基于6种肠集聚性大肠埃希菌毒力基因检测诊断效果评价

Efficiency of six virulence genes detection versus HEp-2 cell adhesion test for diagnosis of enteroaggregative E.coli infection among diarrhea patients

目的对肠集聚性大肠埃希菌(EAEC)分子诊断方法进行性能评价。方法对2015年1月-2019年12月上海市浦东新区腹泻门诊病例粪便样本分别进行大肠埃希菌分离鉴定,并对分离株进行“金标准”HEp-2细胞粘附试验和6种毒力基因检测(aggR、astA、pic、aaiA、aatA、aaiG)。根据不同基因组合,构建诊断方法一(uidA-aggR基因阳性)、诊断方法二(uidA-aggR/astA/pic任一基因阳性)、诊断方法三(uidA-aggR/aatA/aaiG/aaiA任一基因阳性)、诊断方法四(uidA-aggR/astA/aaiA任一基因阳性)、诊断方法五(uidA-aggR/astA/pic/aaiA/aatA/aaiG任一基因阳性)5种分子诊断方法。采用灵敏度、特异性、Kappa值、曲线下面积(AUC)等指标评价其诊断效能。结果在2366例病例样本中分离到1390株大肠埃希菌,19.14%(266/1390)分离株呈AA粘附表型,毒力基因检出率以astA(21.87%,304/1390)最高。以“金标准”、诊断方法一至五进行判定,EAEC检出率分别为11.24%(266/2366)、2.37%(56/2366)、14.84%(351/2366)、9.68%(229/2366)、5.26%(361/2366)和16.48%(390/2366)。诊断方法一特异性最高(99.86%,2097/2100)、灵敏度最低(19.92%,53/266),诊断方法三~五与金标准呈中度一致性(Kappa值为0.58~0.65),诊断方法二~五具有较高诊断价值(AUC为0.806~0.882)。结论基于EAEC毒力基因的分子诊断方法暂不可代替金标准。

Objectives To evaluate the efficiency of detecting six virulence genes of enteroaggregative Escherichia coli(E.coli),abbreviated to EAEC,for diagnosis of EAEC infection among diarrhea patients.Methods Isolation and identification of E.coli strains were conducted for fecal samples collected from 2366 diarrhea patients during January2015 to December 2019 at 12 medical facilities in Pudong New Area of Shanghai municipality.The detection of six virulence genes(aggR,astA,pic,aaiA,aatA,and aaiG)of EAEC and HEp-2 cell adhesion test–the gold standard assay of EAEC were performed on the isolated E.coli strains.The EAEC positivity is determined according to following five positive result combinations of the six virulence genes detection:(1st)both uidA and aggR;(2nd)uidA and any one of aggR/astA/pic;(3rd)uidA and any one of aggR/aatA/aaiG/aaiA;(4th)uidA and any one of aggR/astA/aaiA;and(5th)uidA and any one of aggR/astA/pic/aaiA/aatA/aaiG.Taking the result of HEp-2 cell adhesion test as the gold standard assay,the sensitivity and specificity of the detections of the six virulence genes and their combinations were evaluated using Kappa value,and area under receiver operation curve(AUC).Results For a total of 1390 E.coli strains isolated from all the samples,266(19.14%)were of AA adhesion phenotype and the positivity rate of astA(21.87%,n=304)was the highest among the six virulence genes detected.According to the standard of HEp-2 cell adhesion test,the detected rate(number)of EAEC positive among the samples from the 2366 diarrhea patients were 11.24%(266),2.37%(56),14.84%(351),9.68%(229),5.26%(361)and 16.48%(390/2366)based on the results of the 1st,2nd,3rd,4th,and 5th combination of the six virulence genes detections,respectively,with the highest specificity of 99.86%(2097/2100)and the lowest sensitivity of 19.92%(53/266)for the detection of the 1st combination of the six virulence genes.Moderate consistency was observed between the detection results of the standard assay and the 3rd/4th/5th combination of the six virulence genes detection,with the range of Kappa values of 0.58–0.65 and the AUC was from 0.806 to 0.882 for the2nd/3rd/4th/5th combination of the six virulence genes detection,indicating a high efficiency of the detections for the diagnosis of EAEC infection in diarrhea patients.Conclusion The results of the study suggest that the detection of the six EAEC virulence genes could not substitute HEp-2 cell adhesion test for diagnosing EAEC infection among diarrhea patients.