IFN-γ Assay Research Articles

Relative Importance of Defined Mycobacterium Tuberculosis Antigens in the T Cell Recognition Repertoire of Latently Infected Individuals not Progressing to Active Disease.

In this study, we have mapped the relative importance of well-defined recombinantly expressed Mycobacterium tuberculosis antigens in the T cell recognition repertoire of latently infected individuals not progressing to active disease. Peripheral blood mononuclear cells from healthy latently infected long term non-progressors were screened for antigen-induced proliferation and Th1 cytokine, Interferon- (IFN-γ) responses. The panel of antigens tested showed a clear spectrum of responsiveness and lead to the identification of a subgroup of frequently recognized antigens (MPT59, CFP7, CFP10, CFP21, TB37.6 /PPE68, ESAT-6, MPT51, and DnaK) with a high cellular response level as measured in both proliferation and IFN-γ assays. Among a subgroup of antigens also screened for responses in tuberculosis patients, CFP21 was identified as differentially recognized in non-progressors. For both cellular assays, we found a positive correlation between responder frequency and magnitude of response. A significant correlation between the level of antigen-specific proliferation and INF-γ secretion was also observed. We have identified a defined set of M. tuberculosis antigens frequently recognized by T cells at a high response level from latently infected long term non-progressors which warrant further investigation for a potential role in immune regulation and protection against progression to active disease.

SARS-CoV-2-Specific Immune Cytokine Profiles to mRNA, Viral Vector and Protein-Based Vaccines in Patients with Multiple Sclerosis: Beyond Interferon Gamma.

Disease-modifying therapies (DMTs) impact the cellular immune response to severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) vaccines in patients with multiple sclerosis (pwMS). In this study, we aim to elucidate the characteristics of the involved antigen-specific T cells via the measurement of broad cytokine profiles in pwMS on various DMTs. We examined SARS-CoV-2-specific T cell responses in whole blood cultures characterized by the release of interleukin (IL)-2, IL-4, IL-5, IL-10, IL-13, IL-17A, interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α), as well as antibodies (AB) targeting the SARS-CoV-2 spike protein in pwMS following either two or three doses of mRNA or viral vector vaccines (VVV). For mRNA vaccination non-responders, the NVX-CoV2373 protein-based vaccine was administered, and immune responses were evaluated. Our findings indicate that immune responses to SARS-CoV-2 vaccines in pwMS are skewed towards a Th1 phenotype, characterized by IL-2 and IFN-γ. Additionally, a Th2 response characterized by IL-5, and to a lesser extent IL-4, IL-10, and IL-13, is observed. Therefore, the measurement of IL-2 and IL-5 levels could complement traditional IFN-γ assays to more comprehensively characterize the cellular responses to SARS-CoV-2 vaccines. Our results provide a comprehensive cytokine profile for pwMS receiving different DMTs and offer valuable insights for designing vaccination strategies in this patient population.

The impact of changing the cut-off threshold of the interferon-gamma (IFN-γ) assay for diagnosing bovine tuberculosis in Ireland

In Ireland, the interferon-gamma (IFN-γ) assay is routinely used as an ancillary test interpreted in parallel with the single intradermal comparative tuberculin test (SICTT) to maximize the detection of bovine tuberculosis (bTB) infected animals. Up until 2018, a positive test result was recorded in the IFN-γ ELISA assay following whole blood stimulation with purified protein derivative (PPD)-bovine (B), PPD-avian (A) and nil sample (N), using the interpretation criteria, B-N > 50 optical density units (OD), B > 100 and B-A > 0. Following a review of available data, the threshold of the B-A component changed to B-A > 80. As predicting the impact of changing the cut-off thresholds for the IFN-γ test de novo is challenging, the aims of this study were to follow animals that initially tested negative using the new IFN-γ assay interpretation criteria and investigate their future risk of disclosure with bTB, with a focus on animals that otherwise would have been removed when using the older interpretation criteria (0 < B-A ≤ 80). Enrolled animals (n = 28,669 cattle from 527 herds) were followed up for two years (2019–2021), or to point of bTB detection or death. At the end of follow-up, 1151 (4.0%) of enrolled animals were bTB cases. The majority of these cases were diagnosed using SICTT (80.5%). The cumulative number of positive animals that would have been removed if the old cut-off (0 < B-A ≤ 80) was used amounted to 1680 cattle (5.9% of the enrolled cohort). Of these, 127 (7.5%) were diagnosed with bTB during follow-up. In contrast, 1024 of the 1151 cattle which subsequently tested positive during the study period following a negative IFN-γ test would not have been identified with the old or new IFN-γ cut-off criteria. Survival analysis showed that animals that would have been removed under the old interpretation criteria were at increased risk of a positive diagnosis with bTB during follow-up compared to other test negative animals. A newly developed risk prediction model (using a Cox proportional hazard model) showed that age, animal number of SICTT tests, number of inconclusive SICTT tests, B-A (IFN-γ assay), B-N (IFN-γ assay), animals from store herds and the percentage of the rest of the herd that were positive during the breakdown were statistically significantly associated with bTB detection. However, inclusion of the IFN-γ OD variables did not show added value in terms of prediction performance of the model.

