Prevalence and risk factors of toxigenic Clostridioides difficile asymptomatic carriage in 11 French hospitals

Clostridioides difficile infection (CDI) incidence has increased over the last 20 years. Studies suggest that asymptomatic carriers may be an important reservoir of C. difficile in healthcare settings. We conducted a point prevalence study to estimate the toxigenic C. difficile asymptomatic carriage rate and the associated risk factors in patients >3 years old. Between September 16, 2019 and January 15, 2020, all patients hospitalized in 11 healthcare facilities in the Paris urban area were included in the study. They were screened on the day of the survey for toxigenic C. difficile carriage by rectal swab and interviewed. Isolates were characterized by PCR ribotyping and multiplex PCR targeting toxin genes. A logistic regression model was used to determine the risk factors associated with toxigenic C. difficile asymptomatic carriage using uni- and multivariate analysis in the subpopulation of patients >3 years old. During the study period, 2,389 patients were included and screened. The median age was 62 years (interquartile range 35–78 years) and 1,153 were male (48.3%). Nineteen patients had a previous CDI (0.9%). Overall, 185/2389 patients were positive for C. difficile (7.7%), including 93 toxigenic strains (3.9%): 77 (82.8%) were asymptomatic (prevalence 3.2%) whereas 12 (12.9%) were diarrheic. Prevalences of toxigenic C. difficile were 3.5% in patients >3 years old and 7.0% in ≤3 years old subjects, respectively. Toxigenic strains mainly belonged to PCR ribotypes 106 (n = 14, 15.0%), 014 (n = 12, 12.9%), and 020 (n = 10, 10.8%). Among toxigenic strains, 6 (6.4%) produced the binary toxin. In multivariate analysis, two factors were positively associated with toxigenic C. difficile asymptomatic carriage in patients >3 years old: multidrug-resistant organisms co-carriage [adjusted Odd Ratio (aOR) 2.3, CI 95% 1.2–4.7, p = 0.02] and previous CDI (aOR 5.8, CI 95% 1.2–28.6, p = 0.03). Conversely, consumption of raw milk products were associated with reduced risk of toxigenic C. difficile colonization (aOR 0.5, CI 95% 0.2–0.9, p = 0.01). We showed that there was a low prevalence of asymptomatic toxigenic C. difficile carriage in hospitalized patients. Consumption of raw milk prevents toxigenic C. difficile colonization, probably due to the barrier effect of milk-associated bacteria.


