that HSV reactivation or infection of the upper respiratory tract is frequent among patients in intensive care, and is a risk factor for development of lower respiratory tract infection with this virus, possibly by means of aspiration.

Crowding of severely ill patients in intensive care units has led worldwide to important increases in nosocomial (ICU-related) infections. Moreover, the nature of these hospital-acquired infections is shifting towards Gram-positive microorganisms, yeast and Gram-negative rods, possessing important resistance genes (e.g. extended spectrum beta-lactamases and inducible Enterobacteriaceae). Ceftazidime and aztreonam are loosing their activity against the Gram-negative microorganisms. The fourth generation cephalosporins have an intrinsic high activity against the inducible Enterobacteriaceae. On our Hematology and Intensive Care units, the introduction of cefepime for nosocomial infections led to a remarkable drop in the number of Enterobacter isolates combined with important decreases in Enterobacter resistance towards several antibiotics.

Nucleic acids were extracted from 215 throat swabs from patients with community-acquired pneumonia by the manual Boom extraction, the NucliSens miniMAG and the Qiagen DNA blood kit and amplified respectively by in-house real-time NASBA, NucliSens EasyQ reagents, and real-time PCR for the detection of Mycoplasma pneumoniae and Chlamydophila pneumoniae. Five out of 215 throat swabs were found to be C. pneumoniae positive by all techniques used. A total of 11 out of 215 throat swabs were positive for M. pneumoniae; 10/215 by Qiagen extraction and PCR amplification and 9/215 by NucliSens miniMAG and NucliSens EasyQ amplification. The NucliSens miniMAG and NucliSens EasyQ applications were successfully coupled to detect both organisms in throat swabs.

For the detection of Chlamydia pneumoniae by polymerase chain reaction (PCR) in respiratory samples, an internal control was constructed to monitor the efficiency of amplification in each reaction. The internal control was designed in a way that the same primer pair can be used to amplify the internal control and target DNA. Nasopharyngeal aspirates of children suffering from asthma (> 2 years of age; 24 patients) or bronchiolitis (< 2 years of age; 47 patients) were analysed for the presence of C. pneumoniae, using the internal control in each amplification reaction. Two specimens from asthma patients, both children of 8 years old, were positive for C. pneumoniae. The number of cases studied is too small to draw conclusions regarding the incidence of C. pneumoniae in different age categories in children.

Purpose : In vivo dosimetry has been shown in a number of evaluation studies, generally carried out in larger academic centres, to be a reliable method of checking the overall treatment accuracy. The object of this study was to investigate whether it was possible and useful to perform in vivo dosimetry in a small radiotherapy department and to detect if there were any systematic errors in the overall treatment set-up. Materials and methods : All patients were treated on a cobalt-60 unit equipped with a verification system. Six hundred fifty entrance dose measurements were performed with silicon diodes. Results : The analysis showed a mean deviation of −1.3%. This negative deviation was mainly due to the mean deviation obtained in the treatment of head and neck (−1.6%) or breast (−2.5%) cancer patients. The results for pelvic or lung irradiation showed almost no deviation (−0.8 to 0.43%). Further investigation showed that the negative values for head and neck or breast irradiation were due to the irradiation technique; the lack of scattering material causes a reduction of the dose at the reference point, which is not taken into consideration by the treatment planning system. By performing in vivo dosimetry, we were also able to detect two large errors in 650 measurements and could prevent erroneous treatment. Conclusion : Even when the overall treatment set-up is very accurate, in vivo dosimetry is very useful in a small department since only a small effort can detect and prevent errors.

The nucleotide sequence of the region between the 16S and 23S rRNA genes of the facultative anaerobic bacterium Gardnerella vaginalis has been determined, together with the 5' proximal 500 nucleotides of the 23S rRNA gene. Regions suited for the development of specific, probe-confirmable polymerase chain reaction (PCR) assays were selected. PCR assays were evaluated with respect to sensitivity and specificity, the latter in comparison with a number of G. vaginalis reference strains and closely related species like Bifidobacterium spp. In an initial diagnostic study it appeared that the PCR test detected G. vaginalis in 40% of women irrespective of their clinical status. Ten out of 11 patients suffering from bacterial vaginosis as defined on the basis of clinical parameters were carrying G. vaginalis.

Summary A species-specific PCR assay was developed for the detection of campylobacters important in veterinary medicine, including Campylobacter fetus, Campylobacter hyointestinalis and Campylobacter mucosalis . Specific primers were also developed for the non-pathogenic Campylobacter sputorum . The PCR assays were based on a target sequence which comprises the most variable areas of 23S rDNA. The developed PCR assays were evaluated on a broad selection of Campylobacter strains and phylogenetically related bacteria. Each of the four primer pairs allowed the selective detection of all the strains belonging to the species Campylobacter fetus, Campylobacter hyointestinalis, Campylobacter mucosalis and Campylobacter sputorum .

