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.     

荧光定量PCR测定支气管肺泡灌洗液中TbDNA对涂阴肺结核的诊断价值

目的探讨纤支镜肺泡灌洗液中结核分枝杆菌DNA在涂阴肺结核诊断中的价值。方法应用荧光定量聚合酶链反应（FQ-PCR）技术对300例涂阴肺结核,178例菌阳肺结核及119例肺炎或肺癌患者的纤支镜肺泡灌洗液标本进行结核分枝杆菌DNA检测。结果3种病因肺泡灌洗液结核分枝杆菌DNA阳性检出率分别为：74.3%（223/300）、94.9%（169/178）、17.6%（21/119）。结论荧光定量聚合酶链反应检测肺泡灌洗液结核分枝杆菌DNA,用于肺结核诊断可提高肺结核诊断的敏感性和准确性,明显优于痰抗酸染色,亦较痰结核菌培养诊断灵敏、快速,可作为肺结核诊断较可靠指标。     

支气管肺泡灌洗液多指标联合检测对不典型肺结核的诊断价值探讨

目的探讨支气管肺泡灌洗液（BALF）多指标联合检测对不典型肺结核的诊断价值。方法56例不典型肺结核患者治疗前均进行支气管镜检查,行支气管刷检涂片法、镜检后痰涂片法、BALF涂片法以及联合进行BALF涂片、BALF聚合酶链反应（BALF-PCR）、BALF结核抗原（BALF-TBAG）和BALF腺苷脱氨酶（BALF-ADA）四项指标检测方法,分析不同的检测方法对不典型肺结核诊断的阳性率。结果56例患者行支气管刷检涂片法、镜检后痰涂片法、BALF涂片法的阳性率分别为39.2%,46.4%,75.0%,联合进行BALF涂片、BALF-PCR、BALF-TBAG和BALF-ADA四项指标检测的阳性率为92.8%,与前三种检测方法相比,差异有显著性（P〈0.05）。结论BALF多指标联合检测对不典型肺结核具有较高的诊断价值,特异性高,快速,大大缩短诊断时间,可以明显提高诊断阳性率,是一项较好的组合方式。     

聚合酶链反应检测肺结核患者外周血单个核细胞中结核分 …

探讨外周血单个核细胞中结核分支杆菌ＤＮＡ检测在肺结核诊断中的价值。方法采用改良Ｔｒｉｔｏｎ－ｘ－１００法分离，制备单个核细胞中模板，ＤＮＡ，聚合酶链反应扩增结核分支杆菌２４０ｂｐ基因片段，同时分析了影响ＰＣＲ结果的有关因素。结果８９例肺结核患者的血标本，８４例肺结核患者的痰标本中，结核分支杆菌ＤＮＡ阳性率分别为７３％和５７％；８４例肺结核患者外周血，痰标本配对检测总阳性率可达８７％。     

3479例肺结核病人痰标本抗酸杆菌检查结果分析

目的：探讨肺结核病人痰标本抗酸杆菌检查的影响因素。方法：采用直接厚涂片萋-尼氏法抗酸染色。对临床资料回顾分析。结果：3479例痰标本。抗酸杆菌阳性829例。阳性率23．8％。痰标本的性状、留痰时间对抗酸杆菌检出有很大影响。结论：应提高留痰质量。重视晨痰的留取。     

Use of polymerase chain reaction in the diagnosis of abdominal tuberculosis

BACKGROUND: Early diagnosis and prompt treatment of abdominal tuberculosis is vitally important as it greatly reduces disease and treatment related morbidity and even mortality in extreme cases. A polymerase chain reaction (PCR) test was evaluated for its feasibility as a diagnostic tool in abdominal tuberculosis (TB) in the Indian scenario. METHODS: PCR for the identification of M. tuberculosis amplified a 340 bp nucleotide sequence located within the 38 kDa protein gene of M. tuberculosis. Tissues for processing were obtained from patients suspected to have abdominal TB. These were from various sources such as abdominal lymph nodes, segments of intestine and bowel obtained at various times and in different ways such as laparoscopy, colectomy, bowel and lymph node resection. Fifty such patients had their tissues sent for PCR. RESULTS: PCR results were compared with histopathology (HP). Of the 50 samples, 31 were positive for abdominal TB by HP whereas 30 were positive by PCR. Twenty-four of these were positive for both HP and PCR while of the seven samples positive for HP, five were negative and two gave inhibition by PCR. Six samples negative by HP were positive by PCR. CONCLUSION: This study demonstrates that PCR can be used as an effective tool to diagnose abdominal TB.     

