Tuberculosis (TB) poses a major public health challenge, particularly in children. A substantial proportion of children with TB disease remain undetected and unconfirmed. Therefore, there is an urgent need for a highly sensitive point-of-care test. This study aims to assess the performance of serological assays based on various antigen targets and antibody properties in distinguishing children (0–18 years) with TB disease (1) from healthy TB-exposed children, (2) children with non-TB lower respiratory tract infections, and (3) from children with TB infection.
Methods
The study will use biobanked plasma samples collected from three prospective multicentric diagnostic observational studies: the Childhood TB in Switzerland (CITRUS) study, the Pediatric TB Research Network in Spain (pTBred), and the Procalcitonin guidance to reduce antibiotic treatment of lower respiratory tract infections in children and adolescents (ProPAED) study. Included are children diagnosed with TB disease or infection, healthy TB-exposed children, and sick children with non-TB lower respiratory tract infection. Serological multiplex assays will be performed to identify M. tuberculosis antigen-specific antibody features, including isotypes, subclasses, Fc receptor (FcR) binding, and IgG glycosylation.
Discussion
The findings from this study will help to design serological assays for diagnosing TB disease in children. Importantly, those assays could easily be developed as low-cost point-of-care tests, thereby offering a potential solution for resource-constrained settings.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Background
Diagnosing tuberculosis (TB) in children presents several challenges [1]. TB disease in children is confirmed only in about 50% of patients due to the paucibacillary nature [2, 3]. In the absence of a reliable and easily accessible diagnostic test for screening and confirming TB disease in children, diagnosis typically relies on clinical findings, TB contact history, chest radiography findings, and the results of immune-based TB tests, the Tuberculin skin test (TST) and interferon-γ release assays (IGRA) [4]. However, both immunodiagnostic tests have suboptimal performance and are not well-suited for screening for TB disease [5, 6].
Serological assays have the potential to serve as a screening tool for TB infection and disease in children, especially in resource-limited settings where advanced diagnostic methods are limited. This potential stems from their blood-based nature, thus not requiring sputum collection, and their feasibility to be used as point-of-care tests [7]. However, currently available commercial serological assays are not recommended for clinical use due to their insufficient and variable diagnostic performance, characterised by limited sensitivity, specificity, and susceptibility to cross-reactivity [8, 9]. In a recent narrative review focusing on the diagnostic performance of non-commercial serological assays for TB in children, we found that studies which measured antibodies against only one antigen generally reported relatively high specificity but only achieved limited sensitivity [10]. Higher sensitivity can be achieved when antibodies against multiple targets are measured, and results are interpreted in combination. In addition, emerging evidence suggests that certain antibody properties, such as antibody Fc receptor (FcR) binding profiles [11, 12] and antibody glycosylation patterns [13], can potentially be used to differentiate between TB infection and disease. However, most of those studies have been done in adults, and the evidence in children remains extremely limited.
Anzeige
Methods
Aim
The aim of this study is to evaluate the diagnostic performance of serological assays in detecting children with TB disease, and in distinguishing those subjects from (1) healthy TB-exposed children, (2) children with non-TB lower respiratory tract infection, and (3) children with TB infection.
Study setting and population
This study will utilise plasma samples obtained from three different prospective multicentric observational studies: the Childhood Tuberculosis in Switzerland (CITRUS) study (NCT03044509), the Pediatric TB Research Network in Spain (pTBred), and the Procalcitonin guidance to reduce antibiotic treatment of lower respiratory tract infections in children and adolescents (ProPAED) study (ISRCTN 17,057,980) (Table 1).
