Real-time PCR技术结合浓缩涂片染色法用于结核病快速诊断的研究

目的:评价Real-time PCR技术结合浓缩涂片染色法对结核病快速诊断的应用价值。方法:应用Real-timePCR技术结合浓缩涂片染色法组合测定1 052份临床标本,并与直接涂片法、BACTEC MGIT-960快速培养法结果进行比较。结果:Real-time PCR技术结合浓缩涂片染色法的阳性检出率30.6%,显著高于直接涂片法的19.2%(χ2=36.6,P<0.01),而接近于BACTEC-960培养法的34.9%(χ2=2.5,P>0.05),而且检测时间明显缩短为4 h;以BACTECMGIT-960培养鉴定结果为标准评价,Real-time PCR技术结合浓缩涂片染色法的敏感性为89.7%(280/312),特异性为94.3%(698/740),符合率为93.0%(978/1 052)。结论:Real-time PCR技术结合浓缩涂片染色法用于结核分枝杆菌检测快速简便,具有很高的敏感性和特异性,在结核病早期诊断中有重要的临床价值。     

IFN—γ释放试验（T—SPOT．TB）在结核病诊断中的应用

目的探讨T淋巴细胞感染结核后IFN-γ释放斑点试验fT—SPOT．TB）在结核病诊断中的应用及价值。方法采用T—SPOT．TB方法对疑诊或待排结核患者外周血液中释放IFN-γ的结核杆菌特异性淋巴细胞的测定。结果T—SPOT．TB方法的阳性率是83．1％（89／107）,结核菌素试验（tuberculinskintest,TSTl的阳性率40．2％（43／1071,涂片找抗酸杆菌阳性率是25．6％f21／82）,TB—DNA的阳性率是9．4％（5／531．分枝杆菌分离培养阳性率是21．4％（9／42）。结论T—SPOT．TB方法检测诊断结核性疾病敏感性为83．1％、特异性为95．2％：其阳性率明显高于TST、涂片找抗酸杆菌、TB—DNA、分枝杆菌分离培养,采用该方法检测T淋巴细胞感染结核IFN-γ释放快速敏感．特异性高,在结核性疾病诊断中具有重要价值。     

检测支气管灌洗液中抗酸杆菌的方法学研究

目的探讨捡测支气管灌洗液中抗酸杆菌的最佳方法。方法对155例痰涂片阴性疑似肺结核患者的支气管灌洗液采用涂片、集菌、改良罗氏培养法及Bactec培养法4种不同的检测方法检测抗酸杆菌。结果阳性检出率直接涂片法为2．6%,集菌法为11．6％,改良罗氏培养法为18．1％,Bactec培养法为31．6％;集菌、培养法与直接涂片法抗酸杆菌的阳性检出率比较均有显著性差异（P〈0．05）。结论对疑似肺结核患者支气管灌洗液进行抗酸杆菌检测时,最好采用集菌和培养法同时进行,才能为临床诊治提供最有价值的资料。     

快速离心沉淀集菌涂片法与罗氏培养法检测痰内结核杆菌

直接镜检抗酸杆菌阳性可作为结核病的确诊方法之一,但痰直接涂片敏感性低,已知离心沉淀集菌涂片法可提高痰液抗酸杆菌的检出率。本研究采用快速离心沉淀集菌涂片法检测痰液抗酸杆菌,同时以罗氏培养法为对照,其结果便于其他实验室借鉴与改进。     