263例住院肺结核患者痰涂片抗酸杆菌阳性检出分析

目的了解住院肺结核患者痰涂片抗酸杆菌（AFB）阳性检出特征,为肺结核诊断提供可靠依据。方法对263例住院肺结核患者痰标本直接厚涂片AFB检查,并对涂阳患者进行I豳床分析。结果发现涂片阳性肺结核患者95例,阳性率36．1％。其中AFB（±）7例（7．4％）、AFB（1＋）36例（37．9％）、AFB（2＋）20例（21．0％）、AFB（3＋）13例（13．7％）、AFB（4＋）19例（20．0％）。三个痰标本首次涂片阳性率为65．3％（62／95）,清晨痰标本阳性检出率为61．8％（84／136）,涂阳患者三种不同痰标本和不同送检次数在AFB阳性检出上均有统计学意义（P〈0．01）。与涂阴肺结核比较,涂阳肺结核患者咳嗽、咯血、发热、空洞形成和病灶≥2个肺野等比例分别高达97．9％、41．1％、54．7％、48．4％和82．1％,存在显著性差异（P〈0．05）。结论留取合格痰标本,认真涂片检查,提高痰标本阳性检出率,痰涂片检查符合我国国情,是实施结核病控制项目DOTS策略的关键之一。     

实时荧光定量聚合酶链反应检测支气管肺泡灌洗液中结核分枝杆菌-DNA对肺结核的诊断价值

目的探讨实时荧光定量聚合酶链反应法（FQ-PCR）检测支气管肺泡灌洗液（BALF）中结核分枝杆菌-DNA（TB-DNA）对肺结核的诊断价值。方法肺结核52例（菌阳20例,菌阴32例）,肺炎35例,采用FQ—PCR法检测其支气管肺泡灌洗液中TB—DNA水平。结果52例肺结核组的阳性率为65．4％（菌阳19例,菌阴15例）,35例非肺结核组的阳性率为5．7％,经统计学比较,FQ-PCR法检出结核杆菌阳性率显著高于痰涂片抗酸染色和培养法（P均〈0．05）,FQ-PCR法特异性高。结论FQ-PCR法检测BALF中TB-DNA为痰涂片阴性及无痰患者提供良好的诊断依据。     

聚合酶链反应对菌阳肺结核治疗的监测

目的探讨痰菌阳性肺结核患者在治疗期及停药２年内痰结核分支杆菌及其ＤＮＡ阴转情况与复发的关系以及聚合酶链反应（ＰＣＲ）对菌阳肺结核患者治疗的监测价值。方法用ＰＣＲ技术、涂片及培养法对８７例菌阳肺结核于治疗期每月检测１次，停药期继续随访２年。结果痰结核分支杆菌ＰＣＲ转阴时间通常比涂片和培养迟１～３个月，痰含菌量越多，ＰＣＲ持续阳性时间越长。８７例中１０例（１１％）ＰＣＲ持续阳性１年以上，其中３例（３０％）分别于停药后８、１２、１６个月时复发，１例ＰＣＲ已转阴病例于１８个月时复发。此４例均有痰菌复阳，胸片示病灶增多而再次接受治疗。结论ＰＣＲ用于临床疗效观察比涂片、培养实用，对估计有可能复发的病例有一定帮助。     

抗酸染色对肺结核的诊断价值

目的 了解结核分枝杆菌抗酸染色对肺结核的诊断价值。方法对98例确诊为继发型肺结核和血行播散型肺结核患者,统计分析痰液及纤支镜刷检液涂片抗酸染色情况。结果98例中抗酸染色阳性38例,阴性60例。10例经纤支镜刷检液涂片抗酸染色中,阳性6例。结论抗酸染色是诊断肺结核的常规方法,纤支镜刷检液涂片明显高于痰涂片法。     

涂阴肺结核的诊断与治疗

目的总结涂阴肺结核临床诊断与治疗经验。方法分析2008年-2009年我所治疗的887例涂阴肺结核患者的临床资料。结果临床症状、胸部X线检查（包括胸部CT扫描）、PPD试验、抗结核治疗有效、血结核抗体阳性是诊断本病的主要手段,直接面视下DOTS是确保涂阴肺结核治愈的重要方法。结论涂阴肺结核的诊断有一定的困难,以不住院化疗为主,医护人员直接面视下DOTS。     

