Comparison of polymerase chain reaction amplification of two mycobacterial DNA sequences, IS6110 and the 65kDa antigen gene, in the diagnosis of tuberculosis.

BACKGROUND: Knowledge of the sequences of mycobacterial genes and the availability of DNA amplification techniques have raised the possibility that identification of mycobacterial DNA may offer a rapid and specific diagnostic test for tuberculosis. The correlation between the presence of Mycobacterium tuberculosis DNA and clinical tuberculosis, however, is not known. This study compared the results of polymerase chain reaction amplification of two M tuberculosis DNA sequences, IS6110 and the gene encoding the 65kDa heat shock protein (65kDa Ag), from sputum, bronchoscopy washings, and bronchoalveolar lavage fluid and related these findings to the presence of active and past tuberculosis. METHODS: Highly specific primers were used for amplification of IS6110 and 65kDa Ag DNA. Analysis was performed on one or more samples from 87 patients. RESULTS: IS6110 DNA was identified in samples from all six patients with active tuberculosis, from 15 to 18 patients with past tuberculosis, from five of nine contacts of patients with tuberculosis, and from nine of 54 patients with lung disease unrelated to tuberculosis. The 65kDa Ag DNA was identified in samples from all patients with active and past tuberculosis, from contacts of patients with tuberculosis, and from 14 of 42 patients with non-tuberculous lung diseases. CONCLUSION: These data suggest that the presence of IS6110 DNA correlates more closely with a tuberculosis related diagnosis than that of 65kDa Ag DNA and that both DNAs are found in most subjects with past tuberculosis or contacts of patients with tuberculosis. This may limit the clinical usefulness of these tests.     

Polymerase chain reaction for the detection of Mycobacterium tuberculosis in synovial fluid, tissue samples, bone marrow aspirate and peripheral blood

Extrapulmonary tuberculosis accounts for approximately 10% of tuberculous infections; the musculoskeletal system is involved in a small number of these (10%). Skeletal tuberculosis is an indolent disease, and diagnosis may be delayed. Conventional methods are time consuming and have a low sensitivity rate. In recent years PCR-based protocols raised hopes as a reliable and fast diagnostic tool for extrapulmonary tuberculosis. The authors report the detection of Mycobacterium tuberculosis complex DNA in specimens from six patients using a nested PCR protocol specific for IS6110 insertion element of Mycobacterium tuberculosis complex. Three men and three women are reported with ages ranging from 42 to 68 years. The sites of infection were the knee and shoulder in one case each, the hip in two cases, and the thoracic spine in two cases. Diagnosis was established within three days, and treatment was initiated promptly. PCR is a technically easy approach that can be used as a first step diagnostic tool for early recognition and treatment of bone and joint tuberculosis.     

Value of DNA analysis in addition to cytological testing in the diagnosis of malignant pleural effusions.

BACKGROUND--Aneuploidy appears to be a highly specific marker for cancer, and measurement of cellular DNA content by flow cytometry is rapid and reliable. This study was undertaken to determine if the addition of DNA analysis improved the sensitivity of cytological diagnosis of malignancy in pleural fluid. METHODS--Pleural effusions from 92 patients were studied by cytological examination and flow cytometry. RESULTS--In 41 patients the final diagnosis was malignancy, there were 40 cases of benign effusions including 22 with pleural tuberculosis, and in the remaining 11 patients with biopsy proven cancer the presence of malignant cells was not found by cytological and histological means in the pleural fluid. Aneuploidy and cytological malignancy were found in 14 samples. There were seven cases with abnormal flow cytometry and negative cytological results. In 12 patients the cytological test results were positive but DNA analysis was normal. Thirty six samples of fluid were both diploid and cytologically negative. Of the 22 tuberculous effusions seven contained aneuploid cells. The sensitivity of DNA and cytological analysis was 51.2% and 63.4%, respectively. The specificity of DNA analysis was 74.5%. CONCLUSIONS--DNA analysis of cells in malignant pleural effusions is both less sensitive and specific than the cytological diagnosis. Flow cytometric analysis is not recommended for routine use in the diagnosis of pleural effusions.     

