儿童艰难梭菌相关性腹泻致病基因及临床特点研究

Virulence genes and clinical features of Clostridium difficile-associated diarrhea in children

目的 研究儿童艰难梭菌相关性腹泻（CDAD）的致病基因及临床特点.方法 选择2012年5月至2013年1月首都医科大学附属北京儿童医院121例应用抗生素后出现腹泻的住院患儿为研究对象,用聚合酶链式反应（PCR）方法检测粪便中艰难梭菌致病基因tcdA、tcdB、tcdC、tcdD、tcdE、cdtA、cdtB,并对目标产物进行测序分析,任一致病基因检测阳性者设为CDAD组,致病基因均阴性者设为非CDAD组,了解儿童CDAD的临床特点.结果 入选的121例腹泻患儿中,tcdB基因阳性标本60例（49.6％）,其中tcdA、tcdB均阳性（A＋B＋）12例,tcdA阴性而tcdB阳性（A-B＋）48例,tcdA阳性而tcdB阴性（A＋B-）标本0例,cdtA、cdtB阳性标本0例;在60例tcdB阳性标本中,tcdC、tcdD、tcdE基因阳性分别为24例（40％）、25例（42％）、24例（40％）.tcdA、tcdB、tcdC、tcdD、tcdE基因测序结果与参考序列一致.60例CDAD患儿中,婴幼儿37例（62％）,急性腹泻42例（70％）,伴发症状主要包括发热39例（65％）、贫血40例（67％）、白细胞计数异常36例（60％）、低白蛋白血症30例（50％）、C反应蛋白（CRP）升高25例（42％）.CDAD组CRP[45.0（16.0,89.0）mg/L]明显高于非CDAD组[19.0（14.5,41.5） mg/L],差异有统计学意义（Z=-2.008,P=0.045）;CDAD组白蛋白[35.3（29.7,39.8）g/L]明显低于非CDAD组[38.5（33.9,41.5） g/L],差异有统计学意义（Z=-2.610,P=0.009）.A＋B＋组与A-B＋组患儿性别、年龄、腹泻天数、住院天数、使用抗生素时间及实验室检查差异无统计学意义（P均＞0.05）.结论 本组艰难梭菌致病基因以tcdA阴性而tcdB阳性（A-B＋）多见,儿童CDAD临床表现以急性腹泻伴发热为主,实验室检查可见白细胞计数异常、CRP升高、血红蛋白及白蛋白降低.

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,todD 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.