Screening of probiotic strains to improve visceral hypersensitivity in irritable bowel syndrome by using in vitro and in vivo approaches.

Oral administration of probiotics has been proposed as a promising biotherapy to prevent and treat different diseases related to gastrointestinal disorders, such as irritable bowel syndrome (IBS). Due to the increasing research area on the characterisation of new probiotic bacterial strains, it is necessary to perform suitable in vitro experiments, using pertinent cellular models, in order to establish appropriate readout profiles based on IBS symptoms and subtypes. In this work, a collection of 30 candidate strains, belonging mainly to the Lactobacillus and Bifidobacterium genera, were screened using three different sets of in vitro experiments with different readouts to identify promising probiotic strains with: (1) the ability to inhibit the synthesis of IL-8 production by TNF-α stimulated HT-29 cells, (2) immunomodulatory properties quantified as increased IL-10 levels in peripheral blood mononuclear cell (PBMCs), and (3) the ability to maintain epithelial barrier integrity by increasing the trans-epithelial/endothelial electrical resistance (TEER) values in Caco-2 cells. Based on these criteria, three strains were selected: Lactobacillus gasseri PI41, Lacticaseibacillus rhamnosus PI48 and Bifidobacterium animalis subsp. lactis PI50, and tested in a murine model of low-grade inflammation induced by dinitrobenzene sulfonic acid (DNBS), which mimics some of the symptoms of IBS. Among the three strains, L. gasseri PI41 improved overall host well-being by preventing body weight loss in DNBS-treated mice and restored gut homeostasis by normalising the intestinal permeability and reducing pro-inflammatory markers. Therefore, the potential of this strain was confirmed in a second murine model known to reproduce IBS symptoms: the neonatal maternal separation (NMS) model. The PI41 strain was effective in preventing intestinal permeability and reducing colonic hypersensitivity. In conclusion, the set of in vitro experiments combined with in vivo assessments allowed us to identify a promising probiotic candidate strain, L. gasseri PI41, in the context of IBS.

A predictive score for the result of carbapenem-resistant Enterobacterales and vancomycin-resistant enterococci screening.

BACKGROUND: The duration of extensively drug-resistant bacteria (XDR) carriage depends on several factors for which the information can be difficult to recover. AIM: To determine whether past screening and clinical results of patients can predict the results of subsequent screening. METHODS: In total, 256 patients were retrospectively included from 10 healthcare centres in France from January 2014 to January 2022. We created a predictive clearance score, ranging from -5 to +7, that included the number of XDR species and the type of resistance detected in the sample, as well as the time from the last positive sample, the number of previous consecutive negative samples, and obtaining at least one negative PCR result in the collection. This score could be used for the upcoming rectal screening of a patient carrying an XDR as soon as the last screening sample was negative. FINDINGS: The negative predictive value was >99% for score ≤0. The median time to achieve XDR clearance was significantly shorter for a score of 0 (443 days (259-705)) than that based on previously published criteria. CONCLUSION: This predictive score shows high performance for the assessment of XDR clearance. Relative to previous guidelines, it could help to lift specific infection prevention and control measures earlier. Nevertheless, the decision should be made according to other factors, such as antimicrobial use and adherence to hand hygiene.

Virulence of new variant strains of Clostridium difficile producing only toxin A or binary toxin in the hamster model.

Pathogenesis of Clostridium difficile has been linked to production of toxins, including the large toxins A and B as well as the binary toxin CDT. Until recently, toxin A was only found in combination in clinical strains with the toxin B, unlike toxin B or CDT, which were found alone in toxigenic variants. New toxigenic variants of C. difficile detected in our laboratory from patients with diarrhoea or severe colitis, including a variant producing only toxin A, were tested for virulence in the hamster model, which displays the clinical features of C. difficile disease. Hamsters infected with a strain producing only toxin B induced similar clinical signs, time to death from infection and histologic damage compared to the hypervirulent strain 027. No mortality or clinical signs of infection but caecal histologic damage was found with the variant producing only toxin A. The C. difficile variant strain producing only CDT was able to kill one hamster out of seven; nevertheless, the surviving animals had few alteration of the caecum.