噬菌体生物扩增法检测不同标本对结核病临床诊断的意义

目的探讨噬菌体生物扩增法技术检测不同标本对结核病临床诊断的应用价值。方法采用噬菌体生物扩增法技术对86例活动性肺结核患者痰标本、73例结核性胸膜炎患者胸水、30例肺结核患者肺泡灌洗液、7例结核性脑膜炎患者脑脊液,共计196例结核标本进行检测;对非结核病患者痰标本10例、胸水5例、灌洗液5例,共计20例标本进行检测。每例标本同时进行涂片抗酸染色、罗氏培养。结果86例痰标本、73例胸水、30例肺泡灌洗液、7例脑脊液噬菌体生物扩增法检测阳性率分别为53．49％（46／86）、49．32％（36／73）、60．00％（18／30）、0（0／7）,涂片抗酸染色阳性率分别为24．42％（21／86）、2．74％（2／73）、20．00％（6／30）、0（0／7）,罗氏培养分别为44．19％（38／86）、4．11％（3／73）、36．67％（11／30）、0（0／7）。20例非结核患者标本,噬菌体生物扩增法、涂片抗酸染色、罗氏培养都未检出阳性。结论噬菌体生物扩增法检测结核病标本的阳性率高于涂片和罗氏培养,该方法敏感性高、特异性强、操作简便,对结核病临床诊断有较高的应用价值。     

玻片直接离心集菌涂片法检测脑脊液抗酸杆菌

目前,由于结核性脑膜炎（结脑）的症状多不典型,临床诊断的难度增大,因此需要有效的实验室方法,提供确切的实验诊断依据［１］。检测脑脊液（ＣＳＦ）抗酸杆菌,是诊断结脑的特异性的依据之一,但由于常规的ＣＳＦ抗酸杆菌检测方法（常规法）,敏感性低（约１０％左右）［２］,难以满足诊断的需要,因此研究提高ＣＳＦ抗酸杆菌检测敏感性的方法,具有重要的实用意义。应用玻片直接离心集菌涂片法（玻片离心法）检测ＣＳＦ抗酸杆菌,可以显著提高抗酸杆菌的检出率（＞６５％）,报道如下。一、材料与方法１标本来源：以临床确诊病例的…     

XpertMTB/RIFAssay在结核病诊断中的应用

目的：对 Xpert MTB／RIF 检测结核分枝杆菌（MTB）方法进行临床研究,评价其在结核病诊治中的应用价值。方法应用 Xpert MTB／RIF 法检测结核患者的 MTB,以 BACTEC MGIT 960培养法为参考方法,获得该方法的灵敏度、特异度。结果对123例结核患者的临床样本进行 Xpert MTB／RIF 法检测、BACTEC MGIT 960培养及细菌涂片抗酸染色法检查,其灵敏度和特异度分别为65．9％、47．2％、31．2％和100．0％、96．0％、100．0％。结论Xpert MTB／RIF 检测 MTB 速度快,灵敏度高,特异度强,对结核患者的早期诊断及治疗有着重要的意义。     

回顾性分析噬菌体生物扩增技术在肺结核诊断中的应用

目的评价噬菌体生物扩增技术（PhaB）在肺结核诊断中的应用。方法回顾性分析采用PhaB法、3D培养法、直接涂片法检测373份痰标本的抗酸杆菌结果。结果 PhaB法、3D培养法、直接涂片法检测抗酸杆菌的阳性率分别为53.6%、56.6%及20.1%。以3D培养法结果为标准,直接涂片法的敏感性、特异度、准确性分别为33.2%、95.7%及60.3%;PhaB法的敏感性、特异度、准确性分别为89.1%、93.6%及90.6%。直接涂片法与PhaB法检测结果比较,差异有统计学意义（χ2=94.7,P〈0.01）;PhaB法与3D培养法检测结果比较,差异无统计学意义（χ2=3.46,P〉0.05）。结论 PhaB法检测结核分枝杆菌具有较高的敏感性、特异度、准确性,值得在临床推广。     

夹层杯离心集菌涂片法检测痰液抗酸杆菌的临床应用研究

目的探讨夹层杯离心集菌涂片法检测痰液抗酸杆菌的临床应用价值。方法同时用液基夹层杯离心集菌涂片法、直接涂片法和罗氏培养法对收集的864例晨痰标本进行检测,并比较检测结果。结果夹层杯法、直接法和培养法检测864例晨痰标本的阳性率分别为17.59%、5.67%和18.63%,夹层杯法阳性率明显高于直接涂片法(P<0.05);以培养法为标准,夹层杯法的敏感性、特异性、阳性预期值、阴性预期值及准确性分别为93.16%、99.71%、98.68%、98.46%和98.50%。结论夹层杯离心集菌涂片法是一种阳性率高、更利于标准化和简便化的抗酸杆菌检验方法,其阳性率高于直接涂片法,并与培养法相比有较好的符合率,可能是一种值得推广的检测方法。     

