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Purpose: Clostridioides difficile is the main cause of antibiotic related diarrhea and some ribotypes (RT), such as RT027, RT181 or RT078, are considered high risk clones. A fast and reliable approach for C. difficile ribotyping is needed for a correct clinical approach. This study analyses high-molecular-weight proteins for C. difficile ribotyping with MALDI-TOF MS., Methods: Sixty-nine isolates representative of the most common ribotypes in Europe were analyzed in the 17,000-65,000 m/z region and classified into 4 categories (RT027, RT181, RT078 and 'Other RTs'). Five supervised Machine Learning algorithms were tested for this purpose: K-Nearest Neighbors, Support Vector Machine, Partial Least Squares-Discriminant Analysis, Random Forest (RF) and Light-Gradient Boosting Machine (GBM)., Results: All algorithms yielded cross-validation results > 70%, being RF and Light-GBM the best performing, with 88% of agreement. Area under the ROC curve of these two algorithms was > 0.9. RT078 was correctly classified with 100% accuracy and isolates from the RT181 category could not be differentiated from RT027., Conclusions: This study shows the possibility of rapid discrimination of relevant C. difficile ribotypes by using MALDI-TOF MS. This methodology reduces the time, costs and laboriousness of current reference methods., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Introduction: Clostridioides difficile infection (CDI) is the main cause of nosocomial diarrhoea in developed countries. Recurrent CDI (R-CDI), which affects 20%-30% of patients and significantly increases hospital stay and associated costs, is a key challenge. The main objective of this study was to explore the role of the microbiome and calprotectin levels as predictive biomarkers of R-CDI., Methods: We prospectively (2019-2021) included patients with a primary episode of CDI. Clinical data and faecal samples were collected. The microbiome was analysed by sequencing the hypervariable V4 region of the 16S rRNA gene on an Illumina Miseq platform., Results: We enrolled 200 patients with primary CDI, of whom 54 developed R-CDI and 146 did not. We analysed 200 primary samples and found that Fusobacterium increased in abundance, while Collinsella, Senegalimassilia, Prevotella and Ruminococcus decreased in patients with recurrent versus non-recurrent disease. Elevated calprotectin levels correlated significantly with R-CDI (p=0.01). We built a risk index for R-CDI, including as prognostic factors age, sex, immunosuppression, toxin B amplification cycle, creatinine levels and faecal calprotectin levels (overall accuracy of 79%)., Discussion: Calprotectin levels and abundance of microbial genera such as Fusobacterium and Prevotella in primary episodes could be useful as early markers of R-CDI. We propose a readily available model for prediction of R-CDI that can be applied at the initial CDI episode. The use of this tool could help to better tailor treatments according to the risk of R-CDI., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Vázquez-Cuesta, Lozano García, Fernández, Olmedo, Kestler, Alcalá, Marín, Bermejo, Díaz, Muñoz, Bouza and Reigadas.)

Introduction: Clostridioides difficile infection (CDI) is the main cause of nosocomial diarrhea in developed countries. A key challenge in CDI is the lack of objective methods to ensure more accurate diagnosis, especially when differentiating between true infection and colonization/diarrhea of other causes. The main objective of this study was to explore the role of the microbiome as a predictive biomarker of CDI., Methods: Between 2018 and 2021, we prospectively included patients with CDI, recurrent CDI (R-CDI), non-CDI diarrhea (NO-CDI), colonization by C. difficile , and healthy individuals. Clinical data and fecal samples were collected. The microbiome was analyzed by sequencing the hypervariable V4 region of the 16S rRNA gene on an Illumina Miseq platform. The mothur bioinformatic pipeline was followed for pre-processing of raw data, and mothur and R were used for data analysis., Results: During the study period, 753 samples from 657 patients were analyzed. Of these, 247 were from patients with CDI, 43 were from patients colonized with C. difficile , 63 were from healthy individuals, 324 were from NOCDI, and 76 were from R-CDI. We found significant differences across the groups in alpha and beta diversity and in taxonomic abundance. We identified various genera as the most significant biomarkers for CDI ( Bacteroides, Proteus, Paraprevotella, Robinsoniella ), R-CDI ( Veillonella, Fusobacterium, Lactobacillus, Clostridium sensu stricto I ), and colonization by C. difficile ( Parabacteroides, Faecalicoccus, Flavonifractor, Clostridium XVIII )., Discussion: We observed differences in microbiome patterns between healthy individuals, colonized patients, CDI, R-CDI, and NOCDI diarrhea. We identified possible microbiome biomarkers that could prove useful in the diagnosis of true CDI infections. Further studies are warranted., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Vázquez-Cuesta, Villar, García, Fernández, Olmedo, Alcalá, Marín, Muñoz, Bouza and Reigadas.)

