结核感染T细胞斑点试验对肺外结核诊断价值探讨

目的评价结核感染T细胞斑点试验（T—SPOT．TB）在肺外结核中的诊断价值。方法采用T—SPOT．TB试剂盒对疑诊或待排结核患者外周血中特异性T淋巴细胞进行检测。结果结核感染T细胞斑点试验的对结核病的敏感度、特异度分别为76．7％、84．3％。肺外结核组与肺结核组阳性率分别为92．0％和78．2％,差异有统计学意义（P〈0．05）。该数据显著高于结核菌素试验的31．4％和结核分枝杆菌培养的19．3％,差异有统计学意义（P〈0．05）。结论结核感染T细胞斑点试验是诊断肺外结核的快速敏感方法,值得在临床中推广使用。     

QuantiFERON-TB GOLD ELISA assay for the detection of Mycobacterium tuberculosis-specific antigens in blood specimens of HIV-positive patients in a high-burden country

Tuberculosis is a life-threatening infection worldwide. Despite improvements in therapy, it results in 2 million deaths and 9 million new cases annually. This study evaluated the use of the QuantiFERON-TB GOLD enzyme-linked immunosorbent assay in a high HIV/TB burden setting in an ARV clinic at the Tshwane District Hospital, South Africa. The sensitivity and specificity of the QF assay in the clinic were 30% (9/30) and 63% (19/30), respectively, when compared with the gold standard culture results. Analysis also suggested that the sensitivity of the QuantiFERON assay is determined by a limiting patient CD4 value between 150 and 200.     

Determining Mycobacterium tuberculosis Infection among BCG-Immunised Ugandan Children by T-SPOT.TB and Tuberculin Skin Testing

Background

Children with latent tuberculosis infection (LTBI) represent a huge reservoir for future disease. We wished to determine Mycobacterium tuberculosis (M.tb) infection prevalence among BCG-immunised five-year-old children in Entebbe, Uganda, but there are limited data on the performance of immunoassays for diagnosis of tuberculosis infection in children in endemic settings. We therefore evaluated agreement between a commercial interferon gamma release assay (T-SPOT.TB) and the tuberculin skin test (TST; 2 units RT-23 tuberculin; positive defined as diameter ≥10 mm), along with the reproducibility of T-SPOT.TB on short-term follow-up, in this population.

Methodology/Principal Findings

We recruited 907 children of which 56 were household contacts of TB patients. They were tested with T-SPOT.TB at age five years and then re-examined with T-SPOT.TB (n = 405) and TST (n = 319) approximately three weeks later. The principal outcome measures were T-SPOT.TB and TST positivity. At five years, 88 (9.7%) children tested positive by T-SPOT.TB. More than half of those that were T-SPOT.TB positive at five years were negative at follow-up, whereas 96% of baseline negatives were consistently negative. We observed somewhat better agreement between initial and follow-up T-SPOT.TB results among household TB contacts (κ = 0.77) than among non-contacts (κ = 0.39). Agreement between T-SPOT.TB and TST was weak (κ = 0.28 and κ = 0.40 for T-SPOT.TB at 5 years and follow-up, respectively). Of 28 children who were positive on both T-SPOT.TB tests, 14 (50%) had a negative TST. Analysis of spot counts showed high levels of instability in responses between baseline and follow-up, indicating variability in circulating numbers of T cells specific for certain M.tb antigens.

Conclusions/Significance

We found that T-SPOT.TB positives are unstable over a three-week follow-up interval, and that TST compares poorly with T-SPOT.TB, making the categorisation of children as TB-infected or TB-uninfected difficult. Existing tools for the diagnosis of TB infection are unsatisfactory in determining infection among children in this setting.     

结核感染T细胞斑点试验对肺结核病的临床诊断价值研究

目的：评价结核感染T细胞斑点试验（T-SPOT．TB）对肺结核病的临床诊断价值。方法：选择2012年2月～8月湘雅医院呼吸科住院病人中92例可疑肺结核患者进行T—SPOT．TB检测、结核菌素试验（PPD试验）、结核抗体、血沉及影像学检查及病史收集。分析和比较T．SPOT．TB与传统结核诊断方法的阳性率、特异度、灵敏度并对其检测结果进行相关性分析。结果：92例患者中,48例被确诊为肺结核,其中41例T．SPOT．TB结果阳性,44例非肺结核患者中5例T．SPOT．TB结果阳性。T—SPOT．TB检测的敏感度为85．4％,特异度为88．6％。T-SPOT．TB检测在结核组的阳性检出率（85．4％）显著高于传统检查方法PPD（37．5％,P〈0．01）、结核抗体（16．7％,P〈0．01）、血沉（66．7％,P〈0．05）,在非结核病组中的特异性（88．6％）显著高于血沉（36．6％,P〈0．0J）。PPD与T-SPOT．TB联合可提高诊断结核的阳性率（89．6％）。T．SPOT．TB检测仅与PPD试验的结果存在显著性差异（P〈0．05）。结论：T-SPOT．TB诊断肺结核的敏感性及特异性较传统的PPD实验、结核抗体更高,具有重要的I临床应用价值。     