荧光定量PCR技术检测结核分支杆菌DNA的应用价值

目的评价荧光定量PCR技术检测临床标本中结核分支杆菌的应用价值。方法应用荧光定量PCR法及抗酸染色、结核杆菌培养法,对179例活动性肺结核患者晨痰、123例活动性肺结核患者外周血、34例结核性胸膜炎患者胸水、20例非结核性呼吸系统疾病患者的晨痰标本进行检测。结果179例活动性肺结核患者晨痰、123例活动性肺结核患者外周血、34例结核性胸膜炎患者胸水,涂片抗酸染色阳性率分别为20．67％（37／179）、0．00％、0．00％;细菌培养阳性率分别为41．34％（74／179）、0．00％、2．94％（1／34）;荧光定量PCR技术阳性率分别为64．25％（115／179）、38．21％（47／123）、44．12％（15／34）。3种标本荧光定量PCR技术阳性率高于抗酸染色和结核杆菌培养,经统计学处理发现,差异有统计学意义。用荧光定量PCR技术检测临床标本特异性为95．00％。结论荧光定量PCR技术将PCR扩增、荧光探针杂交及检测一体化,在单一管内完成,具有简便、快速、防污染、敏感性及特异性高等优点,是结核病诊断的有效方法之一。     

噬菌体生物扩增法快速检测脊柱结核脓液中结核分枝杆菌

[目的]探讨噬菌体生物扩增(PhaB)法快速检测脊柱结核脓液中结核分枝杆菌的临床应用价值.[方法]应用直接涂片法,BACTEC MGIT-960法及PhaB法分别检测56例脊柱结核患者的56份脓液标本以及10株结核分枝杆菌标准株,15株非结核分枝杆菌及5株非分枝杆菌.[结果]PhaB法检测10株结核分支杆菌标准株均为阳性,15株非结核分支杆菌和5株非分支杆菌均为阴性.46份BACTEC MGIT-960培养阳性和10份BACTEC MGIT-960培养阴性的标本中,PhaB法分别有42份(91.3%)、3份(30.0%)阳性,检出率分别为82.1%和80.4%(P＞0.05).涂片检查阳性仅13份(23.2%),阴性43份(76.8%),而应用PhaB法可以检测出45份(80.4%)阳性,其中有33份为涂片阴性的标本(P＜0.05).用PhaB法能在18～24 h内得出结果,较BACTEC MGIT-960法提前5～17 d.[结论]PhaB法与直接涂片法、BACTEC MGIT-960法相比,可以简便、快速地检测结核分支杆菌,全程只需1 d,并且具有较高的敏感性和特异性.     

实时荧光定量聚合酶链反应在肺结核诊断中的应用

目的探讨实时荧光定量聚合酶链反应(FQ-PCR)在肺结核诊断中的应用价值。方法应用FQ-PCR对临床诊断肺结核及其他肺部疾病患者的临床标本进行结核分枝杆菌DNA定量检测,同时与涂片抗酸染色、BACTEC 960培养法作比对。结果 189例临床诊断肺结核患者FQ-PCR检测123例阳性,提示敏感度为65.1%(123/189),而涂片抗酸染色、BACTEC 960培养法分别为40.2%(76/189)、63.5%(120/189)。94例其他肺部疾病患者及20例健康对照者FQ-PCR检测均为阴性,特异度为100.0%。结论 FQ-PCR敏感度和特异度均高,检测过程简单、易标准化、全过程仅需4～5h,可用于结核病的快速诊断。     