Clostridium difficile (CD) diagnosis is very varied and under discussion. Different research groups disagree on the clinical significance of patients with negative direct toxin and positive polymerase chain reaction (PCR) or even more with direct toxin and glutamate dehydrogenase (GDH) both negatives, but CD detected by toxigenic culture (TC). The objective was to analyze the characteristics of patients with 3 different diagnostic criteria. We compared these 3 groups of patients: group 1: (GDH+/direct toxin+/PCR+), group 2: (GDH+/direct toxin-/PCR+) and group 3: (GDH-/direct toxin-/PCR not done/TC+). The proportion of patients with CD infection (CDI) (not colonization) for groups 1 to 3 was, respectively, 90.3%, 95.4%, and 59.1%. No differences between severity (40.8%, 38.5%, 27.3%), recurrence (20.3%, 24.1%, 7.6%), or related mortality (12.5%, 5.2%, 0%) were found within the 3 groups of patients. Laboratory clinical results should not be used as the only tool to differentiate CDI versus colonization or severity. We recommend that PCR or a second-look TC be performed on all patients., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objectives: Clostridioides difficile infection has traditionally been considered to be transmitted predominantly within health-care settings. It is not recognized as a pathogen that presents a risk of laboratory acquisition. Data on laboratory contamination and acquisition by laboratory personnel are lacking. Our objective was to assess environmental contamination by C. difficile and its potential for transmission in a clinical microbiology laboratory., Methods: Laboratory surfaces were screened for C. difficile. Samples were taken in areas that handle C. difficile isolates (high-exposure (HE) areas), areas adjacent to HE areas or those processing faecal samples (medium-exposure (ME) areas), and areas that do not process faecal samples or C. difficile isolates (low-exposure (LE) areas). We examined C. difficile carriage (hands/rectal samples) of laboratory workers., Results: A total of 140 environmental samples were collected from two HE areas (n = 56), two ME areas (n = 56) and two LE areas (n = 28). Overall, 37.8% (37/98) of surfaces were contaminated with C. difficile, and 17.3% (17/98) with toxigenic C. difficile (TCD). HE areas were significantly more contaminated with TCD than LE areas (38.1% (16/42) versus 0.0% (0/14), p 0.005) and ME areas (38.1% (16/42) versus 2.4% (1/42), p <0.001). Hands were colonized with TCD in 11.8% (4/34) of cases. We found no rectal carriage of C. difficile., Conclusions: We found a significant proportion of laboratory surfaces to be contaminated with toxigenic C. difficile, as well as hand colonization of laboratory personnel. We recommend specific control measures for high-risk areas and laboratory personnel working in these areas., (Copyright © 2019 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Classification of patients according to their risk of poor outcomes in Clostridioides difficile infection (CDI) would enable implementation of costly new treatment options in a subset of patients at higher risk of poor outcome. In a previous study, we found that low toxin B amplification cycle thresholds (C t ) were independently associated with poor outcome CDI. Our objective was to perform a multicentre external validation of a PCR-toxin B C t as a marker of poor outcome CDI. We carried out a multicentre study (14 hospitals) in which the characteristics and outcome of patients with CDI were evaluated. A subanalysis of the results of the amplification curve of real-time PCR gene toxin B (XpertTM C. difficile) was performed. A total of 223 patients were included. The median age was 73.0 years, 50.2% were female, and the median Charlson index was 3.0. The comparison of poor outcome and non-poor outcome CDI episodes revealed, respectively, the following results: median age (years), 77.0 vs 72.0 (p = 0.009); patients from nursing homes, 24.4% vs 10.8% (p = 0.039); median leukocytes (cells/μl), 10,740.0 vs 8795.0 (p = 0.026); and median PCR-toxin B C t , 23.3 vs 25.4 (p = 0.004). Multivariate analysis showed that a PCR-toxin B C t cut-off <23.5 was significantly and independently associated with poor outcome CDI (p = 0.002; OR, 3.371; 95%CI, 1.565-7.264). This variable correctly classified 68.5% of patients. The use of this microbiological marker could facilitate early selection of patients who are at higher risk of poor outcome and are more likely to benefit from newer and more costly therapeutic options., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Objective: Fecal microbiota transplantation (FMT) is a highly effective therapy for recurrent Clostridium difficile infection (R-CDI). Despite its excellent efficacy, it is still not a routine procedure in most European centers. FMT has not been widely used in Spain to date. We describe our experience with FMT, including a novel approach based on oral fecal capsules., Methods: We analyzed a prospectively recorded case series of patients with R-CDI treated with FMT at a single center (June 2014-July 2017). Primary outcome was defined as resolution of CDI without recurrence in a two-month period. FMT was administered via colonoscopy, nasojejunal tube, or oral capsules. All stool donors were rigorously screened., Results: FMT was performed in 13 patients with R-CDI. Median age was 75.0 years and 76.9% were females. Six FMT were performed via nasojejunal tube, 5 via oral capsules, and 2 by colonoscopy. There were no procedure-related adverse events, except for bacteremia in one patient. During follow-up, R- CDI was observed in one patient at one month after FMT. The primary resolution rate was 83.3% and the overall resolution rate was 91.7%. FMT by capsules achieved a 100% resolution rate, colonoscopy 100%, and nasojejunal tube 80.0%., Conclusions: In our cohort, FMT proved to be safe and effective, even in high risk patients. Oral administration in capsules also proved to be safe, well-tolerated, and highly effective for R-CDI. In our experience, the FMT capsule formulation seems feasible in the routine of a hospital. This administration method will allow FMT to be more widely used., (©The Author 2018. Published by Sociedad Española de Quimioterapia. This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)(https://creativecommons.org/licenses/by-nc/4.0/).)

