Sensitive electrochemiluminescence (ECL) immunoassays for detecting lipoarabinomannan (LAM) and ESAT-6 in urine and serum from tuberculosis patients.

BackgroundTuberculosis (TB) infection was responsible for an estimated 1.3 million deaths in 2017. Better diagnostic tools are urgently needed. We sought to determine whether accurate TB antigen detection in blood or urine has the potential to meet the WHO target product profiles for detection of active TB.Materials and methodsWe developed Electrochemiluminescence (ECL) immunoassays for Lipoarabinomannan (LAM) and ESAT-6 detection with detection limits in the pg/ml range and used them to compare the concentrations of the two antigens in the urine and serum of 81 HIV-negative and -positive individuals with presumptive TB enrolled across diverse geographic sites.ResultsLAM and ESAT-6 overall sensitivities in urine were 93% and 65% respectively. LAM and ESAT-6 overall sensitivities in serum were 55% and 46% respectively. Overall specificity was ≥97% in all assays. Sensitivities were higher in HIV-positive compared to HIV-negative patients for both antigens and both sample types, with signals roughly 10-fold higher on average in urine than in serum. The two antigens showed similar concentration ranges within the same sample type and correlated.ConclusionsLAM and ESAT-6 can be detected in the urine and serum of TB patients, regardless of the HIV status and further gains in clinical sensitivity may be achievable through assay and reagent optimization. Accuracy in urine was higher with current methods and has the potential to meet the WHO accuracy target if the findings can be transferred to a point-of-care TB test

A Novel Sensitive Immunoassay Targeting the 5-Methylthio-d-Xylofuranose-Lipoarabinomannan Epitope Meets the WHO's Performance Target for Tuberculosis Diagnosis.

The only currently commercialized point-of-care assay for tuberculosis (TB) that measures lipoarabinomannan (LAM) in urine (Alere LF-LAM) has insufficient sensitivity. We evaluated the potential of 100 novel monoclonal antibody pairs targeting a variety of LAM epitopes on a sensitive electrochemiluminescence platform to improve the diagnostic accuracy. In the screening, many antibody pairs showed high reactivity to purified LAM but performed poorly at detecting urinary LAM in clinical samples, suggesting differences in antigen structure and immunoreactivity of the different LAM sources. The 12 best antibody pairs from the screening were tested in a retrospective case-control study with urine samples from 75 adults with presumptive TB. The best antibody pair reached femtomolar analytical sensitivity for LAM detection and an overall clinical sensitivity of 93% (confidence interval [CI], 80% to 97%) and specificity of 97% (CI, 85% to 100%). Importantly, in HIV-negative subjects positive for TB by sputum smear microscopy, the test achieved a sensitivity of 80% (CI, 55% to 93%). This compares to an overall sensitivity of 33% (CI, 20% to 48%) of the Alere LF-LAM and a sensitivity of 13% (CI, 4% to 38%) in HIV-negative subjects in the same sample set. The capture antibody targets a unique 5-methylthio-d-xylofuranose (MTX)-dependent epitope in LAM that is specific to the Mycobacterium tuberculosis complex and shows no cross-reactivity with fast-growing mycobacteria or other bacteria. The present study provides evidence that improved assay methods and reagents lead to increased diagnostic accuracy. The results of this work have informed the development of a sensitive and specific novel LAM point-of-care assay with the aim to meet the WHO's performance target for TB diagnosis

Exclusion from Social Relations in Later Life : Micro- and Macro-Level Patterns and Correlations in a European Perspective

Older adults face particular risks of exclusion from social relationships (ESR) and are especially vulnerable to its consequences. However, research so far has been limited to specific dimensions, countries, and time points. In this paper, we examine the prevalence and micro- and macro-level predictors of ESR among older adults (60+) using two waves of data obtained four years apart across 14 European countries in the Survey of Health, Ageing and Retirement in Europe (SHARE). We consider four ESR indicators (household composition, social networks, social opportunities, and loneliness) and link them to micro-level (age, gender, socioeconomic factors, health, and family responsibilities) and national macro-level factors (social expenditures, unmet health needs, individualism, social trust, and institutional trust). Findings reveal a northwest to southeast gradient, with the lowest rates of ESR in the stronger welfare states of Northwest Europe. The high rates of ESR in the southeast are especially pronounced among women. Predictably, higher age and fewer personal resources (socioeconomic factors and health) increase the risk of all ESR dimensions for both genders. Macro-level factors show significant associations with ESR beyond the effect of micro-level factors, suggesting that national policies and cultural and structural characteristics may play a role in fostering sociability and connectivity and, thus, reduce the risk of ESR in later life

Application of multiplexed ion mobility spectrometry towards the identification of host protein signatures of treatment effect in pulmonary tuberculosis.

