Clinical evaluation of Amplicor Mycobacterium detection system for the diagnosis of pulmonary mycobacterial infection using sputum.

SETTING: The Amplicor Mycobacterium detection kit was evaluated for the diagnosis of active pulmonary mycobacterial infection using sputum. OBJECTIVE: To assess the clinical usefulness of the Amplicor Mycobacterium kit for the diagnosis of pulmonary tuberculosis and non-tuberculous mycobacterial infection in the country of medium prevalence. DESIGN: All the patients were diagnosed with bacterial, histopathological, and clinical 'gold standard'. The sensitivity and specificity for diagnosing clinically active pulmonary tuberculosis and Mycobacterium avium and Mycobacterium intracellulare infections were evaluated comparing Amplicor results and clinical diagnosis. RESULTS: A total of 1088 sputum specimens were collected from 780 in and out patients. Mycobacteria were recovered from 339 specimens by culture. The sensitivity and specificity of conventional culture method for the diagnosis of pulmonary tuberculosis were 60.2% and 99.8% respectively based on the number of patients. The figures for Amplicor were 61.8% and 97.4% respectively. There was no statistical significant difference between these methods. In rapidity, the Amplicor was significantly superior to the microscopy method in sensitivity. CONCLUSION: Patients with Amplicor positive and conventional negative result had mostly mycobacteria related diseases. The Amplicor positive result indicated mostly active mycobacterial infection and was clinically useful for rapid diagnosis.     

Comparison of amplicor, in-house polymerase chain reaction, and conventional culture for the diagnosis of tuberculosis in children.

A total of 251 clinical specimens (235 gastric aspirates and 16 bronchoalveolar lavages) from 88 children were prospectively tested in a blinded manner for the presence of Mycobacterium tuberculosis complex, by use of the Amplicor M. tuberculosis test and by means of in-house polymerase chain reaction (PCR). The results were compared with those obtained by conventional culture and by direct microscopy. All of the children underwent extended follow-up to verify or exclude the clinical diagnosis of tuberculosis. The results of the different tests, when compared to the final clinical diagnosis, were a sensitivity of 60% and a specificity of 96.8% for in-house PCR, 44% and 93.7% respectively for the Amplicor test, 44% and 100% for mycobacterial culture and 12% and 100% for microscopy. Amplicor tests presented false-positive findings in children without tuberculous infection. We conclude that both in-house PCR and the Amplicor test are rapid methods that can be helpful for difficult or urgent diagnosis of tuberculosis in children. However, efforts should be aimed toward improvement of the sensitivity and specificity of an easy-to-use PCR kit.     

TaqMan聚合酶链反应技术检测结核分支杆菌DNA及其临？…

目的 探讨ＴａｑＭａｎ聚合酶链反应（ＴａｑＭａｎ－ＰＣＲ）技术在肺结构诊断中的价值。方法 对１６８例活动性肺结核、５７例肺癌患者的痰和外周血及３４－例健康对照外周血,同时应用ＴａｑＭａｎ－ＰＣＲ、ＰＣＲ检测,并与痰涂片法、ＢＡＣＴＥＣ法及改良罗氏培养法结果进行比较。结果 ＴａｑＭａｎ－ＰＣＲ检测痰和外周血总的阳性率分别为５３．０％和６１．３％,显著高于ＰＣＲ、痰涂片法、ＢＡＴＣＴＥＣ法及改良罗氏培     

The Cobas Amplicor MTB test for detection of Mycobacterium tuberculosis complex from respiratory and non-respiratory clinical specimens

The Cobas Amplicor MTB test is a polymerase chain reaction (PCR) technique commonly used for direct detection of Mycobacterium tuberculosis in clinical samples. This assay is only validated for respiratory specimens, but many physicians also request PCR analyses for non-respiratory ones. 877 respiratory and 564 non-respiratory specimens were analysed by this test. Using culture results as standard, the sensitivity, specificity, positive (PPV) and negative predictive values (NPV) of the PCR were, respectively, 97.9%, 100%, 100% and 94.4% for smear-positive respiratory specimens, 68.8%, 99.2%, 87.5% and 97.5% for smear-negative respiratory samples, 57.8%, 98.6%, 78.8% and 96.4% for all non-respiratory specimens, and 42.4%, 98.6%, 66.7% and 96.4% for smear-negative non-respiratory specimens. 154 cerebrospinal fluid samples were analysed and the sensitivity, specificity, PPV and NPV were 55.6%, 97.2%, 55.6% and 97.2%, respectively. These results indicate that the Cobas Amplicor MTB test enables detection of tuberculosis in respiratory specimens, but does not perform well enough in non-respiratory specimens. The method fails particularly in cases where a reliable and rapid test is urgently needed, e.g. in tuberculous meningitis.     