Background: Rifaximin has been proposed as an alternative treatment for specific cases of Clostridium difficile infection (CDI) and intestinal decontamination. Rifaximin-resistant C. difficile has occasionally been reported. Antibiotic susceptibility testing relies on anaerobic agar dilution (reference method), which is cumbersome and not routinely used. There is no commercial test for detection of resistance to rifaximin., Objectives: To assess resistance to rifaximin by C. difficile and to evaluate the correlation between the results of the rifampicin E-test and susceptibility to rifaximin., Methods: We compared the in vitro susceptibility of clinical CDI isolates to rifaximin over a 6-month period using the agar dilution method with susceptibility to rifampicin using the E-test. All isolates were characterized using PCR-ribotyping. Clinical data were recorded prospectively., Results: We recovered 276 consecutive C. difficile isolates and found that 32.2% of episodes were caused by rifaximin-resistant strains. The MICs for rifaximin ranged from <0.0009-256 mg/L, with a geometric mean (GM) of 0.256 mg/L, an MIC 50/90 of 0.015/>256 mg/L. Rifaximin and rifampicin MICs were comparable, and all strains classed as resistant by agar dilution were correctly classified as resistant by E-test. The most common ribotypes were 001 (37.2%), 078/126 (14.3%), and 014 (12.0%). Ribotype 001 exhibited the highest MICs for rifaximin., Conclusions: Resistance to rifaximin was common; resistance rates were higher in ribotype 001 strains. Susceptibility to rifaximin determined by agar dilution correlated with susceptibility to rifampicin determined using the E-test, including rifaximin-resistant strains. Our results suggest that the rifampicin E-test is a valid method for the prediction of rifaximin-resistant C. difficile., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

An outbreak of Clostridium difficile infection (CDI) caused by ribotype 027 (B1/NAP1) began in our hospital in November 2014, and produced 141 episodes in the following months. The aim of this study is to describe this outbreak, assess risk factors for recurrence of CDI-027 and to analyze the implementation of a novel treatment strategy. This is a prospective study of all patients with CDI-027, from November 2014 to November 2015. The epidemiological data were collected daily for each patient. We compared clinical characteristics and treatment between patients with and without recurrence of CDI-027. Interestingly, liver cirrhosis was present in 22% of the patients, and most of them received prophylaxis for hepatic encephalopathy with rifaximin. Patients were also taking antimicrobial drugs (93.6%) and proton pump inhibitors (80.1%). Overall, 27 (23.5%) patients had a first recurrence of CDI-027. Liver cirrhosis increased the risk of recurrence (44.4% vs 14.8%). Patients treated with a prolonged oral vancomycin regimen vs the conventional regimen (oral metronidazole or 10 days of vancomycin) had fewer recurrences (8.6 versus 44.7% [p ≤ 0.01]; OR, 0.91; 95% CI, 0.028-0.294) and less attributable mortality (0% versus 7.1%; p = 0.058). We report an outbreak of CDI-027, mainly in patients with liver cirrhosis. Recurrence of CDI-027 was more common in those patients. A novel approach involving high-dose prolonged vancomycin taper as a first-line treatment, together with a bundle of outbreak measures, seemed to reduce the number of cases of CDI-027, recurrences, and attributable mortality. Nevertheless, this approach warrants further investigation.

Background: Clostridium difficile infection (CDI) causes increased morbidity and mortality. Clinical data cannot clearly predict poor CDI outcome. Data on the value of microbiological predictors is scarce., Objective: To identify early predictors of poor outcome of CDI., Methods: We prospectively included patients with CDI aged >2years. Clinical, immunological (Toxin B IgG/Ig A and Toxin A IgG/Ig A), microbiological factors (bacterial load, toxin quantification, sporulation, germination, and metronidazole susceptibility) were evaluated to identify early independent predictors of poor outcome., Results: We identified 204 cases of CDI; outcome was poor in 22.1%. Advanced age, presence of comorbidities, leukocytosis and high toxigenic C. difficile load were independently associated with poor outcome. We could not demonstrate this correlation for antitoxin antibodies., Conclusion: We identified high bacterial load as a microbiological predictor of poor outcome. We propose this factor to be included in combined clinical and microbiological prediction rules of poor outcome in CDI., (Copyright © 2017 Elsevier Inc. All rights reserved.)