Table 1
Overview of studies from which samples will be used in the described project, including inclusion- and exclusion criteria
Inclusion criteria
Exclusion criteria
Study design
Time
Country
CITRUS
< 18 years
Undergoing evaluation for TB exposure, TB infection, TB disease
- Anti-mycobacterial treatment more than 5 day prior to inclusion
- Previous treatment for TB disease or infection
Multi-centric study with nine centres in Switzerland
Since May 2017 (on going)
Switzerland
pTBred
< 18 years Undergoing evaluation for TB exposure, TB infection, TB disease
- Anti-mycobacterial treatment more than 5 day prior to inclusion
- Previous treatment for TB disease or infection
Multidisciplinary collaborative network
Oct 2019 - Jun 2021
Spain
ProPAED
pulmonary non-TB disease:
< 18 years, presentation with lower respiratory tract infection including fever and cough, regardless of previous antibiotic treatment history
- Severe immunosuppression
- Known HIV infection
- Immunosuppressive treatment
- Neutropenia
- M. tuberculosis infection
- Cystic fibrosis
- Viral laryngotracheitis
- Hospital stay within the previous 14 days
- Other severe infections (e.g., osteomyelitis, endocarditis, or deep tissue abscesses)
Multi-centric study with two emergency departments
Jan 2009 - Feb 2010
Switzerland
CITRUS is a multicentric prospective diagnostic study done at nine centres across Switzerland (Bern, Basel, Zurich, Lausanne, Geneva, Aarau, St. Gallen, Lucerne, Bellinzona). Its primary objective is to evaluate and validate novel immunodiagnostic assays for childhood TB [14, 15]. The study includes children under the age of 18 years, with or without a history of Bacillus Calmette-Guérin (BCG) vaccination, who are undergoing evaluation for TB disease, infection, and exposure. Children who have received any anti-mycobacterial treatment for five days or more before inclusion or who have been previously treated for TB disease or infection are excluded. Recruitment for the CITRUS study began in May 2017 and is currently ongoing.
Anzeige
PTBred is a multidisciplinary collaborative network established in 2014 in Spain, recruiting children < 18 years with TB. Since 2017, different types of samples have been stored in the Biobank of the Gregorio Marañon Hospital or in the individual collection registered as C.0006631 in the National Biobank Collections Registry. For this study, a common protocol for sample processing was implemented in October 2019, including children with children with TB disease, infection, and exposure irrespective of their BCG-vaccination status. The pTBred and CITRUS study follow the same inclusion and exclusion criteria [16].
The ProPAED study collected samples from children and adolescents presenting with fever and cough at two emergency departments in Switzerland (Basel and Aarau), from January 2009 to February 2010. For the ProPAED study, children with severe immunocompromise or known HIV infection, those undergoing immunosuppressive treatment, children with M. tuberculosis infection, neutropenia, cystic fibrosis, viral laryngotracheitis, hospital stay within the preceding 14 days, or other severe infections (e.g., osteomyelitis, endocarditis, or deep tissue abscesses) were excluded [17].
Case definitions
In this study, we will use the published criteria of compound TB case definitions proposed by Graham et al. [18]. Briefly, confirmed TB disease is defined as the presence of bacteriologically confirmed TB disease through culture or nucleic acid amplification tests (NAAT). Unconfirmed TB disease is defined as the absence of bacteriological confirmation in the presence of at least two of the following criteria: symptoms or signs suggestive of TB disease, chest radiograph consistent with TB disease, close TB exposure or immunologic evidence of M. tuberculosis infection, positive response to TB treatment. TB infection is defined as the presence of immunologic evidence of M. tuberculosis infection, including a positive TST of ≥ 5 mm (in accordance with the Swiss and Spanish guidelines [19, 20]) or a positive IGRA without meeting the criteria for confirmed or unconfirmed TB disease. Healthy TB-exposed children are defined as asymptomatic individuals with negative results on IGRA or TST test (single or repeat testing according to age, time since exposure as defined by national guidelines), making them unlikely to have TB. Children with non-TB lower respiratory tract infection will be the sick control group and are defined as presenting with fever (core body temperature ≥ 38.0° C) and at least one symptom (cough, sputum production, pleuritic pain, poor feeding) and at least one sign (tachypnea, dyspnoea, wheezing, late inspiratory crackles, bronchial breathing, pleural rub) lasting for fewer than 14 days.