聚合酶链反应法诊断结核病临床价值探讨

目的 探讨ＰＣＲ法对结核病的诊断价值。方法 用ＰＣＲ法检测痰、纤支镜抽吸物、胸水和血液标本中结核杆菌ＤＮＡ。结果 ＰＣＲ法敏感性为２１．９％，特异性为９２．６％，总的阳性预测值、阴性预测值和诊断效率分别为６４．６％，６５．９％和６５．８％，不同标本敏感性有较大的差异。结论 ＰＣＲ法诊断结核病的敏感性、特异性尚远不能完全满足临床的需要，需进一步完善和改进 。     

噬菌体裂解法与BACTEC-460法在结核分支杆菌快速检测中的比较

目的：比较噬菌体裂解法与BACTEC－460法在结核分支杆菌快速检测及鉴定中的价值。方法：应用噬菌体裂解法和BACTEC－460法分别检测30株结构分支杆菌临床分离株、10株非结核分支杆菌和7株非分支杆菌，以及60份临床确诊的肺结核患者的痰标本，结果：所有结核分支杆菌临床分离株噬菌体裂解试验均为阳性，而非结核分支杆菌和非分支杆菌均为阴性。41份BACTEC－460培养阳性和19份BACTEC－460培养阴性的痰标本中，噬菌体裂试验分别有34份（82．9％）和5份（26．3％）阳性。结论：噬菌体裂解法可以简便，快速地检测结核分支杆菌，并且具有较高的敏感性的特异性。     

Comparative evaluation of the isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN) and Roche Amplicor PCR and culture for detecting Mycobacterium tuberculosis complex in sputum samples

We compared the ability of the newly developed ICAN MTB Detection Kit (TaKaRa Bio Inc.), which uses the Isothermal and Chimeric primer-initiated Amplification of Nucleic acid (ICAN), with that of COBAS Amplicor PCR System (Roche Diagnostics) to directly detect Mycobacterium tuberculosis complex (MTB) in sputum samples. A total of 142 sputum samples from 120 patients were examined in this study. The results were compared with those of acid-fast staining and MGIT liquid culture system (BD) following identification by the probe test (DDH Mycobacteria Kit). A total of 68 specimens were MGIT positive for MTB. In addition, 62 specimens were positive by the combination of staining and MGIT assay for MTB. When compared with that for MGIT, the sensitivity of each assay system was 88.2% for ICAN and 92.6% for COBAS Amplicor, respectively. The specificity of each assay system was 65.7% for ICAN and 62.7% for COBAS Amplicor, respectively. Coincidence between ICAN and COBAS Amplicor assay results was 96.3% (130 of 135 samples). No significant difference was observed between the results of the two assay methods. It is concluded that although both nucleic acid amplification methods are sensitive and specific for the detection of MTB in the respiratory specimens, ICAN system appeared to be more rapid (within 3.5 h from the specimen collection) than Amplicor system. The ICAN system will be useful in clinical laboratories for the rapid detection of MTB without specially programmed thermo-cycler.     

结核抗体lgG/lgM检测在肺结核和肺外结核中的诊断价值

目的 探讨异种血清抗体检测技术在结核病诊断中的应用价值。方法 采用IgG/IgM抗体试剂盒分别检测102例结核病患者(包括73例肺结核和29例肺外结核)、223例其他肺部疾病患者和100例对照者结核感染情况,以临床诊断为标准评价该方法的敏感度和特异性,同时分别与痰菌培养及痰涂片平行检验的结果作比较,统计学分析采用χ²检验。结果 结核抗体IgG/IgM检测结核病患者的敏感度为74.51%、特异度为91.64%。结核抗体IgG/IgM检测肺结核和肺外结核的敏感度分别为82.19%、55.17%,肺内和肺外结核的敏感度差异有统计学意义(P＜0.05),结核抗体lgG/IgM检测结核患者阳性检出率明显高于痰培养法和痰涂片法(P<0.05)。102例结核病患者年龄段分组分析,少年组和老年组检出率分别为58.33%、36%,远低于青年组和中年组的96.15%和89.74%。不同年龄组间进行卡方比较分析显示,P＜0.05,差异有统计学意义。425例标本中,共发现8例非结核分枝杆菌,其中6例胞内分枝杆菌, 2例脓肿分枝杆菌,lgG/lgM抗体检测均为阴性。结论 IgG/IgM血清抗体检测肺内、外结核具有快速方便、经济和较高的敏感度,适合用于临床结核筛查。     

