The validity of HIV testing using sputum from suspected tuberculosis patients, Botswana, 2001.

SETTING: The World Health Organization recommends that sentinel HIV surveillance be conducted on tuberculosis patients. However, serum HIV testing is complicated in the TB clinic context, and may not be acceptable to patients. DESIGN: To determine the utility of the OraQuick HIV-1/2 Assay for the detection of HIV antibodies in sputum, we consecutively enrolled adult in-patients in Botswana who had sputum sent for acid-fast bacilli testing and serum sent for HIV ELISA testing. OraQuick HIV-1/2 Assay was applied to gingival secretions according to manufacturer's guidelines, and was also dipped into sputum specimens. A subset of 60 sputum specimens was also serially tested up to 72 hours after collection. RESULTS: Of 377 patients, 84% were HIV-positive by serum ELISA. Compared with serum ELISA, the OraQuick HIV-1/2 Assay detected HIV in gingival secretions with 98.4% sensitivity and 98.3% specificity (95%CI 97-99 and 92-100, respectively), and 97.1% sensitivity and 98.3% specificity on initial sputum specimens (95%CI 95-99 and 92-100, respectively). OraQuick HIV-1/2 Assay performance on sputum declined slightly when tested up to 72 hours after collection. CONCLUSIONS: When applied to sputum specimens, the OraQuick HIV-1/2 Assay demonstrates sensitivity and specificity comparable to its intended application on gingival secretions. This novel testing method will be valuable in anonymous sentinel HIV surveillance surveys among tuberculosis patients.     

Evaluation of polymerase chain reaction, tuberculostearic acid analysis, and direct microscopy for the detection of Mycobacterium tuberculosis in sputum.

Tuberculosis remains a major global cause of morbidity and mortality. There is an urgent need for improved bacteriologic diagnosis of Mycobacterium tuberculosis infection. Three methods for rapid identification of M. tuberculosis in sputum samples (direct microscopy, gas chromatography-mass spectrometry [GC-MS], and polymerase chain reaction [PCR]), were compared with culture on Lowenstein-Jensen medium. Growth of M. tuberculosis was observed in 38 of 145 sputum samples. Detection of acid-fast bacilli by direct microscopy gave a sensitivity of 66% and a specificity of 100%. Detection of tuberculostearic acid by GC-MS gave a sensitivity of 55% and a specificity of 87%. Amplification by PCR of a fragment of the insertion sequence IS6110 gave a sensitivity of 95% and a specificity of 93% compared with culture and a corrected specificity of 99% compared with both culture and clinical data. This study indicates that PCR can be adapted for clinical use and is the method of choice for rapid diagnosis of pulmonary tuberculosis.     

Method for efficient storage and transportation of sputum specimens for molecular testing of tuberculosis.

SETTING: The polymerase chain reaction (PCR) is a highly sensitive method for the detection of Mycobacterium tuberculosis and is available in most countries, though to a lesser extent in rural areas. OBJECTIVE: To amplify M. tuberculosis DNA sequences of sputum spotted on FTA cards and compare them with the results of microscopic examination among culture-positive samples. DESIGN: A total of 102 sputum specimens of TB patients in treatment were spotted on FTA cards and stored at room temperature until DNA analysis. We assessed the IS6110 region of M. tuberculosis. The efficacy of the PCR assay for the direct detection of M. tuberculosis was evaluated and compared with the results of cultures (Middlebrook 7H9 broth) and smears of fresh sputum specimens. RESULTS: We were able to detect 10 fg/microl of mycobacterial DNA even after 6 months in storage. The PCR sensitivity and specificity using the FTA card system were 82% and 96%, while microscopic examination showed 41% and 95%, respectively. CONCLUSION: The FTA card system for the storage of bacterial DNA from sputum samples should be considered for the molecular diagnosis of tuberculosis. Samples can easily be obtained from geographically isolated populations and shipped by mail for accurate molecular diagnosis.     

Efficacy of Amplicor PCR for the diagnosis of tuberculosis in respiratory specimens other than sputum.

