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ABSTRACT Cervical tuberculous lymphadenitis (CTL), the most frequent extrapulmonary form of tuberculosis, is a serious health problem in Tunisia. CTL diagnosis is challenging mainly due to the paucibacillary nature of the disease and the potential misdiagnosis as cervical non-tuberculous lymphadenitis (CNTL). Here, we evaluated the performance of heparin-binding hemagglutinin (HBHA) interferon-gamma (IFN-γ) release assay (IGRA) for the diagnosis of CTL. In addition, we evaluated granzyme B, granulysin, and perforin release assays as CTL biomarkers and assessed their potential contribution to improve HBHA-IGRA performance. Peripheral blood mononuclear cells from CTL-suspected patients were stimulated with HBHA, early secreted antigenic target 6 (ESAT-6), or purified protein derivative (PPD) for 24 h in the presence of IL-7. Cytotoxic mediators and IFN-γ release were assessed by enzyme-linked immunosorbent assay. Receiver operating characteristic curves were used to evaluate the capacity of HBHA, ESAT-6, and PPD to discriminate between CTL (n = 27) and CNTL (n = 21). After applying bivariate and multivariate analyses, IFN-γ responses to HBHA appeared to offer the best distinction between CTL and CNTL, with an area under the curve of 0.9947, associated with 95.24% and 100% sensitivity and specificity, respectively. A principal component analysis showed clear clustering of the CTL versus the CNTL groups. This clustering was mainly attributed to HBHA-induced IFN-γ, PPD-induced granzyme B, and PPD-induced IFN-γ. These results thus suggest that the HBHA-IGRA provides high diagnostic accuracy for CTL versus CNTL, with high sensitivity and specificity. Combining HBHA-induced IFN-γ and PPD-induced granzyme B improves the accuracy to identify CTL. IMPORTANCE Cervical tuberculous lymphadenitis (CTL), the most frequent extrapulmonary form of tuberculosis, is currently a major health problem in Tunisia and in several regions around the world. CTL diagnosis is challenging mainly due to the paucibacillary nature of the disease and the potential misdiagnosis as cervical non-tuberculous lymphadenitis. This study demonstrates the added value of the heparin-binding hemagglutinin-interferon-gamma release assay as an immunoassay in the context of CTL.

IntroductionThe characterization of B. pertussis (Bp) antigen-specific CD4+ T cell cytokine responses should be included in the evaluation of immunogenicity of pertussis vaccines but is often hindered by the lack of standardized robust assays.MethodsTo overcome this limitation, we developed a two-step assay comprising a short-term stimulation of fresh whole blood with Bp antigens and cryopreservation of the stimulated cells, followed later on by batch-wise intracellular cytokine analysis by flow cytometry. Blood samples collected from recently acellular (aP) vaccine boosted subjects with a whole-cell- or aP-primed background was incubated for 24 hrs with Pertussis toxin, Filamentous hemagglutinin or a Bp lysate (400µl per stimulation). Antigen-specific IFN-γ-, IL-4/IL-5/IL-13-, IL-17A/IL-17F- and/or IL-22-producing CD4+ T cells were quantified by flow cytometry to reveal Th1, Th2, and Th17-type responses, respectively. The frequencies of IFN-γ-producing CD8+ T cells were also analyzed.ResultsWe demonstrate high reproducibility of the Bp-specific whole blood intracellular staining assay. The results obtained after cryopreservation of the stimulated and fixed cells were very well correlated to those obtained without cryopreservation, an approach used in our previously published assay. Optimization resulted in high sensitivity thanks to very low non-specific backgrounds, with reliable detection of Bp antigen-specific Th1, Th2 and Th17-type CD4+ T cells, in the lowest range frequency of 0.01-0.03%. Bp antigen-specific IFN-γ+ CD8+ T lymphocytes were also detected. This test is easy to perform, analyse and interpret with the establishment of strict criteria defining Bp antigen responses.DiscussionThus, this assay appears as a promising test for evaluation of Bp antigen-specific CD4+ T cells induced by current and next generation pertussis vaccines.

