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Objective: Clostridium difficile is an obligate anaerobic Gram-positive bacillus, it can cause Clostridium difficile-associated diarrhea (CDAD). This study aimed to investigate the virulence genes and clinical features of CDAD in children by gene detection., Method: From May 2012 to January 2013, the 121 inpatients in Beijing Children's Hospital who suffered from diarrhea after antibiotics treatment were detected for Clostridium difficile virulence genes including the five genes for pathogenic loci (tcdA, tcdB, tcdC, tcdD, tcdE) and the genes for binary toxin CDT (cdtA and cdtB) using polymerase chain reaction (PCR) in order to research the clinical features of CDAD, and analyze target products by sequencing., Results: In the 121 children with diarrhea, 60 (49.6%) were toxin B-positive,including 12 toxin A-positive and toxin B-positive (A+B+), 48 toxin A-negative but toxin B-positive (A-B+). The toxin A-positive but toxin B-negative (A+B-) specimens or binary toxin (cdtA and cdtB)-positive specimens were not detected. Of 60 tcdB-positive specimens, tcdC, tcdD and tcdE positive specimens were 24 (40%), 25 (42%), 24 (40%), respectively. The sequencing results of tcdA, tcdB, tcdC, tcdD, and tcdE gene were consistent with the reference sequence. In the 60 children with CDAD, infants (≤3 years) accounted for 62% (37/60). The duration of diarrhea was 3-77 days, and 42 (70%) cases had acute diarrhea; 39 (65%) patients had fever, 40 (67%) had anemia, 36 (60%) had abnormal white blood cell count, 30 (50%) had hypoalbuminemia, 25 (42%) had elevated C-reactive protein (CRP). The level of CRP in positive group was significantly higher compared to the negative group (45.0(16.0,89.0) mg/L vs. 19.0(14.5,41.5) mg/L, Z=-2.008, P=0.045). The level of plasma albumin in positive group was significantly lower compared to the negative group (35.3(29.7,39.8) g/L vs. 38.5(33.9,41.5) g/L, Z=-2.610, P=0.009). There were no significant differences in gender, age, duration of diarrhea, hospital staytime, time of using antibiotics and laboratory test between A+B+ group and A-B+ group (all P>0.05)., Conclusion: The main virulence genotype of Clostridium difficile was toxin A-negative but toxin B-positive in this research. The clinical features of CDAD in children were acute diarrhea with fever. Laboratory examination showed that white blood cell count was abnormal, CRP was increased, hemoglobin and plasma albumin were reduced.