聚合酶链反应DNA扩增对活动性肺结核的诊断价值

为探讨聚合酶链反应（ＰＣＲ）ＤＮＡ扩增对诊断活动性肺结核的临床价值，本文用ＰＣＲ技术扩增结核杆菌特有的ＭＰＢ６４蛋白基因序列，对１２１例病人临床标本进行检测。发现３４例活动性肺结核中有２８例ＰＣＲ阳性；疑诊为肺结核者３３例中有１８例阳性；非结核肺疾患５４例中有１０例阳性，其中有肺结核既往史者阳性６例，有结核接触史者阳性３例。提示ＰＣＲ阳性并非为活动性肺结核所特有。     

PCR技术在结核病诊断中的价值

目的探讨结核分支杆菌核酸扩增PCR技术及结核分支杆菌噬菌体生物扩增法对肺结核病及肺外结核患者临床诊断的意义。方法对入选46例菌阳肺结核患者进一步做上述两种方法。结果46例患者PCR阳性为36例（78.3%）噬菌体阳性为11例（23.9%）。结论PCR技术对肺结核病及肺外结核患者的诊断具有一定实际意义。     

结核分枝杆菌效应T细胞检测在诊断活动性结核病中的价值

目的分析结核分枝杆菌效应T细胞检测法在诊断活动性结核病中的价值。方法选取2015年1-12月来自上海市公共卫生临床中心、上海市肺科医院和沈阳市胸科医院的708例活动性结核病患者、289例非结核肺部疾病患者和55名健康志愿者作为研究对象。将55名健康志愿者和289例非结核肺部疾病患者作为非活动性结核组。活动性结核组包括68例肺外结核患者和640例肺结核患者,肺结核并发肺外结核者归属为肺结核患者;在肺结核患者中有230例患者痰标本的细菌学检查呈阳性,410例痰标本细菌学检查阴性。分离研究对象外周血单核细胞,应用结核分枝杆菌效应T细胞检测试剂盒进行检测,评价该检测方法的诊断效能。结果结核分枝杆菌效应T细胞检测试剂盒检测活动性结核组的敏感度为81．78％（579／708）;检测非活动性结核组的特异度为79．36％（273／344）;检测健康志愿者的特异度为89．09％（49／55）。在经检测结果为阳性的650例研究对象中,正确检出阳性579例,阳性预测值为89．08％;在所有检查结果为阴性的402例研究对象中,正确检出阴性273例,阴性预测值为67．91％（273／402）;阳性似然比、阴性似然比和正确诊断效率依次为3．96、0．23和80．99％（852／1052）。检测痰检阳性患者的阳性检出率相对更高,为84．78％（195／230）;检测肺结核并发肺外结核患者的阳性检出率为91．49％（43／47）。结论结核分枝杆菌效应T细胞检测试剂盒对于活动性结核病的诊断具有较高的敏感度和特异度,尤其在肺结核并发肺外结核的患者以及痰检阳性的肺结核患者中效果较好,可用于辅助诊断活动性结核病。     

套式PCR及测序方法用于痰标本中结核分枝杆菌rpoB耐药基因突变检测

目的应用套式PCR—DNA测序方法直接检测痰标本中结核分枝杆菌相关的rpoB基因突变，以期建立一种直接检测分枝杆菌耐利福平的快速方法，并评价其临床应用价值。方法采用套武PCR—DNA测序方法直接检测112例活动性肺结核患者和20例非结核性肺部疾病患者痰、标本中结核分枝杆菌rpoB基因突变情况。同份痰标本同时做涂片抗酸染色，罗氏培养及菌型鉴定。结果112例活动性肺结核患者痰标本套式PCR扩增87例呈阳性，产物DNA测序31例有rpoB基因突变。其中分离出耐利福平株的32例痰中29例发生了基因突变，耐药突变率90．6％（29／32），39例菌阴（涂阴培阴）痰中有2例发生突变。分离出对利福平敏感株的37例痰中未发生突变，20例非结核性肺部疾病患者痰标本套式PCR扩增均为阴性，特异性100％。结论套式PCR—DNA测序可望为直接检测临床痰标本中结核分枝杆菌耐利福平的准确、特异、快速的方法。     

