Exploring the impact of COVID-19 on reported maternal and neonatal complications and access to maternal health care in five government health facilities in Blantyre, Malawi.

BACKGROUND: There is a dearth of studies assessing the effects of SARS-CoV-2 on the healthcare system and access to care, especially in lower- and middle-income countries such as Malawi. We aimed to assess the impacts of COVID-19 on reported maternal and neonatal complications as well as potential changes in maternal care access to care among five primary care health facilities in Blantyre, Malawi. METHODS: This retrospective cohort study assessed maternal and neonatal register data from five participating health centers in Blantyre, Malawi using the Malawi District Health Information Software 2 (DHIS2) to compare outcomes from 15 months before COVID-19 emerged, defined as the pre-Covid period (January 2019 -March 2020) with nine months after COVID-19 (April 2020 -December 2020). RESULTS: There was a significant decrease in reported use of vacuum extraction, which went from <0.01%in the pre-COVID period to 0% in the COVID period (p = 0.01). The proportion of births reporting fetal distress almost tripled from 0.46% to 1.36% (p = 0.001) during the COVID-19 period. Additionally, reported anticonvulsant use significantly increased from 0.01% to 1.2% (p<0.01), and antibiotic use significantly increased from 0.45% to 1.6% (p = 0.01). Asphyxia was the only significant neonatal complication variable reported, increasing from 2.80% to 3.45% (p = 0.01). CONCLUSION: Our findings suggest that significant outcomes were mainly due to the indirect effects of COVID-19 rather than the virus itself. Based on our findings and the contextual qualitative interviews with two Malawian expert midwives, we concluded that mothers may have been affected more due to understaffing and shortage of skilled personnel in the study health facilities. Therefore, the development of highly skilled health workers may contribute to better outcomes, along with adequate staffing and a streamlined referral process.

Longitudinal characterization of circulating neutrophils uncovers phenotypes associated with severity in hospitalized COVID-19 patients.

Mechanisms of neutrophil involvement in severe coronavirus disease 2019 (COVID-19) remain incompletely understood. Here, we collect longitudinal blood samples from 306 hospitalized COVID-19+ patients and 86 controls and perform bulk RNA sequencing of enriched neutrophils, plasma proteomics, and high-throughput antibody profiling to investigate relationships between neutrophil states and disease severity. We identify dynamic switches between six distinct neutrophil subtypes. At days 3 and 7 post-hospitalization, patients with severe disease display a granulocytic myeloid-derived suppressor cell-like gene expression signature, while patients with resolving disease show a neutrophil progenitor-like signature. Humoral responses are identified as potential drivers of neutrophil effector functions, with elevated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific immunoglobulin G1 (IgG1)-to-IgA1 ratios in plasma of severe patients who survived. In vitro experiments confirm that while patient-derived IgG antibodies induce phagocytosis in healthy donor neutrophils, IgA antibodies predominantly induce neutrophil cell death. Overall, our study demonstrates a dysregulated myelopoietic response in severe COVID-19 and a potential role for IgA-dominant responses contributing to mortality.

COVID-19 Seroprevalence in ED Health Care Professionals Study: A Cross-Sectional Study.

INTRODUCTION: ED health care professionals are at the frontline of evaluation and management of patients with acute, and often undifferentiated, illness. During the initial phase of the SARS-CoV-2 outbreak, there were concerns that ED health care professionals may have been at increased risk of exposure to SARS-CoV-2 due to difficulty in early identification of patients. This study assessed the seroprevalence of SARS-CoV-2 antibodies among ED health care professionals without confirmed history of COVID-19 infection at a quaternary academic medical center. METHODS: This study used a cross-sectional design. An ED health care professional was deemed eligible if they had worked at least 4 shifts in the adult emergency department from April 1, 2020, through May 31, 2020, were asymptomatic on the day of blood draw, and were not known to have had prior documented COVID-19 infection. The study period was December 17, 2020, to January 27, 2021. Eligible participants completed a questionnaire and had a blood sample drawn. Samples were run on the Roche Cobas Elecsys Anti-SARS-CoV-2 antibody assay. RESULTS: Of 103 health care professionals (16 attending physicians, 4 emergency residents, 16 advanced practice professionals, and 67 full-time emergency nurses), only 3 (2.9%; exact 95% CI, 0.6%-8.3%) were seropositive for SARS-CoV-2 antibodies. DISCUSSION: At this quaternary academic medical center, among those who volunteered to take an antibody test, there was a low seroprevalence of SARS-CoV-2 antibodies among ED clinicians who were asymptomatic at the time of blood draw and not known to have had prior COVID-19 infection.

