本文關(guān)鍵詞：涂陰肺結(jié)核支氣管肺泡灌洗液和結(jié)核性胸水改良抗酸染色法的建立與應(yīng)用評價　出處：《北京市結(jié)核病胸部腫瘤研究所》2016年博士論文　論文類型：學位論文

  更多相關(guān)文章： 涂陰肺結(jié)核 改良抗酸染色法 直接涂片法 Gene Xpert?MTB/RIF系統(tǒng) 結(jié)核性胸水 改良抗酸染色 結(jié)核分枝桿菌 Gene Xpert?MTB/RIF

【摘要】：目的:本研究旨在傳統(tǒng)抗酸染色法的基礎(chǔ)上利用玻片離心沉淀儀對其進行改良,建立改良抗酸染色法,并評價其在涂陰肺結(jié)核患者支氣管肺泡灌洗液中的應(yīng)用價值。方法:(1)收集痰涂片陰性肺結(jié)核患者的支氣管肺泡灌洗液標本,標本經(jīng)4%Na OH按一定比例消化后,利用玻片離心沉淀儀收集其中的結(jié)核分枝桿菌,待玻片(實驗前經(jīng)多聚賴氨酸處理)干燥后,進行抗酸染色,建立改良抗酸染色法;(2)對收集到的灌洗液標本進行改良羅氏培養(yǎng)、直接涂片法、離心涂片法和改良抗酸染色法檢測,對培養(yǎng)陽性患者的所有灌洗液標本用Gene Xpert?MTB/RIF系統(tǒng)進行檢測,以羅氏培養(yǎng)作為金標準,通過比較不同方法間的差異,探討改良抗酸染色法在涂陰肺結(jié)核患者支氣管肺泡灌洗液中的診斷價值。結(jié)果:共納入379例痰涂片陰性肺結(jié)核患者,其中32例被剔除,被剔除者中5例因診斷不明、15例因資料丟失、12例因標本培養(yǎng)污染而退出分析。最后347例患者中,74例患者改良羅氏培養(yǎng)陽性,基于74例患者進行統(tǒng)計分析,直接涂片法、離心涂片法和改良抗酸染色法敏感性分別為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),改良抗酸染色法較直接涂片法和離心涂片法陽性率高,差異均有統(tǒng)計學意義(P0.05)。直接涂片法、離心涂片法和改良抗酸染色法的特異性分別為100%(n=273/273)、100%(n=273/273)和99.6%(n=272/273)。Gene Xpert?MTB/RIF系統(tǒng)74例培養(yǎng)陽性患者中,陽性67例,陽性率90.5%。74例灌洗液培養(yǎng)陽性患者中共收集到106份標本,基于此106份標本進行統(tǒng)計分析,改良羅氏培養(yǎng)法、直接涂片法、離心涂片法、Gene Xpert?MTB/RIF系統(tǒng)和改良抗酸染色法陽性率分別為76.4%(81/106)、13.2%(14/106)、34%(48/106)、93.4%(99/106)和91.5%(97/106)。改良抗酸染色法與Gene Xpert?MTB/RIF系統(tǒng)的陽性率相近,但遠高于直接涂片法和離心涂片法的陽性率,差異均有統(tǒng)計學意義(P0.05)。對106份標本同時進行三種抗酸染色,每張染色玻片于油鏡下觀察300個視野,直接涂片法和離心涂片法的陽性視野數(shù)分別為34.4±54.0和38.2±36.8,而改良抗酸染色法的陽性視野數(shù)為69.0±67.8,遠高于其它兩種方法,差異均有統(tǒng)計學意義(P0.05)。結(jié)論:(1)利用玻片離心沉淀儀成功建立了涂陰肺結(jié)核支氣管肺泡灌洗液的改良抗酸染色法;(2)改良抗酸染色法明顯提高了涂陰肺結(jié)核支氣管肺泡灌洗液中結(jié)核分枝桿菌的檢出率,對肺結(jié)核的診斷價值較大;(3)改良抗酸染色法簡便、快速、經(jīng)濟、高效,值得臨床推廣應(yīng)用。背景結(jié)核病是單一病原菌引起的感染性疾病中死亡率最高的疾病之一,是人類面臨的重大公共衛(wèi)生和社會問題。我國約有50%的人感染過結(jié)核分枝桿菌,結(jié)核性胸膜炎約占所有類型結(jié)核病5%,占肺結(jié)核病的10%~20%,其發(fā)病率大約為1.25%,據(jù)估算為160萬人,青壯年是易感人群,39歲以下的患者占73%,其中約10%~30%的結(jié)核性胸膜炎患者有胸腔積液出現(xiàn),是臨床上第二大常見的肺外結(jié)核病。結(jié)核性胸膜炎如不能及時診斷和治療,則可能造成胸膜增厚、粘連,甚至可引起肺功能下降而喪失勞動能力,因此及時診治非常重要。胸腔積液是一種常見的臨床問題,結(jié)核病和惡性腫瘤侵及胸膜是出現(xiàn)胸腔積液的兩大主要原因,發(fā)生率分別為49.6%與29.6%。結(jié)核分枝桿菌抗酸染色和培養(yǎng)是診斷結(jié)核性胸膜炎的“金標準”,但結(jié)核性胸水離心涂片法陽性率極低,培養(yǎng)所需時間長,一般在4 w~8 w;PPD試驗檢測結(jié)果不易區(qū)分是否卡介苗接種、MTB潛伏感染及NTM感染;胸膜活檢有創(chuàng)傷性,患者依從性差,診斷的敏感性和陽性率較低,操作耗時費力。結(jié)核性胸膜炎早期診斷困難,而診斷和治療的延遲會導(dǎo)致死亡率的增加。因此,尋求對結(jié)核性胸膜炎快速、高效的診斷方法,在臨床診治工作中仍然是一個挑戰(zhàn)。