Evaluation of an In Vitro Assay for Gamma Interferon Production in Response to Mycobacterium tuberculosis Infections

The tuberculin skin test (TST) is the “gold standard” for detecting infection with Mycobacterium tuberculosis. We compared the TST using purified protein derivative to the QuantiFERON-TB test (QFT). Two groups were examined. Group 1 individuals (n = 66) (low risk) were at low risk for exposure to M. tuberculosis and were not Mycobacterium bovis BCG vaccinated. Group 2 (n = 29) include individuals who were likely to have been exposed to a high prevalence of M. tuberculosis infections and were BCG vaccinated. Group 1 individuals were given a TST. Group 2 individuals were not given a TST because of possible adverse reactions. A 10- to 15-mm indurated area 48 h after TST was considered positive. A positive QFT result was defined as a significant gamma interferon response to M. tuberculosis antigen, Mycobacterium avium antigen, and a nonspecific mitogen stimulus and no response in the negative control. In group 1, 60 of 66 individuals (90.9%) were negative by both methods, and 1 person was positive by both methods. There was one QFT-negative, TST-positive case, one QFT-positive, TST-negative case, and three conditional QFT-positive, TST-negative cases. In group 2, 12 of 29 (41.4%) were positive by QFT and considered likely to be TST positive because of prior BCG vaccination. QFT testing in our low-risk group resulted in an agreement of 96.8%, a sensitivity of 50%, and a specificity of 98.4% compared with TST results. QFT testing with TST in low-risk groups can aid in the detection of latent M. tuberculosis infections.     

Development and Validation of a Gamma Interferon ELISPOT Assay for Quantitation of Cellular Immune Responses to Varicella-Zoster Virus

Cell-mediated immunity appears to be critical for the prevention and control of varicella-zoster virus (VZV) infection and complications arising from zoster. Current assays of VZV-specific cell-mediated immunity are cumbersome or lack sensitivity. We have developed a gamma interferon ELISPOT assay that provides a direct measure of the number of T cells secreting a cytokine following stimulation with antigen. This assay is extremely sensitive and specific, with the ability to detect gamma interferon spot-forming cells (SFC) in the range of 10 to 1,000 SFC per million peripheral blood mononuclear cells (PBMCs). This assay has been validated by demonstrating the following: (i) the response detected is mediated almost entirely by CD4⁺ T cells, (ii) ELISPOT responses from fresh-frozen PBMCs are equivalent to those from freshly isolated cells, (iii) frozen PBMCs can be shipped on dry ice for up to 48 h without loss of activity, (iv) frozen PBMC samples can be stored in liquid nitrogen over long periods (>22 months) without any significant change in response, and (v) the numbers of ELISPOTs counted using a computer-based imaging system are equivalent to those counted by humans but have lower variability. The ability to use frozen cells is facilitated by the use of a recombinant nuclease (Benzonase) that can prevent cell clumping when samples are thawed. Frozen PBMC samples can be cycled through multiple changes in storage between liquid nitrogen and dry ice without any change in response being detected. This facilitates collection of samples at one site and testing performed at a remote location. This VZV ELISPOT assay provides a new versatile tool for monitoring cellular immune responses either during a herpes zoster disease outbreak or following vaccination.     

ELISPOT检测技术在儿童结核病诊断中的应用

为了评价ELISPOT试验在儿童结核病诊断中的应用价值,采用以结核分枝杆菌特异性蛋白ESAT-6和CFP-10(culture filtrate protein-10)为抗原的ELISPOT试验技术,检测了42例非结核性肺疾病患儿和27例活动性结核病患儿体内特异性T淋巴细胞分泌的γ-干扰素水平,评价其在儿童活动性结核病和潜伏结核感染中的敏感性和特异性,并将结果与结核菌素皮试结果(PPD)进行比较;同时分析了该试验的各影响因素.结果显示,ELISPOT试验的敏感性为88.9%,特异性(97.6%)高于PPD(81%,P﹤0.05);与PPD试验结果结合分析,其诊断阳性率为96.3%.此外,ELISPOT试验结果与性别、年龄、BCG接种史、结核病接触史、机体免疫状态、PPD直径和感染部位等影响因素之间的相关性进行了初步探讨.实验结果提示ELISPOT试验适宜作为儿童PPD试验初筛后结核病诊断的重要辅助工具.     

