Detection of Mycobacterium tuberculosis Peptides in the Exosomes of Patients with Active and Latent M. tuberculosis Infection Using MRM-MS

The identification of easily measured, accurate diagnostic biomarkers for active tuberculosis (TB) will have a significant impact on global TB control efforts. Because of the host and pathogen complexities involved in TB pathogenesis, identifying a single biomarker that is adequately sensitive and specific continues to be a major hurdle. Our previous studies in models of TB demonstrated that exosomes, such as those released from infected macrophages, contain mycobacterial products, including many Mtb proteins. In this report, we describe the development of targeted proteomics assays employing multiplexed multiple reaction monitoring mass spectrometry (MRM-MS) in order to allow us to follow those proteins previously identified by western blot or shotgun mass spectrometry, and enhance biomarker discovery to include detection of Mtb proteins in human serum exosomes. Targeted MRM-MS assays were applied to exosomes isolated from human serum samples obtained from culture-confirmed active TB patients to detect 76 peptides representing 33 unique Mtb proteins. Our studies revealed the first identification of bacteria-derived biomarker candidates of active TB in exosomes from human serum. Twenty of the 33 proteins targeted for detection were found in the exosomes of TB patients, and included multiple peptides from 8 proteins (Antigen 85B, Antigen 85C, Apa, BfrB, GlcB, HspX, KatG, and Mpt64). Interestingly, all of these proteins are known mycobacterial adhesins and/or proteins that contribute to the intracellular survival of Mtb. These proteins will be included as target analytes in future validation studies as they may serve as markers for persistent active and latent Mtb infection. In summary, this work is the first step in identifying a unique and specific panel of Mtb peptide biomarkers encapsulated in exosomes and reveals complex biomarker patterns across a spectrum of TB disease states.     

Host Cytokine Responses Induced after Overnight Stimulation with Novel M. tuberculosis Infection Phase-Dependent Antigens Show Promise as Diagnostic Candidates for TB Disease

Background

We previously identified Mycobacterium tuberculosis (M.tb) antigen-induced host markers that showed promise as TB diagnostic candidates in 7-day whole blood culture supernatants. The aim of the present study was to evaluate the utility of these markers further, and cross-compare results with short-term antigen stimulated and unstimulated culture supernatants.

Methods

We recruited 15 culture confirmed TB cases and 15 non-TB cases from a high-TB endemic community in Cape Town, South Africa into a pilot case-control study from an on-going larger study. Blood samples collected from study participants were stimulated with 4 M.tb antigens that were previously identified as promising (ESAT6/CFP10 (early secreted), Rv2029c (latency), Rv2032 (latency) and Rv2389c (rpf)) in a 7-day or overnight culture assay. Supernatants were also collected form the standard QuantiFERON In Tube (QFT-IT) test. The levels of 26 host markers were evaluated in the three culture supernatants using the Luminex platform.

Results

The unstimulated levels of CRP, Serum amyloid P (SAP) and serum amyloid A (SAA) and ESAT-6/CFP-10 specific IP-10 and SAA were amongst the best discriminatory markers in all 3 assays, ascertaining TB with AUC of 72–84%. Four-marker models accurately classified up to 92%, 100% and 100% of study participants in the overnight, 7-day and Quantiferon culture supernatants, respectively, after leave-one-out cross validation.

Conclusion

Unstimulated and antigen-specific levels of CRP, SAA, IP-10, MMP-2 and sCD40L hold promise as diagnostic candidates for TB disease in short-term stimulation assays. Larger studies are required to validate these findings but the data suggest that antigen-specific cytokine production and in particular mutimarker biosignatures might contribute to future diagnostic strategies.     

Novel strategies to identify biomarkers in tuberculosis

The more we learn about the immune response against tuberculosis (TB) and particularly about the features which distinguish protective immunity, disease susceptibility and pathology, the better we can define biomarkers which correlate with these different stages of infection. The most widely used biomarker in TB, which without a doubt is an important component of protective immunity, is IFNgamma secreted by antigen-specific CD4 T-cells. However, the complexity of the immune response against TB makes it more than likely that additional biomarkers are required for a reliable correlate of protection. As a corollary, we assume that a set of biomarkers will be required, termed a biosignature.     

