Molecular epidemiology of SARS-CoV-2, RSV and FLU in children with Severe Acute Respiratory Infection

Covid-19 in children can cause serious cases just like in adults. In our environment, the characterization of respiratory viruses that cause severe acute respiratory infection (SARI) is not known since the beginning of the Covid-19 pandemic. Objective: to evaluate the epidemiological behavior of SARS CoV-2, RSV and FLU as the cause of SARI in patients admitted to the Manuel Ascencio Villarroel Children's Hospital in Cochabamba. Methods: 41 patients up to 5 years of age admitted from June 2021 to June 2022 were included. Polymerase chain reaction (RT-qPCR) was used to detect SARS-CoV-2, RSV and FLU type A and B. Results: RSV was detected in 47.6% of patients, SARS-CoV-2 in 42.9%, and coinfection between SARS CoV-2 and RSV in 9.5%. There were no cases of FLU infection. Fever and cough were reported mainly in 76.2% and 61.9% respectively. 14.3% of patients were admitted to intensive care, two patients died, one with a viral coinfection SARS CoV-2/RSV and the other with simple viral infection by SARS CoV-2. Conclusions: after the easing of restrictions following the start of the Covid-19 pandemic, RSV and SARS CoV-2 were found to be the agents causing SARI with a similar frequency of circulation. Respiratory manifestations are more frequent, showing mostly stable states and favorable recovery in most cases. Constant epidemiological surveillance is necessary given the experience of the Covid-19 pandemic. © 2022 The authors.

Diagnostic Test Kinetics, Infectivity, and Immunological Responses Among Unvaccinated Adults During Acute SARS-CoV-2 Infection

Background. Appropriate diagnostic testing can be used to inform infection control measures and reduce SARS-CoV-2 transmission, yet the test kinetics, infectivity, and immunological responses during acute, non-severe SARS-CoV-2 infection need clarity. Methods. We conducted a prospective cohort study between Nov 2020-July 2021 in Seattle, Washington of 95 unvaccinated, immunocompetent adults with no prior SARS-CoV-2 infection. Nasal swabs (nasopharyngeal and anterior) and blood serum samples were serially collected at six visits over two months. Viral RNA, N and S antigen concentrations, and viral growth/infectivity were measured from nasal samples. Anti-S total antibody and IgG assays were performed on serum. We fit loess curves to quantitative data corresponding to each testing modality by days since symptom onset (DSSO) and compared qualitative test results across time points to demonstrate timedependent agreement of PCR, N antigen, and culture results. Generalized estimating equations were used to approximate relative risk of culture positivity (a proxy for infectiousness) for positive vs. negative test results (antigen and PCR), stratified by presence/ absence of symptoms and DSSO. Sampling Schema Nasal swabs and venous blood were collected at visits 1-4;venous blood only at visits 5-6. All participants were enrolled within 14 days of symptom onset (median: 6) and 7 days of a positive test (median: 4). Results. Infections in this cohort (median age: 29y) were mild (no hospitalization). Median (IQR) time to negative result was 11 (4), 13 (6), and 20 (7) DSSO for culture growth, N antigen, and PCR tests, respectively. Viral RNA quantities declined more slowly than antigen and culturable virus;antibody titers rose rapidly 5-15 DSSO and plateaued 20-30 DSSO. All culture-positive samples collected 0-5 DSSO were positive by PCR, but relative risk of culture positivity (infectiousness) for positive vs. negative PCR results declined 6-10 DSSO. Relative risk of culture positivity for positive vs. negative antigen results was consistently high 0-10 DSSO, with similar results when stratified by presence of symptoms. Diagnostic test kinetics and immunological responses Diagnostic test kinetics and immunological responses measured in adults with non-severe, symptomatic SARS-CoV-2 infection: loess trendlines and 95% confidence intervals are given for SARS-CoV-2 viral load (calculated from PCR Ct value using a calibration curve), TCID50 from viral culture, mean concentrations of nucleocapsid and spike antigen proteins, and anti-S total and IgG antibody concentrations. Conclusion. The results reinforce the importance of molecular PCR testing as a highly sensitive diagnostic tool but with limited utility as an indicator of viral culturability and likely infectiousness. N antigen testing may be a preferable diagnostic test within two weeks of symptom onset, especially 6-10 DSSO, because it more closely correlates with culture growth over the course of infection.

