Nitrous oxide misuse: a clue not to be missed in young patients with venous thromboembolism.

A 27-year-old man presented with altered mental status and unilateral right lower limb swelling. Brain imaging and cerebrospinal fluid analysis were unremarkable. He reported history of nitrous oxide misuse after he recovered from his delirium. The diagnosis of drug induced psychosis was made. The right lower limb swelling was found to be due to extensive deep vein thrombosis. In another case, a 21-year-old woman presented with headache, vomiting and dipoplia. Brain imaging showed extensive cerebral venous sinus thrombosis. She also misused nitrous oxide. Both cases had low-normal vitamin B12 and elevated methylmalonic acid, consistent with nitrous oxide misuse. The woman was found to have elevated homocysteine because of functional vitamin B12 deficiency. Homocysteine was not measured in the man. Raised homocysteine is associated with increased thrombosis risk. Fourteen cases of nitrous oxide misuse associated arterial and venous thrombosis have been reported. These two cases highlighted the importance of inquiring about recreational drug use in young patients who presented with apparently unprovoked venous thromboembolism.

Increased Peripheral Blood Neutrophil Activation Phenotypes and Neutrophil Extracellular Trap Formation in Critically Ill Coronavirus Disease 2019 (COVID-19) Patients: A Case Series and Review of the Literature.

BACKGROUND: Increased inflammation has been well defined in coronavirus disease 2019 (COVID-19), while definitive pathways driving severe forms of this disease remain uncertain. Neutrophils are known to contribute to immunopathology in infections, inflammatory diseases, and acute respiratory distress syndrome, a primary cause of morbidity and mortality in COVID-19. Changes in neutrophil function in COVID-19 may give insight into disease pathogenesis and identify therapeutic targets. METHODS: Blood was obtained serially from critically ill COVID-19 patients for 11 days. Neutrophil extracellular trap formation (NETosis), oxidative burst, phagocytosis, and cytokine levels were assessed. Lung tissue was obtained immediately postmortem for immunostaining. PubMed searches for neutrophils, lung, and COVID-19 yielded 10 peer-reviewed research articles in English. RESULTS: Elevations in neutrophil-associated cytokines interleukin 8 (IL-8) and interleukin 6, and general inflammatory cytokines IFN-inducible protien-19, granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin 1ß, interleukin 10, and tumor necrosis factor, were identified both at first measurement and across hospitalization (P < .0001). COVID-19 neutrophils had exaggerated oxidative burst (P < .0001), NETosis (P < .0001), and phagocytosis (P < .0001) relative to controls. Increased NETosis correlated with leukocytosis and neutrophilia, and neutrophils and NETs were identified within airways and alveoli in lung parenchyma of 40% of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected lungs available for examination (2 of 5). While elevations in IL-8 and absolute neutrophil count correlated with disease severity, plasma IL-8 levels alone correlated with death. CONCLUSIONS: Literature to date demonstrates compelling evidence of increased neutrophils in the circulation and lungs of COVID-19 patients. Importantly, neutrophil quantity and activation correlates with severity of disease. Similarly, our data show that circulating neutrophils in COVID-19 exhibit an activated phenotype with enhanced NETosis and oxidative burst.

Stent-Save a Life international survey on the practice of primary percutaneous coronary intervention during the COVID-19 pandemic.

Aim: To assess the impact of the COVID-19 pandemic on admissions of patients with acute coronary syndromes (ACS) and primary percutaneous coronary intervention (PPCI) in countries participating in the Stent-Save a Life (SSL) global initiative. Methods and Results: We conducted a multicenter observational survey to collect data on patient admissions for ACS, ST-elevation myocardial infarction (STEMI) and PPCI in participating SSL member countries through a period during the COVID-19 outbreak (March and April 2020) compared with the equivalent period in 2019. Of the 32 member countries of the SSL global initiative, 17 agreed to participate in the survey (three in Africa, five in Asia, six in Europe and three in Latin America). Overall reductions of 27.5% and 20.0% were observed in admissions for ACS and STEMI, respectively. The decrease in PPCI was 26.7%. This trend was observed in all except two countries. In these two, the pandemic peaked later than in the other countries. Conclusions: This survey shows that the COVID-19 outbreak was associated with a significant reduction in hospital admissions for ACS and STEMI as well as a reduction in PPCI, which can be explained by both patient- and system-related factors.

