An exploratory deep learning approach to investigate tuberculosis pathogenesis in nonhuman primate model: Combining automated radiological analysis with clinical and biomarkers data.

BACKGROUND: Tuberculosis (TB) kills approximately 1.6 million people yearly despite the fact anti-TB drugs are generally curative. Therefore, TB-case detection and monitoring of therapy, need a comprehensive approach. Automated radiological analysis, combined with clinical, microbiological, and immunological data, by machine learning (ML), can help achieve it. METHODS: Six rhesus macaques were experimentally inoculated with pathogenic Mycobacterium tuberculosis in the lung. Data, including Computed Tomography (CT), were collected at 0, 2, 4, 8, 12, 16, and 20 weeks. RESULTS: Our ML-based CT analysis (TB-Net) efficiently and accurately analyzed disease progression, performing better than standard deep learning model (LLM OpenAI's CLIP Vi4). TB-Net based results were more consistent than, and confirmed independently by, blinded manual disease scoring by two radiologists and exhibited strong correlations with blood biomarkers, TB-lesion volumes, and disease-signs during disease pathogenesis. CONCLUSION: The proposed approach is valuable in early disease detection, monitoring efficacy of therapy, and clinical decision making.

Comparing machine learning screening approaches using clinical data and cytokine profiles for COVID-19 in resource-limited and resource-abundant settings.

Accurate screening of COVID-19 infection status for symptomatic patients is a critical public health task. Although molecular and antigen tests now exist for COVID-19, in resource-limited settings, screening tests are often not available. Furthermore, during the early stages of the pandemic tests were not available in any capacity. We utilized an automated machine learning (ML) approach to train and evaluate thousands of models on a clinical dataset consisting of commonly available clinical and laboratory data, along with cytokine profiles for patients (n = 150). These models were then further tested for generalizability on an out-of-sample secondary dataset (n = 120). We were able to develop a ML model for rapid and reliable screening of patients as COVID-19 positive or negative using three approaches: commonly available clinical and laboratory data, a cytokine profile, and a combination of the common data and cytokine profile. Of the tens of thousands of models automatically tested for the three approaches, all three approaches demonstrated > 92% sensitivity and > 88 specificity while our highest performing model achieved 95.6% sensitivity and 98.1% specificity. These models represent a potential effective deployable solution for COVID-19 status classification for symptomatic patients in resource-limited settings and provide proof-of-concept for rapid development of screening tools for novel emerging infectious diseases.

Complementary and alternative medicines and liver disease.

Complementary and alternative medicines (CAM) include conventional medical treatments. Patients worldwide use CAM at alarming rates; thus, reports of CAM-related DILI have been on the rise. The clinical presentations include asymptomatic liver test abnormalities, acute hepatitis with or without jaundice, acute cholestatic liver disease (bland or with hepatitis), acute liver failure, severe hepatitis with features of portal hypertension, and acute decompensation of known or unknown cirrhosis that can lead to acute-on-chronic liver failure. Acute hepatitis with or without necrosis, hepatocellular and canalicular cholestasis, herb-induced or CAM-triggered autoimmune hepatitis, granulomatous hepatitis, severe steatohepatitis, and vanishing bile duct syndrome are common liver biopsy findings in CAM-DILI. The presence of preexisting liver disease predicts severe liver injury, risk of progression to liver failure, and decreased transplant-free survival in patients with CAM-DILI. This review discusses global epidemiology and trends in CAM-DILI, clinical presentation, assessment and outcomes, commonly emerging threats in the context of hepatotoxic herbs, pragmatic assessment of "liver beneficial" herbs and health care myths, patient communication, regulatory framework, and future directions on research in CAM.

Citizens protein project: A self-funded, transparent, and concerning report on analysis of popular protein supplements sold in the Indian market.