Toxigenic Clostridium difficile carriage in general practice: results of a laboratory-based cohort study.

OBJECTIVES: Reported rates of community-acquired Clostridium difficile infections (CDIs) have been increasing. However, the true burden of the disease in general practice is unknown in France. Our objective was to determine the incidence of toxigenic C. difficile carriage and the percentage of stool samples prescribed by general practitioners (GPs) which contained free C. difficile toxins. METHODS: During an 11-month period, all stool samples submitted for any enteric pathogen detection to 15 different private laboratories in Paris and the surrounding areas were tested for C. difficile, irrespective of the GPs' request. A clinical questionnaire was completed for each patient. Stool samples were screened using a rapid simultaneous glutamate dehydrogenase and toxins A/B detection test: any positive result (glutamate dehydrogenase or toxin) was further confirmed by the stool cytotoxicity assay (CTA) on MRC-5 cells and by toxigenic culture (TC) at a central laboratory. The C. difficile isolates were characterized by PCR ribotyping. RESULTS: A total of 2541 patients (1295 female, 1246 male) were included. The incidences of patients with a positive toxigenic culture and a positive CTA were 3.27% (95% CI 2.61%-4.03%) and 1.81% (95% CI 1.33%-2.41%), respectively. GPs requested C. difficile testing in only 12.93% of the stool samples, detecting 52.30% of all TC-positive patients. The 83 toxigenic C. difficile strains belonged to 36 different PCR ribotypes. CONCLUSIONS: Toxigenic C. difficile carriage is frequent in general practice but remains under-recognized. It may affect young patients without previous antimicrobial therapy or hospitalization.

More than just teddy bears: Unconventional transmission agents in the operating room.

INTRODUCTION: Surgical site infection (SSI) following orthopedic surgery can have a substantial impact on patients and families. The rate remains high, ranging from 0.5% to 8.5% in pediatric spine surgery. It is common to allow children to bring a teddy bear (or similar toy) to the surgical ward to help reduce the stress of surgery. We hypothesize that despite their known benefits for children, teddies would increase the bacterial load in the surgical room. METHODS: A blinded descriptive study was conducted from June 2015 to September 2016. The study included children entering the hospital through the emergency ward for a traumatic cause requiring surgery. Patients admitted for infectious problems and those who had been hospitalized less than 6 months before the inclusion date were excluded. A picture of the teddy was taken and stored in a blind fashion. The AFNOR (Association française de normalisation) standardized rules for bacteriological surface control and the ISO/DIS 14698 protocol were strictly followed. Two independent observers performed blind bacteriologic analyses of the teddy bears with bacteria identification and colony counts. Photos of the teddy bears were then analyzed by two blinded, independent observers: one doctor and one parent from outside the hospital. Cleanliness and fluffiness of the toy was evaluated using a numeric scale. RESULTS: Bacteria were identified on 100% of the 53 teddies included. The mean number of bacteria was 182.5±49.8 CFU/25 cm2. Eight teddies (15.1%) tested positive for potential pathogenic bacteria (two staphylococcus aureus, one acinetobacter ursingii, four acinetobacter baumannii, one pseudomonas stutzeri). Three teddies (5.7%) tested positive for fungi. The median cleanliness score was 2 (interquartile range (IQR)=1) if rated by the doctor and 2 (IQR=1) if rated by the parent. No statistical difference was found between these two values in the global teddy bear population. We found no any statistical link between the number of CFUs and the cleanliness scores given by the doctor. The median fluffiness score given by the parent was 2 (IQR=1). Looking at the correlative CFUs, we found a statistically significant difference between each stage of fluffiness with a higher stage showing higher CFU (P<0.0001). CONCLUSION: Despite their documented benefits for the child, teddy bears are not appropriate in the surgical room.

Environmental contamination by bacteria in hospital washrooms according to hand-drying method: a multi-centre study.