Summary The outer membrane protein (OMP) profiles from 8 Campylobacter jejuni and 5 Campylobacter coli fecal isolates grown under various conditions were compared by SDS-PAGE. The bacteria were grown under usual conditions, in iron-deficient medium (Dip) and on iron-supplemented medium (Fe). The OMP profiles of most bacterial strains grown under usual conditions, or in the Fe-supplemented medium, contained four major bands of approximately 31, 45, 63–66 and 97 kDa, and in addition, a number of minor bands. It was found that OMP from 10 of 13 strains tested and grown on iron deficient medium contained an intensive band of a protein in the molecular weight region of 76 kDa which was lacking in the OMP of bacteria grown in the presence of iron (iron-regulated protein). Sera from 11 children with C. jejuni infection analyzed by Western blot recognized the 76 kDa bands, in contrast to only one out of 10 control sera from healthy children. The Western-blot experiments demonstrated also various bands of other OMP components, both in OMP-Dip and OMP-Fe. The 45 kDa (porin protein) was recognized by all 11 serum samples from C. jejuni -infected patients and in 8 out of 10 control sera. The data suggested that the 76 kDa iron-regulated protein was expressed by bacteria during infection and it stimulated the immune response in children infected with C. jejuni .

The detection of Fructose-6-phosphate phosphoketolase (F6PPK) in cellular extracts, is considered to be the most reliable indication that Gram-positive rod-shaped bacteria belong to the genus Bifidobacterium . Recent phylogenetic studies using 16S rRNA sequencing highlighted the close taxonomic relationship between the species Gardnerella vaginalis and a group containing most of the Bifidobacterium species. In order to further investigate this relationship, we have searched for F6PPK activity in strains identified as ‘true’ G. vaginalis and in other catalase-negative coryneform bacteria, recently described as Actinomyces turicensis -like. Our data show that (i) the enzyme is only present in the G. vaginalis sensu stricto strains (ii) F6PPK can be considered as a taxonomic criterion of both Bifidobacterium and G. vaginalis strains, and not as the specific enzyme of the ‘Bifid-shunt’.

Jenafir House and colleagues (March 28, p 1111)1 elegantly demonstrate the herd protection effect of antimicrobials by treating African children aged 110 years for trachoma. Children who were treated with a quarterly single dose of azithromycin had a significantly lower disease prevalence than did children who, as part of WHO-recommended mass prophylaxis, received a yearly dose. Individuals older than 10 years who did not receive antibiotics also had a two-fold lower infection prevalence than at baseline; however, this reduction was no different from that seen in the population receiving WHO-recommended prophylaxis. This finding is important because adults have more severe disease than children. Children are also the main reservoirs of Streptococcus pneumoniae,2 and, in resource-poor regions where pneumococcal disease often proves lethal in young children, the development of antimicrobial resistance could further reduce the chances of children receiving antibiotics effective against such infections. Azithromycin use has been associated with the emergence of resistance to macrolide antibiotics3 and with the selection of strains multiresistant to â-lactams and cephalosporins.4 These drugs are the mainstay of treatment for severe pneumococcal disease, particularly meningitis. Furthermore, we have shown that administration of azithromycin increases drug resistance in oropharyngeal streptococci and that these changes persist for at least 6 months after treatment.5 Once-a-year mass azithromycin dosing, on the other hand, would potentially allow resistance in commensal streptococci to revert to baseline levels. Since most parts of Africa still await introduction of the pneumococcal vaccine, we urge that facial cleanliness and environmental changes to control trachoma are not forsaken. SMK and SC are funded by Research Foundation Flanders (FWO-V), Belgium. We declare that we have no conflicts of interest.

In a nosocomial outbreak of Campylobacter jejuni infection 11 newborn infants (7 female, 4 male) had meningitis. The outbreak was caused by a single strain of C jejuni, as demonstrated by biotyping (biotype I), serotyping (LAU 7/PEN 18 on heat-stable antigens, a new serotype on heat-labile antigens), and the identical susceptibility pattern and outer-membrane-protein profile on sodium dodecyl sulphate/polyacrylamide gel electrophoresis. Specific antibodies against the outbreak strain (enzyme-linked immunosorbent assay and Western blot) developed in all the babies. They were treated with gentamicin and ampicillin. All but one baby, who had a moderately dilated left lateral ventricle after the meningitis, recovered well. The source of infection could not be clearly determined. Thus, C jejuni can cause serious nosocomial infection; it should be considered as a possible agent of meningitis of unknown origin, particularly in newborn infants and other compromised hosts.