Identification of promising plasma immune biomarkers to differentiate active pulmonary tuberculosis

Although much research has been done related to biomarker discovery for tuberculosis infection, a set of biomarkers that can discriminate between active and latent TB diseases remains elusive. In the current study we correlate clinical aspects of TB disease with changes in the immune response as determined by biomarkers detected in plasma. Our study measured 18 molecules in human plasma in 17 patients with active disease (APTB), 14 individuals with latent tuberculosis infection (LTBI) and 16 uninfected controls (CTRL). We found that active tuberculosis patients have increased plasma levels of IL-6, IP-10, TNF-α, sCD163 and sCD14. Statistical analysis of these biomarkers indicated that simultaneous measurement of sCD14 and IL-6 was able to diagnose active tuberculosis infection with 83% accuracy. We also demonstrated that TNF-α and sCD163 were correlated with tuberculosis severity. We showed that the simultaneous detection of both plasma sCD14 and IL-6 is a promising diagnostic approach to identify APTB, and further, measurement of TNF-α and sCD163 can identify the most severe cases of tuberculosis.     

Highly accurate diagnosis of pleural tuberculosis by immunological analysis of the pleural effusion

Pleural TB is notoriously difficult to diagnose due to its paucibacillary nature yet it is the most common cause of pleural effusions in TB endemic countries such as The Gambia. We identified both cellular and soluble biomarkers in the pleural fluid that allowed highly accurate diagnosis of pleural TB compared to peripheral blood markers. Multi-plex cytokine analysis on unstimulated pleural fluid showed that IP-10 resulted in a positive likelihood ratio (LR) of 9.6 versus 2.8 for IFN-γ; a combination of IP-10, IL-6 and IL-10 resulted in an AUC of 0.96 and positive LR of 10. A striking finding was the significantly higher proportion of PPD-specific IFN-γ+TNF-α+ cell population (PPD-IGTA) in the pleural fluid compared to peripheral blood of TB subjects. Presence of this pleural PPD-IGTA population resulted in 95% correct classification of pleural TB disease with a sensitivity of 95% and specificity of 100%. These data suggest that analysis of the site of infection provides superior diagnostic accuracy compared to peripheral blood for pleural TB, likely due to the sequestration of effector cells at this acute stage of disease.     

Baseline Levels and Temporal Stability of 27 Multiplexed Serum Cytokine Concentrations in Healthy Subjects

BackgroundCytokines are humoral molecules that elicit regulatory function in immunologic pathways. The level and type of cytokine production has become critical in distinguishing physiologic from pathologic immune conditions. Cytokine profiling has become an important biomarker discovery tool in monitoring of the immune system. However, the variations in cytokine levels in individual subjects over time in healthy individuals have not been extensively studied. In this study, we use multiplex bead arrays to evaluate 27 analytes in paired serum samples taken seven days apart from 144 healthy individuals in order to assess variations over a short time period.MethodsFluorescent bead-based immunoassay (Luminex) was used to measure 27 analytes in serum samples. Measurements were performed on matched samples from 144 healthy donors. To assess inter-plate variability, one arbitrarily selected serum sample was analyzed on each of the first ten plates as bridge sample. ResultsUsing the bridge sample, we showed minimal inter-plate variations in the measurement of most analytes. In measurement of cytokines from the 144 patients at two time points, we found that three cytokines (IL-2, IL-15 and GM-CSF) were undetectable and five analytes (RANTES, MCP-1, VEGF, MIP-1β and PDGF-BB) showed significant difference in concentrations at Day 0 compared to Day 7. ConclusionsThe current study demonstrated higher variations in cytokine levels among individuals than were observed for samples obtained one week apart from identical donors. These data suggest that a serum sample from each subject for use as a baseline measurement is a better control for clinical trials rather than sera from a paired cohort.     

The efficacy of a comprehensive lifestyle modification programme based on yoga in the management of bronchial asthma: a randomized controlled trial

Background

Interferon gamma release assays, including the QuantiFERON^® TB Gold In Tube (QFT) have been shown to be accurate in diagnosing Mycobacterium tuberculosis infection. These assays however, do not discriminate between latent TB infection (LTBI) and active TB disease.

Methods

We recruited twenty-three pulmonary TB patients and 34 household contacts from Cape Town, South Africa and performed the QFT test. To investigate the ability of new host markers to differentiate between LTBI and active TB, levels of 29 biomarkers in QFT supernatants were evaluated using a Luminex multiplex cytokine assay.