Psycho-Covid

Introduction: The COVID-19 pandemic generated a health emergency and led to the adoption of different measures, including home quarantine and social isolation, which, as we have seen, has had an impact on the mental health of the majority of citizens, with the possibility of psychiatric disorders appearing. in people without prior mental illness, such as acute decompensations in patients with known disorders, more vulnerable to environmental stressors. Objective(s): Learn and rethink alarm signals in extreme situations such as the one experienced in recent months, as well as observe the impact, negative in many cases, but positive in others, of the patients we treat daily. Method(s): Description through brief clinical cases of the impact of the COVID-19 pandemic on psychotic patients and the decompensation that it has entailed, including due to confinementmeasures and social isolation, associated with over-information through the media, chaos initial and the uncertainty that it caused and the associated fear. Result(s): Restrictions as a result of COVID-19 have played a very relevant role as an external stressor for the appearance of psychopathological alterations, including psychotic symptoms. In addition, people who suffer from psychosis or at risk of psychotic disorder can be especially affected and trigger acute psychopathology with social isolation, loss of daily routines, unemployment, homelessness. Conclusion(s): These cases are an example that shows the need for an early and effective approach to the rise in mental illnesses in circumstances of this caliber.

Psycho-Covid

Introduction The COVID-19 pandemic generated a health emergency and led to the adoption of different measures, including home quarantine and social isolation, which, as we have seen, has had an impact on the mental health of the majority of citizens, with the possibility of psychiatric disorders appearing. in people without prior mental illness, such as acute decompensations in patients with known disorders, more vulnerable to environmental stressors. Objectives Learn and rethink alarm signals in extreme situations such as the one experienced in recent months, as well as observe the impact, negative in many cases, but positive in others, of the patients we treat daily. Methods Description through brief clinical cases of the impact of the COVID-19 pandemic on psychotic patients and the decompensation that it has entailed, including due to confinement measures and social isolation, associated with over-information through the media, chaos initial and the uncertainty that it caused and the associated fear. Results Restrictions as a result of COVID-19 have played a very relevant role as an external stressor for the appearance of psychopathological alterations, including psychotic symptoms. In addition, people who suffer from psychosis or at risk of psychotic disorder can be especially affected and trigger acute psychopathology with social isolation, loss of daily routines, unemployment, homelessness. Conclusions These cases are an example that shows the need for an early and effective approach to the rise in mental illnesses in circumstances of this caliber. Disclosure No significant relationships.

SARS-CoV-2 SPIKE BINDS FIBRINOGEN-INDUCING ABNORMAL INFLAMMATORY BLOOD CLOTS

Background: Life threatening thrombotic events involving both the arterial and venous systems are prominently present in SARS-CoV-2 infected individuals presenting with severe COVID-19. Abnormal clotting also occurs in asymptomatically or mildly infected individuals and in people experiencing post-acute sequelae of SARS-CoV-2 infection (PASC). Clinical management of this clotting disorder has proven difficult in part because these fibrin clots are highly resistant to plasmin-mediated fibrinolysis. Methods: An array of different binding, biochemical, microscopic, and in vivo assays were performed in these studies. All experiments were performed at least three times in triplicate and reported differences were shown to be statistically significant. Results: We find that SARS-CoV-2 Spike directly binds to the terminal clotting factors, fibrinogen and fibrin (Kd of 5.3 μ M and 0.4 μ M respectively). Mixing Spike and plasma accelerates fibrin polymerization. Scanning electron microscopy reveals an abnormal clot structure with finer, denser, and roughened fibrin fibers. Scanning peptide competition assays indicate Spike binds fibrin at three sites: 1) the plasmin cleavage site needed for fibrinolysis;2) a site involved in innate immune signaling via fibrin binding to Complement Receptor 3 (CR3);and 3) a site with no known function. Examination of mice injected 24h earlier with Spike pseudotyped HIV-ΔEnv virions reveals extensive intra-and extravascular fibrin deposition in the lung accompanied by endothelial activation, loss of tight junctions, increased influx of macrophages, and the generation of high levels of reactive oxygen species. This thromboinflammatory response is not observed when Bald virions are injected or when Spike pseudotyped virions are injected into mice lacking fibrinogen. Intriguingly, these Spike-induced proinflammatory effects are blocked by an anti-fibrin monoclonal antibody, 5B8, which interferes with fibrin binding to CR3. Conclusion: Our findings reveal that the SARS-CoV-2 Spike protein binding to fibrinogen/fibrin results in the formation of structurally abnormal, fibrinolysis-resistant blood clots whose inflammatory effects are effectively neutralized by a specific fibrin-targeting monoclonal antibody. While COVID-19 clotting was thought to occur as a result of systemic inflammation, our findings suggest clotting during SARS-CoV-2 infection in fact is a driver of inflammation. Targeting fibrin could lead to novel therapeutic approaches for patients with acute COVID-19 and PASC.

