Diagnostic performance of host protein signatures as a triage test for active pulmonary TB.

The current four-symptom screen recommended by the World Health Organization (WHO) is widely used as screen to initiate diagnostic testing for active pulmonary tuberculosis (TB), yet the performance is poor especially when TB prevalence is low. In contrast, more sensitive molecular tests are less suitable for placement at primary care level in low-resource settings. In order to meet the WHO End TB targets, new diagnostic approaches are urgently needed to find the missing undiagnosed cases. Proteomics-derived blood host biomarkers have been explored because protein detection technologies are suitable for the point-of-care setting and could meet cost targets. This study aimed to find a biomarker signature that fulfills WHO's target product profile (TPP) for a TB screening. Twelve blood-based protein biomarkers from three sample populations (Vietnam, Peru, and South Africa) were analyzed individually and in combinations via advanced statistical methods and machine learning algorithms. The combination of I-309, SYWC and kallistatin showed the most promising results to discern active TB throughout the data sets meeting the TPP for a triage test in adults from two countries (Peru and South Africa). The top-performing individual markers identified at the global level (I-309 and SYWC) were also among the best-performing markers at country level in South Africa and Vietnam. This analysis clearly shows that a host protein biomarker assay is feasible in adults for certain geographical regions based on one or two biomarkers with a performance that meets minimal WHO TPP criteria.

Serum biomarkers for monitoring response to tuberculosis treatment: an assessment of the effect of different covariates among slow and fast treatment responders.

INTRODUCTION: The long duration of tuberculosis treatment, as well as the 2-year post-treatment follow-up period often required for predicting relapse, present a hindrance to drug development and treatment monitoring efforts. Therefore, there is need for treatment response biomarkers to inform treatment time shortening, clinical decision-making, and inform clinical trials. OBJECTIVES: To assess the abilities of serum host biomarkers to predict treatment response among active PTB patients. METHODS: Active pulmonary TB patients (n = 53) as confirmed by sputum MGIT culture were enrolled at a TB treatment centre in Kampala, Uganda. We evaluated concentrations of 27 serum host biomarkers at baseline, month 2, and month 6 following the initiation of anti-tuberculosis treatment using the luminex platform for their ability to predict sputum culture status at month-2 post treatment initiation. RESULTS: There were significant differences in concentrations of IL1ra, IL1ß, IL6, IP10, MCP-1, and IFNγ during treatment. A bio-signature comprising TTP, TNFα, PDGF-BB, IL9, and GCSF best predicted month 2 culture conversion with sensitivity and specificity of 82% (95% CI; 66 -92% and 57 -96% respectively). Slow anti-TB treatment responders had higher pro-inflammatory marker levels during treatment. The strongest correlation was observed between VEGF and IL12p70 (0.94), IL17A and basic FGF (0.92), basic FGF, and IL2 (0.88), and IL10 with IL17A (0.87). CONCLUSION: We identified host biomarkers that predicted early response to PTB treatment, which may be valuable in future clinical trials and treatment monitoring. Similarly, strong correlations between biomarkers provide options for biomarkers substitutions during the development of treatment response monitoring tools or point of care tests.

Field evaluation of a point-of-care triage test for active tuberculosis (TriageTB).

