Background: Awareness and compliance with international guidelines for diagnosis and clinical management of Clostridioides difficile infection (CDI) are unknown.Aim: To compare the awareness and... Show moreBackground: Awareness and compliance with international guidelines for diagnosis and clinical management of Clostridioides difficile infection (CDI) are unknown.Aim: To compare the awareness and compliance with the recommended strategies for diagnosis and clinical management of CDI across Europe in 2018-2019.Methods: Hospital sites and their associated community practices across 12 European countries completed an online survey in 2018-2019, to report on their practices in terms of surveillance, prevention, diagnosis, and treatment of CDI. Responses were collected from 105 hospitals and 39 community general practitioners (GPs).Findings: Hospital sites of 11 countries reported participation in national surveillance schemes compared with six countries for international schemes. The European Society of Clinical Microbiology and Infectious Diseases (ESCMID)-recommended CDI testing meth-odologies were used by 82% (86/105) of hospitals, however countries reporting the highest incidence of CDI used non-recommended tests. Over 75% (80/105) of hospitals were aware of the most recent European CDI treatment guidelines at the time of this survey compared with only 26% (10/39) of surveyed GPs. However, up to 15% (16/105) of hospitals reported using the non-recommended metronidazole for recurrent CDI cases, sites in countries with lower awareness of CDI treatment guidelines. Only 37% (39/105) of hospitals adopted contact isolation precautions in case of suspected CDI.Conclusion: Good awareness of guidelines for the management of CDI was observed across the surveyed European hospital sites. However, low compliance with diagnostic testing guidelines, infection control measures for suspected CDI, and insufficient awareness of treatment guidelines continued to be reported in some countries. 2022 The Author(s). Published by Elsevier Ltd on behalf of The Healthcare Infection Society. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Show less
Rossen, T.M. van; Ooijevaar, R.E.; Vandenbroucke-Grauls, C.M.J.E.; Dekkers, O.M.; Kuijper, E.J.; Keller, J.J.; Prehn, J. van 2022
Objectives: Clostridioides difficile infection (CDI), its subsequent recurrences (rCDIs), and severe CDI (sCDI) provide a significant burden for both patients and the healthcare system. Identifying... Show moreObjectives: Clostridioides difficile infection (CDI), its subsequent recurrences (rCDIs), and severe CDI (sCDI) provide a significant burden for both patients and the healthcare system. Identifying patients diagnosed with initial CDI who are at increased risk of developing sCDI/rCDI could lead to more cost-effective therapeutic choices. In this systematic review we aimed to identify clinical prognostic factors associated with an increased risk of developing sCDI or rCDI.Methods: PubMed, Embase, Emcare, Web of Science and COCHRANE Library databases were searched from database inception through March, 2021. The study eligibility criteria were cohort and caseecontrol studies. Participants were patients >= 18 years old diagnosed with CDI, in which clinical or laboratory factors were analysed to predict sCDI/rCDI. Risk of bias was assessed by using the Quality in Prognostic Research (QUIPS) tool and the Grading of Recommendations Assessment, Development and Evaluation (GRADE) tool modified for prognostic studies. Study selection was performed by two independent reviewers. Overview tables of prognostic factors were constructed to assess the number of studies and the respective effect direction and statistical significance of an association.Results: 136 studies were included for final analysis. Greater age and the presence of multiple comorbidities were prognostic factors for sCDI. Identified risk factors for rCDI were greater age, healthcareassociated CDI, prior hospitalization, proton pump inhibitors (PPIs) started during or after CDI diagnosis, and previous rCDI.Conclusions: Prognostic factors for sCDI and rCDI could aid clinicians to make treatment decisions based on risk stratification. We suggest that future studies use standardized definitions for sCDI/rCDI and systematically collect and report the risk factors assessed in this review, to allow for meaningful metaanalysis of risk factors using data of high-quality trials. (C) 2021 The Author(s). Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases. Show less