套式PCR及测序方法用于痰标本中结核分枝杆菌rpoB耐药基因突变检测

目的应用套式PCR-DNA测序方法直接检测痰标本中结核分枝杆菌相关的rpoB基因突变,以期建立一种直接检测分枝杆菌耐利福平的快速方法,并评价其临床应用价值。方法采用套式PCR-DNA测序方法直接检测112例活动性肺结核患者和20例非结核性肺部疾病患者痰标本中结核分枝杆菌rpoB基因突变情况。同份痰标本同时做涂片抗酸染色,罗氏培养及菌型鉴定。结果112例活动性肺结核患者痰标本套式PCR扩增87例呈阳性,产物DNA测序31例有rpoB基因突变,其中分离出耐利福平株的32例痰中29例发生了基因突变,耐药突变率90.6%(29/32),39例菌阴(涂阴培阴)痰中有2例发生突变。分离出对利福平敏感株的37例痰中未发生突变,20例非结核性肺部疾病患者痰标本套式PCR扩增均为阴性,特异性100%。结论套式PCR-DNA测序可望为直接检测临床痰标本中结核分枝杆菌耐利福平的准确、特异、快速的方法。     

四种结核分枝杆菌检测方法的临床应用评价

目的评估涂片、培养、PCR和增菌PCR检测结核分枝杆菌临床应用价值。方法对124例临床确诊的肺结核、可疑结核患者和非结核病人痰标本的涂片、培养、PCR和增菌PCR四种方法的检测结果进行比较。结果涂片、培养、PCR和4及7d的增菌PCR检测31例临床确诊的肺结核病人痰标本阳性率分别为22.5%、32.2%、54.8%、64.5%和87.1%;检测59例临床可疑肺结核病人痰标本阳性率分别为13.6%、18.6%、28.8%、37.3%和52.5%。比较四种方法的阳性检测率有显著性差异(P<0.05)。检测34例非结核病人痰标本,涂片、培养均为阴性,而PCR和增菌PCR均有1例假阳性,假阳性率2.9%。比较PCR与增菌PCR对菌阳和菌阴病人的痰标本阳性检测率,有显著性差异(P<0.05),而两种方法的假阳性率相同。结论增菌PCR检测结核分枝杆菌具有很高的敏感性和特异性,可作为结核病的有效辅助诊断方法之一。     

结核抗体检测对活动性肺结核的诊断价值评价

目的 评价结核抗体检测对活动性肺结核的诊断价值。方法 收集2016年8月至2017年8月在重庆市公共卫生医疗救治中心结核科住院的495例活动性肺结核患者(观察组)和158例非结核呼吸道疾病患者(对照组),均为综合患者的临床表现、胸部影像、痰细菌病原学检测或诊断性抗结核药物治疗有效等资料临床确诊后进行血清学诊断。分析血清MTB免疫球蛋白G(immunoglobulin G,IgG)、免疫球蛋白M(immunoglobulin M,IgM)、脂阿拉伯甘露聚糖(lipoarabinomannan,LAM),以及相对分子质量16000(以下采用“16kD”表示)和相对分子质量38000(以下采用“38kD”表示)的蛋白抗体的检测资料,以及单独及联合检测不同结核抗原(LAM、38kD和16kD)的结果,评价两组患者结核抗体检测的敏感度、特异度、阳性预测值、阴性预测值,以及对活动性肺结核的诊断效能。结果 495例观察组患者血清结核抗体检测阳性率[68.7%(340/495)]明显高于158例对照组患者[34.8%(55/158)],差异有统计学意义(χ ²=57.50,P<0.01);菌阴肺结核患者血清结核抗体检测阳性率[64.0%(210/328)]明显低于菌阳肺结核患者[77.8%(130/167)],差异有统计学意义(χ ²=9.83,P<0.01)。观察组340例结核抗体阳性患者中,LAM、38kD、IgG抗体联合检测同时均阳性的患者最多[61.8%(210/340)];对照组55例结核抗体阳性患者中,单一IgG抗体阳性最高[67.3%(37/55)]。以临床诊断为标准,血清结核抗体对活动性肺结核的诊断敏感度、特异度、阳性预测值和阴性预测值、总符合率、约登指数分别为68.7%(340/495)、65.2%(103/158)、86.1%(340/395)和39.9%(103/258)、67.8%(443/653)、0.34。 结论 血清结核抗体检测活动性肺结核患者具有较高的阳性率和敏感度,对诊断活动性肺结核具有一定的辅助价值,其中菌阳肺结核患者的检测阳性率高于菌阴肺结核,LAM、38kD和IgG联合检测可提高活动性肺结核的诊断阳性率。     