Objetivos: Avaliar o impacto da pandemia COVID-19 nas admissões de doentes com síndromes coronárias agudas (SCA) e angioplastia coronária primária (PPCI) em países que participam da iniciativa global Stent-Save a Life (SSL). Métodos e resultados: Realizámos estudo observacional multicêntrico para coletar dados sobre admissões de doentes por ACS, STEMI e PPCI nos países participantes no SSL durante um período do surto COVID-19 (março e abril de 2020) em comparação com o período homólogo de 2019. Dos 32 países membros da iniciativa global SSL, 17 aceitaram participar no estudo (3 de África, 5 da Ásia, 6 da Europa e 3 da América Latina (LATAM)). Observámos uma redução global de 27,5% e 20,0% nos internamentos com SCA e STEMI, respetivamente. A diminuição do PPCI foi de 26,7%. Essa tendência foi observada em todos os países, exceto dois. Nestes dois países, a pandemia atingiu o pico mais tarde do que nos restantes. Conclusões: Este estudo mostra que o surto de COVID-19 foi associado a uma redução significativa de admissões hospitalares por SCA e STEMI, bem como uma redução de PPCI, o que pode ser explicado por fatores relacionados com o doente e com o sistema.

Variants in nucleocapsid protein and endoRNase are found to associate with severe COVID-19 in a case-control study in Washington State, USA (preprint)

SARS-CoV-2 is spreading worldwide with continuously evolving variants, some of which occur in the Spike protein and appear to increase the viral transmissibility. However, variants that cause severe COVID-19 or lead to other breakthroughs have not been well characterized. To discover such viral variants, we assembled a cohort of 683 COVID-19 patients; 388 inpatients (“cases”) and 295 outpatients (“controls”) from April to August 2020 using electronically captured COVID test request forms and sequenced their viral genomes. To improve the analytic power, we accessed 7,137 viral sequences in Washington State to filter out viral single nucleotide variants (SNVs) that did not have significant expansions over the collection period. Applying this filter led to the identification of 53 SNVs that were statistically significant, of which 13 SNVs each had 3 or more variant copies in the discovery cohort. Correlating these selected SNVs with case/control status, eight SNVs were found to significantly associate with inpatient status (q-values<0.01). Using temporal synchrony, we identified a four SNV-haplotype (t19839-g28881-g28882-g28883) which was significantly associated with case/control status (Fisher’s exact p=2.84*10 −11 ) that appeared in April 2020, peaked in June, and persisted into January 2021. This association was replicated (OR=5.46, p-value=4.71*10 −12 ) in an independent cohort of 964 COVID-19 patients (June 1, 2020 to March 31, 2021). The haplotype included a synonymous change N73N in endoRNase, and three non-synonymous changes coding residues R203K, R203S and G204R in the nucleocapsid protein. This discovery points to the potential functional role of the nucleocapsid protein in triggering “cytokine storms” and severe COVID-19 that led to hospitalization. The study further emphasizes a need for tracking and analyzing viral sequences in correlations with clinical status.

Coronavirus Pseudotypes for All Circulating Human Coronaviruses for Quantification of Cross-Neutralizing Antibody Responses.

The novel coronavirus SARS-CoV-2 is the seventh identified human coronavirus. Understanding the extent of pre-existing immunity induced by seropositivity to endemic seasonal coronaviruses and the impact of cross-reactivity on COVID-19 disease progression remains a key research question in immunity to SARS-CoV-2 and the immunopathology of COVID-2019 disease. This paper describes a panel of lentiviral pseudotypes bearing the spike (S) proteins for each of the seven human coronaviruses (HCoVs), generated under similar conditions optimized for high titre production allowing a high-throughput investigation of antibody neutralization breadth. Optimal production conditions and most readily available permissive target cell lines were determined for spike-mediated entry by each HCoV pseudotype: SARS-CoV-1, SARS-CoV-2 and HCoV-NL63 best transduced HEK293T/17 cells transfected with ACE2 and TMPRSS2, HCoV-229E and MERS-CoV preferentially entered HUH7 cells, and CHO cells were most permissive for the seasonal betacoronavirus HCoV-HKU1. Entry of ACE2 using pseudotypes was enhanced by ACE2 and TMPRSS2 expression in target cells, whilst TMPRSS2 transfection rendered HEK293T/17 cells permissive for HCoV-HKU1 and HCoV-OC43 entry. Additionally, pseudotype viruses were produced bearing additional coronavirus surface proteins, including the SARS-CoV-2 Envelope (E) and Membrane (M) proteins and HCoV-OC43/HCoV-HKU1 Haemagglutinin-Esterase (HE) proteins. This panel of lentiviral pseudotypes provides a safe, rapidly quantifiable and high-throughput tool for serological comparison of pan-coronavirus neutralizing responses; this can be used to elucidate antibody dynamics against individual coronaviruses and the effects of antibody cross-reactivity on clinical outcome following natural infection or vaccination.

