荧光定量PCR在结核病早期诊断中的临床应用评价

发布时间：2018-04-06 00:25

本文选题：结核分枝杆菌　切入点：FQ-PCR　出处：《新乡医学院》2013年硕士论文

【摘要】：背景结核病(tuberclosis)是严重危害人类健康的慢性传染病,近年来疫情的回升严重威胁着世界各国人民的生命财产安全,已成为全球关注的重大公共卫生问题和社会问题。目前,我国结核病疫情也呈回升趋势,并且重症患者和难治性患者越来越多,呈现出高感染率、高患病率、高死亡率和高耐药率的特点。因此寻找灵敏、特异、高效、实用的结核病诊断方法是实验诊断领域研究的热点,也是结核病早期诊断、彻底治疗、有效预防和控制蔓延的关键。结核病是由结核分枝杆菌感染引起的,实验室分离和检测病原体及其代谢产物是结核病诊断的主要依据。常用的结核杆菌检测方法虽然各有优点,但都有一定的局限性,如容易受其它抗酸性分枝杆菌的干扰,检测灵敏度差、阳性率低、容易造成漏检。特别是培养法作为目前公认的诊断结核分枝杆菌的金标准,仍然存在敏感度低、所需时间过长、对疾病的早期诊断和治疗意义不大等不足。近年来,荧光定量PCR(FQ-PCR)结核杆菌核酸分子(TB-DNA)检测技术的建立为结核病的早期诊治和防控带来了希望。关于FQ-PCR在结核病诊断中的应用研究,目前主要集中在对确诊病人以上几种实验方法的对比方面,在对初次发病的患者的早期诊断及鉴别诊断方面研究较少,对于不同类型结核病的相应标本检测的有效性方面研究也不多,特别是缺乏大样本的临床应用评价。鉴于此,我们开展了本实验。目的完善FQ-PCR法对痰液、胸水、脑脊液和尿液等不同类型标本TB-DNA的检测方法,评价其对肺结核、结核性胸膜炎、结核性脑膜炎和泌尿系结核的早期诊断价值。方法随机选择初次就诊的可疑结核病患者为实验对象,分为肺结核组、结核性胸膜炎组、结核性脑膜炎组和泌尿系结核组,分别收集痰液、胸水、脑脊液和尿液标本,经规范处理后,用FQ-PCR试剂盒进行TB-DNA检测,得到Ct值(cycle threshold),以Ct30并曲线为标准S形曲线的实验结果判为阳性。同时进行抗酸染色、细菌培养等实验室检查,结合临床诊疗过程和随访,确定所选病例的进展。按照各类结核病诊断标准对受试者做出最后诊断。将实验结果与最终诊断进行比对,做出FQ-PCR对各类结核病的早期诊断临床性能评价。结果肺结核组得到有效资料123例,痰液标本FQ-PCR扩增TB-DNA阳性66例,阴性57例；金标准判定为肺结核的93例,不是肺结核的30例。其中真阳性数为64,假阳性数为2,真阴性数为28,假阴性数为29。由此计算出该方法的临床灵敏度尚可,为68.8%,临床特异度较好,为93.3%,总正确度尚可,为74.8%。阳性预测值较好,达96.9%,而阴性预测值较差,仅49.1%。结核性胸膜炎组胸水标本FQ-PCR扩增TB-DNA,其临床灵敏度、临床特异度、阳性预测值和阴性预测值分别为70.4%(38/54)、96.8%(31/32)、97.4%(38/39)、65.9%(31/47)。结核性脑膜炎组脑脊液标本FQ-PCR扩增TB-DNA,其临床灵敏度、临床特异度、阳性预测值和阴性预测值分别为46.9%(31/66)、92.3%(12/13)、96.9%(31/32)、25.5%(12/47)。泌尿系结核组受检的78例疑似病人尿液标本,其临床灵敏度、临床特异度、阳性预测值和阴性预测值分别为38.4%(20/52)、84.6%(22/26)、83.3%(20/24)、40.7%(22/54)。结论 FQ-PCR法检测结核分枝杆菌对结核病的早期诊断具有较高临床价值。痰液和胸水对结核病的诊断特异性好,阳性预测值高,临床灵敏度尚可,但阴性预测值较低,容易出现漏诊。脑脊液和尿液标本对相应结核病的诊断有较好的排除性能,但确诊性能不够好。
[Abstract]:background
Tuberculosis (Tuberclosis) is a chronic infectious disease which seriously endangers human health. In recent years, the epidemic rise is a serious threat to the peoples of the world's life and property safety, has become a major public health problem and social problem of the world. At present, the tuberculosis epidemic situation of China also showed increasing trend, and in patients with severe and refractory patients more and more, showing a high infection rate, high morbidity, high mortality and high resistance rate characteristics. So looking for sensitive, specific, efficient and practical method for diagnosis of tuberculosis is a hot research field of experimental diagnosis, it is early diagnosis and treatment of tuberculosis completely, effectively prevent and control the spread of the key.
Tuberculosis is caused by Mycobacterium tuberculosis infection, laboratory of separation and detection of pathogens and its metabolites is the main basis for the diagnosis of tuberculosis. Mycobacterium tuberculosis detection methods commonly used although each have advantages, but there are some limitations, such as vulnerable to other acid resistant Mycobacterium interference, the detection sensitivity is poor, low positive rate, easy to the cause of the undetected. Especially the culture as the gold standard for the diagnosis of Mycobacterium tuberculosis recognized, there are still low sensitivity, it takes long time to disease, early diagnosis and treatment of significant deficiencies. In recent years, fluorescence quantitative PCR (FQ-PCR) of Mycobacterium tuberculosis DNA (TB-DNA) technique for the early detection of the diagnosis and prevention of tuberculosis has brought hope.
