涂阴肺结核支气管肺泡灌洗液和结核性胸水改良抗酸染色法的建立与应用评价
本文关键词:涂阴肺结核支气管肺泡灌洗液和结核性胸水改良抗酸染色法的建立与应用评价 出处:《北京市结核病胸部肿瘤研究所》2016年博士论文 论文类型:学位论文
更多相关文章: 涂阴肺结核 改良抗酸染色法 直接涂片法 Gene Xpert?MTB/RIF系统 结核性胸水 改良抗酸染色 结核分枝杆菌 Gene Xpert?MTB/RIF
【摘要】:目的:本研究旨在传统抗酸染色法的基础上利用玻片离心沉淀仪对其进行改良,建立改良抗酸染色法,并评价其在涂阴肺结核患者支气管肺泡灌洗液中的应用价值。方法:(1)收集痰涂片阴性肺结核患者的支气管肺泡灌洗液标本,标本经4%Na OH按一定比例消化后,利用玻片离心沉淀仪收集其中的结核分枝杆菌,待玻片(实验前经多聚赖氨酸处理)干燥后,进行抗酸染色,建立改良抗酸染色法;(2)对收集到的灌洗液标本进行改良罗氏培养、直接涂片法、离心涂片法和改良抗酸染色法检测,对培养阳性患者的所有灌洗液标本用Gene Xpert?MTB/RIF系统进行检测,以罗氏培养作为金标准,通过比较不同方法间的差异,探讨改良抗酸染色法在涂阴肺结核患者支气管肺泡灌洗液中的诊断价值。结果:共纳入379例痰涂片阴性肺结核患者,其中32例被剔除,被剔除者中5例因诊断不明、15例因资料丢失、12例因标本培养污染而退出分析。最后347例患者中,74例患者改良罗氏培养阳性,基于74例患者进行统计分析,直接涂片法、离心涂片法和改良抗酸染色法敏感性分别为16.2%(95%CI:12.3,20.0)(n=12/74)、37.8%(95%CI:32.7,42.9)(n=28/74)、87.8%(95%CI:84.4,91.2)(n=65/74),改良抗酸染色法较直接涂片法和离心涂片法阳性率高,差异均有统计学意义(P0.05)。直接涂片法、离心涂片法和改良抗酸染色法的特异性分别为100%(n=273/273)、100%(n=273/273)和99.6%(n=272/273)。Gene Xpert?MTB/RIF系统74例培养阳性患者中,阳性67例,阳性率90.5%。74例灌洗液培养阳性患者中共收集到106份标本,基于此106份标本进行统计分析,改良罗氏培养法、直接涂片法、离心涂片法、Gene Xpert?MTB/RIF系统和改良抗酸染色法阳性率分别为76.4%(81/106)、13.2%(14/106)、34%(48/106)、93.4%(99/106)和91.5%(97/106)。改良抗酸染色法与Gene Xpert?MTB/RIF系统的阳性率相近,但远高于直接涂片法和离心涂片法的阳性率,差异均有统计学意义(P0.05)。对106份标本同时进行三种抗酸染色,每张染色玻片于油镜下观察300个视野,直接涂片法和离心涂片法的阳性视野数分别为34.4±54.0和38.2±36.8,而改良抗酸染色法的阳性视野数为69.0±67.8,远高于其它两种方法,差异均有统计学意义(P0.05)。结论:(1)利用玻片离心沉淀仪成功建立了涂阴肺结核支气管肺泡灌洗液的改良抗酸染色法;(2)改良抗酸染色法明显提高了涂阴肺结核支气管肺泡灌洗液中结核分枝杆菌的检出率,对肺结核的诊断价值较大;(3)改良抗酸染色法简便、快速、经济、高效,值得临床推广应用。背景结核病是单一病原菌引起的感染性疾病中死亡率最高的疾病之一,是人类面临的重大公共卫生和社会问题。我国约有50%的人感染过结核分枝杆菌,结核性胸膜炎约占所有类型结核病5%,占肺结核病的10%~20%,其发病率大约为1.25%,据估算为160万人,青壮年是易感人群,39岁以下的患者占73%,其中约10%~30%的结核性胸膜炎患者有胸腔积液出现,是临床上第二大常见的肺外结核病。结核性胸膜炎如不能及时诊断和治疗,则可能造成胸膜增厚、粘连,甚至可引起肺功能下降而丧失劳动能力,因此及时诊治非常重要。胸腔积液是一种常见的临床问题,结核病和恶性肿瘤侵及胸膜是出现胸腔积液的两大主要原因,发生率分别为49.6%与29.6%。结核分枝杆菌抗酸染色和培养是诊断结核性胸膜炎的“金标准”,但结核性胸水离心涂片法阳性率极低,培养所需时间长,一般在4 w~8 w;PPD试验检测结果不易区分是否卡介苗接种、MTB潜伏感染及NTM感染;胸膜活检有创伤性,患者依从性差,诊断的敏感性和阳性率较低,操作耗时费力。结核性胸膜炎早期诊断困难,而诊断和治疗的延迟会导致死亡率的增加。因此,寻求对结核性胸膜炎快速、高效的诊断方法,在临床诊治工作中仍然是一个挑战。目前亟需研究新的检查方法以提高对结核性胸膜炎的诊断水平。近几年,应用玻片离心沉淀仪的改良抗酸染色技术大大提高了结核性脑膜炎的诊断效率,其原理为首先应用玻片离心沉淀仪浓缩脑脊液中的细胞和结核分枝杆菌,然后进行固定细胞和破坏细胞膜蛋白,再行抗酸染色。由于其浓缩原理较离心沉淀法好,避免了菌的浮力和转移损失的问题,加之收集的菌沉淀面积小,染色后可以同时观察细胞内外的结核分枝杆菌,所以阳性率较离心涂片法提高了将近80%。因此,我们推测玻片离心沉淀仪亦可应用于结核性胸水的诊断,有可能在结核性胸膜炎的早期诊断中发挥重要作用。本研究旨在建立结核性胸水的改良抗酸染色法,并进一步探讨其临床应用价值。目的:在传统抗酸染色的基础上利用玻片离心沉淀仪进行改良,建立胸水改良抗酸染色法,并评价其在结核性胸膜炎中的应用价值。方法:选取2014年6月1日至2015年8月31日于邯郸市传染病医院、河北省胸科医院收治的胸水患者,共纳入实验组结核性胸膜炎患者184例(胸水ADA45U/L),对照组恶性胸水患者43例。建立改良抗酸染色法,对所有胸水分别进行离心涂片法、BACTEC 960培养法、Gene Xpert?MTB/RIF系统和改良抗酸染色法检测,将改良抗酸染色法的结果与其它方法结果进行统计学分析,综合评价其在结核性胸膜炎诊断中的应用价值。结果:共纳入184例结核性胸膜炎患者的胸水标本,5例被剔除的患者中其中1例是由于资料丢失,4例是由于培养污染。最后179例患者中,98例未经过抗结核治疗,81例已经开始抗结核治疗。纳入恶性胸水患者43例,其中3例由于培养污染被剔除。(1)未治疗组和治疗组结核分枝杆菌检出率比较:98例未治疗组,离心涂片法、改良抗酸染色法、BACTEC 960培养法和Gene Xpert?MTB/RIF系统阳性率分别为2.04%(2/98)、33.67%(33/98)、20.41%(20/98)和15.31%(15/98),改良抗酸染色法检测结核分枝杆菌的阳性率较其它三种方法阳性率高,差异有统计学意义(P0.05)。81例已治疗组,离心涂片法、改良抗酸染色法、BACTEC960培养法和Gene Xpert?MTB/RIF系统阳性率分别为1.23%(1/81)、25.93%(21/81)、13.58%(11/81)和8.64%(7/81),改良抗酸染色法较其它三种方法阳性率高,差异有统计学意义(P0.05)。治疗组和未治疗组中同种方法间结核分枝杆菌检出率相比较,差异均无统计学意义(P0.05)。