Age stratification
The study will analyse antibody concentrations and properties in children stratified into distinct age groups: 0 to < 2, 2 to < 5, 5 to < 10, and ≥ 10 years, as proposed by Cuevas et al. [21]. This stratification is crucial due to the differences and dynamics of the nature of TB disease across age. In the youngest age range (infants and children < 2 years old), disseminated diseases and heightened susceptibility to progression from TB infection to TB disease is well-documented [22]. The risks for progression from infection to disease, as well as the subsequent mortality risk following development of disease, consistently declines during childhood, reaching its lowest point between 5 and 10 years of age [23]. Transitioning into adolescence and the onset of puberty, typically beyond the age of 10 years, the phenotype of TB disease becomes more adult-like. Pulmonary TB becomes more prevalent during this phase, contributing to an upsurge in TB-related mortality rates [24, 25].
Selected antigen targets and antibody properties for serological assay
Some previous studies in children have demonstrated improved specificities achieved by combining both protein and glycolipid antigens within serological assays [26‐29]. Furthermore, several studies have illuminated the potential for heightened sensitivity through the combined analysis of multiple antigen targets, effectively overcoming the interindividual heterogeneity of the human humoral immune response to M. tuberculosis [26‐33].
We will analyse antibodies concentrations and properties against single protein antigens, single glycolipid antigens [12, 34‐40], as well as multiple antigens in combination (Table 2). The types of antigens include cell wall fractions, whole cell lysates, and total lipids of M. tuberculosis. The selection of protein antigens is based on results from large protein microarray studies in adults [41‐46], one large multiplex bead-based study in children [31], and published and unpublished data from an adult study performed in the U.K (MIMIC study; personal communication M. Tebruegge) [47]. In order to enhance specificity, the overlap of the antigen targets for M. tuberculosis with Bacillus Calmette-Guérin (BCG) and other non-tuberculous mycobacteria will be reduced.
Table 2
Key protein, glycolipid, and multiple antigens
Type
Name
Rv number/Full name
Protein
FbpC (Ag85C)
Rv0129c
PstS3
Rv0928
PstS1
Rv0934
PapA4
Rv1528
GarA
Rv1827
Apa (Mpt32)
Rv1860
FbpB (Ag85B)
Rv1886c
Mpt63
Rv1926c
Mpt64
Rv1980c
HspX (Acr)
Rv2031c
Acg
Rv2032
Rv2034
Rv2034
Hrp1
Rv2626c
EsxO-EsxP
Rv2346-Rv2347
EspA
Rv3616c
FbpD (Mpt51)
Rv3803c
FbpA (Ag85A)
Rv3804c
EsxB (CFP-10)
Rv3874
EsxA (ESAT-6)
Rv3875
EsxA-EsxB (ESAT6-CFP10)
Rv3875-Rv3874
EspD-EspC
Rv3614-Rv3615
EspB
RV3881c
Ag85 complex
Rv3804c-Rv1886c-Rv0129c
Glycolipid
LAM
Lipoarabinomannan
PDIM
Phthiocerol dimycocerosates
TDM
Trehalose dimycolates
TMM
Trehalose monomycolates
PGL
Phenolic glycolipid
Multiple antigens (H37Rv)
Cell wall fractions
contains proteins and non-protein compounds such as mAGP of M. tuberculosis
Cell membrane fractions
contains the cytoplasmic membrane and components of the outer lipid layer.
Whole cell lysates
contains proteins, lipids and carbohydrates present within the M. tuberculosis bacterial cell
Together with targeted M. tuberculosis antigens, this study will evaluate the following distinct properties of the antibodies: isotypes and their subclasses, FcR binding profiles, and antibody glycosylation patterns (refer to Fig. 1). The rational for this is to obtain further information about the immune response to the antigen. TB disease results from a combination of the mycobacteria infecting and the resulting pathologic immune response. Therefore, antibody concentrations may only reflect on exposure, timepoint, and burden of mycobacteria, whereas additional properties such as FcR may reflect on the fact if the immune response producing tissue damage and pathology or not. This is shown in studies in children with TB disease that have demonstrated the potential enhancement of serological assay sensitivity through the integration of diverse antibody isotypes [48‐50]. Recent advancements in adult research have indicated that an evaluation of certain antibody properties, such as FcRs binding profiles and glycosylation patterns, could potentially enable the differentiation between TB disease and infection [12, 13].
