Hospital-acquired Clostridium difficile infection in Mainland China: A seven-year (2009–2016) retrospective study in a large university hospital

Clostridium difficile infection (CDI) is associated with risk for severe disease and high mortality. Little is known about the extent of hospital-acquired CDI in Mainland China. In this study, we aimed to investigate the annual CDI incidence, bacterial genotypes, risk factors for severe CDI and survival over a 7-year period. A total of 307 hospital-acquired CDI patients were enrolled, and 70.7% of these cases were male. CDI incidence was 3.4 per 10,000 admissions. Thirty-three different sequence types (STs) were identified, among which ST-54 (18.2%), ST-35 (16.6%) and ST-37 (12.1%) were the most prevalent. During the follow-up period, 66 (21.5%) patients developed severe CDI and 32 (10.4%) patients died in 30 days. Multivariate analysis revealed that bloodstream infection, pulmonary infection and C-reactive protein were significantly associated with severe CDI. After adjustment for potential confounders, old age, bloodstream infection, fever, mechanical ventilation, connective tissue disease, macrolide use and hypoalbuminaemia were independently associated with 30-day mortality in patients with CDI. The CDI prevalence has been low and stable in our center, and STs of Clostridium difficile were different from dominant STs in Western countries. Our data emphasize the need of continued education and surveillance of CDI to reduce the CDI burden in China.

Data Collection. The following data of all patients with hospital-acquired CDI were collected through the hospital database, including age, gender, date of onset of diarrhea, prior underlying diseases, prior medication, prior surgery, clinical data, laboratory parameters, in-hospital medications and prognosis. "Prior" refers to within two months before the CDI diagnosis. CDI characteristics at enrollment were also collected. Vital signs and laboratory findings within 12 hours before or 24 hours post-enrollment were tabularized. Charlson comorbidity index score was used to evaluate the basic conditions of underlying diseases 20 . Follow-up clinical data were initiated upon hospital admission. For discharged patients, prognostic information was obtained from medical records, telephone contact, or personal visits. The primary endpoint of the study was 30-day mortality. Statistical Analysis. Annual CDI incidence was calculated as the number of events per 10,000 admissions.
For the assessment of risk factors for severe CDI and 30-day mortality, continuous variables were expressed as median with interquartile range (IQR) or mean ± standard deviation (SD). Continuous data were compared in the univariate analyses by Student t-test or Mann-Whitney's U-test. Nominal variables were expressed as number/percentage and compared using chi-square test. A Cox's proportional hazard model was used to identify the predictors of time-dependent death of CDI patients. Candidate variables (P < 0.10) after a bivariate analysis were entered into a multivariate Cox's model using a backward-forward approach. The survival curves of the different ST groups were plotted through the multivariate Cox's model. In addition, a multivariate logistic regression analysis was performed to assess risk factors for severe CDI, in which the entry and removal probability for the stepwise-backward method was set as 0.05 and 0.10, respectively, and variables with P < 0.05 were retained in the final model. Hosmer-Lemeshow goodness of fit test was performed for logistic regression. Statistical analyses were performed using SPSS (version 23.0; SPSS Inc., Chicago, IL, USA).

Incidence.
During the 85-month study period, 307 of 0.91 million hospital admissions were diagnosed with hospital-acquired CDI (Fig. 1). The average incidence was 3.4 per 10,000 admissions (3.1 per 100,000 patientdays), which varied between 2.5 and 4.3 per 10,000 admissions annually. The average positive rate of toxigenic C. difficile in diarrhea patients was 7.3%, which varied between 5.2% and 8.9% annually (Supplementary Table 1 Both median duration of hospitalization before and after CDI onset was 14 days (IQR, 6-29; 7-31 days). The most frequent comorbidities were liver disease (25.6%) and solid tumor (24.1%). Within eight weeks prior to enrollment, 219 patients (70.0%) were admitted to the hospital at least once, and 16.0% of them had a history of ICU admission prior to developing CDI. The 203 patients (66.1%) received at least 1acid suppression medication, while antibiotic use was noted in 257 patients (83.7%), which were most commonly cephalosporins (50.2%), fluoroquinolones (32.6%), or β-lactam/β-lactamase inhibitors (29.6%). Furthermore, only 110 patients (35.8%) were treated with metronidazole (intravenous or oral) or oral vancomycin that targeted CDI. As the lack of understanding of CDI among clinicians, nearly half of these patients (n = 142, 46.3%) only received antidiarrheal and probiotics treatment; and the remaining 55 patients (17.9%) were not treated at all (Table 1).

Microbiological Characteristics.
A total of 307 toxigenic isolates, isolated from 4,199 stool samples, were available for detailed microbiological characterization. Seventy-two isolates (23.5%) were A− B+ CDT− strains, 13 (4.2%) isolates were A+ B+ CDT+ strains and the rest (222, 72.3%) were A+ B+ CDT− strains. Toxigenic C. difficile strains were analyzed by MLST and divided into 33 different STs. The most common STs are listed in Table 2 Risk Factors for Severe CDI. Sixty-six of 307 CDI episodes were classified as severe CDI (21.5%). The multivariate analyses of risk factors for severe CDI are shown in Table 3 (Candidate variables were selected by univariate logistic regression, Supplementary