RationaleThe monitoring of TB treatments in clinical practice and clinical trials relies on traditional sputum-based culture status indicators at specific time points. Accurate, predictive, blood-based protein markers would provide a simpler and more informative view of patient health and response to treatment.ObjectiveWe utilized sensitive, high throughput multiplexed ion mobility-mass spectrometry (IM-MS) to characterize the serum proteome of TB patients at the start of and at 8 weeks of rifamycin-based treatment. We sought to identify treatment specific signatures within patients as well as correlate the proteome signatures to various clinical markers of treatment efficacy.MethodsSerum samples were collected from 289 subjects enrolled in CDC TB Trials Consortium Study 29 at time of enrollment and at the end of the intensive phase (after 40 doses of TB treatment). Serum proteins were immunoaffinity-depleted of high abundant components, digested to peptides and analyzed for data acquisition utilizing a unique liquid chromatography IM-MS platform (LC-IM-MS). Linear mixed models were utilized to identify serum protein changes in the host response to antibiotic treatment as well as correlations with culture status end points.ResultsA total of 10,137 peptides corresponding to 872 proteins were identified, quantified, and used for statistical analysis across the longitudinal patient cohort. In response to TB treatment, 244 proteins were significantly altered. Pathway/network comparisons helped visualize the interconnected proteins, identifying up regulated (lipid transport, coagulation cascade, endopeptidase activity) and down regulated (acute phase) processes and pathways in addition to other cross regulated networks (inflammation, cell adhesion, extracellular matrix). Detection of possible lung injury serum proteins such as HPSE, significantly downregulated upon treatment. Analyses of microbiologic data over time identified a core set of serum proteins (TTHY, AFAM, CRP, RET4, SAA1, PGRP2) which change in response to treatment and also strongly correlate with culture status. A similar set of proteins at baseline were found to be predictive of week 6 and 8 culture status.ConclusionA comprehensive host serum protein dataset reflective of TB treatment effect is defined. A repeating set of serum proteins (TTHY, AFAM, CRP, RET4, SAA1, PGRP2, among others) were found to change significantly in response to treatment, to strongly correlate with culture status, and at baseline to be predictive of future culture conversion. If validated in cohorts with long term follow-up to capture failure and relapse of TB, these protein markers could be developed for monitoring of treatment in clinical trials and in patient care

Patients with severe COVID-19 do not have elevated autoantibodies against common diagnostic autoantigens

Infection by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative pathogen of coronavirus disease 2019 (COVID-19) presents occasionally with an aberrant autoinflammatory response, including the presence of elevated circulating autoantibodies in some individuals. Whether the development of autoantibodies against self-antigens affects COVID-19 outcomes remains unclear. To better understand the prognostic role of autoantibodies in COVID-19, we quantified autoantibodies against 23 markers that are used for diagnosis of autoimmune disease. To this end, we used serum samples from patients with severe [intensive care unit (ICU)] and moderate (ward) COVID-19, across two to six consecutive time points, and compared autoantibody levels to uninfected healthy and ICU controls. Acute and post-acute serum (from 1 to 26 ICU days) was collected from 18 ICU COVID-19-positive patients at three to six time points; 18 ICU COVID-19-negative patients (sampled on ICU day 1 and 3); 21 ward COVID-19-positive patients (sampled on hospital day 1 and 3); and from 59 healthy uninfected controls deriving from two cohorts. Levels of IgG autoantibodies against 23 autoantigens, commonly used for autoimmune disease diagnosis, were measured in serum samples using MSD® U-PLEX electrochemiluminescence technology (MSD division Meso Scale Discovery®), and results were compared between groups. There were no significant elevations of autoantibodies for any of the markers tested in patients with severe COVID-19. Sample collections at longer time points should be considered in future studies, for assessing the possible development of autoantibody responses following infection with SARS-CoV-2

Viral Antigen and Inflammatory Biomarkers in Cerebrospinal Fluid in Patients With COVID-19 Infection and Neurologic Symptoms Compared With Control Participants Without Infection or Neurologic Symptoms

