Interferon-gamma Test Research Articles

5117 Unusual Sarcoid Presentation as a Case of Hypercalcemia

Abstract Disclosure: M. Salim: None. A. Chauhan: None. S. Avula: None. A. Ahmed: None. K. Zekarias: None. Background: Hypercalcemia may arise from a variety of causes and can pose challenging diagnostic dilemma. Clinical Case: An 89-year-old male presented with worsening malaise related to persistent hypercalcemia. He reported productive cough, polyuria, polydipsia, generalized muscle weakness and weight loss. Physical exam was remarkable for upper and lower extremities weakness. Laboratory tests showed calcium 11.6 (8.8-10.2 mg/dL), phosphorus 2.8 mg/dL (2.5-4.5 mg/dL), albumin 4.2 g/dL (3.5-5.2 g/dL), parathyroid hormone (PTH) 10 pg/mL (15-65 pg/mL), parathyroid hormone-related protein (PTH-rp) 4.5 pmol/L (0.0-2.3 pmol/L), TSH 6.24 (0.30 - 4.20 uIU/mL), free T4 1.10 ng/dL (0.90-1.70 ng/dL), GFR 61 mL/min/1.73m2, 25 OH Vit D 31 ng/mL (20-75 ng/mL), vit D 1,25-dihydroxy 134 pg/mL (19.9-79.3 pg/mL), calcium urine/creatinine 0.54. Hypercalcemia was first noted 11 months before the visit with a level of 14.0 mg/dL (8.8-10.2 mg/dL), phosphorus 3.5 mg/dL, PTH 11, PTH-rp 1.4 pmol/L, 25 OH Vit D 62 ng/mL, Vit D 1,25-dihydroxy 90 pg/mL, angiotensin-1-converting enzyme 12 U/L (16-85 U/L), serum and urine electrophoresis showed no monoclonal gammopathy present, and tuberculosis interferon-gamma test negative. Autoimmune workup was unremarkable, except for weak positive anti-signal recognition particle (SRP) antibodies and anti-Mi-2 antibodies. CT chest/pelvis/abdomen demonstrated peripheral predominant reticular opacities suggestive of interstitial lung disease and no malignant findings. Treatment history included zoledronic acid 11 months before the visit. The patient was referred for a granulomatous disease workup.Upon one month follow-up, the hypercalcemia symptoms were worsening. Repeat calcium was 12.3 mg/dL and fungal serologies were negative. The patient was started on prednisone empirically. PET CT was ordered and revealed patchy inflamed musculature of extremities suspicious of dermatomyositis/polymyositis. Further labs showed unremarkable myomarker panel, u3-Hydroxy-3-Methylglutaryl Coenzyme A reductase (HMGCR) antibody, Anti-cN-1A antibody, ACE 53 U/L, creatine kinase 32 U/L (39-309 U/L), aldolase 5.4 U/L (1.2-7.6 U/L), soluble IL-2R 1,021 U/mL (137-838 U/mL), and lysozyme 3.89 ug/mL (<2.75 ug/mL). A muscle biopsy was done, which showed benign skeletal muscle with patchy myofiber atrophy, patchy interstitial fibrosis, and focal interstitial mild chronic inflammation with no evidence of sarcoid granulomas in the sample. One week lab follow-up post initiation of steroid showed calcium of 10.1 mg/dL and subsequently improvement in his overall labs. Working diagnosis of sarcoid myositis was made. Conclusion: This case report highlights the approach of diagnosing a rare case of hypercalcemia due to sarcoid myositis. In a highly clinical suspicious patient, PET CT scan might be a necessary modality for diagnosis. Presentation: 6/2/2024

Novel SARS-COV2 poly epitope phage-based candidate vaccine and its immunogenicity

Background and purpose: The global emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has prompted widespread concern. Bacteriophages have recently gained attention as a cost-effective and stable alternative for vaccine development due to their adjuvant properties. This study aimed to design and validate a poly epitope composed of viral proteins. Experimental approach: SARS-CoV-2 proteins (spike, nucleocapsid, membrane, envelope, papain-like protease, and RNA-dependent RNA polymerase) were selected for analysis. Immunoinformatic methods were employed to predict B and T cell epitopes, assessing their antigenicity, allergenicity, and toxicity. Epitopes meeting criteria for high antigenicity, non-allergenicity, and non-toxicity were linked to form poly epitopes. These sequences were synthesized and cloned into pHEN4 plasmids to generate Poly1 and Poly2 phagemid vectors. Recombinant Poly1 and Poly2 phages were produced by transforming M13ΔIII plasmids and phagemid vectors into E. coli TG1. Female Balb/c mice were immunized with a cocktail of Poly1 and Poly2 phages, and their serum was collected for ELISA testing. Interferon-gamma (IFN-γ) testing was performed on spleen-derived lymphocytes to evaluate immune system activation. Findings/Results: Recombinant Poly1 and Poly2 phages were produced, and their titer was determined as 1013 PFU/mL. Efficient humoral immune responses and cellular immunity activation in mice were achieved following phage administration. Conclusion and implication: Poly epitopes displayed on phages exhibit adjuvant properties, enhancing humoral and cellular immunity in mice. This suggests that phages could serve as adjuvants to bolster immunity against SARS-Cov-2. Recombinant phages could be applied as effective candidates for injectable and oral vaccine development strategies.

