分子生物学诊断新方法在淋巴结结核诊断中的应用研究

发布时间：2018-08-17 09:30

【摘要】：目的:浅表淋巴结结核目前常用影像学、细菌学培养、常规病理学做为主要的诊断手段,但这些方法在敏感性和特异性方面均不理想。近年来检测结核分枝杆菌特异基因的分子生物学检测方法发展迅速,具有快速、准确等优点。因此本研究通过前瞻性研究,评估了Xpert MTB/RIF新技术、荧光定量PCR技术及高分辨熔解曲线法在浅表淋巴结结核诊断中的应用,并与目前常用的诊断方法进行了对比研究。方法:从2015年3月到2016年1月采用针吸活检和切除活检两种方法连续的收集了86例疑似淋巴结结核标本和13例其他淋巴结疾病标本。收集的标本平均分为两部分,一部分用于Xpert MTB/RIF的检测、BACTEC MGIT960培养和罗氏培养,另一部分用于常规病理学检查、石蜡包埋标本的PCR检测、荧光PCR熔解曲线利福平和异烟肼的耐药检测。结果:临床疑似淋巴结结核的患者81例,Xpert MTB/RIF检测MTBC阳性74例(91.4%),组织标本PCR阳性60例(74%),BACTEC MGIT960培养阳性24例(29.6%),罗培阳性13例(16%),病理形态学诊断淋巴结结核38例(46.9%),所有方法的特异性为100%。分子生物学检测技术敏感性显著高于传统诊断方法,其中XperMTB/RIF是敏感性最高的诊断方法。利福平耐药检测方面,Xpert MTB/RIF检出3例rpoB基因突变病例(3.7%),结核分枝杆菌药敏实验检测到利福平耐药2例(2.4%)这两例与Xpert检测结果符合。异烟肼耐药方面,熔解曲线检测异烟肼耐药突变型7例(8.1%),其中1例有药敏结果。分子生物学技术在耐药检测中显示出更高的灵敏性,且符合率较好,XperMTB/RIF在利福平耐药检测中最为敏感,但无法检测检测异烟肼耐药性,熔解曲线法检出率较低,但同时可以检测利福平和异烟肼的耐药性。结论:分子检测有助于提高淋巴结结核诊断敏感性。分子检测在耐药淋巴结结核诊断中发挥着重要作用,提高了利福平、异烟肼的耐药检测的敏感性。分子生物学、病理学、细菌学联合的诊断新模式可以提高该疾病诊断的敏感性。淋巴结结核患者中,耐药的发生低于肺结核,利福平的耐药与异烟肼的耐药并不是同时发生的,异烟肼耐药多于利福平耐药。
[Abstract]:Objective: at present, imaging, bacteriological culture and routine pathology are the main diagnostic methods for superficial lymph node tuberculosis, but these methods are not satisfactory in sensitivity and specificity. In recent years, molecular biological methods for the detection of Mycobacterium tuberculosis specific genes have been developed rapidly, with the advantages of rapid, accurate and so on. Therefore, this study evaluated the application of new Xpert MTB/RIF technique, fluorescence quantitative PCR technique and high resolution fusion curve method in the diagnosis of superficial lymph node tuberculosis by prospective study, and compared with the current commonly used diagnostic methods. Methods: from March 2015 to January 2016, 86 suspected lymph node tuberculosis specimens and 13 other lymphadenopathy specimens were collected by needle aspiration biopsy and excision biopsy. The collected specimens were divided into two parts on average, one was used for the detection of Xpert MTB/RIF, the other was used for routine pathological examination and PCR detection of paraffin-embedded specimens. Detection of drug resistance of rifampicin and isoniazid in fluorescence PCR melting curve. Results: in 81 cases of suspected lymph node tuberculosis, 74 cases (91.4%) were positive for MTBC by MTB/RIF, 60 cases (74%) were positive for PCR, 24 cases (29.6%) were positive for MGIT960 culture, 13 cases (16%) were positive for ropey, 38 cases (46.9%) were pathomorphological diagnosis of lymph node tuberculosis. The specificity of all methods was 100.1%. The sensitivity of molecular biological detection technique is significantly higher than that of traditional diagnostic methods, and XperMTB/RIF is the most sensitive diagnostic method. In terms of rifampicin resistance, 3 cases (3.7%) of rpoB gene mutation were detected by Xpert MTB/RIF, and 2 cases (2.4%) of rifampicin resistance were detected by Mycobacterium tuberculosis susceptibility test. These two cases were in agreement with the results of Xpert. In the aspect of isoniazid resistance, 7 cases (8.1%) of isoniazid resistance mutants were detected by melting curve. Molecular biology technique showed higher sensitivity in drug resistance detection, and the coincidence rate was good. XperMTB / RIF was the most sensitive in rifampicin resistance detection, but could not detect isoniazid resistance, and the detection rate of fusion curve method was lower. But rifampicin and isoniazid can also be tested for drug resistance. Conclusion: molecular detection is helpful to improve the sensitivity of lymph node tuberculosis diagnosis. Molecular detection plays an important role in the diagnosis of drug-resistant lymph node tuberculosis and improves the sensitivity of rifampicin and isoniazid. A new diagnostic model combined with molecular biology, pathology and bacteriology may enhance the sensitivity of the diagnosis of the disease. In patients with lymph node tuberculosis, the incidence of drug resistance is lower than that of tuberculosis, rifampicin resistance and isoniazid resistance do not occur at the same time, isoniazid resistance is more than rifampicin resistance.
【学位授予单位】：北京市结核病胸部肿瘤研究所
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R52;R440

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