Towards Discovering SARS-CoV-2 Variants of High Consequence Based on Both Surveillance and Electronically Captured Health Data: First Year Experience in Washington State (January 2020-2021) (preprint)

Background: SARS-CoV-2 is continuously evolving with the emergence of variants of interest (VOI) or with variants of concern (VOC). While Variants of High Consequence (VOHC) are well defined, no such variants have been formally documented. Here we propose an integrated strategy and application towards discovering VOHC. Methods: We utilized 7,137 viral sequences collected from COVID-19 cases in Washington State from January 19, 2020 to January 31, 2021, to identify genome-wide viral single nucleotide variants (SNVs). Utilizing a non-parametric regression model, we selected a subset of SNVs that had significant and substantial expansions over the collection period. Further, using unsupervised learning, we identified multiple SNVs forming haplotypes. To evaluate their clinical relevance, we assembled a discovery cohort of COVID-19 cases (388 inpatients and 295 outpatients) to identify SNVs and haplotypes associated with hospitalization status, a proxy for disease severity. A logistic regression model was used to assess associations of SNVs with hospitalization status in the discovery cohort. These results were validated on an independent cohort of 964 genome sequences derived from COVID-19 cases in Washington State from June 1, 2020 to March 31, 2021. Finding: The analysis of the 7,137 sequences led to identification of 107 SNVs that were statistically significant (false positive error rate q-value <0.01) and substantial expansions (maximum value of locally averaged proportions, Pmax>0.10). Forty-one SNVs were considered urgent, because their SNV proportions persisted or expanded above 10% in January 2021, the last month of the current investigation period. Correlating with clinical data, eight SNVs were found to significantly associate with inpatient status (p-values<0.001). By their synchronized dynamics, two SNVs were haplotyped and the mutant haplotype (c15933t-g16968t) was observed among patients in the discovery cohort (Fisher’s exact p=1.53*10-10), and this association was validated in the validation cohort (OR=5.38, p=10-9). Similarly, a haplotype with 4 SNVs (t19839c-g28881a-g28882a-g28883c) was observed only among inpatients (p=1.53*10-10) in the discovery cohort. Discovered haplotypic association was validated in the independent validation cohort (OR=3.69, p-value=3.44*10-10) and was further validated after adjusting for sex, age and collection time (OR=5.46, p-value=4.71*10-12). Interpretation: The mutant haplotype t19839c-g28881a-g28882a-g28883c emerged in April 2020, remained undetected over eight months, and has now begun to re-emerge. Because of its strong association with hospitalization status and re-emergence, this mutant haplotype may be a candidate variant for VOHC, pending further investigation of a) its clinical association with the disease severity, b) asymptomatic transmissibility and/or c) immune evasion to approved vaccines. While preliminary, this result indicates the importance to conduct purpose-driven clinical follow up studies to discover and validate candidate variants for VOHC. Also of interest is the mutant haplotype c15933t-g16968t which expanded in May 2020 but subsided by October 2020. Due to its association with hospitalization, we recommend continued monitoring for re-emergence of this variant and further assessment of viral phenotype.Funding: National Institutes of Health grant R01-GM129325 National Institutes of Health/National Institute of Allergy and Infectious Diseases grant UM1 AI068635Declaration of Interest: The authors declare that they have no competing interests.Ethical Approval: This study was approved by the Human Subject Review Committee at Fred Hutchinson Cancer Research Center (IRB#6007-2043) and by the University of Washington Institutional Review Board (STUDY00000408).

