Diagnosis of Active Tuberculosis in China Using an In-House Gamma Interferon Enzyme-Linked Immunospot Assay

Gamma interferon (IFN-γ) release assays have been proven to be useful in the diagnosis of Mycobacterium tuberculosis infection. Nevertheless, their specificity and sensitivity vary among the different populations studied. Here, we evaluate the value of an in-house IFN-γ enzyme-linked immunospot (ELISPOT) assay in the diagnosis of active tuberculosis (TB) in Shenzhen, China, where the prevalence of tuberculosis is severe and Mycobacterium bovis BCG vaccination is mandatory at birth. A total of 305 patients with active tuberculosis, 18 patients with nontuberculosis lung diseases, and 202 healthy controls were recruited in this study. Among them, 156 individuals were simultaneously tested for IFN-γ responses by the commercial QuantiFERON-TB Gold in-tube (QFT-IT) assay. Tuberculin skin tests (TST) were performed with 202 healthy controls. The overall sensitivities of the ELISPOT and QFT-IT assays for active tuberculosis were 83.60% and 80.85%, respectively; the specificities were 76.6% and 73.26%, respectively. The IFN-γ ELISPOT responses, but not those of the TST, were significantly correlated with TB exposure (r = −0.6040, P < 0.0001). The sensitivities of the ELISPOT assay varied for patients with different forms of tuberculosis, with the highest sensitivity for patients with sputum-positive pulmonary tuberculosis (89.89%) and the lowest for those with tuberculous meningitis (62.5%). In conclusion, the IFN-γ ELISPOT assay is a useful adjunct to current tests for diagnosis of active TB in China. The ELISPOT assay is more accurate than TST in identifying TB infections.Tuberculosis (TB) is a leading cause of morbidity and mortality throughout the world, with 95% of cases and 97% of all deaths occurring in high-prevalence countries, such as China, where the prevalence of active TB is as high as 367/100,000 population (10). For the effective and efficient control of TB in these countries, rapid diagnosis and treatment for active-TB patients are the mainstays of the TB control program. However, the current widely used tests, including acid-fast staining of sputum, mycobacterial culture, and antibody test, are not satisfactory for this purpose (4).Recently, commercial immunodiagnostic tests for TB infection have been introduced. These tests are based on the Mycobacterium tuberculosis-specific antigens early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) and include a whole-blood gamma interferon (IFN-γ) enzyme-linked immunosorbent assay (QuantiFERON-TB Gold in-tube [QFT-IT]; Cellestis Ltd, Victoria, Australia) and an enzyme-linked immunospot (ELISPOT) assay (T-SPOT.TB; Oxford Immunotec, Oxfordshire, United Kingdom). Both tests have shown promising results in the detection of latent TB infection (LTBI) (1, 11). In addition, some clinical data also suggest the potential to use these IFN-γ assays for the differential diagnoses of active tuberculosis (3, 8, 9). However, the sensitivities and specificities of these assays varied among the different populations studied, due mostly to the different HLA genetic backgrounds, the prevalence of TB infection, and the coverage of Mycobacterium bovis BCG vaccination (11).In contrast to the wide use of IFN-γ assays for the diagnosis of M. tuberculosis infection in Europe and America, the utilization of IFN-γ assays in China is scarce, and no data are available to evaluate the diagnostic value of the QFT-IT assay mainly because of the high cost of these kits. Since China ranks second on the list of 22 countries with the highest tuberculosis burden in the world (11), the aim of this study was to evaluate the usefulness of IFN-γ assays in the diagnosis of active tuberculosis in the Chinese population in mainland China. Thus, we developed and evaluated an in-house IFN-γ ELISPOT assay by using recombinant ESAT-6 protein and peptide pools derived from ESAT-6 and CFP-10 for diagnosis of active TB. We also compared the performance of our ELISPOT assay with that of the commercial QFT-IT assay and analyzed the influence of clinical manifestation on the accuracy of the ELISPOT assay.     