A total of 832 respiratory specimens not including the sputum (402 bronchial lavages, 241 bronchial brushing specimens, 136 pumping lavages, 41 pleural effusions, and 12 others) from 462 patients were assayed using the Roche Amplicor Mycobacterium tuberculosis test for amplification and identification of M. tuberculosis, M. avium and M. intracellulare (Amplicor PCR). The results were compared with those obtained using conventional microscopy and cultivation methods. Each patient had little or no sputum and showed an abnormal chest X-ray shadowing of unknown cause. No patients had previously undergone antituberculous therapy. Of the specimens obtained, 24 were both PCR and culture positive, 786 were both PCR and culture negative, 11 were PCR positive and culture negative, and 11 were PCR negative and culture positive. Based on these results, the sensitivity and specificity of Amplicor PCR were determined to be 68.67% and 98.6%, respectively, when compared with culture of respiratory specimens not including the sputum. After correcting for discrepancies due to differences in patient clinical data, the sensitivity of Amplicor PCR was found to be 68.6%, and the specificity to be 99.9%; the corresponding values for culture were 66.7% and 100%, and those for smear were 9.8% and 100%. Thus, Amplicor PCR was shown to possess a similar sensitivity to culture and to be a highly specific technique for the diagnosis of tuberculosis in the respiratory system using non-sputum specimens within hours in patients showing little or no sputum and abnormal chest X-ray shadowing of an indeterminant cause.     

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     

巢式实时荧光定量聚合酶链反应检测结核分枝杆菌DNA的临床应用

目的探讨巢式实时荧光定量PCR（QNRT-PCR）检测结核分枝杆菌（MTB）DNA的临床价值。方法应用SYBR Green I建立检测结核分枝杆菌MTP64基因的QNRT—PCR方法,分别检测24份肺结核患者痰液,27份结核性胸膜炎患者胸水,以及对照组75份痰液和胸水的MTBDNA含量。结果以培养为金标准,QNRT—PCR敏感性、特异性、阳性预测值（PPV）、阴性预测值（NPV）分别为95．83％、61．54％、69．70％和94．12％,结核性胸膜炎的阳性率为70．37％。三种PCR方法阳性率比较,差异具有统计学意义（P〈0．05）,QNRT—PCR与实时荧光定量PCR拷贝数配对检验差异没有统计学意义（P〉0．05）,QNRT—PCR与实时荧光定量PCR的Cr值配对检验差异具有统计学意义（P〈0．01）。结论QNRT—PCR方法可检测痰液和胸水MTB DNA,对含微量MTB DNA样本的高敏感性在结核病的早期诊断中有重要参考价值。     

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.     

Comparative performance of PCR-based assay versus microscopy and culture for the direct detection of Mycobacterium tuberculosis in clinical respiratory specimens in Lebanon.

SETTING: American University of Beirut Medical Center, Lebanon. OBJECTIVE: To assess the performance of a polymerase chain reaction (PCR) using primers that flank 542 bp within IS6110 in Mycobacterium tuberculosis (TB) vs. microscopy and BACTEC culture, in the diagnosis of tuberculosis. DESIGN: A total of 82 clinical respiratory pulmonary specimens and 73 samples from BACTEC vials were tested by the three methods. RESULTS: Of 24 smear-positive culture-positive (SP-CP) and 11 smear-negative culture-positive (SN-CP) TB specimens, PCR detected 83% and 64%, respectively. Among 17 specimens yielding mycobacteria other than tuberculosis (MOTT), the PCR was positive in 33% SP-CP and 14% SN-CP specimens. Among the 73 BACTEC vials, PCR was positive in 36 of 38 (95%) yielding culture-positive TB, and in one of 20 (5%) yielding culture positive MOTT. None of the 30 smear-negative culture-negative (SN-CN) clinical specimens and 15 of the CN vials were positive by PCR. The overall sensitivity of PCR was 77% and 95% for TB detection in respiratory specimens and BACTEC vials, respectively, and the specificity was 94% in both. CONCLUSIONS: Because a substantial number of TB cases are missed, especially in SN-CP specimens, a PCR-based assay utilizing these primers cannot be used reliably, alone, in clinical laboratory diagnosis of mycobacterial respiratory infections.     