Tuberculous pleural effusions: increased culture yield with bedside inoculation of pleural fluid and poor diagnostic value of adenosine deaminase.

A prospective study of 111 adult patients with a pleural effusion was carried out in an area with a high prevalence of tuberculosis to compare the yield of bedside with laboratory inoculation of pleural fluid, and the yield and speed of a radiometric mycobacterial culture system (BACTEC) with that of conventional culture. The use of adenosine deaminase activity in pleural fluid as a diagnostic test for tuberculosis was also evaluated. In the 62 cases of tuberculosis confirmed histologically or by culture, or both, the BACTEC system had the same culture yield as conventional mycobacterial culture (positive in 14 cases-23%), but was significantly faster (18 versus 33 days). Bedside inoculation had a culture yield significantly higher than laboratory inoculation in the 24 patients tested (11 versus four). The remaining three diagnostic categories were malignant (28), miscellaneous (10), and undiagnosed (11). Median adenosine deaminase activity was significantly higher in tuberculous effusions than in any of the other categories, but there was considerable overlap between the groups. It is concluded that the BACTEC system is significantly faster than conventional mycobacterial culture and that bedside inoculation of pleural fluid substantially increases culture yield. Adenosine deaminase does not provide as valuable a diagnostic test of pleural tuberculosis as has been suggested.     

Use of adenosine deaminase as a diagnostic tool for tuberculous pleurisy.

BACKGROUND--A statistical audit of adenosine deaminase (ADA) in pleural effusions was undertaken. METHODS--ADA analysis, cytological and microbiological examinations, and differential cell counts were performed on 462 pleural fluid samples. RESULTS--ADA activity in tuberculous effusions was higher than in any other diagnostic group. At a level of 50 U/l the sensitivity and specificity for the identification of tuberculosis was 90% and 89%, respectively. CONCLUSIONS--ADA activity remains a useful test in the evaluation of pleural effusions.     

Rapid diagnosis of genitourinary tuberculosis by polymerase chain reaction and non-radioactive DNA hybridization

OBJECTIVE: To establish a polymerase chain reaction (PCR) assay for the rapid detection and identification of mycobacteria in urine, and to assess the value of such assay in routine laboratory diagnosis of genitourinary tuberculosis. MATERIALS AND METHODS: Urine specimens from 1000 patients with clinical suspicion of urinary tuberculosis were examined. Two assays for the detection and identification of Mycobacterium tuberculosis (M. tuberculosis) complex and mycobacteria other than tuberculosis (MOTT) by non-radioactive DNA hybridization of PCR-product were applied. The first assay used PCR primers and probe derived from M. tuberculosis species-specific DNA insertion sequence, IS6110. The second utilized mycobacterium genus-specific sequence encoding ribosomal ribonucleic acid (16S rRNA). The results obtained by PCR were compared with those obtained by standard microbiological methods, acid-fast bacilli (AFB) stain and culture. RESULTS: Compared with cultures, the sensitivity of AFB staining was 52.07% and the specificity was 96.7%. In comparison to the results of culture, the overall sensitivity and specificity of the IS6110-PCR assay was 95.59% and 98.12% respectively. While the corresponding results for the 16S rRNA gene-PCR were 87.05% and 98. 9%. CONCLUSION: The high sensitivity and specificity in addition to the potential for rapid detection of mycobacteria, makes this test a useful tool in the clinical management of mycobacterial infection in urine. Urine specimens may contain M. tuberculosis and/or other mycobacteria; therefore, there are advantages in using genus-specific primers in parallel with species-specific primers in PCR assay.     

