患者胃窦黏膜组织幽门螺杆菌real time-PCR检测与分离培养结果的一致性分析

杨雅名 何利华 杨宁敏 宫雅楠 韩秀瑞 范如岳 薛志静 魏永越 张建中

杨雅名, 何利华, 杨宁敏, 宫雅楠, 韩秀瑞, 范如岳, 薛志静, 魏永越, 张建中. 患者胃窦黏膜组织幽门螺杆菌real time-PCR检测与分离培养结果的一致性分析[J]. 疾病监测, 2022, 37(5): 641-645. doi: 10.3784/jbjc.202112020618
引用本文: 杨雅名, 何利华, 杨宁敏, 宫雅楠, 韩秀瑞, 范如岳, 薛志静, 魏永越, 张建中. 患者胃窦黏膜组织幽门螺杆菌real time-PCR检测与分离培养结果的一致性分析[J]. 疾病监测, 2022, 37(5): 641-645. doi: 10.3784/jbjc.202112020618
Yang Yaming, He Lihua, Yang Ningmin, Gong Yanan, Han Xiurui, Fan Ruyue, Xue Zhijing, Wei Yongyue, Zhang Jianzhong. Culture and real time-PCR in detection of Helicobacter pylori in gastric antrum mucosal specimen[J]. Disease Surveillance, 2022, 37(5): 641-645. doi: 10.3784/jbjc.202112020618
Citation: Yang Yaming, He Lihua, Yang Ningmin, Gong Yanan, Han Xiurui, Fan Ruyue, Xue Zhijing, Wei Yongyue, Zhang Jianzhong. Culture and real time-PCR in detection of Helicobacter pylori in gastric antrum mucosal specimen[J]. Disease Surveillance, 2022, 37(5): 641-645. doi: 10.3784/jbjc.202112020618

患者胃窦黏膜组织幽门螺杆菌real time-PCR检测与分离培养结果的一致性分析

doi: 10.3784/jbjc.202112020618
基金项目: 国家科技重大专项(No. 2018ZX10712–001)
详细信息
    作者简介:

    杨雅名,女,新疆维吾尔自治区克拉玛依市人,在读硕士研究生,主要从事幽门螺杆菌临床分离株的耐药现况分析及猪螺杆菌的分离条件探索工作,Email:yangyaming@njmu.edu.cn

    通讯作者:

    张建中,Tel:010–58900757,Email:zhangjianzhong@icdc.cn

  • 中图分类号: R211; R573.6

Culture and real time-PCR in detection of Helicobacter pylori in gastric antrum mucosal specimen

Funds: This study was supported by the fund for National Science and Technology Major Project (No. 2018ZX10712–001)
More Information
  • 摘要:   目的  针对胃黏膜组织标本的幽门螺杆菌(HP)感染诊断的real time-PCR检测和分离培养2种最常用方法进行结果一致性分析,为HP感染诊断方法的选择提供科学依据。  方法  在浙江省4个地区(萧山、温岭、苍南和湖州)各选取1所医院,采集2020年11月16日至12月7日送至杭州致远检验医学研究所进行HP分离培养和耐药性分析的全部患者胃窦部黏膜活检标本,共1 312份。 每份样本经研磨匀浆处理后分为2份,分别用于常规HP分离培养和real time-PCR检测(以cagH为检测靶点),2种方法平行双盲进行。 检测结果的一致性及不同医院来源样本间的差异用χ2检验、Fisher精确检验以及独立样本t检验等统计学方法进行分析。  结果  1 312份样本中,分离培养法和real time-PCR法的阳性率分别为29.34%(385/1 312) 、28.51%(374/1 312),差异无统计学意义(P=0.636)。 来自4所医院的标本,2种方法的检出率差异均无统计学意义(P=0.075),二者在萧山某院、温岭某院、苍南某院、湖州某院样本以及总体样本中的Kappa值分别为0.84、0.89、0.75、0.91、0.85。 real time-PCR法的灵敏度和特异度分别为89.26%、100.00%。 在real time-PCR法检测阳性而培养阴性的样本中,检测Ct值为25.47~34.97,均值为30.90,90%的Ct值均≤34.46,频数最高组为29.00~30.00组。  结论  分离培养法和real time-PCR法在临床胃黏膜标本HP诊断中均可达到较高的检测效能,且2种方法检出结果一致性良好,可应用于HP个体化治疗,以便在做出HP感染诊断的同时提供受检者的药敏检测结果。 标本中HP菌量及标本的保存运输条件可能对结果产生重要影响。
  • 图  1  RT-PCR法检测阳性而分离培养法阴性样本的检测Ct

    Figure  1.  Ct values of RT-PCR single positive samples

    表  1  用于cagH基因的引物和探针

    Table  1.   Primers and probe used for cagH gene

    名称序列(5′~3′)长度
    (bp)
    引物
     cagHF TTATGTTAGAAATCGCTTGAGTGTCA74
     cagHR CGCTTCTCAAATGATACTTAATCAATC
    探针
     cagH-Probe FAM-AGGTGCTAGTAGCTAATC-BHQ1
    下载: 导出CSV