胸腔积液中肿瘤坏死因子和腺苷脱氨酶含量对结核性胸膜炎的诊断意义

目的：探讨肿瘤坏死因子（TNF）和腺苷脱氨酶（ADA）对结核性胸膜炎的诊断价值．方法：ADA采用比色法、TNF采用ELISA法对27例恶性胸腔积液、52例结核性胸腔积液进行检测．结果：结核性胸腔积液ADA和TNF水平都明显高于恶性胸腔积液,差别具有显著性（P＜0.01）．结论：应用TNF和ADA可能有助于结核性胸膜炎的鉴别诊断．     

Selected RD1 Peptides for Active Tuberculosis Diagnosis: Comparison of a Gamma Interferon Whole-Blood Enzyme-Linked Immunosorbent Assay and an Enzyme-Linked Immunospot Assay

We recently set up a gamma interferon (IFN-γ) enzyme-linked immunospot assay (ELISPOT), using selected early secreted antigenic target 6 (ESAT-6) peptides, that appears specific for active tuberculosis (A-TB). However, ELISPOT is difficult to automate. Thus, the objective of this study was to determine if the same selected peptides may be used in a technique more suitable for routine work in clinical laboratories, such as whole-blood enzyme-linked immunosorbent assay (WBE). For this purpose, 27 patients with A-TB and 41 control patients were enrolled. Our WBE, using the already described selected peptides from ESAT-6 plus three new ones from culture filtrate protein 10, was performed, and data were compared with those obtained by ELISPOT. Using our selected peptides, IFN-γ production, evaluated by both WBE and ELISPOT, was significantly higher in patients with A-TB than in controls (P < 0.0001). Statistical analysis showed a good correlation between the results obtained by WBE and ELISPOT (r = 0.80, P < 0.001). To substantiate our data, we compared our WBE results with those obtained by QuantiFERON-TB Gold, a whole-blood assay based on region of difference 1 (RD1) overlapping peptides approved for TB infection diagnosis. We observed a slightly higher sensitivity with QuantiFERON-TB Gold than with our WBE (89% versus 81%); however, our test provided a better specificity result (90% versus 68%). In conclusion, results obtained by WBE based on selected RD1 peptides significantly correlate with those generated by ELISPOT. Moreover, our assay appears more specific for A-TB diagnosis than QuantiFERON-TB Gold, and thus it may represent a complementary tool for A-TB diagnosis for routine use in clinical laboratories.     

Enzyme-Linked Immunospot Assay for Detection of Human Respiratory Syncytial Virus F Protein-Specific Gamma Interferon-Producing T Cells

Respiratory syncytial virus (RSV) causes significant disease in elderly adults, and we have previously reported that individuals 65 years of age and older have reduced RSV F protein-specific gamma interferon (IFN-γ)-producing T cells compared to healthy younger adults. To measure RSV F-specific memory T cell responses in the elderly following infection or vaccination, we optimized and qualified an IFN-γ enzyme-linked immunospot (ELISPOT) assay. Since peripheral blood mononuclear cells (PBMC) from the elderly could be more fragile, we established optimal cryopreservation techniques and minimal viability acceptance criteria. The number of cells per well, types and concentrations of stimulation antigens, and incubation times were evaluated to maximize assay sensitivity and precision. The optimized assay uses 300,000 cells/well, 2 μg/ml of an RSV F peptide pool (RSV Fpp), and incubation for 22 ± 2 h in serum-free CTL-Test medium. The assay was qualified by 3 analysts using 3 RSV F-responding donor PBMC samples (high, medium, and low responders) tested on 5 different assay days. The assay sensitivity or limit of detection (LOD) was determined to be 21 spot-forming cells (SFC) per 10⁶ PBMC, and the lower limit of quantitation (LLOQ) was estimated to be 63 SFC/10⁶ PBMC. The intra- and interassay percent coefficients of variation (CV) were <10.5% and <31%, respectively. The results of the qualification study demonstrate that a robust, precise, and sensitive IFN-γ ELISPOT assay has been developed that is fit for measuring RSV F-specific IFN-γ T cell responses in subjects enrolled in a vaccine clinical trial or in epidemiology studies.     

