恒温扩增荧光检测法检测结核分枝杆菌复合群的应用价值

目的:评价恒温扩增荧光检测法检测痰样本中结核分枝杆菌复合群(Mycobacterium tuberculosis complex, MTBC)的临床应用价值。方法:选择上海市6家结核病定点医院作为研究现场,收集2020年1月至2020年11月各家医院肺科门诊初诊的1848例疑似肺结核患者痰样本,分别进行恒温扩增荧光检测法与传统的痰涂片、BACTEC MGIT 960液体培养(液体培养)及GeneXpert MTB/RIF(简称“GeneXpert”)检测。以液体培养为参照标准,分析比较恒温扩增荧光检测法、痰涂片及GeneXpert的检测效能。结果:在1848例患者痰样本中,恒温扩增荧光检测法、痰涂片、液体培养和GeneXpert检测MTBC的检出率分别为16.7%(309/1848)、14.4%(266/1848)、25.4%(470/1848)和28.7%(530/1848)。以液体培养为参照标准,恒温扩增荧光检测法、痰涂片和GeneXpert检测的敏感度分别为58.94%(277/470)、52.77%(248/470)、80.43%(378/470);特异度分别为97.68%(1...     

利用960系统快速鉴定结核分枝杆菌和非结核分枝杆菌的可行性研究

目的　探讨应用BACTEC MGIT960系统的分枝杆菌生长指示管(Mycobacter Growth Indicator Tube,MGIT)加入对硝基苯甲酸(ρ-Nitro benzoic Acid,PNB)区分结核分枝杆菌复合群(MTBC)与非结核分枝杆菌(NTM)的可行性。方法 用含PNB MGIT检测5株标准分枝杆菌菌株的体外抑菌浓度,并对111株临床分离菌株与传统改良罗氏(Lwenstein-Jensen,L-J)PNB鉴定进行比较。结果 以PNB300μg/ml为界,111株临床分离菌株PNB MGIT法与L-J PNB的菌群鉴定相比较符合率为90.09%;鉴定结果报告平均7.72 d,与L-J PNB法比较有显著性差异,检测时间明显缩短(P<0.001)。结论 应用960系统PNB MGIT法鉴定分枝杆菌菌群的方法快速、准确、实用。     

聚合酶链反应-增强化学发光法快速检测结核杆菌的研究

目的　建立聚合酶链反应 -增强化学发光法 (polymerase chain reaction-enhanced chemilumine cence ,PCR-ECL)快速高灵敏度和高特异性检测结核杆菌的方法。方法 以结核分支杆菌、牛分支杆菌特异性抗原Pab基因的419bp片段为靶序列,PCR体系经优化,直接酶标法标记探针 ,增强化学发光检测(ECL)进行分子杂交。结果 PCR体系对结核分支杆菌、牛分支杆菌、卡介苗的扩增为阳性,PCR体系灵敏度为5fgDNA分子。ECL体系灵敏度为0.5pgDNA分子。采用模拟痰样,可检至10个以下结核杆菌。结论 建立了结核杆菌的PCR-ECL快速检测方法,整个过程可在一天半内完成。     

Evaluation of an immunochromatography test kit for rapid diagnosis of influenza

We assessed the sensitivity and specificity of the Capilia Flu AB rapid diagnosis kit for influenza that utilizes the immunochromatography method. Tested were 114 influenza like illness patients in the 2001/2002 influenza season. We used Capilia Flu AB and Infu A . B Quick, a rapid diagnosis kit based on enzyme immunoassay. As laboratory confirmation tests, influenza virus isolation and polymerase chain reaction (PCR) were done. Those patients with positive results from virus isolation or PCR were regarded as influenza patients. The sensitivities of nasal swab, pharyngeal swab, and nasal wash specimens were 82.8%, 80.0%, and 75.0%, respectively. The specificities of nasal swab, pharyngeal swab, and nasal wash specimens were 95.3%, 93.9%, and 100%, respectively. A total of 20 patients displayed different results in comparison of their nasal and pharyngeal swabs: 15 patients were positive with the nasal swab but negative with the pharyngeal swab and 5 patients were negative with the nasal swab but positive with the pharyngeal swab. Nasal swab would seem to be preferable in terms of sensitivity. The sensitivity and specificity of Capilia Flu AB were a little higher than those of Influ A . B Quick, but with no significant difference. The one-step operation of Capilia Flu AB is easier than the four steps required by Influ A . B Quick, but the time required to make a diagnosis is the same. No significant age related difference in the effectiveness of the kits was found. The Capilia Flu AB rapid diagnosis kit is useful in clinical practice because it has good sensitivity (about 80%) and specificity (about 90%), and it is easy to use.     

