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Background: The aim of this study is to investigate the impact of the intradermal injection of purified protein derivative (PPD) and PPD skin test reactions on the oncological outcomes of patients with non-muscle invasive bladder cancer (NMIBC) treated by trans-urethral resection of bladder tumor (TURBT) and adjuvant intravesical BCG., Methods: The study included 100 consecutive patients with NMIBC prospectively given intradermal PPD 1-2 weeks before starting BCG therapy. Another 100 patients with NMIBC not given intradermal PPD before starting BCG were chosen as a historical control. The control group was chosen to be matching with the study group regarding baseline characteristics. The study group was divided into 2 subgroups with positive and negative reaction to PPD skin test. Oncological outcomes, immunological markers (TNF-α and IL-6) changes and BCG side effects were evaluated., Results: There were no significant differences between patients who received PPD or not regarding the 2-year recurrence free survival (RFS) rates and progression-free survival (PFS) rates and immunological markers changes. The 2-year RFS and PFS rates were significantly higher in patients with positive reactions. Post-induction values of immunological markers increased in all patients with a significant increase in patients with positive reactions. BCG side effects were significantly higher in patients with positive reactions., Conclusions: The intradermal injection of PPD before intravesical BCG has no impact on oncological outcomes of patients with NMIBC treated with TURBT and intravesical BCG. However, the PPD skin test reactions before BCG therapy can predict the oncological outcomes, BCG side effects and the immunological outcomes of patients.

Upon infection with Mycobacterium tuberculosis (Mtb) the host immune response might clear the bacteria, control its growth leading to latent tuberculosis (LTB), or fail to control its growth resulting in active TB (ATB). There is however no clear understanding of the features underlying a more or less effective response. Mtb glycolipids are abundant in the bacterial cell envelope and modulate the immune response to Mtb, but the patterns of response to glycolipids are still underexplored. To identify the CD45 + leukocyte activation landscape induced by Mtb glycolipids in peripheral blood of ATB and LTB, we performed a detailed assessment of the immune response of PBMCs to the Mtb glycolipids lipoarabinomannan (LAM) and its biosynthetic precursor phosphatidyl-inositol mannoside (PIM), and purified-protein derivate (PPD). At 24 h of stimulation, cell profiling and secretome analysis was done using mass cytometry and high-multiplex immunoassay. PIM induced a diverse cytokine response, mainly affecting antigen-presenting cells to produce both pro-inflammatory and anti-inflammatory cytokines, but not IFN-γ, contrasting with PPD that was a strong inducer of IFN-γ. The effect of PIM on the antigen-presenting cells was partly TLR2-dependent. Expansion of monocyte subsets in response to PIM or LAM was reduced primarily in LTB as compared to healthy controls, suggesting a hyporesponsive/tolerance pattern derived from Mtb infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Silva, Sundling, Folkesson, Fröberg, Nobrega, Canto-Gomes, Chambers, Lakshmikanth, Brodin, Bruchfeld, Nigou, Correia-Neves and Källenius.)

In many countries where tuberculosis (TB) is endemic, the Bacillus Calmette-Guérin (BCG) vaccine is given as close to birth as possible to protect infants and children from severe forms of TB. However, BCG has variable efficacy and is not as effective against adult pulmonary TB. At present, most animal models used to study novel TB vaccine candidates rely on the use of adult animals. Human studies show that the infant immune system is different to that of an adult. Understanding how the phenotypic profile and functional ability of the immature host immune system compares to that of a mature adult, together with the subsequent BCG immune response, is critical to ensuring that new TB vaccines are tested in the most appropriate models. BCG-specific immune responses were detected in macaques vaccinated within a week of birth from six weeks after immunization indicating that neonatal macaques are able to generate a functional cellular response to the vaccine. However, the responses measured were significantly lower than those typically observed following BCG vaccination in adult rhesus macaques and infant profiles were skewed towards the activation and attraction of macrophages and monocytes and the synthesis in addition to release of pro-inflammatory cytokines such as IL-1, IL-6 and TNF-α. The frequency of specific immune cell populations changed significantly through the first three years of life as the infants developed into young adult macaques. Notably, the CD4:CD8 ratio significantly declined as the macaques aged due to a significant decrease in the proportion of CD4 + T-cells relative to a significant increase in CD8 + T-cells. Also, the frequency of both CD4 + and CD8 + T-cells expressing the memory marker CD95, and memory subset populations including effector memory, central memory and stem cell memory, increased significantly as animals matured. Infant macaques, vaccinated with BCG within a week of birth, possessed a significantly higher frequency of CD14 + classical monocytes and granulocytes which remained different throughout the first three years of life compared to unvaccinated age matched animals. These findings, along with the increase in monokines following vaccination in infants, may provide an insight into the mechanism by which vaccination with BCG is able to provide non-specific immunity against non-mycobacterial organisms., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sarfas, White, Sibley, Morrison, Gullick, Lawrence, Dennis, Marsh, Fletcher and Sharpe.)

Introduction: The immunogenicity of BCG vaccination in infants differs between populations. We hypothesized that prenatal exposure to mycobacterial antigens might explain the differences in immune responses to BCG seen in other studies of infants in Africa and the United Kingdom (UK) and we explored this in birth cohorts in Uganda and the UK. Materials and Methods: Blood samples were obtained from BCG-immunized infants of mothers with ( n = 110) and without ( n = 121) latent Mycobacterium tuberculosis infection (LTBI) in Uganda and BCG-immunized infants of mothers without LTBI ( n = 25) in the UK at 10 and 52 weeks after birth. Cytokine and chemokine responses to PPD were measured to assess responses to BCG immunization, and to ESAT6/CFP10 to assess exposure to or infection with M. tuberculosis or non-tuberculous mycobacteria (NTM) in 6-day whole blood culture supernatants by a 17-plex Luminex assay. Median responses were compared between Ugandan infants (together, and separated by maternal LTBI status) and UK infants. Results: The IFN-γ response to BCG vaccination was similar between Ugandan and UK infants at 10 and 52 weeks. At week 52, TNF production was marginally higher in Ugandan infants, but after adjusting for multiple comparisons this difference was not significant. At weeks 10 and 52, stimulation of blood with ESAT6/CFP10 produced significantly higher IFN-γ, TNF, IL-12p40, IL-1α, IL-1β, IL-1Ra, IP-10, MIP-1α, MIP-1β, and GM-CSF in Ugandan compared to UK infants. Stimulation of blood with ESAT6/CFP10 produced significantly higher amounts of IL-8 ( p = 0.0001), IL-10 ( p = 0.0022), and IL-13 ( p = 0.0020) in the UK than in Ugandan infants of mothers without LTBI at week 10, but not at week 52. Conclusions: Immune responses to mycobacterial antigens following BCG immunization are similar for PPD, but differ for ESAT6/CFP10, between infants in Uganda and the UK. Neither maternal LTBI nor infant exposure to or infection with mycobacteria impacts the response to BCG. The observed global differences in immune response to BCG immunization are likely to be due to other causes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mawa, Hasso-Agopsowicz, Lubyayi, Nabakooza, Nakibuule, Blitz, Dun, Govind, Kaleebu, Webb, Elliott, Dockrell, Cose and Smith.)

The information generated from the official eradication programmes of caprine tuberculosis (TB) in Castilla y León, Spain, during 2018, has been used to assess the effect of vaccination against paratuberculosis (PTB) and the presence of this infection, on the single intradermal tuberculin (SIT) test results. Data from 121,665 goats belonging to 1936 different herds were analysed using generalized linear models. An epidemiological survey was conducted to know the herd immunization status against PTB and the date of last vaccination. All SIT test-positive animals were further investigated in order to confirm the diagnosis of TB, through bacterial culture, and PTB, by histopathological and qPCR analyses. SIT positivity was found in 39 (2.01%) herds and 507 (0.41%) goats. TB was confirmed by M. caprae or M. bovis isolation in 10 (0.51%) herds and 46 (0.038%) goats. PTB was diagnosed in 13 (33.33%) and 55 (10.84%) of the SIT test-positive herds and goats, respectively. Vaccination against PTB showed a significant influence on the results of the SIT test at herd level, with higher positivity detected among those herds vaccinated. However, this effect was not observed when the total number of animals was considered, where the highest positivity was found in unvaccinated goats. The time elapsed between vaccination and SIT test performance also influenced the results. The strongest effect was found when less than eight months elapsed between performing both activities, and to a lesser extent between 8 and 12 months. Conversely, no positive herds or animals were found when the time elapsed was higher than one year. No significant effect of the presence of PTB was observed. These findings demonstrate that the use of PTB vaccine does not result in false positives to a SIT test at individual level, provided that the time elapsed between the performance of both practices is higher than 12 months., (© 2020 Blackwell Verlag GmbH.)

