Background: Patients receiving B-cell depleting regimens are at increased risk of persistent COVID-19. Selecting effective treatments for these patients becomes challenging with new viral mutations conferring resistance to available targeted therapies.

Plasma RNAemia, delayed antibody responses and inflammation predict COVID-19 outcomes, but the mechanisms underlying these immunovirological patterns are poorly understood. We profile 782 longitudinal plasma samples from 318 hospitalized patients with COVID-19. Integrated analysis using k-means reveals four patient clusters in a discovery cohort: mechanically ventilated critically-ill cases are subdivided into good prognosis and high-fatality clusters (reproduced in a validation cohort), while non-critical survivors segregate into high and low early antibody responders. Only the high-fatality cluster is enriched for transcriptomic signatures associated with COVID-19 severity, and each cluster has distinct RBD-specific antibody elicitation kinetics. Both critical and non-critical clusters with delayed antibody responses exhibit sustained IFN signatures, which negatively correlate with contemporaneous RBD-specific IgG levels and absolute SARS-CoV-2-specific B and CD4+ T cell frequencies. These data suggest that the "Interferon paradox" previously described in murine LCMV models is operative in COVID-19, with excessive IFN signaling delaying development of adaptive virus-specific immunity. The role of IFN signaling in SARS-CoV-2 infection and outcome is still debated. Here, the authors longitudinally profiled plasma samples from hospitalized patients and show that a persistent inflammatory response is linked to delayed generation of adaptive immunity and increased risk of death when coupled with severe infection.

Targeted Next Generation Sequencing (tNGS) and Whole Genome Sequencing (WGS) are increasingly used for genotypic drug susceptibility testing (gDST) of Mycobacterium tuberculosis. Thirty-two multi-drugs resistant and 40 drug susceptible isolates from Madagascar were tested with Deeplex® Myc-TB and WGS using the Mykrobe analysis pipeline. Sixty-four of 72 (89%) yielded concordant categorical gDST results for drugs tested by both assays. Mykrobe didn't detect pncA K96T, pncA Q141P, pncA H51P, pncA H82R, rrs C517T and rpsL K43R mutations, which were identified as minority variants in corresponding isolates by tNGS. One discrepancy (rrs C517T) was associated with insufficient sequencing depth on WGS. Deeplex® Myc-TB didn't detect inhA G-154A which isn't covered by the assay's amplification targets. Despite those targets being included in the Deeplex® Myc-TB assay, a pncA T47A and a deletion in gid were not identified in one isolate respectively. The evaluated WGS and tNGS gDST assays show high but imperfect concordance.

Abstract Background Retrospective and descriptive molecular epidemiology studies have shown that Mycobacterium tuberculosis whole genome sequencing can identify outbreaks and disease transmission events with higher resolution than conventional epidemiological investigations. Those studies have strengthened our understanding of genomic polymorphisms correlating with person-to-person transmission and helped resolve putative transmission clusters. To date, systematic genomic surveillance programs implemented for M. tuberculosis were only implemented in low-incidence settings. The purpose of this study is to determine whether there is an impact of routine M. tuberculosis whole genome sequencing on tuberculosis case detection in a high-incidence setting. Methods A cluster randomized controlled trial will be performed. Forty-eight rural village groups (or Fokontany) in the Vohibato district of Madagascar will be randomized to one of three interventions arms. Arm 1 (standard of care) involves healthcare facility-based passive case detection with smear microscopy testing. Arm 2 (best practice) consists of active case finding and Xpert MTB/RIF Ultra PCR testing followed by household contact investigations. Arm 3 (novel intervention) includes the same interventions as arm 2, with addition of sputum culture and M. tuberculosis whole genome sequencing for all newly diagnosed cases. In arm 3, molecular suggested putative outbreaks are investigated, and additional TB suspects are appropriately tested. The intervention observational period will be 2 years. The primary outcome will be the number of detected cases/100,000/year in each arm after 1 year of intervention. Discussion This study is designed to determine whether there is an impact of prospective whole genome sequencing-based molecular typing on tuberculosis case detection in high-incidence settings. Investigating potential outbreaks and focusing active case finding in spatiotemporal settings where disease transmission is suggested by genomic typing is hypothesized to improve case detection in rural communities. Trial registration ClinicalTrials.gov NCT05406453. Retrospectively registered on June 6, 2022.

