Background: Detection and treatment of individuals with presumed latent tuberculosis (TB) infection (i.e., excluding active disease; LTBI) is imperative to achieve global TB control, as they represent a potential transmission reservoir. However, more sensitive and user–friendly diagnostic tools are needed. Methods: We evaluated the accuracy for TB infection detection of the new VIDAS® TB–IGRA (bioMérieux), a fully automated, single tube (thus eliminating the need for batch testing) overnight incubation assay, compared to the QuantiFERON®–TB Gold Plus (QFT–Plus, QIAGEN), in a global multi–centre cross–sectional study ([NCT04048018][1]) that included patients with TB disease (n=200) or participants at varying levels of TB exposure (n=1460; mixed exposure–risk–population). Results: VIDAS® TB–IGRA identified TB disease with greater sensitivity than QFT–Plus (97.5% vs. 80.7%, P<0.01%), and yielding significantly fewer false–negatives (2.5% vs. 17.5%; P<0.01%) and indeterminate results (1.0% vs. 9.5%; P=0.02%). In the mixed exposure–risk–population, negative (NPA) and positive percent agreement (PPA) were 90.1% (1097/1217) and 92.1% (223/242), respectively. PPA increased with TB–exposure risk (up to 95.7% for high–risk participants), whereas NPA decreased (starting from 96.9% for low–risk participants). Regression analyses revealed that VIDAS® TB–IGRA had a better fit with the risk–exposure gradient than the QFT‑Plus. Specificity in extremely low TB–exposure risk participants (n = 125) was high for both VIDAS® TB–IGRA and QFT–Plus (97.6% vs. 95.2%; P=8.33%). Conclusions: VIDAS® TB–IGRA displayed greater sensitivity than QFT–Plus, had a lower indeterminate rate, correlated better with an exposure gradient, and was highly specific, suggesting that it is a potentially valuable tool for the diagnosis of LTBI. ### Competing Interest Statement All authors had financial support from bioMérieux for performing this study. ### Clinical Trial NCT04048018 ### Funding Statement This study was funded by bioMérieux ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The following gave ethical approval for this work: - The French National Ethics committee (CPP, ref. 2019-A00998-49) - The UK Health Research Authority (Wales Research Ethics Committee 7, REC refs: 19/WA/0284 and 19/WA/0285) - The INMI "L. Spallanzani" (Rome, Italy) Ethics Committee (approval n°35/2019) - The "Comité de Ética en Investigación del Hospital General de Mexicali" (Mexico, ref. 02-01-HGMXL/FMED-UABC-2019-08-29-254) - In South Africa: - Pharma ethics refs. 190822774 and 190822777 - University of Cape Town Human Research Ethics Committee ref. 840/2019 - In USA: - Western Institutional Review Board (WIRB, tracking numbers: 20191965; 20192037; 20192039) - The Rutgers University Institutional Review Board (Refs: Pro2019001840 and Pro2019001936) - The Stanford University Institutional Review Board (eProtocol number: 53485) - The University of Illinois at Chicago Institutional Review Board (research protocol number: 2019-1145) I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT04048018&atom=%2Fmedrxiv%2Fearly%2F2024%2F07%2F05%2F2024.07.03.24309158.atom