Risk-Based Targeting of Animals for Ancillary Testing during a Bovine Tuberculosis Breakdown Is Associated with a Reduced Time to Test Failure: Indirect Evidence of Mycobacterium bovis Exposure?

Bovine tuberculosis (bTB) continues to have significant economic and veterinary health impacts on cattle herds where the disease remains endemic. The continual tailoring of policies to address such maintenance requires an in-depth analysis of national data, underpinning new control strategies. In Ireland, when outbreaks occur, ancillary testing of herd mates deemed to be at the highest risk of exposure to reactors is undertaken using the interferon gamma (GIF) test. This highest risk cohort was hypothesised to be of a higher future risk despite this ancillary testing. We used a dataset from Ireland to model bovine test failure to the comparative tuberculin skin test using a survival analysis (observations: 39,248). Our primary exposure of interest was whether an animal that tested negative had a GIF test after the disclosure of infection within a herd during a bTB breakdown. There was evidence that animals with a negative GIF test during a breakdown had an increased risk of failing a test relative to other animals from the same herds without this exposure. The time to failure was 48.8% (95%CI: 38.3-57.5%) shorter for the exposed group relative to the unexposed group during a two-year follow-up period (2019-2022; time ratio: 0.51; 95%CI: 0.43-0.62; p < 0.001). The results from this study suggest that animals who were GIF-tested, having been deemed to have a higher risk of exposure, subsequently had shorter time-to-test failure periods. The absolute numbers of failure are small (only 2.5% of animals go on to fail during 2-year follow-up). Importantly, however, a high proportion of these high-risk herds included in the dataset failed at least one test at the follow-up (21/54 herds), impacting breakdown duration or recurrence. Such risk-informed targeting of animals could be utilised in future control policies, though further research is warranted.

Differentiation of Bovine Tuberculosis and Paratuberculosis Infections with Antemortem Diagnostic Methods

In this study, based on the results of tuberculin skin tests (Bovine and Avian PPD) used in the antemortem diagnosis and differentiation of Bovine Tuberculosis, the animals in the farms with suspected Tuberculosis were serologically examined to diagnose Paratuberculosis infection and fecal bacterioscopy was performed. In addition, it was aimed to obtain data that will contribute to the eradication studies of Bovine Tuberculosis disease by comparing the antemortem diagnostic methods of Bovine Tuberculosis disease, which is endemic in Türkiye and by determining the sensitivity and specificity values of the interferon gamma (IFN-γ) test. In this context, intradermal tuberculin test was applied to 423 cattle with suspected Tuberculosis in a total of 5 dairy cattle farms, one each from Çankırı, Çorum, Ankara, Eskişehir and Konya regions, and this test was determined as the gold standard method and the sensitivity and specificity of the IFN-γ test were determined as 86% and 97%, respectively. For the diagnosis of Paratuberculosis infection, antibody ELISA, fecal bacterioscopy and IFN-γ ELISA were performed on these animals and the prevalence of these tests were 10.4%, 5.44% and 4.96% respectively and 4 (0.95%) of the cattle were positive for each of the diagnostic methods for Map infection. As a result, it was concluded that IFN-γ test, which gives similar results to intradermal tuberculin test results, can also be used in the antemortem diagnosis of Bovine Tuberculosis. Also, in the comparative intradermal tuberculin test for the diagnosis of Tuberculosis infection, avian PPD positive animals were found to play a decisive role in the detection of nonspecific reactions or Paratuberculosis infected animals, supported by other tests used for the diagnosis of Paratuberculosis.