Background:One in four patients with primary Clostridioides difficile infection (CDI) develops recurrent CDI (rCDI). With every recurrence, the chance of a subsequent CDI episode increases. Early... Show moreBackground:One in four patients with primary Clostridioides difficile infection (CDI) develops recurrent CDI (rCDI). With every recurrence, the chance of a subsequent CDI episode increases. Early identification of patients at risk for rCDI might help doctors to guide treatment. The aim of this study was to externally validate published clinical prediction tools for rCDI.Methods:The validation cohort consisted of 129 patients, diagnosed with CDI between 2018 and 2020. rCDI risk scores were calculated for each individual patient in the validation cohort using the scoring tools described in the derivation studies. Per score value, we compared the average predicted risk of rCDI with the observed number of rCDI cases. Discrimination was assessed by calculating the area under the receiver operating characteristic curve (AUC).Results:Two prediction tools were selected for validation (Cobo 2018 and Larrainzar-Coghen 2016). The two derivation studies used different definitions for rCDI. Using Cobo's definition, rCDI occurred in 34 patients (26%) of the validation cohort: using the definition of Larrainzar-Coghen, we observed 19 recurrences (15%). The performance of both prediction tools was poor when applied to our validation cohort. The estimated AUC was 0.43 [95% confidence interval (CI); 0.32-0.54] for Cobo's tool and 0.42 (95% CI; 0.28-0.56) for Larrainzar-Coghen's tool.Conclusion:Performance of both prediction tools was disappointing in the external validation cohort. Currently identified clinical risk factors may not be sufficient for accurate prediction of rCDI. Show less
Paiva, A.M.O.; Jong, L. de; Friggen, A.H.; Smits, W.K.; Corver, J. 2020
Clostridioides difficile is an anaerobic Gram-positive bacterium that can produce the large clostridial toxins toxin A and toxin B, encoded within the pathogenicity locus (PaLoc). The PaLoc also... Show moreClostridioides difficile is an anaerobic Gram-positive bacterium that can produce the large clostridial toxins toxin A and toxin B, encoded within the pathogenicity locus (PaLoc). The PaLoc also encodes the sigma factor TcdR, which positively regulates toxin gene expression, and TcdC, which is a putative negative regulator of toxin expression. TcdC is proposed to be an anti-sigma factor; however, several studies failed to show an association between the tcdC genotype and toxin production. Consequently, the TcdC function is not yet fully understood. Previous studies have characterized TcdC as a membrane-associated protein with the ability to bind G-quadruplex structures. The binding to the DNA secondary structures is mediated through the oligonucleotide/oligosaccharide binding fold (0B-fold) domain present at the C terminus of the protein. This domain was previously also proposed to be responsible for the inhibitory effect on toxin gene expression, implicating a cytoplasmic localization of the OB-fold. In this study, we aimed to obtain topological information on the C terminus of TcdC and demonstrate that the C terminus of TcdC is located extracellularly. In addition, we show that the membrane association of TcdC is dependent on a membrane-proximal cysteine residue and that mutating this residue results in the release of TcdC from the bacterial cell. The extracellular location of TcdC is not compatible with the direct binding of the OB-fold domain to intracellular nucleic acid or protein targets and suggests a mechanism of action that is different from that of the characterized anti-sigma factors.IMPORTANCE The transcription of C. difficile toxins TcdA and TcdB is directed by the sigma factor TcdR. TcdC has been proposed to be an anti-sigma factor. The activity of TcdC has been mapped to its C terminus, and the N terminus serves as the membrane anchor. Acting as an anti-sigma factor requires a cytoplasmic localization of the C terminus of TcdC. Using cysteine accessibility analysis and a HiBiT-based system, we show that the TcdC C terminus is located extracellularly, which is incompatible with its role as anti-sigma factor. Furthermore, mutating a cysteine residue at position 51 resulted in the release of TcdC from the bacteria. The codon-optimized version of the HiBiT (HiBiT(opt)) extracellular detection system is a valuable tool for topology determination of membrane proteins, increasing the range of systems available to tackle important aspects of C. difficile development. Show less