ELISA法检测痰中抗PPD-lgG对肺结核的诊断价值

为探讨对活动性肺结核方便、有效的诊断方法.应用ELISA法检测86例活动性肺结核、26例肺癌、36例细菌性肺炎患者痰中抗PPD-IgG.结果显示,活动性肺结核痰中抗PPD-IgG与非结核组比较有极显著性差异(P<0.001).该法诊断活动性肺结核的敏感性为81.4%.特异性为100%,阳性预测值为100%,阴性预测值为69.2%.认为是比较理想的新的免疫学诊断方法.     

结核分枝杆菌融合抗原38F和64F诊断活动性肺结核的价值

目的 探讨结核分枝杆菌融合抗原38F和64F对活动性肺结核患者血清中IgG、IgM和IgA抗体检测的诊断价值。 方法 利用结核分枝杆菌抗原优势肽段融合抗原38F和64F,通过ELISA法对223例活动性肺结核患者进行结核分枝杆菌特异性IgG、IgM和IgA抗体水平检测。223例活动性肺结核患者分为三组,第一组为涂片和培养共同阳性组（86例）,第二组为涂片阴性且培养阳性组（51例）,第三组为涂片和培养共同阴性组（86例）。 结果223例活动性肺结核病患者,第一组IgG、IgM和IgA的联合检出率为79.07%（68/86）;第二组IgG、IgM和IgA的联合检出率为62.75%（32/51）;第三组IgG、IgM和IgA的联合检出率为69.77%（60/86）。全部样本血清学方法的检出率为71.75%（160/223）。结论 结核分枝杆菌融合抗原38F和64F对于活动性肺结核具有较高的辅助诊断价值,并且IgG、IgM和IgA抗体联合检测可以提高检出率。     

快速ELISA检测血清抗PPD-IgG对肺结核的诊断价值

目的评价抗PPD－IgG检测对活动性肺结核的诊断价值。方法采用快速酶联免疫吸附法(ELISA)检测340例活动性肺结核、56例非活动性肺结核、88例非结核性病人血清中的抗PPD－IgG。结果活动性肺结核组抗PPD－IgG阳性率为83．8％,其中痰涂片(＋)组为92．2％,涂片(－)组为78．7％。非活动性肺结核组3例阳性,非结核组5例阳性,假阳性率分别为7．1％和5．7％。本法敏感性为83．8％,特异性为93．8％,准确性为86．8％,阳性预测值为96．9％,阴性预测值71．1％。结论。提示血清抗PPD－IgG测定是肺结核病人一项有用的辅助诊断手段。     

Detection of Mycobacterium tuberculosis in bronchoalveolar lavage from patients with sputum smear-negative pulmonary tuberculosis using a polymerase chain reaction assay

Abstract The objective of this study was to evaluate the utility of a polymerase chain reaction (PCR) assay in detecting Mycobacterium tuberculosis in bronchoalveolar lavage (BAL) specimens of patients suspected of having active pulmonary tuberculosis (TB) but who were sputum smear-negative. Patients undergoing investigation for suspected pulmonary TB at the University Hospital, Kuala Lumpur, and who were sputum smear-negative underwent fibreoptic bronchoscopy and BAL. One portion of each lavage specimen was submitted for smear examination for acid-fast bacilli and mycobacterial culture and the other portion assayed by PCR for the presence of a 562-base pair DNA segment belonging to the insertion sequence IS986, unique to the M. tuberculosis complex. As controls, lavage specimens from patients with other lung lesions were also similarly tested. The PCR assay gave a positivity rate of 80.9% (55 of 68) compared with 8.8% of smear examination and 7.4% of culture for detecting M. tuberculosis in BAL specimens. The assay was positive in two of 45 BAL specimens from 35 control subjects. The PCR assay was more sensitive than smear and culture in detecting M. tuberculosis in BAL specimens of patients with sputum smear-negative pulmonary TB.     