BACKGROUND: Hand hygiene is a fundamental component of infection prevention, but few studies have examined whether hand-drying method affects the risk of dissemination of potential pathogens. AIM: To perform a multi-centre, internal-crossover study comparing bacterial contamination levels in washrooms with hand-drying by either paper towels (PT) or jet air dryer (JAD; Dyson). METHODS: A total of 120 sampling sessions occurred over 12 weeks in each of three hospitals (UK, France, Italy). Bacteria were cultured from air, multiple surfaces, and dust. Washroom footfall (patients/visitors/staff) was monitored externally. FINDINGS: Footfall was nine times higher in UK washrooms. Bacterial contamination was lower in PT versus JAD washrooms; contamination was similar in France and the UK, but markedly lower in Italian washrooms. Total bacterial recovery was significantly greater from JAD versus PT dispenser surfaces at all sites (median: 100-300 vs 0-10 cfu; all P < 0.0001). In the UK and France, significantly more bacteria were recovered from JAD washroom floors (median: 24 vs 191 cfu, P < 0.00001). UK meticillin-susceptible Staphylococcus aureus recovery was three times more frequent and six-fold higher for JAD vs PT surfaces (both P < 0.0001). UK meticillin-resistant S. aureus recovery was three times more frequent (21 vs 7 cfu) from JAD versus PT surfaces or floors. Significantly more enterococci and extended-spectrum ß-lactamase (ESBL)-producing bacteria were recovered from UK JAD versus PT washroom floors (P < 0.0001). In France, ESBL-producing bacteria were recovered from dust twice as often during JAD versus PT use. CONCLUSION: Multiple examples of significant differences in surface bacterial contamination, including by faecal and antibiotic-resistant bacteria, were observed, with higher levels in JAD versus PT washrooms. Hand-drying method affects the risk of (airborne) dissemination of bacteria in real-world settings.

Guidance document for prevention of Clostridium difficile infection in acute healthcare settings.

SCOPE: Clostridium difficile infection (CDI) is the most important infective cause of healthcare-associated diarrhoea in high income countries and one of the most important healthcare-associated pathogens in both Europe and the United States. It is associated with high morbidity and mortality resulting in both societal and financial burden. A significant proportion of this burden is potentially preventable by a combination of targeted infection prevention and control measures and antimicrobial stewardship. The aim of this guidance document is to provide an update on recommendations for prevention of CDI in acute care settings to provide guidance to those responsible for institutional infection prevention and control programmes. METHODS: An expert group was set up by the European society of clinical microbiology and infectious diseases (ESCMID) Study Group for C. difficile (ESGCD), which performed a systematic review of the literature on prevention of CDI in adults hospitalized in acute care settings and derived respective recommendations according to the GRADE approach. Recommendations are stratified for both outbreak and endemic settings. QUESTIONS ADDRESSED BY THE GUIDELINE AND RECOMMENDATIONS: This guidance document provides thirty-six statements on strategies to prevent CDI in acute care settings, including 18 strong recommendations. No recommendation was provided for three questions.

How to: Surveillance of Clostridium difficile infections.

BACKGROUND: The increasing incidence of Clostridium difficile infections (CDI) in healthcare settings in Europe since 2003 has affected both patients and healthcare systems. The implementation of effective CDI surveillance is key to enable monitoring of the occurrence and spread of C. difficile in healthcare and the timely detection of outbreaks. AIMS: The aim of this review is to provide a summary of key components of effective CDI surveillance and to provide some practical recommendations. We also summarize the recent and current national CDI surveillance activities, to illustrate strengths and weaknesses of CDI surveillance in Europe. SOURCES: For the definition of key components of CDI surveillance, we consulted the current European Society of Clinical Microbiology and Infectious Diseases (ESCMID) CDI-related guidance documents and the European Centre for Disease Prevention and Control (ECDC) protocol for CDI surveillance in acute care hospitals. To summarize the recent and current national CDI surveillance activities, we discussed international multicentre CDI surveillance studies performed in 2005-13. In 2017, we also performed a new survey of existing CDI surveillance systems in 33 European countries. CONTENT: Key components for CDI surveillance are appropriate case definitions of CDI, standardized CDI diagnostics, agreement on CDI case origin definition, and the presentation of CDI rates with well-defined numerators and denominators. Incorporation of microbiological data is required to provide information on prevailing PCR ribotypes and antimicrobial susceptibility to first-line CDI treatment drugs. In 2017, 20 European countries had a national CDI surveillance system and 21 countries participated in ECDC-coordinated CDI surveillance. Since 2014, the number of centres with capacity for C. difficile typing has increased to 35 reference or central laboratories in 26 European countries. IMPLICATIONS: Incidence rates of CDI, obtained from a standardized CDI surveillance system, can be used as an important quality indicator of healthcare at hospital as well as country level.