Detection of in vitro and in vivo released antigens of diagnostic interest in Mycobacterium tuberculosis by immunoblotting

Identification of in vitro and in vivo released mycobacterial antigens are of considerable interest in diagnosis of Mycobacterium tuberculosis. Isolation of in vitro released antigen from M. tb excretory-secretory culture filtrate protein and in vivo released circulating tuberculous antigen from smear positive pulmonary tuberculosis sera by ammonium sulphate precipitation is reported. The antigens were resolved by SDS-PAGE and immunoblotting was performed using pooled serum of smear positive, smear negative pulmonary tuberculosis sera and normal sera to identify reactive tuberculous antigens. In vitro and in vivo released mycobacterial antigens showed reactivity at 100, 31, 43 and 20 kDa with smear positive and smear negative pulmonary tuberculosis patients. Further, the in vitro released antigen showed strong reactivity exclusively at 55 kDa antigen with smear positive and 24 kDa antigen with smear negative pulmonary tuberculosis sera. In vivo released antigen reacted exclusively at 170 and 16 kDa with smear positive and 19 kDa antigen with smear negative pulmonary tuberculosis patients. Antigens of 24 and 19 kDa which are reactive with sputum negative sera will be of diagnostic interest and need further study in patients with low bacillary load. The in vitro and in vivo released mycobacterial 100, 31,43 and 20 kDa antigens, reactive with patients sera are of diagnostic interest in tuberculosis.     

Circumventing T-cell tolerance to tumour antigens.

During past decades, many attempts have been made to induce or enhance tumour-specific T-cell immunity in cancer patients by vaccination. However, it has become apparent that in a large number of cases the naturally occurring tumour-specific T-cell repertoire is of low affinity and therefore inefficient in mediating tumour rejection. Because of the potential therapeutic value of high affinity TCRs with tumour/lineage specificities, we set out to develop a number of new technologies that can be used to create improved tumour-specific T-cell immunity. These strategies entail: (i) the efficient expansion of low affinity T cells specific for self antigens through the use of variant peptides with improved TCR-binding characteristics; (ii) a retroviral library-based technology to improve the affinity of (self-specific) T-cell receptors in vitro, and (iii) proof of principle for the feasibility of TCR gene transfer as a means to generate T-cell populations with a desired antigen-specificity in vivo. Collectively this toolbox should allow us to create improved T-cell receptors for human tumour antigens, which can subsequently be used to impose tumour-reactivity on to peripheral T cells.     

Post-PCR sterilization: development and application to an HIV-1 diagnostic assay.

We have developed an effective post-PCR sterilization process and have applied the procedure to a diagnostic assay for HIV-1. The method, which is based on isopsoralen photochemistry, satisfies both the inactivation and hybridization requirements of a practical sterilization process. The key feature of the technique is the use of isopsoralen compounds which form covalent photochemical adducts with DNA. These covalent adducts prevent subsequent extension of previously amplified sequences (amplicons) by Taq polymerase. Isopsoralens have minimal inhibitory effect on the PCR, are activated by long wavelength ultraviolet light, provide sufficient numbers of covalent adducts to impart effective sterilization, modify the amplified sequence such that it remains single-stranded, and have little effect on subsequent hybridization. The sterilization procedure can be applied to a closed system and is suitable for use with commonly used detection formats. The photochemical sterilization protocol we have devised is an effective and pragmatic method for eliminating the amplicon carryover problem associated with the PCR. While the work described here is limited to HIV-1, proper use of the technique will relieve the concern associated with carryover for a wide variety of amplicons, especially in the clinical setting.     