Objectives: The genetic diversity of Mycobacterium tuberculosis complex (MTBC) influences the immune response of the host, which may affect the immunodiagnostic tests and biomarker assessment studies used for tuberculosis (TB). This study aimed to determine whether the mycobacterial-antigen-stimulated cytokine responses vary with the genotype of the MTBC infecting the patient., Methods: Eighty-one patients with confirmed active pulmonary TB were recruited, and MTBC clinical strains were isolated from their sputum for bacterial lineage single-nucleotide polymorphism typing. Whole blood was drawn from the patients to measure the purified protein derivative (PPD)-stimulated cytokine responses (GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, TNF-α, IFN-α, IL-12, eotaxin, IL-13, IL-15, IL-17, MIP1-α, MIP1-β, MCP1, IL1RA, IP10, IL2R, MIG) with the Luminex multiplex immunoassay., Results: Of the 24 cytokines studied, three were produced differentially in whole blood dependent on the infecting lineage of MTBC. Decreased production of IL-17 was observed in patients infected with modern lineages compared with patients infected with ancestral lineages (P < 0.01), and production of IFN-γ and IL-2 was significantly decreased in patients infected with lineage 4 strains compared with patients infected with lineage 3 strains (P < 0.05)., Conclusion: MTBC strains belonging to lineage 4 induced a decreased whole-blood PPD-stimulated pro-inflammatory cytokine response., (Copyright © 2021 institut pasteur de madagascar. Published by Elsevier Ltd.. All rights reserved.)

Tuberculin Purified Protein Derivatives (PPDs) exhibit multiple limitations: they are crude extracts from mycobacterial cultures with largely unknown active components; their production depends on culture of mycobacteria requiring expensive BCL3 production facilities; and their potency depends on the technically demanding guinea pig assay. To overcome these limitations, we developed a molecularly defined tuberculin (MDT) by adding further antigens to our prototype reagent composed of ESAT-6, CFP-10 and Rv3615c (DIVA skin test, DST). In vitro screening using PBMC from infected and uninfected cattle shortlisted four antigens from a literature-based list of 18 to formulate the MDT. These four antigens plus the previously identified Rv3020c protein, produced as recombinant proteins or overlapping synthetic peptides, were formulated together with the three DST antigens into the MDT to test cattle experimentally and naturally infected with M. bovis, uninfected cattle and MAP vaccinated calves. We demonstrated significant increases in MDT-induced skin responses compared to DST in infected animals, whilst maintaining high specificity in unvaccinated or MAP vaccinated calves. Further, MDT can also be applied in in vitro blood-based interferon-gamma release assays. Thus, MDT promises to be a robust diagnostic skin and blood test reagent overcoming some of the limitations of PPDs and warrants full validation.

Interferon-γ release assays cannot differentiate latent from active tuberculosis (TB), nor identify the recently infected with increased risk of active disease. The objective of this study was to identify biomarkers of recent infection following exposure to tuberculosis, to increase the positive predictive value for incipient TB. Contacts to patients with pulmonary TB were tested repeatedly with interferon-γ release assays and flow-cytometry. Proliferative CD4 + T cell responses to purified protein derivative (PPD) and 11 M. tuberculosis antigens were analysed. The individual probability of recent and remote infection was estimated using clinical data in a novel mathematical model and compared with CD4 + responses in a prediction model. The most specific prediction of recent infection was high CD4 + proliferative responses to CFP-10 and PPD and a low CD4 + response to ESAT-6. CD4 + proliferative responses to Rec85a, Rec85b and Rv1284 were also observed in recent infection, but did not reach significance in the prediction model. CONCLUSIONS: High CD4 + proliferative responses to CFP-10 and PPD and a low response to ESAT-6 may be used as biomarkers to improve positive predictive values for recent LTBI and thus, increased risk of incipient TB. Rec85a, Rec85b and Rv1284 are also of interest to study further in this context., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Bovine tuberculosis (BTB) caused by Mycobacterium bovis remains a major problem in both the developed and developing countries. Control of BTB in the UK is carried out by test and slaughter of infected animals, based primarily on the tuberculin skin test (PPD). Vaccination with the attenuated strain of the M. bovis pathogen, BCG, is not used to control bovine tuberculosis in cattle at present, due to its variable efficacy and because it interferes with the PPD test. Diagnostic tests capable of Differentiating Infected from Vaccinated Animals (DIVA) have been developed that detect immune responses to M. bovis antigens absent in BCG; but these are too expensive and insufficiently sensitive to be used for BTB control worldwide. To address these problems we aimed to generate a synergistic vaccine and diagnostic approach that would permit the vaccination of cattle without interfering with the conventional PPD-based surveillance. The approach was to widen the pool of M. bovis antigens that could be used as DIVA targets, by identifying antigenic proteins that could be deleted from BCG without affecting the persistence and protective efficacy of the vaccine in cattle. Using transposon mutagenesis we identified genes that were essential and those that were non-essential for persistence in bovine lymph nodes. We then inactivated selected immunogenic, but non-essential genes in BCG Danish to create a diagnostic-compatible triple knock-out ΔBCG TK strain. The protective efficacy of the ΔBCG TK was tested in guinea pigs experimentally infected with M. bovis by aerosol and found to be equivalent to wild-type BCG. A complementary diagnostic skin test was developed with the antigenic proteins encoded by the deleted genes which did not cross-react in vaccinated or in uninfected guinea pigs. This study demonstrates the functionality of a new and improved BCG strain which retains its protective efficacy but is diagnostically compatible with a novel DIVA skin test that could be implemented in control programmes.

Introduction. Current intradermal tuberculin skin tests for latent tuberculosis infection (LTBI) based on purified protein derivative (PPD) have poor specificity. Aims. Developing a better skin test antigen as well as a simple skin patch test may improve and facilitate diagnostic performance. Methodology. Defined recombinant antigens that were unique to Mycobacterium tuberculosis (MTB), including two potential latency-associated antigens (ESAT-6 and Rv2653c) and five DosR-encoded latency proteins (Rv1996, Rv2031c, Rv2032, DevR and Rv3716c), were used as diagnostic skin test reagents in comparison with a standard PPD. The performance of the skin tests based on the detection of delayed-type hypersensitivity (DTH) reaction in guinea pigs sensitized to MTB and M. bovis bacille Calmette-Guérin (BCG) vaccine was evaluated. Results. The latency antigens Rv1996, Rv2031c, Rv2032 and Rv2653c and the ESAT-6 protein elicited less reactive DTH skin responses in MTB-sensitized guinea pigs than those resulting from PPD, but elicited no response in BCG-vaccinated guinea pigs. The remaining two latency antigens (DevR and Rv3716c) elicited DTH responses in both groups of animals, as did PPD. The reactivity of PPD in BCG-vaccinated guinea pigs was greater than that of any of the selected skin test reagents. Using stronger concentrations of selected skin test reagents in the patch test led to increased DTH responses that were comparable to those elicited by PPD in guinea pigs sensitized with MTB. Conclusion. Transdermal application of defined purified antigens might be a promising method for LTBI screening.

Background: Nowadays, several test methods with different useful values are available for diagnosis of the tuberculosis (TB) in non-human primates (NHPs). Despite some limitations of tuberculin skin test (TST), it is still the most commonly used method for TB testing of NHPs., Methods: During this investigation, TST was performed upon three groups of experimentally tuberculin sensitized and one group of non-sensitized vervet monkeys (Chlorocebus pygerythrus) by means of two types of old tuberculin (OT) and two types of purified protein derivative (PPD) tuberculin., Results: The data obtained from this study revealed that PPD tuberculin prepared from both Mycobacterium tuberculosis and Mycobacterium bovis has more advantages over OT in tuberculin testing of the vervet monkeys. The potency of the PPD tuberculin prepared from M bovis was estimated almost twice as much of the M tuberculosis., Conclusions: Intrapalpebral injection of 0.1 mL of a concentration of ≥1 mg/mL of PPD tuberculin prepared from M bovis is the preferred method for TST of vervet monkeys., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Accurate and rapid diagnostic tools are important aspects of managing tuberculosis (TB) cases appropriately. However, the sensitivity and specificity of diagnostic kits based on immune response such as the tuberculin skin test (TST) and interferon gamma release assay (IGRA) are still debated. Thus, the exploration and assessment of specific biomarker-targeted antibodies are needed for the development of an accurate and rapid diagnostic tool. The present study was conducted in patients with a respiratory problem suspected to be TB at Dr. Soetomo Hospital, Surabaya, Indonesia. Among 102 patients tested by GeneXpert and AFB, 59 serum samples were from cases retrospectively determined to have active TB. A total of 102 serum of healthy controls (HC) was also collected. The PPD antigen and the recombinant CFP-10 and ESAT-6 proteins were prepared. Antibody responses against these proteins were evaluated by ELISA. All samples were also screened for the possibility of Mycobacterium avium-intracellulare complex (MAC) infection using Capilla MaC kit. The results showed that TB patients had a significantly higher concentration of IgG antibody in response to PPD than the HC. In addition, the receiver operating characteristic (ROC) curve analysis showed that PPD was acceptable for diagnostic purposes with an AUC value of 0.835 (95% CI 0.770-0.900, p < 0.0001). However, ESAT-6 and CFP-10 had low AUCs, and 32 samples from both groups showed a low concentration of IgA antibody against all antigens. The MAC detection results also showed that the concentration of IgA in the HC group was the highest. The current results indicate that PPD is a better antigen for antibody-based detection of TB than ESAT-6 and CFP-10. Based on the MAC detection assay, 53 people in the HC group were probably infected with rapidly growing nontuberculous mycobacteria (NTM), although antibody response to PPD was low., (Copyright © 2019 Sociedade Brasileira de Infectologia. Published by Elsevier España, S.L.U. All rights reserved.)