(2)四种方法敏感性、特异性比较:以临床诊断为金标准,离心涂片法、改良抗酸染色法、BACTEC 960培养法和Gene Xpert?MTB/RIF系统检测的敏感性分别为1.68%(95%CI:0.00,3.38)、30.17%(95%CI:24.09,36.25)、17.32%(95%CI:12.31,22.33)和12.29%(95%CI:7.94,16.64),特异性分别为100%(40/40)、100%(40/40)、100%(40/40)和97.50%(39/40)。改良抗酸染色法较离心涂片法、BACTEC 960培养法和Gene Xpert?MTB/RIF系统敏感性高,差异有统计学意义(P0.05)。(3)改良抗酸染色法同份单次检测与重复检测检出率比较:实验组179例标本,第一次检测抗酸杆菌阳性为52例,第二次检测抗酸杆菌阳性为53例,第三次检测抗酸杆菌阳性为54例,阳性率分别为29.05%、29.61%和30.17%,三者之间比较无统计学意义(P0.05)。(4)胸水中结核分枝杆菌含量检测结果:在Gene Xpert?MTB/RIF系统阳性的22份标本中,结核分枝杆菌含量极低(CT28)20份,含量低(22CT28)2份。结论:(1)利用玻片离心沉淀仪成功建立了结核性胸腔积液的改良抗酸染色法;(2)结核性胸水中结核分枝杆菌浓度极低,改良抗酸染色法可明显提高结核性胸水的阳性率,比BACTEC 960培养法和Xpert MTB/RIF系统均高,对结核性胸膜炎的诊断价值较大;(3)改良抗酸染色法简便、快速、经济、高效,可作为早期诊断结核性胸膜炎的检查手段,值得临床推广应用。
[Abstract]:Objective: the purpose of this study is to base on the traditional acid fast stain glass centrifugal sedimentation instrument for the improvement, the establishment of the modified Ziehl Neelsen staining method and evaluate the application value of fluid in smear negative pulmonary tuberculosis patients with bronchial alveolar lavage. Methods: (1) bronchoalveolar collected sputum smear negative pulmonary tuberculosis patients lavage specimens samples by 4%Na OH, according to a certain proportion after digestion of Mycobacterium tuberculosis were collected using the slide centrifugal precipitation, the slide (before the experiment with poly lysine treatment) after drying by acid fast stain, a modified acid fast staining method; (2) lavage fluid collected specimens of L-J culture. Direct smear method, detection method and smear method and modified acid fast staining of culture positive patients were all lavage with Gene Xpert? MTB/RIF system were detected with L-J culture as the gold standard, through the comparison of different parties The difference between the study method, modified acid fast staining method in the diagnosis of irrigation lotion smear negative pulmonary tuberculosis patients with bronchial alveolar. Results: 379 cases of smear negative pulmonary tuberculosis patients were included, of which 32 cases were excluded, were removed in 5 cases for diagnosis of unknown, 15 cases due to data loss, 12 cases of specimens from the analysis of culture contamination. Finally, 347 patients, 74 cases of patients with L-J culture positive, statistical analysis was performed in 74 patients based on the method of direct smear, smear method and modified acid fast staining sensitivity was 16.2% (95%CI:12.3,20.0) (n=12/74), 37.8% (95%CI:32.7,42.9) (n=28/74), 87.8% (95%CI:84.4,91.2) (n=65/74), modified acid fast staining method with direct smear method and centrifugal smear positive rate is high, the differences were statistically significant (P0.05). Direct smear method, specific smear method and modified acid fast staining method were 100% (n=273/273), 100% (n= 273/273) and 99.6% (n=272/273).Gene Xpert? MTB/RIF system in 74 cases of culture positive patients, 67 cases were positive, the positive rate of 90.5%.74 cases lavage fluid culture positive patients collected 