Interferon-gamma (IFN-γ) Release Assays Research Articles

Impact of cytotoxic and targeted antineoplastic drugs on the validity of the mitogen-induced interferon-gamma release assay for latent tuberculosis infection: Results of a prospective trial at a comprehensive cancer center

Abstract The T-SPOT.TB test (TS.TB), an interferon-gamma (IFN-γ) release assay (IGRA), is superior in diagnosing latent tuberculosis infection compared with the conventional tuberculin skin test (TST). However, whether cytotoxic chemotherapy and treatment with new-generation antineoplastic monoclonal antibodies affects the TS.TB is not certain. We evaluated the feasibility of using the TS.TB in this population. Sixteen cancer patients at high risk for tuberculosis exposure were prospectively evaluated with the TST and TS.TB. Blood samples were obtained 7.5 ± 89.3 days after the most recent cycle of antineoplastic therapy. Six patients (38%) were febrile within 24 h of blood sampling; high-dose corticosteroid therapy and profound treatment-induced neutropenia were present in 1 patient each. In all patients, TS.TB showed no evidence of latent tuberculosis infection. A robust mitogen-induced IFN-γ response was seen in samples from 14 patients (88%) despite therapy with high-dose corticosteroids, cyclophosphamide, fludarabine, gemtuzumab ozogamicin, and alemtuzumab. The presence of fever or profound neutropenia did not negatively impact mitogen response by peripheral lymphocytes. The 2 patients whose peripheral blood lymphocytes (> 500 cells/ml) failed to generate a cytokine response to ex vivo mitogen stimulation had refractory advanced cancer. Unlike the TST, a negative TS.TB provided interpretable results even in cancer patients undergoing new-generation immunosuppressive therapy.

Interferon gamma release assays and the NICE 2011 guidelines on the diagnosis of latent tuberculosis

In this clinical audit, we assessed retrospectively the current practice of respiratory physicians with respect to interferon gamma (IFNγ) release assay (IGRA) testing for tuberculosis (TB), as recommended by the 2011 National Institute of Health and Care Excellence (NICE) guidelines for the diagnosis and management of TB. All IGRAs requested by respiratory physicians over a 3-year period were identified retrospectively, and both results and clinical indications analysed. Of the total number of IGRAs carried out, 90% formed part of investigations of suspected active TB. However, 89% of the patients had not had a documented Mantoux test and human immunodeficiency virus (HIV) status was unclear in the 35.2% of patients treated for active TB. Of patients with chest X-rays suggestive of TB, 92.3% were treated for active TB. Of the patients under the age of 35 with reactive IGRAs, 84.6% were treated for active or latent TB and 15.4% had justifiable reasons for not receiving chemoprophylaxis. Based on the results of our audit, IGRAs are commonly being utilised for the investigation of active TB, which is contrary to current guidance.

Specific Antibody and Interferon-Gamma Responses Associated with Immunopathological Forms of Bovine Paratuberculosis in Slaughtered Friesian Cattle

Mycobacterium avium subsp. paratuberculosis (MAP) infection causes a chronic granulomatous inflammatory regional enteritis in ruminants. Cell-mediated immune responses are assumed to be protective and therefore, to be associated with its more delimited lesion types, while humoral responses are mainly associated with diffuse histopathological lesions. However, this duality of immune responses has been recently questioned. The aim of this study was to assess the relationship between both types of immunological responses and the type and extension of intestinal lesions and the presence of MAP in bovine tissues. Standard histopathological examinations, two microbiological procedures (culture and real time PCR (rtPCR)), as well as MAP specific antibody and interferon gamma (IFN-γ) release assays (IGRA) were performed on tissues and blood of 333 slaughtered Holstein-Friesian animals. Paratuberculous lesions were observed in 176 (52.9%) of the animals and overall MAP detection rates were estimated at 13.5% and 28.5% for tissue culture and rtPCR, respectively. Unlike the relatively constant non-specific IFN-γ release, both the antibody levels and the specific IFN-γ release significantly increased with tissue damage. Delimited immunopathological forms, which accounted for 93.2% of all forms, were mostly related to positive testing in the IGRA (38.4%) whereas diffuse ones (6.8%) were associated with antibody seropositivity (91.7%). However, since the frequency of positive immune responses in both tests increased as the lesions severity increased, polarization of Th1/Th2 responses was less prominent than expected. MAP was detected in the majority of ELISA-positive animals (culture+: 90%, rtPCR+: 85%) but the bacteria was only confirmed in the 36.1% of IGRA-positive animals by any of the two microbiological tests. In terms of diagnosis, the antibody test was a good indicator of advanced tissue damage (diffuse forms), but the IGRA did not associate well with more delimited forms or with MAP detection.