Longitudinal proteomic analysis of severe COVID-19 reveals survival-associated signatures, tissue-specific cell death, and cell-cell interactions.

Mechanisms underlying severe coronavirus disease 2019 (COVID-19) disease remain poorly understood. We analyze several thousand plasma proteins longitudinally in 306 COVID-19 patients and 78 symptomatic controls, uncovering immune and non-immune proteins linked to COVID-19. Deconvolution of our plasma proteome data using published scRNA-seq datasets reveals contributions from circulating immune and tissue cells. Sixteen percent of patients display reduced inflammation yet comparably poor outcomes. Comparison of patients who died to severely ill survivors identifies dynamic immune-cell-derived and tissue-associated proteins associated with survival, including exocrine pancreatic proteases. Using derived tissue-specific and cell-type-specific intracellular death signatures, cellular angiotensin-converting enzyme 2 (ACE2) expression, and our data, we infer whether organ damage resulted from direct or indirect effects of infection. We propose a model in which interactions among myeloid, epithelial, and T cells drive tissue damage. These datasets provide important insights and a rich resource for analysis of mechanisms of severe COVID-19 disease.

Evaluating the Impact of Language Concordance on Coronavirus Disease 2019 Contact Tracing Outcomes Among Spanish-Speaking Adults in San Francisco Between June and November 2020

We evaluated the impact of language concordance—clinician or public health worker fluency in a patient’s primary language—on coronavirus disease 2019 (COVID-19) contact tracing outcomes among 2668 Spanish-speaking adults in San Francisco. Language concordance was associated with 20% greater odds of COVID-19 testing and 53% greater odds of support service referrals.

Alveolar, Endothelial, and Organ Injury Marker Dynamics in Severe COVID-19.

Rationale: Alveolar and endothelial injury may be differentially associated with coronavirus disease (COVID-19) severity over time. Objectives: To describe alveolar and endothelial injury dynamics and associations with COVID-19 severity, cardiorenovascular injury, and outcomes. Methods: This single-center observational study enrolled patients with COVID-19 requiring respiratory support at emergency department presentation. More than 40 markers of alveolar (including receptor for advanced glycation endproducts [RAGE]), endothelial (including angiopoietin-2), and cardiorenovascular injury (including renin, kidney injury molecule-1, and troponin-I) were serially compared between invasively and spontaneously ventilated patients using mixed-effects repeated-measures models. Ventilatory ratios were calculated for intubated patients. Associations of biomarkers with modified World Health Organization scale at Day 28 were determined with multivariable proportional-odds regression. Measurements and Main Results: Of 225 patients, 74 (33%) received invasive ventilation at Day 0. RAGE was 1.80-fold higher in invasive ventilation patients at Day 0 (95% confidence interval [CI], 1.50-2.17) versus spontaneous ventilation, but decreased over time in all patients. Changes in alveolar markers did not correlate with changes in endothelial, cardiac, or renal injury markers. In contrast, endothelial markers were similar to lower at Day 0 for invasive ventilation versus spontaneous ventilation, but then increased over time only among intubated patients. In intubated patients, angiopoietin-2 was similar (fold difference, 1.02; 95% CI, 0.89-1.17) to nonintubated patients at Day 0 but 1.80-fold higher (95% CI, 1.56-2.06) at Day 3; cardiorenovascular injury markers showed similar patterns. Endothelial markers were not consistently associated with ventilatory ratios. Endothelial markers were more often significantly associated with 28-day outcomes than alveolar markers. Conclusions: Alveolar injury markers increase early. Endothelial injury markers increase later and are associated with cardiorenovascular injury and 28-day outcome. Alveolar and endothelial injury likely contribute at different times to disease progression in severe COVID-19.