Evaluation of a rapid assay for identification of Mycobacterium tuberculosis grown in solid and liquid media

An MPT64 antigen detection assay, the SD Bioline TB Ag MPT64 Rapid kit, was evaluated for the identification of Mycobacterium tuberculosis grown in both solid and liquid media. The rapid test showed positive results in 132 of 133 (99.2%) M. tuberculosis cultures grown on Ogawa medium and 136/143 (95.1%) cultures grown in liquid culture medium, resulting in an overall sensitivity of 97.1%. All 18 non-tuberculous mycobacteria were found to be negative by the rapid test, indicating a specificity of 100%. The rapid test seems useful for the initial confirmation of M. tuberculosis in acidfast bacilli positive cultures.     

快速结核分枝杆菌菌型鉴定方法的应用评估

目的评估MGIT 960液体培养分枝杆菌阳性时,免疫胶体金法MPB64抗原检测及荧光探针PCR法在结核分枝杆菌快速菌型鉴定中的应用价值。方法临床MGIT960TM液体培养得到203例分枝杆菌阳性菌株,平行采用上述两种方法进行结核分枝杆菌菌型鉴定。结果免疫胶体金MPB64抗原检测法灵敏度为97.7%(169/173×100%),特异度为100%(30/30×100%);荧光探针PCR法灵敏度为100%(173/173×100%),特异度为96.7%(29/30×100%)。结论免疫胶体金法检测MPB64抗原及荧光探针PCR法作为液体培养时结核杆菌菌型鉴定的方法均是快速可靠的。免疫胶体金法检测MPB64抗原由于操作简便,实验要求条件低,更易为临床TB实验室接受。     

结核杆菌特异性细胞免疫反应检测试剂的临床试验

目的验证武汉海吉力生物科技有限公司新研制的"结核杆菌特异性细胞免疫反应检测试剂盒(酶联免疫法)"(简称海吉力试剂)的诊断性能,探讨γ-干扰素释放试验在结核病诊断中的价值。方法以北京万泰生物药业有限公司生产的"结核杆菌相关γ-干扰素检测试剂盒(体外酶联免疫法)"(简称万泰试剂)为对照,同时用海吉力试剂对武汉市医疗救治中心、北京老年医院、河北胸科医院1084例受试者的外周血进行检测,比较两种检测试剂盒的一致性、诊断性能。结果海吉力试剂与万泰试剂检测结果的总符合率为90.6%,Kappa值为0.812(P=0.000)。海吉力试剂的敏感性为80.7%,万泰试剂的敏感性为78.6%,两者差异无统计学意义(χ2=1.64,P=0.20),海吉力试剂的特异性为74.1%,万泰试剂的特异性为75.0%,两者差异无统计学意义(χ2=0.09,P=0.76)。对肺外结核、菌阴结核患者,两种试剂敏感性稍高,差异均无统计学意义(P0.05)。结论新研制的海吉力试剂与万泰试剂具有很好的一致性,两者对结核病的诊断效能相当,对肺外结核、菌阴结核患者的诊断价值较高。     

胶体金法结合涂片形态学特征快速鉴别结核分枝杆菌复合群的应用评价

目的 探讨结核分枝杆菌抗原胶体金法结合涂片形态学特征鉴别结核分枝杆菌复合群的应用价值。 方法 采用涂片形态学特征和结核分枝杆菌抗原胶体金法对853株分枝杆菌临床分离株进行鉴别并与传统菌种鉴定结果进行比较。 结果 涂片法鉴别结核分枝杆菌的敏感度为98.4%(750/762),特异度为96.7%(88/91);胶体金试验鉴别结核分枝杆菌的敏感度为99.0%(754/762),特异度为98.9%(90/91);两种方法均为阳性,可报告菌株中含有结核分枝杆菌复合群(MTC),其阳性预测值为100.0%（741/741）;两种方法均为阴性,可报告非结核分枝杆菌(NTM),其阴性预测值为100.0%（87/87）。 结论 结合涂片形态和结核分枝杆菌抗原胶体金两种方法鉴别结核分枝杆菌可以提高检测的准确性,是一种快速、简便,适于各级实验室使用的方法。     

Evaluation of a rapid differentiation test for the Mycobacterium tuberculosis complex by selective inhibition with rho-nitrobenzoic acid and thiophene-2-carboxylic acid hydrazide.