噬菌体生物扩增法、DNA序列分析及单链构象多态性分析检测结核分枝杆菌耐药性

目的　探讨和评价噬菌体生物扩增技术 (phageamplifiedbiologicallyassay,PhaB)用于检测结核分枝杆菌耐药性的应用价值。方法　本研究共选取了 91株利福平 (RFP)耐药株 (其中 82株为耐多药结核分枝杆菌 )、4 2株RFP敏感株、75株氧氟沙星 (OFLX)耐药株、4 0株OFLX敏感株 ,应用PhaB、DNA序列分析法和聚合酶链反应 单链构象多态性分析 (PCR SSCP) 3种检测方法 ,同时对同一菌株应用该 3种方法进行耐药性检测 ,以资分析与评价PhaB法的临床应用价值。结果　以绝对浓度法药敏试验结果为标准 ,检测RFP的药物敏感性 ,PhaB法的准确性为 93% ,敏感性为 92 % ,特异性为95 % ;DNA序列分析法的准确性为 93% ,敏感性为 90 % ,特异性为 10 0 % ;PCR SSCP法的准确性为90 % ,敏感性为 86 % ,特异性为 10 0 %。检测OFLX(OFLX)的药物敏感性 ,PhaB的准确性为 95 % ,敏感性为 95 % ,特异性为 95 % ;DNA序列分析法的准确性为 80 % ,敏感性为 71% ,特异性为 98% ;而PCR SSCP法的准确性为 75 % ,敏感性为 6 3% ,特异性为 98%。结论　PhaB法是一种操作简便、快速、敏感、与绝对浓度法符合率高的结核分枝杆菌耐药性检测方法 ,在 4 8～ 96h内可获得试验结果 ,具有良好的临床应用前景。     

Combined bronchoalveolar lavage and polymerase chain reaction in the diagnosis of pulmonary tuberculosis in smear-negative patients.

SETTING: The polymerase chain reaction (PCR) may be sensitive and specific for the diagnosis of tuberculosis, but most reports are of studies conducted in well-controlled laboratories. A study to evaluate the clinical value of bronchoalveolar lavage (BAL) combined with PCR was necessary. OBJECTIVE: One hundred and thirty one patients were recruited into the study from March 1994 to February 1997. DESIGN: Patients with a positive acid-fast stain on sputum smear were recruited into group A as positive controls, patients with lung cancer and a negative acid-fast stain on sputum smear were put into group B as negative controls, and patients who had clinical symptoms of pulmonary TB without sputum or with negative smear results were the investigating group. PCR was performed on the sputum samples from group A and B patients and on the BAL fluid from those in group C. RESULTS: The sensitivity of PCR was 96% in group A, and the specificity was 100% in group B. The sensitivity of PCR in the BAL fluid from the group C patients was 36% and the specificity was 96%; the positive predictive value was 94% and the negative predictive value was 45%. CONCLUSION: BAL plus PCR is useful in the rapid diagnosis of pulmonary TB in non-productive or smear-negative patients.     

Evaluation of culture and PCR-based assay for detection of Mycobacterium tuberculosis from sputum collected and stored on filter paper

We evaluated the use of culture and PCR-based assay for the direct detection of Mycobacterium tuberculosis (MTB) from sputum collected and stored on filter paper at room temperature for 5 days; the results were compared with those of staining and conventional culture of fresh sputum before storage (the 'gold standard'). Out of 231 sputum specimens examined, MTB was recovered from 124 samples by culture before storage. The culture positivity rate was significantly decreased to 70% after 5 days storage. For PCR assay, a fragment of 377 bp of the IS6110 sequence was amplified and detected using three methods: first PCR product combined with agarose gel electrophoresis (AGE); first PCR product with dot blot hybridization (DBH); nested PCR with AGE. Compared with culture, the sensitivity, specificity, and efficiency for first PCR with AGE were 71.8, 100 and 84.9% respectively; PCR with DBH gave results of 89.5, 96.3 and 92.6% respectively; the same values for nested PCR were 96.0, 97.2, and 96.5% respectively. Of these three methods, nested PCR gave excellent sensitivity and specificity with no significant difference (p = 0.727) from conventional culture. The storage of sputum on filter paper and storage at room temperature for 5 days had no apparent effect on the performance of nested PCR. We propose that this collection and storage method be considered for transporting sputum specimens from peripheral health centers or from the field; specimens may be sent by post to a central point for both culture and PCR analysis by trained technicians supervised in accordance with a well-established quality control system.     