Diagnostic tests based on cell-mediated immunity are used in programs for the control and eradication of bovine tuberculosis (bTB), which is mainly caused by Mycobacterium bovis. Additional serological assays could be performed as an ancillary method to detect an infected animal that fails to produce an immune response against the intradermal reaction (IDR), the official bTB test. In this study, we evaluated the effectiveness of an enzyme-linked immunosorbent assay (ELISA) that uses bovine PPD as a capture antigen as a complement to the IDR in herds with confirmed cases of bTB. The study was conducted in two stages. First, a panel of 200 serum samples was analyzed by ELISA. The sensitivity and specificity obtained were 60% and 99%, respectively. The subsequent stage consisted of evaluating 7,494 bovines from 14 selected dairy farms. The number of animals yielding a IDR negative/ELISA positive result were 200. A necropsy analysis of 33 of these IDR negative/ELISA positive animals revealed that 30 (91%) presented granulomatous lesions and positive M. bovis isolation. This finding confirmed bTB in most cases. Altogether, the results obtained in the present study suggest that the combined use of IDR and ELISA is an effective strategy to improve the control of bTB in endemic herds., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Peritoneal tuberculosis (TB) remains difficult to diagnose because of its non-specific clinical features and the lack of efficient microbiological tests. As delayed diagnosis is associated with high mortality rates, new diagnostic tools are needed., Methods and Findings: We investigated for 24 patients prospectively enrolled with a possible diagnosis of peritoneal TB, the diagnostic value of the analysis of IFN-γ production by peritoneal fluid lymphocytes in response to a short in vitro stimulation with mycobacterial antigens. The patients were classified in two groups: non-TB and confirmed or highly probable TB. Diagnosis of TB was based on microbiological and histopathological criteria and/or a favorable response to anti-TB treatment. The IFN-γ production by peritoneal CD4+ T lymphocytes was analyzed by flow cytometry after an overnight in vitro stimulation with three different mycobacterial antigens, purified protein derivative (PPD), heparin-binding haemagglutinin (HBHA) or early-secreted-antigen-target-6 (ESAT-6). The percentages of PPD-, HBHA- or ESAT-6-induced IFN-γ-producing peritoneal fluid CD4+ T lymphocytes were higher in the TB group than in the non-TB group (p = 0.0007, p = 0.0004, and p = 0.0002 respectively). Based on cut-off values determined by ROC curve analysis of the results from TB and highly probable TB compared to those of non-TB patients, the sensitivity of these three tests was 100% with a specificity of 92%., Conclusions: The analysis of mycobacterial-induced IFN-γ production by peritoneal lymphocytes is a promising tool to reliably and rapidly diagnose peritoneal TB. Further studies should be performed on larger cohorts of patients in high-TB-incidence countries to confirm the clinical value of this new diagnostic approach for peritoneal TB., Competing Interests: The authors have declared that no competing interests exist.

Bovine tuberculosis (bTB) is an important animal and zoonotic disease, which causes severe economic losses. The main focus of this study was to assess the predictive power of previously identified biomarkers of bTB in infected animals that were negative to the tuberculin skin test (TST). We studied 16 animals with bTB, in which the disease was confirmed by necropsy, and 16 healthy animals. The level of expression of ten biomarkers (CXCL9, THBS1, MMP9, IL-22, CXCL10, IFNγ, IL-17, FYVE, CD14, IL-1R) was evaluated by RT-qPCR upon stimulation or not of peripheral blood mononuclear cells with PPDb (purified protein derivative of bovine tuberculin). In this assay, CXCL9, THBS1, MMP9, IL-22 and IFNγ changed their expression level depending on the bTB status. In addition, we evaluated different biomarker candidates simultaneously to infer the animal condition. By performing an analysis with classification trees, we found that the sturdiest combination was IL-22, IFNγ and IL-1R. On the other hand, CXCL10, IFNγ and IL-22's expression distinguished between bTB positive animals that were negative to TST (TST false negative animals) and the bTB negative groups. Thus, these biomarkers are promising candidates to be tested as an ancillary diagnostic assay. In addition, the expression of CXCL10 and IL-22 exhibited also significant differences between the bTB positive animals that were undetectable by IFNγ release assay (IGRA) and TST tests (TST and IGRA false negative animals) and the bTB negative groups. Therefore, CXCL10 and IL-22 constitute candidate biomarkers that could complement the two most widely used diagnostic tests., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: A group of Russian scientists has developed Diaskintest, which comprises Mycobacterium tuberculosis-specific recombinant proteins CFP10-ESAT6, for skin testing (0.2 μg/0.1 ml)., Study Purpose: To evaluate the comparative sensitivity of TST with 2 TU PPD-L and a skin test with tuberculous recombinant allergen (Diaskintest) containing the ESAT6-CFP10 protein in children and adolescents with newly diagnosed active tuberculosis during mass screening in the primary medical service in Moscow., Materials and Methods: The trial was a comprehensive retrospective group study of children and adolescents diagnosed in Moscow with active tuberculosis in 2013-2016, aged 0 to 17 years inclusive., Results: From 441 patients selected for analysis 408 patients had both tests (TST with 2 TU PPD-L and Diaskintest) performed, in 193 patients both tests were given simultaneously, of them 162 patients were BCG-vaccinated. Comparative results of both tests in 408 patients with tuberculosis: at cut-off ≥ 5 mm, both tests has similar sensitivity: Diaskintest 98.3% (95% CI 97.0-99.6%), TST 98.0% (95% CI 96.7-99.4%), at cut-off ≥10 mm, the sensitivity decreases for both tests: Diaskintest 90.0% (95% CI 87.0-93.0%), TST 88.7% (95% CI 85.6-91.9%), but at cut-off ≥ 15 mm, the decrease in sensitivity is statistically significant: for Diaskintest 61.5% (95% CI 56.7-66.3%), and for TST 46.3% (95% CI 41.4-51.3%), p <0.0001. The results of simultaneous setting of tests on different hands in 193 people (including 162 BCG-vaccinated), do not differ from the results for 408 people. The correlation between the results of Diaskintest and TST was significant in all groups., Conclusion: In children and adolescents with active tuberculosis, Diaskintest of 0.2 μg/ml and the Mantoux test with 2 TU PPD-L have high sensitivity (98%) at a cut-off of 5 mm; however, at cut-off ≥ 15 mm sensitivity is significantly reduced, and the decrease is more pronounced in the Mantoux test. The advantage of Diaskintest is that, unlike the Mantoux test, it has high specificity under the conditions of mass BCG vaccination. The test is simple to carry out, and can be used in mass screening., Competing Interests: The authors have declared that no competing interests exist.

Intravesical Bacillus Calmette-Guérin (BCG) immunotherapy preserves the bladder after resection of high-risk non-muscle-invasive bladder cancer (NMIBC). About 30% of patients experience treatment failure, which cannot be predicted a priori and carries a high risk of disease progression. We examined the in vitro tuberculin responsiveness of CD4 + T cells before BCG immunotherapy in 42 patients with high-risk NMIBC. The frequencies and functionalities of cytokine-expressing CD4 + T cells immediately before and after BCG immunotherapy induction were assessed by flow cytometry after overnight tuberculin stimulation. Tuberculin-induced secreted mediators were measured by electrochemiluminescence. We correlated the results with recurrence-free patient survival 6 months after induction. A tuberculin-induced, secreted, IL2 concentration > 250 pg/mL was the best predictor of recurrence-free survival, providing 79% sensitivity, 86% specificity (AUC = 0.852, P = 0.000), and overall correct classification in 78.6% of cases. In 50% of patients later experiencing recurrence, but not in any of the recurrence-free survivors, IL2 secretion was < 120 pg/mL. Other parameters predicting recurrence-free survival included secreted IFNγ (AUC = 0.796, P = 0.002) and the frequencies of TNF-producing (TNF + ) CD4 + T cells (AUC = 0.745, P = 0.010). "Polyfunctional" CD4 + T cells (IFNγ + /IL2 + /TNF + ) were significantly associated with recurrence-free survival (AUC = 0.801, P = 0.002). Thus, the amount of IL2 secretion from CD4 + T cells after overnight in vitro incubation with tuberculin predicted the outcome of BCG immunotherapy. As many as half of potential BCG failures could be identified before induction therapy is begun, enabling better choices regarding treatment. Cancer Immunol Res; 6(10); 1212-9. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Tuberculin skin test (TST) is used most widely for the detection of latent tuberculosis infection (LTBI), even though evidences suggest that it could be underreporting the prevalence of LTBI particularly in high disease-burden settings. We have explored whether in vivo (TST) and in vitro (cell-proliferative) T cell responses to PPD can serve as complementary measures. In addition, we also probed whether in vitro T cell response to cell-membrane antigens (Mem) of Mycobacterium tuberculosis (MTB) can serve as a biomarker for LTBI. Study subjects comprised 43 healthcare workers (HCWs), and 9 smear-positive TB patients served as 'disease control'. To measure proliferative T cell responses, 0.1 ml blood (diluted 1:10) was incubated (5 days) with test or control antigen. Cells were stained with fluorescent antibodies to T cell (CD3+/CD4+/CD8+) surface markers and, after fixation and permeabilization, to nuclear proliferation marker Ki67. Data was acquired on a flow cytometer. HCWs who had an intimate exposure to MTB showed significantly higher TST positivity (85%) than the rest (43%), notwithstanding their BCG vaccination status. The proliferative responses of CD4+ and CD8+ subsets of T cells were comparable. Sixty seven and 100% TST-negative HCWs, respectively, were positive for proliferative T cell response to PPD and MTBMem. Cumulative positivity (TST or in vitro) was 86% with PPD and 100% with MTBMem indicating complementarity of the two responses. As standalone in vitro assay, MTBMem provided a significantly higher positivity (95%) than PPD (67%). T cell responses of TB patients were 'generally' depressed, having implications for the development of immunological assays for 'progressive' LTBI. Altogether, these results demonstrate that in vivo and in vitro T cell responses to PPD are complementary and in vitro response to MTBMem can be developed as a highly sensitive biomarker for LTBI., Competing Interests: The authors have declared that no competing interests exist.