Results

Eight out of 29 biomarkers distinguished active TB from LTBI in a pilot study. Baseline levels of epidermal growth factor (EGF) soluble CD40 ligand (sCD40L), antigen stimulated levels of EGF, and the background corrected antigen stimulated levels of EGF and macrophage inflammatory protein (MIP)-1β were the most informative single markers for differentiation between TB disease and LTBI, with AUCs of 0.88, 0.84, 0.87, 0.90 and 0.79 respectively. The combination of EGF and MIP-1β predicted 96% of active TB cases and 92% of LTBIs. Combinations between EGF, sCD40L, VEGF, TGF-α and IL-1α also showed potential to differentiate between TB infection states. EGF, VEGF, TGF-α and sCD40L levels were higher in TB patients.

Conclusion

These preliminary data suggest that active TB may be accurately differentiated from LTBI utilizing adaptations of the commercial QFT test that includes measurement of EGF, sCD40L, MIP-1β, VEGF, TGF-α or IL-1α in supernatants from QFT assays. This approach holds promise for development as a rapid diagnostic test for active TB.     

The assessment of host and bacterial proteins in sputum from active pulmonary tuberculosis

Pulmonary tuberculosis (TB) is caused by Mycobacterium tuberculosis. The protein composition of sputum may reflect the immune status of the lung. This study aimed to evaluate the protein profiles in spontaneous sputum samples from patients with active pulmonary TB. Sputum samples were collected from patients with pulmonary TB and healthy controls. Western blotting was used to analyze the amount of interleukin 10 (IL-10), interferon-gamma (IFN-γ), IL-25, IL-17, perforin-1, urease, albumin, transferrin, lactoferrin, adenosine deaminase (also known as adenosine aminohydrolase, or ADA), ADA-2, granzyme B, granulysin, and caspase-1 in sputum. Results of detection of IL-10, IFN-γ, perforin-1, urease, ADA2, and caspase-1, showed relatively high specificity in distinguishing patients with TB from healthy controls, although sensitivities varied from 13.3% to 66.1%. By defining a positive result as the detection of any two proteins in sputum samples, combined use of transferrin and urease as markers increased sensitivity to 73.2% and specificity to 71.1%. Furthermore, we observed that the concentration of transferrin was proportional to the number of acid-fast bacilli detected in sputum specimens. Detection of sputum transferrin and urease was highly associated with pulmonary TB infection. In addition, a high concentration of transferrin detected in sputum might correlate with active TB infection. This data on sputum proteins in patients with TB may aid in the development of biomarkers to assess the severity of pulmonary TB.     

Prognostic serum biomarkers in cancer patients with COVID-19: A systematic review

PurposeCancer patients with COVID-19 likely express biomarker changes in circulation. However, the biomarkers used in SARS-CoV-2 infected cancer patients for COVID-19 severity and prognosis are largely unclear. Therefore, this systematic review aims to determine what biomarkers were measured in cancer patients with COVID-19 and their prognostic utility.MethodsA systematic literature review in PubMed, Embase, and Scopus was performed on June 16th, 2021. The search keywords coronavirus, neoplasm, biomarkers, and disease progression were used to filter out 17 eligible studies, which were then carefully evaluated.ResultsA total of 4,168 patients, 16 types of cancer, and 60 biomarkers were included. Seven up-regulated markers, including CRP, d-dimer, ferritin, IL-2R, IL-6, LDH, and PCT, were identified in eligible studies. Albumin and hemoglobin were significantly down-regulated in cancer patients with COVID-19. Moreover, we observed that the SARS-CoV-2 infected cancer patients with lower CRP, ferritin, and LDH levels successfully survived from COVID-19 treatments.ConclusionSeveral important clinical biomarkers, such as CRP, ferritin, and LDH, may serve as the prognostic markers to predict the outcomes following COVID-19 treatment and monitor the deterioration of COVID-19 in cancer patients.     