Glycolysis: An early marker for vancomycin-specific T-cell activation.

Vancomycin, a glycopeptide antibiotic used for Gram-positive bacterial infections, has been linked with drug reaction with eosinophilia and systemic symptoms (DRESS) in HLA-A*32:01-expressing individuals. This is associated with activation of T lymphocytes, for which glycolysis has been isolated as a fuel pathway following antigenic stimulation. However, the metabolic processes that underpin drug-reactive T-cell activation are currently undefined and may shed light on the energetic conditions needed for the elicitation of drug hypersensitivity or tolerogenic pathways. Here, we sought to characterise the immunological and metabolic pathways involved in drug-specific T-cell activation within the context of DRESS pathogenesis using vancomycin as model compound and drug-reactive T-cell clones (TCCs) generated from healthy donors and vancomycin-hypersensitive patients. CD4+ and CD8+ vancomycin-responsive TCCs were generated by serial dilution. The Seahorse XFe96 Analyzer was used to measure the extracellular acidification rate (ECAR) as an indicator of glycolytic function. Additionally, T-cell proliferation and cytokine release (IFN-γ) assay were utilised to correlate the bioenergetic characteristics of T-cell activation with invitro assays. Model T-cell stimulants induced non-specific T-cell activation, characterised by immediate augmentation of ECAR and rate of ATP production (JATPglyc). There was a dose-dependent and drug-specific glycolytic shift when vancomycin-reactive TCCs were exposed to the drug. Vancomycin-reactive TCCs did not exhibit T-cell cross-reactivity with structurally similar compounds within proliferative and cytokine readouts. However, cross-reactivity was observed when analysing energetic responses; TCCs with prior specificity for vancomycin were also found to exhibit glycolytic switching after exposure to teicoplanin. Glycolytic activation of TCC was HLA restricted, as exposure to HLA blockade attenuated the glycolytic induction. These studies describe the glycolytic shift of CD4+ and CD8+ T cells following vancomycin exposure. Since similar glycolytic switching is observed with teicoplanin, which did not activate T cells, it is possible the master switch for T-cell activation is located upstream of metabolic signalling.

Ad5-nCoV Vaccination Could Induce HLA-E Restricted CD8+ T Cell Responses Specific for Epitopes on Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein.

We evaluated cellular immune responses induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines in an immunized population based on HLA-E-restricted CD8+ T cell epitope identification. HLA-E-restricted SARS-CoV-2 CD8+ T cell nonamer peptides were predicted with software. An HLA-E-transfected K562 cell binding assay was used to screen for high-affinity peptides. IFN-γ enzyme-linked immunospot assays were used to identify HLA-E-restricted epitopes. An HLA-E/epitope tetramer was employed to detect the frequencies of epitope-specific CD8+ T cells. Four CD8+ T cell epitopes on the spike protein of SARS-CoV-2 restricted by both HLA-E*0101 and E*0103 were identified. HLA-E-restricted epitope-specific IFN-γ-secreting CD8+ T cell responses could be detected in individuals vaccinated with SARS-CoV-2 vaccines. Importantly, the frequencies of epitope-specific CD8+ T cells in Ad5-nCoV vaccinated individuals were higher than in individuals vaccinated with recombinant protein or inactivated vaccines. Moreover, the frequencies of epitope-specific CD8+ T cells could be maintained for at least 120 days after only one dose of Ad5-nCoV vaccine, while the frequencies of epitope-specific CD8+ T cells decreased in individuals after two doses of Ad5-nCoV vaccine. These findings may contribute to a more comprehensive evaluation of the protective effects of vaccines for SARS-CoV-2; meanwhile, they may provide information to characterize HLA-E-restricted CD8+ T cell immunity against SARS-CoV-2 infection.