Effective disease management of wildlife relies on the strategic application of ante-mortem diagnostic tests for early identification and removal of M. bovis-infected animals. To improve diagnostic performance, interferon-gamma release assays (IGRAs) are often used in conjunction with the tuberculin skin test (TST). Since buffaloes are major maintenance hosts of M. bovis, optimal application of bovine TB diagnostic tests are especially important. We aimed to determine whether the timing of blood collection relative to the TST has an influence on IFN-γ production and diagnostic outcome in African buffaloes. Release of IFN-γ in response to bovine purified protein derivative (PPD), avian PPD and PC-HP ® and PC-EC ® peptides was measured by Bovigam ® and an in-house IGRA in a group of Bovigam ® -positive and - negative buffaloes at the time the TST was performed and three days later. There was significantly lower IFN-γ release in response to these antigens post-TST in Bovigam ® -positive buffaloes, but no significant changes in Bovigam ® -negative buffaloes. Also, a significantly greater proportion of buffaloes were Bovigam ® -positive prior to the TST than three days later. We therefore recommend that blood samples for use in IGRAs be collected prior to or at the time the TST is performed to facilitate the correct identification of greater numbers of IGRA-positive buffaloes., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Accurate diagnosis of tuberculosis in cattle may be compromised in areas where there are high rates of exposure to environmental/non-tuberculous mycobacteria (NTM). This cross reaction of immune responses to Mycobacterium bovis antigens shared with NTMs can result in reduced specificity of commonly used diagnostic tests including tuberculin skin tests and the interferon gamma assay (IFN-ɣ). In this study we assessed the cross-reactive immune responses of M. bovis (infected) and NTM exposed animals to M. bovis and M. avium tuberculin, the ESAT6/CFP10 cocktail antigen, tuberculin derived from cultures of selected NTMs, and a panel of recombinant mycobacterium tuberculosis complex (MTBC) antigens sharing homology with orthologues in NTM. Gamma interferon (IFN-ɣ) responses were measured in whole blood cultures using the IFN-ɣ assay and the IFN-ɣ elispot assay on purified peripheral blood mononuclear cells (PBMC). We observed the expected strong IFN-ɣ response to PPD-B in the M. bovis infected animals that distinguished this group from non-infected NTM exposed cattle. The IFN-ɣ responses to PPD-N (M. nonchromogenicum), were relatively high in both infected and non-infected NTM exposed cattle, but were not significantly different to classify the true infection status of each group. The results indicated that the cross-reactive responses to PPD-B and/or PPD-A with PPD-N, likely arose from prior exposure to environmental non-tuberculous mycobacteria. The IFN-ɣ immune responses to the 10 R-Mag measured by the IFN-ɣ elispot assay revealed that three of the selected antigens, Rv3615 (ESpC), Rv0287 (esxG) and the ESAT6/CFP10, were immunogenic in the infected cattle, and distinguished the infected cattle from the non-infected NTM exposed animals. The combined data of PPDs and R-Mags derived from NTM mycobacteria may prove useful in future development of novel bTB diagnostic tests., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: The WHO strategy for eradication of tuberculosis (TB) by 2035 (The End TB Strategy) is aimed at an early and precise diagnosis and subsequent effective treatment of TB patients. Currently, there is no gold standard for the diagnosis of latent TB infection. This study evaluated the diagnostic capabilities of a new intradermal test using recombinant TB allergen (Diaskintest) compared with tuberculin skin test (TST) and commercial TB interferon-gamma release assays (IGRAs)., Methods: A post-hoc data analysis that involved examining 860 HIV-negative, bacillus Calmette-Guérin (BCG)-vaccinated persons aged 1-65 years who visited the TB health-care institutions of Saint Petersburg to rule out or confirm an active TB was conducted from 2011 to 2016., Results: A high degree of consistency of the Diaskintest results with the enzyme-linked immunospot and QuantiFERON-TB Gold In-Tube test (ELISPOT and QFT) results was observed in the examined pediatric population (n = 696), with a Diaskintest cutoff ≥5 mm: the kappa consistency indices were 1.000 and 0.937, for ELISPOT and QFT, respectively. A high sensitivity of Diaskintest, comparable with the IGRA tests, was observed in patients with a confirmed TB diagnosis in all age groups. The sensitivity of Diaskintest in patients of the TB/MTB + group aged 18 years and older was 88.7%; of ELISPOT, 90.6%; of QFT, 87.0%. The conducted analysis has shown a high concordance of results of the commercial TB tests in adult HIV-negative patients (n = 164) with a Diaskintest cutoff ≥5 mm: the kappa indices were 0.805 and 0.636 (Diaskintest vs. ELISPOT and QFT, respectively) among BCG-vaccinated people., Conclusion: According to the WHO recommendations, replacing the TST by IGRAs is not recommended as a public health intervention in resource-constrained settings because the IGRA tests are more costly and technically complex to conduct than the TST. Diaskintest has comparable complexity to the TST and its performance is close to that of IGRA in a BCG-vaccinated population. Thus, our study demonstrates that replacing the TST by Diaskintest can be recommended as a public health intervention in resource-constrained and universal BCG vaccination settings., Competing Interests: There are no conflicts of interest.

A functional genomics approach was used to examine the immune response for transcriptional profiling of PBMC M. bovis infected cattle and healthy control cattle to stimulation with bovine tuberculin (purified protein derivative PPD-b). Total cellular RNA was extracted from non-challenged control and M. bovis challenged MDM for all animals at intervals of 6 h post-challenge, in response to in-vitro challenge with M. bovis (multiplicity of infection 2:1) and prepared for global gene expression analysis using the Agilent Bovine (V2) Gene Expression Microarray, 8 × 60 K. The pattern of expression of these genes in PPD bovine stimulated PBMC provides the first description of an M. bovis specific signature of infection that may provide insights into the molecular basis of the host response to infection. Analysis of these mapped reads showed 2450 genes (1291 up regulated and 1158 down regulated) 462 putative natural antisense transcripts (354 up-regulated and 108 down regulated) that were differentially expressed based on sense and antisense strand data, respectively (adjusted P-value ≤ 0.05). The results provided enrichment for genes involved top ten up regulated and down regulated panel of genes, including transcription factors proliferation of T and B lymphocytes. The highest differentially-expressed genes were associated to immune and inflammatory responses, immunity, differentiation, cell growth, apoptosis, cellular trafficking and regulation of lipolysis and thermogenesis. Microarray results were confirmed in infected cattle by RT qPCR to identify potential biomarkers TLR2, CD80, NFKB1, IL8, CXCL6 and ADORA3 of bovine tuberculosis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Little is known regarding the restoration of the specific immune response after combined antiretroviral therapy (cART) and anti-tuberculosis (TB) therapy introduction among TB-HIV patients. In this study, we examined the immune response of TB-HIV patients to Mycobacterium tuberculosis (Mtb) antigens to evaluate the response dynamics to different antigens over time. Moreover, we also evaluated the influence of two different doses of efavirenz and the factors associated with immune reconstitution., Methods: This is a longitudinal study nested in a clinical trial, where cART was initiated during the baseline visit (D0), which occurred 30 ± 10 days after the introduction of anti-TB therapy. Follow-up visits were performed at 30, 60, 90 and 180 days after cART initiation. The production of IFN-γ upon in vitro stimulation with Mtb antigens purified protein derivative (PPD), ESAT-6 and 38 kDa/CFP-10 using ELISpot was examined at baseline and follow-up visits., Results: Sixty-one patients, all ART-naïve, were selected and included in the immune reconstitution analysis; seven (11.5%) developed Immune Reconstitution Inflammatory Syndrome (IRIS). The Mtb specific immune response was higher for the PPD antigen followed by 38 kDa/CFP-10 and increased in the first 60 days after cART initiation. In multivariate analysis, the variables independently associated with increased IFN-γ production in response to PPD antigen were CD4 + T cell counts <200 cells/mm 3 at baseline, age, site of tuberculosis, 800 mg efavirenz dose and follow-up CD4 + T cell counts. Moreover, the factors associated with the production of IFN-γ in response to 38 kDa/CFP-10 were detectable HIV viral load (VL) and CD4 + T cell counts at follow-up visits of ≥200 cells/mm 3 ., Conclusions: These findings highlight the differences in immune response according to the specificity of the Mtb antigen, which contributes to a better understanding of TB-HIV immunopathogenesis. IFN-γ production elicited by PPD and 38 kDa/CFP-10 antigens have a greater magnitude compared to ESAT-6 and are associated with different factors. The low response to ESAT-6, even during immune restoration, suggests that this antigen is not adequate to assess the immune response of immunosuppressed TB-HIV patients.