Protein powders, including those containing herbal and dietary supplements such as vitamins, minerals, and other natural or synthetic ingredients, can be associated with hepatotoxicity. Protein supplements are often mislabeled and deceptive in their contents. In this self-funded transparent study, we extensively analyzed popular protein supplements in India to identify potential hepatotoxic substances based on industrial standards. All products underwent extensive analysis, including total protein content, fungal aflatoxin detection, pesticide residue estimation, heavy metal quantification, steroid detection, and complete organic and inorganic profiling, according to industry standards. Most protein supplements did not meet the labeled and advertised protein content, while certain brands surpassed the stated levels, raising concerns about potential "protein/amino-spiking." In addition, the major brands contained detectable fungal toxins and pesticide residues. Furthermore, many major formulations contained harmful heavy metals such as lead and arsenic, and some featured hepatoxic herbal extracts, particularly green tea extract, turmeric, Garcinia cambogia, and Ashwagandha. Indian-made products were inferior to those manufactured by multinational companies. The presence of various potentially toxic compounds, such as cycloheptatriene, benzene derivatives, toluene, and isopropyl alcohol, within a nonstandardized and unregulated diverse ingredient mix added to the overall concern. We demonstrate that the protein-based herbal and dietary supplement industry requires stringent scrutiny, regulation, and basic safety studies before being marketed. Manufacturers must consider reducing "ingredient complexities" of their protein powders to prevent adverse interactions between herbal and nonherbal components in consumers. Manufacturers must avoid using known toxic ingredients to reduce the avoidable disease burden within the public community.

BCG as an Innovative Option for HCC Treatment: Repurposing and Mechanistic Insights.

This study investigates Bacillus Calmette-Guérin (BCG) as a potential treatment for hepatocellular carcinoma (HCC), a condition often associated with unfavorable treatment outcomes. Exploiting BCG's recognized immune-boosting properties, preclinical trials are conducted using HCC mice, with a single subcutaneous dose of BCG administered post-tumor formation. Results indicate that BCG treatment effectively diminishes tumor burden and extends survival in both male and female HCC mice. Positive influences on hepatic fibrosis and metabolism are observed, leading to a reduction in lipid levels. Spatial analysis underscores BCG's tumor-specific effects, inducing the enrichment of metabolic pathways and inhibiting various cancer-related pathways. Furthermore, BCG promotes immune cell infiltration, including CD4+, CD8+ T cells, and M1 macrophages, in both v-akt murine thymoma viral oncogene homolog 1(AKT)/neutoblastoma RAS viral oncogene homolog (RAS) and ß-catenin positive HCC models. Interestingly, blocking T cells, trained immunity, and Interferon-γ (IFN-γ) function reverses BCG's anti-HCC effects. In conclusion, BCG emerges as a promising treatment option for HCC, characterized by a favorable safety profile and efficacy in inhibiting fibrosis, improving metabolism, and engaging both trained immunity and T cells in therapeutic mechanisms.

Dynamics of temporal immune responses in nonhuman primates and humans immunized with COVID-19 vaccines.

We assessed the humoral immune responses to a COVID-19 vaccine in a well-controlled rhesus macaque model compared to humans immunized with two mRNA vaccines over several months post-second dose. The plasma IgG levels against seven coronaviruses (including SARS-CoV-2) and antibody subtypes (IgG 1-4 and IgM) against SARS-CoV-2 were evaluated using multiplex assays. The neutralization capacity of plasma antibodies against the original SAR-CoV-2 isolate and nine variants was evaluated in vaccinated humans and non-human primates. Immunization of macaques and humans with SARS-CoV-2 vaccines induced a robust neutralizing antibody response. In non-SIV-infected adult macaques immunized with an adenoviral vector expressing S-RBD (n = 7) or N protein (n = 3), elevated levels of IgG and neutralizing antibodies were detected 2 weeks post-second dose. Immune responses to the S-RBD vaccine in SIV-infected adult macaques (n = 2) were similar to the non-SIV-infected animals. Adult humans immunized with Pfizer (n = 35) or Moderna (n = 18) vaccines developed IgG and neutralizing antibodies at 4 weeks post-second dose. In both vaccine groups, IgG 1 was the predominant subtype, followed by IgG 3. The IgG levels, including total and IgG 1,2,3 elicited by the Moderna vaccine, were significantly higher than the corresponding levels elicited by the Pfizer vaccine at 4 weeks post-second dose. A significant correlation was observed between the plasma total IgG antibody levels and neutralization titers in both macaques and humans. Furthermore, broad-spectrum neutralization antibodies against several variants of SARS-CoV-2 were detected in the plasma of both macaques and humans after two vaccinations.

