HBHA-IGRA and cytotoxic mediators release assays for the diagnosis of cervical tuberculous lymphadenitis.

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.

Analysis of a Combined HBHA and ESAT-6-Interferon-γ-Release Assay for the Diagnosis of Tuberculous Lymphadenopathies.

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.

A semi high-throughput whole blood-based flow cytometry assay to detect and monitor Bordetella pertussis-specific Th1, Th2 and Th17 responses.

Introduction: The 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. Methods: To 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. Results: We 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. Discussion: Thus, this assay appears as a promising test for evaluation of Bp antigen-specific CD4+ T cells induced by current and next generation pertussis vaccines.

Chemerin plasma levels are increased in COVID-19 patients and are an independent risk factor of mortality.

Background: Chemerin 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. Methods: Blood 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). Results: 21 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<0.0001). Moreover, they were higher in deceased patients compared to survivors (p<0.05). Logistic univariate regression and multivariate analysis demonstrated that chemerin level at day 14 of admission was an independent risk factor for death. Accordingly, chemerin levels correlated with inflammatory biomarkers such as C-reactive protein and tumor necrosis factor α. Finally, IHC analysis revealed a strong expression of ChemR23 on smooth muscle cells and chemerin on myofibroblasts in advanced acute respiratory distress syndrome (ARDS). Discussion: Increased plasma chemerin levels are a marker of severity and may predict death of COVID-19 patients. However, multicentric studies are needed, before chemerin can be considered as a biomarker of severity and death used in daily clinical practice. Further studies are also necessary to identify the precise mechanisms of the chemerin/ChemR23 system in ARDS secondary to viral pneumonia.

Immuno-Diagnosis of Active Tuberculosis by a Combination of Cytokines/Chemokines Induced by Two Stage-Specific Mycobacterial Antigens: A Pilot Study in a Low TB Incidence Country.

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.

Distinct Expression Patterns of Interleukin-22 Receptor 1 on Blood Hematopoietic Cells in SARS-CoV-2 Infection.

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.

Impact of Maternal Pertussis Antibodies on the Infants' Cellular Immune Responses.

INTRODUCTION: Maternal antibody interference of the infant's humoral immune responses raises some concern to the strategy of maternal Tdap (tetanus, diphtheria, acellular pertussis [aP]) vaccination. This study assessed the impact of maternal Tdap antibodies on the infant's pertussis-specific T lymphocyte responses following infant vaccination with an aP containing vaccine, in a term and preterm born cohort. METHODS: Heparin samples (±0.5 mL) were conveniently drawn from infants of a Belgian prospective cohort study (N = 79, NCT02511327), including Tdap vaccinated (Boostrix®) and nonvaccinated women (no Tdap vaccine in the last 5 years) that delivered at term or prematurely. Sampling was performed before and 1 month after primary (8-12-16 weeks) and booster vaccination (13 or 15 months) with DTaP-IPV-HB-PRP~T vaccine (Hexyon®). Pertussis toxin (PT)-specific CD3+, CD3+ CD4+ and CD3+ CD8+ lymphoblasts and their cytokine secretions were measured using a flow cytometric assay on whole blood (FASCIA) and multiplex technology (Meso Scale Discovery), respectively. RESULTS: In total, 57% of all infants were considered PT-specific CD3+ CD4+ lymphoblasts responders after primary and booster vaccination, whereas 17% were CD3+ CD8+ lymphoblast responders. Interferon (IFN)-γ, interleukin (IL)-13, IL-17A, and IL-5 cytokine secretions after primary and booster vaccination were indicative of a mixed T helper (Th) 1/Th2/Th17 cell profile. Lymphoblast and cytokine levels were comparable between term and preterm infants. Nonresponders for IL-13 after booster vaccination had higher maternal PT immunoglobulin G (IgG) levels at birth when compared to responders. CONCLUSIONS: Term and preterm born infants are capable of inducing Th1, Th2, and Th17 responses after aP vaccination, yet maternal vaccination modulate these responses. Evaluation of this effect in larger trials is needed.

Differential expression of maturation and activation markers on NK cells in patients with active and latent tuberculosis.

NK cells were recently suggested to be important for the initial control of M. tuberculosis infection. The phenotypes of the 3 main NK blood subsets, CD56bright , CD56dim , and CD56neg cells, were characterized by flow cytometry in a cohort of 81 prospectively enrolled subjects (21 untreated patients with active tuberculosis -aTB-, 35 latently TB infected -LTBI- subjects, and 25 non-infected controls), using 9 different mAbs added to whole blood. Compared to LTBI subjects, patients with aTB had lower proportions of total NK cells, lower proportions and numbers of CD56neg cells expressing early maturation markers (CD161, NKp30, NKp46), but higher density of NKp30 and NKp46 expression on both CD56neg and CD56dim subsets, associated with higher expression of granzymes A/B. They also had higher proportions of activated CD69pos cells within all 3 NK cell subsets and, the percentage of CD69pos CD56dim cells among CD69pos and/or NKG2Cpos NK cells was identified as a potential biomarker to discriminate aTB from LTBI. LTBI subjects were in contrast characterized by higher expression of late maturation markers (CD57, KIR molecules) on the CD56neg subset, by higher proportions of NKG2Cpos KIRpos CD56dim NK cells, and by higher in vitro IFN-γ production than patients with aTB. Thus, the in-depth phenotypic characterization of blood NK cell subsets provides new insights on possible functional modifications and the potential role of NK cells in the control of M. tuberculosis infection in humans.