目前亟需研究新的檢查方法以提高對結(jié)核性胸膜炎的診斷水平。近幾年,應(yīng)用玻片離心沉淀儀的改良抗酸染色技術(shù)大大提高了結(jié)核性腦膜炎的診斷效率,其原理為首先應(yīng)用玻片離心沉淀儀濃縮腦脊液中的細胞和結(jié)核分枝桿菌,然后進行固定細胞和破壞細胞膜蛋白,再行抗酸染色。由于其濃縮原理較離心沉淀法好,避免了菌的浮力和轉(zhuǎn)移損失的問題,加之收集的菌沉淀面積小,染色后可以同時觀察細胞內(nèi)外的結(jié)核分枝桿菌,所以陽性率較離心涂片法提高了將近80%。因此,我們推測玻片離心沉淀儀亦可應(yīng)用于結(jié)核性胸水的診斷,有可能在結(jié)核性胸膜炎的早期診斷中發(fā)揮重要作用。本研究旨在建立結(jié)核性胸水的改良抗酸染色法,并進一步探討其臨床應(yīng)用價值。目的:在傳統(tǒng)抗酸染色的基礎(chǔ)上利用玻片離心沉淀儀進行改良,建立胸水改良抗酸染色法,并評價其在結(jié)核性胸膜炎中的應(yīng)用價值。方法:選取2014年6月1日至2015年8月31日于邯鄲市傳染病醫(yī)院、河北省胸科醫(yī)院收治的胸水患者,共納入實驗組結(jié)核性胸膜炎患者184例(胸水ADA45U/L),對照組惡性胸水患者43例。建立改良抗酸染色法,對所有胸水分別進行離心涂片法、BACTEC 960培養(yǎng)法、Gene Xpert?MTB/RIF系統(tǒng)和改良抗酸染色法檢測,將改良抗酸染色法的結(jié)果與其它方法結(jié)果進行統(tǒng)計學分析,綜合評價其在結(jié)核性胸膜炎診斷中的應(yīng)用價值。結(jié)果:共納入184例結(jié)核性胸膜炎患者的胸水標本,5例被剔除的患者中其中1例是由于資料丟失,4例是由于培養(yǎng)污染。最后179例患者中,98例未經(jīng)過抗結(jié)核治療,81例已經(jīng)開始抗結(jié)核治療。納入惡性胸水患者43例,其中3例由于培養(yǎng)污染被剔除。(1)未治療組和治療組結(jié)核分枝桿菌檢出率比較:98例未治療組,離心涂片法、改良抗酸染色法、BACTEC 960培養(yǎng)法和Gene Xpert?MTB/RIF系統(tǒng)陽性率分別為2.04%(2/98)、33.67%(33/98)、20.41%(20/98)和15.31%(15/98),改良抗酸染色法檢測結(jié)核分枝桿菌的陽性率較其它三種方法陽性率高,差異有統(tǒng)計學意義(P0.05)。81例已治療組,離心涂片法、改良抗酸染色法、BACTEC960培養(yǎng)法和Gene Xpert?MTB/RIF系統(tǒng)陽性率分別為1.23%(1/81)、25.93%(21/81)、13.58%(11/81)和8.64%(7/81),改良抗酸染色法較其它三種方法陽性率高,差異有統(tǒng)計學意義(P0.05)。治療組和未治療組中同種方法間結(jié)核分枝桿菌檢出率相比較,差異均無統(tǒng)計學意義(P0.05)。(2)四種方法敏感性、特異性比較:以臨床診斷為金標準,離心涂片法、改良抗酸染色法、BACTEC 960培養(yǎng)法和Gene Xpert?MTB/RIF系統(tǒng)檢測的敏感性分別為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),特異性分別為100%(40/40)、100%(40/40)、100%(40/40)和97.50%(39/40)。改良抗酸染色法較離心涂片法、BACTEC 960培養(yǎng)法和Gene Xpert?MTB/RIF系統(tǒng)敏感性高,差異有統(tǒng)計學意義(P0.05)。(3)改良抗酸染色法同份單次檢測與重復(fù)檢測檢出率比較:實驗組179例標本,第一次檢測抗酸桿菌陽性為52例,第二次檢測抗酸桿菌陽性為53例,第三次檢測抗酸桿菌陽性為54例,陽性率分別為29.05%、29.61%和30.17%,三者之間比較無統(tǒng)計學意義(P0.05)。(4)胸水中結(jié)核分枝桿菌含量檢測結(jié)果:在Gene Xpert?MTB/RIF系統(tǒng)陽性的22份標本中,結(jié)核分枝桿菌含量極低(CT28)20份,含量低(22CT28)2份。結(jié)論:(1)利用玻片離心沉淀儀成功建立了結(jié)核性胸腔積液的改良抗酸染色法;(2)結(jié)核性胸水中結(jié)核分枝桿菌濃度極低,改良抗酸染色法可明顯提高結(jié)核性胸水的陽性率,比BACTEC 960培養(yǎng)法和Xpert MTB/RIF系統(tǒng)均高,對結(jié)核性胸膜炎的診斷價值較大;(3)改良抗酸染色法簡便、快速、經(jīng)濟、高效,可作為早期診斷結(jié)核性胸膜炎的檢查手段,值得臨床推廣應(yīng)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.

【學位授予單位】：北京市結(jié)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】：R521

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