SETTING: Mycobacterial growth in media to which inhibitory substances are added has been used in species identification. Growth of the Mycobacterium tuberculosis complex (MTC) is inhibited by rho-nitrobenzoic acid (PNB), whereas non-tuberculous mycobacteria (NTM) are resistant. Thiophene-2-carboxylic acid hydrazide (TCH) is useful in the differentiation of MTC when performed together with other tests. OBJECTIVE: To develop a test using PNB or TCH added to culture medium, and to evaluate its usefulness in the screening of mycobacteria isolates. DESIGN: In 2001, PNB testing was performed in 109 M. tuberculosis strains identified by Instituto Adolfo Lutz (IAL) and 52 NTM strains from the institute's culture collection. The drugs were added to L?wenstein-Jensen (LJ) medium and to BBL-MGIT. RESULTS: Species differentiation of MTC with the MGIT/TCH method was similar to that observed using the conventional LJ/TCH method. The accuracy of the MGIT/PNB method to differentiate NTM and MTC strains was 99.4%. The BBL-MGIT system allowed presumptive identification in 3-11 days, compared to > or =12 days with LJ medium. CONCLUSION: A simple, low-cost test using growth inhibitors may be incorporated into a modern, safe and quick methodology enabling differentiation of MTC and NTM.     

Evaluation of nutrient agar for the culture of Mycobacterium tuberculosis using the microcolony detection method

We evaluated nutrient agar using the microcolony detection method for the recovery of Mycobacterium tuberculosis on 37 acid-fast bacilli (AFB) positive sputum specimens, and compared it with conventional L?wenstein-Jensen (LJ) medium. Nutrient agar detected 35 isolates compared to 34 on LJ medium. The mean time to detection of mycobacteria on nutrient agar and LJ medium was respectively 9.6 and 21.4 days. The contamination rate on nutrient agar and LJ medium was respectively 5.4% and 2.7%. Nutrient agar detects M. tuberculosis more rapidly than LJ medium, and could be an economical, rapid culture method in resource-poor settings, provided our findings are confirmed by further studies.     

Study of detailed conditions in DNA probe test by use of Gen-Probe Rapid Diagnostic System for identification of Mycobacterium avium complex and Mycobacterium tuberculosis complex

In order to improve feasibility of technical procedures in Gen Probe Rapid Diagnostic System (Gen Probe Inc., San Diego, CA, U.S.A.) for identification of Mycobacterium avium complex (MAC) and M. tuberculosis complex (MTC), we studied several test conditions in the DNA probe testing, such as stability of test bacterial suspension, optimal duration of bacterial cultivation, the number of organisms in test bacterial suspension required for accurate determination, and so on. With respect to concentration of organisms (MAC and MTC) in test bacterial suspension (0.1ml), we found that 5-fold dilution as well as 5-fold condensation of the standard bacterial suspension (McFarland No.1) gave substantially the same result as in the case where bacterial suspension at the standard concentration was used. This indicates that the test bacterial suspensions (0.1ml) containing either 1.5 X 10(7)-5 X 10(8) of MAC or 3 X 10(5)-8 X 10(6) of MTC are available for the DNA probe testing. Test bacterial suspension at McFarland No.1 prepared from fresh cultures (3-4 week-old) could be stored either at -80, -20 or 4 degrees C at least for 17 weeks without significant loss of reactivity to M. avium, M. intracellulare and MTC DNA probes. In this case, stability of DNA probe-reactivity was preserved in the following order: MTC, M. avium and M. intracellulare. Concerning the age of bacterial cultures, at least 16-week-old cultures of MAC and MTC after initial appearance of cell growth on 1% Ogawa's egg media were sufficiently reactive to either MAC or MTC DNA probe. In this case, MTC showed most stable reactivity during the course of long-term cultivation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of the alkaline-phosphatase labeled DNA probes for Mycobacterium tuberculosis and Mycobacterium avium complex]

Non-radioisotopic, alkaline phosphatase-labeled DNA (AP-DNA) probe tests for the identification of Mycobacterium tuberculosis complex (Mtb) and Mycobacterium avium complex (MAC) were evaluated. The overall agreement, sensitivity and specificity of the AP-DNA probes for Mtb and MAC were 100% respectively compared with the conventional biochemical method. Because the procedure is rapid (it can be completed approximately 120 min), safe (it does not use radioisotopes) and convenient (it does not need the special equipment to be performed), it can be easily performed in any clinical laboratory.     