We compared the performance of the new chromogenic medium ChromID C. difficile with that of CLO agar. ChromID C. difficile agar is a sensitive medium that can accelerate the presumptive identification of C. difficile, however ribotype 023 might go undetected when using this chromogenic medium., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Objectives: Prediction of patients with poor outcome is necessary in order to plan the proper management of Clostridium difficile infection (CDI); however, clinical criteria are insufficient. In a previous study, we observed that high toxigenic C. difficile cfu stool counts at diagnosis were associated with a poor outcome. Our objective was to investigate the role of the PCR toxin B amplification cycle threshold (Ct) in the prediction of CDI poor outcome and to derive and validate a high-risk prediction rule using this marker., Methods: We prospectively included patients with CDI (derivation cohort, January 2013 to June 2014; and validation cohort, December 2014 to May 2015), who were followed for at least 2 months after their last episode/recurrence. All samples were tested with Xpert™ C. difficile., Results: For the derivation cohort (n = 129) toxin B Ct was independently associated with poor outcome (P < 0.001). The receiver operating characteristic (ROC) curve yielded an AUC of 0.816. Using a cut-off of <23.5 cycles for high risk of poor outcome, the diagnostic accuracy was 81.4%, the sensitivity was 46.5% (95% CI 32.5-61.1) and the specificity was 98.8% (95% CI 93.7-99.8). For the validation cohort (n = 170), the diagnostic accuracy was 81.8%, the sensitivity was 88.4% (95% CI 75.5-94.9) and the specificity was 79.5% (95% CI 71.7-85.6). The ROC curve yielded an AUC of 0.857., Conclusions: Low toxin B Ct values from samples collected at the initial moment of diagnosis appears to be a strong marker for poor outcome. This available test may identify, at an early stage, patients who are at higher risk of a poor outcome CDI., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: Clostridium difficile infection (CDI) is the leading cause of hospital-acquired diarrhoea in developed countries. Although an optimal diagnosis is crucial, laboratory diagnostics remain challenging. Currently, the reference methods are direct cytotoxicity assay and toxigenic culture; however there is controversy in the interpretation of discordant results of these tests., Objective: The aim of our study was to determine the clinical significance of detecting C. difficile only by toxigenic culture with a negative direct cytotoxicity assay., Methods: We conducted a prospective study in which patients aged >2 years with CDI were enrolled and monitored at least 2 months after their last episode. Samples were tested by both cytotoxicity assay and toxigenic culture., Results: During the 6-month study period, we identified 169 episodes meeting CDI criteria that had been tested by both assays, out of which 115 were positive for both cytotoxicity assay and toxigenic culture, and 54 CDI episodes (31.9%) were positive only by toxigenic culture. Overall, patients median age was 71.3, 50.9% were male and the most frequent underlying disease was malignancy. The comparison of CDI episodes positive for both assays and by toxigenic culture only revealed the following, respectively: mild CDI (77.4% vs 94.4%; p = 0.008), severe CDI (21.7% vs 5.6%; p = 0.008), severe complicated (0.9% vs 0.0%; p = 1.000), pseudomembranous colitis (1.7% vs 1.9% p = 1.000), recurrence (17.4% vs 14.8%; p = 0.825), overall mortality (8.7% vs 7.4%; p = 1.000) and CDI related mortality (2.6% vs 0%; p = 0.552)., Conclusion: CDI episodes positive by cytotoxicity assay were more severe than those positive only by toxigenic culture, however there were a significant proportion of CDI cases (31.9%) that would have been missed if only cytotoxicity had been considered as clinically significant for CDI treatment, including severe CDI cases. Our data suggest that a positive test by toxigenic culture with a negative result for cytotoxicity should not be interpreted as colonization., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Binary toxin (BT) has been associated with strains causing more severe Clostridium difficile infection (CDI), such as ribotype 027. Data on the outcome of patients having BT present in ribotypes other than 027 are scarce. Our objective was to investigate the association between BT isolates and outcome of CDI in a non-027 ribotype setting. We prospectively included CDI episodes (January-June 2013 and March-June 2014) from symptomatic patients aged >2 years. Epidemiological and clinical data were recorded. BT genes were detected using multiplex PCR. During the study period, we identified 326 episodes of CDI, of which 319 were available for molecular analysis. Of these, 54 (16·9%) were caused by C. difficile strains with BT. Most (90·7%) isolates with BT were ribotype 078/126. CDI patients with BT-positive strains did not differ from those with BT-negative strains in terms of recurrence (13·0% vs. 15·5%, P = 0·835), treatment failure (0·0% vs. 2·3%, P = 0·594), overall mortality (11·1% vs. 9·1%, P = 0·612), or CDI-related mortality (0·0% vs. 1·9%, P = 0·612). Multivariate regression revealed no association between BT and poor outcome. In conclusion, in a non-027 setting, we found that most BT isolates were 078/126 and were not associated with poor outcome.