Assessing the impact of various tuberculin PPD brands on bovine tuberculosis diagnosis

Although several brands of tuberculin purified protein derivatives (PPDs) are available for diagnosing bovine tuberculosis (bTB), comparative studies to determine their diagnostic accuracy are infrequent. In Ecuador we compared two different PPD brands for bTB diagnosis using skin testing and measuring skin thickness increase. Additionally, we evaluated four PPD brands, including those used for skin testing, in the Bovine Tuberculosis Interferon Gamma Test (IFN-γ test) measuring IFN-γ induction in whole blood. The study included 17 naturally tuberculosis-infected PPD and IFN-γ test positive bovines. Both the field and laboratory results showed significant differences in classifying the 17 bovines as bTB positive or negative. We hypothesize that several factors, such as the genetic background of the cows, sensitization to environmental mycobacteria, M. bovis strains involved in the bTB infection, and the manufacturing procedures of the PPDs, could have influenced the immune reaction toward the different tuberculin PPD brands. Our study emphasizes the necessity for comparative studies aimed at determining the diagnostic accuracy of PPD brands for bTB diagnosis as well as the development of standardized methods for PPD production and potency determination.

A decade of tuberculosis eradication programs in the Mediterranean water buffalo (Bubalus bubalis) in South Italy: Are we heading toward eradication?

The water buffalo (Bubalus bubalis) is susceptible to bovine tuberculosis (TB), which receives increased attention in areas where buffalo breeding is prevalent, such as in Southern Italy, especially in the Campania region, where 70% of the buffalo stock is bred. Since 2012, TB testing in buffalo herds has been conducted using the Single Intradermal Test (SIT), with the Comparative Intradermal test (CIT) used in cases of inconclusive results. From 2012 to 2016, the interferon-gamma (IFN-γ) test was occasionally employed experimentally in herds with TB outbreaks to expedite eradication efforts. A local TB eradication program was implemented in officially TB-free buffalo herds between 2017 and 2019. This program involves initial screening with SIT, followed by confirmatory tests, including CIT and IFN-γ, for positive reactions. Since June 2019, the IFN-γ test has replaced the CIT in officially TB-free herds upon positive SIT reactions. Additionally, in suspected and confirmed TB-outbreak herds, the IFN-γ test was used at the discretion of the competent authority. Between 2017 and 2019, approximately 295,000 buffaloes in Campania were screened annually with in vivo tests provided by TB eradication programs. During this period, 32,040 animals from 855 herds were tested using the IFN-γ test and 4,895 tested positive. Since 2020, the use of IFN-γ testing has increased, and has become a prerequisite for the acquisition of TB-free status and is being systematically applied for TB outbreak-extinction procedures. The test was performed in all breeding buffaloes in cases of doubtful SIT results in TB-free herds and when TB lesions are detected at slaughter in animals from TB-free herds. This combined approach helped detect more TB outbreaks, and thereby led to a reduction in the TB prevalence and incidence rates. By 2022, the prevalence had decreased to 1.56%, and the incidence had decreased to 0.73%, after the increased use of the IFN-γ test. This study highlights the effectiveness of implemented strategies in reducing TB in this region. Overall, the data demonstrate the successful impact of TB eradication measures and surveillance activities in reducing bubaline TB prevalence and incidence in the Campania region.

Enferplex bovine TB antibody test and bovine TB diagnosis: letter to the editor

Bovine tuberculosis is usually diagnosed using tuberculin skin and interferon gamma tests. However, it is clear these tests miss infected animals due to poor sensitivity. The Enferplex Bovine TB antibody test has been validated by the World Organisation for Animal Health as fit for purpose in diagnosing bovine TB. A recent paper by Madden and colleagues (Veterinary Research Communications published online 17 August 2023) presented data on the future risk of Enferplex test antibody positive animals developing bovine TB. We argue in this communication that this does not make sense. Also, the study design did not include measuring antibodies at the point of censure of the animals and hence the survival analysis performed was meaningless. Most significantly, the study misses the point that skin and interferon gamma tests fail to detect a significant proportion of infected animals identified by the Enferplex test.

Relationship between ambient temperature at sampling and the interferon gamma test result for bovine tuberculosis in cattle