Anzeige
×
As a quality control and potential normalisation variable, we will measure the total antibody concentration of each isotype and the total antibody concentration binding to distinct FcRs.
Sample preparation
Upon plasma sample collection, preservation is ensured through storage in a − 80 °C freezer until the initiation of laboratory assays. Customised multiplex antigen-coupled beads will be produced to evaluate antigen-specific antibodies concentrations and properties in plasma samples. The protein antigens will be coupled to carboxylated beads through covalent NHS-ester linkages, using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and Sulfo-NHS (Thermo Scientific), following the manufacturer’s recommendations [51, 52]. Glycan antigen LAM, single lipid antigens (e.g., TDM and TMM), and multiple lipid antigen from Mycobacterium tuberculosis total lipids will be modified using 4-(4,6-dimethoxy [1, 3, 5] triazin-2-yl)-4-methyl-morpholinium (DMTMM) dissolved in ethanol and conjugated beads following the COOH-DMTMM method [53].
The antigen-specific antibodies concentrations and properties will be measured using different PE-labelled detection antibodies as follows: for the isotypes and subclasses, PE-coupled detection antibodies (anti-IgG, anti-IgA, anti-IgM, anti-IgG1, anti-IgG2, anti-IgG3 and anti-IgG4) at a concentration of 1 µg/mL; [52] for the FcR binding profiles, FcRs (FcγRIIIa/CD16a, FcγRIIIb/CD16b, FcγRIIa/CD32a H167, FcγRIIb/CD32b, FcγRI/CD64 from R&D Systems) will be labelled with PE and added to the samples at a concentration of 1 µg/mL; and for the glycosylation profiles, PE-labelled lectins (SNA for sialic acid, ECL for galactose, LCA for fucose and PHA-E for N-acetylglucosamine) will be used at a concentration of 20 µg/mL. After 2 h of incubation at room temperature, the beads will be washed with PBS-0.05% Tween20, and PE signal will be measured using xMAP technology. (refer to Fig. 2)
Anzeige
×
Data management
All data will be securely entered and shared through password-protected and encrypted systems to uphold the confidentiality of health-related personal information. Adhering to Swiss legal requirements for data protection (Ordinance HRO Art. 5), our procedures for storing biological samples and handling health data are meticulously governed. Coding mechanisms and personalised logins are implemented to grant exclusive access to the study database and source documents for authorised personnel, thereby preventing third-party disclosure. Unique identification numbers are assigned to the biological samples and health-related personal data.
Data analysis
Descriptive statistics, including mean, median, standard deviation, and interquartile range, will be used to summarise antibody concentrations stratified by diagnostic group (TB disease, TB infection, healthy TB-exposed controls, and non-TB lower respiratory tract infections) and age groups (< 2 years, 2 to < 5 years, 5 to < 10 years, and ≥ 10 years). Antigen-specific antibody concentrations will be analysed in relation to the total (nonspecific) antibody concentrations. Comparisons between groups will be made using t-tests or Mann-Whitney U tests if normality assumptions are not met. Children with TB disease and infection will be compared with the following groups: all other remaining children combined, healthy TB-exposed children, and children with non-TB lower respiratory tract infections.
To assess the performance of each individual antigen specific antibody feature as a diagnostic assay, sensitivity and specificity will be calculated based on cut-off values determined by the highest Youden’s index. Receiver operating characteristic (ROC) analysis will be performed, and area under the curve (AUC) will be calculated (confidence interval will be determined using the DeLong method).