QUANTITATIVE REAL-TIME PCR (Q-PCR) FOR SPUTUM SMEAR DIAGNOSIS OF PULMONARY TUBERCULOSIS AMONG PEOPLE WITH HIV/AIDS

Objective: To assess quantitative real-time polymerase chain reaction (q-PCR) for the sputum smear diagnosis of pulmonary tuberculosis (PTB) in patients living with HIV/AIDS with a clinical suspicion of PTB. Method: This is a prospective study to assess the accuracy of a diagnostic test, conducted on 140 sputum specimens from 140 patients living with HIV/AIDS with a clinical suspicion of PTB, attended at two referral hospitals for people living with HIV/AIDS in the city of Recife, Pernambuco, Brazil. A Löwenstein-Jensen medium culture and 7H9 broth were used as gold standard. Results: Of the 140 sputum samples, 47 (33.6%) were positive with the gold standard. q-PCR was positive in 42 (30%) of the 140 patients. Only one (0.71%) did not correspond to the culture. The sensitivity, specificity and accuracy of the q-PCR were 87.2%, 98.9% and 95% respectively. In 39 (93%) of the 42 q-PCR positive cases, the CT (threshold cycle) was equal to or less than 37. Conclusion: q-PCR performed on sputum smears from patients living with HIV/AIDS demonstrated satisfactory sensitivity, specificity and accuracy, and may therefore be recommended as a method for diagnosing PTB.     

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 。     

结核分枝杆菌IS6110序列在石蜡组织中的检测、克隆与测序

目的：探讨套式-聚合酶链反应（Nested-PCR)检测石蜡组织中结核分枝杆菌的特异性和敏感性。方法：采用套式-PCR检测石蜡包埋组织中结核菌复合体特异插入序列IS6110，并对部分标本的PCR产物进行克隆和测序。结果：31例结核标本石蜡组织检出结核菌DNA共28例，套式-PCR的敏感度为90.3%,特异度为100%。阳性预测值为100%。随机选取两例PCR产物没是序结果与结核菌标准株H37Rv同源性分别为97%和95.3%。结论：套式-PCR检测常规石蜡包埋组织中结核菌IS6110序列具有特异性强和敏感性高的特点，可应用于临床诊断，尤其是对那些常规苏木精-伊红染色和抗酸染色无法确诊的病例更具意义。     

PCR-荧光探针法检测临床痰标本结核分枝杆菌的可靠性研究

目的 评价PCR-荧光探针法快速检测Mtb和非结核分枝杆菌（NTM）的临床应用价值。方法 应用分枝杆菌核酸检测试剂对1015 例痰标本和56株临床分离株进行检测,与Mtb核酸扩增（PCR）荧光（目前临床上认可惟一同类产品）检测结果进行比较。采用SPSS 11.5统计软件进行数据统计处理,采用χ²检验,以P＜0.05为差异有统计学意义。结果 PCR-荧光探针法检测痰标本分枝杆菌灵敏度50.3%（373/741）,特异度98.9%（271/274）;菌阳肺结核标本检测灵敏度97.0%（257/265）,菌阴肺结核标本检测灵敏度24.4%（116/476）;1015份痰标本中检测出11例非结核分枝杆菌（NTM）核酸阳性标本（占1.1%）,经16S rDNA 测序确认,准确率100.0%。随机数字量表法抽取138例标本重复进行第2次检测,符合率100.0%。对56株临床分离株（1株Mtb临床分离株,55株NTM临床分离株）检测,准确率100.0%。对临床需要的226份标本同时与达安公司试剂盒检测结果比较,总体符合率95.1%（215/226）,经统计学分析,两者之间差异无统计学意义（χ²=2.98,P=0.226）。结论 PCR-荧光探针法可快速检测和区分痰标本Mtb与NTM,具有临床推广应用价值。     