Introduction
Clostridioides difficile is the leading cause of healthcare-associated diarrhea, accounting for almost half of all nosocomial gastrointestinal infections in hospitals in Europe (1). Exposure to C. difficile can lead to asymptomatic carriage or C. difficile infection (CDI) with a wide range of clinical presentations and outcomes (from mild diarrhea to severe colitis and death). CDI is associated with a substantial morbidity and mortality. In France, the extra cost of CDI in public acute-care hospitals was estimated to 163.1 million per year (2). Prevention of CDI stills remains challenging in both acute care and long-term care facilities. The CDI incidence in hospitalized patients has increased over the last 20 years in France (3). The asymptomatic C. difficile colonization rate is frequent although highly variable across studies (range: 6-18%) (4)(5)(6)(7)(8). Main risk factors for asymptomatic C. difficile carriage included previous antibiotic treatment, use of gastric acid suppression therapy, prior hospitalization and history of CDI (4,9,10). Risk factors described in the literature vary widely, depending especially on the sampling technique, the C. difficile detection method, the population targeted and the epidemiology situation (outbreak versus endemicity). Colonization is a prerequisite to infection, and asymptomatic carriage is associated with a higher risk of CDI (11). Moreover, asymptomatic carriers of toxigenic strains are an important and hidden reservoir of C. difficile in healthcare settings and could play a key role in the transmission of this sporeforming micro-organism (12). A recent study showed that the environment of asymptomatic C. difficile carriers is as contaminated as that of symptomatic CDI patients (13). A quasi-experimental controlled study revealed that identification and isolation of C. difficile carriers was associated with a decreased incidence of CDI (14). However, screening asymptomatic carrier is not currently recommended in infection control guidelines (15). The aim of the study was to evaluate the prevalence of toxigenic C. difficile asymptomatic carriage and its associated risk factors in order to provide insights into this potential reservoir in healthcare facilities.
We conducted a serial cross-sectional survey of C. difficile carriage between September 16, 2019 and January 15, 2020. All patients hospitalized more than 24 h on a given day in one of the 11 facilities (except patients from psychiatric units) who agreed to participate in the survey were eligible to inclusion. A non-opposition form was obtained from each patient included in the study (or from parents/ guardians for pediatric patients). Clinical wards and eligible patients were informed by information notes according to the protocol.
Six nurses were specifically recruited and trained to implement the protocol. They interviewed the patients and collected the data. The questionnaire (available as Supplementary Table S1) was completed by the nurses and included demographic data, life conditions, previous treatments and food consumption.
For all included patients, a screening sample was taken using an eSwab ® system (COPAN, Brescia, Italy) by the nurse or by the patients themselves. The sample was collected by rectal swabbing, by soaking the swab in the stools or by swabbing an ostomy. All samples were sent to a central microbiology laboratory for subsequent analysis.
Plates were incubated for 48 h at 37°C in an anaerobic atmosphere. Suspicious colonies (based on black coloration and the morphological aspect) were analyzed using matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (Brucker Daltonics, Germany). DNA extraction was performed from colonies grown on Brucella agar (bioMérieux ® , Marcy-l'Etoile, France) using the InstaGene Matrix™ kit (Bio-Rad ® , Marnes-la-Coquette, France).
Strains were characterized by a previously described multiplex PCR assay detecting tpi, tcdA, tcdB, tcdC, cdtA, and cdtB genes coding for Frontiers in Medicine 03 frontiersin.org the triose phosphate isomerase, toxin A, toxin B, TcdC and the two components of the binary toxin, respectively (17). Isolates were typed by capillary gel electrophoresis-based PCR ribotyping on an ABI 3500 sequencer (Applied Biosystems, Foster City, CA, United States), using primers described by Bidet et al. (18). After DNA amplification, each PCR product was diluted at 1/200. One microliter of this dilution was mixed with 10.5 μL formamide and 0.5 μL GeneScan LIZ600 (Applied Biosystems ® , Foster City, United States). The banding patterns were analyzed with the GeneMapper software (Thermo Fisher Scientific, Villebon-sur-Yvette, France). PCR ribotypes (RT) were assigned using the Webribo database. 1 ESwab ® samples were also plated onto specific chromogenic culture media: chromID ® CARBA SMART, chromID ® VRE, and chromID ® ESBL-PE (bioMérieux, Craponne, France) to search for the presence of MDRO co-carriage.

Statistical analysis
Prevalence of C. difficile carriage (including toxigenic and non-toxigenic strains) was reported in the overall study population, by hospitals, by specialties, by age and by time from admission. Results were expressed as median [interquartile range (IQR)] for continuous variables and N (%) for categorical variables.
Risk factors for toxigenic C. difficile asymptomatic carriage in patients >3 years old were investigated by uni-and multivariate analysis. Asymptomatic patients with toxigenic C. difficile were compared with asymptomatic non-C. difficile carriers in patient >3 years old. Asymptomatic patients with toxigenic C. difficile was defined as patient without diarrhea (<3 stools per day for 48 h) and with a rectal sample positive for a toxigenic C. difficile isolate. Patients with diarrhea on the day of inclusion were excluded from the analysis. A logistic regression analysis was used to determine the risk factors associated with asymptomatic carriage of toxigenic C. difficile. Variables with a p < 0.20 in univariable analysis were included in the multivariate logistic regression model. A backward stepwise approach was used to identify independent predictors. Adjusted odds ratios (aOR) and 95% confidence intervals (95% CIs) were calculated for all variables. p < 0.05 were considered statistically significant. Statistical analysis was made using Stata ® software, v15 (StataCorp, College Station, Texas, United States).

Risk factors for asymptomatic toxigenic Clostridioides difficile carriage in patients >3 years old
Risk factors for toxigenic C. difficile asymptomatic carriage in patients >3 years old were investigated by uni-and multivariate analysis: 61 toxigenic C. difficile asymptomatic carriage patients were compared with 1724 asymptomatic non-carriers of C. difficile (Table 3).
In univariate analysis, six factors were significantly associated with toxigenic C. difficile asymptomatic carriage: male gender, comorbidity, previous CDI, MDRO co-carriage, vegan or vegetarian and consumption of raw milk products. Other factors, e.g., previous antimicrobial treatment, use of proton-pump inhibitor, laxatives or Frontiers in Medicine 05 frontiersin.org anti acids or hospitalization during the last 12 months were more frequently found in C. difficile carriers than controls but the difference was not significant. In multivariate analysis, only two factors remained significantly associated with toxigenic C. difficile asymptomatic carriage: MDRO co-carriage (aOR 2.3, CI95% 1.2-4.7, p = 0.02) and previous CDI (aOR 5.8, CI95% 1.2-28.6, p = 0.03). Conversely, consumption of raw milk products were associated with reduced risk of toxigenic C. difficile colonization (aOR 0.5, CI95% 0.2-0.9, p = 0.01).