How to: diagnose infection caused by Clostridium difficile.

BACKGROUND: Clostridium difficile is recognized as the major agent responsible for nosocomial diarrhoea. In the context of recent increase in the incidence and severity of C. difficile infections (CDI), an accurate diagnosis is essential for optimal treatment and prevention, but continues to be challenging. AIMS: The present article reviews each key step of CDI diagnosis including stool selection, methods and strategies used, and interpretation of the results. SOURCES: The most recent guidelines for CDI diagnosis published by scientific societies were reviewed. CONTENT: CDI diagnosis is based on clinical presentation and laboratory tests confirming the presence of toxigenic strain or toxins in stools. Stool selection is crucial and can be improved by implementing rejection criteria and a strict policy for appropriate testing. Multiple laboratory tests detecting different targets (free toxin or presence of a potentially toxigenic strain) are commercially available. However, none of these tests combine high sensitivity and specificity to diagnose CDI, low hands-on time and low cost. An optimized diagnosis can be achieved by implementing a two- or three-step algorithm. Algorithms currently recommended by the ESCMID comprise a screening test with high sensitivity followed by a more specific test to detect free toxins. Presence of free toxins in stools has been shown to better correlate with severe outcome whereas nucleic acid amplification tests may lead to an over-diagnosis by detecting asymptomatic carriers of a toxigenic strain. IMPLICATION: To date, no single test can accurately diagnose CDI. Guidelines from the ESCMID recommend a two- or three-step algorithm for optimal CDI detection.

Excess mortality between 2007 and 2014 among patients with Clostridium difficile infection: a French health insurance database analysis.

BACKGROUND: The impact of Clostridium difficile infection (CDI) on mortality is controversial. AIM: To assess excess mortality due to CDI in France. METHOD: Two cohorts of patients with CDI and a cohort of matched controls were extracted from a 1% representative sample of subjects covered by the general health insurance system in France (Echantillon Généraliste de Bénéficiaires database, 660,000 patients). The CDI patients were hospitalized with CDI as a principal diagnosis or an associated diagnosis between 2007 and 2014, but not in 2006. Controls were patients hospitalized between 2007 and 2014 but not hospitalized with CDI between 2006 and 2014. The one-year incidence of deaths between 2007 and 2014 was estimated and compared with that of a propensity score (PS)-matched control group with no CDI (two controls per case). The PS was calculated with the following variables: age; sex; Charlson Comorbidity Index score; duration of stay; year of index stay; and main comorbidities. Cox and Poisson models were used to estimate the increased risk of death while adjusting for PS. Sensitivity analyses (timeframe, diarrhoea, recurrent hospitalization for CDI) were used to explore the robustness of the results. FINDINGS: In total, 482 patients who had been infected with C. difficile were matched with 964 controls. A significantly higher risk of death was observed among the subjects with CDI, with a non-adjusted hazard ratio of 1.65 [95% confidence interval (CI) 1.33-2.04] and an adjusted ratio of 1.58 (95% CI 1.27-1.97). The adjusted relative risk of death was 1.78 (95% CI 1.18-2.70]) at 28 days, 1.52 (95% CI 1.17-1.98) at three months, 1.52 (95% CI 1.20-1.93) at six months and 1.64 (95% CI 1.32-2.03) at 12 months. Sensitivity analyses produced similar results; the hazard ratio ranged from 1.53 to 1.86, and was always statistically significant. CONCLUSION: CDI is responsible for excess mortality after taking age, sex, comorbidities and length of hospital stay into account.