Bovine tuberculosis (bTB) is a disease of significant economic and zoonotic importance, therefore, optimising tests for the identification of Mycobacterium bovis infected cattle is essential. The Interferon Gamma (IFN-γ) Release Assay (IGRA) can diagnose M. bovis infected cattle at an early stage, is easy to perform and can be used alongside skin tests for confirmatory purposes or to increase diagnostic sensitivity. It is known that IGRA performance is sensitive to environmental conditions under which samples are taken and transported. In this study, the association between the ambient temperature on the day of bleeding and the subsequent IGRA result for bTB was quantified using field samples from Northern Ireland (NI). Results of 106,434 IGRA results (2013–2018) were associated with temperature data extracted from weather stations near tested cattle herds. Model dependent variables were the levels of IFN-γ triggered by avian purified protein derivative (PPDa), M. bovis PPD (PPDb), their difference (PPD(b-a)) as well as the final binary outcome (positive or negative for M. bovis infection). IFN-γ levels after both PPDa and PPDb stimulation were lowest at the extremes of the temperature distribution for NI. The highest IGRA positive probability (above 6%) was found on days with moderate maximum temperatures (6–16 °C) or moderate minimum temperatures (4–7 °C). Adjustment for covariates did not lead to major changes in the model estimates. These data suggest that IGRA performance can be affected when samples are taken at high or low temperatures. Whilst it is difficult to exclude physiological factors, the data nonetheless supports the temperature control of samples from bleeding through to laboratory to help mitigate post-collection confounders.

Diagnostic accuracy of the Enferplex Bovine Tuberculosis antibody test in cattle sera

Bovine tuberculosis is a contagious bacterial disease of worldwide economic, zoonotic and welfare importance caused mainly by Mycobacterium bovis infection. Current regulatory diagnostic methods lack sensitivity and require improvement. We have developed a multiplex serological test for bovine tuberculosis and here we provide an estimate of the diagnostic accuracy of the test in cattle. Positive and negative reference serum samples were obtained from animals from Europe and the United States of America. The diagnostic specificity estimate was 98.4% and 99.7% using high sensitivity and high specificity settings of the test respectively. Tuberculin boosting did not affect the overall specificity estimate. The diagnostic sensitivity in samples from Mycobacterium bovis culture positive animals following tuberculin boosting was 93.9%.The relative sensitivity following boosting in tuberculin test positive, lesion positive animals and interferon gamma test positive, lesion positive animals was 97.2% and 96.9% respectively. In tuberculin test negative, lesion positive animals and in interferon gamma test negative, lesion positive animals, the relative sensitivity following tuberculin boosting was 88.2% and 83.6% respectively. The results show that the test has high diagnostic sensitivity and specificity and can detect infected animals that are missed by tuberculin and interferon gamma testing.

2143. Contact Investigation after Exposure to Active Pulmonary Tuberculosis in the Pediatric Cancer Center

Abstract Background The prevalence of tuberculosis (TB) in Korean children has decreased due to universal BCG vaccination and the national TB elimination project. However, exposure to TB in children has been consistently reported. There have been few reports of TB exposure in the pediatric hematology-oncology ward. Methods A mother of a pediatric cancer patient was diagnosed with active TB with a cavity. Contact investigation was performed on patients, their parents, and healthcare workers who were exposed to the index case. The criteria for close contact were exposure in the same space with the index patient for more than 8 hours continuously or a total of 40 hours in case of multiple exposures. The initial assessment was performed as soon as the index case was identified. The second evaluation was conducted 8–10 weeks after the initial exposure. Both tuberculin skin test (TST), TB Specific Interferon-Gamma (T-SPOT) tests, and chest radiography were used in immunocompromised pediatric patients. For immunocompetent adults, the QuantiFERON-TB Gold (QFT-G) test and chest radiography were performed. Results Total 23 patients and 29 parents were exposed. The median exposure duration of patients was 3 days (1–8 days). The initial evaluation did not detect TB infection in pediatric patients. However, at the second evaluation, five patients (17.4%) were diagnosed with latent TB infection (LTBI, three with positive TST and two with positive T-SPOT), and two patients (8.7 %) were diagnosed with active TB infection (lymphadenitis and pulmonary TB). Among the 29 parents, 11 individuals (37.9%) were diagnosed with LTBI at the initial evaluation, which was considered as preexisting LTBI before this exposure. No additional case of TB infection was identified in the second evaluation. Among exposed 24 healthcare workers, two (9.1%) were eventually confirmed as newly diagnosed LTBI. Conclusion TB exposure in pediatric hematology-oncology ward and outpatient chemotherapy clinic caused active TB or LTBI cases with an attack rate of 26% in children. The high LTBI rate, up to 38% among parents even before this exposure, needs further intervention such as routine LTBI evaluation and treatment for families of pediatric cancer patients to prevent a similar event. Disclosures Joon-sik Choi, MD, MS, Ministry of Trade, industry and Energy, Republic of Korea: Grant/Research Support Yae-Jean Kim, MD, PhD, Janssen: Grant/Research Support|Korean Society of Pediatric Infectious Diseases: Grant/Research Support|Ministry of Trade, Industry and Energy: Grant/Research Support|MSD: Grant/Research Support.