BACKGROUND: To improve tuberculosis (TB) diagnosis, the World Health Organisation (WHO) has called for a non-sputum based triage test to focus TB testing on people with a high likelihood of having active pulmonary tuberculosis (TB). Various host or pathogen biomarker-based testing devices are in design stage and require validity assessment. Host biomarkers have shown promise to accurately rule out active TB, but further research is required to determine generalisability. The TriageTB diagnostic test study aims to assess the accuracy of diagnostic test candidates, as well as field-test, finalise the design and biomarker signature, and validate a point-of-care multi-biomarker test (MBT). METHODS: This observational diagnostic study will evaluate sensitivity and specificity of biomarker-based diagnostic candidates including the MBT and Xpert® TB Fingerstick cartridge compared with a gold-standard composite TB outcome classification defined by symptoms, sputum GeneXpert® Ultra, smear and culture, radiological features, response to TB therapy and presence of an alternative diagnosis. The study will be conducted in research sites in South Africa, Uganda, The Gambia and Vietnam which all have high TB prevalence. The two-phase design allows for finalisation of the MBT in Phase 1 in which candidate host proteins will be evaluated on stored serum from Asia, South Africa and South America and on fingerstick blood from 50 newly recruited participants per site. The MBT test will then be locked down and validated in Phase 2 on 250 participants per site. DISCUSSION: By targeting confirmatory TB testing to those with a positive triage test, 75% of negative GXPU may be avoided, thereby reducing diagnostic costs and patient losses during the care cascade. This study builds on previous biomarker research and aims to identify a point-of-care test meeting or exceeding the minimum World Health Organisation target product profile of a 90% sensitivity and 70% specificity. Streamlining TB testing by identifying individuals with a high likelihood of TB should improve TB resources use and, in so doing, improve TB care. TRIAL REGISTRATION: NCT04232618 (clinicaltrials.gov) Date of registration: 16 January 2020.

The Intestinal Commensal, Bacteroides fragilis, Modulates Host Responses to Viral Infection and Therapy: Lessons for Exploration during Mycobacterium tuberculosis Infection.

The gut microbiota has emerged as a critical player in host health. Bacteroides fragilis is a prominent member of the gut microbiota within the phyla Bacteroidetes. This commensal bacterium produces unique capsular polysaccharides processed by antigen-presenting cells and activates CD4+ T cells to secrete inflammatory cytokines. Indeed, due to their immunomodulatory functions, B. fragilis and its capsular polysaccharide-A (PSA) are arguably the most explored single commensal microbiota/symbiotic factor. B. fragilis/PSA has been shown to protect against colitis, encephalomyelitis, colorectal cancer, pulmonary inflammation, and asthma. Here, we review recent data on the immunomodulatory role of B. fragilis/PSA during viral infections and therapy, B. fragilis PSA's dual ability to mediate pro-and anti-inflammatory processes, and the potential for exploring this unique characteristic during intracellular bacterial infections such as with Mycobacterium tuberculosis. We also discuss the protective roles of single commensal-derived probiotic species, including B. fragilis in lung inflammation and respiratory infections that may provide essential cues for possible exploration of microbiota based/augmented therapies in tuberculosis (TB). Available data on the relationship between B. fragilis/PSA, the immune system, and disease suggest clinical relevance for developing B. fragilis into a next-generation probiotic or, possibly, the engineering of PSA into a potent carbohydrate-based vaccine.

Evaluation of host biomarkers for monitoring treatment response in spinal tuberculosis: A 12-month cohort study.

BACKGROUND: Monitoring treatment response is an important precaution in spinal tuberculosis (TB), particularly when the condition was clinically diagnosed rather than bacteriologically confirmed and when drug susceptibility testing was not performed. Conventional monitoring measures have limitations and there is a need for favourable alternatives. Therefore, this study aimed to investigate changes in immune biomarkers over the course of treatment for spinal TB and to compare these responses to the conventional monitoring measure, erythrocyte sedimentation rate (ESR). METHODS: Patients with spinal TB were recruited from a tertiary hospital in the Western Cape, South Africa, and provided blood samples at 0, 3, 6, 9 and 12 months of TB treatment. Blood samples were analysed for ESR, using standard techniques, and for 19 cytokines, using a multiplex platform. Changes in ESR and cytokine levels were investigated using a mixed model ANOVA and Least Significant Difference post-hoc testing. RESULTS: Twenty-six patients with spinal TB were included in the study although only fifteen remained in follow-up at 12 months. Seven biomarkers changed significantly over the course of treatment (CRP, Fibrinogen, IFN-γ, Ferritin, VEGF-A, ApoA1 and NCAM, p < 0.01) with a further three showing a strong trend towards change (CCL1, CXCL9 and GDF-15, 0.05 ≥ p ≤ 0.06). Responsive biomarkers could be approximately grouped according to patterns of progressive, initial or delayed change. ESR performed similarly to CRP, Fibrinogen and IFN-γ with all showing significant decreases between 0, 6 and 12- months of treatment. Individual ESR responses were variable. DISCUSSION: Individual ESR responses may be unreliable and support the investigation of multi-marker approaches to evaluating treatment response in spinal TB. Biomarkers of treatment response identified in the current study require validation in a larger study, which may also incorporate aspects such as evaluating biomarkers within the first week of treatment and the inclusion of a healthy control group.