Direct detection by ligase chain reaction of Mycobacterium tuberculosis complex in pleural fluid

Our purpose was to investigate the utility of the DNA amplification by ligase chain reaction (LCR) for the direct detection of Mycobacterium tuberculosis complex (MTC) in pleural fluid specimens from patients suspected for tuberculous pleural effusion. We have used the LCx M. tuberculosis kit (Abbott) which uses the amplification of the gene that encodes for antigen b. We have examined 81 pleural fluid specimens by isolation (on L?wenstein-Jensen medium and MB/BacT system) and by LCR. Out of 10 positive specimens in culture, 4 were also positive by LCR; out of 71 negative specimens in culture, 8 were positive by LCR. We have re-evaluated the LCR results according to the clinical diagnosis, sustained by the successful therapy, and to the pathological diagnosis on the pleural biopsy. The sensitivity and specificity of LCR in the diagnosis of tuberculous pleural effusion were 31.5% and 100%. This commercial LCR kit is a rapid, specific, but less sensitive test for the routine diagnosis of the tuberculous pleural effusion.     

Interferon gamma for diagnosing tuberculous pleural effusions

BACKGROUND: A study was undertaken to evaluate the diagnostic value of pleural fluid concentrations of interferon gamma (IFN-gamma) as a marker of tuberculosis. METHODS: Patients admitted to King Chulalongkorn Memorial Hospital between April 1997 and January 1998 with a lymphocytic exudative pleural effusion were enrolled into the study. The pleural fluids were examined for cytology, staining for acid fast bacilli, and mycobacterial culture. Pathological examination and mycobacterial culture were performed on each pleural biopsy specimen. The diagnosis of tuberculosis was made when one of the following criteria was met: (1) Mycobacterium tuberculosis was isolated from either the pleural fluid or pleural tissue; (2) granulomas were demonstrated in the pleural tissue which stained positive for acid fast bacilli (AFB); or (3) in the presence of granulomas negative on staining for AFB in pleural tissue there was a response to antituberculous treatment on follow up. All pleural fluid samples were stored at -70 degrees C and the IFN-gamma level was measured by immunoassay. Analysis was made using sensitivity, specificity, and likelihood ratio for a positive test result. The best cut off point was determined by the highest likelihood ratio and receiver operating characteristic curve. RESULTS: A total of 66 patients were enrolled and tuberculosis was confirmed in 39 of them. The diagnoses in the non-tuberculous group included malignancy (15), paramalignancy (11), and chronic pleuritis secondary to infective endocarditis (1). The mean (SE) IFN-gamma level in the pleural fluid was significantly higher in the tuberculous group than in the non-tuberculous group (1493.3 (131.3) pg/ml versus 80.1 (50.4) pg/ml, p<0.001). The overlap between the two groups was minimal. At the cut off value of 240 pg/ml the sensitivity was 94.9% (95% CI 86.6 to 100), the specificity was 96.3% (95% CI 89.2 to 100), and the likelihood ratio for a positive test result was 25.6. CONCLUSIONS: The pleural fluid concentration of IFN-gamma is a good and useful diagnostic marker of tuberculosis presenting as a lymphocytic exudative pleural effusion.     

Value of adenosine deaminase in the diagnosis of tuberculous pleural effusions in young patients in a region of high prevalence of tuberculosis.

BACKGROUND--Pleural biopsy is usually considered important for the diagnosis of pleural effusions, especially for distinguishing between tuberculosis and neoplasia, even though tuberculous pleural fluid contains sensitive biochemical markers. In regions with a high prevalence of tuberculosis, and in patient groups with a low risk of other causes of pleurisy, the positive predictive value of these markers is increased. The criteria for performing a pleural biopsy under these circumstances have been investigated, using adenosine deaminase (ADA) as a pleural fluid marker for tuberculosis. METHODS--One hundred and twenty nine patients with a pleural effusion aged < or = 35 years (mean (SD) 25.2 (4.9) years) were studied. Seventy three were men. Eighty one effusions (62.8%) were tuberculous, 12 (9.3%) parapneumonic, and 10 (7.7%) neoplastic, five were caused by pulmonary thromboembolism, four by systemic lupus erythematosus, seven by empyema, three following surgery, one was the result of asbestosis, and one of nephrotic syndrome. In five cases no definitive diagnosis was reached. ADA levels were determined by the method of Galanti and Giusti. RESULTS--The diagnostic yield of procedures not involving biopsy was 94.5% (122/129). Pleural biopsy provided a diagnosis in a further two cases, but not in the remaining five. All tuberculous cases had pleural fluid levels of ADA of > 47 U/l (mean (SD) 111.1 (36.6) U/l). The only other cases in which ADA exceeded this level were six of the seven patients with empyema. Cytological examination of the pleural fluid diagnosed eight of the 10 neoplastic cases, compared with six diagnosed by pleural biopsy. CONCLUSIONS--In a region with a high prevalence of tuberculosis procedures not involving pleural biopsy have a very high diagnostic yield in patients with a pleural effusion aged < or = 35 years, making biopsy necessary only in cases in which pleural levels of ADA are below 47 U/l, pleural fluid cytology is negative and, in the absence of a positive basis for some other diagnosis, neoplasia is suspected.     