Mycobacterium tuberculosis DNA detection in soluble fraction of urine from pulmonary tuberculosis patients.

SETTING: A tertiary care and research institution in Italy. BACKGROUND: Small DNA fragments from cells dying throughout the body have been detected in urine (transrenal DNA [Tr-DNA]). OBJECTIVE: To test the hypothesis that Mycobacterium tuberculosis Tr-DNA could be detected in the urine of pulmonary tuberculosis (TB) patients. DESIGN: We studied 43 patients with culture-confirmed pulmonary TB with no evidence of extra-pulmonary involvement, 10 patients with pulmonary diseases other than TB and 13 healthy controls. DNA was extracted from urine and analysed by semi-nested polymerase chain reaction (PCR). RESULTS: M. tuberculosis-specific sequences were found in the urine of 34 of 43 (79%) TB patients studied, whereas all controls were negative. The transrenal nature of M. tuberculosis DNA was demonstrated by two lines of evidence: first, separate analysis of supernatants and sediments from eight of the study patients found seven positive supernatants but only two matched positive sediments. Second, M. tuberculosis-specific sequences were amplified by semi-nested PCR with primers designed for short but not large amplicons. CONCLUSION: Small M. tuberculosis DNA fragments may be detected in the urine of a significant proportion of patients with pulmonary TB. If these observations are confirmed by larger studies, Tr-DNA technology could represent a new approach for detecting pulmonary M. tuberculosis infection.     

Value of smear and PCR in bronchoalveolar lavage fluid in culture positive pulmonary tuberculosis.

At present, further investigations are needed in patients with suspected pulmonary tuberculosis (TB) and either negative sputum smear or without sputum. The aim of the present study was to analyse the yield of bronchoalveolar lavage fluid (BALF) smear and PCR in patients with confirmed pulmonary TB. Patients with a positive culture for Mycobacterium tuberculosis complex in sputum or BALF were analysed over 5 yrs. In total, 90 out of 230 (39%) patients with culture-positive pulmonary TB had a positive sputum smear, and 120 patients underwent bronchoscopy. BALF smear was positive in 56 (47%), BALF PCR in 93 (78%) patients, and BALF smear and/or PCR was positive in 83%. In total, 71 patients who underwent bronchoscopy and had complete clinical records were further analysed. BALF (smear or Mycobacterium tuberculosis complex-PCR) allowed a rapid diagnosis in 10 (59%) out of 17 patients who had a negative sputum smear, and 49 (91%) out of 54 patients without sputum production. Of these 71 patients, 12 (17%) were only culture positive. Rapid diagnosis of pulmonary TB by smear and/or PCR was made in 190 out of 210 patients (90%) in sputum or BALF. In conclusion, combined use of bronchoalveolar lavage fluid smear and Mycobacterium tuberculosis complex-PCR has a good diagnostic yield in patients with sputum smear-negative tuberculosis or without sputum production.     

Diagnosis of intestinal tuberculosis using a monoclonal antibody to Mycobacterium tuberculosis

AIM: To investigate the utility of immunohistochemical (IHC) staining with an antibody to Mycobacterium tuberculosis (M. tuberculosis) for the diagnosis of intestinal tuberculosis (TB).METHODS: We retrospectively identified 10 patients (4 males and 6 females; mean age = 65.1 ± 13.6 years) with intestinal TB. Clinical characteristics, including age, gender, underlying disease, and symptoms were obtained. Chest radiograph and laboratory tests, including sputum Ziehl-Neelsen (ZN) staining, M. tuberculosis culture, and sputum polymerase chain reaction (PCR) for tubercle bacilli DNA, as well as Tuberculin skin test (TST) and QuantiFERON-TB gold test (QFT), were examined. Colonoscopic records recorded on the basis of Sato’s classification were also reviewed, in addition to data from intestinal biopsies examined for histopathological findings, including hematoxylin and eosin staining, and ZN staining, as well as M. tuberculosis culture, and PCR for tubercle bacilli DNA. For the present study, archived formalin-fixed paraffin-embedded (FFPE) intestinal tissue samples were immunohistochemically stained using a commercially available species-specific monoclonal antibody to the 38-kDa antigen of the M. tuberculosis complex. These sections were also stained with the pan-macrophage marker CD68 antibody.RESULTS: From the clinical data, we found that no patients were immunocompromised, and that the main symptoms were diarrhea and weight loss. Three patients displayed active pulmonary TB, six patients (60%) had a positive TST, and 4 patients (40%) had a positive QFT. Colonoscopic findings revealed that all patients had type 1 findings (linear ulcers in a circumferential arrangement or linear ulcers arranged circumferentially with mucosa showing multiple nodules), all of which were located in the right hemicolon and/or terminal ileum. Seven patients (70%) had concomitant healed lesions in the ileocecal area. No acid-fast bacilli were detected with ZN staining of the intestinal tissue samples, and both M. tuberculosis culture and PCR for tubercle bacilli DNA were negative in all samples. The histopathological data revealed that tuberculous granulomas were present in 4 cases (40%). IHC staining in archived FFPE samples with anti-M. tuberculosis monoclonal antibody revealed positive findings in 4 patients (40%); the same patients in which granulomas were detected by hematoxylin and eosin staining. M. tuberculosis antigens were found to be mostly intracellular, granular in pattern, and primarily located in the CD68⁺ macrophages of the granulomas.CONCLUSION: IHC staining with a monoclonal antibody to M. tuberculosis may be an efficient and simple diagnostic tool in addition to classic examination methods for the diagnosis of intestinal TB.     