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。     

rRNA扩增方法对结核分枝杆菌临床诊断作用的评估

目的 评价rRNA扩增方法 在临床应用的效果。 方法 选取到北京胸科医院、山东省胸科医院、河南疾控中心结核病防治所3个机构就诊的肺结核可疑症状者及健康志愿者的痰标本为研究对象,共纳入551例痰标本,对每例痰标本均进行涂片显微镜检查、罗氏培养、rRNA扩增试验以及实时荧光定量PCR。与罗氏培养方法 比较分析rRNA扩增方法 的敏感性、特异性、阳性预测值和阴性预测值以及与实时荧光定量PCR扩增方法 的一致性。 结果rRNA扩增方法 的敏感性为98.5%,特异性为95.0%,阳性预测值为95.0%,阴性预测值为98.5%,rRNA扩增方法 在涂阳标本和涂阴标本间的敏感度和特异度差异均有统计学意义（χ²=9.60,P=0.002;χ²=79.80,P<0.01）。与PCR检测结果的一致性为93.8%,涂阳和涂阴标本中2种方法 的一致性差异有统计学意义（χ²=4.45,P=0.035）。 结论 rRNA扩增方法 的敏感度和特异度均较好,且能明显缩短诊断时间,该方法 是一种较有前景的结核病实验室诊断方法 。     

Detection of Mycobacterium tuberculosis from sputum specimens using one-tube nested PCR

One-tube nested PCR was developed for diagnosis of pulmonary tuberculosis using sequences based on thel6SrRNA gene. The usage of primers 16SOL, 16SOR, 16SIL and 16SIR with optimized conditions could detect 555 bp DNA band from 21 species, 41 strains of mycobacteria and one isolate of Nocardia asteroides. It also revealed a specific 306 bp DNA band from 59 strains of M. tuberculosis complex. Cross amplification was observed in M. marinum, M. ulcerans and a few isolates of M. fortuitum complex. The developed method could detect as little as 100 fg of M. tuberculosis DNA. The PCR mixtures could be stored at 0 degrees C for 2 months or at -20 degrees C for at least 20 months without decrease in sensitivity. Using one-tube nested PCR for detection of M. tuberculosis compared with acid fast staining and culture results from 153 sputum specimens revealed 88.6% sensitivity and 89.2% specificity in smear positive specimens and 93.2% sensitivity and 85.0% specificity in culture positive specimens.     

实时荧光PCR检测儿童肺结核粪便中Mtb-DNA的效果评价

目的 应用实时荧光PCR快速检测肺结核患儿粪便中Mtb-DNA,并初步评估其临床效果。 方法 收集肺结核住院儿童粪便标本76份,应用实时荧光PCR检测Mtb-DNA,检测结果与20例其他呼吸系统疾病患儿的粪便实时荧光PCR检测Mtb-DNA、76例粪便抗酸杆菌涂片检查以及41例患儿痰标本的涂片和(或)培养、实时荧光PCR检测结果进行比较。 结果 粪便实时荧光PCR检测敏感度达到23.68%(18/76),特异度为100.00％(20/20),肺结核患儿粪便PCR阳性率［23.68%(18/76)］明显高于涂片阳性率［6.58%(5/76)］,41例患儿中痰涂片和(或)培养阳性例数(15例)和痰PCR检测阳性例数(18例)高于粪便PCR检测阳性例数(11例)。 结论 实时荧光PCR检测儿童粪便Mtb-DNA,是一种特异度高、比较敏感、非侵入性且相对安全的儿童肺结核快速诊断方法。     

Evaluation of a polymerase chain reaction for the diagnosis of tuberculosis

A polymerase chain reaction for the specific detection of Mycobacterium tuberculosis has been developed and evaluated for clinical applicability. Primers were designed to amplify a 240 base pair region in the MPB 64 protein coding gene (nts 460-700). From among 15 different DNA templates tested (including 10 species of mycobacteria) PCR amplified the DNA from M. tuberculosis complex only, demonstrating its exquisite specificity. Sensitivity studies using serial ten-fold dilutions of M. tuberculosis bacilli determined the limit of detectability to be 10 organisms. A total of 143 clinical specimens were analysed. This consisted of 26 known non-tuberculous specimens (control group) and 117 specimens received at the Tuberculosis Diagnostic Service of AIIMS (test group). None of the specimens in the control group was positive by PCR. Out of 117 specimens in the test group, 19 were culture positive for mycobacteria and 17 of these isolates were identified as M. tuberculosis. All the specimens from which M. tuberculosis was grown were also PCR positive. The remaining two isolates were identified as mycobacteria other than M. tuberculosis and these two specimens were PCR negative. An additional 14 culture negative specimens were PCR positive yielding an overall M. tuberculosis positivity rate of 26.5% (31/117) compared to 14.5% (17/117) by culture. The superior sensitivity of PCR over culture was more evident in non-pulmonary cases where PCR picked up 10 cases in addition to three culture positives out of 69 specimens. On the other hand, out of 48 pulmonary specimens only four cases in addition to 14 culture positives were picked up by PCR.     