The diagnosis of a latent tuberculosis infection (LTBI) is based on detection of immunity against Mycobacterium tuberculosis (Mtb). The tuberculin skin test (TST), the Quantiferon (QFT) and a prolonged lymphocyte stimulation test using either ESAT-6/CFP-10 (LST-EC) or PPD (LST-PPD) were evaluated in a cohort of 495 individuals, suspected to have LTBI, in a low endemic country. While the TST and LST-PPD were both positive in the majority (75%) of individuals, only one third responded in the LST-EC and in the QFT. The choice for LTBI treatment was significantly associated with ESAT6/CFP10 recognition, however the LST-EC detected considerably more individuals (21%) with immunity against Mtb, who might also be at risk for development of active TB, although none of them did during follow up. Follow-up for 2 years showed 7% conversions and 32% reversions for the QFT. The LST-EC showed higher conversion rates (∼45%), although the percentage of individuals positive in the LST-EC did not change significantly within the follow-up period. LTBI treatment did not alter immune recognition of Mtb antigens. In conclusion, the sensitivity of tests for detection of cellular immunity to Mtb specific antigens depends on test methodology and may vary considerably over time in a low endemic region., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Bacille Calmette-Guérin (BCG) vaccination is known to cause false-positive tuberculin skin test (TST) results from cross-reactions with mycobacterial antigens. However, the duration of BCG vaccination influence on the TST is poorly characterized. The objective of this study was to assess the long-term effect of BCG vaccination on TST reactivity., Methods: Data on TST reactivity were prospectively collected during 1935 to 1947 as part of a clinical trial among Native Americans/Alaskan Natives and were retrospectively collected thereafter between 1948 and 1998. TST induration of ≥ 10 mm was defined as a positive reaction. Kaplan-Meier analysis and multivariate Cox regression were used to compare the time to TST conversion and reversion between the BCG and placebo groups., Results: BCG vaccination after infancy was associated with an increased risk of TST reactivity in the first 15 years after vaccination (adjusted hazard ratio [HR], 2.33). This association remained during the interval 16 to 55 years after vaccination, although the effect was attenuated (adjusted HR, 1.26). Age at vaccination modestly impacted the effect of BCG on TST results in the first 15 years. Positive TST results among the BCG-vaccinated group were more likely to revert to negative results during the first 15 years but not in the latter period., Conclusions: This study provides evidence that BCG vaccination after infancy may influence TST results beyond the 10-year period conventionally accepted by the Centers for Disease Control and Prevention (CDC), extending up to 55 years after vaccination. This suggests that BCG vaccination should be taken into account when interpreting TST results regardless of the time elapsed since vaccination., (Published by Elsevier Inc.)

The measurement of bovine interferon-gamma (IFN-γ) forms the basis of a diagnostic test for bovine tuberculosis where Mycobacterium bovis sensitised effector T cells produce IFN-γ following in vitro stimulation with tuberculin antigens. In cattle infected with M. bovis it is also known that the anti-inflammatory IL-10 cytokine can inhibit in vitro production of IFN-γ leading to a reduced response in the IFN-γ diagnostic test. In order to investigate this in greater detail, whole blood samples from tuberculin skin test positive and negative cattle were stimulated with bovine and avian tuberculin antigens and in parallel with a neutralising anti-IL-10 monoclonal antibody. The results showed that IFN-γ protein levels increased when IL-10 activity was suppressed by Anti - IL-10. By using a standard diagnostic interpretation, the elevated levels of IFN-γ were shown to change the level of agreement between the performance of the single intradermal comparative tuberculin test (SICTT) and IFN-γ assay, depending on the tuberculin treatment. A transcriptomic analysis using RT-qPCR investigated the influence of IL-10 activity on expression of a suite of cytokine genes (IFNG, IL12B, IL10 and CXCL10) associated with antigen-stimulated production of IFN-γ. The IFNG and IL12B genes both experienced significant increases in expression in the presence of Anti-IL-10, while the expression of IL10 and CXCL10 remained unaffected., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Bovine tuberculosis (bTB) is usually diagnosed in vivo and ex vivo on the basis of delayed hypersensitivity reactions with a complex pool of antigens named bovine tuberculin (PPDB). The IFN-γ release assay (IGRA) for bTB is a blood-based assay that improves detection of infected cattle at early stages that escape skin testing. Improvements to IFN-γ testing with specific proteins have been performed to increase sensitivity. DosR regulon-related antigens are well known mycobacterial proteins expressed during the non-replicative phases of infection, this has been useful to improve the diagnosis of subclinical forms of TB in suspected individuals. Transcripts of DosR genes mb2054c, mb2057c, and mb2660c have been identified by our group in lymph nodes of IFN-γ test negative cattle. This led us to hypothesize that DosR-related proteins may potentiate the IFN-γ response to PPDB in animals with a false negative IFN-γ test, making evident subclinical infection. Three hundred animals were evaluated by means of IGRA and post-mortem microbiological analysis of tissue samples to validate M. bovis infection. We found that 176 out of 300 animals showed an overall increased OD in complemented IGRA with two purified protein cocktails in comparison to PPDB alone, and were scrutinized for a subclinical infection; thirty percent when PPDB was supplemented with a cocktail of four DosR antigens, and 70% when PPDB was supplemented with a cocktail of six antigens (four DosR and two RD1 antigens). Forty five animals showed a substantial IFN-γ overproduction but remained negative, and 40 animals changed the result to a positive test. Only 18 out of 176 IFN-γ high producing animals were also positive to M. bovis isolation. Fifty seven animals with no visible lesions at slaughter and with a negative IGRA test result contained M. bovis DNA in tissue samples. In conclusion, Mb1762c, Mb2054c, Mb2057c, and Mb2660c have the potential to increase sensitivity of the IFN-γ in vitro test for bTB diagnosis when supplemented to PPDB., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Cutaneous leishmaniasis (CL) heals spontaneously within several weeks or months, but, in rare cases, CL-active lesions last for many years. In this study, we assessed cell-mediated immunity in non-healing CL through the measurement of three pro-inflammatory cytokines: Interferon-γ (IFN-γ), IL-17a and CXCL-11. For this, 32 patients afflicted with healing or non-healing CL were recruited in this study. Peripheral blood mononuclear cells (PBMCs) of every patient were treated with three antigens: purified protein derivative (PPD), soluble Leishmania antigen (SLA) and phytohaemagglutinin (PHA). Cytokine quantification was performed using enzyme-linked immunosorbent assay (ELISA) method. Results of our study showed that neither cytokine produced in the presence of a PPD stimulator (as an irrelevant antigen) significantly differed between the healing and non-healing groups (P-value ≥0.05 for all of them). However, IFN-γ, CXCL-11 and IL-17a levels produced in the presence of PHA or SLA were significantly higher within the healing than in the non-healing group (P-value <0.01 for all of them). It seems that appropriate levels of IFN-γ, as well as IL-17a and CXCL-11, contribute to the control of Leishmania infection., (© 2017 The Foundation for the Scandinavian Journal of Immunology.)

Tuberculosis (TB) causes significant morbidity and mortality on a global scale. The African region has 24% of the world's TB cases. TB overlaps with other infectious diseases such as malaria and HIV, which are also highly prevalent in the African region. TB is a leading cause of death among HIV-positive patients and co-infection with HIV and TB has been described as a syndemic. In view of the overlapping epidemiology of these diseases, it is important to understand the dynamics of the immune response to TB in the context of co-infection. We investigated the cytokine response to purified protein derivative (PPD) in peripheral blood mononuclear cells from TB patients co-infected with HIV or malaria and compared it to that of malaria- and HIV-free TB patients. A total of 231 subjects were recruited for this study and classified into six groups; untreated TB-positive, TB positive subjects on TB drugs, TB- and HIV-positive, TB- and malaria-positive, latent TB and apparently healthy control subjects. Our results demonstrate maintenance of interferon (IFN)-γ production in HIV and malaria co-infected TB patients in spite of lower CD4 counts in the HIV-infected cohort. Malaria co-infection caused an increase in the production of the T helper type 2 (Th2)-associated cytokine interleukin (IL)-4 and the anti-inflammatory cytokine IL-10 in PPD-stimulated cultures. These results suggest that malaria co-infection diverts immune response against M. tuberculosis towards a Th-2/anti-inflammatory response which might have important consequences for disease progression., (© 2016 British Society for Immunology.)