Interferon Gamma Release Assays for Diagnosis of Pleural Tuberculosis: a Systematic Review and Meta-Analysis

The role of interferon gamma release assays (IGRAs), although established for identifying latent tuberculosis, is still evolving in the diagnosis of active extrapulmonary tuberculosis. We systematically evaluated the diagnostic performance of blood- and pleural fluid-based IGRAs in tuberculous pleural effusion (TPE). We searched the PubMed and Embase databases for studies evaluating the use of commercially available IGRAs on blood and/or pleural fluid samples for diagnosing TPE. The quality of the studies included was assessed through the QUADAS-2 tool. The pooled estimates of sensitivity and specificity with 95% confidence intervals (95% CI) were generated using a bivariate random-effects model and examined using forest plots and hierarchical summary receiver operating characteristic (HSROC) curves. Indeterminate IGRA results were included for sensitivity calculations. Heterogeneity was explored through subgroup analysis and meta-regression based on prespecified covariates. We identified 19 studies assessing the T.SPOT.TB and/or QuantiFERON assays. There were 20 and 14 evaluations, respectively, of whole-blood and pleural fluid assays, involving 1,085 and 727 subjects, respectively. There was only one good-quality study, and five studies used nonstandard assay thresholds. The pooled sensitivity and specificity for the blood assays were 0.77 (95% CI, 0.71 to 0.83) and 0.71 (95% CI, 0.65 to 0.76), respectively. The pooled sensitivity and specificity for the pleural fluid assays were 0.72 (95% CI, 0.55 to 0.84) and 0.78 (95% CI, 0.65 to 0.87), respectively. There was considerable heterogeneity; however, multivariate meta-regression did not identify any covariate with significant influence. There was no publication bias for blood assays. We conclude that commercial IGRAs, performed either on whole-blood or pleural fluid samples, have poor diagnostic accuracy in patients suspected to have TPE.     

Diagnosis of Congenital Toxoplasmosis by Using a Whole-Blood Gamma Interferon Release Assay