BackgroundChemerin is an extracellular protein with chemotactic activities and its expression is increased in various diseases such as metabolic syndrome and inflammatory conditions. Its role in lung pathology has not yet been extensively studied but both known pro- and anti-inflammatory properties have been observed. The aim of our study was to evaluate the involvement of the chemerin/ChemR23 system in the physiopathology of COVID-19 with a particular focus on its prognostic value.MethodsBlood samples from confirmed COVID-19 patients were collected at day 1, 5 and 14 from admission to Erasme Hospital (Brussels – Belgium). Chemerin concentrations and inflammatory biomarkers were analyzed in the plasma. Blood cells subtypes and their expression of ChemR23 were determined by flow cytometry. The expression of chemerin and ChemR23 was evaluated on lung tissue from autopsied COVID-19 patients by immunohistochemistry (IHC).Results21 healthy controls (HC) and 88 COVID-19 patients, including 40 in intensive care unit (ICU) were included. Plasma chemerin concentration were significantly higher in ICU patients than in HC at all time-points analyzed (p

Background and Objectives: The incidence of tuberculosis lymphadenopathy (TBLA) is increasing, and diagnostic procedures lack sensitivity and are often highly invasive. TBLA may be asymptomatic, and differential diagnosis with other adenopathies (ADPs) is difficult. We evaluated a blood-cell interferon-γ release assay (IGRA) with two different stage-specific mycobacterial antigens for the differential diagnosis of ADP suspected of mycobacterial origin. Methods: Twenty-one patients were included and divided into three groups: (1) cervical/axillar ADP (n = 8), (2) mediastinal ADP (n = 10), and (3) disseminated ADP (n = 3). The mycobacterial antigens used for the IGRA were the heparin-binding haemagglutinin (HBHA) and the early-secreted antigenic target-6 (ESAT-6), a latency-associated antigen and a bacterial replication-related antigen, respectively. Diagnosis of TBLA based on microbiological results and/or response to anti-TB treatment was obtained for 15 patients. Results: An IGRA profile highly suggestive of active TB (higher IFN-γ response to ESAT-6 compared to HBHA) was found for 3/6 TBLA patients from group 1, and for all the TBLA patients from groups 2 and 3, whereas this profile was not noticed in patients with a final alternative diagnosis. Conclusion: These results highlight the potential value of this combined HBHA/ESAT-6 IGRA as a triage test for the differential diagnosis of ADP.

Active tuberculosis (aTB) remains a major killer from infectious disease, partially due to delayed diagnosis and hence treatment. Classical microbiological methods are slow and lack sensitivity, molecular techniques are costly and often unavailable. Moreover, available immuno-diagnostic tests lack sensitivity and do not differentiate between aTB and latent TB infection (LTBI). Here, we evaluated the performance of the combined measurement of different chemokines/cytokines induced by two different stage-specific mycobacterial antigens, Early-secreted-antigenic target-6 (ESAT-6) and Heparin-binding-haemagglutinin (HBHA), after a short in vitro incubation of either peripheral blood mononuclear cells (PBMC) or whole blood (WB). Blood samples were collected from a training cohort comprising 22 aTB patients, 22 LTBI subjects and 17 non-infected controls. The concentrations of 13 cytokines were measured in the supernatants. Random forest analysis identified the best markers to differentiate M. tuberculosis-infected from non-infected subjects, and the most appropriate markers to differentiate aTB from LTBI. Logistic regression defined predictive abilities of selected combinations of cytokines, first on the training and then on a validation cohort (17 aTB, 27 LTBI, 25 controls). Combining HBHA- and ESAT-6-induced IFN-γ concentrations produced by PBMC was optimal to differentiate infected from non-infected individuals in the training cohort (100% correct classification), but 2/16 (13%) patients with aTB were misclassified in the validation cohort. ESAT-6-induced-IP-10 combined with HBHA-induced-IFN-γ concentrations was selected to differentiate aTB from LTBI, and correctly classified 82%/77% of infected subjects as aTB or LTBI in the training/validation cohorts, respectively. Results obtained on WB also selected ESAT-6- and HBHA-induced IFN-γ concentrations to provided discrimination between infected and non-infected subjects (89%/90% correct classification in the training/validation cohorts). Further identification of aTB patients among infected subjects was best achieved by combining ESAT-6-induced IP-10 with HBHA-induced IL-2 and GM-CSF. Among infected subjects, 90%/93% of the aTB patients were correctly identified in the training/validation cohorts. We therefore propose a two steps strategy performed on 1 mL WB for a rapid identification of patients with aTB. After elimination of most non-infected subjects by combining ESAT-6 and HBHA-induced IFN-γ, the combination of IP-10, IL-2 and GM-CSF released by either ESAT-6 or HBHA correctly identifies most patients with aTB.