术前短期化疗对脊柱结核手术安全性的实验研究

目的通过巢式聚合酶链式反应（polymerase chain reaction,PCR）动态检测脊柱结核患者围手术期外周血的MTB-DNA的IS6110基因的含量,评估术前短期化疗对脊柱结核患者的手术安全性。方法根据IS6110基因设计两对特异性硫化修饰引物,结合高效保真聚合酶建立的巢式PCR,动态检测25例术前短期化疗的脊柱结核患者术前1天、术后1天、术后7天、术后14天外周血MTB-DNA的IS6110基因的含量变化,对外周血扩增产物IS6110基因含量进行比较分析。结果脊柱结核患者围手术期外周血IS6110基因含量变化的两种趋势：（1）15例患者伴随术后结核病灶的清除,外周血中的结核杆菌IS6110基因的从术后1天就开始逐渐减少;（2）10例患者术后1天时外周血中结核杆菌IS6110基因有短暂回升,但与术前1天外周血中结核杆菌IS6110基因含量比较无显著性差异（P〉0.05）,随着术后持续抗痨治疗,结核杆菌IS6110基因含量在术后1～2周均减少,均未发生结核杆菌血行播散并发症。结论如果脊柱结核患者一般情况好,术前短期化痨后行手术治疗安全有效。     

Diagnosis of tuberculosis by detection of mycobacterial antigen in pleural effusions and ascites

We describe a method for the diagnosis of pleural and peritoneal tuberculosis by the detection of tuberculous antigens using an enzyme-linked immunosorbent assay. Eleven tuberculous pleural fluid and 10 tuberculous ascitic fluid samples were studied by this technique, using 10 non-tuberculous pleural fluid and 14 non-tuberculous ascitic fluid samples as controls. An absorbance value of 0.3 was found to separate the tuberculous groups from their controls to a statistically significant extent (ascitic fluid P less than 0.05; pleural fluid P less than 0.01).     

Diagnostic potential of multi‐targeted LAMP (loop‐mediated isothermal amplification) for osteoarticular tuberculosis

Delay in diagnosing osteoarticular tuberculosis (OATB) contributes significantly to morbidity by causing disfiguration and neurological sequelae. The delay caused by conventional culture and the expertise and expense involved in other nucleic acid based tests, make LAMP (loop‐mediated isothermal amplification) assay a favorable middle path. We evaluated LAMP assay using IS6110 and MPB64 for rapid diagnosis of OATB by comparing with IS6110 PCR and culture. LAMP assay was performed on 140 synovial fluid and pus samples (10 culture‐positive proven cases, 80 culture‐negative probable cases, and 50 negative controls) using three set of primer pairs each for IS6110 and MPB64. LAMP assay, using two‐target approach, had an overall sensitivity and specificity of 90% and 100% in detecting OATB. Sensitivity of IS6110 PCR, IS6110 LAMP, and MPB64 LAMP was 80%, 100%, and 100%, respectively, for confirmed cases and 72.5%, 81.75%, and 86.25%, respectively, for probable cases. Six additional cases were picked using two‐target approach. LAMP assay utilizing IS6110 and MPB64 is a cost‐effective technique for an early and reliable diagnosis of OATB. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:361–365, 2017.

     

Comparison of polymerase chain reaction for IS6110 and Amplicor in the diagnosis of tuberculosis.