Validation of host cerebrospinal fluid protein biomarkers for early diagnosis of tuberculous meningitis in children: a replication and new biosignature discovery study.

Purpose: The diagnosis of tuberculous meningitis (TBM) in children is often delayed due to diagnostic difficulties. New tools are urgently needed to improve the diagnosis of the disease in this vulnerable group. The present study aimed to validate the accuracy of recently identified host cerebrospinal (CSF) biomarkers as candidates for the diagnosis of TBM in children.Materials and methods: We collected CSF samples from 87 children aged 3 months to 13 years, that were consecutively admitted at a tertiary hospital in Cape Town, South Africa, on suspicion of having TBM. We evaluated the concentrations of 67 selected host protein biomarkers using a multiplex platform.Results: Previously identified 3-marker (VEGF-A + IFN-γ + MPO) and 4-marker (IFN-γ + MPO + ICAM-1 + IL-8) signatures diagnosed TBM with AUCs of 0.89 (95% CI, 0.81-0.97) and 0.87 (95% CI, 0.79-0.95) respectively; sensitivities of 80.6% (95% CI, 62.5-92.5%) and 81.6% (95% CI, 65.7-92.3%), and specificities of 86.8% (71.9-95.6%) and 83.7% (70.4-92.7%) respectively. Furthermore, a new combination between the analytes (CC4b + CC4 + procalcitonin + CCL1) showed promise, with an AUC of 0.98 (95% CI, 0.94-1.00).Conclusions: We have shown that the accuracies of previously identified candidate CSF biomarkers for childhood TBM was reproducible. Our findings augur well for the future development of a simple bedside test for the rapid diagnosis of TBM in children.

Tuberculous Meningitis: Pathogenesis, Immune Responses, Diagnostic Challenges, and the Potential of Biomarker-Based Approaches.

Tuberculous meningitis (TBM) is the most devastating form of tuberculosis (TB), causing high mortality or disability. Clinical management of the disease is challenging due to limitations of the existing diagnostic approaches. Our knowledge on the immunology and pathogenesis of the disease is currently limited. More research is urgently needed to enhance our understanding of the immunopathogenesis of the disease and guide us toward the identification of targets that may be useful for vaccines or host-directed therapeutics. In this review, we summarize the current knowledge about the immunology and pathogenesis of TBM and summarize the literature on existing and new, especially biomarker-based, approaches that may be useful in the management of TBM. We identify research gaps and provide directions for research which may lead to the development of new tools for the control of the disease in the near future.

Serum cytokine levels associated with myocardial injury in systemic lupus erythematosus.