Lipid Mediators and Cytokines/Chemokines Display Differential Profiles in Severe versus Mild/Moderate COVID-19 Patients.

Host immune responses play a key role in COVID-19 pathogenesis. The underlying phenomena are orchestrated by signaling molecules such as cytokines/chemokines and lipid mediators. These immune molecules, including anti-SARS-CoV-2 antibodies, interact with immune cells and regulate host responses, contributing to inflammation that drives the disease. We investigated 48 plasma cytokines/chemokines, 21 lipid mediators, and anti-S protein (RBD) antibodies in COVID-19 patients (n = 56) and non-COVID-19 respiratory disease controls (n = 49), to identify immune-biomarker profiles. Cytokines/chemokines (IL-6, CXCL-10 (IP-10), HGF, MIG, MCP-1, and G-CSF) and lipid mediators (TxB2, 11-HETE, 9-HODE, 13-HODE, 5-HETE, 12-HETE, 15-HETE, 14S-HDHA, 17S-HDHA, and 5-oxo ETE) were significantly elevated in COVID-19 patients compared to controls. In patients exhibiting severe disease, pro-inflammatory cytokines/chemokines (IL-6, CXCL-10, and HGF) and anti-SARS-CoV-2 antibodies were significantly elevated. In contrast, lipid mediators involved in the reduction/resolution of inflammation, in particular, 5-HETE, 11-HETE, and 5-oxoETE, were significantly elevated in mild/moderate disease. Taken together, these immune-biomarker profiles provide insight into immune responses related to COVID-19 pathogenesis. Importantly, our findings suggest that elevation in plasma concentrations of IL-6, CXCL-10, HGF, and anti-SARS-CoV-2 antibodies can predict severe disease, whereas elevation in lipid mediators peaks early (compared to cytokines) and includes induction of mechanisms leading to reduction of inflammation, associated complications, and maintenance of homeostasis.

Ashwagandha-induced liver injury-A case series from India and literature review.

BACKGROUND: Ashwagandha herb is commonly used in Ayurveda and a "fad" dietary supplement for a host of indications based on low levels of evidence. Recently, ashwagandha was implicated in multiple reports of herb-induced liver injury (HILI), mainly from the United States. We present the first, and currently largest, series of ashwagandha-HILI from multiple centers in India. METHODS: We retrospectively analyzed the respective institutional electronic medical records for ashwagandha-HILI. Patients consuming ashwagandha as part of multiherbal formulations or along with other known hepatotoxic supplements or medicines were excluded. All patients underwent a detailed diagnostic workup to exclude competing causes reasonably. Where possible, the implicated herbal formulation was retrieved and subjected to chemical analysis. RESULTS: Out of 23 patients with liver injury from ashwagandha (January 2019 to December 2022), we report 8 patients with single-ingredient formulation-related HILI. Study cohort was male predominant, and cholestatic hepatitis was the commonest presentation. Five patients had underlying chronic liver disease; 3 presented with acute-on-chronic liver failure, and all 3 died on follow-up. In others, the liver injury was prolonged, nonetheless self-limiting. Liver biopsy revealed cholestatic features predominantly with hepatocellular necrosis and lymphocyte/eosinophil predominant portal-based inflammation. One patient progressed to chronic HILI. Chemical analysis revealed only natural phytochemicals without adulteration or contamination. CONCLUSIONS: Ashwagandha-HILI presents with cholestatic hepatitis and can lead to the syndrome of acute-on-chronic liver failure with high mortality in those with pre-existing liver disease. Educating the public on avoiding the use of potentially toxic and unrecommended herbal supplements can help mitigate the avoidable liver disease burden in the community.

Toxic hepatitis-associated aplastic anaemia after dual homeopathic remedies and Gymnema sylvestre use.

Hepatitis-associated aplastic anaemia (HAAA) is a rare condition characterised by onset of acute hepatitis which is followed by development of severe pancytopenia due to bone marrow failure within 6 months. This syndrome can be precipitated by acute viral infections, but the aetiology remains unknown in the majority. Drug-induced HAAA is extremely rare and has been reported with nutritional and dietary supplements in current literature. We report the first cases of ayurvedic herbal and homeopathic remedies-associated HAAA in two patients which proved fatal in both. Evaluation of patients with acute hepatitis and severe pancytopenia must include a detailed evaluation for complementary and alternative medicine use.