涂阴老年不典型肺结核的多种CT影像表现

目的探讨涂阴老年不典型肺结核的多种CT表现。方法收集我院住院的经临床治疗、痰检及纤支镜证实的涂阴老年不典型肺结核30例,就其CT表现回顾性分析。结果 1大片状肺实变影3例(占10%);2单发球形或肿块2例(占6.7%):1例结核球;1例肿块;3多发结节及肿块:2例(占6.7%);4间质改变11例(占36.7%);5空洞征3例:薄壁空洞1例(占3.3%),厚壁空洞2例(占6.7%);6树芽征6例(占20%);7磨玻璃影3例(占10%)。结论胸部CT扫描可识别涂阴老年不典型肺结核的多种征象,判断病变的性质和程度等方面具有较高的敏感度,有助于评价和监测不典型继发性肺结核的诊断、征象的转归等演变过程,为临床提供不可或缺的支持。     

实时荧光核酸恒温扩增技术在肺结核诊断中的价值分析

目的评价实时荧光核酸恒温扩增技术(SAT-TB)在肺结核诊断中的价值。方法纳入1121例非相同患者痰标本,其中26例为非结核分枝杆菌。570例肺结核为观察组,525例其他肺部疾病患者为对照组。对治疗前的痰标本分别进行抗酸杆菌涂片、培养及鉴定和SAT法检测。阳性检出率的比较使用卡方检验。结果临床诊断肺结核570例,痰培养结核菌阳性率46.5%(265/570),SAT法检测阳性率46.7%(266/570),痰涂片阳性率34.4%(196/570),SAT与涂片法阳性率的差异有统计学意义(χ2=17.83,P0.01)。在涂阴肺结核中,SAT阳性率达19.5%。以培养阳性作为金标准,SAT法诊断结核病的灵敏度为89.4%,特异度96.3%,阳性似然比为24.2,阴性似然比为0.11,一致率是94.6%,阳性预测值是88.4%,阴性预测值为96.6%。在非结核分枝杆菌肺病中SAT检测阳性率为零。结论 SAT-TB法在肺结核的早期诊断中是一种快速、准确、敏感的方法,值得推广。     

初治涂阳肺结核诊治延迟230例分析

目的了解初治涂阳肺结核患者诊治延迟的时间及原因。方法对我院2003年至2005年接诊的初治涂阳肺结核病人诊治延迟时间和原因进行分析。结果230例初治涂阳肺结核就医、确诊、发现诊断延迟的中位时间分别为60．6d、31．5d和96d。就医延迟主要原因是病人出现症状不去就医或自行服药。确诊延迟与病人临床表现不典型、医生对结核病认识不足有关。结论涂阳肺结核病人诊治延迟的现象仍然比较严重,应大力宣传和普及防痨知识,加强公共卫生机构筛查和检出肺结核患者的力度。     

The rapid diagnosis of smear-negative pulmonary tuberculosis: a cost-effectiveness analysis

OBJECTIVE: The prompt diagnosis of smear-negative pulmonary tuberculosis (PTB) is a clinical challenge. It may be achieved by a number of tests which have varying accuracies, costs and degrees of invasiveness. The objective of this study was to compare the cost-effectiveness of clinical judgement (empirical), the Roche Cobas amplicor assay for Mycobacterium tuberculosis (amplicor), acid-fast staining of bronchoalveolar lavage specimens (BAL), nucleic acid amplification tests of bronchoalveloar lavage specimens for M. tuberculosis (BAL + NAA), computed tomography (CT) and amplicor assay followed by BAL. METHODOLOGY: The range of predictive values of the various strategies were derived from published data and a new study of 441 consecutive adult patients with suspected smear-negative PTB prospectively stratified into three pretest risk groups: low, intermediate and high. The cost-effectiveness was evaluated with a decision tree model (DATA software). RESULTS: The incidence of PTB was 5.7% (4% culture positive) for the whole group, 95% in the high-risk group, 0.9% in the low-risk group and 3.4% in the intermediate-risk group. The sensitivity of the empirical approach was 49% and of the amplicor assay was 44%. Patient outcomes were expressed as life expectancy for the base case of a 58-year-old man with a pretest probability of 5.7%. At this low pretest risk the differences in life expectancies between tests was < 0.1 years and the empirical approach incurred the lowest cost. Sensitivity analysis at increasing pretest risks showed better life expectancies (approximately 1 years) for CT scan and test combinations than empirical and amplicor for additional costs of US$243-US$309. Bronchoalveolar lavage had the worst overall cost-effectiveness. CONCLUSIONS: We conclude that the pretest risk of active PTB was a key determinant of test utility; that the AMPLICOR assay was comparable to clinical judgement; that BAL was the least useful test; and that with increasing risks, CT scan and test combinations performed better. Further studies are needed to better define patients with intermediate risk for PTB and to directly compare the cost-effectiveness of more sensitive nucleic acid amplification tests such as the enhanced Gen Probe, CT scan and test combinations/sequences in these patients.     