BACTEC MGIT-960快速检测分枝杆菌的临床应用研究

目的探讨BACTEC MGIT-960全自动分枝杆菌检测技术在结核病诊治中的应用价值。方法用BACTEC MGIT-960对不同种类标本进行结核杆菌分离培养及药敏试验,并与改良罗氏培养法和BACTEC-460系统进行比较。结果BACTEC-960全自动分枝杆菌检测技术初代分离阳性时间平均为9.8天,药敏平均为7.5天,明显短于改良罗氏培养(28.5天和22.1天),与BACTEC-460(8.8天和6.5天)结果接近。486份各类标本总阳性率46.50%,其中痰标本为54.02%。BACTEC MGIT-960药敏试验结果,与BACTEC-460、L-J法总符合率分别为98.54%和95.79%。结论BACTEC MGIT-960全自动分枝杆菌检测技术是目前一种比较理想的快速检测结核菌的方法。     

The evaluation of FASTPlaqueTB test for the rapid diagnosis of tuberculosis

FASTPlaqueTB (Biotec Laboratories Ltd., Ipswich, UK) is a rapid test which utilizes bacteriophage amplification technology for the detection of viable Mycobacterium tuberculosis in clinical specimens. We evaluated performance of the FASTPlaqueTB test by comparing with BACTEC 460 TB culture system (Becton Dickinson Co., Maryland, USA), polymerase chain reaction (PCR) and acid fast bacilli (AFB) smear methods. We investigated 192 sputum specimens collected from the patients suspected of having pulmonary TB by AFB smear, BACTEC 460 TB culture system, PCR and FASTPlaqueTB test. The sensitivity of AFB smear, PCR and FASTPlaqueTB test were 57.8%, 84.4% and 87.5% respectively when we accepted BACTEC 460 TB culture system as gold standard. We conclude that FASTPlaqueTB test has a good potential for rapid diagnosis of Mycobacterium tuberculosis as a result of the evaluation of these three tests by comparison to the BACTEC 460 TB culture system.     

Reduction in turnaround time for laboratory diagnosis of pulmonary tuberculosis by routine use of a nucleic acid amplification test

Every first diagnostic specimen from suspected patients with pulmonary TB was tested by a nucleic acid amplification test (NAAT) to determine the reduction in turnaround time (TAT) for detecting Mycobacterium tuberculosis (MTB) that was possible under normal laboratory operating conditions. NAAT (Gen-Probe Mycobacterium tuberculosis Direct Testtrade mark) was performed on the first specimen and liquid culture (BACTEC 460), solid culture (Lowenstein-Jensen [LJ] agar and selective 7H11 [7H11S] agar), and fluorescent acid-fast bacilli (AFB) smear were performed on all 3 specimens from each patient. Eighty-one (10.2%) of 797 patients tested were diagnosed with pulmonary TB. The sensitivity of NAAT, BACTEC, LJ, 7H11S, and smear for the first specimen was 90%, 85%, 67%, 53%, and 58%, respectively, whereas the sensitivity for the series of 3 specimens was 90%, 95%, 74%, 74%, and 70%, respectively. Positive predictive value was 100% for all tests except AFB smear, which was 79%. The time to detect 75% of all TB cases was 4 days for NAAT and 21 days for liquid culture; other tests had a sensitivity of less than 75%. Identification and testing every first diagnostic specimen by NAAT has the potential to reduce the overall TAT for laboratory TB diagnosis by approximately 2 weeks.     

支气管镜结合BD-960分枝杆菌培养及药物敏感系统对门诊肺结核病的诊断意义

目的 探讨支气管镜检查结合BD-960分枝杆菌培养/药敏系统(BACTEC MGIT-960)时肺结核的诊断价值.方法 回顾性分析行BACTEC MGIT-960培养检测的279例门诊肺结核患者的临床资料,通过对比不同标本取材方式及检测方法,了解支气管镜检查结合BACTEC MGIT-960培养检测对肺结核的诊断阳性率.结果 支气管镜组92例通过支气管镜检查刷检涂片阳性率26.0%(24/92)、灌洗液行BACTEC MGIT-960培养检测阳性率为47.8%(44/92),常规留痰组187例行涂片抗酸染色阳性率23.0%(43/187)、BACTEC MGIT-960培养检测阳性率为33.7%(63/187).结论 支气管镜检查结合BACTEC MGIT-960培养检测可明显提高门诊肺结核病的确诊率.     