Peripheral γδ T Cells Regulate Neutrophil Expansion and Recruitment in Experimental Psoriatic Arthritis.

OBJECTIVE: This study was undertaken to identify the mechanistic role of γδ T cells in the pathogenesis of experimental psoriatic arthritis (PsA). METHODS: In this study, we performed interleukin-23 (IL-23) gene transfer in wild-type (WT) and T cell receptor δ-deficient (TCRδ-/- ) mice and conducted tissue phenotyping in the joint, skin, and nails to characterize the inflammatory infiltrate. We further performed detailed flow cytometry, immunofluorescence staining, RNA sequencing, T cell repertoire analysis, and in vitro T cell polarization assays to identify regulatory mechanisms of γδ T cells. RESULTS: We demonstrated that γδ T cells support systemic granulopoiesis, which is critical for murine PsA-like pathology. Briefly, γδ T cell ablation inhibited the expression of neutrophil chemokines CXCL1 and CXCL2 and neutrophil CD11b+Ly6G+ accumulation in the aforementioned PsA-related tissues. Although significantly reduced expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-17A was detected systemically in TCRδ-/- mice, no GM-CSF+/IL-17A+ γδ T cells were detected locally in the inflamed skin or bone marrow in WT mice. Our data showed that nonresident γδ T cells regulate the expansion of an CD11b+Ly6G+ neutrophil population and their recruitment to joint and skin tissues, where they develop hallmark pathologic features of human PsA. CONCLUSION: Our findings do not support the notion that tissue-resident γδ T cells initiate the disease but demonstrate a novel role of γδ T cells in neutrophil regulation that can be exploited therapeutically in PsA patients.

Automated machine learning for endemic active tuberculosis prediction from multiplex serological data.

Serological diagnosis of active tuberculosis (TB) is enhanced by detection of multiple antibodies due to variable immune responses among patients. Clinical interpretation of these complex datasets requires development of suitable algorithms, a time consuming and tedious undertaking addressed by the automated machine learning platform MILO (Machine Intelligence Learning Optimizer). MILO seamlessly integrates data processing, feature selection, model training, and model validation to simultaneously generate and evaluate thousands of models. These models were then further tested for generalizability on out-of-sample secondary and tertiary datasets. Out of 31 antigens evaluated, a 23-antigen model was the most robust on both the secondary dataset (TB vs healthy) and the tertiary dataset (TB vs COPD) with sensitivity of 90.5% and respective specificities of 100.0% and 74.6%. MILO represents a user-friendly, end-to-end solution for automated generation and deployment of optimized models, ideal for applications where rapid clinical implementation is critical such as emerging infectious diseases.

Immune response dynamics in COVID-19 patients to SARS-CoV-2 and other human coronaviruses.

COVID-19 serological test must have high sensitivity as well as specificity to rule out cross-reactivity with common coronaviruses (HCoVs). We have developed a quantitative multiplex test, measuring antibodies against spike (S) proteins of SARS-CoV-2, SARS-CoV, MERS-CoV, and common human coronavirus strains (229E, NL63, OC43, HKU1), and nucleocapsid (N) protein of SARS-CoV viruses. Receptor binding domain of S protein of SARS-CoV-2 (S-RBD), and N protein, demonstrated sensitivity (94% and 92.5%, respectively) in COVID-19 patients (n = 53), with 98% specificity in non-COVID-19 respiratory-disease (n = 98), and healthy-controls (n = 129). Anti S-RBD and N antibodies appeared five to ten days post-onset of symptoms, peaking at approximately four weeks. The appearance of IgG and IgM coincided while IgG subtypes, IgG1 and IgG3 appeared soon after the total IgG; IgG2 and IgG4 remained undetectable. Several inflammatory cytokines/chemokines were found to be elevated in many COVID-19 patients (e.g., Eotaxin, Gro-α, CXCL-10 (IP-10), RANTES (CCL5), IL-2Rα, MCP-1, and SCGF-b); CXCL-10 was elevated in all. In contrast to antibody titers, levels of CXCL-10 decreased with the improvement in patient health suggesting it as a candidate for disease resolution. Importantly, anti-N antibodies appear before S-RBD and differentiate between vaccinated and infected people-current vaccines (and several in the pipeline) are S protein-based.

