Human intestinal microbiome: Role in health and disease.

The study of the microbiota and the microbiome, and specifically the intestinal one, has determined great interest due to the possible association of their alterations with numerous diseases. These include entities as diverse as Crohn's disease, autism, diabetes, cancer or situations as prevalent today as obesity. In view of this situation, different recommendations have been performed regarding the use of probiotics, prebiotics, and postbiotics as modulators of the microbiota and the microbiome, seeking both preventive and therapeutic effects, and faecal material transfer (FMT) is proposed as an alternative. The latter has emerged as the only proven beneficial intervention on the intestinal microbiome, specifically in the treatment of recurrent colitis associated with Clostridioides difficile (R-CDI). In the rest of the entities, the lowering of laboratory costs has favored the study of the microbiome, which is resolved by delivering reports with catalogs of microorganisms, metabolites or supposed biomarkers without consensus on their composition associated with healthy or diseased microbiota and the disease. There is still insufficient evidence in any disease for interventions on the microbiome beyond FMT and R-CDI. Multi- and multi-disciplinary work with extensive research and the application of artificial intelligence in this field may shed light on the questions raised currently. Ethical issues must also be resolved in light of possible interventions within the umbrella of personalized medicine.

Role of Clostridioides difficile in hospital environment and healthcare workers.

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.

Recommendations for the diagnosis and treatment of Clostridioides difficile infection: An official clinical practice guideline of the Spanish Society of Chemotherapy (SEQ), Spanish Society of Internal Medicine (SEMI) and the working group of Postoperative Infection of the Spanish Society of Anesthesia and Reanimation (SEDAR).

This document gathers the opinion of a multidisciplinary forum of experts on different aspects of the diagnosis and treatment of Clostridioides difficile infection (CDI) in Spain. It has been structured around a series of questions that the attendees considered relevant and in which a consensus opinion was reached. The main messages were as follows: CDI should be suspected in patients older than 2 years of age in the presence of diarrhea, paralytic ileus and unexplained leukocytosis, even in the absence of classical risk factors. With a few exceptions, a single stool sample is sufficient for diagnosis, which can be sent to the laboratory with or without transportation media for enteropathogenic bacteria. In the absence of diarrhoea, rectal swabs may be valid. The microbiology laboratory should include C. difficile among the pathogens routinely searched in patients with diarrhoea. Laboratory tests in different order and sequence schemes include GDH detection, presence of toxins, molecular tests and toxigenic culture. Immediate determination of sensitivity to drugs such as vancomycin, metronidazole or fidaxomycin is not required. The evolution of toxin persistence is not a suitable test for follow up. Laboratory diagnosis of CDI should be rapid and results reported and interpreted to clinicians immediately. In addition to the basic support of all diarrheic episodes, CDI treatment requires the suppression of antiperistaltic agents, proton pump inhibitors and antibiotics, where possible. Oral vancomycin and fidaxomycin are the antibacterials of choice in treatment, intravenous metronidazole being restricted for patients in whom the presence of the above drugs in the intestinal lumen cannot be assured. Fecal material transplantation is the treatment of choice for patients with multiple recurrences but uncertainties persist regarding its standardization and safety. Bezlotoxumab is a monoclonal antibody to C. difficile toxin B that should be administered to patients at high risk of recurrence. Surgery is becoming less and less necessary and prevention with vaccines is under research. Probiotics have so far not been shown to be therapeutically or preventively effective. The therapeutic strategy should be based, rather than on the number of episodes, on the severity of the episodes and on their potential to recur. Some data point to the efficacy of oral vancomycin prophylaxis in patients who reccur CDI when systemic antibiotics are required again.

Faecal microbiota transplantation for recurrent Clostridioides difficile infection: experience with lyophilized oral capsules.