Faecal lactoferrin and calprotectin in patients with Clostridium difficile infection: a case-control study.

Calprotectin and lactoferrin are released by the gastrointestinal tract in response to infection and mucosal inflammation. Our objective was to assess the usefulness of quantifying faecal lactoferrin and calprotectin concentrations in Clostridium difficile infection (CDI) patients with or without free toxins in the stools. We conducted a single-centre 22-month case-control study. Patients with a positive CDI diagnosis were compared to two control groups: group 1 = diarrhoeic patients negative for C. difficile and matched (1:1) to CDI cases on the ward location and age, and group 2 = diarrhoeic patients colonised with a non-toxigenic strain of C. difficile. Faecal lactoferrin and calprotectin concentrations in faeces were determined for patients with CDI and controls. Of 135 patients with CDI, 87 (64.4%) had a positive stool cytotoxicity assay (free toxin) and 48 (35.6%) had a positive toxigenic culture without detectable toxins in the stools. The median lactoferrin values were 26.8 µg/g, 8.0 µg/g and 15.8 µg/g in CDI patients and groups 1 and 2, respectively. The median calprotectin values were 218.0 µg/g, 111.5 µg/g and 111.3 µg/g, respectively. Among patients with CDI, faecal lactoferrin and calprotectin levels were higher in those with free toxins in their stools (39.2 vs. 10.2 µg/g, p = 0.003 and 274.0 vs. 166.0 µg/g, p = 0.051, respectively). Both faecal calprotectin and lactoferrin were higher in patients with CDI, especially in those with detectable toxin in faeces, suggesting a correlation between intestinal inflammation and toxins in stools.

Variability in testing policies and impact on reported Clostridium difficile infection rates: results from the pilot Longitudinal European Clostridium difficile Infection Diagnosis surveillance study (LuCID).

Lack of standardised Clostridium difficile testing is a potential confounder when comparing infection rates. We used an observational, systematic, prospective large-scale sampling approach to investigate variability in C. difficile sampling to understand C. difficile infection (CDI) incidence rates. In-patient and institutional data were gathered from 60 European hospitals (across three countries). Testing methodology, testing/CDI rates and case profiles were compared between countries and institution types. The mean annual CDI rate per hospital was lowest in the UK and highest in Italy (1.5 vs. 4.7 cases/10,000 patient bed days [pbds], p < 0.001). The testing rate was highest in the UK compared with Italy and France (50.7/10,000 pbds vs. 31.5 and 30.3, respectively, p < 0.001). Only 58.4 % of diarrhoeal samples were tested for CDI across all countries. Overall, only 64 % of hospitals used recommended testing algorithms for laboratory testing. Small hospitals were significantly more likely to use standalone toxin tests (SATTs). There was an inverse correlation between hospital size and CDI testing rate. Hospitals using SATT or assays not detecting toxin reported significantly higher CDI rates than those using recommended methods, despite testing similar testing frequencies. These data are consistent with higher false-positive rates in such (non-recommended) testing scenarios. Cases in Italy and those diagnosed by SATT or methods NOT detecting toxin were significantly older. Testing occurred significantly earlier in the UK. Assessment of testing practice is paramount to the accurate interpretation and comparison of CDI rates.

European Society of Clinical Microbiology and Infectious Diseases: update of the diagnostic guidance document for Clostridium difficile infection.