OBJECTIVES: To identify cytokines, markers of endothelial activation [soluble vascular cell adhesion molecule-1 (sVCAM-1)] and myocyte strain [soluble ST2 (sST2)] associated with myocardial injury (MInj) in SLE, classified by cardiac magnetic resonance (CMR) criteria. METHODS: CMR was performed on patients with SLE, identifying stages of MInj (inflammation and necrosis or fibrosis). Data captured included: clinical assessment, laboratory and serological analyses, cytokine (IL-1ß, IL-1Ra, IL-2, IL-6, IL-10, IL-17, IL-18, TNF-alpha), sVCAM-1 and sST2 levels. Cytokines were compared with regard to SLE features and evidence of CMR MInj. Predictors of CMR MInj were determined through regression analyses. RESULTS: Forty-one patients with high disease activity (SLEDAI-2K: 13; IQR: 3-17) were included. SLE features included: LN (n = 12), neurolupus (n = 6) and clinical lupus myocarditis (LM) (n = 6). Nineteen patients had CMR evidence of MInj. Patients with a SLEDAI-2K ≥ 12 had higher sVCAM-1 (P = 0.010) and sST2 (P = 0.032) levels. Neurolupus was associated with higher IL-1Ra (P = 0.038) and LN with lower IL-1Ra (P = 0.025) and sVCAM-1 (P = 0.036) levels. Higher IL-1Ra (P = 0.012), IL-17 (P = 0.045), IL-18 (P = 0.003), and sVCAM-1 (P = 0.062) levels were observed in patients with CMR MInj compared with those without. On multivariable logistic regression, IL-1Ra predicted CMR inflammation and fibrosis/necrosis (P < 0.005) while anti-Ro/SSA [odds ratio (OR): 1.197; P = 0.035] and the SLE damage index (OR: 4.064; P = 0.011) predicted fibrosis/necrosis. CONCLUSION: This is a novel description of associations between cytokines and SLE MInj. IL-18 and IL-1Ra were significantly higher in patients with MInj. IL-1Ra independently predicted different stages of CMR MInj. Exploration of the role of these cytokines in the pathogenesis of SLE MInj may promote targeted therapies for LM.

The gut microbiome in tuberculosis susceptibility and treatment response: guilty or not guilty?

Although tuberculosis (TB) is a curable disease, it remains the foremost cause of death from a single pathogen. Globally, approximately 1.6 million people died of TB in 2017. Many predisposing factors related to host immunity, genetics and the environment have been linked to TB. However, recent evidence suggests a relationship between dysbiosis in the gut microbiome and TB disease development. The underlying mechanism(s) whereby dysbiosis in the gut microbiota may impact the different stages in TB disease progression, are, however, not fully explained. In the wake of recently emerging literature, the gut microbiome could represent a potential modifiable host factor to improve TB immunity and treatment response. Herein, we summarize early data detailing (1) possible association between gut microbiome dysbiosis and TB (2) the potential for the use of microbiota biosignatures to discriminate active TB disease from healthy individuals (3) the adverse effect of protracted anti-TB antibiotics treatment on gut microbiota balance, and possible link to increased susceptibility to Mycobacterium tuberculosis re-infection or TB recrudescence following successful cure. We also discuss immune pathways whereby the gut microbiome could impact TB disease and serve as target for clinical manipulation.

Application of Cerebrospinal Fluid Host Protein Biosignatures in the Diagnosis of Tuberculous Meningitis in Children from a High Burden Setting.

BACKGROUND: The diagnosis of tuberculous meningitis (TBM) especially in children is challenging. New tests are urgently needed for the diagnosis of the disease, especially in resource-limited settings. METHODS: We collected cerebrospinal fluid (CSF) samples from children presenting with symptoms requiring investigation for meningitis at a tertiary hospital in Cape Town, South Africa. Children were later classified as TBM or no TBM using published case definitions. Using a multiplex platform, we investigated the concentrations of biomarkers comprising a previously established 3-marker biosignature (VEGF, IL-13, and LL-37) and other potentially useful host biomarkers as diagnostic candidates for TBM. FINDINGS: Out of 47 children, age, 3 months to 13 years, 23 were diagnosed with TBM and six (16%) were HIV-infected. We validated the previously identified CSF biosignature (sensitivity of 95.7% (95% CI, 79.0-99.2%) and specificity of 37.5% (95% CI, 21.2-57.3%)). However, substitution of IL-13 and LL-37 with IFN-γ and MPO, respectively, resulted in improved accuracy (area under the ROC curve (AUC) = 0.97, 95% CI, 0.92-1.00, up to 91.3% (21/23) sensitivity and up to 100% (24/24) specificity). An alternative four-marker biosignature (sICAM-1, MPO, CXCL8, and IFN-γ) also showed potential, with an AUC of 0.97. CONCLUSION: We validated a previously identified CSF biosignature and showed that refinement of this biosignature by incorporation of other biomarkers diagnosed TBM with high accuracy. Incorporation of these biomarkers into a point-of-care or bedside diagnostic test platform may result in the improved management of TBM in children.