A multicenter study of Clostridium difficile infection (CDI) performed during 2008 in Spain revealed that two of every three episodes went undiagnosed or were misdiagnosed owing to nonsensitive diagnostic tests or lack of clinical suspicion and request. Since then, efforts have been made to improve the diagnostic tests used by laboratories and to increase the awareness of this disease among both clinicians and microbiologists. Our objective was to evaluate the impact of these efforts by assessing the current magnitude of underdiagnosis of CDI in Spain using two point-prevalence studies performed on one day each in January and July of 2013. A total of 111 Spanish laboratories selected all unformed stool specimens received for microbiological diagnosis on these days, and toxigenic culture was performed at a central reference laboratory. Toxigenic isolates were characterized both pheno- and genotypically. The reference laboratory detected 103 episodes of CDI in patients aged 2 years or more. Half (50.5 %) of the episodes were not diagnosed in the participating laboratories, owing to insensitive diagnostic tests (15.5 %) or the lack of clinical suspicion and request (35.0 %). The main ribotypes were 014, 078/126, 001/072, and 106. Ribotype 027 caused 2.9 % of all cases. Despite all the interventions undertaken, CDI remains a highly neglected disease because of the lack of sensitive diagnostic tests in some institutions and, especially, the absence of clinical suspicion, mainly in patients with community-associated CDI. Toxigenic C. difficile should be routinely sought in unformed stools sent for microbiological diagnosis, regardless of their origin.

Objectives: Clostridium difficile infection (CDI) is the leading cause of hospital-acquired diarrhoea in developed countries. Metronidazole and vancomycin are the mainstay of treatment, although they are associated with treatment failure and recurrence. Novel agents have emerged to address these shortcomings. We investigated the in vitro activity of a novel agent, surotomycin (formerly CB-183,315), and seven other antimicrobial agents against clinical C. difficile isolates., Methods: Antimicrobial susceptibility to surotomycin, fidaxomicin, metronidazole, vancomycin, clindamycin, rifaximin, moxifloxacin and tigecycline was determined for 100 contemporary clinical isolates of C. difficile collected in 2013. MICs were determined by agar dilution according to CLSI procedures. In addition, 10 strains with reduced susceptibility to metronidazole (n = 6) and vancomycin (n = 4) were also tested. Strains were PCR ribotyped., Results: The MICs of surotomycin for the 100 isolates ranged from ≤0.06 to 2 mg/L, with a geometric mean (GM) of 0.31 mg/L and an MIC50/90 of 0.25/0.5 mg/L. The MIC range of surotomycin was 0.25-1 mg/L (GM = 0.45 mg/L) for isolates with reduced metronidazole susceptibility and 0.125-0.5 mg/L (GM = 0.25 mg/L) for isolates with reduced vancomycin susceptibility. The three most common ribotypes were 001 (31.0%), 014/020 (17.0%) and 078/126 (17.0%). Ribotype 014/020 exhibited the lowest MICs of surotomycin (GM = 0.22 mg/L); the highest MICs were for ribotype 078/126 (GM = 0.72 mg/L)., Conclusions: Surotomycin exhibited potent in vitro activity against all the isolates tested, including those with elevated metronidazole and vancomycin MICs. The potential role of this agent in the treatment of CDI requires further clinical evaluation., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Purpose: Despite the high concentration of patients with known risk factors for Clostridium difficile infection (CDI) in intensive care units (ICUs), data on ICU patients are scarce. The aim of this study was describe the incidence, clinical characteristics, and evolution of CDI in critically ill patients., Materials and Methods: From 2003 to 2012, adult patients admitted to an ICU (A-ICU) and positive for CDI were included and classified as follows: pre-ICU, if the positive sample was obtained within ±3 days of ICU admission; in-ICU, if obtained after 3 days of ICU admission and up to 3 days after ICU discharge., Results: We recorded 4095 CDI episodes, of which 328 were A-ICU (8%). Episodes of A-ICU decreased from 19.4 to 8.7 per 10000 ICU days of stay (P < .0001). Most A-ICU CDIs (66.3%) were mild to moderate. Pre-ICU episodes accounted for 16.2% and were more severe complicated than in-ICU episodes (11% vs 0%; P = .020). Overall mortality was 28.6%, and CDI-attributable mortality was only 3%., Conclusion: The incidence of A-ICU CDI has decreased steadily over the last 10 years. A significant proportion of A-ICU CDI episodes are pre-ICU and are more severe than in-ICU CDI episodes. Most episodes of A-ICU CDI were nonsevere, with low associated mortality., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Clostridium difficile infection (CDI) is the leading cause of hospital-acquired diarrhoea in developed countries, however a high proportion of CDI episodes go undiagnosed, either because physicians do not request identification of toxigenic C. difficile or microbiologists do not perform the appropriate tests., Objective: To investigate the clinical characteristics of patients with CDI within a non-selected population and to determine risk factors for clinical underdiagnosis., Methods: We conducted a prospective study in which systematic testing for toxigenic C. difficile on all diarrhoeic stool samples was performed regardless of the clinician's request. Patients aged >2 years positive for toxigenic C. difficile and diarrhoea were enrolled (Jan-June 2013) and monitored at least 2 months after their last episode., Results: We identified 204 cases of CDI, of which three-quarters were healthcare-associated. Most cases were mild to moderate (83.8%), the recurrence rate was 16.2%, and CDI-related mortality was low (2.5%). A significant proportion (12.7%) of CDI cases would have been missed owing to lack of clinical suspicion. Community-acquired cases and young age were risk factors for clinical underdiagnosis., Conclusion: Our data support the introduction of a systematic search for toxigenic C. difficile in all diarrhoeic stools from inpatients and outpatients older than 2 years., (Copyright © 2014 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)