BACKGROUND: Polymerase chain reaction (PCR) amplification of Mycobacterium tuberculosis DNA offers the potential of a sensitive and specific diagnostic test for tuberculosis. To evaluate this technique from the clinician's perspective, samples were collected from patients with chronic respiratory disease and the sensitivity and specificity of a newly introduced commercially available PCR kit (Amplicor) was compared with that of an established method to detect the target sequence IS6110. METHODS: Sputum or bronchial washings from patients with active tuberculosis, previously treated tuberculosis or other selected respiratory illnesses were analysed by both techniques and their sensitivity and specificity determined. RESULTS: Amplicor was more specific than IS6110 in the diagnosis of active infection (98% versus 79%). Both techniques were equally sensitive (92%). CONCLUSION: These results suggest that analysis of respiratory samples by Amplicor PCR in inner city populations of patients has greater specificity for a diagnosis of active tuberculosis than PCR for IS6110, and thus Amplicor PCR may aid the clinician in making a diagnosis of active tuberculosis.     

PCR diagnosis on formalin-fixed, paraffin-embedded tissues with acid-fast stain and culture negativity in chronic dialysis patients of cervico-mediastinal tuberculous lymphadenitis

Background: Bacteriologic studies often provide negative results in tuberculous infection, and do not favour early diagnosis. Polymerase chain reaction (PCR) is known to diagnose tuberculosis quickly. With this in mind, we used PCR to detect mycobacterial DNA on formalin-fixed, paraffin-embedded tissues with acid-fast stain and culture negativity in two dialysis patients with cervico-mediastinal lymphadenopathy. Methods: Sections of neck lymph nodes were cut at two different levels. At each level, two semi-adjacent sections with a thickness of 5 &mgr;m each were cut using standard microtomes with disposable blades. The first section mounted on a glass slide was stained b Ziehl-Neelsen, and the second section was examined by PCR based on a 123 bp fragment of IS6110 that is specific for the Mycobacterium tuberculosis complex. Results: The histology of lymph nodes disclosed inflammatory necrotizing granulomas, but acid-fast stain for M. tuberculosis was negative in the two patients. DNA of M. tuberculosis was detected in lymph node samples from each patient by PCR on the IS6110 element and by dot-blot hybridization. Conclusions: PCR assay is a potentially useful approach for early and rapid diagnosis of tuberculous lymphadenitis in chronic dialysis patients, since mycobacterial staining and culture often provide negative results.     

Mycobacterium tuberculosis DNA in tissues affected by sarcoidosis

BACKGROUND: Although some studies have reported the presence of Mycobacterium tuberculosis (MTb) DNA in tissues affected by sarcoidosis, the data are conflicting. The aim of this study was to collect prospectively tissue from patients with sarcoidosis in whom tuberculosis had been excluded, and to use polymerase chain reaction (PCR) to search for DNA sequences specific for MTb. METHODS: Fresh tissue samples (node or lung biopsy) taken from 23 patients with newly diagnosed sarcoidosis, 10 with other respiratory disease, and four patients with culture positive tuberculosis were analysed using PCR to amplify a 123 bp fragment of IS6110, the insertion element present in MTb, and nested PCR to further amplify an 85 bp sequence within the 123 bp product. DNA was also extracted from formalin fixed tissue from eight additional patients with sarcoidosis. RESULTS: MTb DNA was not detected in any of the tissue samples from patients with sarcoidosis or other respiratory disease but was found in all four patients with tuberculosis. CONCLUSIONS: This study has shown the absence of MTb DNA in lymph node and lung biopsy samples from patients with sarcoidosis. MTb is therefore unlikely to be a factor in the pathogenesis of this disease.     