In 2009 the first European Society of Clinical Microbiology and Infectious Diseases (ESCMID) guideline for diagnosing Clostridium difficile infection (CDI) was launched. Since then newer tests for diagnosing CDI have become available, especially nucleic acid amplification tests. The main objectives of this update of the guidance document are to summarize the currently available evidence concerning laboratory diagnosis of CDI and to formulate and revise recommendations to optimize CDI testing. This update is essential to improve the diagnosis of CDI and to improve uniformity in CDI diagnosis for surveillance purposes among Europe. An electronic search for literature concerning the laboratory diagnosis of CDI was performed. Studies evaluating a commercial laboratory test compared to a reference test were also included in a meta-analysis. The commercial tests that were evaluated included enzyme immunoassays (EIAs) detecting glutamate dehydrogenase, EIAs detecting toxins A and B and nucleic acid amplification tests. Recommendations were formulated by an executive committee, and the strength of recommendations and quality of evidence were graded using the Grades of Recommendation Assessment, Development and Evaluation (GRADE) system. No single commercial test can be used as a stand-alone test for diagnosing CDI as a result of inadequate positive predictive values at low CDI prevalence. Therefore, the use of a two-step algorithm is recommended. Samples without free toxin detected by toxins A and B EIA but with positive glutamate dehydrogenase EIA, nucleic acid amplification test or toxigenic culture results need clinical evaluation to discern CDI from asymptomatic carriage.

Comparison of a novel chemiluminescent based algorithm to three algorithmic approaches for the laboratory diagnosis of Clostridium difficile infection.

BACKGROUND: Rapid commercial assays, including nucleic acid amplification tests and immunoassays for Clostridium. difficile toxins, have replaced the use of older assays. They are included in a two-step algorithm diagnosis, including first the detection of the glutamate dehydrogenase (GDH) as a screening method and second the detection of toxins as a confirmatory method. Although assays that detect the presence of free toxins in feces are known to lack sensitivity, they are preferable to confirm infection. We evaluated the accuracy of the chemiluminescence-based method detecting C. difficile GDH and free toxins A/B (DiaSorin algorithm) to an enzyme-immunoassay (EIA) for GDH with a molecular toxins test (Meridian algorithm), EIA-GDH and an EIA-toxins A/B algorithm (Alere algorithm) with and without toxigenic culture for confirmation. FINDINGS: A total of 468 diarrhoeal and loose stool samples were included in the study. A positive result was defined by a positive GDH and a positive toxin test. Discordant samples were resolved using an enriched toxigenic culture considered as the reference method. After resolution, the DiaSorin algorithm showed a high sensitivity (86.7 %) compared to that of the Alere algorithm with (60.0 %) and without (50.0 %) confirmation by culture and was as sensitive as the Meridian algorithm (90.0 %), while the specificities were similar: 99.1, 99.5, 99.5 and 98.9 %, respectively. CONCLUSIONS: The DiaSorin algorithm was as sensitive as an algorithm including nucleic acid amplification test for toxins. Chemiluminescence toxin-enhanced signal assay compensates the lack of sensitivity usually observed for EIA tests for toxins.

Hospital cost of Clostridium difficile infection including the contribution of recurrences in French acute-care hospitals.

BACKGROUND: The impact of Clostridium difficile infection (CDI) on healthcare costs is significant due to the extra costs of associated inpatient care. However, the specific contribution of recurrences has rarely been studied. AIM: The aim of this study was to estimate the hospital costs of CDI and the fraction attributable to recurrences in French acute-care hospitals. METHODS: A retrospective study was performed for 2011 on a sample of 12 large acute-care hospitals. CDI costs were estimated from both hospital and public insurance perspectives. For each stay, CDI additional costs were estimated by comparison to controls without CDI extracted from the national DRG (diagnosis-related group) database and matched on DRG, age and sex. When CDI was the primary diagnosis, the full cost of stay was used. FINDINGS: A total of 1067 bacteriological cases of CDI were identified corresponding to 979 stays involving 906 different patients. Recurrence(s) were identified in 118 (12%) of these stays with 51.7% of them having occurred within the same stay as the index episode. Their mean length of stay was 63.8 days compared to 25.1 days for stays with an index case only. The mean extra cost per stay with CDI was estimated at €9,575 (median: €7,514). The extra cost of CDI in public acute-care hospitals was extrapolated to €163.1 million at the national level, of which 12.5% was attributable to recurrences. CONCLUSION: The economic burden of CDI is substantial and directly impacts healthcare systems in France.