NEUROPILIN-1 MEDIATES SARS-CoV-2 INFECTION of ASTROCYTES PROMOTING NEURON DYSFUNCTION

Background: SARS-CoV-2 primarily infects the lung but may also damage other organs including the brain, heart, kidney, and intestine. Central nervous system (CNS) disorders include loss of smell and taste, headache, delirium, acute psychosis, seizures, and stroke. Pathological loss of gray matter occurs in SARS-CoV-2 infection but it is unclear whether this is due to direct viral infection, indirect effects associated with systemic inflammation, or both. Methods: We used iPSC-derived brain organoids and primary human astrocytes from cerebral cortex to study direct SARS-CoV-2 infection, as confirmed by Spike and Nucleocapsid immunostaining and RT-qPCR. siRNAs, blocking antibodies, and small molecule inhibitors were used to assess SARS-CoV-2 receptor candidates. Bulk RNA-seq, DNA methylation seq, and Nanostring GeoMx digital spatial profiling were utilized to identify virus-induced changes in host gene expression. Results: Astrocytes were robustly infected by SARS-CoV-2 in brain organoids while neurons and neuroprogenitor cells supported only low-level infection. Based on siRNA knockdowns, Neuropilin-1, not ACE2, functioned as the primary receptor for SARS-CoV-2 in astrocytes. The endolysosomal two-pore channel protein, TPC, also facilitated infection likely through its regulatory effects on endocytosis. Other alternative receptors, including the AXL tyrosine kinase, CD147, and dipeptidyl protease 4 (DPP4), did not function as SARS-CoV-2 receptors in astrocytes. SARS-CoV-2 infection dynamically induced type I, II, and III interferons, and genes involved in Toll-like receptor signaling, MDA5 and RIG-I sensing of double-stranded RNA, and production of inflammatory cytokines. Genes activating apoptosis were also increased. Down-regulated genes included those involved in water, ion and lipid transport, synaptic transmission, and formation of cell junctions. Epigenetic analyses revealed transcriptional changes related to DNA methylation states, particularly decreased DNA methylation in interferon-related genes. Long-term viral infection of brain organoids resulted in progressive neuronal degeneration and death. Conclusion: Our findings support a model where SARS-CoV-2 infection of astrocytes produces a panoply of changes in the expression of genes regulating innate immune signaling and inflammatory responses. Deregulation of these genes in astrocytes produces a microenvironment within the CNS that ultimately disrupts normal neuron function, promoting neuronal cell death and CNS deficits.