Discussion
In this retrospective study, a standard diagnosis procedure was used to determine hospital-acquired CDI frequencies between 2009 and 2016 in our center. Our analysis revealed an annually stable, low CDI prevalence in our center, and the average CDI rate was 3.4 per 10,000 admissions or 7.3% among diarrhea patients. We also analyzed the STs of all isolates, and the dominant STs were different from that reported in North America and Europe.
A recent meta-analysis of CDI incidence in Mainland China suggested that the pooled incidence of toxigenic C. difficile among patients with diarrhea was 14%, but it varied significantly from 23% to 3% in different regions 11 .  We have reason to believe that a CDI diagnosis may have been missed in a portion of patients with diarrhea because of the poor awareness of CDI among physicians, representing the main reason for the relatively low CDI incidence. In addition, a portion of patients who developed CDI after discharge within 28 days were not included due to incomplete clinical data. Another possible reason was the different dominant CD strains compared in other countries.
Age appears to be important for developing CDI. The median age among patients in this study was 56 years, which was slightly younger than that reported in Western countries [6][7][8] . However, this study clearly revealed that the age composition of CDI patients was biased toward an older population: more than half of the cases were 50 years or older, and one in four cases was 70 years or older. The association of old age with CDI may suggest that weakened immunity is a condition for CDI.
Another finding in this study was that 70.7% of CDI patients were male, and 9.2% (20/217) of these male patients died within 30 days, which was in contrast with the findings reported in Western countries, where females were dominant among CDI patients 6,7 . The population of CDI patients was also not predominant female in several studies from East Asian countries, we suggest that the difference in CDI patient gender may be related to geographical location, genetic disposition and diet habits [28][29][30] .
Furthermore, as high as 83.7% of CDI patients had a history of previous use of antibiotics in this study, which was in line with the rates reported by others 7,27 , suggesting that the use of antibiotics may have caused imbalance of bacterial flora in the gut. It was interesting to note that the highest CDI occurred in patients admitted to the Internal Medicine Department, since these patients were most likely exposed to antibiotics for long periods and had a long hospital stay.
Due to the lack of awareness of CDI among physicians, the treatment of diarrhea did not follow the CDI treatment guidelines. Interestingly, when 30-day mortality was correlated with the treatment, mortality was 9.9% (14/142) in patients who accepted symptomatic treatment and 9.1% (5/55) in patients with no therapy; and both were lower than the overall mortality. The lower mortality in patients without treatment may suggest mild CDI, and only severe diarrhea brought the attention of physicians to possible CDI and initiated the CDI treatment. Furthermore, among patients who accepted symptomatic treatment, 100 of 142 (70.4%) patients received probiotics. Most of these probiotics contained clostridium butyricum, which has been shown to be effective in restoring CDI affected colonies both in vitro and in vivo [31][32][33] .
In this study, ST-54 (RT 012) and ST-35 (RT 046) were the most prevalent, followed by ST-37 (RT 017) and ST-3 (RT 001), which differed from that identified by Bauer and colleagues 7     Marjolein and colleagues 6 reported that CDT+ strains accounted for 23% (90/389) of the total isolates, and 19/90 (21.1%) of these isolates were RT 027. However, CDT+ strains in our study were rare (4.2%, 13/307), and most of the strains were ST-5 (9/13, 69.2%). Similarly, Rupnik and colleagues 35 only found five isolates (1.6%) positive for binary toxin among the 310 isolates obtained from nine hospitals in Japan and Korea. In the present study, only a single isolate for ST-1 (RT 027) and ST-11 (RT 078) was identified, respectively, in 2012 and 2016. Due to the association of ST-1 with the CDI outbreak, the CDI incidence in the Estrie region of Quebec increased from 22.2 per 100,000 population in 1991 to 92.2 per 100,000 population in 2003 36 . This first detection of ST-1 in our hospital alerts us that this type is frequently associated with CDI outbreaks and dismal prognosis in the Western world 2 .
Furthermore, as many as 21.1% of cases were diagnosed as severe CDI, and this was in line with the reported percentages in literatures 8,37 . Several studies had identified pulmonary infection as independent risk factors for severe CDI, and other risk factors were mentioned occasionally 27 . CDI patients that were accompanied by concomitant bloodstream and pulmonary infection may have weakened immunity, aggravating the CDI disease. High CRP likely reflects the severity of colonic inflammation, which represents a candidate marker for prognosis. Strain types have been suggested as an additional factor for CDI severity. In the present study, the disease in patients with ST-81 (5/13, 38.5%) and ST-5 (3/9, 33.3%) infection was likely more severe, compared with other STs.
Most risk factors identified in the present study for 30-day mortality were consistent with previous studies 37 . For instance, a pervious study suggested that CDI-related mortality occurred mainly within 30 days after onset, and was low after 90 days, as demonstrated by long-term follow-ups 6 . The 30-day CDI-related mortality in this cohort was 10.4%, which was comparable to the reported mortalities in North America and Europe 6,7,38,39 . It was noted in our study that CDT+ strains or ST-5 infected patients were predisposed to higher mortality than other STs. Thus, different extents of pathologic changes caused by different strains may have different impacts on  Table 3). mortality. Interestingly, A+ B+ strains were associated with more fatal outcomes than A− B+ strains, except for ST-3. This difference in mortality between A− B+ and A+ B+ strains requires further investigation. In addition, differences in the severity of the primary disease in each CDI patient in hospitals may primarily contribute to death. Unexpectedly, we found that connective tissue disease was associated with increased mortality. Patients with connective tissue disease may have immune dysfunction, which may have made it difficult to contain CDI. Few limitations in this study warrants discussion. First, only 30-day follow-ups were carried out, since many of the patients in this cohort resided outside of Hangzhou metropolitan boundaries. Second, our results derived a single-center study, and the main findings require verification through multicenter long-term studies in the future.
In conclusion, hospital-acquired CDI frequency in our region appears to be relatively moderate, and no clear spike was observed during the 7-year period. CDI outcomes appeared to be less severe compared to other regions. The strain types of C. difficile infection in our center also markedly differed from the strains reported from Western countries. We identified bloodstream infection and connective tissue disease as independent risks for severe CDI and 30-day mortality.