The new pandemic virus SARS-CoV-2 is characterized by uncontrolled hyper-inflammation in severe cases. As the IL-22/IL-22R1 axis was reported to be involved in inflammation during viral infections, we characterized the expression of IL-22 receptor1, IL-22 and IL-22 binding protein in COVID-19 patients. Blood samples were collected from 19 non-severe and 14 severe patients on the day they presented (D0), at D14, and six months later, and from 6 non-infected controls. The IL-22R1 expression was characterized by flow cytometry. Results were related to HLA-DR expression of myeloid cells, to plasma concentrations of different cytokines and chemokines and NK cells and T lymphocytes functions characterized by their IFN-γ, IL-22, IL-17A, granzyme B and perforin content. The numbers of IL-22R1+ classical, intermediate, and non-classical monocytes and the proportions of IL-22R1+ plasmacytoid DC (pDC), myeloid DC1 and DC2 (mDC1, mDC2) were higher in patients than controls at D0. The proportions of IL-22R1+ classical and intermediate monocytes, and pDC and mDC2 remained high for six months. High proportions of IL-22R1+ non-classical monocytes and mDC2 displayed HLA-DRhigh expression and were thus activated. Multivariate analysis for all IL-22R1+ myeloid cells discriminated the severity of the disease (AUC=0.9023). However, correlation analysis between IL-22R1+ cell subsets and plasma chemokine concentrations suggested pro-inflammatory effects of some subsets and protective effects of others. The numbers of IL-22R1+ classical monocytes and pDC were positively correlated with pro-inflammatory chemokines MCP-1 and IP-10 in severe infections, whereas IL-22R1+ intermediate monocytes were negatively correlated with IL-6, IFN-α and CRP in non-severe infections. Moreover, in the absence of in vitro stimulation, NK and CD4+ T cells produced IFN-γ and IL-22, and CD4+ and CD8+ T cells produced IL-17A. CD4+ T lymphocytes also expressed IL-22R1, the density of its expression defining two different functional subsets. In conclusion, we provide the first evidence that SARS-CoV-2 infection is characterized by an abnormal expression of IL22R1 on blood myeloid cells and CD4+ T lymphocytes. Our results suggest that the involvement of the IL-22R1/IL-22 axis could be protective at the beginning of SARS-CoV-2 infection but could shift to a detrimental response over time.

BackgroundSurfactant protein D (SP-D) and pulmonary club cell protein 16 (CC-16) are called “pneumoproteins” and are involved in host defense against oxidative stress, inflammation, and viral outbreak. This study aimed to determine the predictive value of these pneumoproteins on the incidence of acute respiratory distress syndrome (ARDS) or death in patients with coronavirus disease-2019 (COVID-19).MethodsThis retrospective study included 87 patients admitted to an emergency department. Blood samples were collected on three time points (days 1, 5, and 14 from hospital admission). SP-D and CC-16 serum levels were determined, and univariate and multivariate analyses considering confounding variables (age, body mass index, tobacco use, dyspnea, hypertension, diabetes mellitus, neutrophil-to-lymphocyte ratio) were performed.ResultsBased on the multivariate analysis, SP-D level on D1 was positively and slightly correlated with subsequent development of ARDS, independent of body mass index, dyspnea, and diabetes mellitus. CC-16 level on D1 was modestly and positively correlated with fatal outcome. A rise in SP-D between D1 and D5 and D1 and D14 had a strong negative association with incidence of ARDS. These associations were independent of tobacco use and neutrophil-to-lymphocyte ratio.ConclusionsOverall, our data reveal that increase in SP-D levels is a good prognostic factor for patients with COVID-19, and that initial CC-16 levels correlated with slightly higher risk of death. SP-D and CC-16 may prove useful to predict outcomes in patients with COVID-19.

Abstract Background Patients with end-stage renal disease undergoing chronic hemodialysis (HD) are at high risk to develop tuberculosis (TB) associated with a high mortality rate. TB diagnosis is often delayed due to non-specific symptoms, frequent extra-pulmonary manifestations, and rare microbiological confirmation. This case report illustrates the clear added value of combined interferon-γ -release assays (IGRA) in response to different mycobacterial antigens for an early diagnosis of TB in HD patients. Case presentation We report the case of an Egyptian patient under chronic HD treatment, who presented with recurrent episodes of fever and myalgia of unknown origin, associated with an important inflammatory syndrome. These episodes resolved partially or completely within less than 1 month without any treatment but recurred 10 times within 3 years. Chest Computed Tomography and 18F-fluorodeoxyglucose Positron Emission Tomography/Computed Tomography (18FDG PET-CT) demonstrated several active mediastinal lymphadenopathies. TB was the first suspected diagnosis but cultures and polymerase chain reaction (PCR) remained negative on a mediastinal lymph node aspiration. In contrast, the results from two different IGRA performed on blood were highly suggestive of TB disease. Several granulomas, some of them with central non-caseating necrosis, were demonstrated on a pulmonary nodule obtained by thoracoscopic resection, but PCR and culture remained negative for M. tuberculosis. Three years after the initial symptoms, a new PET-CT revealed a retro-clavicular lymphadenopathy in addition to the mediastinal lymphadenopathies, and the M. tuberculosis culture performed on the resected lymphadenopathy was positive. Antibiotic treatment for TB was started and resulted in a clear improvement of the patient’s clinical condition, allowing him to successfully receive a renal graft. Conclusions In view of the high frequency of TB in patients undergoing chronic HD and of the limitations of the classical diagnosis procedures, nephrologists have to diagnose TB mostly on clinical suspicion. We demonstrate here that the use of a combined IGRA to two different mycobacterial antigens may significantly raise the index of suspicion and help clinicians to decide starting anti-TB treatment in HD patients.