In Vitro Immunomodulation of a Whole Blood IFN-γ Release Assay Enhances T Cell Responses in Subjects with Latent Tuberculosis Infection

BackgroundActivation of innate immunity via pathogen recognition receptors (PRR) modulates adaptive immune responses. PRR ligands are being exploited as vaccine adjuvants and as therapeutics, but their utility in diagnostics has not been explored. Interferon-gamma (IFN-γ) release assays (IGRAs) are functional T cell assays used to diagnose latent tuberculosis infection (LTBI); however, novel approaches are needed to improve their sensitivity.MethodsIn vitro immunomodulation of a whole blood IGRA (QuantiFERON®-TB GOLD In-Tube) with Toll-like receptor agonists poly(I:C), LPS, and imiquimod was performed on blood from subjects with LTBI and negative controls.ResultsIn vitro immunomodulation significantly enhanced the response of T cells stimulated with M. tuberculosis antigens from subjects with LTBI but not from uninfected controls. Immunomodulation of IGRA revealed T cell responses in subjects with LTBI whose T cells otherwise do not respond to in vitro stimulation with antigens alone. Similar to their in vivo functions, addition of poly(I:C) and LPS to whole blood induced secretion of inflammatory cytokines and IFN-α and enhanced the surface expression of antigen presenting and costimulatory molecules on antigen presenting cells.ConclusionsIn vitro immunomodulation of whole blood IGRA may be an effective strategy for enhancing the sensitivity of T cells for diagnosis of LTBI.

Interferon gamma mRNA quantitative real-time polymerase chain reaction for the diagnosis of latent tuberculosis: a novel interferon gamma release assay

The interferon gamma (IFN-γ) release assay (IGRA) is widely used as a diagnostic method for latent tuberculosis infection (LTBI). The QuantiFERON-TB Gold and QuantiFERON-TB Gold In-tube (QFT-IT) tests measure plasma IFN-γ levels using enzyme-linked immunosorbent assay (ELISA), and T-SPOT.TB counts IFN-γ–producing cells using enzyme-linked immunosorbent spot assay. IFN-γ mRNA was evaluated as an indicator of IGRA in comparison with QFT-IT IFN-γ ELISA in 46 subjects with active TB and in 73 at low risk for TB. Significant IFN-γ mRNA expression was detected from 30 min and peaked 4 h after stimulation with MTB antigens or mitogen. This was defined as the optimal time point for IFN-γ mRNA real-time polymerase chain reaction (PCR). The sensitivities of IFN-γ mRNA real-time PCR and IFN-γ ELISA were 84.8% (39/46) and 89.1% (41/46), respectively (no significant difference). Although the specificities of IFN-γ ELISA was 4.1% higher than that of IFN-γ mRNA real-time PCR (60.3% versus 56.2%), the difference was not statistically significant. The overall agreement between IFN-γ mRNA real-time PCR and IFN-γ ELISA was 79.8% (kappa = 0.475). Whilst there was no difference in the performance of IFN-γ mRNA real-time PCR and IFN-γ ELISA, IFN-γ mRNA real-time PCR was superior to IFN-γ ELISA in terms of the time required for detection of MTB infection.