In many Gram-positive bacteria, the general stress response is regulated at the transcriptional level by the alternative sigma factor sigma B (sigma(B)). In C. difficile, sigma(B) has been... Show moreIn many Gram-positive bacteria, the general stress response is regulated at the transcriptional level by the alternative sigma factor sigma B (sigma(B)). In C. difficile, sigma(B) has been implicated in protection against stressors such as reactive oxygen species (ROS) and antimicrobial compounds. Here, we used an anti-us antibody to demonstrate time-limited overproduction of sigma(B) in C difficile despite its toxicity at higher cellular concentrations. This toxicity eventually led to the loss of the plasmid used for anhydrotetracycline-induced sigma(B) gene expression. Inducible sigma(B) overproduction uncouples sigma(B) expression from its native regulatory network and allows for the refinement of the previously proposed sigma(B) regulon. At least 32% of the regulon was found to consist of genes involved in the response to reactive radicals. Direct gene activation by C. difficile 0 8 was demonstrated through in vitro runoff transcription of specific target genes (cd0350, cd3614, cd3605, and cd2963). Finally, we demonstrated that different antimicrobials and hydrogen peroxide induce these genes in a manner dependent on this sigma factor, using a plate-based luciferase reporter assay. Together, our work suggests that lethal exposure to antimicrobials may result in the formation of toxic radicals that lead to sigma(B)-dependent gene activation.IMPORTANCE Sigma B is the alternative sigma factor governing stress response in many Gram-positive bacteria. In C. difficile, a sigB mutant shows pleiotropic transcriptional effects. Here, we determine genes that are likely direct targets of sigma(B) by evaluating the transcriptional effects of sigma(B) overproduction, provide biochemical evidence of direct transcriptional activation by sigma(B), and show that sigma(B)-dependent genes can be activated by antimicrobials. Together, our data suggest that sigma(B) is a key player in dealing with toxic radicals. Show less
Gut microbiota composition in patients with Clostridioides difficile colonization is not well investigated. We aimed to identify bacterial signatures associated with resistance and susceptibility... Show moreGut microbiota composition in patients with Clostridioides difficile colonization is not well investigated. We aimed to identify bacterial signatures associated with resistance and susceptibility to C. difficile colonization (CDC) and infection (CDI). Therefore, gut microbiota composition from patients with CDC (n = 41), with CDI (n = 41), and without CDC (controls, n = 43) was determined through 16S rRNA gene amplicon sequencing. Bacterial diversity was decreased in CDC and CDI patients (p < 0.01). Overall microbiota composition was significantly different between control, CDC, and CDI patients (p = 0.001). Relative abundance of Clostridioides (most likely C. difficile) increased stepwise from controls to CDC and CDI patients. In addition, differential abundance analysis revealed that CDI patients' gut microbiota was characterized by significantly higher relative abundance of Bacteroides and Veillonella than CDC patients and controls. Control patients had significantly higher Eubacterium hallii and Fusicatenibacter abundance than colonized patients. Network analysis indicated that Fusicatenibacter was negatively associated with Clostridioides in CDI patients, while Veillonella was positively associated with Clostridioides in CDC patients. Bacterial microbiota diversity decreased in both CDC and CDI patients, but harbored a distinct microbiota. Eubacterium hallii and Fusicatenibacter may indicate resistance against C. difficile colonization and subsequent infection, while Veillonella may indicate susceptibility to colonization and infection by C. difficile. Show less
Two hundred and fifty-three non-duplicate toxigenic Clostridium difficile isolates, collected from February 2012 to December 2014, were evaluated for phenotypic resistance to ten antimicrobial... Show moreTwo hundred and fifty-three non-duplicate toxigenic Clostridium difficile isolates, collected from February 2012 to December 2014, were evaluated for phenotypic resistance to ten antimicrobial drugs with the E-test gradient diffusion method. All strains of C. difficile were susceptible to metronidazole, vancomycin, and tigecycline. The metronidazole MIC values of the hyperepidemic PCR-ribotypes RT027 and RT176 were higher than those of non-epidemic PCR-ribotypes (p < 0.05, as evidenced by Mann-Whitney U test). In contrast, vancomycin susceptibility did not differ between hyperepidemic and non-epidemic strains, although the difference was almost significant (p = 0.065). Clostridium difficile RT027 and RT176 isolates could be assessed to five and four different susceptibility patterns, respectively, representing various combinations of resistance to different antimicrobial classes. A single point mutation (Thr82Ile) in