Gamma-interferon assay for the ancillary diagnosis of bovine tuberculosis in dairy cattle in urban and adjacent areas of Dhaka city, Bangladesh.

Bovine tuberculosis (bTB) is an infectious disease of cattle, mainly caused by Mycobacterium bovis. This study aimed to evaluate the efficacy of the interferon-gamma (IFN-γ) assay and single-intradermal comparative tuberculin test (SICTT) in detecting bTB. In an earlier study, 150 positive, 83 inconclusive, and 480 negative animals from 24 cattle herds were screened using SICTT. From these groups, 125 positive, 17 inconclusive, and six negative animals were subsequently verified using the IFN-γ assay. Single-intradermal comparative tuberculin test outcomes were interpreted according to standard guidelines, whereas blood samples were collected and stimulated with purified protein derivatives. Sandwich enzyme-linked immunosorbent assay was used to measure secreted IFN-γ. Concordant and Bayesian latent class analyses were performed to evaluate test performance. Results from the IFN-γ assay revealed that 83.2%, 64.7%, and 16.67% of the animals were positive in the SICTT-positive, inconclusive, and negative animal categories, respectively. Sensitivity (SE) and specificity (SP) of SICTT were 83.9% (95% confidence interval [CI]: 77.4-90.1) and 95.7% (95% CI: 86.9-99.7), respectively. Sensitivity and SP for the IFN-γ assay were 78.9% (95% CI: 71.9-85.4) and 83.9% (65.9-95.9), respectively. The use of both tests in parallel increases the SE of bTB detection (~94%), compared with SICTT alone. Use of the IFN-γ assay with SICTT in parallel, predominantly on cattle demonstrating an inconclusive SICTT outcome, boosts bTB detection rate in low resource settings.

Investigation of the association between the Enferplex bovine tuberculosis antibody test and the future risk of bovine tuberculosis in irish cattle in infected herds: a pilot field study

The Single Intradermal Comparative Tuberculin Test (SICTT) and the interferon-gamma (IFN-γ) assay are the approved diagnostic tests for bovine tuberculosis (bTB) in Ireland. The aim of this pilot study was to explore if there was any added diagnostic benefit from applying the Enferplex bTB test (an antibody test) in severe bTB herd breakdowns after the removal of cattle that had tested positive to the SICTT and the IFN-γ test. In addition to the normal bTB testing and management protocols, the animals in these herds that tested negative to SICTT and the IFN-γ test were followed forward for a period of two years. All animals were tested by Enferplex at enrolment. The time to subsequent bTB detection (diagnosed with SICTT/IFN-γ tests or detection of visible lesions at routine slaughter) for animals that tested positive or negative to the Enferplex bTB test at the start of the study was compared using Kaplan–Meier survival curves and Cox based survival models. Of the 484 enrolled animals (from 11 herds), 171 (35.3%) and 151 (31.1%) initially tested positive in the Enferplex assay under the high sensitivity and high specificity interpretation settings respectively. The results of the survival analysis showed that there was no difference in the survival time to a positive diagnosis with bTB during the follow-up period between animals initially classified as positive and negative by the Enferplex test. Further research is warranted to explore the potential benefit of using the Enferplex test in other scenarios.

Novel insights on the therapeutic effect of levamisole on the chronic toxoplasmosis in mice model.

Seventy-five Me49 Toxoplasma gondii infected Swiss albino mice were divided into five groups; (GI): noninfected control group; (GII): infected untreated control group; (GIII): infected- LEVA treated group; (GIV): infected and received combination of spiramycin and LEVA and (GV): infected-spiramycin treated group. The impact was assessed through brain cyst count by Quantitative Real-Time Polymerase Chain Reaction (PCR), interferon gamma (IFN-γ) assay, histopathological study, and total blood counts. The progression of chronic toxoplasmosis could only be partially controlled by using either levamisole or spiramycin as a separate drug. The combined spiramycin and levamisole treatment significantly decreased the burden of Toxoplasma brain cyst, increased IFN-γ level, total blood parameters and improved the histopathological features especially at the late stage of infection. Levamisole effectively modulated Toxoplasma-induced immune responses, resulting in chronic toxoplasmosis remission. Further clinical trials will be needed to study the effect of these combination in HIV/AIDS (human immunodeficiency virus) patients with toxoplasmosis.

