Efficiency of six virulence genes detection versus HEp-2 cell adhesion test for diagnosis of enteroaggregative E.coli infection among diarrhea patients
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摘要:
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。 Abstract: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 2 366 diarrhea patients during January 2015 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 1 390 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 2 366 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% (2 097/2 100) 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 the 2nd/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. -
图 1 EAEC检测流程图
Figure 1. Workflow for detection of EAEC in fecal samples from diarrhea patients
图 2 各诊断方法ROC曲线
Figure 2. ROC curves for combination-specific six virulence genes detections
表 1 EAEC特征基因引物序列、扩增产物长度及扩增条件
Table 1. Primer sequences, amplification products sizes and reaction conditions for detections of six virulence genes of EAEC
特征基因 引物序列（5'-3'） 产物长度（bp） 扩增程序 uidA （F）TGATCGCGGTGTCAGTTCTTT；（R） ATTGCCCGGCTTTCTTGTA 269 预变性94℃ 5 min；
变性94℃ 60 s，
退火58℃ 90 s，
延伸72℃ 60 s，
循环30次；
延伸72℃ 5 minaggR （F） CGATACATTAAGACGCCTAAAG；（R）TCTGATACATTAAATTCATCTGC 400 pic （F） AGCCGTTTCCGCAGAAGCC；（R） AAATGTCAGTGAACCGACGATTGG 1111 astA （F） TGCCATCAACACAGTATATCCG；（R） ACGGCTTTGTAGTCCTTCCAT 102 aaiA （F） CCCACGACCAGATAACG；（R） GTTTTCAGGATTGCCATTAG 476 aatA （F） CTGGCGAAAGACTGTATCAT；（F） CAATGTATAGAAATCCGCTGTT 630 aaiG （F） GGGAGTGTTTCAGTCTGGAC；（R） ATTTGTCACAAGCTCAGCAT 782 
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表 2 EAEC检测分子诊断方法分类
Table 2. Combinations of six virulence genes to be detected for identification of EAEC in stool samples from diarrhea patients
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表 3 上海市浦东新区 2366例腹泻病例不同年龄组、性别、发病时间分布情况
Table 3. Gender, season, and year distribution by age groups for 2 366 diarrhea patients surveyed d in Pudong New Area of Shanghai
人口学特征 0 ～5岁 6～20岁 21～50岁 51～65岁 ≥ 66岁 合计 n % n % n % n % n % n % 性别 男性 479 37.05 118 9.13 434 33.57 168 12.99 94 7.27 1293 54.65 女性 313 29.17 75 6.99 355 33.08 207 19.29 123 11.46 1073 45.35 季节 春季 177 32.24 57 10.38 184 33.52 85 15.48 46 8.38 549 23.20 夏季 219 34.22 44 6.88 213 33.28 103 16.09 61 9.53 640 27.05 秋季 245 36.84 54 8.12 199 29.92 103 15.49 64 9.62 665 28.11 冬季 151 29.49 38 7.42 193 37.70 84 16.41 46 8.98 512 21.64 年份（年） 2015 107 20.34 36 6.84 212 40.30 108 20.53 64 11.97 526 22.23 2016 155 31.12 26 5.18 178 35.94 80 16.06 59 11.85 498 21.05 2017 164 38.32 35 8.18 138 32.24 52 12.15 39 9.11 428 18.09 2018 203 41.43 42 8.57 140 28.57 76 15.51 29 5.92 490 20.71 2019 163 38.44 54 12.74 121 28.54 59 13.92 27 6.37 424 17.92
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表 4 不同年龄组腹泻病例6种EAEC毒力基因检出情况
Table 4. Age group-specific detection rates of six EAEC virulence genes in 2 366 diarrhea patients
年龄组（岁） aggR astA pic aaiA aaiG aatA 0～5 n 24 73 31 54 6 35 % 5.41 16.44 6.98 12.16 1.35 7.88 6～20 n 7 21 6 10 1 10 % 6.80 20.39 5.83 9.71 0.97 9.71 21～50 n 17 12 22 56 10 46 % 3.52 2.48 4.55 11.59 2.07 9.52 51～65 n 8 60 13 22 2 24 % 3.59 26.91 5.83 9.87 0.9 10.76 ≥ 66 n 0 28 6 12 5 8 % 0.00 20.44 4.38 8.76 3.65 5.84 合计 n 56 304 78 154 24 123 % 4.03 21.87 5.61 11.08 1.73 8.85 χ2 值 703.15 3.01 1.94 3.43 P 值 < 0.05 a < 0.05 0.56 0.75 0.34 a 0.49 注：n为阳性数，%为检出率。a Fisher精确检验，仅有P 值，无χ2 值。
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表 5 EAEC毒力基因型分布情况
Table 5. Strain type-specific number of genotypes and positive strains for the six virulence genes of detected EAEC
菌株类别 毒力基因型数量 菌株数 % 优势型别 V1 6 201 51.54 astA V2 10 89 22.82 aaiA-aatA V3 11 57 14.62 astA-aaiA-aatA V4 6 29 7.44 astA-pic-aaiA-aatA V5 3 14 3.59 aggR-astA-pic-aaiA-aatA 注：根据携带毒力基因种类数量，将菌株定义为V1～V5五类，%为构成比。
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表 6 不同诊断方法间结果比较及一致性评价
Table 6. Consistency among combination-specific six virulence genes detection and HEp-2 cell adhesion test in diagnosis of EAEC infection among diarrhea patients
分子诊断方法 灵敏度（%） 特异性（%） Kappa 值 a 金标准 诊断方法一 诊断方法二 诊断方法三 诊断方法四 诊断方法一 19.92 99.86 0.30 诊断方法二 73.30 92.43 0.58 0.24 诊断方法三 61.91 97.19 0.65 0.37 0.61 诊断方法四 78.20 92.91 0.61 0.24 0.95 0.70 诊断方法五 84.21 92.71 0.64 0.22 0.94 0.70 0.95 注：P 均 < 0.05。
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