106 specimens, 106 specimens of the statistical analysis based on the modified culture method, direct smear, smear method, Gene Xpert? MTB/RIF system and modified acid fast staining positive rate were 76.4% (81/106), 13.2% (14/106), 34% (48/106), 93.4% (99/106) and 91.5% (97/106). Gene Xpert method and modified acid fast staining? The positive rate of MTB/RIF system is similar, but the positive rate is much higher than the direct smear and smear method the differences were statistically significant (P0.05). Of the 106 samples and three kinds of acid fast stain, oil microscope 300 fields each stained glass slides, the number of positive view of direct smear and smear method were 34.4 + 54 and 38.2 + 36.8, and improved resistance The number of positive vision acid staining was 69 + 67.8, far higher than the other two methods, the differences were statistically significant (P0.05). Conclusion: (1) sedimentograph successfully established a modified acid fast smear negative pulmonary tuberculosis in bronchoalveolar lavage fluid staining using centrifugal slide; (2) improved anti acid staining method significantly increased the detection rate of smear negative pulmonary tuberculosis mycobacterium tuberculosis in bronchoalveolar lavage fluid in the diagnosis value of pulmonary tuberculosis; (3) modified acid fast staining method is simple, rapid, economical, efficient, worthy of clinical application. The background of TB disease mortality is one of the infectious diseases caused by pathogenic bacteria in the single highest. Is a major public health and social problems in the world. In China there are about 50% people are infected with Mycobacterium tuberculosis, tuberculous pleurisy accounted for about 5% of all types of tuberculosis, 10%~20% tuberculosis, the incidence rate is about 1.25%, according to estimates For 1 million 600 thousand people, young adults are susceptible. The patients under the age of 39 accounted for 73%, of which about 10%~30% of the patients with tuberculous pleurisy with pleural effusion, second is clinically common tuberculosis. Tuberculous pleurisy as not timely diagnosis and treatment, it may cause pleural thickening, adhesion, even due to the decline of lung function and loss of ability to work, so the timely diagnosis and treatment is very important. Pleural effusion is a common clinical problem, tuberculosis and malignant tumor and pleural invasion is the two major causes of pleural effusion, the incidence rates were 49.6% and 29.6%. Mycobacterium tuberculosis by acid fast staining and culture is the gold "the diagnosis of tuberculous pleurisy the standard, but the positive rate of pleural fluid smear method is extremely low, training required a long time, generally in 4 w~8 W; PPD test results are not easy to distinguish whether BCG vaccination, latent infection of MTB and NTM. Dye; pleural biopsy is invasive and poor patient compliance, diagnostic sensitivity and positive rate is low, the operation is