Psoriasis is a skin inflammatory condition for which significant progress has been made in its management by the use of targeted biological drugs. Detection of latent M. tuberculosis infection (LTBI) is mandatory before starting biotherapy that is associated with reactivation risk. Together with evaluation of TB risk factors and chest radiographs, tuberculin skin tests (TST) and/or blood interferon-γ-release assays (IGRA), like the QuantiFERON (QFT), are usually performed to diagnose M. tuberculosis infection. Using this approach, 14/49 psoriatic patients prospectively included in this study were identified as LTBI (14 TST+, induration size ≥ 10mm, 8 QFT+), and 7/14 received prophylactic anti-TB treatment, the other 7 reporting past-treatment. As the specificity and sensitivity of these tests were challenged, we evaluated the added value of an IGRA in response to a mycobacterial antigen associated with latency, the heparin-binding haemagglutinin (HBHA). All but one TST+ patient had a positive HBHA-IGRA, indicating higher sensitivity than the QFT. The HBHA-IGRA was also positive for 12/35 TST-QFT- patients. Measurement for 15 psoriatic patients (12 with HBHA-IGRA+) of 8 chemokines in addition to IFN-γ revealed a broad array of HBHA-induced chemokines for TST+QFT- and TST-QFT- patients, compared to a more restricted pattern for TST+QFT+ patients. This allowed us to define subgroups within psoriatic patients characterized by different immune responses to M. tuberculosis antigens that may be associated to different risk levels of reactivation of the infection. This approach may help in prioritizing patients who should receive prophylactic anti-TB treatment before starting biotherapies in order to reduce their number.

Diagnosis of tuberculosis (TB) in children remains challenging due to unspecific clinical presentation and low bacillary load. In low TB incidence countries, most cases are diagnosed by a contact screening strategy after exposure to an index TB case. Due to the severity of TB in young children, the priority is to determine whether a child is infected or not, whereas differential diagnosis between active TB (aTB) and latent TB constitutes a second step. In Belgium, a low TB incidence country, we prospectively included 47 children with a defined M. tuberculosis infection status (12 children with aTB, 18 with latent TB, and 17 uninfected) (exploratory cohort), and determined the optimal combinations of cytokines secreted by their peripheral blood mononuclear cells in response to a 5-days in vitro stimulation with four different mycobacterial antigens, in an attempt to classify the children according to their infectious status. Correct identification of all infected children was obtained by several combinations of two purified protein derivative (PPD)-induced cytokines (IFN-γ and either GM-CSF, MIP-1α, sCD40L or TNF-α), or by combining PPD-induced IFN-γ with culture-filtrate protein-10 (CFP-10)-induced TNF-α. Alternatively, combining CFP-10-induced TNF-α and IP-10 with heparin-binding haemagglutinin (HBHA)-induced-IFN-γ was more effective in testing recently BCG-vaccinated children or those suspected to be infected with non-tuberculous mycobacteria, providing a correct classification of 97% of the M. tuberculosis-infected children. This combination also correctly classified 98% of the children from a validation cohort comprising 40 M. tuberculosis infected children and 20 non-infected children. Further differentiation between aTB and children with latent TB was more difficult. Combining ESAT-6-induced MIP1-α and IP-10, CFP-10-induced MIG, and HBHA-induced MIG provided a correct classification of 77% of the children from the exploratory cohort but only of 57.5% of those from the validation cohort. We conclude that combining the measurement of 2–4 cytokines induced by three different mycobacterial antigens allows an excellent identification of M. tuberculosis-infected children, whereas differentiating children with aTB from those with latent TB remains far from perfect.