Toxins A and B are the main virulence factors of Clostridium difficile and are the targets for molecular diagnostic tests. Here, we describe a new toxin A-negative, toxin B-positive, binary toxin CDT (Clostridium difficile transferase)-negative (A(-) B(+) CDT(-)) toxinotype (XXXII) characterized by a variant type of pathogenicity locus (PaLoc) without tcdA and with atypical organization of the PaLoc integration site., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

We studied the molecular mechanisms of linezolid resistance in 9 isolates of toxigenic Clostridium difficile with high linezolid MICs. The activity of linezolid was determined against 891 clinical isolates of toxigenic C. difficile. The MIC50 and MIC90 of linezolid were 0.75 μg/ml and 1.5 μg/ml, respectively. Nine strains (1%) showed high linezolid MICs (6 μg/ml to 16 μg/ml) and also were resistant to clindamycin, erythromycin, and chloramphenicol. These strains were selected for molecular studies: sequencing of domain V of the 23 rRNA gene, detection of the cfr methyltransferase gene, and sequencing of the ribosomal protein genes rplC and rplD. Molecular relatedness between strains was assessed using PCR ribotyping and MLVA (multilocus variable-number tandem-repeat analysis) typing. The strains belonged to ribotypes 001 (2/9), 017 (6/9), and 078 (1/9). MLVA showed that strains of ribotype 001 and 017 belonged to the same clonal complex in each ribotype. We did not detect mutations in the 23S rRNA gene. The cfr gene was detected in 7 of 9 strains. Sequencing of cfr amplicons revealed a similarity of 100% to a fragment of transposon Tn6218 of C. difficile, which was annotated as a putative chloramphenicol/florfenicol resistance protein. We were unable to detect mechanisms of resistance to linezolid in the 2 strains belonging to ribotype 001. While the relevance of our results lies in the detection of the cfr gene as a possible mechanism of resistance to linezolid in C. difficile, our findings should be assessed by further investigations to characterize these possible cfr genes and their contribution to linezolid resistance., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

We compared two multistep diagnostic algorithms based on C. Diff Quik Chek Complete and, as confirmatory tests, GenomEra C. difficile and Xpert C. difficile. The sensitivity, specificity, positive predictive value, and negative predictive value were 87.2%, 99.7%, 97.1%, and 98.3%, respectively, for the GenomEra-based algorithm and 89.7%, 99.4%, 95.5%, and 98.6%, respectively, for the Xpert-based algorithm. GenomEra represents an alternative to Xpert as a confirmatory test of a multistep algorithm for Clostridium difficile infection (CDI) diagnosis., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Introduction: Clostridium difficile ribotype 027 (Cd027) has caused outbreaks in the United States, Canada, and Europe since 2001. In Spain, the importance of Cd027 is still unknown. In 2007, we began active surveillance of Cd027 to determine its incidence in our hospital., Methods: From January 2007 to April 2012, isolates of C. difficile by multiplex PCR were studied to detect toxin genes. Binary toxin-positive isolates were characterized using PCR-ribotyping. Cd027 were further characterized by toxino-typing, sequencing of tcdC gene, and MLVA (multilocus-variable-number-tandem-repeat-analysis)., Results: Only 8 strains were Cd027 from 3666 isolates of C. difficile analyzed during the study period. These strains were isolated from 4 patients: a Spanish patient previously hospitalized in the UK, a pregnant laboratory technician, a British tourist, and a Spanish patient without epidemiological antecedents for acquiring Cd027. MLVA typing of Cd027 isolates revealed 4 different patterns. The first patient had 2 episodes of diarrhea caused by different Cd027. The strains from the first episode of patient 1 and the strain from patient 2 were grouped in the same clonal cluster (these cases were previously published as laboratory transmission), while strains from patients 3 and 4 were genetically unrelated to each other, and to the strains from patients 1 and 2., Conclusion: We report the first finding of an autochthonous case of non-severe Cd027 infection. Our results indicate that Cd027 diarrhea is uncommon in our area, and it appears mainly as imported cases. MLVA typing enables us to distinguish different genotypes among our Cd027 isolates., (Copyright © 2013 Elsevier España, S.L. y Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.)

