【摘要】：目的:淺表淋巴結結核目前常用影像學、細菌學培養(yǎng)、常規(guī)病理學做為主要的診斷手段,但這些方法在敏感性和特異性方面均不理想。近年來檢測結核分枝桿菌特異基因的分子生物學檢測方法發(fā)展迅速,具有快速、準確等優(yōu)點。因此本研究通過前瞻性研究,評估了Xpert MTB/RIF新技術、熒光定量PCR技術及高分辨熔解曲線法在淺表淋巴結結核診斷中的應用,并與目前常用的診斷方法進行了對比研究。方法:從2015年3月到2016年1月采用針吸活檢和切除活檢兩種方法連續(xù)的收集了86例疑似淋巴結結核標本和13例其他淋巴結疾病標本。收集的標本平均分為兩部分,一部分用于Xpert MTB/RIF的檢測、BACTEC MGIT960培養(yǎng)和羅氏培養(yǎng),另一部分用于常規(guī)病理學檢查、石蠟包埋標本的PCR檢測、熒光PCR熔解曲線利福平和異煙肼的耐藥檢測。結果:臨床疑似淋巴結結核的患者81例,Xpert MTB/RIF檢測MTBC陽性74例(91.4%),組織標本PCR陽性60例(74%),BACTEC MGIT960培養(yǎng)陽性24例(29.6%),羅培陽性13例(16%),病理形態(tài)學診斷淋巴結結核38例(46.9%),所有方法的特異性為100%。分子生物學檢測技術敏感性顯著高于傳統(tǒng)診斷方法,其中XperMTB/RIF是敏感性最高的診斷方法。利福平耐藥檢測方面,Xpert MTB/RIF檢出3例rpoB基因突變病例(3.7%),結核分枝桿菌藥敏實驗檢測到利福平耐藥2例(2.4%)這兩例與Xpert檢測結果符合。異煙肼耐藥方面,熔解曲線檢測異煙肼耐藥突變型7例(8.1%),其中1例有藥敏結果。分子生物學技術在耐藥檢測中顯示出更高的靈敏性,且符合率較好,XperMTB/RIF在利福平耐藥檢測中最為敏感,但無法檢測檢測異煙肼耐藥性,熔解曲線法檢出率較低,但同時可以檢測利福平和異煙肼的耐藥性。結論:分子檢測有助于提高淋巴結結核診斷敏感性。分子檢測在耐藥淋巴結結核診斷中發(fā)揮著重要作用,提高了利福平、異煙肼的耐藥檢測的敏感性。分子生物學、病理學、細菌學聯(lián)合的診斷新模式可以提高該疾病診斷的敏感性。淋巴結結核患者中,耐藥的發(fā)生低于肺結核,利福平的耐藥與異煙肼的耐藥并不是同時發(fā)生的,異煙肼耐藥多于利福平耐藥。
[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.
【學位授予單位】：北京市結核病胸部腫瘤研究所
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R52;R440

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