Congenital toxoplasmosis in newborns is generally subclinical, but infected infants are at risk of developing ocular lesions. Diagnosis at birth relies mainly on serological tests. Cell-mediated immunity plays the major role in resistance to infection but is not routinely investigated for diagnostic purposes. Here, we describe a simple test based on the gamma interferon (IFN-γ) response after stimulation of whole blood by crude parasitic antigens. One milliliter of heparinized blood was centrifuged; plasma was kept for routine serological tests, and pellets were resuspended in culture medium. After 24 h of culture in the presence of crude Toxoplasma gondii antigen, the cells were centrifuged and the supernatant was assayed for IFN-γ. For 62 infants under 1 year of age born to mothers who were infected during pregnancy, the sensitivity and specificity of the test were 94% (with positive results for 16 of 17 infected infants) and 98% (with negative results for 44 of 45 uninfected infants), respectively. The false-negative result was for a treated baby who gave positive results after the withdrawal of treatment. The false positive was obtained for a 3-month-old baby. For a cohort of 124 congenitally infected patients between 1 and 30 years of age, the sensitivity of the assay was 100%. We present a simple test based on IFN-γ secretion to assess cell-mediated immunity in toxoplasmosis. As only 1 ml of blood is required to investigate humoral and cellular immunity, our assay is well adapted for the study of congenital toxoplasmosis in infants. Using purified antigens or recombinant peptides may improve the test performance.Toxoplasma gondii, a ubiquitous intracellular protozoan parasite, is an important cause of morbidity and mortality in congenitally infected individuals. Maternal infection may have serious consequences for the fetus (10). In other cases, infected newborns appear to be totally asymptomatic at birth but are at risk of developing retinal diseases during childhood or adolescence (26). For these patients, the diagnosis of the disease relies mainly on the detection of specific antibodies. Toxoplasma-specific immunoglobulin M (IgM) and IgA, which do not cross the placenta, are considered to be good markers of congenital infection. However, gestational age at maternal infection affects test performance (25), and at birth, the tests cannot detect more than 75% of infected babies (20). Because Toxoplasma-specific IgG crosses the placenta, its presence in the blood of newborns cannot be considered a marker of congenital infection. Maternally transferred IgG usually disappears within 6 to 12 months (20). Therefore, uninfected infants born to mothers who seroconverted during pregnancy have to undergo regular sampling for serological testing for 1 year before congenital toxoplasmosis can be ruled out (16). Clinicians are seeking valid indicators of congenital infection to improve clinical decision making. T. gondii infection results in long-lasting cell-mediated immunity which is highly dependent upon the effector activity of T lymphocytes that produce gamma interferon (IFN-γ) (6). Surprisingly, few studies have investigated the potential role of cell immunity in diagnosis of the disease. Data in the literature are contradictory. An absence of stimulation of lymphocytes by T. gondii antigen in congenitally infected children has been reported previously (18, 27). Recently, Guglietta et al. (13), using synthetic peptides, detected age-related impairment of the specific T-cell response to parasitic antigen in congenital infections. Conversely, a recent publication reported that evaluation of T-cell immunity is important for an early and accurate diagnosis of congenital toxoplasmosis (3). By detecting CD25 expression by flow cytometry, we demonstrated previously that specific cell immunity is detectable in almost all infected patients, including newborns (11). In this study, we evaluate the performance of a whole-blood IFN-γ release assay for the diagnosis of congenital toxoplasmosis. In this clinical setting, the quantity of blood required is limited, and we therefore looked at the possibility of separating plasma from blood cells in order to conduct serological tests (the “gold standard”) and the IFN-γ assay with the same sample.     

结核性胸膜炎的诊断进展

结核性胸膜炎是我国渗出性胸腔积液的最常见原因,本文对结核性胸膜炎各项诊断指标及诊断方法作一综述。     

DNA定量分析检测对胸腔积液的诊断价值研究

目的 探讨细胞DNA定量分析在不明原因胸水诊断中的应用价值。方法 收集本院2015年9月~2016年3月122例胸腔积液患者抽取胸水,标本制片后分别经巴氏染色行脱落细胞学检查,Feulgen染色后行细胞DNA定量分析。结果 122例胸腔积液中有56例恶性胸腔积液,66例良性胸腔积液。细胞学检测恶性胸水敏感度为82.14%,特异性为98.48%,阳性预测值97.87%,阴性预测值86.67%。细胞DNA定量分析检测恶性胸水敏感度为80.36%,特异度为90.91%,阳性预测值88.24%,阴性预测值84.51%。两种方法诊断结果一致性一般（Kappa=0.473,P＜0.001）,差异无统计学意义（P=1.0）。结论 DNA定量分析检测是一种较敏感而特异的肺癌的筛查技术,DNA异倍体有望作为良恶性胸水诊断有价值的标志物。     