A fluorescence polarization assay using recombinant protein ESAT-6 for the detection of antibodies against pathogenic Mycobacterium bovis in bovine.

Bovine tuberculosis (bTB) is a major bacterial disease that causes significant economic disruption across the globe. Our study was based on using a fluorescence polarization assay (FPA) that used fluorescein-labeled ESAT-6 protein to detect Mycobacterium bovis antibodies in bovine serum. The ESAT-6 protein was used in a FPA. Positive TB reactors were determined by the comparative intradermal test (CID) and interferon gamma test (IFN-γ). Antibodies against M. bovis were detected using a fluorescein isothiocyanate (FITC) labeled tracer and a whole culture FITC labeled tracer in the positive cattle. Of the 192 animals tested for bTB, 37 were found to be positive by either the CID or IFN-γ assays. Using the mP values from five culture-positive serum samples, a cutoff value of more than >127 mp provided the best discrimination between positive reactors and negative bTB animals. The ESAT-6 results of FPA in comparison with CID results revealed sensitivity of 92.9% and specificity of 64.6%, and in comparison with results IFN-γ, showed sensitivity of 95.7% and specificity of 49%. FPA using FITC labelled ESAT-6 as a tracer has better sensitivity (95.7%) and specificity (49.1%) than IFN-γ test in humoral immune response in animals. This work revealed that the ESAT-6 protein as an antigen can be used in diagnosing bTB using a practical and sensitive humoral test.

Detection of tuberculosis in goats using anti-mortem techniques

A total of 50 goats of smallholders and different farms from in and around Nagpur were investigated for tuberculosis using comparative intradermal tuberculin test (CITT) and IFN-γ assay. CITT was performed using avian and bovine PPD and IFN-γ assay by Mycobacterium bovis gamma interferon test kit. Overall, 4% (2/50) goats were found inconclusive by CITT and 4% positive by IFN-γ assay. However, in goats which found inconclusive in CITT, only those turned positive in IFN-γ assay. Hence, when CITT and IFN- γ used together lead to more accurate screening for tuberculosis in animals.

Quantitative decision making in animal health surveillance: Bovine Tuberculosis Surveillance in Belgium as case study.

Despite eradication and control measures applied across Europe, bovine tuberculosis (bTB) remains a constant threat. In Belgium, after several years of official bTB-free status, routine movement testing, as currently practiced, revealed itself inadequate to detect some herds affected by sporadic breakdowns. The aim of this study was to assess different surveillance system components that strike a balance between cost and effectiveness and to identify sustainable alternatives, which substantiate a bTB-free claim while ensuring early detection and acceptance by various animal health stakeholders. For this pupose, a stochastic iteration model was used to simulate the current surveillance system's expected performance in terms of detection sensitivity and specificity. These results were then descriptively compared with observed field results. Second, the cost and effectiveness of simulated alternative surveillance components were quantified. Sensitivity analyses were performed to measure key assumptions' impacts (i.e. regarding diagnostic tests and true prevalence). The results confirmed discrepancies between the observed and simulated expected performance of bTB surveillance in Belgium. Second, simulated alternatives showed that interferon gamma (IFN-γ) and serological testing with antibody-enzyme linked immunosorbent assay (Ab-ELISA) targeting at-risk herds would enable an increase in the overall cost effectiveness (sensitivity and specificity) of the Belgian bTB surveillance system. Sensitivity analyses showed that results remained constant despite the modification of some key assumptions. While the performance of the ongoing bTB surveillance system in Belgium was questionable at the time of the study, this exercise highlighted that not only sensitivity but specificity also are key drivers of surveillance performance. The quantitative approach, taking into consideration various stakeholders' needs and priorities, revealed itself to be a useful tool in allowing evidence-based decision making for future tuberculosis surveillance in Belgium, in line with the international standards.

Early Detection of Mycobacterium avium subsp. paratuberculosis Infected Cattle: Use of Experimental Johnins and Innovative Interferon-Gamma Test Interpretative Criteria.