Plasma Linoleate Diols Are Potential Biomarkers for Severe COVID-19 Infections.

Polyunsaturated fatty acids are metabolized into regulatory lipids important for initiating inflammatory responses in the event of disease or injury and for signaling the resolution of inflammation and return to homeostasis. The epoxides of linoleic acid (leukotoxins) regulate skin barrier function, perivascular and alveolar permeability and have been associated with poor outcomes in burn patients and in sepsis. It was later reported that blocking metabolism of leukotoxins into the vicinal diols ameliorated the deleterious effects of leukotoxins, suggesting that the leukotoxin diols are contributing to the toxicity. During quantitative profiling of fatty acid chemical mediators (eicosanoids) in COVID-19 patients, we found increases in the regioisomeric leukotoxin diols in plasma samples of hospitalized patients suffering from severe pulmonary involvement. In rodents these leukotoxin diols cause dramatic vascular permeability and are associated with acute adult respiratory like symptoms. Thus, pathways involved in the biosynthesis and degradation of these regulatory lipids should be investigated in larger biomarker studies to determine their significance in COVID-19 disease. In addition, incorporating diols in plasma multi-omics of patients could illuminate the COVID-19 pathological signature along with other lipid mediators and blood chemistry.

Gut microbiome dysbiosis and correlation with blood biomarkers in active-tuberculosis in endemic setting.

Tuberculosis (TB) is the largest infectious disease with 10 million new active-TB patients and1.7 million deaths per year. Active-TB is an inflammatory disease and is increasingly viewed as an imbalance of immune responses to M. tb. infection. The mechanisms of a switch from latent infection to active disease is not well worked out but a shift in the immune responses is thought to be responsible. Increasingly, the role of gut microbiota has been described as a major influencer of the immune system. And because the gut is the largest immune organ, we aimed to analyze the gut microbiome in active-TB patients in a TB-endemic country, Pakistan. The study revealed that Ruminococcacea, Enetrobactericeae, Erysipelotrichaceae, Bifidobacterium, etc. were the major genera associated with active-TB, also associated with chronic inflammatory disease. Plasma antibody profiles against several M. tb. antigens, as specific biomarkers for active-TB, correlated closely with the patient gut microbial profiles. Besides, bcoA gene copy number, indicative of the level of butyrate production by the gut microbiome was five-fold lower in TB patients compared to healthy individuals. These findings suggest that gut health in TB patients is compromised, with implications for disease morbidity (e.g., severe weight loss) as well as immune impairment.

Evaluation of multi-antigen serological screening for active tuberculosis among people living with HIV.

Better triage tests for screening tuberculosis (TB) disease are needed for people living with HIV (PLHIV). We performed the first evaluation of a previously-validated 8-antigen serological panel to screen PLHIV for pulmonary TB in Kampala, Uganda. We selected a random 1:1 sample with and without TB (defined by sputum culture) from a cohort of PLHIV initiating antiretroviral therapy. We used a multiplex microbead immunoassay and an ensemble machine learning classifier to determine the area under the receiver operating characteristic curve (AUC) for Ag85A, Ag85B, Ag85C, Rv0934-P38, Rv3881, Rv3841-BfrB, Rv3873, and Rv2878c. We then assessed the performance with the addition of four TB-specific antigens ESAT-6, CFP-10, Rv1980-MPT64, and Rv2031-HSPX, and every antigen combination. Of 262 participants (median CD4 cell-count 152 cells/µL [IQR 65-279]), 138 (53%) had culture-confirmed TB. The 8-antigen panel had an AUC of 0.53 (95% CI 0.40-0.66), and the additional 4 antigens did not improve performance (AUC 0.51, 95% CI 0.39-0.64). When sensitivity was restricted to ≥90% for the 8- and 12-antigen panel, specificity was 2.2% (95% CI 0-17.7%) and 8.1% (95% CI 0-23.9%), respectively. A three-antigen combination (Rv0934-P38, Ag85A, and Rv2031-HSPX) outperformed both panels, with an AUC of 0.60 (95% CI 0.48-0.73), 90% sensitivity (95% CI 78.2-96.7%) and 29.7% specificity (95% CI 15.9-47%). The multi-antigen panels did not achieve the target accuracy for a TB triage test among PLHIV. We identified a new combination that improved performance for TB screening in an HIV-positive sample compared to an existing serological panel in Uganda, and suggests an approach to identify novel antigen combinations specifically for screening TB in PLHIV.