T细胞斑点试验反应强度在诊断活动性结核病中的价值

目的探讨结核感染T细胞免疫斑点试验的反应强度与活动性结核病的关系。方法选取2012年6月1日至2014年12月31日期间在重庆医科大学附属第一医院呼吸科342例的住院患者,所有住院患者均进行了结核感染T细胞斑点试验(T-SPOT.TB)。分析T-SPOT.TB对结核病诊断的敏感性、特异性、阳性预测值、阴性预测值;同时比较在活动性肺内结核病组、活动性肺外结核病组及陈旧性结核病组的T-SPOT.TB的免疫强度。结果 T-SPOT.TB检测结核病的敏感性、特异性、阳性预测值、阴性预测值分别为:87.67%,79.19%,64.00%,93.84%。在活动性肺内结核病组中T-SPOT.TB免疫斑点数为抗原A(10,36),抗原B(11,50),在活动性肺外结核病组中为抗原A(15,50),抗原B(15,40);在陈旧性结核病组中为抗原A(6,20),抗原B(6,30)。比较活动性肺内结核病组、肺外结核病组及陈旧性结核病组T-SPOT.TB的免疫斑点数,发现活动性肺内结核病组与活动性肺外结核病组免疫强度强于陈旧性结核病组(H_A=0.015,H_B=0.012,P0.05;H_A=0.006,H_B=0.006,P0.05)。结论 T-SPOT.TB对结核病的诊断有较高的灵敏度和特异性,T-SPOT.TB阴性时可有效的排除结核分枝杆菌的感染,阳性时有利于辅助诊断活动性结核病。     

Epstein-Barr virus-transformed B cells process and present Mycobacterium tuberculosis particulate antigens to T-cell clones

We have analyzed the presentation of mycobacterial antigens by Epstein-Barr virus-transformed human B (EBV-B) cells to mycobacteria-specific T-cell clones and lines, and to purified resting T cells. EBV-B cells were able to process and present not only soluble forms of antigen, such as PPD and the expressate preparation of M. tuberculosis strain H37Rv, but also particulate forms of antigen, such as whole mycobacterial H37Rv or M. bovis organisms. Electron microscopy studies demonstrated the capacity of EBV-B cells to phagocytose mycobacterial cells in 18 hr and pulsing experiments confirmed that an 18-hr of incubation is required for an efficient processing and presentation of mycobacterial determinants to T cells. The processing of whole-H37Rv particulate antigen by EBV-B cells was inhibited by the lysosomotrophic compound chloroquine and by high doses of irradiation. Finally, the analysis of the presentation of soluble and particulate mycobacterial antigens by PPD-positive and PPD-negative EBV-B cell clones has shown a preferential presentation of both forms of antigen by PPD-positive EBV-B clones.     

Whole blood assay to access T cell-immune responses to Mycobacterium tuberculosis antigens in healthy Brazilian individuals

The production of interferon gamma (IFNgamma) guarantees effective T cell-mediated immunity against Mycobacterium tuberculosis infection. In the present study, we simply compare the in vitro immune responses to Mycobacterium antigens in terms of IFNg production in a total of 10 healthy Brazilian volunteers. Whole blood and mononuclear cells were cultivated in parallel with PPD, Ag85B, and M. bovis hsp65, and five-days supernatants were harvested for cytokine detection by ELISA. The inter-assay result was that the overall profile of agreement in response to antigens was highly correlated (r(2) = 0.9266; p = 0.0102). Potential analysis is in current progress to dictate the usefulness of this method to access the immune responses also in tuberculosis patients and its contacts.     

Use of T-SPOT.TB in latent tuberculosis infection diagnosis in general and immunosuppressed populations

Immunosuppressed patients have a nine-fold greater risk of developing active tuberculosis (TB) disease given the latent TB infection than the general population. Few data are available on the predictivity of T-SPOT.TB in immunosuppressed patients. We had a T-SPOT.TB determination and a TST from 197 immunosuppressed haematological patients and 324 community contacts of infectious TB cases. In the general population, TST was positive in 275 (84.9%), T-SPOT.TB in 167 (51.5%) (p < 0.0001). In immunosuppressed patients, TST was positive in 34 (17.3%), T-SPOT.TB in 70 (35.5%). T-SPOT.TB is not influenced by immunosuppression and even an indeterminate result may yield useful information on patient's anergy.     

Autoimmunity to glial antigens in vitro.

Lymph nodes cells and spleen cells in a 50:50 mixture from Fischer 344 rats were cultured on syngeneic glial cells and fibroblasts. In the glia cultures, but not in the fibroblast cultures, the lymphocytes were stimulated to a vivid blast transformation and mitotic activity with a peak after 7 days, after which they reverted to small- and medium-sized lymphocytes. The stimulated lymphoid cells were not cytotoxic to glial cells when tested in a microcytotoxicity assay. A fraction of the lymphoblasts and their progeny (approximately 4%) took a positive intracytoplasmic stain for γ-globulin immunoglobulin G after direct immunofluorescence. Efforts to demonstrate immunoglobulin produced and released into the culture medium by the stimulated cells were negative. The findings may indicate that among the lymphoid cells responding to the glial antigens under these conditions, there are suppressor cells that abrogate the killer cell effect.     