Longitudinal Characterization of Circulating Neutrophils Uncovers Distinct Phenotypes Associated with Disease Severity in Hospitalized COVID-19 Patients (preprint)

Multiple studies have identified an association between neutrophils and COVID-19 disease severity; however, the mechanistic basis of this association remains incompletely understood. Here we collected 781 longitudinal blood samples from 306 hospitalized COVID-19 + patients, 78 COVID-19 - acute respiratory distress syndrome patients, and 8 healthy controls, and performed bulk RNA-sequencing of enriched neutrophils, plasma proteomics, cfDNA measurements and high throughput antibody profiling assays to investigate the relationship between neutrophil states and disease severity or death. We identified dynamic switches between six distinct neutrophil subtypes using non-negative matrix factorization (NMF) clustering. At days 3 and 7 post-hospitalization, patients with severe disease had an enrichment of a granulocytic myeloid derived suppressor cell-like state gene expression signature, while non-severe patients with resolved disease were enriched for a progenitor-like immature neutrophil state signature. Severe disease was associated with gene sets related to neutrophil degranulation, neutrophil extracellular trap (NET) signatures, distinct metabolic signatures, and enhanced neutrophil activation and generation of reactive oxygen species (ROS). We found that the majority of patients had a transient interferon-stimulated gene signature upon presentation to the emergency department (ED) defined here as Day 0, regardless of disease severity, which persisted only in patients who subsequently died. Humoral responses were identified as potential drivers of neutrophil effector functions, as enhanced antibody-dependent neutrophil phagocytosis and reduced NETosis was associated with elevated SARS-CoV-2-specific IgG1-to-IgA1 ratios in plasma of severe patients who survived. In vitro experiments confirmed that while patient-derived IgG antibodies mostly drove neutrophil phagocytosis and ROS production in healthy donor neutrophils, patient-derived IgA antibodies induced a predominant NETosis response. Overall, our study demonstrates neutrophil dysregulation in severe COVID-19 and a potential role for IgA-dominant responses in driving neutrophil effector functions in severe disease and mortality.Funding:We acknowledge support from the Ragon Institute of MGH, MIT and Harvard, the Massachusetts Consortium on Pathogen Readiness (MassCPR), the NIH (3R37AI080289-11S1, R01AI146785, U19AI42790-01, U19AI135995-02, U19AI42790-01, 1U01CA260476 – 01, CIVIC75N93019C00052, T32 GM007592), the American Lung Association, and the MGH Executive Committee on Research. A.-C.V. acknowledges funding support from the COVID-19 Clinical Trials Pilot grant from the Executive Committee on Research at MGH, a COVID-19 Chan Zuckerberg Initiative grant (2020-216954), and funds from the Manton Foundation and the Klarman Family Foundation.Declaration of Interests: M.S.F receives funding from Bristol-Myers Squibb. G.A. is a founder of Seromyx Systems Inc. N.H. holds equity in Biontech and holds equity in and advises Danger Bio. Ethics Approval Statement: The study was approved by the Mass General Brigham Institutional Review Board under protocol 2017P001681, with an approval for a waiver of informed consent in compliance with the 45CFR 46, 2018 Common rule.

