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Objective: We analyze the characteristics of Clostridioides difficile ( C. difficile ) infection among diarrhea patients in Kunming from 2018 to 2020 and provide evidence for follow-up surveillance and prevention. Methods: A total of 388 fecal samples of diarrhea patients from four sentinel hospitals in Yunnan Province from 2018 to 2020 were collected. Real-time quantitative PCR was used to detect the fecal toxin genes of C. difficile . The positive fecal samples isolated the bacteria, and isolates were identified by mass spectrometry. The genomic DNA of the strains was extracted for multi-locus sequence typing (MLST). The fecal toxin, strain isolation, and clinical patient characteristics, including co-infection with other pathogens, were analyzed. Results: Among the 388 fecal samples, 47 samples with positive reference genes of C. difficile were positive, with a total positive rate of 12.11%. There were 4 (8.51%) non-toxigenic and 43 (91.49%) toxigenic ones. A total of 18 strains C. difficile were isolated from 47 positive specimens, and the isolation rate of positive specimens was 38.30%. Among them, 14 strains were positive for tcdA , tcdB , tcdC , tcdR, and tcdE . All 18 strains of C. difficile were negative for binary toxins. The MLST results showed 10 sequence types (ST), including 5 strains of ST37, accounting for 27.78%; 2 strains of ST129, ST3, ST54, and ST2, respectively; and 1 strain of ST35, ST532, ST48, ST27, and ST39, respectively. Fecal toxin gene positive ( tcdB +) results were statistically associated with the patient's age group and with or without fever before the visit; positive isolates were only statistically associated with the patient's age group. In addition, some C. difficile patients have co-infection with other diarrhea-related viruses. Conclusions: The infection of C. difficile in diarrhea patients in Kunming is mostly toxigenic strains, and the high diversity of strains was identified using the MLST method. Therefore, the surveillance and prevention of C. difficile should be strengthened.

Clostridioides difficile is a key pathogen of antibiotic related diarrhea and hospital associated infection, causing several outbreaks in Europe and North Americans and resulting in severe disease burden. However, the standardized diagnostic principle and detection specifications in C . difficile infection (CDI) survey are limited in China, and the infection rate and disease burden of CDI in China are unclear. Therefore, National Institute for Communicable Disease Control and Prevention,National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, together with another 11 institutions, draft the group standard entitled "Diagnosis of Clostridium difficile infection (T/CPMA 008-2020)" of Chinese Preventive Medicine Association. Based on the principle of "legality, scientificity, advancement, and feasibility", this standard clarifies risk factors, diagnosis principles, diagnoses and differential diagnoses in order to improve the accuracy of CDI diagnosis in clinical practice, guide the surveillance for CDI, and understand the infection rate and disease burden of CDI in China .