Background: Cytokines play an important role in cell-mediated immune responses against Mycobacterium tuberculosis (Mtb) infection. Cytokine profile specifically associated with active tuberculosis (ATB) patients, subjects with latent tuberculosis infection (LTBI) and non-infected individuals remains to be determined., Methods: We enrolled a total of 92 subjects including patients with ATB (n = 25), LTBI (n = 36) and healthy controls (HC, n = 31) to investigate the cytokine production by peripheral blood mononuclear cells after Mtb purified protein derivative (PPD) stimulation which was evaluated by a beads-based multiplex assay system., Results: The production of IL-1β, IL-2, IL-6, IL-10, IL-17, G-CSF, IFN-γ, IP-10, MIP-1α and TNF-α was abundantly induced by PPD in all three groups. The levels of IL-2, IL-10, IFN-γ, IP-10 and TNF-α were significantly higher in LTBI group than in ATB group. The combination of PPD-stimulated IL-2 and IL-10 accurately identified 84.0% of ATB and 88.9% of LTBI. We validated the use of PPD-stimulated IL-2 and IL-10 test combined with T-SPOT.TB test in a cohort of 44 subjects with TB suspicion. The sensitivity and specificity of the combined test were 83.3% and 92.3%, respectively. The PPD-stimulated IL-2/IFN-γ ratio (p < 0.001) in LTBI subjects was significantly higher than in active TB patients., Conclusion: Our study identified cytokine patterns characteristic of ATB and LTBI. Cytokines such as IL-2 and IL-10 may serve as biomarkers for distinguishing ATB from LTBI and healthy control and may contribute to intervention and improvement in TB diagnosis., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Sporadic cases of bovine tuberculosis (bTB) have been reported in warthogs in Southern Africa and confirmed through mycobacterial culture. However, there are no validated ante-mortem tests currently available for bTB in warthogs. In this study, we evaluated the use of three serological assays for the detection of Mycobacterium bovis infection in warthogs; an indirect enzyme-linked immunosorbent assay (ELISA) using bovine purified protein derivative (PPD b ) as a capture antigen (indirect PPD ELISA), as well as two commercial assays, the TB ELISA-VK ® and DPP ® VetTB Assay. Test performance of these assays was compared using sera from 35 warthogs of known Mycobacterium bovis infection status. All three assays were able to distinguish M. bovis-infected from uninfected individuals with high sensitivity (Se) and specificity (Sp) (indirect PPD ELISA Se: 88%, Sp: 89%; TB ELISA-VK ® 88%, 79%; DPP ® VetTB Assay 75%, 89%, respectively). The assays performed very similarly and the ELISA assays showed the greatest agreement (κ=0.89). These results indicate that M. bovis-infected warthogs develop measurable pathogen-specific humoral responses which can be used to distinguish them from uninfected animals. Therefore, serological assays have value as ante-mortem bTB diagnostic tests in warthogs., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Although eosinophils are inflammatory cells, there is increasing attention on their immunomodulatory roles. For example, murine eosinophils can present antigen to CD4 + T helper (Th) cells, but it remains unclear whether human eosinophils also have this ability. This study determined whether human eosinophils present a range of antigens, including allergens, to activate Th cells, and characterized their expression of MHC class II and co-stimulatory molecules required for effective presentation. Human peripheral blood eosinophils purified from non-allergic donors were pulsed with the antigens house dust mite extract (HDM), Timothy Grass extract (TG) or Mycobacterium tuberculosis purified protein derivative (PPD), before co-culture with autologous CD4 + Th cells. Proliferative and cytokine responses were measured, with eosinophil expression of HLA-DR/DP/DQ and the co-stimulatory molecules CD40, CD80 and CD86 determined by flow cytometry. Eosinophils pulsed with HDM, TG or PPD drove Th cell proliferation, with the response strength dependent on antigen concentration. The cytokine responses varied with donor and antigen, and were not biased towards any particular Th subset, often including combinations of pro- and anti-inflammatory cytokines. Eosinophils up-regulated surface expression of HLA-DR/DP/DQ, CD80, CD86 and CD40 in culture, increases that were sustained over 5 days when incubated with antigens, including HDM, or the major allergens it contains, Der p I or Der p II. Human eosinophils can, therefore, act as effective antigen-presenting cells to stimulate varied Th cell responses against a panel of antigens including HDM, TG or PPD, an ability that may help to determine the development of allergic disease., (© 2016 John Wiley & Sons Ltd.)

Background: Vaccination with Bacillus Calmette Guerin (BCG) protects infants against childhood tuberculosis however the immune mechanisms involved are not well understood. Further elucidation of the infant immune response to BCG will aid with the identification of immune correlates of protection against tuberculosis and with the design of new improved vaccines. The purpose of this study was to investigate BCG-induced CD4+ T-cell responses in blood samples from infants for cytokine secretion profiles thought to be important for protection against tuberculosis and compare these to PBMC-mediated in vitro mycobacterial growth inhibition., Methods: Blood from BCG-vaccinated or unvaccinated infants was stimulated overnight with Mycobacterium tuberculosis (M. tb) purified protein derivative (PPD) or controls and intracellular cytokine staining and flow cytometry used to measure CD4+T-cell responses. PBMC cryopreserved at the time of sample collection were thawed and incubated with live BCG for four days following which inhibition of BCG growth was determined., Results: PPD-specific IFNγ+TNFα+IL-2+CD4+T-cells represented the dominant T-cell response at 4monthsand1yearafter infant BCG. These responses were undetectable in age-matched unvaccinated infants. IL-17+CD4+T-cells were significantly more frequent in vaccinated infants at 4monthsbut not at 1-year post-BCG. PBMC-mediated inhibition of mycobacterial growth was significantly enhanced at 4monthspost-BCG as compared to unvaccinated controls. In an analysis of all samples with both datasets available, mycobacterial growth inhibition correlated significantly with the frequency of polyfunctional (IFNγ+TNFα+IL-2+) CD4+T-cells., Conclusions: These data suggest that BCG vaccination of infants induces specific polyfunctional T-helper-1 and T-helper-17 responses and the ability, in the PBMC compartment, to inhibit the growth of mycobacteria in vitro. We also demonstrate that polyfunctional T-helper-1 cells may play a role in growth inhibition as evidenced by a significant correlation between the two., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Following exposure to TB, contacts are screened to target preventive treatment at those at high risk of developing TB. The UK has recently revised its recommendations for screening and now advises a 5 mm tuberculin skin test (TST) cut-off irrespective of age or BCG status. We sought to evaluate the impact of BCG on TST responses in UK children exposed to TB and the performance of different TST cut-offs to predict interferon γ release assay (IGRA) positivity., Methods: Children <15 years old were recruited from 11 sites in the UK between January 2011 and December 2014 if exposed in their home to a source case with sputum smear or culture positive TB. Demographic details were collected and TST and IGRA undertaken. The impact of BCG vaccination on TST positivity was evaluated in IGRA-negative children, as was the performance of different TST cut-offs to predict IGRA positivity., Results: Of 422 children recruited (median age 69 months; IQR: 32-113 months), 300 (71%) had been vaccinated with BCG. BCG vaccination affected the TST response in IGRA-negative children less than 5 years old but not in older children. A 5 mm TST cut-off demonstrated good sensitivity and specificity in BCG-unvaccinated children, and an excellent negative predictive value but was associated with low specificity (62.7%; 95% CI 56.1% to 69.0%) in BCG-vaccinated children. For BCG-vaccinated children, a 10 mm cut-off provided a high negative predictive value (97.7%; 95% CI 94.2% to 99.4%) with the positive predictive value increasing with increasing age of the child., Discussion: BCG vaccination had little impact on TST size in children over 5 years of age. The revised TST cut-off recommended in the recent revision to the UK TB guidelines demonstrates good sensitivity but is associated with impaired specificity in BCG-vaccinated children., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

The Toll-like and IL-1 family receptors play critical roles in innate and adaptive immunity against intracellular pathogens. Although previous data demonstrated the importance of TLRs and IL-1R signaling events for the establishment of an effective immune response to mycobacteria, the possible function of the adaptor molecule IL-1R-associated kinase (IRAK)-4 against this pathogen has not been addressed. In this study, we determined the role of IRAK-4 in signaling pathways responsible for controlling mycobacterial infections. This kinase is important for the production of IL-12 and TNF-α by macrophages and dendritic cells exposed to mycobacteria. Moreover, Mycobacterium bovis-infected IRAK-4-knockout macrophages displayed impaired MAPK and NF-κB activation. IL-1β secretion and caspase-1 activation were also dependent on IRAK-4 signaling. Mice lacking IRAK-4 showed increased M. bovis burden in spleen, liver, and lungs and smaller liver granulomas during 60 d of infection compared with wild-type mice. Furthermore, 80% of IRAK-4(-/-) mice succumbed to virulent M. tuberculosis within 100 d following low-dose infection. This increased susceptibility to mycobacteria correlated with reduced IFN-γ/TNF-α recall responses by splenocytes, as well as fewer IL-12p70-producing APCs. Additionally, we observed that IRAK-4 is also important for the production of IFN-γ by CD4(+) T cells from infected mice. Finally, THP-1 cells treated with an IRAK-4 inhibitor and exposed to M. bovis showed reduced TNF-α and IL-12, suggesting that the results found in mice can be extended to humans. In summary, these data demonstrate that IRAK-4 is essential for innate and adaptive immunity and necessary for efficient control of mycobacterial infections., (Copyright © 2016 by The American Association of Immunologists, Inc.)

The most widely used ante-mortem diagnostic tests for tuberculosis in cattle are the tuberculin skin test and the interferon-gamma (IFN-γ) release assay, both of which measure cell-mediated immune responses to Mycobacterium bovis infection. However, limitations in the performance of these tests results in a failure to identify all infected animals. In attempting to increase the range of diagnostic tests for tuberculosis, measurement of the cytokine IP-10 in antigen-stimulated blood has previously been shown to improve the detection of M. tuberculosis and M. bovis infection, in humans and African buffaloes (Syncerus caffer), respectively. In the present study, 60 cattle were identified by the single intradermal comparative tuberculin test as tuberculosis reactors (n = 24) or non-reactors (n = 36) and the release of IFN-γ and IP-10 in antigen-stimulated whole blood from these animals was measured using bovine specific ELISAs. There was a strong correlation between IP-10 and IFN-γ production in these samples. Moreover, measurement of the differential release of IP-10 in response to stimulation with M. bovis purified protein derivative (PPD) and M. avium PPD distinguished between reactor and non-reactor cattle with a sensitivity of 100% (95% CI, 86%-100%) and a specificity of 97% (95% CI, 85%-100%). These results suggest that IP-10 might prove valuable as a diagnostic biomarker of M. bovis infection in cattle.