Discussion
In this large French multicenter point-prevalence study, a low prevalence of toxigenic C. difficile carriage was observed among hospitalized patients (3.9%). These results provide valuable epidemiological insight into the understanding of CDI, a frequent and severe condition in hospitalized patients. Asymptomatic carriers of C. difficile have been shown to play a role in the transmission of CDI (11,19) and can contaminate the hospital environment by spores. Moreover, asymptomatic carriers after discharge from hospital could also be a source of community-associated CDI cases (20, 21).
Our results are in the lower range of asymptomatic carriage previously reported in hospitalized patients (6-18%) and also lower than the prevalence of 5.8% found in another recent French multicenter study (4)(5)(6)(7)(8). This result may reflect differences in studied populations, or local epidemiology such as hospital outbreaks. It can also be explained by different microbiological techniques used to isolate C. difficile. We used rectal swabbing and direct culture whereas others used stool samples and enriched culture (4).
Children ≤3 years old were frequently found positive for C. difficile (22.4%) including with toxigenic C. difficile (7.0%) but the presence of C. difficile in this population has often been considered as non-pathogenic (22). Our results are in agreement with previous studies where a high colonization rate in children less than 2 years old was observed, ranging from 17 to 70% and mainly due to nontoxigenic strains (22-26). In contrast, prevalence was the lowest in parturient (0.7%), reflecting asymptomatic carriage in the community (27).
We identified two factors positively associated with toxigenic C. difficile carriage in subjects >3 years old. A previous CDI episode was already known as a risk factor (28,29), and had the highest impact in our study (aOR 5.7, CI95% 1.2-28.1, p = 0.03). The other associated factor was MDRO co-carriage. This risk factor may be a marker of a prolonged hospitalization and antibiotic exposure. It may also reflect the high level of healthcare worker contacts linked to inter-individual transmission of pathogens.
In contrast, consumption of raw milk products reduced risk of toxigenic C. difficile carriage, probably due to the barrier effect of milk-associated bacteria. A meta-analysis suggested that probiotic prophylaxis may be a useful and safe CDI prevention strategy (30).
Another striking finding of this study was the lack of significant association between C. difficile colonization and antimicrobials or proton-pump inhibitor therapy. Use of antibiotics and proton-pump inhibitors, by disrupting the intestinal microbiota, were previously associated with C. difficile colonization and infection (31, 32). However, in a recent meta-analysis including 1,588 asymptomatic patients colonized with C. difficile, antibiotics in the previous 3 months was not associated with significant effects on risk of colonization (9).
The results of the strain typing analysis indicated a large diversity of strains and the distribution of RT was consistent with previous investigations. For several years, RTs 014 and 020 have been predominant in epidemiological studies in France or in Europe. They represented 18.7 and 21.9% of C. difficile strains in   (36). In a recent French multicenter study, RT 014/020 was the most common toxigenic strain in C. difficile This study has some limitations. First, the cross-sectional survey design did not allow us to investigate the dynamics of C. difficile colonization (acquisition, persistence and clearance of carriage). Second, generalizing our results is difficult because only 55.7% of eligible patients were included and screened. Third, the screening method for carriage was not the most sensitive since we used rectal swabbing rather than stool specimens and there was no broth enrichment step during culture.
A strength of our study was the inclusion of a large number of hospitalized patients from 11 facilities, without selecting patients at hospital admission or from high-risk units, thus reducing the impact of center effect. We also explored many risk factors including food consumption in the context of one health.

Conclusion
In conclusion, the prevalence of C. difficile toxigenic carriage was low in patients >3 years old (3.5%), but higher in ≤3 years old (7.0%) and in patients hospitalized >3 days (4.6%). These patients and may therefore represent a potential reservoir for CDIs.

Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics statement
The studies involving human participants were reviewed and approved by the AP-HP project CODBAHRE no. 180561; IDRCB no. 2019-A01226-51. Written informed consent to participate in this study was provided by the participants or their legal guardian/ next of kin.