Alloreactive T cells to Assess Acute Rejection Risk in Kidney Transplant Recipients.

Samples from 103 consecutive kidney transplant recipients (2018-2019) were obtained pretransplantation and at time of for-cause biopsy sampling within 6 mo of transplantation. The number of donor-reactive interferon gamma (IFN-γ) and interleukin (IL)-21-producing memory T cells was analyzed by enzyme-linked immunosorbent spot (ELISPOT) assay. Of the 63 patients who underwent a biopsy, 25 had a biopsy-proven acute rejection (BPAR; 22 aTCMR and 3 aAMR), 19 had a presumed rejection, and 19 had no rejection. Receiver operating characteristic analysis showed that the pretransplant IFN-γ ELISPOT assay distinguished between patients who later developed BPAR and patients who remained rejection-free (area under the curve [AUC] 0.73; sensitivity 96% and specificity 41%). Both the IFN-γ and IL-21 assays were able to discriminate BPAR from other causes of transplant dysfunction (AUC 0.81; sensitivity 87% and specificity 76% and AUC 0.81; sensitivity 93% and specificity 68%, respectively). This study validates that a high number of donor-reactive memory T cells before transplantation is associated with the development of AR after transplantation. Furthermore, it demonstrates that the IFN-γ and IL-21 ELISPOT assays are able to discriminate between patients with AR and patients without AR at the time of biopsy sampling.

Immunogenicity of a Third Dose of BNT162b2 Vaccine among Lung Transplant Recipients-A Prospective Cohort Study.

Two doses of mRNA SARS-CoV-2 vaccines elicit an attenuated humoral immune response among immunocompromised patients. Our study aimed to assess the immunogenicity of a third dose of the BNT162b2 vaccine among lung transplant recipients (LTRs). We prospectively evaluated the humoral response by measuring anti-spike SARS-CoV-2 and neutralizing antibodies in 139 vaccinated LTRs ~4-6 weeks following the third vaccine dose. The t-cell response was evaluated by IFNγ assay. The primary outcome was the seropositivity rate following the third vaccine dose. Secondary outcomes included: positive neutralizing antibody and cellular immune response rate, adverse events, and COVID-19 infections. Results were compared to a control group of 41 healthcare workers. Among LTRs, 42.4% had a seropositive antibody titer, and 17.2% had a positive t-cell response. Seropositivity was associated with younger age (t = 3.736, p < 0.001), higher GFR (t = 2.355, p = 0.011), and longer duration from transplantation (t = -1.992, p = 0.024). Antibody titer positively correlated with neutralizing antibodies (r = 0.955, p < 0.001). The current study may suggest the enhancement of immunogenicity by using booster doses. Since monoclonal antibodies have limited effectiveness against prevalent sub-variants and LTRs are prone to severe COVID-19 morbidity, vaccination remains crucial for this vulnerable population.

Accuracy of Tests for Diagnosis of Animal Tuberculosis: Moving Away from the Golden Calf (and towards Bayesian Models)

The last decades have seen major efforts to develop new and improved tools to maximize our ability to detect tuberculosis-infected animals and advance towards the objective of disease control and ultimately eradication. Nevertheless, there is still uncertainty regarding test performance due to the wide range of specificity and especially sensitivity estimates published in the scientific literature. Here, we performed a systematic review of the literature on studies that evaluated the performance of tuberculosis diagnostic tests used in animals through Bayesian Latent Class Models (BLCMs), which do not require the application of a (fallible) reference procedure to classify animals as infected with tuberculosis or not. BLCM-based sensitivity and specificity estimates deviated from those obtained using a reference procedure for certain antemortem tests: an overall lower sensitivity of skin tests and serology and a higher sensitivity of interferon-gamma (IFN-γ) assays was reported. In the case of postmortem diagnostic tests, sensitivity estimates from BLCMs were similar to estimates from studies based on other methodologies. For specificity, the range of BLCM-based estimates was narrower than those based on a reference test, reaching values close to 100% (but lower in the case of IFN-γ assays). In conclusion, Bayesian methods have been increasingly applied for the evaluation of tuberculosis diagnostic tests in animals, yielding results that differ (sometimes substantially) from previously reported test performance in the literature, particularly for in vivo tests and sensitivity estimates. Newly developed models that allow adjustment for relevant factors (e.g., age, breed, region, and herd size) can contribute to the generation of more unbiased estimates of test performance. Nevertheless, although BLCMs for tuberculosis do not require the use of an imperfect reference procedure and are therefore not influenced by its limited performance, they require careful implementation, and transparent systematic reporting should be the norm.