Importance: Neurologic symptoms are common in COVID-19, but the central nervous system (CNS) pathogenesis is unclear, and viral RNA is rarely detected in cerebrospinal fluid (CSF). / Objective: To measure viral antigen and inflammatory biomarkers in CSF in relation to neurologic symptoms and disease severity. Design, Setting, and / Participants: This cross-sectional study was performed from March 1, 2020, to June 30, 2021, in patients 18 years or older who were admitted to Sahlgrenska University Hospital, Gothenburg, Sweden, with COVID-19. All patients had CSF samples taken because of neurologic symptoms or within a study protocol. Healthy volunteer and prepandemic control groups were included. / Exposure: SARS-CoV-2 infection. / Main Outcomes and Measures: Outcomes included CSF SARS-CoV-2 nucleocapsid antigen (N-Ag) using an ultrasensitive antigen capture immunoassay platform and CSF biomarkers of immune activation (neopterin, β2-microglobulin, and cytokines) and neuronal injury (neurofilament light protein [NfL]). / Results: Forty-four patients (median [IQR] age, 57 [48-69] years; 30 [68%] male; 26 with moderate COVID-19 and 18 with severe COVID-19 based on the World Health Organization Clinical Progression Scale), 10 healthy controls (median [IQR] age, 58 [54-60] years; 5 [50%] male), and 41 patient controls (COVID negative without evidence of CNS infection) (median [IQR] age, 59 [49-70] years; 19 [46%] male) were included in the study. Twenty-one patients were neuroasymptomatic and 23 were neurosymptomatic (21 with encephalopathy). In 31 of 35 patients for whom data were available (89%), CSF N-Ag was detected; viral RNA test results were negative in all. Nucleocapsid antigen was significantly correlated with CSF neopterin (r = 0.38; P = .03) and interferon γ (r = 0.42; P = .01). No differences in CSF N-Ag concentrations were found between patient groups. Patients had markedly increased CSF neopterin, β2-microglobulin, interleukin (IL) 2, IL-6, IL-10, and tumor necrosis factor α compared with controls. Neurosymptomatic patients had significantly higher median (IQR) CSF interferon γ (86 [47-172] vs 21 [17-81] fg/mL; P = .03) and had a significantly higher inflammatory biomarker profile using principal component analysis compared with neuroasymptomatic patients (0.54; 95% CI, 0.03-1.05; P = .04). Age-adjusted median (IQR) CSF NfL concentrations were higher in patients compared with controls (960 [673-1307] vs 618 [489-786] ng/L; P = .002). No differences were seen in any CSF biomarkers in moderate compared with severe disease. / Conclusions and Relevance: In this study of Swedish adults with COVID-19 infection and neurologic symptoms, compared with control participants, viral antigen was detectable in CSF and correlated with CNS immune activation. Patients with COVID-19 had signs of neuroaxonal injury, and neurosymptomatic patients had a more marked inflammatory profile that could not be attributed to differences in COVID-19 severity. These results highlight the clinical relevance of neurologic symptoms and suggest that viral components can contribute to CNS immune responses without direct viral invasion

Dietary L-Tryptophan Consumption Determines the Number of Colonic Regulatory T cells and Susceptibility to Colitis via GPR15

Environmental factors are the major contributor to the onset of immunological disorders such as ulcerative colitis. However, their identities remain unclear. Here, we discover that the amount of consumed L-Tryptophan (L-Trp), a ubiquitous dietary component, determines the transcription level of the colonic T cell homing receptor, GPR15, hence affecting the number of colonic FOXP3+ regulatory T (Treg) cells and local immune homeostasis. Ingested L-Trp is converted by host IDO1/2 enzymes, but not by gut microbiota, to compounds that induce GPR15 transcription preferentially in Treg cells via the aryl hydrocarbon receptor. Consequently, two weeks of dietary L-Trp supplementation nearly double the colonic GPR15+ Treg cells via GPR15-mediated homing and substantially reduce the future risk of colitis. In addition, humans consume 3–4 times less L-Trp per kilogram of body weight and have fewer colonic GPR15+ Treg cells than mice. Thus, we uncover a microbiota-independent mechanism linking dietary L-Trp and colonic Treg cells, that may have therapeutic potential