ADA、ACE、LDH、CEA的联合检测在结核性和恶性胸腔积液中的鉴别诊断价值

目的测定血清和胸水中腺苷脱氨酶（ADA）、血管紧张素转化酶（ACE）、乳酸脱氢酶（LDH）与癌胚抗原（CEA）的水平,探讨其指标联合检测对结核性和恶性胸水的鉴别诊断意义。方法对临床已确诊的72例胸腔积液患者（结核性40例,恶性32例）的胸水和血清分别采用酶免疫法和化学发光法进行ADA、ACE、LDH和CEA含量测定。结果结核性胸水中ADA的含量为（60.2±20.10）U/L,ACE的含量为（35±9.6）U/L,LDH的含量为（338±41）U/L,CEA的含量为（12.8±5.82）μg/L;在恶性胸水中,ADA为（11.02±5.23）U/L,ACE为（16±11.0）U/L,LDH为（379±69.0）U/L,CEA为（39.9±19.7）μg/L。结核性胸水ADA和ACE含量较恶性胸水组明显增高（P〈0.01）,CEA在恶性胸水中含量较结核性胸水组明显增高（P〈0.01）。胸水中ADA和ACE的检测对结性性胸膜积液诊断的敏感性分别为84.3%、87.5%,特异性分别为87.5%、80.0%;而胸水中LDH和CEA的检测对恶性胸膜积液诊断的敏感分别为84.3%、75.0%,特异性分别为80.0%、93.0%。四项指标联合检测敏感性性为78.1%,特异性为97.5%,较单一指标的特异性高。结论胸水中ADA、ACE、LDH和CEA的联合检测对结核性和恶性胸水的鉴别诊断具有一定价值,有助于临床胸水性质的诊断。     

Chlamydia pneumoniae Induces Interferon Gamma Responses in Peripheral Blood Mononuclear Cells in Children with Allergic Asthma

Respiratory infections caused by Chlamydia pneumoniae have been associated with exacerbations of asthma. Cell‐mediated immunity (CMI) is critical for maintaining immunity. We compared interferon (IFN)‐γ responses in C. pneumoniae‐infected peripheral blood mononuclear cells (PBMC) in paediatric patients ± asthma. Presence of C. pneumoniae was tested from asthma patients (N = 17) and non‐asthmatic controls (N = 16) (PCR). PBMC were infected for 1 h ± C. pneumoniae AR‐39 (MOI = 0.1) and cultured for 48 h. IFN‐γ levels were measured in supernatants (ELISA). C. pneumoniae‐IgG antibodies in serum were determined (MIF). All subjects tested negative for C. pneumoniae (PCR). C. pneumoniae‐induced IFN‐γ production in vitro was more prevalent in asthma compared with non‐asthma; levels of IFN‐γ were higher in asthma compared with non‐asthma (P = 0.003). There was no association between recent respiratory infection and positive IFN‐γ responses. These data show that C. pneumoniae modulates IFN‐γ responses in patients with asthma, even in absence of active infection.     

Short-Term Reproducibility of a Commercial Interferon Gamma Release Assay

Interferon gamma release assays (IGRAs) have been shown to be sensitive and highly specific for the detection of immune memory against Mycobacterium tuberculosis. Little is known about the reproducibility and within-person variability of these assays. Various aspects of short-term reproducibility of a commercial IGRA, the QuantiFERON-TB Gold In-Tube (QFT-IT) assay, were assessed. The QFT-IT assay was performed twice within 3 days in 27 health care workers in Cape Town, South Africa. Two sets of tests were performed by different operators on day 1, and one set was performed on day 3. Aspects such as interoperator, intraoperator, day-to-day variability, and test-retest variability as well as different the storage methods of plasma were investigated. Seventeen of 27 (63%) of participants had at least one positive QFT-IT text; six had discordant results. The agreement of all aspects studied was high, with kappa values between 0.82 and 1.00 for dichotomous measures, and interclass correlations (ICC) of 0.809 to 0.965 were observed for continuous gamma interferon (IFN-γ) measures. The variability of the magnitude of response was highest comparing measures obtained from individuals on different days (ICC of 0.809). The magnitude of the IFN-γ responses between assays performed for individual participants was variable, with ranges from 0.03 to 11 IU/ml, resulting is discordant results for five participants. The results indicate that the QFT-IT assay is a robust and highly reproducible assay. Considerable intraindividual variability occurs in the magnitude of IFN-γ responses, which may influence the interpretation of serial measures.Commercial T-cell-based interferon gamma release assays (IGRAs) have been shown to be sensitive and highly specific for the detection of Mycobacterium tuberculosis infection (19). IGRAs have recently been incorporated into international guidelines for tuberculosis (TB) screening and diagnosis in several countries including in the United States, Canada, United Kingdom, Germany, and France, either as a confirmatory test for a positive tuberculin skin test (TST) or as replacement for the TST (2, 4, 8, 13, 15). It has further been suggested that IGRAs could be used for the serial measurement of gamma interferon (IFN-γ) responses to detect M. tuberculosis infection in high-risk populations such as health care workers and as a tool to monitor the response to treatment in individuals with active TB disease (measured through a decline in IFN-γ responses) (1, 3, 6, 10, 13, 17).Despite the increased use and availability of IGRAs, there are limited published data regarding the reproducibility of the two currently commercial assays, the QuantiFERON-TB Gold In-Tube (QFT-IT; Cellestis, Australia) and the T-SPOT.TB (Oxford Immunotec, United Kingdom) tests. In two recent publications, test-retest variability and within-person reproducibility of the QFT-IT assay were assessed over a period of 12 days and 3 months, respectively, focusing on test agreement, conversions, and reversions (20, 22). Little is known about the short-term within-person variation in T-cell IFN-γ responses. These could be nonspecific but may be important in the interpretation of serial measures and the definition of test conversion and reversion, especially if the risk of intercurrent M. tuberculosis exposure is low (14, 18).In addition to the need for data guiding the interpretation of serial QFT-IT measures, there are additional aspects of the QFT-IT test that require investigation. Although testing of samples by enzyme-linked immunosorbent assay (ELISA) is traditionally performed in duplicate or triplicate, the manufacturers of the QFT-IT assay recommend testing of a single sample per stimulation condition, and limited data are provided regarding test-retest variability. The robustness of these test measures could also be influenced by additional laboratory factors including interoperator and intraoperator variability and storage practices. We conducted a study to investigate the short-term reproducibility of the QFT-IT assay for the detection of M. tuberculosis infection.     