the gyrA gene was detected in 11 (78.6%) of 14 isolates with high level of resistance to ciprofloxacin and moxifloxacin and four different types of single point mutations (Arg447Lys, Ser416Ala, Asp426Val, Asp426Asn) in the gyrB gene were detected in 4 strains, also with high level of resistance to ciprofloxacin and moxifloxacin. Four different point mutations were detected in the rpoB gene in 21 rifampicin-resistant strains of which one has not been reported previously, GIn489Leu. This study demonstrates the presence of multidrug-resistant C. difficile strains in Polish hospitals over the study period, irrespective of geographical location or reference level of the hospital. (C) 2020 Elsevier Ltd. All rights reserved. Show less
Objectives: Clostridium difficile is a major global human pathogen divided into five clades, of which Glade 3 is the least characterized and consists predominantly of PCR ribotype (RT) 023 strains.... Show moreObjectives: Clostridium difficile is a major global human pathogen divided into five clades, of which Glade 3 is the least characterized and consists predominantly of PCR ribotype (RT) 023 strains. Our aim was to analyse and characterize this Glade.Methods: In this cohort study the clinical presentation of C. difficile RT023 infections was analysed in comparison with known 'hypervirulent' and non-hypervirulent strains, using data from the Netherlands national C. difficile surveillance programme. European RT023 strains of diverse origin were collected and whole-genome sequenced to determine the genetic similarity between isolates. Distinctive features were investigated and characterized.Results: Clinical presentation of C. difficile RT023 infections show severe infections akin to those seen with 'hypervirulent' strains from clades 2 (RT027) and 5 (RT078) (35%, 29% and 27% severe CDI, respectively), particularly with significantly more bloody diarrhoea than RT078 and non-hypervirulent strains (RT023 8%, other RTs 4%, p 0.036). The full genome sequence of strain CD305 is presented as a robust reference. Phylogenetic comparison of CD305 and a further 79 previously uncharacterized European RT023 strains of diverse origin revealed minor genetic divergence with >99.8% pairwise identity between strains. Analyses revealed distinctive features among Glade 3 strains, including conserved pathogenicity locus, binary toxin and phage insertion toxin genotypes, glycosylation of S-layer proteins, presence of the RT078 four-gene trehalose cluster and an esculinase-negative genotype.Conclusions: Given their recent emergence, virulence and genomic characteristics, the surveillance of Glade 3 strains should be more highly prioritized. (C) 2019 The Authors. Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases. Show less
Jovanovic, M.; Dorp, S.M. van; Drakulovic, M.; Papic, D.; Pavic, S.; Jovanovic, S.; ... ; Kuijper, E.J. 2020
Clostridium (Clostridioides) difficile infections (CDIs) are among the most frequent healthcare-associated infections in Serbia. In 2013, Serbia participated in the European Clostridium difficile... Show moreClostridium (Clostridioides) difficile infections (CDIs) are among the most frequent healthcare-associated infections in Serbia. In 2013, Serbia participated in the European Clostridium difficile Infection Surveillance Network (ECDIS-Net) who launched a pilot study to enhance laboratory capacity and standardize surveillance for CDI. Two clinics of Clinical Center of Serbia [Clinic for Infectious and Tropical Diseases (CITD) and Clinic of Orthopedic Surgery and Traumatology (COT)] from Belgrade and one general hospital from another metropolitan area of Serbia, Laice, participated. During a period of 3 months in 2013, all patients with diagnosed CDI were included. The CDI incidence rates in CITD, COT, and General Hospital Uzice were 19.0, 12.2, and 3.9 per 10,000 patient-days, respectively. In total, 49 patients were enrolled in the study with average age of 72 years. A complicated course of CDI was found in 14.3% of all patients. Six (12.2%) of 49 patients died, but not attributable to CDI. Of 39 C. difficile isolates, available for ribotyping, 78.9% belonged to ribotype 027; other PCR ribotypes were 001, 015, 002, 005, 010, 014, and 276. Antimicrobial susceptibility testing revealed low levels of MIC50 and MIC90 for metronidazole (0.5 mu g/ml both) and vancomycin (0.25 and 0.5 mu g/ml), while 28 strains of ribotype 027 were resistant to moxifloxacin with MIC >= 4 mu g/ml. National surveillance is important to obtain more insight in the epidemiology of CDI and to compare the results with other European countries. This study by ECDIS-Net gives bases for a national surveillance of CDI in Serbia. Show less