Suitability of saliva for Tuberculosis diagnosis: comparing with serum.

BACKGROUND: In the search for fast, simple and better ways for diagnosis of tuberculosis (TB), there is need to discover and evaluate new biomarkers that are found in samples other than sputum to determine their effectiveness. This study examined the utility of saliva vis-a-vis serum by evaluating levels of biomarkers found in saliva and serum from TB suspects. METHODS: Study enrolled tuberculosis suspects. Sputum MGIT was used as the gold standard for active TB. Quantiferon gold-In tube assay was done to identify exposure to Mycobacterium tuberculosis (M.tb). Multiplex assay was run for 10 markers using a 10 plex customized kit from Bio-Rad Laboratories. RESULTS: There was a significant difference between saliva and serum marker levels. Saliva had significantly higher levels of GM-CSF and VEGF. Serum had higher levels of MIP-1a, b, TNF-a, G-CSF and IFN-g. Serum levels of IL-6, VEGF and TNF-a were significantly different between participants with active TB disease and those with other respiratory diseases. CONCLUSION: Salivary TB biomarkers are worth the search to evaluate their ability to differentiate between TB disease states for generation of a non invasive point of care test for TB diagnosis.

Centella asiatica enhances hepatic antioxidant status and regulates hepatic inflammatory cytokines in type 2 diabetic rats.

CONTEXT: Neutralizing the over-activation of oxidative stress and inflammation remains an important goal in the management of type 2 diabetes mellitus (T2DM). Centella asiatica (L.) Urban (Apiaceae) (CA) has been used in traditional folklore in Africa and Asia to treat various ailments including diabetes. OBJECTIVE: We investigated the hepatic antioxidant and anti-inflammatory potential of methanol extract of CA leaves in T2DM. MATERIALS AND METHODS: T2DM was induced in male Sprague-Dawley rats with 10% fructose in drinking water for 14 days followed by a single intraperitoneal injection of streptozotocin (40 mg/kg b.wt). Hepatic oxidant/antioxidant status was assessed by measuring the concentrations of malondialdehyde (MDA), ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC), Trolox equivalent antioxidant capacity (TEAC), reduced glutathione (GSH) and activities of glutathione S-transferase (GST) and glutathione peroxidase (GPX). The concentrations of cytokines IL-1ß, IL-4, IL-6, IL-10, MCP-1 and TNF-α in the liver were determined. RESULTS: Diabetes increased MDA formed (47%) and reduced FRAP (20%), TEAC (15%), GSH levels (32%), significantly; decreased GST and GPX activities in the liver and elevated levels of cytokines studied. Treatment of diabetic rats with 500 mg/kg b.wt CA for 14 days decreased MDA (44%); elevated FRAP (15%) and GSH (131%) levels and increased the activities of GST and GPX by 16%. Hepatic concentrations of IL-1ß, MCP-1 and TNF-α in DCA group were reduced to 68%, 75% and 63% of DC values, respectively. CONCLUSIONS: The antioxidant and anti-inflammatory properties of CA may protect tissues such as the liver from diabetes-induced oxidative damage.

Diagnostic performance of a seven-marker serum protein biosignature for the diagnosis of active TB disease in African primary healthcare clinic attendees with signs and symptoms suggestive of TB.