Induction of an antibody response in vitro against synthetic antigens in guinea pigs. I. Culture and assay conditions

Guinea pig spleen and lymph node cells were found to produce anti-2,4-dinitrophenyl (Dnp) oligolysine PFC in vivo against 2,4-dinitrophenyl-β-alanyl glycyl glycyl (Dagg-SRBC) but not against trinitrophenyl-SRBC target indicator cells. Furthermore, when sensitized spleen cells or their purified B-cell fractions were cocultured with primed peritoneal exudate lymphocytes (PEL) but not splenic T cells they were able to generate a secondary PFC response in vitro to the synthetic antigens, Dnp oligolysines. PFC were not induced in vitro if these same cultures were pulsed with short-chain peptides (five lysines) or the complex antigen, dinitrophenyl-bovine γ-globulin (DnpBGG). Con A was able to substitute for PEL in triggering spleen cells to mount a secondary in vitro PFC response to homologous Dnp oligolysines. More importantly, the Con A-aided spleen cell cultures were not induced above background values when challenged in vitro with heterologous Dnp oligolysines. This study suggests that spleen cells may lack a nonspecific signal for the development of a secondary in vitro PFC response.     

A three-way comparison of tuberculin skin testing, QuantiFERON-TB gold and T-SPOT.TB in children

Background

There are limited data comparing the performance of the two commercially available interferon gamma (IFN-γ) release assays (IGRAs) for the diagnosis of tuberculosis (TB) in children. We compared QuantiFERON-TB gold In Tube (QFT-IT), T-SPOT.TB and the tuberculin skin test (TST) in children at risk for latent TB infection or TB disease.

Methods and Findings

The results of both IGRAs were compared with diagnosis assigned by TST-based criteria and assessed in relation to TB contact history. Results from the TST and at least one assay were available for 96 of 100 children. Agreement between QFT-IT and T-SPOT.TB was high (93% agreement, κ = 0.83). QFT-IT and T-SPOT.TB tests were positive in 8 (89%) and 9 (100%) children with suspected active TB disease. There was moderate agreement between TST and either QFT-IT (75%, κ = 0.50) or T-SPOT.TB (75%, κ = 0.51). Among 38 children with TST-defined latent TB infection, QFT-IT gold and T-SPOT.TB assays were positive in 47% and 39% respectively. Three TST-negative children were positive by at least one IGRA. Children with a TB contact were more likely than children without a TB contact to have a positive IGRA (QFT-IT LR 3.9; T-SPOT.TB LR 3.9) and a positive TST (LR 1.4). Multivariate linear regression analysis showed that the magnitude of both TST induration and IGRA IFN-γ responses was significantly influenced by TB contact history, but only the TST was influenced by age.

Conclusions

Although a high level of agreement between the IGRAs was observed, they are commonly discordant with the TST. The correct interpretation of a negative assay in a child with a positive skin test in clinical practice remains challenging and highlights the need for longitudinal studies to determine the negative predictive value of IGRAs.     

In vitro affinity maturation of an anti-PSA antibody for prostate cancer diagnostic assay

Prostate-specific antigen (PSA) is a serum marker that is widely used for the diagnosis of prostatic diseases. Various subforms of free PSA, which are associated with prostate cancer differently, have been identified in sera. Thus, specific detection of certain subforms could permit discrimination between benign and malignant cases. Although the monoclonal antibody 5D3D11 displays the desired selectivity, its relative weak binding affinity prevents its development into an effective diagnostic tool. The directed-evolution strategy presented here succeeds in enhancing affinity and immunoassay sensitivity while maintaining selectivity.Starting without structural data, we constructed four independent phage-display single-chain variable fragment (scFv) libraries targeting hot spots from CDR-L1, H1, H2, and H3. Mutations derived from each library were combined, yielding further affinity gains. This constitutes the first demonstration of additivity for independently selected complementarity-determining region (CDR) hot-spot mutations. The X-ray structure of the Fab′ 5D3D11-PSA complex (after it became available) inspired the design of two new libraries targeting CDR-L3 that resulted in other higher-affinity variants. Attempts at combining the new variants with previous ones did not result in further gains, suggesting that mutations from the two strategies provide alternative but noncomplementary solutions for affinity enhancement of 5D3D11. The results can be interpreted to provide a plausible explanation for the observed lack of additivity.Finally, with respect to the wild-type scFv, the best binders show an enhancement of sensitivity in sandwich immunoassay. Its ability to discriminate between prostate cancer sera and benign prostatic hyperplasia sera has now been confirmed through the dosage of 63 patients.     