Variable pharmacokinetics of rifampin in tuberculosis (TB) treatment can lead to poor outcomes. Urine spectrophotometry is simpler and more accessible than recommended serum-based drug monitoring, but its optimal efficacy in predicting serum rifampin underexposure in adults with TB remains uncertain. Adult TB patients in New Jersey and Virginia receiving rifampin-containing regimens were enrolled. Serum and urine samples were collected over 24 h. Rifampin serum concentrations were measured using validated liquid chromatography-tandem mass spectrometry, and total exposure (area under the concentration-time curve) over 24 h (AUC(0-24)) was determined through noncompartmental analysis. The Sunahara method was used to extract total rifamycins, and rifampin urine excretion was measured by spectrophotometry. An analysis of 58 eligible participants, including 15 (26%) with type 2 diabetes mellitus, demonstrated that urine spectrophotometry accurately identified subtarget rifampin AUC(0-24) at 0-4, 0-8, and 0-24 h. The area under the receiver operator characteristic curve (AUC ROC) values were 0.80 (95% CI 0.67-0.90), 0.84 (95% CI 0.72-0.94), and 0.83 (95% CI 0.72-0.93), respectively. These values were comparable to the AUC ROC of 2 h serum concentrations commonly used for therapeutic monitoring (0.82 [95% CI 0.71-0.92], P = 0.6). Diabetes status did not significantly affect the AUC ROCs for urine in predicting subtarget rifampin serum exposure (P = 0.67-0.92). Spectrophotometric measurement of urine rifampin excretion within the first 4 or 8 h after dosing is a simple and cost-effective test that accurately predicts rifampin underexposure. This test provides critical information for optimizing tuberculosis treatment outcomes by facilitating appropriate dose adjustments.

ObjectivePharmacokinetic variability drives tuberculosis (TB) treatment outcomes but measurement of serum drug concentrations for personalised dosing is inaccessible for children in TB-endemic settings. We compared rifampin urine excretion for prediction of a serum target associated with treatment outcome.DesignProspective diagnostic accuracy study.SettingInpatient wards and outpatient clinics, northern Tanzania.PatientsChildren aged 4–17 years were consecutively recruited on initiation of WHO-approved treatment regimens.InterventionsSamples were collected after directly observed therapy at least 2 weeks after initiation in the intensive phase: serum at pre-dose and 1, 2 and 6 hours post-dose, later analysed by liquid chromatography-tandem mass spectrometry for calculation of rifampin total exposure or area under the concentration time curve (AUC0-24); urine at post-dose intervals of 0–4, 4–8 and 8–24 hours, with rifampin excretion amount measured onsite by spectrophotometry.Main outcome measuresReceiver operating characteristic (ROC) curve for percentage of rifampin dose excreted in urine measured by spectrophotometry to predict serum rifampin AUC0–24target of 31.7 mg*hour/L.Results89 children, 52 (58%) female, with median age of 9.1 years, had both serum and urine collection. Only 59 (66%) reached the serum AUC0–24target, reflected by a range of urine excretion patterns. Area under the ROC curve for percentage of rifampin dose excreted in urine over 24 hours predicting serum AUC0–24target was 69.3% (95% CI 56.7% to 81.8%), p=0.007.ConclusionsUrine spectrophotometry correlated with a clinically relevant serum target for rifampin, representing a step toward personalised dosing for children in TB-endemic settings.

Importance: Accurate diagnosis of tuberculosis (TB) infection can be achieved with interferon gamma ; release assays. Their performance can be improved by utilizing fully automated, single-patient formats. Objective: Establish clinical thresholds for a new interferon gamma; release assay, the VIDAS(R) TB-IGRA, and compare diagnostic performance in detecting tuberculosis infection and disease with the established QuantiFERON-TB Gold Plus (QFT-Plus). Design: Preliminary diagnostic performance study (October 2nd, 2019 to February 20th, 2020). Setting: Multicenter. Participants: Participants were divided into TB disease, high-risk, and low-risk populations. The confirmed TB disease population included 107 patients. The high-risk population included 162 individuals with flagged risk factors on a questionnaire but without objective clinical confirmation of TB. The Low-risk population included 117 healthy blood donors from the French National Blood Bank. Exposures: Tuberculosis. Main Outcomes and Measures: Positive and negative percent agreement (PPA, NPA) were determined between the VIDAS(R) TB-IGRA and QFT-Plus. In the TB disease and low-risk populations, sensitivity was also measured against bacterial culture and PCR. Results: The VIDAS(R) TB-IGRA produced fewer indeterminate results than the QFT-Plus (1/107 vs. 23/107) in the TB disease population. One analysis included indeterminate results as false negatives (94 positives and 10 false negatives vs. 56 positives and 48 false negatives), and the VIDAS(R) TB-IGRA exhibited higher sensitivity than the QFT-Plus (90.4% vs. 53.8%) (p<0.0001). Another analysis excluded indeterminate results (76 positives and 4 false negatives vs. 55 positives and 25 false negatives), and the VIDAS(R) TB-IGRA again exhibited higher sensitivity than the QFT-Plus (95.0% vs. 68.8%) (p<0.0001). A 98.2% PPA was calculated between the two tests with this dataset. In the high-risk population, the VIDAS(R) TB-IGRA exhibited a strong PPA (94.4%) with the QFT-Plus. However, a lower than expected NPA was observed (85.2%). In the low-risk population, the VIDAS(R) TB-IGRA demonstrated high specificity (94.9%) and a strong NPA (98.2%) with the QFT-Plus. Conclusions and Relevance: The fully automated VIDAS(R) TB-IGRA is a promising diagnostic test for both TB infection and disease. It exhibits higher sensitivity while maintaining specificity and produces fewer indeterminate interpretations. Its easy-to-use, single-patient format may lead to increased TB testing to help with the worldwide eradication of the disease.