Anzeige
In subsequent analyses, we aim to evaluate the combined interpretation of antigen-specific antibodies concentrations and properties using different strategies:
Strategy one involves defining cut-off values based on a specificity of ≥ 98%, in accordance with the minimal WHO’s TPP requirement for a biomarker-based detection test. We will calculate the corresponding sensitivity. Similarly, we will determine cut-off values based on a sensitivity of ≥ 66% and calculate the corresponding specificity. To assess the combined interpretation of multiple antigen targets, the test for a specific antibody or antibody property will be scored positive if at least one antibody level against a specific antigen exceeds the cut-off value in an individual’s plasma sample, and negative if all antibody levels against all antigens in a plasma sample are below the cut-off values.
Another strategy for the combined interpretation of multiple antibody concentrations and properties will involve feature selection using the least absolute shrinkage and selection operator (LASSO). This approach will help identifying the most informative features that could be used in diagnostic assays. To validate the predictive power of the selected features (k features), we will train and evaluate an additional model using only those k features. In a further step, we will include the selection of antibody concentrations and properties in the training of the model. By performing feature selection using LASSO, we aim to maximize prediction performance using all features and select the k most informative features after the training stage. This procedure is based on the concept that selecting the most informative features from a well-performing prediction model will also yield a well-performing prediction model when one only has access to the selected subset of features. Recent advances in machine learning research will enable us to incorporate feature subset selection directly into the training step of a model [54, 55]. Therefore, we optimise not only the prediction performance but also the subset selection of k features during training. The choice of subset size, k, should be based on external constraints. The diverse sensitivities and specificities observed in paediatric TB serological tests make a precise sample size determination challenging. To estimate the sample size for our experiments, we used data generated from a cohort of adults with latent infection (n = 20) and active pulmonary disease (n = 22) from South Africa [56]. For the analysis of 75 antibody features, linear regression was conducted to assess the association between diagnosis and antibody feature, while controlling for age and gender. For the thirteen features exceeding a false discovery rate threshold of 10%, the partial correlation coefficient of 0.50 or higher was observed between diagnosis and antibody feature. Using this estimate as the effect size of biologically active antibody features, 68 individuals in an independent cohort (34 LTB, 34 ATB) would provide a statistical power of 80% to observe significant differences in top antibody features between tuberculosis infection and disease at an alpha level of 0.0005. This alpha level represents the threshold for significance required by the Bonferroni-Holm correction method, set at 0.0005 to accommodate the testing of 100 antibody features.
Publication and dissemination policy
Findings of this study will be disseminated through peer-reviewed journals, scientific conferences, and other relevant platforms. Participants will receive a summary of the results. All scientific data generated from this project will be made available as soon as possible, and no later than the time of publication or the end of the funding period, whichever comes first. The data and related metadata underlying reported findings will be deposited in a public data repository. A data access committee will support third parties who wish to perform further research with the data. Data will be curated in the repository following accepted standards and a persistent identifier, a DOI, is created for each data set published. If intellectual property is developed, dissemination of data will occur after appropriate protections for intellectual property are put in place.
Discussion
The development of reliable point-of-care tests for detecting TB infection and disease in children is crucial. Serological assays offer a promising approach, as they may be used in a point-of-care test format, making them suitable for widespread implementation in diverse settings [7]. However, there are several hurdles that need to be addressed to advance the development of TB serological assays. One challenge is the incomplete understanding of the immunogenic properties of the numerous potential antigens of M. tuberculosis, including proteins and glycolipids [57]. Our study has four main strengths. First, our study will evaluate antibodies against a broad range of protein antigens [41, 45, 46, 58], as well as glycolipids that are believed to play a crucial role int the pathogenesis of M. tuberculosis [59, 60].