Classification and characterisation of extracellular vesicles-related tuberculosis subgroups and immune cell profiles

Around the world, tuberculosis (TB) remains one of the most common causes of morbidity and mortality. The molecular mechanism of Mycobacterium tuberculosis (Mtb) infection is still unclear. Extracellular vesicles (EVs) play a key role in the onset and progression of many disease states and can serve as effective biomarkers or therapeutic targets for the identification and treatment of TB patients. We analysed the expression profile to better clarify the EVs characteristics of TB and explored potential diagnostic markers to distinguish TB from healthy control (HC). Twenty EVs-related differentially expressed genes (DEGs) were identified, and 17 EVs-related DEGs were up-regulated and three DEGs were down-regulated in TB samples, which were related to immune cells. Using machine learning, a nine EVs-related gene signature was identified and two EVs-related subclusters were defined. The single-cell RNA sequence (scRNA-seq) analysis further confirmed that these hub genes might play important roles in TB pathogenesis. The nine EVs-related hub genes had excellent diagnostic values and accurately estimated TB progression. TB's high-risk group had significantly enriched immune-related pathways, and there were substantial variations in immunity across different groups. Furthermore, five potential drugs were predicted for TB using CMap database. Based on the EVs-related gene signature, the TB risk model was established through a comprehensive analysis of different EV patterns, which can accurately predict TB. These genes could be used as novel biomarkers to distinguish TB from HC. These findings lay the foundation for further research and design of new therapeutic interventions aimed at treating this deadly infectious disease.     

Interleukin-10 (IL-10) gene polymorphism as a potential host susceptibility factor in tuberculosis

Several genes encoding for different cytokines may play crucial roles in host susceptibility to tuberculosis (TB), since the cytokine production capacity varies among individuals and depends on the cytokine gene polymorphism. The association of the cytokine gene polymorphisms with the development of TB was investigated in this study. DNA samples were obtained from a Turkish population of 81 patients with the different clinical forms of TB, and 50 healthy control subjects. All genotyping (IL-6, IL-10, IFN-gamma, TGF-beta and TNF-alpha) experiments were performed using sequence-specific primers PCR (PCR-SSP). Analysis of allele frequencies showed that IL-10 -1082 G allele frequency was significantly more common in TB patients than healthy controls (37.7% vs 23.0%, p: 0.014). No statistically significant differences were observed between the different clinical forms of the disease. These results suggest that the polymorphisms in IL-10 gene may affect susceptibility to TB and increase risk of developing the disease. To confirm the biological significance of our results, further studies should be performed on other population groups.     

C-Reactive Protein (CRP), Interferon Gamma-Inducible Protein 10 (IP-10), and Lipopolysaccharide (LPS) Are Associated with Risk of Tuberculosis after Initiation of Antiretroviral Therapy in Resource-Limited Settings

ObjectiveThe association between pre-antiretroviral (ART) inflammation and immune activation and risk for incident tuberculosis (TB) after ART initiation among adults is uncertain.DesignNested case-control study (n = 332) within ACTG PEARLS trial of three ART regimens among 1571 HIV-infected, treatment-naïve adults in 9 countries. We compared cases (participants with incident TB diagnosed by 96 weeks) to a random sample of controls (participants who did not develop TB, stratified by country and treatment arm).MethodsWe measured pre-ART C-reactive protein (CRP), EndoCab IgM, ferritin, interferon gamma (IFN-γ), interleukin 6 (IL-6), interferon gamma-inducible protein 10 (IP-10), lipopolysaccharide (LPS), soluble CD14 (sCD14), tumor necrosis factor alpha (TNF-α), and CD4/DR+/38+ and CD8/DR+/38+ T cells. Markers were defined according to established cutoff definitions when available, 75th percentile of measured values when not, and detectable versus undetectable for LPS. Using logistic regression, we measured associations between biomarkers and incident TB, adjusting for age, sex, study site, treatment arm, baseline CD4 and log10 viral load. We assessed the discriminatory value of biomarkers using receiver operating characteristic (ROC) analysis.ResultsSeventy-seven persons (4.9%) developed incident TB during follow-up. Elevated baseline CRP (aOR 3.25, 95% CI: 1.55–6.81) and IP-10 (aOR 1.89, 95% CI: 1.05–3.39), detectable plasma LPS (aOR 2.39, 95% CI: 1.13–5.06), and the established TB risk factors anemia and hypoalbuminemia were independently associated with incident TB. In ROC analysis, CRP, albumin, and LPS improved discrimination only modestly for TB risk when added to baseline routine patient characteristics including CD4 count, body mass index, and prior TB.ConclusionIncident TB occurs commonly after ART initiation. Although associated with higher post-ART TB risk, baseline CRP, IP-10, and LPS add limited value to routine patient characteristics in discriminating who develops active TB. Besides determining ideal cutoffs for these biomarkers, additional biomarkers should be sought that predict TB disease in ART initiators.     