BACKGROUND:While the biomarkers of COVID-19 severity have been thoroughly investigated, the key biological dynamics associated with COVID-19 resolution are still insufficiently understood. MAIN BODY:We report a case of full resolution of severe COVID-19 due to convalescent plasma transfusion in a patient with underlying multiple autoimmune syndrome. Following transfusion, the patient showed fever remission, improved respiratory status, and rapidly decreased viral burden in respiratory fluids and SARS-CoV-2 RNAemia. Longitudinal unbiased proteomic analysis of plasma and single-cell transcriptomics of peripheral blood cells conducted prior to and at multiple times after convalescent plasma transfusion identified the key biological processes associated with the transition from severe disease to disease-free state. These included (i) temporally ordered upward and downward changes in plasma proteins reestablishing homeostasis and (ii) post-transfusion disappearance of a particular subset of dysfunctional monocytes characterized by hyperactivated Interferon responses and decreased TNF-α signaling. CONCLUSIONS:Monitoring specific subsets of innate immune cells in peripheral blood may provide prognostic keys in severe COVID-19. Moreover, understanding disease resolution at the molecular and cellular level should contribute to identify targets of therapeutic interventions against severe COVID-19.

While the biomarkers of COVID-19 severity have been thoroughly investigated, the key biological dynamics associated with COVID-19 resolution are still insufficiently understood. We report a case of full resolution of severe COVID-19 due to convalescent plasma transfusion. Following transfusion, the patient showed fever remission, improved respiratory status, and rapidly decreased viral burden in respiratory fluids and SARS-CoV-2 RNAemia. Longitudinal unbiased proteomic analysis of plasma and single-cell transcriptomics of peripheral blood cells conducted prior to and at multiple times after convalescent plasma transfusion identified the key biological processes associated with the transition from severe disease to disease-free state. These included (i) temporally ordered upward and downward changes in plasma proteins reestablishing homeostasis and (ii) post-transfusion disappearance of a subset of monocytes characterized by hyperactivated Interferon responses and decreased TNF-α signaling. Monitoring specific dysfunctional myeloid cell subsets in peripheral blood may provide prognostic keys in COVID-19.

Monitoring the burden and spread of infection with the new coronavirus SARS-CoV-2, whether within small communities or in large geographical settings, is of paramount importance for public health purposes. Serology, which detects the host antibody response to the infection, is the most appropriate tool for this task, since virus-derived markers are most reliably detected during the acute phase of infection. Here we show that our ELISA protocol, which is based on antibody binding to the Receptor Binding Domain (RBD) of the S1 subunit of the viral Spike protein expressed as a novel fusion protein, detects antibody responses to SARS-CoV-2 infection and COVID-19 vaccination. We also show that our ELISA is accurate and versatile. It compares favorably with commercial assays widely used in clinical practice to determine exposure to SARS-CoV-2. Moreover, our protocol accommodates use of various blood- and non-blood-derived biospecimens, such as breast milk, as well as dried blood obtained with microsampling cartridges that are appropriate for remote collection. As a result, our RBD-based ELISA protocols are well suited for seroepidemiology and other large-scale studies requiring parsimonious sample collection outside of healthcare settings.