Paratuberculosis (PTB), also known as Johne's disease, is a chronic proliferative enteritis of ruminants caused by Mycobacterium avium subsp.paratuberculosis (MAP). To date, PTB diagnosis, based on serology, fecal culture, and real-time polymerase chain reaction, has identified animals in advanced stages of infection. To detect MAP infection in animals earlier, the interferon-gamma (IFN-γ) test may be applied. This assay detects cytokines produced by T-lymphocytes of infected subjects after stimulation with purified protein derivatives (PPDs), extracted from Mycobacterium bovis (MB) and from M. avium (MA). The study involved three bovine herds: one PTB-infected herd, one PTB-free herd, and one with an outbreak of bovine tuberculosis. The IFN-γ test was performed on 235 animals, using bovine PPD (PPDB), avian PPD (PPDA), and three experimental PPD Johnins (PPDJs) extracted from a synthetic liquid medium culture of MAP (PPDJ A, B, and C), to assess early MAP detection and avoid false reactions to MB. Furthermore, IFN-γ results were evaluated using 12 interpretative criteria (ICs), based on the differences and ratio between PPD optical density (OD) and IFN-γ basal OD values after lymphocytic stimulation. IC accuracy was expressed as area under the receiver operating characteristic curve. Through a longitudinal study, PPDJs proved to be specific and sensitive in the detection of MAP-infected animals. Among the evaluated ICs, six showed the best performance in terms of accuracy (p < 0.0001), highlighting PTB subclinical infections. In particular, the two best criteria reached sensitivity values of 100% [confidence interval (CI) 95%, 94.1–100%] with a specificity of 91.8% (CI 95%, 81.9–97.3%) and sensitivity levels of 80.6% (CI 95%, 69.1–89.2%) with a specificity of 100% (CI 95%, 94.1–100%). Thus, the IFN-γ assay proved to be a useful diagnostic tool to identify early subclinical MAP-infected animals, in order to manage infected cattle or those exposed to MAP and to monitor younger calves within a herd. Furthermore, the IFN-γ test can be considered an additional test to avoid the introduction of MAP-infected animals, especially in herds where disease has already been eradicated and preservation of the health status is required to maintain the PTB certification level.

Tuberculosis and the Heart.

Acquired tuberculosis continues to be a challenge worldwide. Although tuberculosis has been considered a global public health emergency, it remains poorly controlled in many countries. Despite being primarily a pulmonary disease, tuberculosis could involve the heart. This systematic review is part of the "Neglected Tropical Diseases and Other Infectious Diseases Involving the Heart" (the NET‐Heart Project) initiative from the Interamerican Society of Cardiology. This project aims to review the cardiovascular involvement of these heterogeneous diseases, advancing original algorithms to help healthcare providers diagnose and manage cardiovascular complications. In tuberculosis, pericardium involvement is relatively common, especially in AIDS, and tuberculosis is the most common cause of constrictive pericarditis in endemic countries. Myocarditis and aortitis by tuberculosis are rare. Clinical manifestations of cardiovascular involvement by tuberculosis differ from those typically found for bacteria or viruses. Prevailing systemic symptoms and the pericarditis diagnostic index should be taken into account. An echocardiogram is the first step for diagnosing cardiovascular involvement; however, several image modalities can be used, depending on the suspected site of infection. Adenosine deaminase levels, gamma interferon, or polymerase chain reaction testing could be used to confirm tuberculosis infection; each has a high diagnostic performance. Antituberculosis chemotherapy and corticosteroids are treatment mainstays that significantly reduce mortality, constriction, and hospitalizations, especially in patients with HIV. In conclusion, tuberculosis cardiac involvement is frequent and could lead to heart failure, constrictive pericarditis, or death. Early detection of complications should be a cornerstone of overall management.

CTL ELISPOT Assay and T Cell Detection.

Enzyme-linked immune absorbent spot (Elispot) is a quantitative method for measuring relevant parameters of T-cell activation. The sensitivity of Elispot allows the detection of low-frequency antigen-specific T-cells that secrete cytokines and effector molecules, such as granzyme B and perforin. Cytotoxic T-cell (CTL) studies have taken advantage with this high-throughput technology by providing insights of quantity and immune kinetics. Accuracy, sensitivity, reproducibility, and robustness of Elispot resulted in a wide range of applications in research as well as in diagnostic field. Actually, CTL monitoring by Elispot is a gold standard for the evaluation of antigen-specific T-cell immunity in clinical trials and vaccine candidates where the ability to detect rare antigen-specific T-cells is of relevance for immune diagnostic. The most utilized Elispot assay is the Interferon-gamma (IFN-γ) test, a marker for CD8+ CTL activation, but Elispot can be also used to distinguish different subsets of activated T-cells by using other cytokines such as T-helper (Th) 1 type cells (characterized by the production of IFN-γ, IL-2, IL-6, IL-12, IL-21 and TNF-α), Th2 (producing cytokines like IL-4, IL-5, IL-10 and IL-13), and Th17 (IL-17) cells.The reliability of Elispot generated data, by the evaluation of T-cell frequency recognizing individual antigen/peptide, is the core of this method currently applied widely to investigate specific immune responses in cancer, infections, allergies, and autoimmune diseases. The Elispot Assay is competing with other methods measuring single-cell cytokine production, e.g., intracellular cytokine by FACS or Milteny cytokine secretion assay. Other types of lymphocyte frequency and function assays include limiting dilution assay (LDA), cytotoxic T-cell assay (CTL), and tetramer staining. Compared with respect to sensitivity the Elispot Assay is outranking other methods to define frequency of antigen-specific lymphocytes. The method described herein would like to offer helpful and clear protocols for researchers that apply Elispot. IFN-γ and Perforin Elispot assays will be described.