Introduction: Belgium is a country with low incidence of tuberculosis (TB) and a very low number of TB cases in children. Children in contact with an adult smear-positive TB case are at high risk of transmission. Early diagnosis is important as young children have a significant predisposition of developing TB disease. In this paper, we describe two outbreaks after exposure to, respectively, two teachers with smear-positive pulmonary TB: one in a primary school, a nursery teacher, and another in a private language school.Methods: An exposure investigation was carried out in both index cases household and school, according to the stone-in-the-pond principle. The tuberculin skin test (TST) was used a screening tool. The time elapsed between TB diagnosis in the index case and contact investigation was, respectively, 1 and 3 weeks. If this initial test was negative, it was repeated after a “window period” of ≥8 weeks.Results: Index cases showed a transmission rate of, respectively, 13 and 40% in their classes at school, defined as casual contacts. The proximity of contact increased the risk of infection. TB disease was observed in, respectively, 4 and 11% of all the casual contacts; all of them were children younger than 5 years old. TB-infected and children with active TB disease had good compliance with recommended treatment. Uptake of chemoprophylaxis during the “window period” was poor, respectively, only 32–42%, in children under 5 years with an initially negative TST.Discussion: The World Health Organization recommends to screen all young children (

CD4+ T cells comprise multiple functionally distinct cell populations that play a key role in immunity. Despite blood monitoring of CD4+ T-cell subsets is of potential clinical utility, no standardized and validated approaches have been proposed so far. The aim of this study was to design and validate a single 14-color antibody combination for sensitive and reproducible flow cytometry monitoring of CD4+ T-cell populations in human blood to establish normal age-related reference values and evaluate the presence of potentially altered profiles in three distinct disease models—monoclonal B-cell lymphocytosis (MBL), systemic mastocytosis (SM), and common variable immunodeficiency (CVID). Overall, 145 blood samples from healthy donors were used to design and validate a 14-color antibody combination based on extensive reagent testing in multiple cycles of design–testing–evaluation–redesign, combined with in vitro functional studies, gene expression profiling, and multicentric evaluation of manual vs. automated gating. Fifteen cord blood and 98 blood samples from healthy donors (aged 0–89 years) were used to establish reference values, and another 25 blood samples were evaluated for detecting potentially altered CD4 T-cell subset profiles in MBL (n = 8), SM (n = 7), and CVID (n = 10). The 14-color tube can identify ≥89 different CD4+ T-cell populations in blood, as validated with high multicenter reproducibility, particularly when software-guided automated (vs. manual expert-based) gating was used. Furthermore, age-related reference values were established, which reflect different kinetics for distinct subsets: progressive increase of naïve T cells, T-helper (Th)1, Th17, follicular helper T (TFH) cells, and regulatory T cells (Tregs) from birth until 2 years, followed by a decrease of naïve T cells, Th2, and Tregs in older children and a subsequent increase in multiple Th-cell subsets toward late adulthood. Altered and unique CD4+ T-cell subset profiles were detected in two of the three disease models evaluated (SM and CVID). In summary, the EuroFlow immune monitoring TCD4 tube allows fast, automated, and reproducible identification of ≥89 subsets of CD4+ blood T cells, with different kinetics throughout life. These results set the basis for in-depth T-cell monitoring in different disease and therapeutic conditions.

Introduction: Interferon Gamma Release Assay (IGRA) has proven to be a useful test to evaluate the immune response to Mycobacterium tuberculosis antigens in children over the age of 5 years as an alternative to tuberculin skin testing (TST). Much less is known about its performance in younger children, who are at higher risk for developing tuberculosis (TB) disease after exposure. We aimed to evaluate the accuracy of using IGRA in TB screening in this population.Methods: Children below the age of 5 years at high risk for TB infection were prospectively enrolled, to compare the performance of TST and the QuantiFERON-TB Gold-In-Tube test (QFT). Children were treated in accordance with the diagnosis made at baseline and followed-up for 12 months.Results: We included a total of 60 children of which 97 blood samples were available for analysis. There was 90.72% agreement between TST and QFT (Kappa test 0.59, moderate agreement). With TST as a reference, the QFT positive predictive value was 0.72 and the negative predictive value 0.93. Discordant results were observed with 6% TST+/QFT– paired tests. When we restricted the comparison of TST and QFT to non-BCG-vaccinated children, the degree of agreement was more substantial (95%, Kappa test 0.75) and the negative predictive value was 0.99. We observed 3% discordant TST–/QFT+ results. All children with active TB disease had concordant positive QFT results, with QFT values above 4.00 IU/ml.Conclusion: In a low TB prevalence country, serial testing of QFT was found to produce a moderate agreement with TST results. False positive QFT results would have been eliminated by using a higher cutoff without misdiagnosing the children with TB disease. Some of the false negative QFT results could be explained by false positive TST results on consecutive testing. For now the most prudent approach would be to consider discordant QFT–/TST+ results as false negative QFT results, taking into account the young age of our population and the potential risk for evolution to active TB disease.