RSPO3 impacts body fat distribution and regulates adipose cell biology in vitro

Fat distribution is an independent cardiometabolic risk factor. However, its molecular and cellular underpinnings remain obscure. Here we demonstrate that two independent GWAS signals at RSPO3, which are associated with increased body mass index-adjusted waist-to-hip ratio, act to specifically increase RSPO3 expression in subcutaneous adipocytes. These variants are also associated with reduced lower-body fat, enlarged gluteal adipocytes and insulin resistance. Based on human cellular studies RSPO3 may limit gluteofemoral adipose tissue (AT) expansion by suppressing adipogenesis and increasing gluteal adipocyte susceptibility to apoptosis. RSPO3 may also promote upper-body fat distribution by stimulating abdominal adipose progenitor (AP) proliferation. The distinct biological responses elicited by RSPO3 in abdominal versus gluteal APs in vitro are associated with differential changes in WNT signalling. Zebrafish carrying a nonsense rspo3 mutation display altered fat distribution. Our study identifies RSPO3 as an important determinant of peripheral AT storage capacity

Absence of N addition facilitates B cell development, but impairs immune responses

The programmed, stepwise acquisition of immunocompetence that marks the development of the fetal immune response proceeds during a period when both T cell receptor and immunoglobulin (Ig) repertoires exhibit reduced junctional diversity due to physiologic terminal deoxynucleotidyl transferase (TdT) insufficiency. To test the effect of N addition on humoral responses, we transplanted bone marrow from TdT-deficient (TdT−/−) and wild-type (TdT+/+) BALB/c mice into recombination activation gene 1-deficient BALB/c hosts. Mice transplanted with TdT−/− cells exhibited diminished humoral responses to the T-independent antigens α-1-dextran and (2,4,6-trinitrophenyl) hapten conjugated to AminoEthylCarboxymethyl-FICOLL, to the T-dependent antigens NP19CGG and hen egg lysozyme, and to Enterobacter cloacae, a commensal bacteria that can become an opportunistic pathogen in immature and immunocompromised hosts. An exception to this pattern of reduction was the T-independent anti-phosphorylcholine response to Streptococcus pneumoniae, which is normally dominated by the N-deficient T15 idiotype. Most of the humoral immune responses in the recipients of TdT−/− bone marrow were impaired, yet population of the blood with B and T cells occurred more rapidly. To further test the effect of N-deficiency on B cell and T cell population growth, transplanted TdT-sufficient and -deficient BALB/c IgMa and congenic TdT-sufficient CB17 IgMb bone marrow were placed in competition. TdT−/− cells demonstrated an advantage in populating the bone marrow, the spleen, and the peritoneal cavity. TdT deficiency, which characterizes fetal lymphocytes, thus appears to facilitate filling both central and peripheral lymphoid compartments, but at the cost of altered responses to a broad set of antigens

Early Low-Titer Neutralizing Antibodies Impede HIV-1 Replication and Select for Virus Escape

Single genome sequencing of early HIV-1 genomes provides a sensitive, dynamic assessment of virus evolution and insight into the earliest anti-viral immune responses in vivo. By using this approach, together with deep sequencing, site-directed mutagenesis, antibody adsorptions and virus-entry assays, we found evidence in three subjects of neutralizing antibody (Nab) responses as early as 2 weeks post-seroconversion, with Nab titers as low as 1∶20 to 1∶50 (IC50) selecting for virus escape. In each of the subjects, Nabs targeted different regions of the HIV-1 envelope (Env) in a strain-specific, conformationally sensitive manner. In subject CH40, virus escape was first mediated by mutations in the V1 region of the Env, followed by V3. HIV-1 specific monoclonal antibodies from this subject mapped to an immunodominant region at the base of V3 and exhibited neutralizing patterns indistinguishable from polyclonal antibody responses, indicating V1–V3 interactions within the Env trimer. In subject CH77, escape mutations mapped to the V2 region of Env, several of which selected for alterations of glycosylation. And in subject CH58, escape mutations mapped to the Env outer domain. In all three subjects, initial Nab recognition was followed by sequential rounds of virus escape and Nab elicitation, with Nab escape variants exhibiting variable costs to replication fitness. Although delayed in comparison with autologous CD8 T-cell responses, our findings show that Nabs appear earlier in HIV-1 infection than previously recognized, target diverse sites on HIV-1 Env, and impede virus replication at surprisingly low titers. The unexpected in vivo sensitivity of early transmitted/founder virus to Nabs raises the possibility that similarly low concentrations of vaccine-induced Nabs could impair virus acquisition in natural HIV-1 transmission, where the risk of infection is low and the number of viruses responsible for transmission and productive clinical infection is typically one