Objective: To characterise the delayed-type hypersensitivity (DTH) skin reaction to repeated challenges of keyhole limpet hemocyanin (KLH) and tuberculin purified protein derivative (PPD) in healthy volunteers, as a potential model to test T cell-targeted investigational agents., Subjects, Treatment and Methods: Forty-nine subjects received either KLH, PPD, or PBS repeat skin challenges, and clinical assessments including induration, erythema and Laser Doppler Imaging. Skin biopsies or suction blisters were taken after challenge to investigate the cellular infiltrate of the challenge site, the T cell activation status, as determined by LAG-3 expression, and, specifically for the blister, the concentrations of inflammatory cytokines. Point estimates, estimates of variation and corresponding 95% confidence intervals were constructed for each type of challenge and timepoint., Results: The DTH response could be measured at 48 and 120 h post-KLH and PPD challenge with induration, erythema and Laser Doppler Imaging, with 48 h post-challenge demonstrating the peak of the response. PPD was well tolerated in subjects after multiple challenges, however, a significant number of KLH-treated subjects demonstrated an injection site reaction 6-7 days following the SC injection. PPD demonstrated a boost effect on the second challenge as measured by increased induration, where as this was not noted consistently for KLH. Compared to unchallenged and PBS control-injected skin, increased T cell numbers were detected in the challenge site by both the skin suction blister and biopsy technique, at either time point following KLH or PPD challenge. Use of the T cell activation marker LAG-3 demonstrated the activated phenotype of these cells. In skin blisters, higher numbers of LAG-3+ T cells were detected at 48 h post-challenge, whereas in the biopsies, similar numbers of LAG-3+ cells were observed at both 48 and 120 h. Analysis of blister T cell subpopulations revealed some differences in phenotypes between the time points and between the CD4 and CD8 T cells. Blister cytokine analysis revealed a pro-inflammatory dominated signature in PPD-challenged skin., Conclusions: In summary, our data support the use of a repeat KLH and PPD DTH challenge in clinical trials and that the clinical measures of induration and to a lesser extent erythema are appropriate to monitor the clinical DTH response. Both the blister and biopsy can be utilised to assess and quantify activated T cells and at the dose used, PPD was better tolerated than KLH and hence may be optimal for future studies.

Orally delivered mycobacterial antigens may not sensitize the immunized animals causing a positive tuberculin skin test response. As the first step to address this critical issue, we characterized the response of farmed red deer (Cervus elaphus) to orally delivered heat-inactivated Mycobacterium bovis. Thirty-two adult red deer hinds from a farm known to be free of tuberculosis (TB) were randomly assigned to two different treatment groups, immunized (n=24) and control (n=8). Immunized hinds were dosed orally with 2 ml of PBS containing 6 × 10(6) heat-inactivated M. bovis. The mean skin test response of immunized deer to both avian purified protein derivative (aPPD) and bovine PPD (bPPD) was consistently lower in immunized than in control hinds. One year after immunization, immunized hinds had a significant reduction in the skin test response to aPPD and in the ELISA antibody levels against both aPPD and bPPD (24-36% reduction; P<0.05). By contrast, no significant change was observed in the skin test response to phytohaemagglutinin, or in the ELISA antibody levels against the M. bovis specific antigen MPB70. The mRNA levels for C3, IFN-γ and IL-1β and serum protein levels for IFN-γ and IL-1β did not vary between immunized and control deer. However, serum C3 protein levels were significantly higher (P=0.001) in immunized than in control deer six months after immunization. These results confirm that oral heat-inactivated M. bovis does not sensitize farmed red deer and therefore does not cause false-positive responses in the tuberculin skin test. The absence of sensitization in orally immunized deer opens the possibility of testing the vaccine in deer and possibly other ruminants without the risk of causing false-positive reactions in TB-tests. This study also provided the first evidence that orally-delivered inactivated mycobacterial antigens elicit some kind of immune response in a ruminant., (Copyright © 2016 Elsevier B.V. All rights reserved.)

HIV-infected persons are at greater risk of developing tuberculosis (TB) even before profound CD4 loss occurs, suggesting that HIV alters CD4(+) T cell functions capable of containing bacterial replication. An effective immune response to Mycobacterium tuberculosis most likely relies on the development of a balanced CD4 response, in which distinct CD4(+) Th subsets act in synergy to control the infection. To define the diversity of M. tuberculosis-specific CD4(+) Th subsets and determine whether HIV infection impacts such responses, the expression of lineage-defining transcription factors T-bet, Gata3, RORγt, and Foxp3 was measured in M. tuberculosis-specific CD4(+) T cells in HIV-uninfected (n = 20) and HIV-infected individuals (n = 20) with latent TB infection. Our results show that, upon 5-d restimulation in vitro, M. tuberculosis-specific CD4(+) T cells from healthy individuals have the ability to exhibit a broad spectrum of Th subsets, defined by specific patterns of transcription factor coexpression. These transcription factor profiles were skewed in HIV-infected individuals where the proportion of T-bet(high)Foxp3(+) M. tuberculosis-specific CD4(+) T cells was significantly decreased (p = 0.002) compared with HIV-uninfected individuals, a change that correlated inversely with HIV viral load (p = 0.0007) and plasma TNF-α (p = 0.027). Our data demonstrate an important balance in Th subset diversity defined by lineage-defining transcription factor coexpression profiles that is disrupted by HIV infection and suggest a role for HIV in impairing TB immunity by altering the equilibrium of M. tuberculosis-specific CD4(+) Th subsets., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Setting: Intradermal injection using a syringe and needle is generally accepted as the most accurate method for the tuberculin skin test (TST). However, the Mantoux technique using a conventional needle is often difficult to perform reliably, affecting testing results and safety., Objective: We evaluated the efficacy and safety of a novel intradermal injection device, the MicronJet600(TM) microneedle, compared with conventional injection in terms of skin reactivity to the TST., Design: A prospective, open-label clinical study was conducted. The TST was administered by both methods in the same subject. For pain assessment, participants filled in a visual analogue scale (VAS) after each TST. Any side effects due to TST or injections were observed., Results: TST reaction rates (cut-off ⩾5 mm) from microneedles and needles were respectively 44.0% and 47.2%, with no significant difference between the two. Furthermore, agreement of positivity between the two methods was excellent with both 5 mm and 10 mm cut-off values. However, the level of pain experienced when microneedles were used for TST was significantly lower than with conventional needles. No adverse effects were attributed to the MicronJet device., Conclusion: The novel microneedle device used for TST in this study was effective, safe and less painful in healthy adult volunteers.

Several lines of evidence have recently suggested that natural killer (NK) cells develop immunological memory against viral infections. However, there is no apparent evidence that NK cells acquire specific memory against Mycobacterium bovis bacillus Calmette-Guérin (BCG), the only currently licensed vaccine for preventing tuberculosis. In the present study, we investigated whether murine splenic NK cells can be activated by BCG in a dendritic cell (DC)-independent or -dependent manner, and furthermore examined whether these NK cells acquire specific memory following BCG vaccination. NK cells isolated from spleens of BCG-immunized mice produced interferon (IFN)γ through direct BCG stimulation in the absence of antigen-presenting cells; however, NK cells from control animals similarly directly responded to BCG, and the response level was not statistically significant between the immunized and the naïve NK cells. When purified NK cells that had been exposed to BCG were cocultured with RAW murine macrophages infected with BCG, the antibacterial activity of the macrophages was strongly enhanced; however, its level was similar to that by naïve NK cells, which had not been exposed to BCG. When splenocytes harvested from BCG-immunized mice were stimulated with purified protein derivative (PPD) derived from Mycobacterium tuberculosis, a specific IFNγ response was clearly observed, mainly attributed to NK cells and memory CD4+ T cells. To investigate whether these NK cells as well as the T cells are activated by cell-cell interaction with DCs presenting mycobacterial antigens, NK cells isolated from BCG-immunized mice were cocultured with splenocytes harvested from naïve mice in the presence of PPD stimulation. However, no IFNγ response was found in the NK cells. These results suggest that murine splenic NK cells do not develop BCG-specific immunological memory in either a DC-independent or -dependent manner.

A new class of highly antigenic, MHC-II-restricted mycobacterial lipopeptides that are recognized by CD4-positive T lymphocytes of Mycobacterium tuberculosis-infected humans has recently been described. To investigate the relevance of this novel class of mycobacterial Ags in the context of experimental bacille Calmette-Guérin (BCG) vaccination, Ag-specific T cell responses to mycobacterial lipid and lipopeptide-enriched Ag preparations were analyzed in immunized guinea pigs. Lipid and lipopeptide preparations as well as complex Ag mixtures, such as tuberculin, mycobacterial lysates, and culture supernatants, all induced a similar level of T cell proliferation. The hypothesis that lipopeptide-specific T cells dominate the early BCG-induced T cell response was corroborated in restimulation assays by the observation that Ag-expanded T cells specifically responded to the lipopeptide preparation. A comparative analysis of the responses to Ag preparations from different mycobacterial species revealed that the antigenic lipopeptides are specific for strains of the M. tuberculosis complex. Their intriguing conservation in pathogenic tuberculous bacteria and the fact that these highly immunogenic Ags seem to be actively released during in vitro culture and intracellular infection prompt the urgent question about their role in the fine-tuned interplay between the pathogen and its mammalian host, in particular with regard to BCG vaccination strategies., (Copyright © 2016 by The American Association of Immunologists, Inc.)