Background:Mycobacterial culture is routinely performed to diagnose tuberculosis (TB) in Canada. Globally, meta-analyses suggest that up to 2% of positive cultures are falsely positive for Mycobacterium tuberculosis due to laboratory cross-contamination. Five patients from distinct clinical institutions in Montréal were diagnosed with culture-positive TB as their clinical samples were processed in a centralized mycobacteria laboratory. Cross-contamination was suspected due to culture positivity in an organ donor with low TB pre-test probability. We describe a TB pseudo-outbreak due to laboratory cross-contamination and assess the role of conventional typing (i.e., mycobacterial interspersed repetitive unit variable number of tandem repeats [MIRU-VNTR]) and whole-genome sequencing (WGS) in supporting the investigation. Methods:Patients' epidemiological risk factors and clinical presentations were reviewed. The trajectories of pre- and per-analytic samples were retraced to identify potential cross-contamination events. Tuberculosis isolates were characterized by MIRU-VNTR and WGS using Oxford Nanopore Technology (ONT). The bioinformatic pipeline tbpore (v0.7.1) cluster was used for phylogenetic analyses. Results:Two patients had previous exposure to endemic settings and clinical symptoms compatible with TB. Culture media inoculation overlapped in time for four patients, including one with suspected pulmonary cavitary disease and an organ donor whose organs had been transplanted in three different receivers. The MIRU-VNTR and WGS typing confirmed isolates from those four patients to be identical. Conclusion:Clinical, laboratory and molecular typing data, including results from ONT sequencing, were considered sufficiently robust to confirm laboratory cross-contamination and TB therapy was discontinued including in all organ transplant recipients.

Objectives: We evaluated the added value of infection control-guided, on demand, and locally performed severe acute respiratory coronavirus virus 2 (SARS-CoV-2) genomic sequencing to support outbreak investigation and control in acute-care settings.Design and setting: This 18-month prospective molecular epidemiology study was conducted at a tertiary-care hospital in Montreal, Canada. When nosocomial transmission was suspected by local infection control, viral genomic sequencing was performed locally for all putative outbreak cases. Molecular and conventional epidemiology data were correlated on a just-in-time basis to improve understanding of coronavirus disease 2019 (COVID-19) transmission and reinforce or adapt control measures.Results: Between April 2020 and October 2021, 6 outbreaks including 59 nosocomial infections (per the epidemiological definition) were investigated. Genomic data supported 7 distinct transmission clusters involving 6 patients and 26 healthcare workers. We identified multiple distinct modes of transmission, which led to reinforcement and adaptation of infection control measures. Molecular epidemiology data also refuted (n = 14) suspected transmission events in favor of community acquired but institutionally clustered cases.Conclusion: SARS-CoV-2 genomic sequencing can refute or strengthen transmission hypotheses from conventional nosocomial epidemiological investigations, and guide implementation of setting-specific control strategies. Our study represents a template for prospective, on site, outbreak-focused SARS-CoV-2 sequencing. This approach may become increasingly relevant in a COVID-19 endemic state where systematic sequencing within centralized surveillance programs is not available.Trial registration: clinicaltrials.gov identifier: NCT05411562

BackgroundPrevention and management of COVID-19 nosocomial outbreaks is essential to protect patients and healthcare workers, and to maintain clinical care activities. The COVID-19 pandemic exposed challenges faced by conventional hospital epidemiology for identification of healthcare associated infections.MethodsThis is a prospective molecular epidemiology study based at a tertiary care hospital in Montreal, Canada. When nosocomial transmission was suspected by local infection control teams’ investigations, SARS-CoV-2 viral genomic sequencing was performed locally for all putative outbreak cases. Molecular and conventional epidemiology data were rapidly produced and correlated just-in-time to improve understanding of COVID-19 transmission and reinforce or adapt control measures.FindingsBetween April 2020 and October 2021, six outbreaks including 59 nosocomial infections as per the epidemiological definition were investigated. Genomic data supported seven distinct transmission clusters involving 6 patients and 26 healthcare workers. Multiple distinct modes of transmission were identified leading to reinforcement and adaptation of infection control measures. Molecular epidemiology data also refuted (n=14) suspected transmission envents in favor of community acquired but institutionnaly clustered cases.InterpretationOur findings confirm that SARS-CoV-2 genomic sequencing can refute or strenghten transmission hypotheses from conventional nosocomial epidemiological investigations, and guide implementation of setting-specific control strategies. Our study represents a template for prospective, on site, outbreak-focused SARS-CoV-2 sequencing, an approach which may become increasingly relevant in a COVID endemic state where systematic sequencing within centralized surveillance programs is not available.Trial Registration Details: This study protocol was made publicly available on ClinicalTrials.gov under ID NCT05411562.Funding Information: Fonds de Recherche Santé Québec - Réseau SIDA-Maladies Infectieuses (Ref.70142). Centre Hospitalier de l’Université de Montréal (CHUM). Declaration of Interests: All other authors declare no competing interests. Although many co-authors participate on local and national COVID-19 response advisory committees, the presented views on COVID-19 diagnostics and outbreak management are those of the authors and not those of the Public Agency of Canada (PHAC), the Quebec National Institute of Public Health (INSPQ), the Quebec Ministry 450 of Health and Social Services (MSSS) or Quebec Nosocomial Infections Committee (CINQ). SGL is supported by the Fonds de Recherche Santé Québec. CT is supported by the Pfizer-Université de Montréal Chair on HIV translational research. Ethics Approval Statement: This study was performed using clinical information and samples provided to our IPAC service and clinical microbiology laboratory as part of routine outbreak investigations. The study did not require individual informed consent but was approved by CHUM’s Ethic Commitee (Ref. 2021- 9253, 20.270).

