Simplified urine-based method to detect rifampin underexposure in adults with tuberculosis: a prospective diagnostic accuracy study.

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 (AUC0-24) 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 AUC0-24 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.

Rifampin urinary excretion to predict serum targets in children with tuberculosis: a prospective diagnostic accuracy study.

OBJECTIVE: Pharmacokinetic 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. DESIGN: Prospective diagnostic accuracy study. SETTING: Inpatient wards and outpatient clinics, northern Tanzania. PATIENTS: Children aged 4-17 years were consecutively recruited on initiation of WHO-approved treatment regimens. INTERVENTIONS: Samples 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 MEASURES: Receiver operating characteristic (ROC) curve for percentage of rifampin dose excreted in urine measured by spectrophotometry to predict serum rifampin AUC0-24 target of 31.7 mg*hour/L. RESULTS: 89 children, 52 (58%) female, with median age of 9.1 years, had both serum and urine collection. Only 59 (66%) reached the serum AUC0-24 target, 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 AUC0-24 target was 69.3% (95% CI 56.7% to 81.8%), p=0.007. CONCLUSIONS: Urine spectrophotometry correlated with a clinically relevant serum target for rifampin, representing a step toward personalised dosing for children in TB-endemic settings.

Longitudinal Analysis of Biologic Correlates of COVID-19 Resolution: Case Report.

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.

Biologic correlates of beneficial convalescent plasma therapy in a COVID-19 patient reveal disease resolution mechanisms.

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.

Highly versatile antibody binding assay for the detection of SARS-CoV-2 infection and vaccination.

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 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.

Highly versatile antibody binding assay for the detection of SARS-CoV-2 infection.

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.

Vaccination boosts protective responses and counters SARS-CoV-2-induced pathogenic memory B cells.

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.

Active Tuberculosis Is Characterized by Highly Differentiated Effector Memory Th1 Cells.

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.