METAGENOMIC INTERROGATION OF RESPIRATORY AND GUT MICROBIAL COMMUNITIES, HOST IMMUNE RESPONSE, AND RISK OF SEVERE COVID-19

Background: Human-associated microbial communities have been linked to host immune response to respiratory viral infections. Prior investigations have observed shifts in the composition of the gut or respiratory microbiome in severe COVID-19. However, there has been no comprehensive metagenomic evaluation of the interaction between lower respiratory and gut microbiomes and host immune factors in COVID-19. Methods: From April 2020 to May 2021, we prospectively enrolled 153 hospitalized patients with mild (n=12), moderate (n=65), and severe (n=76) COVID-19 infection categorized using established clinical criteria. We longitudinally collected stool (n=270) for metagenomic profiling, and in a subset, we generated comprehensive host-microbiome-molecular profiles by collecting sputum metagenomes (n=87 participants with 212 samples) and blood cytokine levels (n=109 with 181 samples) weekly until hospital discharge. We performed omnibus testing of overall gut and respiratory community structure, species-level differential abundance testing using mixed effects modeling accounting for repeated sampling, hierarchical clustering of paired gut and respiratory metagenomic profiles, and multi-omic machine learning classification of disease severity. Results: Patients with severe COVID-19 tended to be older, were more frequently male, had higher rates of overweight/obesity, and a greater mean Charlson Comorbidity Index. Patients with severe COVID-19 infection had significantly decreased stool and respiratory microbiome a-diversity irrespective of antibiotic administration. COVID severity accounted for a small proportion of variance in stool (R2=2.4%, p=0.002) and sputum (R2=4.4%, p= 0.03) profiles. Hierarchical clustering of paired gut and respiratory samples from patients with severe COVID revealed the joint expansion of oral-typical taxa typically present during systemic inflammation (i.e., increases in Streptococcus and Peptostreptococus spp. in both gut and sputum). A pro-inflammatory milieu defined by a composite elevation of circulating plasma cytokines (e.g., IL-6, TNF-a, and IL-29 among others) were linked to broad microbial excursions in community structure for both stool and sputum as measured by Bray-Curtis distances. A random forest classifier incorporating either stool or sputum taxonomic features and accounting for age, sex, body mass index, and recent antibiotic use achieved excellent classification of biospecimens from patients with severe vs. non-severe COVID patients (AUROC > 0.80). Conclusions: Alterations of the gut and respiratory microbiome were associated with differences in host immune response and COVID-19 disease severity. Further studies are needed to identify the potential role of human-associated microbial communities as a biomarker for poor patient outcomes in COVID-19 who may warrant escalated levels of care.(Figure Presented) Fig. 1. (A) Using unsupervised feature selection (species abundance > 0.001) inclusive of taxa differentially abundant by non-parametric Wilcoxon rank-sum testing (nominal p-value < 0.05), (B) we performed random forest classification using a twice-repeated 5-fold crossvalidation scheme to predict COVID-19 disease severity from shotgun metagenomic stool profiles (C) yielding an AUROC of 0.91.

A safety study of intermittent versus continuous inhaled no therapy in spontaneously breathing covid-19 patients: A randomized controlled trial

Introduction: Nitric oxide (NO) inhalation (iNO) therapy has been approved as a salvage therapy in patients with acute respiratory distress syndrome. However, the optimal patterns of therapeutic administration and iNO efficacy in COVID-19 patients with hypoxemia have not been studied so far. Hypothesis: A combination of high-concentration intermittent iNO treatment with continuous lowconcentration NO gas supplementation between therapeutic high-dose iNO sessions may be a safe approach in COVID-19 patients with hypoxemia compared with the high-dose spike therapy alone. Methods: A single-center randomized controlled trial examined the safety of administration of intermittent high-dose iNO therapy versus the intermittent therapy in the presence of continuous low-dose iNO in spontaneously breathing COVID-19 patients (www.clinicaltrials.gov #NCT04476992). The study comprised adult patients tested positive for the COVID-19 by RT-PCR, who required oxygen and was admitted to the respiratory hospital. After attrition, a total of 20 patients were enrolled and randomized to the group of intermittent high-dose iNO treatment combined with continuous low-dose NO supplementation (group 1) or the group of intermittent high-dose iNO treatment(group 2). Control group (group 3) comprised retrospective patients who received conventional treatment for COVID-19 in hospital. Results: Administration of intermittent high-dose iNO therapy combined with continuous low-dose NO supplementation significantly improved physiological interactions in the heart-lung blood circulation system and was not associated with the extrapulmonary organ dysfunction. Safety parameters are presented in Table 1. Conclusions: High-dose intermittent iNO therapy combined with continuous low-dose NO gas supplementation may be considered a safe adjuvant modality of treatment for COVID-19 in spontaneously breathing hypoxemic patients.