BACKGROUND: User-friendly, rapid, inexpensive yet accurate TB diagnostic tools are urgently needed at points of care in resource-limited settings. We investigated host biomarkers detected in serum samples obtained from adults with signs and symptoms suggestive of TB at primary healthcare clinics in five African countries (Malawi, Namibia, South Africa, The Gambia and Uganda), for the diagnosis of TB disease. METHODS: We prospectively enrolled individuals presenting with symptoms warranting investigation for pulmonary TB, prior to assessment for TB disease. We evaluated 22 host protein biomarkers in stored serum samples using a multiplex cytokine platform. Using a pre-established diagnostic algorithm comprising of laboratory, clinical and radiological findings, participants were classified as either definite TB, probable TB, questionable TB status or non-pulmonary TB. RESULTS: Of the 716 participants enrolled, 185 were definite and 29 were probable TB cases, 6 had questionable TB disease status, whereas 487 had no evidence of TB. A seven-marker biosignature of C reactive protein, transthyretin, IFN-γ, complement factor H, apolipoprotein-A1, inducible protein 10 and serum amyloid A identified on a training sample set (n=491), diagnosed TB disease in the test set (n=210) with sensitivity of 93.8% (95% CI 84.0% to 98.0%), specificity of 73.3% (95% CI 65.2% to 80.1%), and positive and negative predictive values of 60.6% (95% CI 50.3% to 70.1%) and 96.4% (95% CI 90.5% to 98.8%), respectively, regardless of HIV infection status or study site. CONCLUSIONS: We have identified a seven-marker host serum protein biosignature for the diagnosis of TB disease irrespective of HIV infection status or ethnicity in Africa. These results hold promise for the development of a field-friendly point-of-care screening test for pulmonary TB.

Host biomarkers detected in saliva show promise as markers for the diagnosis of pulmonary tuberculosis disease and monitoring of the response to tuberculosis treatment.

BACKGROUND: There is an urgent need for new tools for the rapid diagnosis of tuberculosis (TB) disease in resource-constrained settings. Tests based on host immunological biomarkers maybe useful, especially if based on easily available samples. We investigated host biomarkers detected in saliva samples from individuals with suspected pulmonary TB disease, as tools for the diagnosis of TB disease and monitoring of the response to treatment. METHODS: We collected saliva samples from 104 individuals that presented with symptoms requiring investigation for TB disease at a primary health care clinic in the outskirts of Cape Town, South Africa, prior to assessment for TB disease. We evaluated the concentrations of 33 host markers in stored saliva samples using a multiplex cytokine platform. Using a combination of clinical, radiological and laboratory results and a pre-established diagnostic algorithm, participants were later classified as having TB disease or other respiratory diseases (ORD). The diagnostic potentials of individual analytes were analysed by the receiver operator characteristics curve approach while the predictive abilities of combinations of analytes for TB disease were analysed by general discriminant analysis, with leave-one-out cross validation. RESULTS: Of the 104 individuals enrolled, 32 were pulmonary TB cases. There were significant differences in the levels of 10 of the markers investigated between the patients with TB disease and those with ORDs. However, the optimal diagnostic biosignature was a seven-marker combination of salivary CRP, ferritin, serum amyloid P, MCP-1, alpha-2-macroglobulin, fibrinogen and tissue plasminogen activator. This biosignature diagnosed TB disease with a sensitivity of 78.1% (95% CI, 59.6-90.1%) and specificity of 83.3% (95% CI, 72.3-90.7%) after leave-one-out cross validation. When compared to baseline levels, the concentrations of 9 markers including granzyme A, MCP-1, IL-1ß, IL-9, IL-10, IL-15, MIP-1ß, ferritin and serum amyloid A changed significantly by months 2 or 6 after initiation of TB treatment, thereby indicating that they might be useful in monitoring the response to TB treatment. CONCLUSION: We have identified candidate biomarkers in saliva, which may be useful in the diagnosis of TB disease and monitoring of the response to TB treatment. These results require further validation in larger studies.

Host immune response to tuberculous meningitis.

BACKGROUND: Tuberculous meningitis (TBM) is a severe complication of tuberculosis predominantly affecting young children. Early treatment is vital to prevent morbidity and mortality, emphasizing the importance of early diagnosis. The lack of sensitive methods for early diagnosis is the most common cause of delay. Attempts have been made to develop simplified tests for tuberculosis, but their diagnostic power remains poor. The clinical picture of TBM is mainly driven by the host's immune response to Mycobacterium tuberculosis; therefore, identification of disease-specific biomarkers may have diagnostic and therapeutic value and improve our understanding of its pathogenesis. METHODS: We investigated disease-specific biomarkers of childhood TBM in a cohort of children aged 3 months-13 years with symptoms and signs suggestive of meningitis. Cerebrospinal fluid (CSF) and serum from 56 patients with and 55 patients without TBM were assessed for 28 soluble mediators. RESULTS: Unsupervised hierarchical clustering analysis revealed a disease-specific pattern of biomarkers for TBM relative to other types of meningitis. A biomarker-based diagnostic prediction model for childhood TBM based on CSF concentrations of interleukin 13 (cutoff value, 37.26 pg/mL), vascular endothelial growth factor (cutoff value, 42.92 pg/mL), and cathelicidin LL-37 (cutoff value, 3221.01 pg/mL) is presented with a sensitivity of 0.52 and a specificity of 0.95. CONCLUSIONS: These data highlight the potential of biosignatures in the host's CSF for diagnostic applications and for improving our understanding of the pathogenesis of TBM to discover strategies to prevent immunopathological sequelae.