Mycobacterium Tuberculosis-Specific Memory NKT Cells in Patients with Tuberculous Pleurisy

Natural killer T (NKT) cells from mouse and human play a protective role in the immune responses against the infection of Mycobacterium tuberculosis. However, the characteristic of CD3⁺TCRvβ11⁺ NKT cells at the local site of M. tuberculosis infection remains poorly defined. In the present study, we found that the numbers of CD3⁺TCRvβ11⁺ NKT cells in pleural fluid mononuclear cells (PFMCs) were significantly lower than those in peripheral blood mononuclear cells (PBMCs). However, CD3⁺TCRvβ11⁺ NKT cells from PFMCs spontaneously expressed high levels of CD69 and CD25 and effector memory phenotypes of CD45RO^highCD62L^lowCCR7^low. After stimulation with the antigens of M. tuberculosis, CD3⁺TCRvβ11⁺ NKT cells from PFMCs produced high levels of IFN-γ. Sorted CD3⁺TCRvβ11⁺ NKT cells from PFMCs cultured with antigen presenting cells (APCs) produced IFN-γ protein and mRNA. The production of IFN-γ could be completely inhibited by AG490 and Wortmannin. In addition, CD3⁺TCRvβ11⁺ NKT cells from PFMCs expressed higher levels of Fas (CD95), FasL (CD178) and perforin but lower levels of granzyme B compared with those from PBMCs. Taken together, our data demonstrated for the first time that M. tuberculosis-specific CD3⁺TCRvβ11⁺ NKT cells participated in the local immune responses against M. tuberculosis through the production of IFN-γ and the secretion of cytolytic molecules.     

AM、CYFRA21-1、NSE和CEA联合检测对恶性与结核性胸腔积液的鉴别诊断价值

用RIA检测了52例(男32例, 女20例)结核性胸腔积液组和74例(男49例,女25例)恶性胸腔积液组的肾上腺髓质素(AM)、CYFRA21-1、NSE和CEA水平,结果表明: 恶性胸腔积液组四项肿瘤标志物含量均显著高于结核性胸腔积液组, 四项联合检测可进一步提高诊断敏感性和准确性至90.5%和92.9%, 有显著性差异(P＜0.01).结论: 胸腔积液AM、 CYFRA21-1、NSE和CEA联合检测对鉴别恶性与结核性胸腔积液有实用价值, 可显著提高恶性胸腔积液的阳性诊断率.     