Background: Improved diagnostic tests are needed for the early identification of Mycobacterium tuberculosis-infected young children exposed to an active TB (aTB) index case. We aimed to compare the diagnostic accuracy of new blood-based tests to that of the tuberculin skin test (TST) for the identification of all infected children and for a potential differentiation between aTB and latent TB infection (LTBI).Methods: 144 children exposed to a patient with aTB were included, and those who met all inclusion criteria (130/144) were classified in three groups based on results from classical investigations: non-infected (NI: n = 69, 53%, median age 10 months), LTBI (n = 28, 22%, median age 96 months), aTB disease (n = 33, 25%, median age 24 months). The first whole blood assay consisted of a 7-days in vitro stimulation of blood with four different mycobacterial antigens (40 μl/condition), followed by flow cytometric measurement of the proportions of blast cells appearing among lymphocytes as a result of their specific activation. Thresholds of positivity were determined by Receiver Operating Characteristic (ROC) curve analysis (results of NI children vs. children with LTBI/aTB) in order to identify infected children in a first stage. Other cut-offs were determined to discriminate subgroups of infected children in a second step (results from children with aTB/LTBI). Analysis of blood monocytes and dendritic cell subsets was performed on 100 μl of blood for 25 of these children as a second test in a pilot study.Results: Combining the results of the blast-induced CD3+ T lymphocytes by Heparin-Binding Haemagglutinin and by Culture Filtrate Protein-10 identified all but one infected children (sensitivity 98.2% and specificity 86.9%, compared to 93.4 and 100% for the TST). Further identification among infected children of those with aTB was best achieved by the results of blast-induced CD8+ T lymphocytes by purified protein derivative (sensitivity for localized aTB: 61.9%, specificity 96.3%), whereas high proportions of blood type 2 myeloid dendritic cells (mDC) were a hallmark of LTBI.Conclusions: New blood-based tests requiring a very small volume allow the accurate identification of M. tuberculosis-infected young children among exposed children and are promising to guide the clinical classification of children with aTB or LTBI.

BackgroundPeritoneal 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 findingsWe 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%.ConclusionsThe 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.

To advance research and development of improved pertussis vaccines, new immunoassays are needed to qualify the outcome of Bordetella pertussis (Bp) specific CD4+ T-cell differentiation. Here, we applied a recently developed whole blood assay to evaluate Bp specific CD4+ T-cell responses. The assay is based on intracellular cytokine detection after overnight in vitro Bp antigen stimulation of diluted whole blood. We show for the first time that CD4+ T-cell memory of Th1, Th2, and Th17 lineages can be identified simultaneously in whole blood. Participants ranging from 7 to 70 years of age with different priming backgrounds of whole-cell pertussis (wP) and acellular pertussis (aP) vaccination were analyzed around an acellular booster vaccination. The assay allowed detection of low frequent antigen-specific CD4+ T-cells and revealed significantly elevated numbers of activated and cytokine-producing CD4+ T-cells, with a significant tendency to segregate recall responses based on primary vaccination background. A stronger Th2 response hallmarked an aP primed cohort compared to a wP primed cohort. In conclusion, analysis of Bp specific CD4+ T-cell responses in whole blood showed separation based on vaccination background and provides a promising tool to assess the quantity and quality of CD4+ T-cell responses induced by vaccine candidates.

Tuberculosis (TB) in young children differs from adult TB in that the risk of rapid progression to active TB (aTB) is higher in children than in adults. The reasons for this increased risk are not fully understood. Early differentiation remains difficult between children at risk to develop aTB from those who will remain healthy and develop a latent TB infection (LTBI). Biomarkers to differentiate aTB from LTBI in children, especially in very young children, are urgently needed. To identify M. tuberculosis-specific functional T cell subsets related to clinical manifestations in children, we enrolled 87 children exposed to M. tuberculosis. After standard clinical assessment, the children were classified as aTB, LTBI, or uninfected. Their CD4+ T cell cytokine profiles (IFN-γ, TNF-α, IL-2, IL-17) were analyzed at the single-cell level by flow cytometry after stimulation with three mycobacterial antigens, purified protein derivative (PPD), early-secreted-antigenic target-6 (ESAT-6), or heparin-binding hemagglutinin (HBHA). This approach identified age-related discriminative markers between aTB and LTBI. Whereas among the 3- to 15-year-old children, an excellent discrimination between aTB and LTBI was provided by comparing the ratio between the proportions of ESAT-6-induced IFN-γsingle+ and ESAT-6-induced TNF-αsingle+CD4+ T lymphocytes, this was not the case for children younger than 3 years. By contrast, in this group (