Consistency ofMycobacterium tuberculosis-Specific Interferon-Gamma Responses in HIV-1-Infected Women during Pregnancy and Postpartum

Background. We determined the consistency of positive interferon-gamma (IFN-γ) release assays (IGRAs) to detect latent TB infection (LTBI) over one-year postpartum in HIV-1-infected women. Methods. Women with positive IGRAs during pregnancy had four 3-monthly postpartum IGRAs. Postpartum change in magnitude of IFN-γ response was determined using linear mixed models. Results. Among 18 women with positive pregnancy IGRA, 15 (83%) had a subsequent positive IGRA; 9 (50%) were always positive, 3 (17%) were always negative, and 6 (33%) fluctuated between positive and negative IGRAs. Women with pregnancy IGRA IFN-γ>8 spot forming cells (SFCs)/well were more likely to have consistent postpartum IGRA response (odds ratio: 10.0; 95% confidence interval (CI): 0.9–117.0). Change in IFN-γ response over postpartum was 10.2 SFCs/well (95% CI: −1.5–21.8 SFCs/well). Conclusion. Pregnancy positive IGRAs were often maintained postpartum with increased consistency in women with higher baseline responses. There were modest increases in magnitude of IGRA responses postpartum.

Interferon-gamma inducible protein 10 as a biomarker for active tuberculosis and latent tuberculosis infection in children: A case–control study

Interferon-gamma (IFN-γ) release assays (IGRAs) are suboptimally sensitive to diagnose tuberculosis (TB) and latent TB infection (LTBI) in young children. In this study we compared Mycobacterium tuberculosis antigen-stimulated IFN-γ inducible protein 10 (IP-10) responses in children with active TB and LTBI to responses from children with non-tuberculous mycobacterial (NTM) lymphadenopathy and respiratory tract infection (RTI). We also assessed test agreement between IP-10 and the QuantiFERON(®)-TB Gold In-Tube (QFT-IT) test results, and investigated whether IP-10 release upon mitogen stimulation is associated with age. We recruited 48 children (median age 54 months) diagnosed in Germany with either active TB (n = 11), LTBI (n = 14), NTM lymphadenopathy (n = 8), or common RTI (n = 15). IFN-γ levels were measured using the QFT-IT. These plasma supernatants were used to determine IP-10 concentrations using an in-house enzyme-linked immunosorbent assay (ELISA). The median antigen-stimulated IP-10 levels in children with active TB, LTBI, NTM lymphadenopathy, and RTI were 12,702 pg/ml, 9109 pg/ml, 97 pg/ml, and 84 pg/ml, respectively. We observed a strong correlation between IP-10 and IFN-γ plasma concentration in children with active TB and LTBI (r(2) = 0.69). Overall agreement between IP-10 and QFT-IT assays was high (kappa = 0.95). IP-10 levels after mitogen stimulation showed no association with age. IP-10 and IFN-γ were both induced with antigen stimulation in blood from children in the TB and LTBI groups, in contrast to the NTM and RTI groups. Compared to IFN-γ the IP-10 levels were higher and IP-10 was released independently of age. IP-10 therefore may represent an additional biomarker in the paediatric population.

Reversion and conversion of Mycobacterium tuberculosis IFN-γ ELISpot results during anti-tuberculous treatment in HIV-infected children