BACKGROUND: Faecal microbiota transplantation (FMT) is a highly effective approach for refractory and recurrent Clostridioides difficile infection (CDI). Despite its excellent efficacy, FMT is not yet a routine procedure in most centres. There is very little experience with FMT based on lyophilized capsules, and data from European institutions are lacking. This article describes our experience with FMT to treat recurrent CDI using lyophilized oral capsules. METHODS: A prospectively recorded single-centre case series of patients with recurrent CDI who underwent FMT between January 2018 and May 2019 were analysed. The primary outcome was defined as resolution of CDI without recurrences over a two-month period. Overall resolution was defined as resolution of diarrhoea without recurrence of CDI within two months after a further cycle of FMT. The FMT process involved oral ingestion of four or five lyophilized capsules in a single dose. All stool donors were rigorously screened. FINDINGS: FMT was performed in 32 patients. Primary cure was achieved in 81.3% of patients, and the overall cure rate was 87.5%. FMT via lyophilized capsules was well tolerated. No FMT procedure-related adverse events and no further complications were observed for lyophilized-capsule FMT. CONCLUSIONS: This initial clinical experience suggests that FMT based on oral lyophilized preparations is a safe, well-tolerated, and highly effective treatment for recurrent CDI. Administration of oral lyophilized capsules seems feasible in hospital routine and will enable FMT to be more widely used.

Clostridioides difficile contamination in a clinical microbiology laboratory?

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.

Prediction of poor outcome in Clostridioides difficile infection: A multicentre external validation of the toxin B amplification cycle.

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 (Ct) were independently associated with poor outcome CDI. Our objective was to perform a multicentre external validation of a PCR-toxin B Ct 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 Ct, 23.3 vs 25.4 (p = 0.004). Multivariate analysis showed that a PCR-toxin B Ct 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.

Evaluation of Clostridium difficile Infection with PET/CT Imaging in a Mouse Model.

PURPOSE: Existing clinical or microbiological scores are not sensitive enough to obtain prompt identification of patients at risk of complicated Clostridium difficile infection (CDI). Our aim was to use a CDI animal model to evaluate 2-deoxy-2-[18F]fluoro-D-glucose positron emission tomography ([18F]FDG-PET) as a marker of severe course of infection. PROCEDURES: CDI was induced with cefoperazone for 10 days followed by clindamycin 1 day before C. difficile inoculation. Mice were divided into wild type (n = 6), antibiotic without infection (AC n = 4), h001-infected (n = 5, ribotype 001), and h027-infected (n = 5, ribotype 027). Two days after inoculation, [18F]FDG-PET was acquired. Weight, general animal condition, and survival were monitored daily for 9 days. RESULTS: h001 group showed symptoms for 4 days with 0 % mortality and a similar colon uptake than control animals (h001 0.52 ± 0.20, WT 0.42 ± 0.07, and AC 0.36 ± 0.06). The h027 group showed symptoms up to 7 days, with 66.7 % of mortality 4 days after infection, and significantly higher colon uptake (0.93 ± 0.38, p < 0.05). Clinical score was associated to colon and cecum uptake (rho = 0.78, p = 0.0001) (rho = 0.73, p = 0.0003). CONCLUSION: High toxin producer ribotype 027 induced more severe CDI infections, correlating with higher colon and cecum [18F]FDG uptake. Colon uptake may purportedly serve as early predictor of CDI severity.

Fecal microbiota transplantation for recurrent Clostridium difficile infection: Experience, protocol, and results.

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.

Rifaximin-resistant Clostridium difficile strains isolated from symptomatic patients.

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 MIC50/90 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.

An outbreak of Clostridium difficile PCR ribotype 027 in Spain: risk factors for recurrence and a novel treatment strategy.

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.

Clinical, immunological and microbiological predictors of poor outcome in Clostridium difficile infection.

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.

C. difficile PCR-ribotype 023 might go undetected when using ChromId C. difficile agar.

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.

New direct-acting antivirals for patients with chronic HCV infection: can we monitor treatment using an HCV core antigen assay?

We evaluated the diagnostic usefulness of an HCV core antigen (HCV-Ag) assay in HCV-infected patients undergoing treatment with direct-acting antivirals. We analyzed 103 samples from 28 patients. Compared with RT-PCR, sensitivity was 96.2% and specificity was 100%. The correlation between techniques was excellent (Pearson coefficient: 0.871). HCV-Ag proved to be useful in patients with sustained viral response and in patients who experienced treatment failures.

Toxin B PCR cycle threshold as a predictor of poor outcome of Clostridium difficile infection: a derivation and validation cohort study.

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.

Clinical significance of direct cytotoxicity and toxigenic culture in Clostridium difficile infection.

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.

Role of binary toxin in the outcome of Clostridium difficile infection in a non-027 ribotype setting.

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.