Vogelzang, E.H.; Lankelma, J.M.; Mansfeld, R. van; Prehn, J. van; Houdt, R. van 2020
A proportion of patients suspected of Clostridium difficile infection are unnecessarily placed in contact isolation. By introducing a random-access glutamate dehydrogenase (GDH) test for C.... Show moreA proportion of patients suspected of Clostridium difficile infection are unnecessarily placed in contact isolation. By introducing a random-access glutamate dehydrogenase (GDH) test for C. difficile, we aimed to reduce isolation time. In addition, we investigated whether the result of the toxin A&B enzyme immunoassay (EIA) was associated with the decision to initiate antibiotic treatment against C. difficile. This retrospective pre- and post-implementation study was from June 3, 2016, to June 4, 2018. Pre-implementation, only a NAAT was performed. In the post-implementation period, a GDH test was performed; if positive, a toxin A&B EIA followed the same day and subsequently a NAAT. Contact isolation for CDI was discontinued when the GDH test was negative. Median time in isolation was 50.8 h pre-implementation (n = 189) versus 28.0 h post-implementation (n = 119), p < 0.001. The GDH test had a negative predictive value of 98.8% (95% CI 97.9-99.4). In 7/31 (22.6%) patients with a positive NAAT and GDH test and a negative toxin A&B EIA, no antibiotics against C. difficile were initiated versus 4/28 (14.3%) patients who were NAAT, GDH and toxin A&B EIA positive. Introducing a random-access screening test resulted in a significant decrease in patient isolation time. The GDH test had a high negative predictive value making it suitable to determine whether contact isolation can be discontinued. Furthermore, the result of a toxin A&B EIA had limited added value on the percentage of patients in whom antibiotic treatment against C. difficile was initiated. Show less
The carriage of two important pathogens of pigs, that is enterotoxigenic Escherichia coli (ETEC) and Clostridioides difficile, was investigated in 104 cloacal samples from wild griffon vultures ... Show moreThe carriage of two important pathogens of pigs, that is enterotoxigenic Escherichia coli (ETEC) and Clostridioides difficile, was investigated in 104 cloacal samples from wild griffon vultures (Gyps fulvus) fed on pig carcasses at supplementary feeding stations (SFS), along with their level of antimicrobial resistance (AMR). E. coli was isolated from 90 (86.5%) samples, but no ETEC was detected, likely because ETEC fimbriae confer the species specificity of the pathogen. Resistance to at least one antimicrobial agent was detected in 89.9% of E. coli isolates, with AMR levels being extremely high (>70%) for tetracycline and streptomycin and very high (>50%) for ampicillin and sulfamethoxazole-trimethoprim. Resistance to other critically important antimicrobials such as colistin and extended-spectrum cephalosporins was 2.2% and 1.1%, respectively, and was encoded by the mcr-1 and bla(SHV-12) genes. Multidrug resistance was displayed by 80% of the resistant E. coli, and bla(SHV-12) gene shared plasmid with other AMR genes. In general, resistance patterns in E. coli from vultures mirrored those found in pigs. Clostridioides difficile was detected in three samples (2.9%); two of them belonged to PCR ribotype 078 and one to PCR ribotype 126, both commonly found in pigs. All C. difficile isolates were characterized by a moderate-to-high level of resistance to fluoroquinolones and macrolides but susceptible to metronidazole or vancomycin, similar to what is usually found in C. difficile isolates from pigs. Thus, vultures may contribute somewhat to the environmental dissemination of some pig pathogens through their acquisition from pig carcasses and, more importantly, of AMR for antibiotics of critical importance for humans. However, the role of vultures would likely be much lesser than that of disposing pig carcasses at the SFS. The monitoring of AMR, and particularly of colistin-resistant and ESBL-producing E. coli, should be considered in pig farms used as sources of carcasses for SFS. Show less