五种结核病实验室检测方法对结核病诊断价值的效果评价

目的评价涂片法、BACTECMGIT960快速培养法、改良罗氏培养法、荧光定量PCR法和斑点免疫层析法在结核病实验室快速诊断方法中的作用和地位。方法对1260例结核病患者（结核病组）和100例非结核病患者（非结核病组）的各类标本采用涂片法、快速培养法、改良罗氏培养法、荧光定量PCR法进行检测,同时分离患者外周血血清进行结核抗体检测,并对这五种检测方法的结果进行分析比较。结果五种实验室检测方法对结核病组阳性标本和非结核病组阴性标本的检测结果差异均有统计学意义（χ2=466．31,χ2=216．14,P均〈0．05）。对于结核病组的涂阳和涂阴标本,快速培养法和荧光定量PCR法检测的灵敏度及准确度均高于其他三种方法,而涂片法、改良罗氏培养法、快速培养法的特异性均为100．0％,高于荧光定量PCR法和斑点免疫层析法;快速培养法的阳性检出率均较改良罗氏培养法高,差异均有统计学意义（χ2=3387．00,χ2=4233．00,P均〈0．05）,平均培养时间也较改良罗氏培养法短;但快速培养法与荧光定量PCR法的阳性检出率差异均无统计学意义（P均〉0．05）。结论BACTECMGIT960快速培养法和荧光定量PCR法是诊断结核病快速、有效的检测方法,其阳性检出率高,且能缩短培养时间,BACTECMGIT960快速检测系统还能进行快速药物敏感性试验,为结核病的快速诊断提供依据。     

Usefulness of induced sputum and fibreoptic bronchoscopy specimens in the diagnosis of pulmonary tuberculosis

We investigated the diagnostic value of induced sputum (IS) and bronchial lavage (BL) specimens in patients with suspected pulmonary tuberculosis who had negative spontaneous sputum specimens or who were unable to produce sputum spontaneously. IS specimens and BL specimens obtained using flexible fibreoptic bronchoscopy from 55 patients were evaluated for the presence of acid-fast bacilli (AFB) and cultured for Mycobacterium tuberculosis. Positive results were found with IS smear in 23 patients, BL smear in 26 patients, and IS or BL culture in 42 patients. Culture of BL specimens had a higher sensitivity than IS or BL smears or culture of IS specimens. The highest sensitivity rate was obtained with a positive BL or IS culture (86%). For early diagnosis (a positive IS or BL smear), the sensitivity was 57%. IS has a higher sensitivity rate than spontaneous sputum for the detection of tuberculosis, and fibreoptic bronchoscopy is useful for the early diagnosis of tuberculosis when AFB are not detected in spontaneous or induced sputum specimens.     

Evaluation of the BACTEC MGIT 960 system for the recovery of mycobacteria

We evaluated the BACTEC MGIT 960 system, which is a fully automated, non-invasive, continuous monitoring system for the growth and detection of mycobacteria. Including respiratory and other specimens, 1,742 specimens were processed and inoculated into the BACTEC MGIT 960 and the BACTEC 460 TB Systems, as well as onto Lowenstein-Jensen (L-J) media. A total of 104 isolates of mycobacteria were recovered from all culture systems. This included Mycobacterium tuberculosis complex, Mycobacterium avium-intracellulare (MAI) complex and other mycobacteria (MOTT). The isolation rates for M. tuberculosis complex and MAI complex were comparable for the BACTEC 460 (54.8% and 13.5%) and the BACTEC MGIT 960 (51.9% and 13.5%). The overall isolation rate was less for BACTEC MGIT 960 (76.9%) which was due to lesser number of MOTT isolates recovered from this system. The mean times to detection (TTD) for all mycobacteria were 9.3 days for the BACTEC MGIT, 14.6 days for the BACTEC 460 and 21.6 days for L-J. A significant difference was observed when TTD was tested in relation to degree of positivity in smears, with the BACTEC MGIT maintaining the short TTD even with less number of bacilli in the smear. The contamination rates were, 6.4% for BACTEC MGIT, 2.9% for BACTEC 460 and 12.1% for L-J medium. The BACTEC MGIT 960 system shows performances comparable to the BACTEC 460 and seems to be a dependable, user friendly system.