Performance of Multiplex Cytokine Assays in Serum and Saliva among Community-Dwelling Postmenopausal Women

Multiplexing arrays increase the throughput and decrease sample requirements for studies employing multiple biomarkers. The goal of this project was to examine the performance of Multiplex arrays for measuring multiple protein biomarkers in saliva and serum. Specimens from the OsteoPerio ancillary study of the Women’s Health Initiative Observational Study were used. Participants required the presence of at least 6 teeth and were excluded based on active cancer and certain bone issues but were not selected on any specific condition. Quality control (QC) samples were created from pooled serum and saliva. Twenty protein markers were measured on five multiplexing array panels. Sample pretreatment conditions were optimized for each panel. Recovery, lower limit of quantification (LLOQ) and imprecision were determined for each analyte. Statistical adjustment at the plate level was used to reduce imprecision estimates and increase the number of usable observations. Sample pre-treatment improved recovery estimates for many analytes. The LLOQ for each analyte agreed with manufacturer specifications except for MMP-1 and MMP-2 which were significantly higher than reported. Following batch adjustment, 17 of 20 biomarkers in serum and 9 of 20 biomarkers in saliva demonstrated acceptable precision, defined as <20% coefficient of variation (<25% at LLOQ). The percentage of cohort samples having levels within the reportable range for each analyte varied from 10% to 100%. The ratio of levels in saliva to serum varied from 1∶100 to 28∶1. Correlations between saliva and serum were of moderate positive magnitude and significant for CRP, MMP-2, insulin, adiponectin, GM-CSF and IL-5. Multiplex arrays exhibit high levels of analytical imprecision, particularly at the batch level. Careful sample pre-treatment can enhance recovery and reduce imprecision. Following statistical adjustments to reduce batch effects, we identified biomarkers that are of acceptable quality in serum and to a lesser degree in saliva using Multiplex arrays.     

A Multi-Analyte Assay for the Non-Invasive Detection of Bladder Cancer

Accurate urinary assays for bladder cancer (BCa) detection would benefit both patients and healthcare systems. Through genomic and proteomic profiling of urine components, we have previously identified a panel of biomarkers that can outperform current urine-based biomarkers for the non-invasive detection of BCa. Herein, we report the diagnostic utility of various multivariate combinations of these biomarkers. We performed a case-controlled validation study in which voided urines from 127 patients (64 tumor bearing subjects) were analyzed. The urinary concentrations of 14 biomarkers (IL-8, MMP-9, MMP-10, SDC1, CCL18, PAI-1, CD44, VEGF, ANG, CA9, A1AT, OPN, PTX3, and APOE) were assessed by enzyme-linked immunosorbent assay (ELISA). Diagnostic performance of each biomarker and multivariate models were compared using receiver operating characteristic curves and the chi-square test. An 8-biomarker model achieved the most accurate BCa diagnosis (sensitivity 92%, specificity 97%), but a combination of 3 of the 8 biomarkers (IL-8, VEGF, and APOE) was also highly accurate (sensitivity 90%, specificity 97%). For comparison, the commercial BTA-Trak ELISA test achieved a sensitivity of 79% and a specificity of 83%, and voided urine cytology detected only 33% of BCa cases in the same cohort. These datashow that a multivariate urine-based assay can markedly improve the accuracy of non-invasive BCa detection. Further validation studies are under way to investigate the clinical utility of this panel of biomarkers for BCa diagnosis and disease monitoring.     