Towards Discovering SARS-CoV-2 Variants of High Consequence Based on Both Surveillance and Electronically Captured Health Data: First Year Experience in Washington State (January 2020-2021) (preprint)

Background: SARS-CoV-2 is continuously evolving with the emergence of variants of interest (VOI) or with variants of concern (VOC). While Variants of High Consequence (VOHC) are well defined, no such variants have been formally documented. Here we propose an integrated strategy and application towards discovering VOHC.Methods: We utilized 7,137 viral sequences collected from COVID-19 cases in Washington State from January 19, 2020 to January 31, 2021, to identify genome-wide viral single nucleotide variants (SNVs). Utilizing a non-parametric regression model, we selected a subset of SNVs that had significant and substantial expansions over the collection period. Further, using unsupervised learning, we identified multiple SNVs forming haplotypes. To evaluate their clinical relevance, we assembled a discovery cohort of COVID-19 cases (388 inpatients and 295 outpatients) to identify SNVs and haplotypes associated with hospitalization status, a proxy for disease severity. A logistic regression model was used to assess associations of SNVs with hospitalization status in the discovery cohort. These results were validated on an independent cohort of 964 genome sequences derived from COVID-19 cases in Washington State from June 1, 2020 to March 31, 2021.Finding: The analysis of the 7,137 sequences led to identification of 107 SNVs that were statistically significant (false positive error rate q-value <0.01) and substantial expansions (maximum value of locally averaged proportions, Pmax>0.10). Forty-one SNVs were considered urgent, because their SNV proportions persisted or expanded above 10% in January 2021, the last month of the current investigation period. Correlating with clinical data, eight SNVs were found to significantly associate with inpatient status (p-values<0.001). By their synchronized dynamics, two SNVs were haplotyped and the mutant haplotype (c15933t-g16968t) was observed among patients in the discovery cohort (Fisher’s exact p=1.53*10-10), and this association was validated in the validation cohort (OR=5.38, p=10-9). Similarly, a haplotype with 4 SNVs (t19839c-g28881a-g28882a-g28883c) was observed only among inpatients (p=1.53*10-10) in the discovery cohort. Discovered haplotypic association was validated in the independent validation cohort (OR=3.69, p-value=3.44*10-10) and was further validated after adjusting for sex, age and collection time (OR=5.46, p-value=4.71*10-12). Interpretation: The mutant haplotype t19839c-g28881a-g28882a-g28883c emerged in April 2020, remained undetected over eight months, and has now begun to re-emerge. Because of its strong association with hospitalization status and re-emergence, this mutant haplotype may be a candidate variant for VOHC, pending further investigation of a) its clinical association with the disease severity, b) asymptomatic transmissibility and/or c) immune evasion to approved vaccines. While preliminary, this result indicates the importance to conduct purpose-driven clinical follow up studies to discover and validate candidate variants for VOHC. Also of interest is the mutant haplotype c15933t-g16968t which expanded in May 2020 but subsided by October 2020. Due to its association with hospitalization, we recommend continued monitoring for re-emergence of this variant and further assessment of viral phenotype.Funding Information: National Institutes of Health grant R01-GM129325. National Institutes of Health/National Institute of Allergy and Infectious Diseases grant UM1 AI068635Declaration of Interests: None to declare. Ethics Approval Statement: This study was approved by the Human Subject Review Committee at Fred Hutchinson Cancer Research Center (IRB#6007-2043) and by the University of Washington Institutional Review Board (STUDY00000408).

Fibrinolytic Strategy for ST-Segment-Elevation Myocardial Infarction: A Contemporary Review in Context of the COVID-19 Pandemic.

The ongoing coronavirus disease 2019 pandemic has resulted in additional challenges for systems designed to perform expeditious primary percutaneous coronary intervention for patients presenting with ST-segment-elevation myocardial infarction. There are 2 important considerations: the guideline-recommended time goals were difficult to achieve for many patients in high-income countries even before the pandemic, and there is a steep increase in mortality when primary percutaneous coronary intervention cannot be delivered in a timely fashion. Although the use of fibrinolytic therapy has progressively decreased over the last several decades in high-income countries, in circumstances when delays in timely delivery of primary percutaneous coronary intervention are expected, a modern fibrinolytic-based pharmacoinvasive strategy may need to be considered. The purpose of this review is to systematically discuss the contemporary role of an evidence-based fibrinolytic reperfusion strategy as part of a pharmacoinvasive approach, in the context of the emerging coronavirus disease 2019 pandemic.