结核性胸膜炎患者腺苷脱氨酶与总蛋白、乳酸脱氢酶及其比值的相关性分析

目的 探讨胸水与血清中的腺苷脱氨酶(ADA)与总蛋白(TP)、乳酸脱氢酶(LDH)及其比值在结核性胸膜炎患者中的相关性.方法 随机抽取2014年上半年本院收治的结核性胸膜炎患者50例(结核组),肝性胸水(对照组)20例,进行胸水和血清的ADA、TP、LDH检测,计算其相应的比值,分析胸水和血清中ADA、TP、LDH及其比值之间的相关性.结果 结核组中ADA、TP、LDH含量及其比值明显高于对照组,差异具有统计学意义(P＜0.05).结核组的胸水ADA与TP、LDH呈直线正相关(P＜0.01);血清ADA与TP呈直线正相关(P＜0.01),与LDH无相关(P＞0.05);胸水ADA与血清ADA的比值(PADA/SADA)与胸水LDH与血清LDH的比值(PLDH/SLDH)呈正相关(P＜0.01),与胸水TP与血清TP比值(PTP/STP)无相关(P＞0.05).结论 胸水与血清中的ADA、TP、LDH及其比值在结核性胸膜炎诊断中具有重要的临床意义.     

可曲式内科胸腔镜在结核性胸膜炎诊断中的价值

目的 探讨可曲式内科胸腔镜在提高结核性胸膜炎诊断准确性方面的应用价值.方法 回顾性分析2012年1月至2014年12月确诊的60例结核性胸膜炎患者的胸水结核分枝杆菌培养、结核分枝杆菌实时荧光定量聚合酶链反应(TB-PCR)和胸腔镜的检查结果.结果 60例结核性胸膜炎患者中胸腔镜胸膜活检阳性率85.00％ (51/60),明显高于培养法的1.67％ (1/60)和TB-PCR法的8.33％ (5/60),差异均有统计学意义(P ＜0.001).结论 可曲式内科胸腔镜检查在结核性胸膜炎的诊断中有重要价值.     

Release of Interleukin 2 and Gamma Interferon by Peripheral Mononuclear Cells in Human Schistosoma mansoni Infection Normalizes after Chemotherapy

Interleukin 2 (IL-2) and gamma interferon (IFN-gamma) were determined in supernatants of mitogen- and antigen-driven cell cultures from patients with hepatosplenic or intestinal schistosomiasis. Skin reactivity was tested using a panel of eight recall antigens. Results were compared with those of uninfected local controls. In both schistosomiasis groups, IL-2 activity was reduced before treatment. In less than one third of the patients, schistosomal antigens elicited detectable IL-2 activity. IFN-gamma production was reduced more severely in hepatosplenic cases, in particular after stimulation by anti-CD3 monoclonal antibodies. After anti-schistosomal therapy with praziquantel, mitogen-induced IL-2 and IFN-gamma activities became normal within 3 months in intestinal schistosomiasis, and within 6 months in the hepatosplenic patient group. Results of in vivo delayed-type hypersensitivity tests paralleled those of in vitro lymphokine production. In conclusion, evidence is presented for severe, antigen-unspecific suppression of lymphokine production and skin reactivity against recall antigens. Anti-parasitic chemotherapy is shown to reverse the impairment of cell-mediated immune responses at the cytokine level.     