应用磁纳米捕获-PCR技术检测痰液结核分枝杆菌的研究

目的采用磁纳米捕获技术富集痰液中的结核分枝杆菌,以提高PCR检测的灵敏度,快速诊断结核病。方法以普通PCR为对照,采用磁纳米捕获技术富集187份结核和非结核呼吸系统疾病患者痰标本中的结核分枝杆菌,进行PCR检测。结果 152份肺结核患者痰标本41份(27.0%)涂片抗酸染色阳性,72份(47.4%)普通PCR检测阳性,126份(82.9%)磁纳米捕获-PCR检测阳性。35份非结核呼吸系统疾病患者,痰标本中抗酸染色均为阴性,1份普通PCR和磁纳米捕获-PCR检测均阳性,该患者临床诊断肺部感染合并陈旧性肺结核。结论采用磁纳米捕获技术富集痰液中的结核分枝杆菌,可显著提高PCR检测的灵敏度。     

AmpliSensor—聚合酶链反应定量检测肺结核患者外周血结 …

目的 探讨ＡｍｐｌｉＳｅｎｓｏｒ－聚合酶链反应定量检测外周血中结核分支杆菌ＤＮＡ在肺结核的应用价值。方法 采用ＱｌＡａｍｐ和ＡｃｕＰｕｒｅ法提取，制备全血中模板ＴＢ－ＤＮＡ，应用ＡｍｐｌｉＳｅｎｓｏｒ－ＰＣＲ定量检测，并与ＩＳ６１１０－单管巢式聚合酶链反应（ＳＮ－ＰＣＲ）作比较。结果２００例肺结核患者的血液标本中，两种方法测得结核分支杆菌ＤＮＡ的阳性率分别为６０．５％、６３．５％。８５例非结核肺病     

The IS6110 repetitive DNA element of Mycobacterium tuberculosis is not detected in exhaled breath condensate of patients with active pulmonary tuberculosis

BACKGROUND: A large tertiary referral hospital in inner-city Chicago. OBJECTIVES: To determine whether the IS6110 repetitive DNA element of Mycobacterium tuberculosis is detected in exhaled breath condensate of patients with newly diagnosed active pulmonary tuberculosis. METHODS: Ten hospitalized patients with positive Ziehl-Neelson-stained sputum smears were studied. Concurrent sputum cultures for mycobacteria were performed as well. Exhaled breath condensate was collected from each patient within 6 days of initiating antituberculosis chemotherapy (median 1.5 days). These samples were analyzed by polymerase chain reaction (PCR) using primers designed to amplify the IS6110 DNA fragment of M. tuberculosis. Exogenous M. tuberculosis DNA was added to exhaled breath condensate samples to detect PCR inhibitors. Concurrent cultures of exhaled breath condensate for mycobacteria were performed. RESULTS:M. tuberculosis was identified in 9 of 10 sputum cultures. One isolate was identified as Mycobacterium kansasii. The IS6110 repetitive DNA element of M. tuberculosis was not detected in any of the 10 exhaled breath condensate samples. Exogenous M. tuberculosis DNA added to these samples elicited the characteristic band pattern of M. tuberculosis on agarose gel electrophoresis. No PCR inhibitors were detected. Cultures of exhaled breath condensate showed no growth of mycobacteria. CONCLUSIONS: The IS6110 repetitive DNA element of M. tuberculosis is not detected in exhaled breath condensate of patients with newly diagnosed active pulmonary tuberculosis.     