PCR can help early diagnosis of pulmonary tuberculosis

One hundred and fifty-one patients, clinically suspected for pulmonary tuberculosis (age: 31 +/- 13 years, male/female: 112/39), were investigated to evaluate the diagnostic potential of polymerase chain reaction (PCR) based detection of the Mycobacterium tuberculosis complex in sputum. The diagnostic efficacy of PCR was compared with culture of Mycobacterium tuberculosis on egg-based Lowenstein-Jensen modified medium. PCR detected 71.5% (108/151), whereas culture detected 66.2% (100/151) of the clinically suspected patients. There was a significant association between the results of PCR and culture (chi2 = 59.524, p < 0.001). However, 23.2% (35/151) samples were found negative in both culture and PCR. Considering culture as the gold standard, the sensitivity of the PCR was 92%. and its specificity 70%. This lower apparent specificity may be due to the higher sensitivity of 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。     

Effectiveness of the BDProbeTec ET system for detection of Mycobacterium tuberculosis complex in sputum and bronchoalveolar lavage specimens

ObjectiveThe diagnostic efficacy of the BDProbeTEC ET Mycobacterium tuberculosis (MTB) complex direct detection assay (DTB) performed on bronchoalveolar lavage (BAL) specimens and sputum smears was compared with acid-fast bacilli (AFB) smear microscopy.MethodAFB smear microscopy, DTB and culture results of 286 patients with pulmonary tuberculosis were retrospectively reviewed. A total of 120 patients provided expectorated sputum samples, and 166 patients provided BAL specimens. Culture results and clinical diagnosis were used as gold standards.ResultsThe sensitivity and specificity of the DTB assay in detecting MTB in sputum specimens was significantly higher compared to AFB smear microscopy (83.7% and 82.4%, vs. 75.6%, and 41.2%, respectively). The sensitivity and specificity of the DTB assay in detecting MTB in sputum samples was 77.2% and 100% compared to clinical diagnosis, while AFB smear had a sensitivity and specificity of 70.3% and 26.3%, respectively. Compared to culture, DTB had a sensitivity and specificity of 82.8% and 93.2%, respectively, in detecting MTB from BAL specimens; AFB smear had a sensitivity and specificity of 41.9% and 87.7%, respectively. Compared to clinical diagnosis, DTB had a sensitivity and specificity of 67.2% and 100%, respectively, in detecting MTB from BAL specimens; AFB smear had a sensitivity and specificity of 34.8% and 79.5%, respectively.ConclusionsThe superior performance of the DTB assay relative to AFB smear microscopy makes it a valuable tool to enable early diagnosis of MTB, thereby improving patient care and reducing transmission.     

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.     

Informative value of polymerase chain reaction in the diagnosis of a tuberculous process

The informative value of the polymerase chain reaction (PCR) in the diagnosis of a tuberculous process in patients with pulmonary and extrapulmonary tuberculosis was studied. Sputum, urine, pleural and spinal fluids, and other specimens from 111 patients and 14 healthy individuals were tested. An analysis of sputum from 58 persons clinically diagnosed as having tuberculosis showed the following comparative sensitivity of the used methods: 82.8%, PCR; 65.5%, bacterioscopy; and 67.2%, inoculation. The detection rate of specific DNA from different samples was the following: 82.8% from the sputum, 66.6% from urine, and 54.5% from pleural fluid. The informative value of PCR in 14 patients with osteoarticular tuberculosis and in 39 with urinary tuberculosis was 42.8 and 40.6%, respectively. Thus, the application of PCR to a comprehensive examination of patients with tuberculosis of different sites is justifiable.