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。     

Evaluation of the Idaho Technology LightCycler PCR for the direct detection of Mycobacterium tuberculosis in respiratory specimens.

SETTING: The rapid detection of Mycobacterium tuberculosis (TB) in clinical samples is an important goal. The LightCycler heralds an advance in thermal cycle technology combining rapid cycle DNA amplification with fluorimetry, eliminating the need to perform amplification and product analysis separately. OBJECTIVES: To evaluate the LightCycler for direct detection of M. tuberculosis complex in respiratory specimens. To evaluate a DNA extraction method based on Chelex 100 resin, heating and ultrasonication for the prevention of endogenous inhibitions in respiratory samples. DESIGN: DNA was extracted from sputum samples using the Chelex method and polymerase chain reaction (PCR) for TB performed with the LightCycler. RESULTS: For 88 sputum samples positive by microscopy and culture for M. tuberculosis, 95% were PCR-positive. None of the five sputum samples that were smear-negative but culture-positive for M. tuberculosis, the 79 culture-negative sputum samples and the 29 sputum samples that were culture-positive for mycobacteria other than TB yielded positive PCR results. PCR inhibitors were not detected in any of the samples. CONCLUSION: The LightCycler proved a simple, reproducible and rapid system, reducing the time to result from weeks (culture) or days (conventional PCR) to hours. The Chelex 100 resin method produced good results for the smear-positive specimens. However, a larger study is required to determine the efficacy of the method with smear-negative specimens and for specimens known to contain endogenous inhibitors.     

The validity of acid-fast smears of gastric aspirates as an indicator of pulmonary tuberculosis.

SETTING: A tuberculosis referral hospital in Canada. OBJECTIVE: To determine the validity of acid-fast (AFB) smears of gastric aspirates (GA) in the diagnosis of pulmonary tuberculosis, and to assess the prevalence of nontuberculous mycobacteria (NTM) in GA isolates from such patients. DESIGN: A retrospective case review of our experience with AFB smears (Kinyoun) and cultures of GA and sputum over a 3-year period. RESULTS: From 1994 to 1996 inclusive, 1155 GA were performed in 889 patients. Mycobacteria were cultured from 109 (9%) GA. Thirteen of these were positive on smear (sensitivity 19%). All GA that were positive on smear were culture positive for Mycobacterium tuberculosis. There were no false positive smears (specificity 100%). The sensitivity and specificity of the sputum smear were 45% and 99%, respectively. Of the 96 culture positive, smear negative GA, 54 grew M. tuberculosis and 42 grew an NTM. Of 13 patients who had sputum and GA studied coincidentally, and in whom the sputum was both smear and culture positive, the GA culture was positive in 13 and the smear was positive in eight (66%). CONCLUSION: AFB smear of GA is a relatively insensitive but highly specific indicator of pulmonary tuberculosis warranting institution of antituberculosis treatment. Gastric AFB smear positivity appears to reflect a high bacillary burden within the respiratory tract.     

Evaluations of MTD and Amplicor Mycobacterium for direct detection of Mycobacteria from clinical specimens]