A Subgroup of Latently Mycobacterium tuberculosis Infected Individuals Is Characterized by Consistently Elevated IgA Responses to Several Mycobacterial Antigens.

Elevated antibody responses to Mycobacterium tuberculosis antigens in individuals with latent infection (LTBI) have previously been linked to an increased risk for progression to active disease. Studies in the field focussed mainly on IgG antibodies. In the present study, IgA and/or IgG responses to the mycobacterial protein antigens AlaDH, NarL, 19 kDa, PstS3, and MPT83 were determined in a blinded fashion in sera from 53 LTBI controls, 14 healthy controls, and 42 active TB subjects. Among controls, we found that elevated IgA levels against all investigated antigens were not randomly distributed but concentrated on a subgroup of <30%-with particular high levels in a small subgroup of ~5% comprising one progressor to active TB. Based on a specificity of 100%, anti-NarL IgA antibodies achieved with 78.6% sensitivity the highest accuracy for the detection of active TB compared to healthy controls. In conclusion, the consistently elevated IgA levels in a subgroup of controls suggest higher mycobacterial load, a risk factor for progression to active TB, and together with high IgG levels may have prognostic potential and should be investigated in future large scale studies. The novel antigen NarL may also be promising for the antibody-based diagnosis of active TB cases.

Beyond the IFN-γ horizon: biomarkers for immunodiagnosis of infection with Mycobacterium tuberculosis.

Latent infection with Mycobacterium tuberculosis (LTBI) is defined by the presence of M. tuberculosis-specific immunity in the absence of active tuberculosis. LTBI is detected using interferon-γ release assays (IGRAs) or the tuberculin-skin-test (TST). In clinical practice, IGRAs and the TSTs have failed to distinguish between active tuberculosis and LTBI and their predictive value to identify individuals at risk for the future development of tuberculosis is limited. There is an urgent need to identify biomarkers that improve the clinical performance of current immunodiagnostic methods for tuberculosis prevention, diagnosis and treatment monitoring. Here, we review the landscape of potential alternative biomarkers useful for detection of infection with M. tuberculosis. We describe what individual markers add in terms of specificity for active/latent infection, prediction of progression to active tuberculosis and immunodiagnostic potential in high-risk groups' such as HIV-infected individuals and children.

Bifunctional T-cell-derived cytokines for the diagnosis of tuberculosis and treatment monitoring.

BACKGROUND: Diagnosis and treatment monitoring of patients with tuberculosis remain challenging. OBJECTIVE: We have evaluated whether Mycobacterium-specific interferon (IFN)-γ and interleukin (IL)-2 bifunctional cytokine immune response assays improve the diagnosis of and correlate to treatment response in pulmonary tuberculosis. METHODS: Early secretory antigenic target (ESAT)6/culture filtrate protein 10 (CFP10), microsomal triglyceride transfer protein 65 (MTP65) and the purified protein derivative (PPD) tuberculin-specific immune profiles were investigated in peripheral blood mononuclear cells from 19 patients with culture-confirmed tuberculosis and 23 healthy community controls (HCCs; 82.6% with latent M. tuberculosis infection) using a novel fluorescence-based dual-colour enzyme-linked immunospot (EliSpot) technology (FluoroSpot). RESULTS: The frequency of ESAT6/CFP10-induced IFN-γ+IL-2- producing cells was elevated (p < 0.001), whereas the percentages of specific IFN-γ-IL-2+ (p = 0.002) and IFN-γ+IL-2+ double producing cells (p = 0.037) were diminished in tuberculosis patients in comparison to HCCs. A 3-host marker model using a combination of those IFN-γ and IL-2 single-cell responses showed 93.8% sensitivity and 77.8% specificity for tuberculosis. During tuberculosis treatment, the PPD-induced immune responses shifted from an IFN-γ+IL-2- dominated profile towards a balance of IFN-γ-IL-2+ and IFN-γ+IL-2+ double producing cells (all p ≤ 0.05). CONCLUSIONS: The addition of antigen-specific IL-2 production to IFN-γ responses by EliSpot in IFN-γ release assays increases diagnostic sensitivity for active tuberculosis.