Iso-FRET: an isothermal competition assay to analyze quadruplex formation in vitro

Algorithms have been widely used to predict G-quadruplexes (G4s)-prone sequences. However, an experimental validation of these predictions is generally required. We previously reported a high-throughput technique to evidence G4 formation in vitro called FRET-MC. This method, while convenient and reproducible, has one known weakness: its inability to pin point G4 motifs of low thermal stability. As such quadruplexes may still be biologically relevant if formed at physiological temperature, we wanted to develop an independent assay to overcome this limitation. To this aim, we introduced an isothermal version of the competition assay, called iso-FRET, based on a duplex-quadruplex competition and a well-characterized bis-quinolinium G4 ligand, PhenDC3. G4-forming competitors act as decoys for PhenDC3, lowering its ability to stabilize the G4-forming motif reporter oligonucleotide conjugated to a fluorescence quencher (37Q). The decrease in available G4 ligand concentration restores the ability of 37Q to hybridize to its FAM-labeled short complementary C-rich strand (F22), leading to a decrease in fluorescence signal. In contrast, when no G4-forming competitor is present, PhenDC3 remains available to stabilize the 37Q quadruplex, preventing the formation of the F22 + 37Q complex. Iso-FRET was first applied to a reference panel of 70 sequences, and then used to investigate 23 different viral sequences.     

The development of an enzyme-linked immunosorbent assay for measuring the potency of vaccines containing Clostridium chauvoei antigens

Current standards (British Pharmacopeia (Veterinary) 1985) for vaccines containing Clostridium chauvoei require a potency test based on a challenge assay in guinea-pigs. Animal welfare and cost considerations favour the development of alternatives. Most veterinary clostridial vaccines are multi component, requiring assays in rabbits to test the potency of components other than C. chauvoei. We describe the application of an ELISA to measure the response to C. chauvoei vaccines in rabbits. The antigen is a sonicated extract of C. chauvoei strain CH4, intended to include a mixture of cellular and soluble antigens. The rabbit response to more than 70 vaccines containing C. chauvoei has been assessed against a reference serum which has been assigned an arbitrary potency of 100 units ml-1. The antibody titres of rabbit sera have been compared with the results of guinea-pig challenge potency tests on the same vaccines. The pass level in the guinea-pig potency test is equivalent to a rabbit ELISA titre of 50 units ml-1.     

Immunoreactivity of five antigens of Mycobacterium tuberculosis in patients attending a public health care facility in an area with high endemicity for TB

The objective of this study was to evaluate people attending a primary health clinic in Rio de Janeiro, Brazil for immunoreactivity to five Mycobacterium tuberculosis antigens, as these antigens are markers of immune response and factors associated with active TB. The serum antibody titers of different categories of patients (defined by microbiological and radiological characteristics and by response to therapy on follow‐up) to 38 kDa, 16 kDa, MPT64, ESAT‐6 and MT10.3 antigens were determined blind with ELISA. Positive tests to each antigen were defined with ROC analysis. OR were calculated for factors associated with humoral response in patients with active TB. A total of 201 patients underwent serological testing. Patients with confirmed active TB responded more frequently to MPT64 (44%), 16 kDa (37.7%) and 38 kDa (36.1%). ESAT‐6 and MT10.3 were also able to distinguish people in TB groups from controls. TB infected subjects responded less frequently to ESAT‐6 and MT10.3 (3.7% and 11%, respectively). Sensitivity and specificity to all antigens combined were 58.4% and 60.7%, respectively. Reactivity to 38 kDa and to MPT64 was more likely among alcohol users OR 2.61 (95%CI;1.05–6.94) and OR 3.27 (95%CI;1.33–8.15), respectively. 16 kDa antigen elicited a more protective response among smokers, OR 0.29 (95%CI; 0.10–0.83). It was concluded that reactivity to all antigens tested represented markers of active disease. ESAT‐6 and MT10.3 could not be identified as markers of TB infection in this community. Sensitivity was higher to all antigens combined, but at a cost of lower specificity. Interestingly, among factors associated with positive immunoreactivity, alcohol use and smoking seem to polarize the humoral response in different directions. This finding deserves further investigation.