Optimization of a Whole-Blood Gamma Interferon Assay for Detection of Mycobacterium bovis-Infected Cattle

Antigens of Mycobacterium bovis elicit a cell-mediated immune response upon intradermal injection in cattle. In vitro, such antigens stimulate the production of gamma interferon (IFN-γ) by bovine T cells in whole-blood culture (IFN-γ assay). We have analyzed various parameters of the in vitro IFN-γ assay, ranging from blood sampling to execution of the IFN-γ test, in view of potential simplifications of the assay. Here, we show that IFN-γ responses may be reduced under certain animal handling/holding conditions and that a delayed time from blood collection to culture may lead to a reduced in vitro IFN-γ response. Delayed initiation of culture in a purified-protein-derivative-based assay (24 h compared to 8 h after blood collection), however, resulted in a significant improvement of specificity (97% compared to 85%), whereas there was only a modest reduction of sensitivity (from 96% to 90%), which was statistically not significant. Furthermore, we show that the stimulation temperature needs to be 33°C or higher; that carbon dioxide is not required for stimulation; and that various plate formats, ranging from 24 to 96 wells per plate, can be utilized. The produced IFN-γ is stable at 4°C for 28 days as well as after repeated freeze-thaw cycles. Thus, stimulation of samples may be initiated in the field without the need for a carbon dioxide source, and bovine IFN-γ is stable under various routine laboratory temperature scenarios. These findings demonstrate opportunities for improvements in the bovine IFN-γ test platform and flexibilities in test application.Bovine tuberculosis (TB), caused by Mycobacterium bovis, has an important and adverse effect on socioeconomic conditions, public health, and trade of animals and animal products (2). Eradication of bovine TB in cattle is based on detection and slaughter of infected animals or whole herds. The standard antemortem screening test for detection of TB is the intradermal tuberculin skin test (i.e., intradermal injection of tuberculin eliciting a cell-mediated immune response [CMI] at the site, which in turn leads to skin thickening). As an alternative, the CMI can be measured in vitro by stimulating blood cells with tuberculin, which in turn leads to production of gamma interferon (IFN-γ), which can then be quantified by an enzyme-linked immunosorbent assay (ELISA; Bovigam IFN-γ assay) (15).The Bovigam assay constitutes a laboratory-based TB test and is widely used complementarily to the tuberculin skin test (4, 11), as it offers national TB control programs and industry an additional tool for curtailing the spread of TB in cattle and other Bovidae. The assay critically depends on the sample quality, culture conditions, and quality control of stimulation reagents. The CMI, both in vivo and in vitro, may be negatively affected by stress or corticosteroid application (5). Thus, parallel stimulation of blood leukocytes with mitogen or superantigen in the IFN-γ assay is commonly used as an indicator of sample quality and potential for underlying CMI suppression, thereby reducing the risk of false-negative test results. Conditions such as the anticoagulant used for blood collection, the temperature and time of blood storage, and the culture duration also affect IFN-γ production in whole-blood culture (8). Furthermore, delays in culture setup, initial high sample temperatures (2 h at 37°C, followed by 22 h at 22°C, prior to 24 h of culture at 37°C), or a low sample temperature (4°C) diminishes IFN-γ responses with samples from M. bovis-infected cattle (14). Thus, sample quality, as affected by pre- and postcollection parameters, affects the accuracy of the IFN-γ test. Once the sample reaches the laboratory, additional variables, such as the culture plate format, the culture conditions, the antigens used for TB-specific stimulation, the nonspecific stimulation control reagents, and the cutoff for the final test interpretation, may also influence test performance. While the ability to modify IFN-γ test parameters offers the possibility to adapt the assay more closely to the needs within a TB program, this option also provides challenges to ensure standardization of testing procedures and quality assurance. Indeed, variations in assay protocols, with both the skin test and the IFN-γ test, have resulted in disparate results in test accuracy between studies (reviewed in reference 4).We therefore analyzed the capacities of animals under various field conditions to produce IFN-γ. Furthermore, we evaluated different parameters of the in vitro assay, including the influence of time to culture initiation, culture vessel geometry, the cell culture temperature, the need for carbon dioxide (generally used in tissue culture to stabilize pH), the animal holding conditions, and the stability of the produced IFN-γ under various standard storage scenarios. All of these parameters were analyzed in view of defining a range of possible conditions and potentially simplifying the assay for use in bovine TB eradication programs.