艰难梭菌培养联合酶法检测毒素蛋白(A/B)的临床应用价值探索

Value exploration of Clostridium difficile toxin A/B detection by combination methods of culture and ELFA

目的通过对艰难梭菌培养及毒素检测方法比较,建立艰难梭菌培养联合酶法检测毒素蛋白(A/B)的检测流程,评估其临床应用。方法收集 2015 年-2017 年住院腹泻患者粪便标本 852份,采用显色培养法( ChromID^TM)培养艰难梭菌,阳性菌株用酶联免疫荧光法检测其毒素蛋白A/B( Clostridium difficile toxins A and B,CDAB);用酶联免疫荧光法检测毒素蛋白A/B及阳性菌株,用PCR方法检测其tcdA和(或)tedB基因进行比较,运用检出率、灵敏度、特异度、阳性预测值和阴性预测值进行效能评价。结果852 例粪便标本经显色培养共分离菌108株,粪便直接酶法A/B毒素蛋白检出率为5.99%(51/852);阳性菌株联合酶法检测A/B毒素蛋白检出率为10. 92%(93/852);108株阳性菌株联合PCR方法93株表达tcdA^+tcdB^+;6株表达tcdA^-tcdB^+;9株未表达,为tedA^-tcdB^-;阳性菌株用酶法检测毒素蛋白A/B结果93株阳性,15株阴性,且93株阳性经PCR检测基因均表达tedA^+tcdB^+;以PCR检测艰难梭菌毒素基因作为参考方法,培养阳性菌株联合酶法、粪便直接酶法检测毒素蛋白A/B的敏感性、特异性、阳性预测值、阴性预测值分别为93. 94%、100%、100%.60%和51.52%、100%、100%、15. 79%;致性检验Kappa值分别为0.721和0. 150。结论显色培养后阳性菌株联合酶法检测艰难梭菌毒素蛋白A/B可以提高艰难梭菌检出率,与PCR毒素基因检测有较好一致性,操作简单,具有较好的临床应用价值。

Objective To establish the detection procedure of C. ifcile toxin A/ B by comparing the method of chromogenic cul- ture with enzyme-linked fluorescence assay ( ELFA) and the toxin detection methods, S0 as to evaluale the clinical application. Methods 825 stool specimens of inpatients with diaurhea were colleted during 2015-2017. Clostridlium dificile was culured by ChromID^TM, and the positive strains of toxin protein A/B (Clostridium dificile toxins A and B, CDAB) were detected by enzymelinked imnunolluorescence aussay;loxin protein A was delected by enzynne-linked immunofluorescence assay. tedA and/or tedB genes of the positive strains were deteted by PCR and compared. EFicacy evaluation was performed using detection rate, sensitivity, speeificity, positive predictive value,and negative predictive value. Results 108 strains of C. dificile were isolated from 852 specimens by chromogenic culture mcthod, the CDAB direct detection rate in stool specimens by ELIFA was 5. 99%(51/852);the CDAB dctcction rate of combinating the chromogenic culture and ELIFA was 10. 92%( 93/852);Of the 108 C. difficile strains, 93 strains carried todA and tcdB genes;6 strains carried todB only;tedA and tedR genes were absent in 9 strains;93 strains were CDAB positive and 15 strains were CDAB negative by ELIFA;and 93 strains with CDAB contained tedA aund tedB genes by PCR. CDAB gene was detected by PCR as a reference,the sensitivity,specifeity, positive predictive value and negutive predietive value of CDAB were deteeted by combinating the bacterial culture, ELIFA and stool ELIFA were 93. 94%, 100%,100%,60%,51. 52%,100%,100% and 15. 79%,respectively. The Kappa values in interrater reliability were 0.721 and 0.150, respectively. Conclusion The combination of the methods by chromogenic culture and ELIFA can improve the deteetion rate of CDAB, and the new method showed well consistency with toxin gene detection by PCR;on account of the easy operation aund low cosls;the method showed great significance in diarrhea diagnosis and preferuble clinical application valuc.