Study on Application of FQ-PCR in the diagnosis of tuberculosis, mainly focuses on the comparison of patients above several experimental methods, for the first time in the early onset of the patient's diagnosis and differential diagnosis of small, for the study of the corresponding samples of different types of tuberculosis of the effectiveness is not much, especially the lack of clinical evaluation large sample. In view of this, we carry out this experiment.
objective
To improve the FQ-PCR method of sputum, pleural effusion, cerebrospinal fluid detection method and urine samples of different types of TB-DNA, the evaluation of pulmonary tuberculosis, tuberculous pleurisy, early diagnosis of tuberculous meningitis and urinary tuberculosis.
Method
Random selection of initial treatment of suspected tuberculosis patients as the experimental object, divided into pulmonary tuberculosis group, tuberculous pleurisy group, tuberculous meningitis group and urinary tuberculosis group were collected sputum, pleural effusion, cerebrospinal fluid and urine samples after standard treatment, using the FQ-PCR kit for detection of TB-DNA, Ct value (cycle threshold), with Ct30 and standard curve S curve. The experimental results were considered positive. Acid fast staining at the same time, bacterial culture and laboratory examination, combined with clinical diagnosis and follow-up process, determine the progress of selected cases. According to the diagnostic criteria of all kinds of nodes make the final diagnosis of the disease subjects to compare the experimental results. With the final diagnosis, make FQ-PCR of tuberculosis early diagnosis clinical performance evaluation.
Result
Pulmonary tuberculosis group effective 123 cases sputum samples were amplified by FQ-PCR TB-DNA was positive in 66 cases, 57 cases were negative; the gold standard for determination of 93 cases of pulmonary tuberculosis, 30 cases of pulmonary tuberculosis is not. The true positive rate was 64, false positive rate was 2, true negative rate was 28, the false negative number is 29. calculate the clinical sensitivity is acceptable, the method was 68.8%, the clinical specificity was 93.3%, the total is good, the accuracy is better, up to 96.9%, the positive predictive value of 74.8%., while the negative predictive value is poor, only 49.1%. group of tuberculous pleurisy pleural effusion specimens of FQ-PCR amplification of TB-DNA, the clinical sensitivity, clinical specificity, positive predictive value and negative predictive value were 70.4% (38/54), 96.8% (31/32), 97.4% (38/39), 65.9% (31/47). Tuberculous meningitis cerebrospinal fluid specimens was amplified by FQ-PCR TB-DNA, the clinical clinical sensitivity, specificity, positive predictive value and negative predictive value were 46.9% (31/66), 92.3% (12/13 ), 96.9% (31/32), 25.5% (12/47). Urinary tuberculosis group examined 78 patients with suspected urine specimens, the clinical clinical sensitivity, specificity, positive predictive value and negative predictive value were 38.4% (20/52), 84.6% (22/26), 83.3% (20/24), 40.7% (22/54).
conclusion
Has high clinical value in early diagnosis of tuberculosis detection of Mycobacterium tuberculosis by FQ-PCR. Sputum and pleural effusion diagnosis specificity of tuberculosis, the positive predictive value of high sensitivity, clinical acceptable, but the negative predictive value is low, prone to misdiagnosis. Cerebrospinal fluid and urine specimens for diagnosis of tuberculosis has overcome the corresponding performance. But the diagnosis performance is not good enough.

【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R52

【共引文献】