The ligase chain reaction as a primary screening tool for the detection of culture positive tuberculosis

BACKGROUND: The ligase chain reaction Mycobacterium tuberculosis assay uses ligase chain reaction technology to detect tuberculous DNA sequences in clinical specimens. A study was undertaken to determine its sensitivity and specificity as a primary screening tool for the detection of culture positive tuberculosis. METHODS: The study was conducted on 2420 clinical specimens (sputum, bronchoalveolar lavage fluid, pleural fluid, urine) submitted for primary screening for Mycobacterium tuberculosis to a regional medical microbiology laboratory. Specimens were tested in parallel with smear, ligase chain reaction, and culture. RESULTS: Thirty nine patients had specimens testing positive by the ligase chain reaction assay. Thirty two patients had newly diagnosed tuberculosis, one had a tuberculosis relapse, three had tuberculosis (on antituberculous therapy when tested), and three had healed tuberculosis. In the newly diagnosed group specimens were smear positive in 21 cases (66%), ligase chain reaction positive in 30 cases (94%), and culture positive in 32 cases (100%). Using a positive culture to diagnose active tuberculosis, the ligase chain reaction assay had a sensitivity of 93.9%, a specificity of 99.8%, a positive predictive value of 83.8%, and a negative predictive value of 99.9%. CONCLUSIONS: This study is the largest clinical trial to date to report the efficacy of the ligase chain reaction as a primary screening tool to detect Mycobacterium tuberculosis infection. The authors conclude that ligase chain reaction is a useful primary screening test for tuberculosis, offering speed and discrimination in the early stages of diagnosis and complementing traditional smear and culture techniques.     

Pleural fluid pH: diagnostic, therapeutic, and prognostic value

Measurement of pleural fluid pH has diagnostic, therapeutic, and prognostic implications in exudative pleural effusions (Table II). A parapneumonic effusion with a pleural fluid pH below 7.2 indicates an empyema is forming which necessitates chest tube drainage in all patients, whereas a pleural fluid pH over 7.3 does not require drainage. If the pH of a parapneumonic effusion is 7.2 to 7.3, serial pleural fluid pH measurements with clinical observation will help to determine the need for chest tube drainage. A pleural fluid glucose level of below 60 mg/dl and a lactic dehydrogenase level over 1,000 IU/dl in conjunction with a pleural fluid pH of 7.2 to 7.3 indicate an impending empyema. These findings are consistent with our clinical experience in patients with parapneumonic effusion. Tuberculous pleural effusions had a pleural fluid pH below 7.4 in all reported patients. This pH may be of value in distinguishing tuberculous pleural effusions from recent malignant effusions, which tend to have a higher pleural fluid pH, particularly if used in conjunction with other pleural fluid values, cell counts, and other clinical parameters. In patients with malignant pleural effusions, a pH of less than 7.3 is usually seen in those effusions present for several months and is associated with a lower glucose level and a higher white cell count and lactic dehydrogenase level. Results of cytologic study of the pleural fluid and pleural biopsy are often positive, there is poor response to sclerosing agents, and the prognosis is poor. A rheumatoid pleural effusion most often has a pleural fluid pH below 7.3. A pleural fluid pH below 6 is seen almost exclusively in esophageal rupture but rarely with empyemas, whereas a pleural fluid pH below 7 occurs in esophageal rupture, empyema, and rheumatoid pleural effusions. In pleural effusions secondary to congestive heart failure, the pH is almost always greater than 7.4 unless systemic acidemia coexists, in which case the pleural fluid pH is within 0.04 units of the simultaneous arterial pH. The major value of pleural fluid pH is to determine the need for chest tube drainage in parapneumonic effusions and to determine the response to sclerosing agents in patients with malignant pleural effusions. As with all diagnostic tests, the results should be interpreted in the context of other diagnostic tests of the pleural fluid and clinical aspects before diagnostic or therapeutic decisions are made.     

Associate investigations: detection of tuberculosis infections in children resulting in discovery of undiagnosed tuberculosis in adults

The authors present the design and implementation of associate investigations of young children with positive tuberculin skin test results. Case study analysis of an associate investigation was done using epidemiologic surveillance techniques, medical interviewing, sociogram mapping, tuberculin skin testing, radiographic evidence, and bacteriologic analysis. Deoxyribonucleic acid fingerprinting of the Mycobacterium tuberculosis isolates using a standardized IS6110-based restriction fragment length polymorphism analysis and IS6110-independent DNA spoligotyping methods was done to track and identify specific bacterial strains. Deoxyribonucleic acid fingerprinting and spoligotyping done on isolates obtained from family members demonstrated same-strain transmission of M. tuberculosis. Three adults with active pulmonary disease and six individuals with latent tuberculosis (TB) were discovered during this investigation. The arrival of a family member from Mexico who had the same strain suggests that the source case lives in Mexico. A child with positive tuberculin skin test results indicates recent and potentially ongoing transmission of TB in the community. Targeted tuberculin skin testing performed on high-risk groups by primary care physicians allows for detection of TB infections. When TB infections are discovered in children, associate investigations can result in the discovery of undiagnosed adult cases and prevent further transmission within the community.     