SARS-CoV-2 whole genome sequencing is a molecular biology tool performed to support many aspects of the response to the pandemic. Freezing of primary clinical nasopharyngeal swabs and shipment to reference laboratories is usually required for sequencing. Cobas PCR Media transport medium facilitates high throughput SARS-CoV-2 RT-PCR analyses on cobas platforms. The manufacturer doesn't recommend freezing this transport medium because of risks of degrading molecular templates and impairing test results. Our objective was to compare the quality and results of SARS-CoV-2 genomic sequencing when performed on fresh or frozen samples in cobas PCR Media. Viral genome sequencing was performed using Oxford Nanopore Technologies MinION platform. Sequencing performance, quality and results did not significantly differ between fresh and frozen samples (n = 10). Freezing of cobas PCR Media does not negatively affect SARS-CoV-2 RNA sequencing results and it is therefore a suitable transport medium for outsourcing sequencing analyses to reference laboratories.

Despite advances in COVID-19 management, identifying patients evolving toward death remains challenging. To identify early predictors of mortality within 60 days of symptom onset (DSO), we performed immunovirological assessments on plasma from 279 individuals. On samples collected at DSO11 in a discovery cohort, high severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral RNA (vRNA), low receptor binding domain–specific immunoglobulin G and antibody-dependent cellular cytotoxicity, and elevated cytokines and tissue injury markers were strongly associated with mortality, including in patients on mechanical ventilation. A three-variable model of vRNA, with predefined adjustment by age and sex, robustly identified patients with fatal outcome (adjusted hazard ratio for log-transformed vRNA = 3.5). This model remained robust in independent validation and confirmation cohorts. Since plasma vRNA’s predictive accuracy was maintained at earlier time points, its quantitation can help us understand disease heterogeneity and identify patients who may benefit from new therapies.

ABSTRACT Despite advances in COVID-19 management, it is unclear how to recognize patients who evolve towards death. This would allow for better risk stratification and targeting for early interventions. However, the explosive increase in correlates of COVID-19 severity complicates biomarker prioritisation. To identify early biological predictors of mortality, we performed an immunovirological assessment (SARS-CoV-2 viral RNA, cytokines and tissue injury markers, antibody responses) on plasma samples collected from 144 hospitalised COVID-19 patients 11 days after symptom onset and used to test models predicting mortality within 60 days of symptom onset. In the discovery cohort (n=61, 13 fatalities), high SARS-CoV-2 vRNA, low RBD-specific IgG levels, low SARS-CoV-2-specific antibody-dependent cellular cytotoxicity, and elevated levels of several cytokines and lung injury markers were strongly associated with increased mortality in the entire cohort and the subgroup on mechanical ventilation. Model selection revealed that a three-variable model of vRNA, age and sex was very robust at identifying patients who will succumb to COVID-19 (AUC=0.86, adjusted HR for log-transformed vRNA=3.5; 95% CI: 2.0-6.0). This model remained robust in an independent validation cohort (n=83, AUC=0.85). Quantification of plasma SARS-CoV-2 RNA can help understand the heterogeneity of disease trajectories and identify patients who may benefit from new therapies.