CXCL1: A new diagnostic biomarker for human tuberculosis discovered using Diversity Outbred mice

More humans have died of tuberculosis (TB) than any other infectious disease and millions still die each year. Experts advocate for blood-based, serum protein biomarkers to help diagnose TB, which afflicts millions of people in high-burden countries. However, the protein biomarker pipeline is small. Here, we used the Diversity Outbred (DO) mouse population to address this gap, identifying five protein biomarker candidates. One protein biomarker, serum CXCL1, met the World Health Organization’s Targeted Product Profile for a triage test to diagnose active TB from latent M.tb infection (LTBI), non-TB lung disease, and normal sera in HIV-negative, adults from South Africa and Vietnam. To find the biomarker candidates, we quantified seven immune cytokines and four inflammatory proteins corresponding to highly expressed genes unique to progressor DO mice. Next, we applied statistical and machine learning methods to the data, i.e., 11 proteins in lungs from 453 infected and 29 non-infected mice. After searching all combinations of five algorithms and 239 protein subsets, validating, and testing the findings on independent data, two combinations accurately diagnosed progressor DO mice: Logistic Regression using MMP8; and Gradient Tree Boosting using a panel of 4: CXCL1, CXCL2, TNF, IL-10. Of those five protein biomarker candidates, two (MMP8 and CXCL1) were crucial for classifying DO mice; were above the limit of detection in most human serum samples; and had not been widely assessed for diagnostic performance in humans before. In patient sera, CXCL1 exceeded the triage diagnostic test criteria (>90% sensitivity; >70% specificity), while MMP8 did not. Using Area Under the Curve analyses, CXCL1 averaged 94.5% sensitivity and 88.8% specificity for active pulmonary TB (ATB) vs LTBI; 90.9% sensitivity and 71.4% specificity for ATB vs non-TB; and 100.0% sensitivity and 98.4% specificity for ATB vs normal sera. Our findings overall show that the DO mouse population can discover diagnostic-quality, serum protein biomarkers of human TB.     

Evaluation of matricellular proteins in systemic and local immune response to Mycobacterium tuberculosis infection

Matricellular proteins such as osteopontin (OPN), galectin‐9 (Gal‐9), and tenascin‐C (TN‐C) are expressed not only under normal physiological conditions, but also during infection, inflammation and tumorigenesis. Plasma concentrations of matricellular proteins were studied to determine their diagnostic value as potential markers of tuberculosis (TB) activity. It was found that concentrations of OPN and TN‐C were higher in patients with active TB than in healthy controls and individuals with latent infection. Moreover, LTBI patients had higher concentrations of OPN than did healthy controls. Gal‐9 concentrations did not differ significantly between groups. Concentrations of matricellular proteins were higher in pleural fluid than in the plasma of patients with TB. Expression of matricellular proteins was also investigated in TB granulomas and other granulomatous diseases. Positive OPN and Gal‐9 staining was observed in TB and sarcoidosis granulomas, but not in Crohn disease granulomas. The fibrotic ring around granulomas stained positive for TN‐C in TB and sarcoidosis, but not in Crohn disease. Of the three matricellular proteins studied, OPN and TN‐C may serve as reliable plasma markers for monitoring TB activity, whereas Gal‐9 seems to be expressed more at the site of infection than in the systemic circulation.     

Salivary Biomarkers for Detection of Systemic Diseases

Background and Objective

Analysis of inflammatory biomarkers in saliva could offer an attractive opportunity for the diagnosis of different systemic conditions specifically in epidemiological surveys. The aim of this study was to investigate if certain salivary biomarkers could be used for detection of common systemic diseases.

Materials and Methods

A randomly selected sample of 1000 adults living in Skåne, a county in the southern part of Sweden, was invited to participate in a clinical study of oral health. 451 individuals were enrolled in this investigation, 51% women. All participants were asked to fill out a questionnaire, history was taken, a clinical examination was made and stimulated saliva samples were collected. Salivary concentrations of IL-1β, -6, -8, TNF-α, lysozyme, MMP-8 and TIMP-1 were determined using ELISA, IFMA or Luminex assays.

Results

Salivary IL-8 concentration was found to be twice as high in subjects who had experience of tumour diseases. In addition, IL-8 levels were also elevated in patients with bowel disease. MMP-8 levels were elevated in saliva from patients after cardiac surgery or suffering from diabetes, and muscle and joint diseases. The levels of IL-1β, IL-8 and MMP-8, as well as the MMP-8/TIMP-1 ratio were higher in subjects with muscle and joint diseases.

Conclusion

Biomarkers in saliva have the potential to be used for screening purposes in epidemiological studies. The relatively unspecific inflammatory markers used in this study can not be used for diagnosis of specific diseases but can be seen as markers for increased systemic inflammation.