Evaluation of the Tuberculin Gamma Interferon Assay: Potential To Replace the Mantoux Skin Test

We evaluated an in vitro test of cell-mediated immunity, the tuberculin gamma interferon assay, QuantiFERON-TB (QIFN), in 455 individuals from three groups: group I, 237 immigrants from high-risk countries; group II, 127 health care workers undergoing Mantoux testing; group III, 91 patients being investigated for possible active tuberculosis (79 patients) or Mycobacterium avium-Mycobacterium intracellulare complex infection (12 patients). The QIFN results were compared either to those of the Mantoux test or to microbiological and clinical diagnosis, as appropriate. In each group the correlation between the diameter of induration for the skin test and the magnitude of QIFN response was significant and of moderate strength (Spearman's rank correlation coefficient; rho = 0.59 to 0.61; P < 0.001). For group I, the agreement between QIFN and Mantoux results was 89% for Mantoux-negative and 64% for Mantoux-positive individuals. For group II, when >/=10-mm-diameter induration was taken as positive, the agreement was 81% for Mantoux-negative and 67% for Mantoux-positive individuals. For group III, agreement was 81% for Mantoux-negative and 86% for Mantoux-positive patients. For patients being evaluated for active tuberculosis, the performance of the Mantoux test was not statistically different from that of the QIFN assay. In patients with active tuberculosis, the assay had a sensitivity of 77%, not significantly higher for extrapulmonary than pulmonary cases (83% versus 74%). QIFN sensitivity was not significantly different for smear-negative or smear-positive cases (80% versus 71%). The QIFN assay is a potential replacement for the Mantoux test. The acceptability of these performance values and those of similar evaluations will determine the place this test will have in detecting evidence of mycobacterial 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.     

Production of Gamma Interferon in Mls Disparate Interactions

The murine T-cell clone E11 isolated from a primary H-2k histocompatible one-way mixed lymphocyte culture of B10.BR anti C3H/Tif spleen cells was used to study multidirectional interactions in strong stimulatory Mls disparate responses. Several parameters have recently been studied, and proliferation of T cells upon stimulation by macrophages or B cells of Mls-disparate stimulator cells, induction of differentiation of B cells of stimulator strains but inhibition of their macrophage differentiation, and the inhibition by E11 T cells of the production of a mitogenic mediator by Mls-disparate spleen cells have been found. As shown in this paper, these phenomena can be explained by an Mlsa, d, e specific induction of gamma-interferon (IFN-gamma) production in the responder B10.BR (Mlsb) E11 T cells. It is suggested that IFN-gamma, as a regulator of feedback mechanisms plays a critical role in Mls disparate T-cell stimulation.     

Interferon Alpha Treatment of Patients with Impaired Interferon Gamma Signaling

Patients with deficiency in the interferon gamma receptor (IFN-γR) are unable to respond properly to IFN-γ and develop severe infections with nontuberculous mycobacteria (NTM). IFN-γ and IFN-α are known to signal through STAT1 and activate many downstream effector genes in common. Therefore, we added IFN-α for treatment of patients with disseminated mycobacterial disease in an effort to complement their IFN-γ signaling defect. We treated four patients with IFN-γR deficiency with adjunctive IFN-α therapy in addition to best available antimicrobial therapy, with or without IFN-γ, depending on the defect. During IFN-α treatment, ex vivo induction of IFN target genes was detected. In addition, IFN-α driven gene expression in patients’ cells and mycobacteria induced cytokine response were observed in vitro. Clinical responses varied in these patients. IFN-α therapy was associated with either improvement or stabilization of disease. In no case was disease exacerbated. In patients with profoundly impaired IFN-γ signaling who have refractory infections, IFN-α may have adjunctive anti-mycobacterial effects.     