Mycobacterium microti Interferes with Bovine Tuberculosis Surveillance.

Mycobacterium microti, a member of the Mycobacterium tuberculosis complex, was originally described as the cause of tuberculosis in wild rodents. However, in the last few years, an increasing number of cases have been reported in wildlife (wild boars and badgers) and livestock (goat and cattle) in the frame of bovine tuberculosis (bTB) surveillance program, demonstrating the risk of interference with bTB diagnosis in France. In 2019, we detected four cattle infected with M. microti, from three different herds in three different distant regions. For all these cases, ante-mortem diagnosis by the skin test (single intradermal comparative cervical tuberculin (SICCT)) was positive. Confirmation of M. microti infection was based on molecular tests, i.e., specific real-time PCR and spoligotyping. These results highlight a non-negligible risk of interference in the bTB diagnosis system and raise concern about the reliability of diagnostic tests used for bTB surveillance. The use of highly specific tests, like the interferon gamma test (IFN-γ) employed in France or new synthetic specific tuberculins for skin testing could alternatively be used to accurately identify M. bovis (or Mycobacterium caprae) infection at ante-mortem examination. At post-mortem diagnosis, the use of specific molecular tools should be considered to accurately distinguish pathogens within the MTBC and to avoid misleading bTB diagnosis.

Interpretation of serial interferon-gamma test results to measure new tuberculosis infection among household contacts in Zambia and South Africa

BackgroundA more stringent QuantiFERON-TB Gold In-Tube (QFT) conversion (from negative to positive) definition has been proposed to allow more definite detection of recent tuberculosis (TB) infection. We explored alternative conversion definitions to assist the interpretation of serial QFT results and estimate incidence of TB infection in a large cohort study.MethodsWe used QFT serial results from TB household contacts aged ≥15 years, collected at baseline and during two follow-up visits (2006–2011) as part of a cohort study in 24 communities in Zambia and South Africa (SA). Conversion rates using the manufacturers’ definition (interferon-gamma (IFN-g) < 0.35 to ≥0.35, ‘def1’) were compared with stricter definitions (IFN-g < 0.2 to ≥0.7 IU/ml, ‘def2’; IFN-g < 0.2 to ≥1.05 IU/ml, ‘def3’; IFN-g < 0.2 to ≥1.4 IU/ml, ‘def4’). Poisson regression was used for analysis.ResultsOne thousand three hundred sixty-five individuals in Zambia and 822 in SA had QFT results available. Among HIV-negative individuals, the QFT conversion rate was 27.4 per 100 person-years (CI:22.9–32.6) using def1, 19.0 using def2 (CI:15.2–23.7), 14.7 using def3 (CI:11.5–18.8), and 12.0 using def4 (CI:9.2–15.7). Relative differences across def1-def4 were similar in Zambia and SA. Using def1, conversion was less likely if HIV positive not on antiretroviral treatment compared to HIV negative (aRR = 0.7, 95%CI = 0.4–0.9), in analysis including both countries. The same direction of associations were found using def 2–4.ConclusionHigh conversion rates were found even with the strictest definition, indicating high incidence of TB infection among household contacts of TB patients in these communities. The trade-off between sensitivity and specificity using different thresholds of QFT conversion remains unknown due to the absence of a reference standard. However, we identified boundaries within which an appropriate definition might fall, and our strictest definition plausibly has high specificity.