Diagnosis and genotyping of Helicobacter pylori by polymerase chain reaction of bacterial DNA from gastric juice

BACKGROUND: Efficient and accurate detection of Helicobacter pylori infection as well as identification of virulence-associated alleles are important for the treatment of gastroduodenal diseases caused by this gastric pathogen. The present study was performed to test the efficiency of gastric juice polymerase chain reaction (PCR) method for the rapid detection of H. pylori infection and to determine the bacterial genotypes without the need for culture, which is often not feasible especially in developing countries. METHODS: DNA was extracted from gastric juice samples collected from 45 subjects and was used to amplify urease B gene (ureB) for H. pylori. Results obtained from this method were further confirmed by rapid urease test (RUT), histology and culture. Genotypes of the infected strains predicted from gastric juice PCR were compared to the genotype data obtained from the isolated strains. RESULTS: Among 45 cases, 32 were positive by RUT, 37 by histological examination, 25 by gastric juice PCR method, while culture yielded positive results for 19 samples. Except for one case, all the 19 culture-positive strains gave the same genotype with the gastric juice PCR result. It was found that the gastric juice PCR is more efficient for detection of multiple-strain infection as compared to genotype data obtained from strains isolated as pooled culture. CONCLUSIONS: This moderately sensitive technique could be employed with good efficiency, particularly in cases where it is difficult to obtain biopsy. Moreover, with this method bacterial genotype could be obtained.     

含左氧氟沙星化疗方案治疗初治涂阳肺结核疗效研究

目的观察左氧氟沙星化疗方案治疗初治涂阳肺结核的短期和长期疗效。方法将60例初治涂阳肺结核患者随机分实验组和观察组,实验组在观察组的基础上于前两个月加用左氧氟沙星。结果实验组在前两个月强化期内痰菌阴转率为86.7%,明显高于观察组的63.3%。病灶吸收率为86.7%,亦明显优于60.0%。但在巩固期随着治疗的延长,差异愈小,在疗程结束时并无显著性差异。结论含左氧氟沙星化疗方案治疗初治涂阳肺结核只能缩短痰菌阴转和短期内病灶吸收时间,但对整个疗程而言并无确切意义。对初治涂阳肺结核的治疗不提倡加用左氧氟沙星。     

荧光定量PCR技术在肺结核诊断中的临床应用研究

目的评价荧光定量PCR技术在肺结核病诊断中的应用价值。方法采用荧光定量PCR、金胺O染色荧光镜检和培养法同时检测860例肺结核患者的痰标本,对检测结果进行比较。结果荧光定量PCR检测灵敏度可达10~100 cfu/ml,重复性好,对其他呼吸道病原体检测结果均为阴性。肺结核患者痰标本荧光定量PCR检测阳性率要高于金胺O染色荧光镜检和培养法的阳性率,差异有统计学意义(P0.01)。结论应用荧光定量PCR方法检测结核杆菌,具有特异、灵敏、简便、快速的特点,可作为结核病诊断的方法之一。     

实时多聚酶链反应检测支气管分泌物在涂阴结核病快速诊断中的应用

背景:实时定量PCR技术在微生物包括分枝杆菌检测领域应用越来越广泛。目的:评价在痰标本涂片抗酸染色无法获得诊断信息时(如没有痰或痰涂片阴性),RT-PCR在结核病诊断中的作用。设计:我们分析了从2006年1月至2008年4月之间连续纳入的为诊断结核病而进行的支气管分泌物RT-PCR检测的病人。所有病人均接收了计算机断层扫描(CT)、支气管镜和组织活检确诊结核病,对收集的支气管分泌物进行了微生物学和RT-PCR检测。最终的确诊基于微生物学或临床病理学的标准。结果:有136例患者最终得到了诊断结果,其中77例(包括65例培养阳性病例)诊断为结核。RT-PCR在确诊病人中的阳性率是51.9%(40/77)。与支气管分泌物抗酸染色相比,更多的病人(20.8%)通过RT-PCR被检测出来(40比20,P<0.001)。结合支气管分泌物抗酸染色和RT-PCR,77例结核确诊病例中的44例(57.1%)在几天之内被确诊。结论:支气管分泌物的RT-PCR检测在涂阴或无痰结核病人的快速诊断中是一种有用的方法。