Gamma Interferon Is Dispensable for Neopterin Production In Vivo

Previous studies have indicated that neopterin is synthesized in vitro by human monocyte-derived macrophages and dendritic cells upon stimulation with gamma interferon (IFN-γ). Neopterin production under specific conditions in vitro has also been obtained upon stimulation with IFN-α and/or IFN-β. However, it is unknown if any IFN-γ-independent neopterin synthesis is possible in vivo. In the present study we investigated the serum neopterin concentrations in patients affected by the syndrome of Mendelian susceptibility to mycobacterial disease (MSMD). Indeed, this syndrome is characterized by deeply impaired or absent IFN-γ production or function due to severe mutations in molecules involved in IFN-γ/interleukin-12 (IL-12)/IL-23-dependent pathway. Serum neopterin levels were measured by an enzyme-linked immunosorbent assay in 27 patients with MSMD. We found that serum neopterin levels are elevated in the complete absence of IFN-γ activity due either to a complete deficiency of its receptor or to deleterious mutations of IL-12 or its receptor. These data clearly indicate that, as reported from in vitro studies, other stimuli are able to induce neopterin synthesis in vivo. Consequently, neopterin cannot be used as means of diagnosis of MSMD due to IFN-γ-, IL-12-, and IL-23-dependent pathway defects.     

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.     

A New Cell Enzyme-Linked Immunosorbent Assay Demonstrates Gamma Interferon Suppression by Beta Interferon in Multiple Sclerosis

Multiple sclerosis (MS) is a demyelinating disorder of the central nervous system of unknown etiology. Immune mechanisms involving the proinflammatory cytokine gamma interferon (IFN-γ) are believed to play an important role in the pathogenesis of MS. IFN-β-1b has been introduced as a treatment for MS and was found to reduce the number and severity of clinical exacerbations. To examine the influence of IFN-β-1b on myelin basic protein (MBP)-specific and phytohemagglutinin-induced IFN-γ production, we developed a cell-released capturing enzyme-linked immunosorbent assay (CRC-ELISA), which rapidly measures spontaneous and antigen- or mitogen-induced cellular IFN-γ production. CRC-ELISA documented a significant MBP-specific T-cell response in the blood of untreated MS patients, as assessed by IFN-γ production. This response was suppressed in MS patients treated with IFN-β-1b. The present work confirms in vivo the in vitro suppressive effects of IFN-β-1b on IFN-γ production in MS. Moreover, it provides a powerful new technique for detection of cytokines.     

Gamma Interferon Immunospot Assay of Pleural Effusion Mononuclear Cells for Diagnosis of Tuberculous Pleurisy

Diagnosis of tuberculous pleurisy remains a challenge in the clinic. In this study, we evaluated the usefulness of a previously developed Mycobacterium tuberculosis antigen-specific gamma interferon enzyme-linked immunospot (ELISPOT) assay in the diagnosis of tuberculous pleurisy by testing a cohort of 352 patients with pleural effusion. We found that M. tuberculosis antigen-specific gamma interferon-producing cells were enriched four to five times in pleural fluid compared with their levels in peripheral blood from patients with tuberuclous pleurisy assayed in parallel. The sensitivity, specificity, positive predictive value, and negative predictive value of the pleural fluid mononuclear cell ELISPOT assay for the diagnosis of tuberculous pleurisy were 95.7%, 100%, 100%, and 81.0%, respectively. In comparison, the sensitivity and specificity of the ELISPOT assay using peripheral blood mononuclear cells were 78.3% and 86.3%, respectively. The sensitivity and specificity of the pleural fluid adenosine deaminase activity test were 55.5% and 86.3%, respectively. These results demonstrate that the M. tuberculosis antigen-specific ELISPOT assay performed on pleural fluid mononuclear cells provides an accurate, rapid diagnosis of tuberculous pleurisy.     