Information on recurrent Clostridium difficile infections (rCDI) in children is rare and limited, especially community acquired (CA-CDI).This study was designed to identify risk factors for rCA-CDI... Show moreInformation on recurrent Clostridium difficile infections (rCDI) in children is rare and limited, especially community acquired (CA-CDI).This study was designed to identify risk factors for rCA-CDI in Serbian pediatric population. The study group included 71 children (aged from 1 to 14 years) with a first episode of CDI. Data were collected from 56 (78.87%) children with only one episode of CA-CDI and from 15 (21.13%) children with rCA-CDI were mutually compared. The following parameters were found to be statistically significantly more frequent in the children with rCA-CDI group (p < 0.05); leukemia as underlying disease, treatment with immunosuppressive and-or cytostatic drugs, and treatment with antibiotics. Similarly, previously visits to outpatient facilities, daycare hospitals and hospitals were also associated with rCDI. Analysis of clinical symptoms and laboratory parameters, revealed a statistically significant association of the severity of the first episode of CDI (determined by an increase in body temperature, higher maximum WBC and higher CRP) with development of a rCDI. Ribotype (RT) 027 was more common in children with rCA-CDI (66.7%, p = 0.006). During the seven-year research period, we found a rate of rCA-CDI rate in children of 21.13%. Our study identified several parameters statistically significantly more frequently in children with rCA-CDI. The obtained results will serve as a basis for future larger studies, but new prospective, studies are necessary to build a prediction model of rCDI in children that can be used to guide the treatment to prevent rCDI. Show less
Terveer, E.M.; Fallon, M.; Kraakman, M.E.M.; Ormond, A.; Fitzpatrick, M.; Caljouw, M.A.A.; ... ; Fitzpatrick, F. 2019
The Gram-positive enteropathogen Clostridioides difficile (Clostridium difficile) is the major cause of healthcare-associated diarrhoea and is also an important cause of community-acquired... Show moreThe Gram-positive enteropathogen Clostridioides difficile (Clostridium difficile) is the major cause of healthcare-associated diarrhoea and is also an important cause of community-acquired infectious diarrhoea. Considering the burden of the disease, many studies have employed whole-genome sequencing of bacterial isolates to identify factors that contribute to virulence and pathogenesis. Though extrachromosomal elements (ECEs) such as plasmids are important for these processes in other bacteria, the few characterized plasmids of C. difficile have no relevant functions assigned and no systematic identification of plasmids has been carried out to date. Here, we perform an in silico analysis of publicly available sequence data to show that similar to 13% of all C. difficile strains contain ECEs, with 1-6 elements per strain. Our approach identifies known plasmids (e.g. pCD6, pCD630 and cloning plasmids) and six novel putative plasmid families. Our study shows that plasmids are abundant and may encode functions that are relevant for C. difficile physiology. The newly identified plasmids may also form the basis for the construction of novel cloning plasmids for C. difficile that are compatible with existing tools. Show less
Czepiel, J.; Drozdz, M.; Pituch, H.; Kuijper, E.J.; Perucki, W.; Mielimonka, A.; ... ; Biesiada, G. 2019
Introduction: Clostridium difficile (C. difficile) is a major nosocomial infectious agent in hospitals. Previous studies have addressed the high proportion of infection episodes that are overlooked... Show moreIntroduction: Clostridium difficile (C. difficile) is a major nosocomial infectious agent in hospitals. Previous studies have addressed the high proportion of infection episodes that are overlooked in health care facilities.Objective: the main aim of this study was to characterize C. difficile clinical cases that occurred in a secondary care hospital during a five-month period.Material and methods: for this purpose, a total of 137 stool samples from the same number of patients with diarrhea were analyzed for the presence of C. difficile by culture techniques. An enzyme immunoassay (EIA) test for the detection of C. difficile and its toxins was also used in 50 cases (36.5%) for diagnostic purposes.Results: a total of 14 (10.2%) C. difficile isolates were obtained, of which nine (64.3%) were toxigenic. A mean incidence of 3.2 episodes of C. difficile infections (CDI) per 10,000 patients-days was estimated for the study period. Around 56% of the CDI cases were determined as hospital-acquired, whereas 44% originated in the community. Among these, only two episodes (22.2%) were detected in the hospital by the EIA test, which indicated that the hospital CDI detection protocol needed to be revised. One unusual C. difficile isolate was negative for all toxin genes examined and also for the non-toxigenic strain assay, which highlights the need to perform genome sequencing to study its pathogenicity locus insertion site organization. A stable metronidazole-resistant C. difficile strain and three strains showing multidrug resistance were detected in this study, suggesting that C. difficile antimicrobial susceptibility surveillance programs should be established in this health-care facility. Show less