Baseline and end-of-treatment host serum biomarkers predict relapse in adults with pulmonary tuberculosis.

BACKGROUND: There is a need for new tools for monitoring of the response to TB treatment. Such tools may allow for tailored treatment regimens, and stratify patients initiating TB treatment into different risk groups. We evaluated combinations between previously published host biomarkers and new candidates, as tools for monitoring TB treatment response, and prediction of relapse. METHODS: Serum samples were collected at multiple time points, from patients initiating TB treatment at research sites situated in South Africa (ActionTB study), Brazil and Uganda (TBRU study). Using a multiplex immunoassay platform, we evaluated the concentrations of selected host inflammatory biomarkers in sera obtained from clinically cured patients with and without subsequent relapse within 2 years of TB treatment completion. RESULTS: A total of 130 TB patients, 30 (23%) of whom had confirmed relapse were included in the study. The median time to relapse was 9.7 months in the ActionTB study (n = 12 patients who relapsed), and 5 months (n = 18 patients who relapsed) in the TBRU study. Serum concentrations of several host biomarkers changed during TB treatment with IL-6, IP-10, IL-22 and complement C3 showing potential individually, in predicting relapse. A six-marker signature comprising of TTP, BMI, sICAM-1, IL-22, IL-1ß and complement C3, predicted relapse, prior to the onset of TB treatment with 89% sensitivity and 94% specificity. Furthermore, a 3-marker signature (Apo-CIII, IP-10 and sIL-6R) predicted relapse in samples collected at the end of TB treatment with sensitivity of 71% and specificity of 74%. A previously identified baseline relapse prediction signature (TTP, BMI, TNF-ß, sIL-6R, IL-12p40 and IP-10) also showed potential in the current study. CONCLUSION: Serum host inflammatory biomarkers may be useful in predicting relapse in TB patients prior to the initiation of treatment. Our findings have implications for tailored patient management and require prospective evaluation in larger studies.

Differential expression of host biomarkers in saliva and serum samples from individuals with suspected pulmonary tuberculosis.

The diagnosis of tuberculosis remains challenging in individuals with difficulty in providing good quality sputum samples such as children. Host biosignatures of inflammatory markers may be valuable in such cases, especially if they are based on more easily obtainable samples such as saliva. To explore the potential of saliva as an alternative sample in tuberculosis diagnostic/biomarker investigations, we evaluated the levels of 33 host markers in saliva samples from individuals presenting with pulmonary tuberculosis symptoms and compared them to those obtained in serum. Of the 38 individuals included in the study, tuberculosis disease was confirmed in 11 (28.9%) by sputum culture. In both the tuberculosis cases and noncases, the levels of most markers were above the minimum detectable limit in both sample types, but there was no consistent pattern regarding the ratio of markers in serum/saliva. Fractalkine, IL-17, IL-6, IL-9, MIP-1 ß , CRP, VEGF, and IL-5 levels in saliva and IL-6, IL-2, SAP, and SAA levels in serum were significantly higher in tuberculosis patients (P < 0.05). These preliminary data indicate that there are significant differences in the levels of host markers expressed in saliva in comparison to those expressed in serum and that inflammatory markers in both sample types are potential diagnostic candidates for tuberculosis disease.