Preliminary Evaluation of Whole-Blood Gamma Interferon Release for Clinical Assessment of Cellular Immunity in Patients with Active Coccidioidomycosis

Assessment of the cellular immune response in coccidioidomycosis has epidemiologic and prognostic importance. Measurement of delayed-type hypersensitivity to skin testing has been used in the past to determine cellular immunity in coccidioidomycosis. However, no skin tests are currently available in the United States. Assay of gamma interferon (IFN-γ) release in whole blood in response to incubation with antigen has been used to assess cellular immunity in tuberculosis. We used a similar assay using the coccidioidal antigen preparation T27K to measure the in vitro cellular immune responses among a cohort of 69 subjects with active coccidioidomycosis. IFN-γ release was bimodal, with concentrations above and below 5 IU/ml. Using multivariate logistic regression, underlying disease and disseminated or chronic pulmonary coccidioidomycosis was significantly associated with the release of IFN-γ at a concentration of <5 IU/ml (P = 0.02 or 0.05, respectively). In addition, the release IFN-γ concentration was <5 IU/ml in all subjects with a clinical severity score of ≥6 (P = 0.02). The release IFN-γ concentration correlated with expression of CD69 on T lymphocytes in an in vitro assay using T27K as the antigen (Spearman's rho = 0.59; P < 0.01). These results suggest that the IFN-γ release assay with T27K as the antigen may be a useful clinical test for assessing cellular immunity in patients with active coccidioidomycosis.     

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.     

Two Patients with Complete Defects in Interferon Gamma Receptor-Dependent Signaling

Unusual susceptibility to mycobacterial infections can be caused by deleterious mutations in genes that encode the interferon-γ receptor 1 chain. Such mutations hamper the activation of macrophages by a type 1 immune response and result in enhanced survival of intracellular pathogens. We here report two patients with unusual mycobacterial infections, both diagnosed with homozygous deleterious interferon-γ receptor 1 gene mutations. Patient 1 became ill after Bacillus Calmette–Guérin vaccination at the age of 9 months and died at the age of 18 months. She carried a homozygous C71Y mutation in the extracellular part of the mature interferon-γ receptor 1 protein, resulting in the lack of detectable protein expression and absence of interferon-γ dependent signaling. Patient 2 became ill at the age of 3 years, is still alive at 19 years of age, and has suffered from five successive infection episodes with atypical mycobacteria. A homozygous splice-site mutation in intron 3 was identified, resulting in the deletion of exon 3 at the mRNA level and consequently a truncated interferon-γ receptor 1 protein with absence of the transmembrane domain. Protein expression and interferon-γ dependent signaling were not detectable. Financial support: Revolving Fund 2000 of the Erasmus MC Rotterdam, Rotterdam, The Netherlands.     

Evaluation of Gamma Interferon and Antibody Tuberculosis Tests in Alpacas

We describe the performance of cell-based and antibody blood tests for the antemortem diagnosis of tuberculosis (TB) in South American camelids (SAC). The sensitivity and specificity of the gamma interferon (IFN-γ) release assay, two lateral flow rapid antibody tests (Stat-Pak and Dual Path Platform [DPP]), and two enzyme-linked immunosorbent assay (ELISA)-based antibody tests (Idexx and Enferplex) were determined using diseased alpacas from Mycobacterium bovis culture-confirmed breakdown herds and TB-free alpacas from geographical areas with no history of bovine TB, respectively. Our results show that while the sensitivities of the IFN-γ and antibody tests were similar (range of 57.7% to 66.7%), the specificity of the IFN-γ test (89.1%) was lower than those of any of the antibody tests (range of 96.4% to 97.4%). This lower specificity of the IFN-γ test was at least in part due to undisclosed Mycobacterium microti infection in the TB-free cohort, which stimulates a positive purified protein derivative (PPD) response. The sensitivity of infection detection could be increased by combining two antibody tests, but even the use of all four antibody tests failed to detect all diseased alpacas. These antibody-negative alpacas were IFN-γ positive. We found that the maximum sensitivity could be achieved only by the combination of the IFN-γ test with two antibody tests in a “test package,” although this resulted in decreased specificity. The data from this evaluation of tests with defined sensitivity and specificity provide potential options for antemortem screening of SAC for TB in herd breakdown situations and could also find application in movement testing and tracing investigations.     