Second, to overcome the challenge of potential cross-reactivity of antibodies detected in a serological assay for TB with BCG- and non-tuberculous mycobacteria-antigens [25], we will include a large range of antibodies and reduced the overlap between M. tuberculosis and BCG/non-tuberculous mycobacteria-antigens selected. Third, there exists substantial interindividual heterogeneity in the antibody response to M. tuberculosis [61, 62]. Different individuals may react to different antigens, resulting in relatively low sensitivity but good specificity for each individual antigen serological assays [30, 31, 49]. To account for this heterogeneity, our analysis includes multiple antigen targets, such as cell wall fractions and total lipids, and aims at a combined interpretation of these parameters.
Finally, we will evaluate specific antibody properties, such as antibody isotypes, glycosylation patterns, and FcR binding profiles [12]. So far, IgG is the most extensively studied isotype and has shown the most promising results for use in diagnostic assays to detect TB disease in children. Other isotypes, such as IgA, have gained attention more recently, as these have a protective role in human and animal studies in preventing TB infection [63, 64]. Glycosylation of the Fc region affects the binding affinity of the antibody to the FcRs. Notably, distinct glycosylation patterns have been associated with various stages of TB disease and infection [11]. Lastly, our data analysis is stratified across distinct age groups to accommodate the dynamic nature of TB disease during various developmental stages of children.
The findings of our study will improve our understanding of the human humoral immune response to M. tuberculosis infection and disease and holds the potential to pave the way for designing antibody-based assays with high performance characteristic for use in children.
Acknowledgements
We thank the local Principal Investigators of the CITRUS study: Sara Bernhard, Lisa Kottanattu, Andrea Duppenthaler, Anne Morand, Jürg Barben, Christoph Berger, Christa Relly, Isabelle Rochat, Marie Rohr, as well as of Noemi Meier and Andrea Marten for their contribution to plasma sample collection. Our gratitude extends to the investigators of the ProPAED study: Gurli Baer, Jan Bonhoeffer, Philipp Baumann, Michael Buettcher, Ulrich Heininger, Gerald Berthet, Julia Schäfer, Heiner Bucher, Daniel Trachsel, Jaques Schneider, Muriel Gambon, Diana Reppucci, Jessica Bonhoeffer, Jody Stähelin-Massik, Philipp Schuetz, Beat Mueller, Gabor Szinnai, and Urs Schaad. We also appreciate the efforts of the recruiters of the pTBred network: Mar Santos Sebastián, Marisa Navarro, Elena Rincón, Jesús Saavedra, David Aguilera, and the laboratory and biobank manager Andrea López Suarez. Special thanks go to the children and their parents for their essential participation in this study.
Declarations
Ethics approval and consent to participate
The studies involving human participants were reviewed and approved by the Ethikkommission Nordwestschweiz (ref: EKNZ 2016 − 01094) for the CITRUS study, by the Ethics Committee of Basel (ref: EKBB 369/08) for the ProPAED study, and by the Gregorio Marañón Ethics Committee (code 359/21) for the pTBred network. Written informed consent to participate in this study was provided by the legal guardian or next of kin of the participants.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Bei Menschen mit Typ-2-Diabetes sind die Chancen, einen Myokardinfarkt zu überleben, in den letzten 15 Jahren deutlich gestiegen – nicht jedoch bei Betroffenen mit Typ 1.
Ob Patienten und Patientinnen mit neu diagnostiziertem Blasenkrebs ein Jahr später Bedauern über die Therapieentscheidung empfinden, wird einer Studie aus England zufolge von der Radikalität und dem Erfolg des Eingriffs beeinflusst.
„Kalte“ Tumoren werden heiß – CD28-kostimulatorische Antikörper sollen dies ermöglichen. Am besten könnten diese in Kombination mit BiTEs und Checkpointhemmern wirken. Erste klinische Studien laufen bereits.
Eine perioperative Therapie mit Nivolumab reduziert das Risiko für Rezidive und Todesfälle bei operablem NSCLC im Vergleich zu einer alleinigen neoadjuvanten Chemotherapie um über 40%. Darauf deuten die Resultate der Phase-3-Studie CheckMate 77T.
Update Innere Medizin
Bestellen Sie unseren Fach-Newsletter und bleiben Sie gut informiert.