IMMUNE RECONSTITUTION INFLAMMATORY SYNDROME: THE RISK OF ADALIMUMAB CESSATION IN TB TREATMENT

SESSION TITLE: Medical Student/Resident Chest Infections Posters SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: October 18-21, 2020 INTRODUCTION: Pharmacologic inhibition of tumor necrosis factor-alpha (TNF-a) is a mainstay in the treatment of autoimmune disease. Guidelines recommend starting TNF-a antagonists after a negative screening test result for tuberculosis (TB) infection is confirmed, due to the increased risk of reactivation TB in an immunosuppressed host. We present the case of a patient on adalimumab who developed extrapulmonary TB and immune reconstitution inflammatory syndrome (IRIS) after starting TB treatment. CASE PRESENTATION: A 67-year-old woman with a history of psoriasis, on adalimumab therapy for one year, presented to our hospital with chills, fevers, and a non-productive cough for one month. She denied hemoptysis and night sweats but acknowledged a 30-pound weight loss over the prior two months. On presentation, she was afebrile, normotensive, saturating 95% on room air. Chest X-ray was notable for bilateral miliary nodules and CT scan showed extensive miliary lesions in her lungs and spleen. She was admitted to the medicine service and put on airborne isolation. Interferon-gamma release assay (IGRA) and testing for HIV were negative. Induced sputum was collected, acid-fast bacilli (AFBs) were noted on smear microscopy, and PCR-based assay confirmed presence of Mycobacterium tuberculosis (MTb). Mycobacterial blood cultures were sent. Anti-TB therapy was started, and she was discharged to home isolation. One month later, she re-presented with dyspnea on exertion. Repeat CT scan showed a large left pleural effusion and a small pericardial effusion. AFB stain and culture of pleural fluid was negative. Repeat sputum AFB stain and culture was also negative, and MTb was not detected by PCR. Prior blood cultures resulted as positive for MTb. She was discharged home after clinical improvement with resolution of her dyspnea. DISCUSSION: TB is one of the top 10 causes of death worldwide. 20% of patients with active TB can have false negative IGRAs, especially if immunosuppressed. IRIS, most often seen in HIV+ patients, can develop with TB treatment initiation. Paradoxical worsening of clinical status occurs as the depressed immune system begins to recover. Patients on TNF-a antagonists are also at risk for IRIS, more commonly in lung and extrapulmonary TB. Thus, the combination of TNF-a antagonist discontinuation and TB treatment placed our patient at heightened risk for IRIS. CONCLUSIONS: This case demonstrates TB dissemination in an immunocompromised patient on a TNF-a antagonist who developed IRIS with adalimumab cessation and TB treatment initiation. Among commonly used biologic agents, adalimumab has been associated with a greater risk for both TB infection and IRIS development after starting TB treatment. Clinicians should be aware of this risk and monitor patients accordingly once treatment is initiated. Reference #1: World Health Organization. Global tuberculosis report 2019. Geneva, Switzerland: World Health Organization; 2019. Reference #2: Dobler, C. C. (2016). Biologic agents and tuberculosis. Microbiology Spectrum, Dec; 4(6). Reference #3: Cheng, V. C. et al. Clinical spectrum of paradoxical deterioration during antituberculosis therapy in non-HIV-infected patients. Eur. J. Clin. Microbiol. Infect. Dis. 21, 803–809 (2002). DISCLOSURES: No relevant relationships by Mark Adelman, source=Web Response No relevant relationships by Vickie Kassapidis, source=Web Response

Assessment of the cost-effectiveness of alternative bovine tuberculosis surveillance protocols in French cattle farms using the mixed interferon gamma test

Periodic screening in farms, using intradermal cervical comparative tuberculin test (ICCT), is a component of the French ante mortem surveillance of bovine tuberculosis (bTB). Previous studies have estimated the cost-effectiveness of the French mandatory bTB screening protocols. In these protocols, a second ICCT (ICCT2) is performed 42 days after the first one (ICCT1), either on the entire herd (strict protocol) or in series on animals with non-negative results (reactors) to ICCT1 (compliant protocol). The 42-days interval reduced protocols' cost-effectiveness. To minimize this interval, we suggested two alternative protocols, in which a mixed interferon gamma test (IFNMIX), with better sensitivity than ICCT2 and comparable specificity, replaces the ICCT2, and is carried out directly after the ICCT1. In the strict alternative protocol, reactors to ICCT1 are culled to perform laboratory analyses (PCR, bacteriology). Negative results to these analyses imply the IFNMIX testing of the entire herd. In the compliant alternative protocol, only reactor(s) to ICCT1 are tested with IFNMIX, and animals with positive results to IFNMIX are culled for laboratory analyses. We evaluated these protocols through scenario tree modelling.The estimated cost-effectiveness indexes showed that the compliant alternative protocol was the most efficient. The strict protocols (mandatory and alternative) were never the most efficient, but were the most effective. Therefore, using IFNMIX instead of ICCT2 may be useful in reducing the costs of the compliant protocol used when the probability of infection is considered low. The strict alternative protocol may become more attractive would IFNMIX's price decreased.