Diagnostic performance of blood inflammatory markers for tuberculosis screening in people living with HIV.

BACKGROUND: Approaches to screening for active tuberculosis (TB) among people living with HIV are inadequate, leading to missed diagnoses and poor implementation of preventive therapy. METHODS: Consecutive HIV-infected adults hospitalized at Mulago Hospital (Kampala, Uganda) between June 2011 and July 2013 with a cough ≥ 2 weeks were enrolled. Patients underwent extensive evaluation for pulmonary TB. Concentrations of 43 cytokines/chemokines were measured at the same time point as C-reactive protein (CRP) in banked plasma samples using commercially-available multiplex kits. Advanced classification algorithms were used to rank cytokines/chemokines for their ability to identify TB, and to model the specificity of the top-ranked cytokines/chemokines individually and in combination with sensitivity constrained to ≥ 90% as recommended for TB screening. RESULTS: The median plasma level of 5 biomarkers (IL-6, INF-γ, MIG, CRP, IL-18) was significantly different between patients with and without TB. With sensitivity constrained to 90%, all had low specificity with IL-6 showing the highest specificity (44%; 95% CI 37.4-49.5). Biomarker panels were found to be more valuable than any biomarker alone. A panel combining IFN-γ and IL-6 had the highest specificity (50%; 95% CI 46.7-53.3). Sensitivity remained high (>85%) for all panels among sputum smear-negative TB patients. CONCLUSIONS: Direct measurement of unstimulated plasma cytokines/chemokines in peripheral blood is a promising approach to TB screening. Cytokine/chemokine panels retained high sensitivity for smear-negative TB and achieved improved specificity compared to individual cytokines/chemokines. These markers should be further evaluated in outpatient settings where most TB screening occurs and where other illnesses associated with systematic inflammation are less common.

Evaluation of antibody responses to panels of M. tuberculosis antigens as a screening tool for active tuberculosis in Uganda.

BACKGROUND: Improved systematic screening of high-risk groups is a key component of the tuberculosis (TB) elimination strategy endorsed by the World Health Organization (WHO). We used a multiplex microbead immunoassay to measure antibody responses to 28 M. tuberculosis (M.tb) antigens, and assessed whether combinations of antibody responses achieve accuracy thresholds required for a TB screening test. METHODS: A random selection of plasma samples obtained from consecutive HIV-negative adults who were admitted to Mulago Hospital in Kampala, Uganda with cough ≥2 weeks' but <6 months' duration were analyzed for serological response to 28 M.tb antigens using an in-house multiplex microbead immunoassay. We compared the median difference of the antibody response to each antigen between patients with and without culture-confirmed TB, ranked each antigen according to variable importance (VIM), and assessed the sensitivity and specificity of combinations of antibody responses using an advanced classification algorithm, SuperLearner. RESULTS: Among the 237 patients included in the analysis, 119 (50%) were female, median age was 32 years (IQR 25, 46), and 113 (48%) had TB. Median antibody levels to eight antigens were significantly different between patients with and without TB. A panel including eight of the top ranked antigens had a sensitivity of 90.6% (95% CI 89.4, 93.8) and a specificity of 88.6% (95% CI 78.2, 97.6) (Ag85B, Ag85A, Ag85C, Rv0934-P38, Rv3881, BfrB, Rv3873, and Rv2878c). With sensitivity constrained to be >90%, specificity remained close to 70% with as few as 3 antigens included in the panels. CONCLUSIONS: Measuring antibody responses to combinations of antigens could facilitate TB screening and should be further evaluated in populations being targeted for systematic screening.