Gamma Interferon Release Assay for Monitoring of Treatment Response for Active Tuberculosis: an Explosion in the Spaghetti Factory

Few studies have correlated the results of interferon (gamma interferon) release assays (IGRAs) with known markers of tuberculosis (TB) treatment response. We report the results of serial QuantiFERON-TB gold in-tube assay (QFT) testing on 149 patients with active tuberculosis and correlate the results with smear and culture conversion. We show that QFT results do not offer much value for treatment monitoring of TB disease.     

Gamma Interferon Inhibits Production of Anti-OspA Borreliacidal Antibody In Vitro

The ability of a Lyme borreliosis vaccine to induce and maintain sustained levels of borreliacidal antibody is necessary for prolonged protection against infection with Borrelia burgdorferi. Vaccination against infection with B. burgdorferi could be improved by determining the mechanism(s) that influences the production of protective borreliacidal antibody. Borreliacidal antibody was inhibited in cultures of lymph node cells obtained from C3H/HeJ mice vaccinated with formalin-inactivated B. burgdorferi and cultured with macrophages and B. burgdorferi and treated with recombinant gamma interferon (rIFN-γ). The suppression of production of outer surface protein A (OspA) borreliacidal antibody by rIFN-γ was not affected by the time of treatment. In addition, treatment with rIFN-γ inhibited the production of other anti-B. burgdorferi antibodies. By contrast, treatment of cultures of immune lymph node cells with anti-IFN-γ marginally increased the production of borreliacidal antibody and enhanced the production of other antibodies directed against B. burgdorferi. These results show that IFN-γ does not play a major role in the production of anti-OspA borreliacidal antibody. Additional studies are needed to determine which cytokine(s) will enhance production of borreliacidal antibody.     

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.     

Clinical and Diagnostic Developments of a Gamma Interferon Release Assay for Use in Bovine Tuberculosis Control Programs

Currently, the Bovigam assay is used as an official supplemental test within bovine tuberculosis control programs. The objectives of the present study were to evaluate two Mycobacterium bovis-specific peptide cocktails and purified protein derivatives (PPDs) from two sources, liquid and lyophilized antigen preparations. PPDs and peptide cocktails were also used for comparison of a second-generation gamma interferon (IFN-γ) release assay kit with the currently licensed first-generation kit (Bovigam; Prionics AG). Three strains of M. bovis were used for experimental challenge: M. bovis 95-1315, M. bovis Ravenel, and M. bovis 10-7428. Additionally, samples from a tuberculosis-affected herd (i.e., naturally infected) were evaluated. Robust responses to both peptide cocktails, HP (PC-HP) and ESAT-6/CFP10 (PC-EC), and the PPDs were elicited as early as 3 weeks after challenge. Only minor differences in responses to Commonwealth Serum Laboratories (CSL) and Lelystad PPDs were detected with samples from experimentally infected animals. For instance, responses to Lelystad M. avium-derived PPD (PPDa) exceeded the respective responses to the CSL PPDa in M. bovis Ravenel-infected and control animals. However, a 1:4 dilution of stimulated plasma demonstrated greater separation of PPDb from PPDa responses (i.e., PPDb minus PPDa) with the use of Lelystad PPDs, suggesting that Lelystad PPDs provide greater diagnostic sensitivity than CSL PPDs. The responses to lyophilized and liquid antigen preparations did not differ. Responses detected with first- and second-generation IFN-γ release assay kits (Bovigam) did not differ throughout the study. In conclusion, antigens may be stored in a lyophilized state without loss in potency, PC-HP and PC-EC are dependable biomarkers for aiding in the detection of bovine tuberculosis, and second-generation Bovigam kits are comparable to currently used kits.