Community-acquired pneumonia (CAP) is a major cause of morbidity in children. This study estimated the proportion of children with pneumococcal CAP among children hospitalised with CAP in Belgium and describes the causative serotype distribution after implementation of the 7-valent pneumococcal conjugate vaccine. Children 0-14 years hospitalised with X-ray-confirmed CAP were prospectively enrolled in a multicentre observational study. Acute and convalescent blood samples were collected. Pneumococcal aetiology was assessed by conventional methods (blood or pleural fluid cultures with Quellung reaction capsular typing or polymerase chain reaction [PCR] in pleural fluid), and recently developed methods (real-time PCR in blood and World Health Organization-validated serotype-specific serology). A total of 561 children were enrolled. Pneumococcal aetiology was assessed by conventional methods in 539, serology in 171, and real-time PCR in blood in 154. Pneumococcal aetiology was identified in 12.2% (66/539) of the children by conventional methods alone but in 73.9% by the combination of conventional and recently developed methods. The pneumococcal detection rate adjusted for the whole study population was 61.7%. Serotypes 1 (42.3%), 5 (16.0%), and 7F(7A) (12.8%) were predominant. In conclusion, Streptococcus pneumoniae remains the predominant bacteria in children hospitalised for CAP in Belgium after implementation of 7-valent pneumococcal conjugate vaccine, with non-vaccine-serotypes accounting for the majority of cases. The use of recently developed methods improves diagnosis of pneumococcal aetiology.

BACKGROUND: Patients with end-stage renal disease (ESRD) and latently infected with Mycobacterium tuberculosis (LTBI) are at higher risk to develop tuberculosis (TB) than healthy subjects. Interferon-gamma release assays (IGRAs) were reported to be more sensitive than tuberculin skin tests for the detection of infected individuals in dialysis patients. METHODS: On 143 dialysis patients prospectively enrolled, we compared the results from the QuantiFERON®-TB Gold assay (QFT), to those of an IGRA in response to in vitro stimulation of circulating mononuclear cells with the mycobacterial latency antigen Heparin-Binding Haemagglutinin purified from Mycobacterium bovis BCG (native HBHA, nHBHA). RESULTS: Seven patients had a past history of active TB and 1 had an undetermined result with both IGRAs. Among the other 135 patients, 94 had concordant results with the QFT and nHBHA-IGRA, 40.0% being negative and therefore not latently infected, and 29.6% being positive and thus LTBI. Discrepant results between these tests were found for 36 patients positive only with the nHBHA-IGRA and 5 only with the QFT. CONCLUSIONS: The nHBHA-IGRA is more sensitive than the QFT for the detection of LTBI dialysis patients, and follow-up of the patients will allow us to define the clinical significance of discrepant results between the nHBHA-IGRA and the QFT.

Infant CD4+ T-cell responses to bacterial infections or vaccines have been extensively studied, whereas studies on CD8+ T-cell responses focused mainly on viral and intracellular parasite infections. Here we investigated CD8+ T-cell responses upon Bordetella pertussis infection in infants, children, and adults and pertussis vaccination in infants. Filamentous hemagglutinin-specific IFN-γ secretion by circulating lymphocytes was blocked by anti-MHC-I or -MHC-II antibodies, suggesting that CD4+ and CD8+ T lymphocytes are involved in IFN-γ production. Flow cytometry analyses confirmed that both cell types synthesized antigen-specific IFN-γ, although CD4+ lymphocytes were the major source of this cytokine. IFN-γ synthesis by CD8+ cells was CD4+ T cell dependent, as evidenced by selective depletion experiments. Furthermore, IFN-γ synthesis by CD4+ cells was sometimes inhibited by CD8+ lymphocytes, suggesting the presence of CD8+ regulatory T cells. The role of this dual IFN-γ secretion by CD4+ and CD8+ T lymphocytes in pertussis remains to be investigated.