Longitudinal characterization of circulating neutrophils uncovers distinct phenotypes associated with disease severity in hospitalized COVID-19 patients (preprint)

Multiple studies have identified an association between neutrophils and COVID-19 disease severity; however, the mechanistic basis of this association remains incompletely understood. Here we collected 781 longitudinal blood samples from 306 hospitalized COVID-19+ patients, 78 COVID-19- acute respiratory distress syndrome patients, and 8 healthy controls, and performed bulk RNA-sequencing of enriched neutrophils, plasma proteomics, cfDNA measurements and high throughput antibody profiling assays to investigate the relationship between neutrophil states and disease severity or death. We identified dynamic switches between six distinct neutrophil subtypes using non-negative matrix factorization (NMF) clustering. At days 3 and 7 post-hospitalization, patients with severe disease had an enrichment of a granulocytic myeloid derived suppressor cell-like state gene expression signature, while non-severe patients with resolved disease were enriched for a progenitor-like immature neutrophil state signature. Severe disease was associated with gene sets related to neutrophil degranulation, neutrophil extracellular trap (NET) signatures, distinct metabolic signatures, and enhanced neutrophil activation and generation of reactive oxygen species (ROS). We found that the majority of patients had a transient interferon-stimulated gene signature upon presentation to the emergency department (ED) defined here as Day 0, regardless of disease severity, which persisted only in patients who subsequently died. Humoral responses were identified as potential drivers of neutrophil effector functions, as enhanced antibody-dependent neutrophil phagocytosis and reduced NETosis was associated with elevated SARS-CoV-2-specific IgG1-to-IgA1 ratios in plasma of severe patients who survived. In vitro experiments confirmed that while patient-derived IgG antibodies mostly drove neutrophil phagocytosis and ROS production in healthy donor neutrophils, patient-derived IgA antibodies induced a predominant NETosis response. Overall, our study demonstrates neutrophil dysregulation in severe COVID-19 and a potential role for IgA-dominant responses in driving neutrophil effector functions in severe disease and mortality.

Early immune response in mice immunized with a semi-split inactivated vaccine against SARS-CoV-2 containing S protein-free particles and subunit S protein (preprint)

The development of a vaccine against COVID-19 is a hot topic for many research laboratories all over the world. Our aim was to design a semi-split inactivated vaccine offering a wide range of multi-epitope determinants important for the immune system including not only the spike (S) protein but also the envelope, membrane and nucleocapsid proteins. We designed a semi-split vaccine prototype consisting of S protein-depleted viral particles and free S protein. Next, we investigated its immunogenic potential in BALB/c mice. The animals were immunized intradermally or intramuscularly with the dose adjusted with buffer or addition of aluminum hydroxide, respectively. The antibody response was evaluated by plasma analysis at 7 days after the first or second dose. The immune cell response was studied by flow cytometry analysis of splenocytes. The data showed a very early onset of both S protein-specific antibodies and virus-neutralizing antibodies at 90% inhibition regardless of the route of vaccine administration. However, significantly higher levels of neutralizing antibodies were detected in the intradermally (geometric mean titer - GMT of 7.8 {+/-} 1.4) than in the intramuscularly immunized mice (GMT of 6.2 {+/-} 1.5). In accordance with this, stimulation of cellular immunity by the semi-split vaccine was suggested by elevated levels of B and T lymphocyte subpopulations in the murine spleens. These responses were more predominant in the intradermally immunized mice compared with the intramuscular route of administration. The upward trend in the levels of plasmablasts, memory B cells, Th1 and Th2 lymphocytes, including follicular helper T cells, was confirmed even in mice receiving the vaccine intradermally at a dose of 0.5 g. We demonstrated that the semi-split vaccine is capable of eliciting both humoral and cellular immunity early after vaccination. Our prototype thus represents a promising step toward the development of an efficient anti-COVID-19 vaccine for human use.