BackgroundRecent interest has focused on the potential use of serial interferon gamma (IFN-γ) release assay (IGRA) measurements to assess the response to anti-tuberculous (TB) treatment. The kinetics of IFN-γ responses to Mycobacterium tuberculosis (MTB) antigens in HIV-infected children during treatment have not however been previously investigated.MethodsIFN-γ responses to the MTB antigens, ESAT-6, CFP-10 and PPD were measured by an enzyme-linked immunospot assay (IFN-γ ELISpot) at presentation and at one, two and six months after starting anti-tuberculous treatment in HIV-infected children with definite or probable TB. Responses at different time points were compared using a Mann-Whitney U test with paired data analysed using the Wilcoxon signed rank test. A Fisher's exact or Chi-squared test was used to compare proportions when test results were analysed as dichotomous outcomes.ResultsOf 102 children with suspected TB, 22 (21%) had definite TB and 24 (23%) probable TB. At least one follow up IFN-γ ELISpot assay result was available for 31 (67%) of the 46 children. In children with definite or probable TB in whom the IFN-γ ELISpot assay result was positive at presentation, anti-tuberculous treatment was accompanied by a significant decrease in both the magnitude of the IFN-γ response to individual or combined MTB-specific antigens (ESAT-6 median 110 SFCs/106 PBMC (IQR 65-305) at presentation vs. 15 (10-115) at six months, p = 0.04; CFP-10 177 (48-508) vs. 20 (5-165), p = 0.004, ESAT-6 or CFP-10 median 250 SFCs/106 PBMC (IQR 94-508) vs. 25 (10-165), p = 0.004) and in the proportion of children with a positive IFN-γ ELISpot assay (Fisher's exact test: ESAT-6 15/0 vs 5/11, p = 0.0002, CFP-10 22/0 vs 8/17, p = 0.0001, ESAT-6 or CFP-10 22/0 vs. 9/17, p= 0.002). However almost half of the children had a positive IFN-γ ELISpot assay after six months of anti-tuberculous treatment. In addition, there was conversion of the IFN-γ ELISpot assay result during anti-tuberculous therapy in six of 12 children in whom the initial IFN-γ ELISpot assay was negative.ConclusionsIn HIV-infected children with definite or probable TB, anti-tuberculosis treatment is accompanied by a reduction in the magnitude of the IFN-γ ELISpot response to MTB-antigens. However, serial IFN-γ ELISpot measurements appear to have limited clinical utility in assessing a successful response to anti-tuberculous treatment in HIV infected children.

Diagnosing Latent Tuberculosis Infection in the HIV Era

Tuberculin skin testing (TST) and Interferon-gamma (IFNγ)release assays (IGRAs) are presently the only available assays for the detection of Mycobacterium tuberculosis infected individuals. IGRAs might progressively replace TST, as numerous published reports establish their higher specificity and similar sensitivity when tested in BCG vaccinated, immunocompetent individuals or in populations who may have been in contact with atypical mycobacteria. However, few published reports have commented on their role in TB diagnosis in immunocompromised individuals (HIV, immunosuppressive therapy, cancer…). It is the purpose of this report to review IGRAs published studies in HIV individuals in endemic and non endemic area for tuberculosis (TB). IGRAs were tested in the presence or absence of active TB but correlated to duration of exposure. In newly diagnosed active TB, IGRAs demonstrated a similar sensitivity to TST. In TB non infected individuals, TST and IGRAs also gave similar values when categorization of individuals was correlated to the risk of infection. A higher number of positive IGRAs was observed in individuals from TB endemic areas, in similar proportions to immunocompetent individuals. Comparison between the two IGRAs: QuantiFERON-TB Gold® (QF-TB, Cellestis, Australia) and T-SPOT-TB® (Oxford Immunotec, UK), and against TST, in the same HIV population demonstrates a higher sensitivity of T-SPOT-TB and TST than QF-TB. Indeterminate results, which correspond to the absence of a positive T-cell IFNγ response towards phytohemaglutinin (PHA), is a key point when comparing both IGRAs. This PHA control is indicative of the level of immunosuppression observed in the tested individual. QF-TB seems to present, in HIV populations, more indeterminate results than T-SPOT-TB. The calibration and/or concentration of PBMC on nitrocellulose membrane for the T-SPOT-TB, as compared to a whole blood assay, might explain this difference, with less indeterminate results with the T-SPOT-TB assay. Neither assay is able to differentiate active TB from latent TB infection (LTBI). Several laboratories have tried new antigenic epitopes to solve this issue. It is of importance that these studies need to be repeated on a larger scale by others to validate their results. Two blood assays might add information characterising the evolution from LTBI to active TB: either by losing protective immunity, as demonstrated by the whole blood killing assay, or by evaluating the kinetics of the antibodies synthesized against M. tuberculosis specific antigens. In conclusion, longitudinal studies are still needed to validate IGRAs and other assays and to define their respective predictive values.