time-consuming. Difficulties in early diagnosis of tuberculosis pleurisy, and diagnosis and treatment delay will lead to increased mortality. Therefore, seeking for tuberculous pleurisy rapid diagnosis method, high efficiency, in the clinical diagnosis and treatment work is still a challenge. The new method of examination need to study in order to improve the diagnostic level of tuberculous pleurisy. In recent years, the application of glass centrifugal sedimentation instrument modified acid fast staining technique can greatly improve the efficiency of diagnosis of tuberculous meningitis, the principle for the first precipitation and mycobacterial cells were concentrated in the cerebrospinal fluid of application slide centrifugation, then fixed cells and destroy the cell membrane protein, and acid fast staining. Because of its principle is concentrated by centrifugal sedimentation, to avoid bacteria buoyancy and transfer loss. In addition, the collection of bacteria precipitation area, Mycobacterium tuberculosis can be observed at the same time inside and outside cells after staining, so the positive rate than smear method increased by nearly 80%. so, we speculate that the diagnosis of slide centrifugal precipitation can also be applied to the instrument of tuberculous pleural effusion, may play an important role in the early diagnosis of tuberculous pleurisy. The purpose of this study is to establish. The modified acid fast staining method of tuberculous pleural effusion, and to discuss its clinical value. Objective: to improve the precipitation apparatus using centrifugal slide based on traditional acid fast stain on the establishment of hydrothorax modified acid fast staining method, and to evaluate its application value in the diagnosis of tuberculous pleurisy. Methods: June 1, 2014 to August 31, 2015 in Handan city hospital for infectious diseases, patients with pleural effusion from the Chest Hospital of Hebei Province, a total of 184 cases of experimental group patients with tuberculous pleurisy (hydrothorax ADA45U/L), The control group of 43 cases of malignant pleural effusion. The establishment of the modified Ziehl Neelsen staining method for all pleural effusion were smear method, 960 BACTEC culture method, Gene Xpert? MTB/RIF system and modified acid fast staining method, the modified acid fast staining results with other methods of statistical analysis and comprehensive evaluation of its application value in the the diagnosis of tuberculous pleurisy. Results: 184 cases of patients with tuberculous pleurisy pleural effusion specimens were included, 5 cases were excluded in 1 patients which is due to data loss, 4 cases due to culture pollution. Finally in 179 cases, 98 cases without anti tuberculosis treatment, 81 cases of tuberculosis has begun the treatment of malignant pleural effusion patients. In 43 cases, including 3 cases of pollution due to the train being removed. (1) treatment group and non treatment group of Mycobacterium