RAPPID: a platform of ratiometric bioluminescent sensors for homogeneous immunoassays (preprint)

Heterogeneous immunoassays such as ELISA have become indispensable in modern bioanalysis, yet translation into easy-to-use point-of-care assays is hindered by their dependence on external calibration and multiple washing and incubation steps. Here, we introduce RAPPID (Ratiometric Plug-and-Play Immunodiagnostics), a "mix-and-measure" homogeneous immunoassay platform that combines highly specific antibody-based detection with a ratiometric bioluminescent readout that can be detected using a basic digital camera. The concept entails analyte-induced complementation of split NanoLuc luciferase fragments, photoconjugated to an antibody sandwich pair via protein G adapters. We also introduce the use of a calibrator luciferase that provides a robust ratiometric signal, allowing direct in-sample calibration and quantitative measurements in complex media such as blood plasma. We developed RAPPID sensors that allow low-picomolar detection of several protein biomarkers, anti-drug antibodies, therapeutic antibodies, and both SARS-CoV-2 spike protein and anti-SARS-CoV-2 antibodies. RAPPID combines ratiometric bioluminescent detection with antibody-based target recognition into an easy-to-implement standardized workflow, and therefore represents an attractive, fast, and low-cost alternative to traditional immunoassays, both in an academic setting and in clinical laboratories for point-of-care applications.

Plasma proteomics reveals tissue-specific cell death and mediators of cell-cell interactions in severe COVID-19 patients (preprint)

COVID-19 has caused over 1 million deaths globally, yet the cellular mechanisms underlying severe disease remain poorly understood. By analyzing several thousand plasma proteins in 306 COVID-19 patients and 78 symptomatic controls over serial timepoints using two complementary approaches, we uncover COVID-19 host immune and non-immune proteins not previously linked to this disease. Integration of plasma proteomics with nine published scRNAseq datasets shows that SARS-CoV-2 infection upregulates monocyte/macrophage, plasmablast, and T cell effector proteins. By comparing patients who died to severely ill patients who survived, we identify dynamic immunomodulatory and tissue-associated proteins associated with survival, providing insights into which host responses are beneficial and which are detrimental to survival. We identify intracellular death signatures from specific tissues and cell types, and by associating these with angiotensin converting enzyme 2 (ACE2) expression, we map tissue damage associated with severe disease and propose which damage results from direct viral infection rather than from indirect effects of illness. We find that disease severity in lung tissue is driven by myeloid cell phenotypes and cell-cell interactions with lung epithelial cells and T cells. Based on these results, we propose a model of immune and epithelial cell interactions that drive cell-type specific and tissue-specific damage in severe COVID-19.

Looking for pathways related to COVID-19 phenotypes: Confirmation of pathogenic mechanisms by SARS-CoV-2 - Host interactome. (preprint)

In the last months, many studies have clearly described several mechanisms of SARS-CoV-2 infection at cell and tissue level. Host conditions and comorbidities were identified as risk factors for severe and fatal disease courses, but the mechanisms of interaction between host and SARS-CoV-2 determining the grade of COVID-19 severity, are still unknown. We provide a network analysis on protein-protein interactions (PPI) between viral and host proteins to better identify host biological responses, induced by both whole proteome of SARS-CoV-2 and specific viral proteins. A host-virus interactome was inferred on published PPI, using an explorative algorithm (Random Walk with Restart) triggered by all the 28 proteins of SARS-CoV-2, or each single viral protein one-by-one. The functional analysis for all proteins, linked to many aspects of COVID-19 pathogenesis, allows to identify the subcellular districts, where SARS-CoV-2 proteins seem to be distributed, while in each interactome built around one single viral protein, a different response was described, underlining as ORF8 and ORF3a modulated cardiovascular diseases and pro-inflammatory pathways, respectively. Finally, an explorative network-based approach was applied to Bradykinin Storm, highlighting a possible direct action of ORF3a and NS7b to enhancing this condition. This network-based model for SARS-CoV-2 infection could be a framework for pathogenic evaluation of specific clinical outcomes. We identified possible host responses induced by specific proteins of SARS-CoV-2, underlining the important role of specific viral accessory proteins in pathogenic phenotypes of severe COVID-19 patients.