Persistent memory despite rapid contraction of circulating T Cell responses to SARS-CoV-2 mRNA vaccination.

While antibodies raised by SARS-CoV-2 mRNA vaccines have had compromised efficacy to prevent breakthrough infections due to both limited durability and spike sequence variation, the vaccines have remained highly protective against severe illness. This protection is mediated through cellular immunity, particularly CD8+ T cells, and lasts at least a few months. Although several studies have documented rapidly waning levels of vaccine-elicited antibodies, the kinetics of T cell responses have not been well defined. Interferon (IFN)-γ enzyme-linked immunosorbent spot (ELISpot) assay and intracellular cytokine staining (ICS) were utilized to assess cellular immune responses (in isolated CD8+ T cells or whole peripheral blood mononuclear cells, PBMCs) to pooled peptides spanning spike. ELISA was performed to quantitate serum antibodies against the spike receptor binding domain (RBD). In two persons receiving primary vaccination, tightly serially evaluated frequencies of anti-spike CD8+ T cells using ELISpot assays revealed strikingly short-lived responses, peaking after about 10 days and becoming undetectable by about 20 days after each dose. This pattern was also observed in cross-sectional analyses of persons after the first and second doses during primary vaccination with mRNA vaccines. In contrast, cross-sectional analysis of COVID-19-recovered persons using the same assay showed persisting responses in most persons through 45 days after symptom onset. Cross-sectional analysis using IFN-γ ICS of PBMCs from persons 13 to 235 days after mRNA vaccination also demonstrated undetectable CD8+ T cells against spike soon after vaccination, and extended the observation to include CD4+ T cells. However, ICS analyses of the same PBMCs after culturing with the mRNA-1273 vaccine in vitro showed CD4+ and CD8+ T cell responses that were readily detectable in most persons out to 235 days after vaccination. Overall, we find that detection of spike-targeted responses from mRNA vaccines using typical IFN-γ assays is remarkably transient, which may be a function of the mRNA vaccine platform and an intrinsic property of the spike protein as an immune target. However, robust memory, as demonstrated by capacity for rapid expansion of T cells responding to spike, is maintained at least several months after vaccination. This is consistent with the clinical observation of vaccine protection from severe illness lasting months. The level of such memory responsiveness required for clinical protection remains to be defined.

Correlation of angiogenic growth factors and inflammatory cytokines with the clinical phenotype of ocular tuberculosis.

To determine the correlation of angiogenic growth factors and inflammatory cytokines with the clinical phenotype of ocular tuberculosis (OTB). Vitreous fluid was analysed for cytokines in patients with OTB and non-OTB uveitis using multiplex fluorescent bead-based flow cytometric assay. The clinical phenotypes were recorded and correlated with vitreous biomarkers. Vitreous humour from OTB patients had elevated levels of interleukin-10 (IL-10), IL-17-A, interferon-gamma (IFN-γ), and tumour necrosis factor-alpha (TNF-α). Angiopoietin (Ang-2) levels were higher in the panuveitis phenotype. OTB posterior uveitis phenotype had relatively higher vascular endothelial growth factor (VEGF) levels and lower fibroblast growth factor (FGF) levels. Additionally, eyes with choroiditis and vasculitis had elevated levels of VEGF and Ang-2 with FGF downregulation. Both IFN-γ and IL-10 were upregulated in the choroiditis phenotype of OTB. Angiogenic growth factors and inflammatory cytokines were altered in the vitreous humour of OTB patients. IFN-γ, VEGF, and IL-10 levels are increased in choroiditis and vasculitis phenotypes. Receiver operating characteristic (ROC) curve analysis further emphasized the importance of the IFN-γ assay in the diagnosis of OTB.

RIG-I and TLR-7/8 agonists as combination adjuvant shapes unique antibody and cellular vaccine responses to seasonal influenza vaccine.