Field evaluation of a blood based test for active tuberculosis in endemic settings.

Over 9 million new active tuberculosis (TB) cases emerge each year from an enormous pool of 2 billion individuals latently infected with Mycobacterium tuberculosis (M. tb.) worldwide. About 3 million new TB cases per year are unaccounted for, and 1.5 million die. TB, however, is generally curable if diagnosed correctly and in a timely manner. The current diagnostic methods for TB, including state-of-the-art molecular tests, have failed in delivering the capacity needed in endemic countries to curtail this ongoing pandemic. Efficient, cost effective and scalable diagnostic approaches are critically needed. We report a multiplex TB serology panel using microbead suspension array containing a combination of 11 M.tb. antigens that demonstrated overall sensitivity of 91% in serum/plasma samples from TB patients confirmed by culture. Group wise sensitivities for sputum smear positive and negative patients were 95%, and 88%, respectively. Specificity of the test was 96% in untreated COPD patients and 91% in general healthy population. The sensitivity of this test is superior to that of the frontline sputum smear test with a comparable specificity (30-70%, and 93-99%, respectively). The multiplex serology test can be performed with scalability from 1 to 360 patients per day, and is amenable to automation for higher (1000s per day) throughput, thus enabling a scalable clinical work flow model for TB endemic countries. Taken together, the above results suggest that well defined antibody profiles in blood, analyzed by an appropriate technology platform, offer a valuable approach to TB diagnostics in endemic countries.

Development of immune-biomarkers of pulmonary tuberculosis in a rabbit model.

Tuberculosis (TB) causes extensive morbidity and mortality worldwide with approximately 10 million new cases of active disease emerging mostly from a pool of two billion individuals latently infected with Mycobacterium tuberculosis (M. tb) every year. The underlying host immune responses that drive M. tb infection to active disease or latency are not well understood. We propose that identification and characterization of host immune biomarkers will be helpful to better understand the mechanisms that drive this process, and may, in addition, lead to the development of better diagnostic tools for TB. We have previously reported the profiles of plasma immune biomarkers in pulmonary TB patients in endemic countries, and in M. tb-infected nonhuman primates. However, biomarker profiling for a cost-effective and user-friendly animal model relevant to human disease, such as rabbit, has not been developed. One challenge in the analysis of circulating cytokines/chemokines for rabbit model of TB is the limited availability of validated immune-reagents. Here we report the use of a commercially available multiplex microbead human cytokine/chemokine panels as development platform for rabbit immune reagents. The results demonstrate their utility to determine circulating analytes and define their profiles related to TB in the rabbit model. In addition, we report the profiles of circulating anti-M. tb antibodies in the plasma of rabbits with active pulmonary TB. These studies show that the pattern of expression of circulating immune biomarkers correlate with TB pathology in rabbits, and are similar to those defined in pulmonary TB patients.

Gender biased immune-biomarkers in active tuberculosis and correlation of their profiles to efficacy of therapy.

Active pulmonary TB is an inflammatory disease and is increasingly viewed as an imbalance of immune responses to Mycobacterium tuberculosis (M. tb.) infection. In addition, this immune imbalance may be gender biased (males have a higher prevalence of TB) but reasons for such bias are uncertain. We hypothesized that studies on profiles of immune-biomarkers will not only provide insight into molecular basis of gender bias but may also help identify biomarkers to monitor efficacy of TB therapy. We examined 10 plasma cytokine/chemokine/growth-factor and 8 antibody (against 8 M. tb. antigens) biomarkers (elevated in TB patients) by multiplex microbead immunoassays. In addition, we examined these biomarkers in patients under anti-tuberculosis therapy (ATT). The results showed that female patients contained significantly higher levels of CXCL9 (MIG) and CXCL10 (IP-10), while males contained higher levels of PDGF-BB. In contrast, more males than females contained antibodies against several antigens. Our results also show that there are progressive and substantial decreases in plasma levels of CXCL9, CXCL10, PDGF-BB, IFNγ, and IL-18, correlating with treatment success. Our results suggest that studies on gender bias in immunebiomarkers will enhance understanding of host responses in TB and would be valuable as biomarkers for monitoring efficacy of ATT.