Influence of HLA-DR antigens on lymphocyte response to Mycobacterium tuberculosis culture filtrate antigens and mitogens in pulmonary tuberculosis.

SETTING: Influence of HLA-DR antigens and lymphocyte responses in pulmonary TB patients. OBJECTIVE: To elucidate the role of HLA-DR genes/gene products on lymphocyte responses to Mycobacterium tuberculosis antigens and mitogens, the present study was carried out in pulmonary tuberculosis during active and cured stage of the disease. DESIGN: Serological determination of HLA-DR antigens was carried out in 50 active TB patients, 44 cured TB patients and 58 normal healthy control subjects. The influence of HLA-DR antigens on peripheral blood lymphocyte responses to M. tuberculosis culture filtrate antigens and mitogens such as phytohaemagglutinin (PHA) and concanavalin-A (Con-A) was studied in the patients as well as normal healthy control subjects. RESULTS: Of all the DR antigens studied, patients (active TB and cured TB) with DR2 antigen showed an increased lymphocyte response (stimulation index) to a higher dose of antigenic (10 micrograms/ml) stimulation. A significantly lower lymphocyte response to antigen and mitogens was seen in HLA-DR3 positive normal healthy subjects than non-DR3 (DR3 negative) subjects. CONCLUSION: The present study suggests that HLA-DR genes/gene products may be playing an immunoregulatory role in eliciting an immune response against M. tuberculosis antigens and mitogens induced lymphocyte response in pulmonary TB patients and normal healthy subjects.     

Accuracy of an interferon-gamma release assay to detect active pulmonary and extra-pulmonary tuberculosis.

OBJECTIVE: To examine the performance of an interferon-gamma (IFN-gamma) release assay (QuantiFERON-TB 2G assay [QFT-G]) to detect Mycobacterium tuberculosis infection in a Japanese general hospital, for the diagnosis of active pulmonary tuberculosis (PTB) and extra-pulmonary tuberculosis (EPTB). DESIGN: We prospectively examined the performance of QFT-G in 194 patients suspected of active TB. Diagnosis was confirmed by 1) positive M. tuberculosis cultures, or 2) clinical manifestations or laboratory or pathological findings consistent with active TB and response to specific therapy. RESULTS: Three patients with indeterminate QFT-G results were excluded. Among the remaining 191 patients, 77 had active TB. When the cut-off concentration of IFN-gamma was set at 0.35 IU/ml, as recommended by the manufacturer, the assay was positive in 69 patients and negative in 122. The sensitivity of the assay was 76.6% in all patients, 74.5% in the 47 patients with PTB and 80.0% in the 30 patients with EPTB. The overall specificity of the assay was 91.2%. CONCLUSION: Although the specificity of the QFT-G to detect active TB was high and its sensitivity low, it was as accurate for the detection of active EPTB as for PTB when the 0.35 IU/ml INF-gamma cut-off concentration was used.     

Do mitochondrial DNA haplogroups play a role in susceptibility to tuberculosis?

BACKGROUND AND OBJECTIVES: Mitochondrial DNA has a unique role in ATP production and subsequent mitochondrial reactive oxygen species (ROS) production in eukaryotic cells and there is a potential role for ROS and oxygen burst against Mycobacterium tuberculosis, an intracellular pathogen. This study aimed to determine whether the frequency of different mitochondrial haplogroups was significantly different in patients with tuberculosis (TB) compared with a normal population. METHODS: Mitochondrial DNA haplogroups M, N, J and K were studied by PCR-restriction fragment length polymorphism and sequencing. Cases were 54 patients with confirmed smear positive pulmonary TB. Controls were 256 healthy persons. RESULTS: There were no statistically significant differences between those with TB and the control group. CONCLUSIONS: There was no statistically significant association between mtDNA haplogroups and the presence of TB infection.