Influenza vaccine effectiveness could be improved by combination with an adjuvant with the potential to enhance the host-vaccine response both quantitatively and qualitatively. The goal of this study was to explore a RIG-I agonist (SDI-nanogel) and a TLR7/8 agonist (Imidazoquinoline (IMDQ)-PEG-Chol) as adjuvants, when co-administered with a licensed quadrivalent inactivated influenza vaccine (QIV), and to determine the role of these adjuvants in directing helper T (Th) cell responses for their role in the immunoglobulin (Ig) class switching. Administration of QIV with the two adjuvants, individually or combined, resulted in enhanced HA-specific serum ELISA IgG titers, serum hemagglutination inhibition (HAI) titers and splenic T cell responses as examined by IFN-γ and IL-4 enzyme-linked immunosorbent spot (ELISPOT) assays, 4-weeks post-prime and post-boost vaccination in BALB/c mice. While QIV+SDI-nanogel largely induced antigen-specific IgG1 responses, QIV+IMDQ-PEG-Chol predominantly induced IgG2a antibody isotypes post-prime vaccination, suggesting efficient induction of Th2 (IL-4) and Th1 (IFN-γ) responses, respectively. Combination of the two adjuvants not only skewed the response completely towards IgG2a, but also resulted in induction of HAI titers that outperformed groups that received single adjuvant.Moreover, enhanced IgG2a titers correlate with antibody-mediated cellular cytotoxicity (ADCC) that targets both the highly conserved H1 hemagglutination (HA) stalk domain and N1 neuraminidase (NA). A booster vaccination with QIV+IMDQ-PEG-Chol resulted in a more balanced IgG1/IgG2a response in animals primed with QIV+IMDQ-PEG-Chol but increased only IgG2a titers in animals that received the combination adjuvant during prime vaccination, suggesting that class switching events in germinal centers during the prime vaccination contribute to the outcome of booster vaccination. Importantly, IMDQ-PEG-Chol, alone or in combination, always outperformed the oil-in-water control adjuvant Addavax. Vaccine-induced antibody and T cell responses correlated with protection against lethal influenza virus infection. This study details the benefit of adjuvants that target multiple innate immune receptors to shape the host vaccine response.

Tuberculosis Treatment Response Monitoring by the Phenotypic Characterization of MTB-Specific CD4+ T-Cells in Relation to HIV Infection Status.

HIV infection causes systemic immune activation, impacts TB disease progression and hence may influence the diagnostic usability of Mycobacterium tuberculosis-specific T cell profiling. We investigated changes of activation and maturation markers on MTB-specific CD4+ T-cells after anti-tuberculosis treatment initiation in relation to HIV status and the severity of lung impairment. Thawed peripheral blood mononuclear cells from TB patients with (n = 27) and without HIV (n = 17) were analyzed using an intracellular IFN-γ assay and flow cytometry 2 and 6 months post-TB treatment initiation. H37Rv antigen was superior to the profile MTB-specific CD4+ T-cells phenotype when compared to PPD and ESAT6/CFP10. Regardless of HIV status and the severity of lung impairment, activation markers (CD38, HLA-DR and Ki67) on MTB-specific CD4+ T-cells declined after TB treatment initiation (p < 0.01), but the expression of the maturation marker CD27 did not change over the course of TB treatment. The MTB-specific T cell phenotype before, during and after treatment completion was similar between people living with and without HIV, as well as between subjects with severe and mild lung impairment. These data suggest that the assessment of activation and maturation markers on MTB-specific CD4+ T-cells can be useful for TB treatment monitoring, regardless of HIV status and the severity of lung disease.

Evaluation of Interleukin 2, Interleukin 10, and Interferon Gamma Among Pregnant Women Co-infected With HIV and Plasmodium falciparum

Background: In pregnancy, the body undergoes modulation of pro-inflammation responses to ensure fetal survival, and these adjustments may decrease maternal immune defenses and promote infections. Objectives: This study was conducted to determine interleukin 2 (IL-2), IL-10, and interferon gamma (IFN-γ) among pregnant women co-infected with HIV and Plasmodium falciparum in Edo State, Nigeria. Methods: This study was conducted at the Central Hospital, Benin City, Edo State. A total of 200 participants were enrolled, consisting of 150 pregnant women co-infected with HIV and P. falciparum and 50 non-pregnant women (as controls) who were neither infected with HIV nor P. falciparum. Venous blood specimen was collected for P. falciparum detection, CD4 T-lymphocyte estimation, and IL-2, IL-10, and IFN-γ assays using standard methods. Results: Among HIV-infected pregnant women, IL-2 (P = 0.0070), IL-10 (P = 0.0179) and IFN-γ (P = 0.1564) values were lower in primiparous women compared with multiparous women. HIV-infected pregnant women who were married had significantly higher IL-2 (P = 0.0085) and IFN-γ levels (P = 0.0332) compared with single women, while marital status did not affect the IL-10 level of pregnant women infected with HIV. Only the IL-2 levels of the HIV-infected pregnant women increased significantly (P = 0.0012) with increasing trimester. The tested cytokine levels were lower in those with malaria compared with those without malaria among HIV-infected pregnant women. Conclusions: The HIV status and not malaria infection affects the cytokine levels of pregnant women co-infected with HIV and malaria.