Tuberculosis Risk Stratification of Psoriatic Patients Before Anti-TNF-α Treatment.

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.

Specific Host Signatures for the Detection of Tuberculosis Infection in Children in a Low TB Incidence Country.

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.

SP-D and CC-16 Pneumoproteins' Kinetics and Their Predictive Role During SARS-CoV-2 Infection.

BACKGROUND: Surfactant 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). METHODS: This 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. RESULTS: Based 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. CONCLUSIONS: Overall, 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.

Early diagnosis of miliary tuberculosis in a hemodialysis patient by combining two interferon-γ-release assays: a case report.

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.

Uncovering Distinct Primary Vaccination-Dependent Profiles in Human Bordetella Pertussis Specific CD4+ T-Cell Responses Using a Novel Whole Blood Assay.

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.

Highlights of the 12th International Bordetella Symposium.

To commemorate the 100th anniversary of the Nobel prize being awarded to Jules Bordet, the discoverer of Bordetella pertussis, the 12th International Bordetella Symposium was held from 9 to 12 April 2019 at the Université Libre de Bruxelles, where Jules Bordet studied and was Professor of Microbiology. The symposium attracted more than 300 Bordetella experts from 34 countries. They discussed the latest epidemiologic data and clinical aspects of pertussis, Bordetella biology and pathogenesis, immunology and vaccine development, and genomics and evolution. Advanced technological and methodological tools provided novel insights into the genomic diversity of Bordetella and a better understanding of pertussis disease and vaccine performance. New molecular approaches revealed previously unrecognized complexity of virulence gene regulation. Innovative insights into the immune responses to infection by Bordetella resulted in the development of new vaccine candidates. Such discoveries will aid in the design of more effective approaches to control pertussis and other Bordetella-related diseases.

Tuberculosis Transmission in a Primary School and a Private Language School. An Estimation of Infectivity.

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 (<5 years old) who have close contact with a person affected by pulmonary TB and to initiate Latent tuberculosis infection treatment even before infection can be demonstrated, after ruling out active TB disease. Despite this knowledge, a small percentage of the children younger than 5 years with no proof of infection was treated with the proposed chemoprophylactic treatment, in both cases. Conclusion: This exposure investigation of two teachers detects high transmission among family contacts and school casual contacts. Recommendations for chemoprophylactic treatment in children <5 years showed low compliance, reflecting the difficulty of communication to staff, parents, and children in a school outbreak. It is essential to develop a new approach for this vulnerable group of patients. This approach could be improved, applied, and evaluated by National TB Control Programs, involving public and private health services. Public health authorities play a role in raising public awareness about the risks of TB for young children.

Natural T Cell Epitope Containing Methyl Lysines on Mycobacterial Heparin-Binding Hemagglutinin.

T cell epitopes are mostly nonmodified peptides, although posttranslationally modified peptide epitopes have been described, but they originated from viral or self-proteins. In this study, we provide evidence of a bacterial methylated T cell peptide epitope. The mycobacterial heparin-binding hemagglutinin (HBHA) is a protein Ag with a complex C-terminal methylation pattern and is recognized by T cells from humans latently infected with Mycobacterium tuberculosis By comparing native HBHA with recombinant HBHA produced in Mycobacterium smegmatis (rHBHA-Ms), we could link antigenic differences to differences in the methylation profile. Peptide scan analyses led to the discovery of a peptide containing methyl lysines recognized by a mAb that binds to native HBHA ∼100-fold better than to rHBHA-Ms This peptide was also recognized by T cells from latently infected humans, as evidenced by IFN-γ release upon peptide stimulation. The nonmethylated peptide did not induce IFN-γ, arguing that the methyl lysines are part of the T cell epitope.

Age Distribution of Multiple Functionally Relevant Subsets of CD4+ T Cells in Human Blood Using a Standardized and Validated 14-Color EuroFlow Immune Monitoring Tube.

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.

The effect of maternal antibodies on the cellular immune response after infant vaccination: A review.

During the last few decades, maternal immunization as a strategy to protect young infants from infectious diseases has been increasingly recommended, yet some issues have emerged. Studies have shown that for several vaccines, such as live attenuated, toxoid and conjugated vaccines, high maternal antibody titers inhibit the infant's humoral immune response after infant vaccination. However, it is not clear whether this decreased antibody titer has any clinical impact on the infant's protection, as the cellular immune responses are often equally important in providing disease protection and may therefore compensate for diminished antibody levels. Reports describing the effect of maternal antibodies on the cellular immune response after infant vaccination are scarce, probably because such studies are expensive, labor intensive and utilize poorly standardized laboratory techniques. Therefore, this review aims to shed light on what is currently known about the cellular immune responses after infant vaccination in the presence of high (maternal) antibody titers both in animal and human studies. Overall, the findings suggest that maternally derived antibodies do not interfere with the cellular immune responses after infant vaccination. However, more research in humans is clearly needed, as most data originate from animal studies.

Contribution of QuantiFERON-TB Gold-in-Tube to the Diagnosis of Mycobacterium tuberculosis Infection in Young Children in a Low TB Prevalence Country.

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.

Identification of Mycobacterium tuberculosis Infection in Infants and Children With Partial Discrimination Between Active Disease and Asymptomatic Infection.

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.