Integrating pocus education with critical care in the era of distance learning

Learning Objectives: Teach the use of POCUS in critically ill patients with respect to image acquisition, image interpretation, and clinical decision-making in the setting of distance learning. : Introduction: Point-of-care ultrasound (POCUS) is integral to Emergency Medicine Residency training and often a fundamental component of a senior medical student EM rotation. The Covid-19 pandemic has dramatically limited in-person instruction and necessitated innovative methods of ultrasound education. Using video-conferencing software, we created a novel simulation experience that integrates POCUS into the core EM content delivery of a virtual EM sub-internship. Curricular Design: Following a brief didactic session, a group of 20 sub-interns was divided into 5 “breakout rooms,” each with 1 resident facilitator. The students then progressed through 4 critical care cases in slide format. For each case, students were able to choose from a variety of diagnostic and therapeutic options, and when the students selected POCUS, they then chose both the order and anatomic region of the scans. Images of normal and abnormal findings were provided in GIF format as they pertained to the given case. After verbalizing and interpreting the findings, students could then perform additional scans or interventions until the patient was stabilized. Following the initial session, some ambiguity was added to the vignettes to increase the number of scans typically performed prior to intervening. Impact/Effectiveness: Based on post-session feedback, students felt this approach was highly effective in helping interpret POCUS images and apply the information to a clinical scenario. Mean Likert scale feedback on organization, applicability to clinical practice, and effectiveness was 4.92 out of 5 for each of the categories based on 25 total responses. Written feedback revealed students would have preferred less didactic time and more time with cases. Qualitatively, this feedback did not notably differ from similar in-person sessions held previously. Similar approaches could be used to teach these skills to providers of all levels from the next room or a location across the world.

HUMAN PRE-IMPLANTATION EMBRYOS ARE PERMISSIVE TO SARS-COV-2 ENTRY

Objective: To determine whether human pre-implantation embryos have the potential to be infected by SARS-CoV-2, the virus responsible for COVID-19. Design: Assessment of expression levels of SARS-CoV-2 entry mediators in human embryo biopsies by RNAseq analysis, and infection of cultured embryos with SARS-CoV-2 Spike glycoprotein pseudotyped reporter virions expressing green fluorescent protein (GFP). Materials and Methods: Trophectoderm biopsies from blastocyst-stage embryos (n=24) were processed for RNAseq using a commercial kit and sequenced;results were analyzed for expression of factors implicated in SARS-CoV-2 cellular entry. For viral infection experiments, blastocyst-stage embryos (n=94) were hatched from zonas mechanically, and infected by spinoculation with GFP-reporter virions pseudotyped with the SARS-CoV-2 Spike glycoprotein (required for SARS-CoV-2 entry). Embryos were subsequently monitored for fluorescence at 24-48 hours post-infection. Various control conditions were used as specified in the ‘results’ section. A mixed population of euploid, aneuploid, and untested embryos used in the study were from IVF treatment, donated to research by signed informed consent. The project was approved by an Institutional Review Board. Results: Cells collected from blastocyst-stage embryos robustly expressed the canonical SARS-CoV-2 entry receptor ACE2 and the putative activator protease TMPRSS2, in addition to other reported entry factors. Embryos exposed to reporter virions pseudotyped with SARS-CoV-2 Spike glycoprotein displayed robust GFP signal, often in numerous cells with cytoplasmic localization. Specificity was confirmed by the absences of fluorescence in embryos treated with virions lacking the Spike glycoprotein (“bald” virus), or when embryos were spinoculated with media alone in the absence of virus. Embryos exposed to Spike glycoprotein-positive reporter virus in the presence of neutralizing anti Spike- or anti-ACE2-blocking antibodies exhibited negligible GFP signal, while control monoclonal IgG antibody-treated embryos maintained GFP expression. These results implicated the canonical Spike-ACE2 axis in the viral entry. Lastly, embryos exposed to reporter virions pseudotyped with Spike glycoprotein of SARS-CoV-1 (which also enters cells via ACE2) displayed GFP fluorescence, while embryos exposed to reporter viruses pseudotyped with Spike glycoprotein of MERS (which utilizes Dipeptidyl Peptidase IV (DPP4) instead of ACE2) resulted in no fluorescence. Conclusions: Our results indicate that cells present in pre-implantation embryos are permissive to the canonical Spike-ACE2 viral entry mechanism utilized by SARS-CoV-2. These results encourage further investigation into the potential of SARS-CoV-2 infection in human embryos and may have wider implications in natural conception and ART practice.