Comparative analysis of intradermal tuberculin test and γ-interferon assay for diagnosis of bovine tuberculosis

The present study was carried out in and around Anand district of Gujarat with the aim to assess the sensitivity and specificity of IFN-γ assay as compared to intradermal tuberculin test for diagnosis of bovine tuberculosis during the period 2011-12. The prevalence of bovine TB in the cattle was 26.19% by TST and 38.49% by IFN- γ assay. Breed wise, highest prevalence of bovine TB was found in Gir (38.96%), followed by Triple cross (23.95%) and Kankrej (16.45%) by TST; while by IFN- γ assay, highest prevalence was observed in Triple cross (42.70%). Age wise, highest prevalence of bovine TB was found in adults (37.90%) followed by calves (26.66%) and heifers (11.22%) by TST; while by IFN-γ assay, highest prevalence was observed in calves (66.66%) followed by adults (44.35%) and heifers (22.44%). Sex wise, more prevalence of bovine TB was found in males (56.25%) than in females (21.81%) by TST; while by IFN-γ assay, more prevalence was observed in females (39.25%) than in males (31.25%). Sensitivity and specificity of TST in detecting bovine TB were 27.27% and 57.52% respectively, compared to IFN-γ assay. ELISPOT assay showed 34.78% animals were found positive for bovine TB. IFN-γ assay showed better sensitivity in detecting bovine TB at younger age compared to TST, and hence can be useful in timely removal of the infected animals from the herd.

Evaluation of an ELISA for the diagnosis of bovine tuberculosis using milk samples from dairy cows in China

Bovine tuberculosis (bTB) is an important zoonotic disease in developing countries including China. Lack of rapid and simple detection methods for surveillance affects efficient control and eradication of this disease. The objective of this study was to determine the feasibility of using an individual and bulk milk sample ELISA based on the fusion protein of four specific antigens (CFP10/ESAT-6/MPB70/MPB83) of Mycobacterium bovis for the rapid detection and surveillance of bTB in dairy cattle herds and to identify individual animal factors that affect its results. The study population consisted of 388 dairy cows from four herds in Henan province, China. The serum sample ELISA (cut-off value 30%) and milk sample ELISA (cut-off value 10%) were shown to have good agreement, while, there was fair agreement between a blood sample IFN-γ in vitro release assay and the milk sample ELISA (cut-off value 10%) (Cohen’s kappa statistic of 0.492 and 0.306, respectively). The sensitivity and specificity for the milk sample ELISA using a S/P% cut-off value of 10%, as estimated using a Bayesian latent class analysis model, was 48.4% (95%CI: 31.0 – 85.2) and 97.1% (95%CI: 92.2 – 99.7), respectively. Although both ELISAs had comparable sensitivities and specificities, the milk sample ELISA had a greater discriminatory ability, as measured by the area under the curve (ROC), than the serum sample ELISA with the difference of their AUCs being − 0.069 (95%PCI: −0.121 to −0.021). Further, it was estimated that the lower limit of detection at an animal level lacto-prevalence might be 1.1% with this milk ELISA using bulk milk samples. Additionally, a mixed linear regression model, incorporating herd as a random effect variable, was constructed to investigate the association between natural log-transformed bTB sample-to-positive ratio (LnS/P%) and milk data. Across the model, natural log-transformed Somatic Cell Count (SCC) and parity had a positive effect on the LnS/P% of bTB antibody response, and milk urea nitrogen had a curvilinear association with LnS/P%. In conclusion, a milk ELISA for bTB antibodies, especially bulk tank milk antibodies, could be used as an efficient tool for rapid screening in surveillance, which might be followed by confirmatory testing with IFN-γ assay in dairy cattle, during the implementation of bTB control programmes. Relevant milk factors should be considered to adjust the S/P% with the aim of classifying the infection status of animals and herds as accurately as possible.

Significant difference in Th1/Th2 paradigm induced by tuberculosis-specific antigens between IGRA-positive and IGRA-negative patients.