    表  2  不同医院来源病例幽门螺杆菌检测结果

    Table  2.   Detection results of H. pylori in specimens from different hospitals

     医院不同性别病例数(%)年龄(岁)培养法阳性率
    (%)
    RT-PCR法阳性率
    (%)
    χ2P双阴性率
    (%)
    男性女性
    萧山某院47.4652.5450.10±12.0727.8926.660.2490.61869.49
    温岭某院48.8651.1447.84±12.4828.5427.630.0900.76469.63
    苍南某院50.9449.0649.70±12.9431.1336.790.7570.38460.38
    湖州某院46.2253.7849.87±13.9438.6634.450.4530.50161.34
     合计48.0951.9149.29±12.4929.3428.510.2240.63668.06
    下载: 导出CSV

    表  3  分离培养法与real time-PCR法检测结果的一致性检验

    Table  3.   Consistency test for culture and real time-PCR

     医院RT-PCR法
    检测样本
    (份)
    分离培养法检测样本(份)KappaP检出
    不一致率
    (%)
    阳性数阴性数
    萧山某院156170.840.2806.47
    25451
    温岭某院11380.890.5034.57
    12305
    苍南某院3090.750.14611.32
    364
    湖州某院4100.910.0634.20
    573
     合计340340.850.2606.02
    45893
    下载: 导出CSV
  • [1] Kotilea K, Bontems P, Touati E. Epidemiology, diagnosis and risk factors of Helicobacter pylori infection[M]//Kamiya S, Backert S. Helicobacter Pylori in Human Diseases. Cham: Springer, 2019: 17–33. DOI: 10.1007/5584_2019_357.
    [2] Kotilea K, Bontems P, Touati E. Epidemiology, diagnosis and risk factors of Helicobacter pylori infection[M]//Kamiya S, Backert S. Helicobacter Pylori in Human Diseases. Cham: Springer, 2019: 17–33. DOI: 10.1056/NEJMra020542.
    [3] Hunt RH, Xiao SD, Megraud F, et al. Helicobacter pylori in developing countries. World gastroenterology organisation global guideline[J]. J Gastrointestin Liver Dis, 2011, 20(3): 299–304.
    [4] Ren S, Cai PP, Liu YQ, et al. Prevalence of Helicobacter pylori infection in China: a systematic review and meta-analysis[J]. J Gastroenterol Hepatol, 2022, 37(3): 464–470. DOI:  10.1111/jgh.15751.
    [5] O'Connor A, O'Morain CA, Ford AC. Population screening and treatment of Helicobacter pylori infection[J]. Nat Rev Gastroenterol Hepatol, 2017, 14(4): 230–240. DOI:  10.1038/nrgastro.2016.195.
    [6] 谢川, 吕农华. 中国幽门螺杆菌感染的现状[J]. 疾病监测,2018,33(4):272–275. DOI:10.3784/j.issn.1003−9961.2018.04.004.

    Xie C, Lyu NH. Helicobacter pylori infection in China[J]. Dis Surveill, 2018, 33(4): 272–275. DOI: 10.3784/j.issn.1003−9961.2018.04.004.
    [7] Patel SK, Pratap CB, Jain AK, et al. Diagnosis of Helicobacter pylori: what should be the gold standard?[J]. World J Gastroenterol, 2014, 20(36): 12847–12859. DOI:  10.3748/wjg.v20.i36.12847.
    [8] Peng XH, Song ZQ, He LH, et al. Gastric juice-based real-time PCR for tailored Helicobacter pylori treatment: a practical approach[J]. Int J Med Sci, 2017, 14(6): 595–601. DOI:  10.7150/ijms.18996.
    [9] Hedman J, Knutsson R, Ansell R, et al. Pre-PCR processing in bioterrorism preparedness: improved diagnostic capabilities for laboratory response networks[J]. Biosecur Bioterror, 2013, 11 Suppl 1: S87-101. DOI:  10.1089/bsp.2012.0090.
    [10] 李红, 杨天阔, 申亚琳, 等. 幽门螺杆菌药敏检测方法研究进展[J]. 中华医学杂志,2020,100(30):2393–2396. DOI:10.3760/cma.j.cn112137−20200525−01648.