In many settings, adults with active or latent tuberculosis will also be coinfected with helminths. Our study aimed to investigate how anthelmintic treatment modulates antimycobacterial immunity, in a setting where helminth reinfection should not occur. We investigated the potential impact of helminth infection on immune responses to Mycobacterium tuberculosis (Mtb) in patients with latent Mtb infection with or without helminth infection (Strongyloides or Schistosoma), and tested T-cell responses before and after anthelmintic treatment. The study was performed in migrants resident in the United Kingdom, where reexposure and reinfection following anthelmintic treatment would not occur. The frequency of CD4(+) IFN-γ(+) T cells was measured following stimulation with Mtb Purified Protein Derivative or ESAT-6/CFP-10 antigen, and concentrations of IFN-γ in culture supernatants measured by ELISA and multiplex bead array. Helminth infection was associated with a lower frequency of CD4(+) IFN-γ(+) T cells, which increased following treatment. Patients with helminth infection showed a significant increase in CD4(+) FoxP3(+) T cells (Treg) compared to those without helminth infection. There was a decrease in the frequency of Treg cells, and an associated increase in CD4(+) IFN-γ(+) T cells after the anthelmintic treatment. Here, we show a potential role of Treg cells in reducing the frequency and function of antimycobacterial CD4(+) IFN-γ(+) T cells, and that these effects are reversed after anthelmintic treatment., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objectives: To determine the effect of anti-tuberculin antibodies in the T-cell proliferation in response to tuberculin and Candida antigens in individuals with different levels of tuberculosis (TB) risk., Methods: Sixteen high-risk TB individuals, 30 with an intermediate TB risk (group A), and 45 with a low TB risk (group B), as well as 49 control individuals, were studied. Tuberculin skin test (TST) results were analyzed and serum levels of antibodies (IgG and IgM) against purified protein derivative (PPD) were measured by ELISA. Tuberculin and Candida antigens were used to stimulate T-cell proliferation in the presence of human AB serum or autologous serum., Results: High levels of anti-tuberculin IgG antibodies were found to be significantly associated with the blocking of T-cell proliferation responses in cultures stimulated with tuberculin but not with Candida antigens in the presence of autologous serum. This phenomenon was particularly frequent in high-risk individuals with high levels of anti-tuberculin IgG antibodies in the autologous serum when compared to the other risk groups, which exhibited lower levels of anti-tuberculin antibodies., Conclusions: Although cellular immunity plays a central role in the protection against TB, humoral immunity is critical in the control of Mycobacterium tuberculosis infection in high-risk individuals with latent TB infection., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Recent evidence indicates that the prevalence of diseases caused by nontuberculous mycobacteria (NTM) has been increasing in both human and animals. In this study, antibody profiles of NTM in rhesus monkeys (Macaca mulatta) were determined and compared with those of monkeys infected with Mycobacterium tuberculosis complex (MTBC). Antibodies against 10 M. tuberculosis proteins, purified protein derivative (PPD), and mammalian old tuberculin (MOT) were detected in 14 monkeys naturally infected with NTM by indirect ELISA. Sera from 10 monkeys infected with MTBC and 10 healthy monkeys were set as controls. All antigens showed high serological reactivities to MTBC infections and low reactivities in healthy monkeys. NTM infections showed strong antibody responses to MOT and PPD; moderate antibody responses to 16kDa, U1, MPT64L, 14kDa, and TB16.3; and low antibody responses to 38kDa, Ag85b, CFP10, ESAT-6, and CFP10-ESAT-6. According to the criteria of MTBC, only CFP10, ESAT-6, and CFP10-ESAT-6 showed negative antibody responses in all NTM infections. Taken together, these results suggest that positive results of a PPD/MOT-based ELISA in combination with results of antibodies to M. tuberculosis-specific antigens, such as CFP10 and ESAT-6, could discriminate NTM and MTBC infections. Two positive results indicate an MTBC infection, and a negative result for an M. tuberculosis-specific antigen may preliminarily predict an NTM infection.

In this study, we used ELISPOT to quantify frequencies of bronchoalveolar lavage (BAL) and peripheral blood T cells capable of producing IFNγ in response to PPD, antigen 85B, and Mtb-specific antigens CFP-10 and ESAT-6 in individuals with latent tuberculosis infection (LTBI) and Mtb-naïve controls. Compared to peripheral blood, BAL cells of LTBI subjects displayed significant enrichment for T cells responding to PPD, antigen 85B, and CFP-10, but not to ESAT-6. Baseline BAL cells of LTBI subjects displayed significant production of Mig (CXCL9) in response to PPD, antigen 85B, and CFP-10 as well. These findings suggest that enrichment for Mtb-specific T cells within BAL is not unique to active pulmonary tuberculosis and may, to the contrary, contribute to protection from re-infection in Mtb immune individuals., (Published by Elsevier Ltd.)

Objective: To test the hypothesis that having a scar and a positive tuberculin skin test (TST) response after vaccination with Bacille Calmette-Guérin (BCG) is associated with reduced infant mortality., Methods: We studied cohorts of 2709 normal-birthweight (NBW) and 1102 low-birthweight (LBW) infants in Guinea-Bissau. Children were enrolled in randomised trials between year 2002 and 2008 and received BCG vaccination at birth. BCG scars and TST responses were assessed at 2 and 6 months of age. The infants were followed for mortality to 12 months of age, and survival was analysed using Cox regression., Results: At age 2 months, 88% of NBW children and 91% of LBW children had a BCG scar, and 36% and 17% had a TST response, respectively. The LBW infants had nearly twofold higher mortality (4.5%) than the NBW infants (2.8%) between 2 and 12 months of age. In the LBW cohort, the adjusted mortality rate ratio (MRR) comparing children with a BCG scar with those without was 0.42 (95% CI = 0.19; 0.93). There was a similar tendency for TST positivity: MRR = 0.47 (95% CI = 0.14; 1.54). For LBW children who had both a positive TST reaction and a scar, the MRR was 0.22 (95% CI = 0.05; 0.87). For NBW children, a scar and a positive TST were associated with 20% reductions in mortality, which did not reach statistical significance., Conclusion: We confirmed previous observations that having a scar and a TST response after BCG vaccination is associated with lower mortality risk. The possibility of revaccinating scar-negative children should be considered., (© 2015 John Wiley & Sons Ltd.)

Bovine tuberculosis (BTB) represents not only a significant economic concern, but also an important public health problem. Currently, interferon-gamma (IFN-γ) release assays (IGRAs) are widely used as an adjunct to the tuberculin test (TST) for the diagnosis of BTB. A great number of international studies have demonstrated that the sensitivity of the IFN-γ assay, which uses purified protein derivatives (PPDs) as diagnostic reagents, is superior to that of the TST. However, there are concerns about its specificity, largely because of the cross reactivity of common antigens shared by pathogenic and non-pathogenic mycobacterial species. The use of pathogen-specific antigens theoretically offers the most effective way to improve the specificity of IGRAs. In this study, we evaluated the potential utility of 13 purified recombinant putative antigens, which are highly specific to the Mycobacterium tuberculosis complex, as diagnostic reagents in IGRAs. A CFP-10-ESAT-6 fusion protein (abbreviated CE) displayed the greatest potential, whereas four region of difference 2 (RD2) antigens, especially Rv1985c were identified as potential candidate antigens, and can be included in an IGRA cocktail, together with CE as stimulators in the IFN-γ release assay for the diagnosis of BTB., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Setting: Public human immunodeficiency virus (HIV) clinic and tuberculosis (TB) clinics in Kampala, Uganda., Objective: To examine TB-specific CD4 T-cell single and polyfunctional cytokine correlates of clinical diagnostic tests for latent tuberculous infection (LTBI) in HIV-1-infected subjects., Design: Thirty antiretroviral therapy-naïve HIV-1-infected adults without active TB disease underwent clinical tuberculin skin test (TST), interferon-gamma release assay (IGRA), and in vitro flow cytometry analysis on cells stimulated with purified protein derivative (PPD) and TB antigens early secreted antigenic target 6 + culture filtrate protein 10 (EC) for frequencies of interleukin (IL) 2, IL-17, interferon-gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) expressing cells., Results: PPD-specific CD4 T-cell expression of TNF-α and IFN-γ was higher in the TST-positive than in the TST-negative group. EC-specific CD4 T-cell expression of TNF-α and IL-2 was higher in the TST+ group than in the TST- group. Expression of both PPD and EC-specific expression of IL-2, IFN-γ and TNF-α were greater in IGRA-positive than in IGRA-negative subjects. The TST+ group exhibited greater polyfunctionality than the TST- group. All cytokine combinations that contained TNF-α correlated strongly with TST size., Conclusion: While IL-2, IFN-γ and TNF-α correlate with clinical tests of LTBI, TNF-α is the dominant cytokine correlating with both TST size and magnitude of IGRA response.