二代测序技术应用于脑脊液检测在结核性脑膜炎中的早期诊断价值

目的评价二代测序技术应用于脑脊液检测在结核性脑膜炎（TBM）患者中的早期诊断价值。 方法前瞻性纳入2018年2月2日至2018年8月2日于山东省胸科医院就诊的临床怀疑TBM的患者共50例,并跟踪随访其诊疗结局。送检脑脊液标本均进行二代测序,测序所得原始序列与病原微生物数据库进行对比得到最终结果。二代测序结果以检测到结核分枝杆菌复合群唯一比对序列为阳性,未检测到唯一比对序列为阴性。以符合脑脊液结核分枝杆菌培养阳性、涂片阳性、Xpert MTB/RIF检测阳性及结核分枝杆菌核酸检测阳性等4项中至少1项即为确诊TBM患者;临床可疑TBM且抗结核治疗有效为临床诊断患者;有其他病原学依据或临床排除TBM者为非TBM患者。分析二代测序在TBM早期诊断中的敏感性和特异度。 结果确诊为TBM患者22例中Xpert MTB/RIF检测阳性13例,培养阳性6例,结核分枝杆菌核酸PCR检测阳性5例,临床诊断为TBM患者12例,非TBM患者16例。在确诊及临床诊断患者中,二代测序技术检测到结核分枝杆菌复合群系列20例,敏感性为58.8%（20/34）,特异度为100%（16/16）。在确诊患者中,二代测序的敏感性为63.6%（14/22）;在同步进行结核分枝杆菌培养、Xpert MTB/RIF检测与二代测序的50例标本中,以临床诊断为标准,3种方法的特异度均为100%（16/16）;传统方法、Xpert MTB/RIF检测及二代测序的敏感性分别为29.4%（10/34）、38.2（13/24）和58.8（20/34）,前两种检测方法与二代测序敏感性差异均有统计学意义（McNemar检验：χ² = 8.333、P = 0.013,χ² = 8.333、P = 0.065）。传统方法与二代测序联合检测的敏感性高达82.4%（28/34）。 结论二代测序技术能够较快速地检测脑脊液中的结核分枝杆菌复合群,且其敏感性和特异度均较高,可作为TBM的早期诊断指标。二代测序联合传统检测方法可提高检出率。     

γ-干扰素释放试验联合胸膜组织活检用于诊断结核性胸膜炎的评估

目的：评价全血γ-干扰素释放试验联合胸膜活检对结核性胸腔积液诊断的应用价值。方法应用全血γ-干扰素释放试验QuantiFERON-TB Gold In Tube（QFT-GIT）对79例胸腔积液患者进行检测,其中结核性胸腔积液患者45例,非结核性胸腔积液患者34例,结核合并肿瘤患者1例。同时,对其中27例患者进行胸腔镜下胸膜组织活检。结果结核性胸腔积液组,QFT-GIT的阳性率为91.1%（41/45）,非结核性胸腔积液组的阳性率为26.5%（9/34）。QFT-GIT试验诊断结核性胸腔积液的敏感性为91.1%（41/45）,特异性为73.5%（25/34）,阳性预测值为82.0%,阴性预测值为89.3%;胸膜组织活检诊断结核性胸膜炎的敏感性为96.3%（26/27）,特异性为100.0%（12/12）。联合胸膜活检诊断结核性胸膜炎的敏感性为95.5%（42/44）,特异性为94.1%（32/34）。结论全血γ-干扰素释放试验QFT-GIT联合胸膜组织活检用于诊断结核性胸腔积液具有较高的敏感性和特异性,在我国具有较高的临床应用价值。