Clostridium difficile is an emerging pathogen for humans and animals and there is concern about the possibility that livestock might serve as a reservoir of epidemic strains. In Spain, ribotype 078 is one of the most prevalent in human episodes of C. difficile infection, but the distribution of this and other ribotypes in animals is yet unknown. We present the first report on the ribotype distribution and antimicrobial susceptibility of C. difficile in swine in Spain. A total of 144 isolates were PCR ribotyped, and their MIC values for 13 antimicrobial agents were determined using the Etest. Toxins A and B production was assessed using a commercial immunoassay and, in the case of toxin B, a specific cytotoxicity test. Our results show a high prevalence of the toxigenic 078 ribotype (94.4%) and multidrug resistance (49.3%) among the studied isolates. A minority of isolates (5.6%) belonged to a mostly non-toxinogenic ribotype. All isolates were resistant to the fluoroquinolone ciprofloxacin, but susceptible to daptomycin, linezolid, meropenem, rifampicin, teicoplanin, tigecycline, metronidazole and vancomycin. Resistance to clindamycin, ertapenem, erythromycin and moxifloxacin was common (≥27.8% in all cases). Resistance rates for the different antibiotics tested were in all cases independent from the ribotype of isolates and the host's condition (diarrheic or non-diarrheic), but erythromycin and moxifloxacin resistance was associated with the geographic origin of isolates. Metronidazole heteroresistance was found among animal isolates of C. difficile. Our results highlight the role of livestock as a potential source of epidemic multidrug resistant strains in Spain., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Underdiagnosis of Clostridium difficile infection (CDI) because of lack of clinical suspicion or the use of non-sensitive diagnostic techniques is a known problem whose real magnitude has not yet been quantified. In order to estimate the extent of this underdiagnosis, we performed C. difficile cultures on all unformed stool specimens sent-irrespective of the type of request-to a series of laboratories in Spain on a single day. The specimens were cultured, and isolates were characterized at a central reference laboratory. A total of 807 specimens from 730 patients aged ≥ 2 years were selected from 118 laboratories covering 75.4% of the Spanish population. The estimated rate of hospital-acquired CDI was 2.4 episodes per 1000 admissions or 3.8 episodes per 10,000 patient-days. Only half of the episodes occurred in patients hospitalized for >2 days. Two of every three episodes went undiagnosed or were misdiagnosed, owing to non-sensitive diagnostic tests (19.0%) or lack of clinical suspicion and request (47.6%; mostly young people or non-hospitalized patients). The main ribotypes were 014/020 (20.5%), 001 (18.2%), and 126/078 (18.2%). No ribotype 027 strains were detected. Strains were fully susceptible to metronidazole and vancomycin. CDI was underdiagnosed in diarrhoeic stools in a high proportion of episodes, owing to the use of non-sensitive techniques or lack of clinical suspicion, particularly in people aged <65 years or patients with community-acquired diarrhoea. C. difficile toxins should be routinely sought in unformed stools of any origin sent for microbiological diagnosis. The ribotype 027 clone has not yet disseminated in Spain., (© 2012 The Authors. Clinical Microbiology and Infection © 2012 European Society of Clinical Microbiology and Infectious Diseases.)