MTD (GEN-PROBE AMPLIFIED MYCOBACTERIUM TUBERCULOSIS DIRECT TEST) for Mycobacterium tuberculosis, and Amplicor Mycobacterium for Mycobacteria (AMP-M. tb for M. tuberculosis, AMP-M. av for M. avium and AMP-M. in for M. intracellulare) were used for the detection of relevant Mycobacterium. Their sensitivity and specificity were evaluated. Total 244 clinical specimens including 164 sputa were examined by the above two tests. The results were compared with those obtained by the conventional methods. Of 244 samples, number of the M. tuberculosis positive samples by microscopy, cultural test, MTD and AMP-M. tb were 32, 33, 38 and 35, respectively. Among 33 culture positive samples, 25 were MTD positive and 26 were AMP-M. tb positive. Therefore, sensitivity of MTD and AMP-M. tb were 75.8% and 78.8%, and their specificity were 93.8% and 95.7%, respectively. When only sputa were used for the tests as the clinical specimens, both sensitivity of MTD and AMP-M. tb were increased to 94.4%. For MAC, positive samples of M. avium complex by culture, M. avium by AMP-M. av and M. intracellulare by AMP-M. in were 13, 16, and 8, respectively. Sensitivity and specificity of AMP-M. av/M. in were 100% and 95.2%, respectively. Clinical findings of the patients whose MTD tests were positive but negative by culture were reexamined. Three of 9 specimens were also positive in AMP-M. tb. From the records of the isolations of tubercle bacilli or other important pathogens from the other kind of clinical specimens, smear tests and patients' response to tuberculosis chemotherapy, four of 9 specimens were confirmed as true positive, three were suspected as positive, and two other specimens were false positive which might be caused by contamination. From these observations, it could be concluded that MTD and AMP-M. tb are more sensitive than conventional culture method, and MTD is more sensitive than AMP-M. tb but needs more careful treatment to avoid the contamination.     

Laboratory diagnosis of pulmonary tuberculosis in TB and HIV endemic settings and the contribution of real time PCR for M. tuberculosis in bronchoalveolar lavage fluid

Background Tuberculosis (TB) in Africa is increasing because of the human immunodeficiency virus (HIV) epidemic, and in HIV/AIDS patients it presents atypically. Pulmonary tuberculosis (PTB) in Africa is mainly diagnosed clinically, by chest radiograph or by sputum smear for acid fast bacilli (AFB). Methods We evaluated in 120 HIV‐infected patients with chest infection the diagnostic accuracy of AFB smear of sputum and bronchoalveolar lavage (BAL) fluid, sputum Mycobacterium tuberculosis (MTB) culture, real‐time PCR and MycoDot^® serological test, using MTB culture of BAL fluid as gold standard. We correlated PCR cycle threshold values (C_T) to the culture results. Retrospectively, we evaluated the development of active TB in patients with positive PCR but negative culture. Results Culture of BAL fluid identified 28 patients with PTB. Fifty‐six patients could not produce adequate sputum. Sputum AFB smear and the serological test had sensitivities of 66.7% and 0%, respectively. PCR with C_T 40 was positive in 73 patients, 27 of whom were also TB culture positive (96.4% sensitivity and 52.3% specificity of PCR). PCR with C_T 32 had sensitivity of 85.7% and specificity of 90.9% to diagnose PTB in BAL. No patients with positive PCR but negative culture developed active TB during 18 months follow‐up. Conclusion In these HIV‐infected patients, AFB smear and serology had very low sensitivities. PCR of BAL with C_T value 32 had improved specificity to diagnose active PTB. A prospective follow‐up study is warranted in TB/HIV endemic settings, applying real time PCR to both sputum and BAL.     

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。     

Detection of Mycobacterium tuberculosis by a polymerase chain reaction colorimetric dot-blot assay.

SETTING: A public health laboratory in a tuberculosis-endemic region in Brazil. OBJECTIVE: To evaluate the accuracy of a combined polymerase chain reaction (PCR) colorimetric dot-blot protocol for Mycobacterium tuberculosis detection in clinical samples in a public health laboratory. DESIGN: Eighty clinical samples (13 cerebrospinal fluid, 31 induced sputum, 17 expectorated sputum, eight bronchoalveolar lavage and 11 pleural fluid) were assayed with the developed protocol. The accuracy of polymerase chain reaction (PCR) dot-blot methodology was compared to PCR agarose gel electrophoresis (PCR-AG) using as a gold standard the bacteriological result (culture and biochemical identification) combined with clinical follow-up. One internal region of the IS6110 repetitive element of the M. tuberculosis complex was selected for amplification and the amplified product transferred to nylon membranes to be detected by biotinylated DNA probe. RESULTS: Overall sensitivity and specificity obtained were respectively 90% and 97% for PCR-AG and 95% and 97% for the PCR dot-blot. Among the 56 respiratory specimens, the sensitivity and specificity results for PCR-AG were respectively 88% and 95%, and for PCR dot-blot they were 94% and 95%. Among the 24 non-respiratory specimens the sensitivity and specificity results were respectively 83% and 100% for PCR-AG, and 100% and 100% for the PCR dot-blot protocol. CONCLUSION: The results demonstrated that the PCR dot-blot assay may be helpful in the diagnosis of tuberculosis, and feasible even in resource-poor countries.