BACKGROUND: Most individuals infected with Mycobacterium tuberculosis develop latent tuberculosis infection (LTBI). Some may progress to active disease and would benefit from preventive treatment yet no means currently exists to predict who will reactivate. Here, we provide an approach to stratify LTBI based on IFN-γ responses to two antigens, the recombinant Early-Secreted Antigen Target-6 (rESAT-6) and the latency antigen Heparin-Binding Haemagglutinin (HBHA). METHODS: We retrospectively analyzed results from in-house IFN-γ-release assays with HBHA (HBHA-IGRA) and rESAT-6 (rESAT-6-IGRA) performed during a 12-year period on serial blood samples (3 to 9) collected from 23 LTBI subjects in a low-TB incidence country. Both the kinetics of the absolute IFN-γ concentrations secreted in response to each antigen and the dynamics of HBHA/rESAT-6-induced IFN-γ concentrations ratios were examined. RESULTS: This analysis allowed the identification among the LTBI subjects of three major groups. Group A featured stable HBHA and rESAT-6-IGRA profiles with an HBHA/rESAT-6 ratio persistently higher than 1, and with high HBHA- and usually negative rESAT-6-IGRA responses throughout the study. Group B had changing HBHA/rESAT-6 ratios fluctuating from 0.0001 to 10,000, with both HBHA and rESAT-6 responses varying over time at least once during the follow-up. Group C was characterized by a progressive disappearance of all responses. CONCLUSIONS: By combining the measures of IFN-γ concentrations secreted in response to an early and a latency antigens, LTBI subjects can be stratified into different risk groups. We propose that disappearing responses indicate cure, that persistent responses to HBHA with HBHA/rESAT-6 ratios ≥ 1 represent stable LTBI subjects, whereas subjects with ratios varying from >1 to

A number of different interferon-gamma ELISpot protocols are in use in laboratories studying antigen-specific immune responses. It is therefore unclear how results from different assays compare, and what factors most significantly influence assay outcome. One such difference is that some laboratories use a short in vitro stimulation period of cells before they are transferred to the ELISpot plate; this is commonly done in the case of frozen cells, in order to enhance assay sensitivity. Other differences that may be significant include antibody coating of plates, the use of media with or without serum, the serum source and the number of cells added to the wells. The aim of this paper was to identify which components of the different ELISpot protocols influenced assay sensitivity and inter-laboratory variation. Four laboratories provided protocols for quantifying numbers of interferon-gamma spot forming cells in human peripheral blood mononuclear cells stimulated with Mycobacterium tuberculosis derived antigens. The differences in the protocols were compared directly. We found that several sources of variation in assay protocols can be eliminated, for example by avoiding serum supplementation and using AIM-V serum free medium. In addition, the number of cells added to ELISpot wells should also be standardised. Importantly, delays in peripheral blood mononuclear cell processing before stimulation had a marked effect on the number of detectable spot forming cells; processing delay thus should be minimised as well as standardised. Finally, a pre-stimulation culture period improved the sensitivity of the assay, however this effect may be both antigen and donor dependent. In conclusion, small differences in ELISpot protocols in routine use can affect the results obtained and care should be given to conditions selected for use in a given study. A pre-stimulation step may improve the sensitivity of the assay, particularly when cells have been previously frozen.

BACKGROUND: The detection of latent tuberculosis infection (LTBI) is a major component of tuberculosis (TB) control strategies. In addition to the tuberculosis skin test (TST), novel blood tests, based on in vitro release of IFN-gamma in response to Mycobacterium tuberculosis-specific antigens ESAT-6 and CFP-10 (IGRAs), are used for TB diagnosis. However, neither IGRAs nor the TST can separate acute TB from LTBI, and there is concern that responses in IGRAs may decline with time after infection. We have therefore evaluated the potential of the novel antigen heparin-binding hemagglutinin (HBHA) for in vitro detection of LTBI. METHODOLOGY AND PRINCIPAL FINDINGS: HBHA was compared to purified protein derivative (PPD) and ESAT-6 in IGRAs on lymphocytes drawn from 205 individuals living in Belgium, a country with low TB prevalence, where BCG vaccination is not routinely used. Among these subjects, 89 had active TB, 65 had LTBI, based on well-standardized TST reactions and 51 were negative controls. HBHA was significantly more sensitive than ESAT-6 and more specific than PPD for the detection of LTBI. PPD-based tests yielded 90.00% sensitivity and 70.00% specificity for the detection of LTBI, whereas the sensitivity and specificity for the ESAT-6-based tests were 40.74% and 90.91%, and those for the HBHA-based tests were 92.06% and 93.88%, respectively. The QuantiFERON-TB Gold In-Tube (QFT-IT) test applied on 20 LTBI subjects yielded 50% sensitivity. The HBHA IGRA was not influenced by prior BCG vaccination, and, in contrast to the QFT-IT test, remote (>2 years) infections were detected as well as recent (