tuberculosis detection rate: 98 cases of untreated group, smear method, modified acid fast staining method, BACTEC 960. The positive rate of Xpert method and Gene? MTB/RIF system were 2.04% (2/98), 33.67% (33/98), 20.41% (20/98) and 15.31% (15/98), modified acid fast staining positive rate of detection of Mycobacterium tuberculosis was higher than that of the other three methods, the difference was statistically significant (P0.05).81 cases of treatment group. Smear method, modified acid fast staining method, BACTEC960 culture and Gene Xpert? MTB/RIF positive rate system were 1.23% (1/81), 25.93% (21/81), 13.58% (11/81) and 8.64% (7/81), modified acid fast staining positive rate method than the other three methods, the difference was statistically significant (P0.05) treatment. Group and non treatment group in the same way between Mycobacterium tuberculosis detection rate comparison, there were no significant differences (P0.05). (2) the sensitivity of the four methods, to compare the specificity in clinical diagnosis as the gold standard, centrifugal smear method, modified acid fast staining method, 960 BACTEC culture and Gene Xpert? MTB/RIF The sensitivity of detection system were 1.68% (95%CI:0.00,3.38), 30.17% (95%CI:24.09,36.25), 17.32% (95%CI:12.31,22.33) and 12.29% (95%CI:7.94,16.64), the specificity was 100% (40/40), 100% (40/40), 100% (40/40) and 97.50% (39/40). Compared with the modified acid fast staining smear method, BACTEC method and culture 960 Gene Xpert? MTB/RIF system with high sensitivity, the difference was statistically significant (P0.05). (3) modified acid fast staining method with a detection rate of single detection and repeat detection: 179 cases in the experimental group were the first detection of acid fast bacilli positive in 52 cases, second detection of acid fast bacilli positive in 53 cases, third detection acid fast bacilli positive in 54 cases, the positive rates were 29.05%, 29.61% and 30.17%, between the three was not statistically significant (P0.05). (4) Mycobacterium tuberculosis in the pleural fluid content detection results in Gene Xpert? MTB/RIF 22 samples were positive in tuberculosis Very low levels of Mycobacterium (CT28) 20 (22CT28), low content of 2. Conclusion: (1) sedimentograph successfully established tuberculous pleural effusion modified acid fast staining method using centrifugal slide; (2) tuberculous Mycobacterium tuberculosis in the pleural fluid concentration is very low, can significantly improve the positive rate of tuberculous pleurisy water modified acid fast staining method and Xpert culture, MTB/RIF system is higher than BACTEC 960, greater diagnostic value of tuberculosis pleurisy; (3) modified acid fast staining method is simple, rapid, economic, efficient and can be used in early diagnosis of tuberculous pleurisy examination means, it is worthy of clinical application.
【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】:R521
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