    Li H, Yang TK, Shen YL, et al. Research progress in antibiotic sensitivity testing assays for Helicobacter pylori[J]. Natl Med J China, 2020, 100(30): 2393–2396. DOI: 10.3760/cma.j.cn112137−20200525−01648.
    [11] Malfertheiner P, Megraud F, O'Morain CA, et al. Management of Helicobacter pylori infection—the Maastricht V/Florence Consensus Report[J]. Gut, 2017, 66(1): 6–30. DOI: 10.1136/gutjnl−2016−312288.
    [12] Park SA, Ko A, Lee NG. Stimulation of growth of the human gastric pathogen Helicobacter pylori by atmospheric level of oxygen under high carbon dioxide tension[J]. BMC Microbiol, 2011, 11: 96. DOI: 10.1186/1471−2180−11−96.
    [13] Schabereiter-Gurtner C, Hirschl AM, Dragosics B, et al. Novel real-time PCR assay for detection of Helicobacter pylori infection and simultaneous clarithromycin susceptibility testing of stool and biopsy specimens[J]. J Clin Microbiol, 2004, 42(10): 4512–4518. DOI: 10.1128/JCM.42.10.4512−4518.2004.
    [14] Mikula M, Dzwonek A, Jagusztyn-Krynicka K, et al. Quantitative detection for low levels of Helicobacter pylori infection in experimentally infected mice by real-time PCR[J]. J Microbiol Methods, 2003, 55(2): 351–359. DOI: 10.1016/s0167−7012(03)00166−0.
    [15] Lascols C, Lamarque D, Costa JM, et al. Fast and accurate quantitative detection of Helicobacter pylori and identification of clarithromycin resistance mutations in H. pylori isolates from gastric biopsy specimens by real-time PCR[J]. J Clin Microbiol, 2003, 41(10): 4573–4577. DOI: 10.1128/jcm.41.10.4573−4577.2003.
    [16] Kobayashi D, Eishi Y, Ohkusa T, et al. Gastric mucosal density of Helicobacter pylori estimated by real-time PCR compared with results of urea breath test and histological grading[J]. J Med Microbiol, 2002, 51(4): 305–311. DOI: 10.1099/0022−1317−51−4−305.
    [17] He Q, Wang JP, Osato M, et al. Real-time quantitative PCR for detection of Helicobacter pylori[J]. J Clin Microbiol, 2002, 40(10): 3720–3728. DOI: 10.1128/jcm.40.10.3720−3728.2002.
    [18] 宋函憶, 姚鑫洁, 郑宇芪, 等. 幽门螺杆菌药敏检测方法比较及个体化治疗200例临床分析[J]. 中国实用内科杂志,2019,39(9):813–816. DOI: 10.19538/j.nk2019090117.

    Song HY, Yao XJ, Zheng YQ, et al. Comparison of antibiotic susceptibility tests of Helicobacter pylori and individualized therapy in 200 cases: a clinical analysis[J]. Chin J Pract Intern Med, 2019, 39(9): 813–816. DOI:  10.19538/j.nk2019090117.
    [19] Acharya KR, Dhand NK, Whittington RJ, et al. PCR Inhibition of a quantitative PCR for detection of Mycobacterium avium subspecies paratuberculosis DNA in feces: diagnostic implications and potential solutions[J]. Front Microbiol, 2017, 8: 115. DOI:  10.3389/fmicb.2017.00115.
    [20] Sidstedt M, Rådström P, Hedman J. PCR inhibition in qPCR, dPCR and MPS—mechanisms and solutions[J]. Anal Bioanal Chem, 2020, 412(9): 2009–2023. DOI: 10.1007/s00216−020−02490−2.
    [21] 刘国栋. 基于胃黏膜多重Real time PCR的幽门螺杆菌根除药物选择技术的建立与评价[D]. 北京: 中国疾病预防控制中心, 2011.

    Liu GD. Establishment and evaluation of multi Real time PCR of gastric biopsy for Helicobacter pylori eradication drugs selection[D]. Beijing: Chinese Center for Disease Control and Prevention, 2011.
    [22] Atkinson NSS, Braden B. Helicobacter pylori infection: diagnostic strategies in primary diagnosis and after therapy[J]. Dig Dis Sci, 2016, 61(1): 19–24. DOI: 10.1007/s10620−015−3877−4.
    [23] Pouw RE, Barret M, Biermann K, et al. Endoscopic tissue sampling - Part 1: upper gastrointestinal and hepatopancreatobiliary tracts. European Society of Gastrointestinal Endoscopy (ESGE) Guideline[J]. Endoscopy, 2021, 53(11): 1174–1188. DOI: 10.1055/a−1611−5091.
    [24] Malaty HM, Graham DY. Importance of childhood socioeconomic status on the current prevalence of Helicobacter pylori infection[J]. Gut, 1994, 35(6): 742–745. DOI:  10.1136/gut.35.6.742.
    [25] Yu H, Liu Y, Jiang SJ, et al. Serum pepsinogen II levels are doubled with Helicobacter pylori infection in an asymptomatic population of 40, 383 Chinese subjects[J]. Medicine (Baltimore) , 2021, 100(27): e26562. DOI:  10.1097/MD.0000000000026562.
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  • 收稿日期:  2021-12-02
  • 网络出版日期:  2022-04-19
  • 刊出日期:  2022-05-31

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