False negative interferon-γ release assay (IGRA) results constitute the major dilemma for the diagnosis of tuberculosis (TB) infections. Herein, we conducted a cohort study to compare the host immunological response to TB-specific antigens between active TB patients with positive and negative IGRA results and control groups. A total of 274 laboratory-confirmed TB patients were included in our analysis, consisting of 221 were IGRA positive and 53 were IGRA negative. Patients with the elderly were identified as an independent risk factor for negative IGRA results. In addition, the elevated level of IL-4 and the decreased levels of IFN-γ, IL-2, IL-6, IL-1β, and IL-12 in IGRA negative TB relative to IGRA positive TB group, demonstrating a significant difference in Th1/Th2 paradigm between two groups. The IFN-γ&IL-2 based assay could correctly identify 247 out of 307 MTB-infected individuals [271 TB patients and 36 individuals with latent TB infection (LTBI)], demonstrating a sensitivity of 80.5%. Then the IFN-γ and IL-4 were applied to distinguish healthy control and IGRA-negative group. When using the stepwise algorithm, the sensitivity for detecting Mycobacterium tuberculosis (MTB) infections was significantly increased from 80.5% to 89.6%. Additionally, patients with negative IGRA results had a conversion to culture-negative status longer than those with positive IGRA results. In conclusion, a stepwise algorithm outperforms IGRA assays to accurately identify MTB infections by the combination IFN-γ, IL-2, and IL-4. Further study is needed to evaluate the accuracy of our diagnostic algorithm in the LTBI population.

Bovine Tuberculosis Epidemiology in Cameroon, Central Africa, Based on the Interferon-Gamma Assay.

Despite sub-Saharan Africa (SSA) accounting for ~20% of the global cattle population, prevalence estimates and related risk factors of bovine tuberculosis (bTB) are still poorly described. The increased sensitivity of the IFN-γ assay and its practical benefits suggest the test could be useful to investigate bTB epidemiology in SSA. This study used a population-based sample to estimate bTB prevalence, identify risk factors and estimate the effective reproductive rate in Cameroonian cattle populations. A cross-sectional study was conducted in the North West Region (NWR) and the Vina Division (VIN) of Cameroon in 2013. A regional stratified sampling frame of pastoral cattle herds produced a sample of 1,448 cattle from 100 herds. In addition, a smaller cross-sectional study sampled 60 dairy cattle from 46 small-holder co-operative dairy farmers in the NWR. Collected blood samples were stimulated with bovine and avian purified protein derivatives, with extracted plasma screened using the IFN-γ enzyme-linked immunosorbent assay (Prionics Bovigam®). Design-adjusted population prevalences were estimated, and multivariable mixed-effects logistic regression models using Bayesian inference techniques identified the risk factors for IFN-γ positivity. Using the IFN-γ assay, the prevalence of bTB in the dairy cattle was 21.7% (95% CI: 11.2–32.2). The design-adjusted prevalence of bTB in cattle kept by pastoralists was 11.4% (95% CI: 7.6–17.0) in the NWR and 8.0% (95% CI: 4.7–13.0) in the VIN. A within-herd prevalence estimate for pastoralist cattle also supported that the NWR had higher prevalence herds than the VIN. Additionally, the estimates of the effective reproductive rate Rt were 1.12 for the NWR and 1.06 for the VIN, suggesting different transmission rates within regional cattle populations in Cameroon. For pastoral cattle, an increased risk of IFN-γ assay positivity was associated with being male (OR = 1.89; 95% CI:1.15–3.09), increasing herd size (OR = 1.02; 95% CI:1.01–1.03), exposure to the bovine leucosis virus (OR = 2.45; 95% CI: 1.19–4.84) and paratuberculosis (OR = 9.01; 95% CI: 4.17–20.08). Decreased odds were associated with contacts at grazing, buffalo (OR = 0.20; 95% CI: 0.03–0.97) and increased contact with other herds [1–5 herds: OR = 0.16 (95% CI: 0.04–0.55); 6+ herds: OR = 0.18 (95% CI: 0.05–0.64)]. Few studies have used the IFN-γ assay to describe bTB epidemiology in SSA. This study highlights the endemic situation of bTB in Cameroon and potential public health risks from dairy herds. Further work is needed to understand the IFN-γ assay performance, particularly in the presence of co-infections, and how this information can be used to develop control strategies in the SSA contexts.