At our institution, the prevalence of clinical isolates of Clostridium difficile with resistance to metronidazole is 6.3%. We observed that initial metronidazole MICs of 16 to 64 mg/liter against toxigenic, primary fresh C. difficile isolates, as determined by agar dilution, decreased to 0.125 mg/liter after the isolates were thawed. In this study, we examined the possibility of heterogeneous or inducible resistance. Totals of 14 metronidazole-resistant and 10 metronidazole-susceptible clinical isolates of toxigenic C. difficile were studied. The isolates were investigated for the presence of nim genes by PCR. After the isolates were thawed, susceptibility testing was done by agar dilution, by disc diffusion using a 5-mug metronidazole disc, and by the Etest method. An experiment for determining the effect of prolonged exposure to metronidazole was applied to all resistant isolates and to susceptible control strains. None of the isolates presented the nim genes. All initially metronidazole-resistant C. difficile isolates became susceptible after thawing; however, they presented slow-growing subpopulations within the inhibition zones of both the disk and the Etest strip. All metronidazole-susceptible isolates remained homogeneously susceptible by both methods. After prolonged exposure in vitro to metronidazole, no zone of inhibition was found around the 5-microg disk in any of the metronidazole-resistant isolates, and the MICs as determined by the Etest method ranged from 0.125 to >256 mg/liter, with colonies growing inside the inhibition zone. Our results indicate that (i) resistance to metronidazole was not due to the presence of nim genes, (ii) resistance to metronidazole in toxigenic C. difficile isolates is heterogeneous, and (iii) prolonged exposure to metronidazole can select for in vitro resistance. We recommend routine performance of the disk diffusion method (5-microg metronidazole disk) with primary fresh C. difficile isolates in order to ensure that metronidazole-heteroresistant populations do not go undetected.

We evaluated the in vitro activity of ramoplanin, an antimicrobial compound that inhibits cell wall synthesis by acting at the level of lipid intermediate formation, against Clostridium difficile. We included strains with reduced susceptibilities to vancomycin (vancomycin-intermediate [Van(i)] strains) or with resistance to metronidazole (Mtz(r)), in order to assess the potential utility of ramoplanin for the treatment of C. difficile-associated diarrhea. We tested the activity of ramoplanin against a total of 105 nonduplicate clinical isolates of toxigenic C. difficile, including 8 Van(i) isolates and 6 Mtz(r) isolates, obtained from our laboratory. Ramoplanin was active against all strains tested at concentrations ranging from 0.03 to 0.5 microg/ml (MICs at which 50 and 90% of isolates were inhibited, 0.25 microg/ml; geometric mean MIC, 0.22 microg/ml). All isolates, independently of their levels of susceptibility to vancomycin or metronidazole, were considered susceptible to ramoplanin (MICs, < or =0.5 microg/ml).

Pulsed-field gel electrophoresis (PFGE) is the 'gold standard' technique for bacterial typing and has proved to be discriminatory and reproducible for typing Clostridium difficile. Nevertheless, a high proportion of strains are non-typable by this technique due to the degradation of the DNA during the process. The introduction of several modifications in the PFGE standard procedure increased typability from 40% (90 isolates) to 100% (220 isolates) while maintaining the high degree of discrimination and reproducibility of the technique.

The aim of this study was to evaluate the toxigenic status of circulating strains of Clostridium difficile in a large teaching hospital. Overall 220 isolates were studied of which 199 (90.5 %) produced both large clostridial toxins detected by conventional methods. Ten more strains (4.5 %) had toxin A and B genes detectable by PCR. Eleven (5.0 %) variant strains (A- B+) were detected among the isolates studied and 10 strains (4.5 %) had the binary toxin genes (cdtA and cdtB).

Clostridioides difficile infection (CDI) was traditionally considered to be transmitted within healthcare environment, from other patients or healthcare workers (HCW). Recently, this idea has been challenged. Our objective was to determine the extent of C. difficile contamination in hospital environment with a simplified method for C. difficile recovery. Environmental samples were taken from rooms of patients positive for CDI (Case) and negative for toxigenic C. difficile (Control). Environmental sampling was performed at the time a fecal sample was taken for CDI diagnosis, 48 h after, and 10 days after. HCW hands were also sampled. A total of 476 environmental samples were collected, 246 samples from "Case" rooms and 230 from "Control". Overall, 15.34% of environmental samples were positive for toxigenic C. difficile (TCD), 20.72% of "Case" rooms samples and 9.57% of the samples from "Control" rooms (p = 0.001). When samples from "Case" rooms were analyzed by sampling time, at diagnosis 52.94% were positive, 38.46% were positive at 48 h after symptom resolution and 23.07% were positive after course of treatment. Overall, the most contaminated site corresponded to the bathroom tap, followed by the toilet. We recovered TCD from alcohol-based dispensers and from 4.2% of HCW hands. We found a high proportion of surfaces contaminated with TCD, as well as hand colonization. Notably, even after isolation measures were terminated, there was still TCD contamination. • Description of a simple one step method for laboratory processing of Polywipes sponges. • A significant proportion of hospital surfaces were contaminated with toxigenic C. difficile , as well as hand colonization. • We documented C. difficile contamination of alcohol gel dispensers. [ABSTRACT FROM AUTHOR]