Much is to be learned about the interface between immune responses to SARS-CoV-2 infection and vaccination. We monitored immune responses specific to SARS-CoV-2 Spike Receptor-Binding-Domain (RBD) in convalescent individuals for eight months after infection diagnosis and following vaccination. Over time, neutralizing antibody responses, which are predominantly RBD specific, generally decreased, while RBD-specific memory B cells persisted. RBD-specific antibody and B cell responses to vaccination were more vigorous than those elicited by infection in the same subjects or by vaccination in infection-naïve comparators. Notably, the frequencies of double negative B memory cells, which are dysfunctional and potentially pathogenic, increased in the convalescent subjects over time. Unexpectedly, this effect was reversed by vaccination. Our work identifies a novel aspect of immune dysfunction in mild/moderate COVID-19, supports the practice of offering SARS-CoV-2 vaccination regardless of infection history, and provides a potential mechanistic explanation for the vaccination-induced reduction of "Long-COVID" symptoms.

Since human infection with coronavirus SARS-CoV-2 was first detected in December 2019, we are still developing an understanding of the nature and duration of protection against this infection. Most neutralizing antibodies, which are a key component of the protective response against SARS-CoV-2 infection, target the Receptor Binding Domain (RBD) of the Spike glycoprotein and critically prevent binding of the virus to the host cell receptor, and viral entry. Thus, it is of vital importance to monitor the presence of neutralizing antibodies and RBD-specific B cells that are key for rapid production of protective antibodies upon reinfection with SARS-COV2 infection. In this study, we developed a multicolor flow cytometric assay to enumerate the RBD-specific memory B cell and memory B cell subsets. We collected peripheral blood cells and plasma from 22 subjects 1–2 months since COVID-19 diagnosis (early time point - ET) and then again after 5–7 months (late time point – LT). Comparing the data collected from these two time points, we observed a significant decrease in plasma blasts and double-negative memory B cell and an increase in the IgG+ switched-memory B cells and decrease in the IgM+ switched-memory B cells at LT relative to ET. We concurrently observed a trend toward decreased anti-RBD IgG titers over time. When we tested plasma neutralizing activity employing ACE2-expressing HeLa cell lines infected with mNeonGreen(mNG)SARS-CoV-2, we also observed reduced neutralizing antibody titers over time. Thus, a correlation exists between titers of RBD-specific IgG antibody and neutralizing titers. In addition, the presence of RBD-specific B-cell memory in circulating blood is a strong indication of a durable protective response.

Despite advances in diagnosing latent Mycobacterium tuberculosis infection (LTBI), we still lack a diagnostic test that differentiates LTBI from active tuberculosis (TB) or predicts the risk of progression to active disease. One reason for the absence of such a test may be the failure of current assays to capture the dynamic complexities of the immune responses associated with various stages of TB, since these assays measure only a single parameter (release of IFN-γ) and rely on prolonged (overnight) T cell stimulation. We describe a novel, semi-automated RNA flow cytometry assay to determine whether immunological differences can be identified between LTBI and active TB. We analyzed antigen-induced expression of Th1 cytokine mRNA after short (2- and 6-h) stimulation with antigen, in the context of memory T cell immunophenotyping. IFNG and TNFA mRNA induction was detectable in CD4+ T cells after only 2 h of ex vivo stimulation. Moreover, IFNG- and TNFA-expressing CD4+ T cells (Th1 cells) were more frequent in active TB than in LTBI, a difference that is undetectable with conventional, protein-based cytokine assays. We also found that active TB was associated with higher ratios of effector memory to central memory Th1 cells than LTBI. This effector memory phenotype of active TB was associated with increased T cell differentiation, as defined by loss of the CD27 marker, but not with T cell exhaustion, as determined by PD-1 abundance. These results indicate that single-cell-based, mRNA measurements may help identify time-dependent, quantitative differences in T cell functional status between latent infection and active tuberculosis.