One-tube multiplex PCR method for rapid identification of mycobacterium tuberculosis

A rapid, inexpensive, simple, and accurate multiplex polymerase chain reaction (PCR) was developed in a single tube for identification of Mycobacterium tuberculosis. Assessment of sensitivity and specificity of simple PCR was performed with 116 strains of M. tuberculosis complex (MTC) and 144 strains of nontuberculous mycobacteria (NTM) compared with the biochemical method. Specific amplification of KS4, MTC-specific DNA fragment, was found in 98% (114/116) of MTC and not detected in 99% (143/144) of NTM. Amplification of the mtp40 gene revealed 95% sensitivity (100/105 strains of M. tuberculosis) and 77% specificity (not found in 119/155 mycobacterial strains). A multiplex PCR method based on the combination of KS4- and mtp40-derived primers was used for identification of M. tuberculosis. Crude DNA from slow growing mycobacteria with cream rough colonies that showed both 768-bp amplified product for KS4 and 396-bp for mtp40 was identified as M. tuberculosis whereas that from MTC gave only the 768-bp product.     

Evaluation of FASTPlaqueTB-RIF, a rapid, manual test for the determination of rifampicin resistance from Mycobacterium tuberculosis cultures.

SETTING: Two Mycobacteriology Reference Laboratories in Johannesburg (Laboratory 1) and Cape Town (Laboratory 2), South Africa. OBJECTIVE: To determine the ability of the FASTPlaqueTB-RIF test to correctly identify rifampicin susceptibility on strains of Mycobacterium tuberculosis cultured on solid media. DESIGN: A comparative study of FASTPlaqueTB-RIF and conventional drug susceptibility methods, with selection bias to include sufficient rifampicin resistant strains. RESULTS: Rifampicin susceptibility results were available for 191 strains of M. tuberculosis. Eighty-one strains were found to be rifampicin resistant and 110 strains were rifampicin susceptible by conventional methods. The sensitivity, specificity and overall accuracy for the FASTPlaqueTB-RIF were 100%, 97% and 98% at Laboratory 1, and 100%, 94% and 97% at Laboratory 2. CONCLUSION: FASTPlaqueTB-RIF offers a performance comparable to the gold standard proportion methods of rifampicin susceptibility testing, as well as the advantage of the speed of results that the newer methods deliver, without the need for specialised equipment. This makes FASTPlaqueTB-RIF a rapid test for rifampicin resistance suitable for widespread application.     

快速免疫色谱实验诊断可疑肺结核病人的评价

目的评价基于结核分支杆菌38KDa等五种抗原的快速免疫色谱实验(澳大利亚ICT TB)对可疑肺结核的临床诊断价值?方法以初诊可疑肺结核患者125例为受试对象,同时做痰涂片?培养,血清ICT TB以及胸片检查?结果总的敏感性为45%?特异性为100%?结论此试验不能完全代替痰涂片抗酸染色检查,但二者联合使用,可使敏感性提高到63.6%.     

环介导等温扩增法快速检测结核分枝杆菌的初步观察

目的建立1种用于结核分枝杆菌快速检测的LAMP技术平台,用于结核分枝杆菌的鉴定。方法使用了11株标准菌株,建立了一套用于结核分枝杆菌快速检测的平台,对灵敏度和特异性进行优化,随后使用47株临床分离株,和13株食源性致病菌对建立的方法进行了验证。结果经过标准菌株建立并优化检测平台,经过优化的LAMP反应体系检出限为1pg的模板DNA,在使用47株临床分离株,和13株食源性致病菌对方法进行验证,发现LAMP特异性为97.4%。结论通过这项研究发现LAMP应用于结核分枝杆菌的快速检测,具有检出浓度低、特异性强和检测周期短的优点。