Breathing nitric oxide at high dose in severe-critical COVID-19 during pregnancy: A case series

Introduction: Pregnant patients with a severe form of COVID-19 are at increased risk of maternal and fetal complications. Nitric Oxide (NO) gas is a selective pulmonary vasodilator currently approved to treat newborns with pulmonary hypertension. Inhaled NO has been safely used in patients with severe pneumonia and for cases of pregnant patients with pulmonary hypertension. The antimicrobial effect of NO has been confirmed against bacteria and viruses. In vitro study demonstrated a dose-dependent effect of NO against SARS-CoV-1 and 2. We hypothesize that breathing NO at 160-200 ppm twice daily for 30 minutes in spontaneously breathing pregnant patients might provide a safe and effective treatment for COVID-19. Methods: We retrospectively reviewed the data of 6 pregnant patients hospitalized for COVID-19 treated with inhaled NO. Nitric Oxide was delivered at 160- 200 ppm for 30 minutes twice daily until resolution of respiratory symptoms or negative RT-PCR for SARS-CoV- 2. Demographic and clinical data were collected to assess cardiopulmonary function and safety during the treatment. For safety, we focused on the values of blood methemoglobin (MetHb) and inhaled Nitrogen Dioxide (NO2). Data regarding newborn delivery and health, and 28 days outcomes of mothers and babies were collected. Results: Six pregnant patients were admitted with a severe (2 patients) or critical (4 patients) form of COVID-19 and received inhaled NO therapy between April and June 2020. Two pregnant patients were in the second trimester, while 4 were in the third trimester. A total of 39 treatments were administered. No adverse events were reported relating to NO administration. MetHb peaked at 2.5% (1.95%-3%, safety limit =5%) and inhaled NO2 remained below the safety limit of 2ppm. The patients remained hemodynamically stable;cardiac ultrasound performed in three patients did not detect any rebound pulmonary hypertension after NO interruption. Oxygen saturation improved in hypoxemic patients after the initiation of NO (Figure-1). All patients experienced a reduction in respiratory rate (by a median 4.5breaths/min after NO initiation). Three patients delivered a total of 4 babies (negative for SARS-CoV-2) while the other 3 remained pregnant after hospitalization (gestational age 22-26-33weeks) with normal follow-ups. Five mothers out of 6 tested negative for COVID-19 28 days after hospitalization. Conclusion: Nitric oxide gas at 160-200 ppm was safely administered to pregnant patients with severe-critical COVID-19, improved oxygenation and reduced respiratory rate in all 6 patients. The clinical effectiveness shown suggests inhaled high dose NO as a therapeutic novel therapy for COVID-19 in pregnancy.

Identifying clinical and biochemical phenotypes in acute respiratory distress syndrome secondary to coronavirus disease- 2019

RationaleAcute respiratory distress syndrome (ARDS) secondary to Coronavirus Disease-2019 (COVID-19) is characterized by substantial heterogeneity in clinical, biochemical, and physiological characteristics. However, the pathophysiology of severe COVID-19 infection is poorly understood. Among “classical' ARDS cohorts, previous studies established two predominant biological phenotypes - patients with and without evidence of a hyperinflammatory response - with important prognostic and therapeutic implications. The phenotypic profile of COVID-19 associated ARDS remains unknown. Methods We used latent class modeling via a multivariate mixture model to identify phenotypes from clinical and biochemical data collected from 263 patients admitted to Massachusetts General Hospital intensive care unit with COVID-19-associated ARDS between March 13 and August 2, 2020. Classdefining variables included demographic features, respiratory parameters, hematologic and inflammatory biomarkers, and markers of end-organ function. Interleukin-6 (IL-6) and fibrinogen levels, which were available for n = 53 and n = 189 patients, respectively, were incorporated post-hoc. Results We identified two distinct latent classes representing 74.4% (Class 1, n = 193) and 26.6% (Class 2, n = 70) of the cohort, respectively. Posterior probability of class assignment was high (median 98.2%, IQR [98.0%, 100%]). To understand each class's distinguishing biological features, we compared the standardized mean of the continuous class-defining variables (Fig. 1A). The minority phenotype (class 2, n = 70, 26.6%) demonstrated increased markers of vascular dysregulation, with mild relative hyper-inflammation and dramatically increased markers of end-organ dysfunction (e.g., creatinine, troponin). There was little distinction according to respiratory parameters. The class 2 phenotype was characterized by significantly decreased fibrinogen and increased IL- 6 compared to Class 1 (Fig. 1B), even though these variables were not used in the statistical inference. Furthermore, the 28-day mortality among the class 2 phenotype was more than double that of the class 1 phenotype (40.0% vs. 23.3%, OR 2.3, 95% CI [1.3, 4.1]). Conclusion We identified distinct phenotypic profiles in COVID-19 associated ARDS, with little variation according to respiratory physiology but with important variation according to systemic and extra-pulmonary markers. Phenotypic identity was highly associated with shortterm mortality risk. The class 2 phenotype exhibited prominent signatures of vascular dysregulation, suggesting that vascular dysfunction may play an important role in the clinical progression of severe COVID-19-related disease.

Ideation and Assessment of a Novel Nitric Oxide Delivery System for Spontaneously Breathing Subjects S. Gianni1

Rationale High dose nitric oxide (NO) produces broad antimicrobial activity and several clinical trials are now testing the efficacy of NO inhalation on patients infected by SARS CoV-2. Nitrogen dioxide (NO2) is formed by the reaction between NO and oxygen, and when combined with water in the airways, NO2, forms nitric acid, leading to a caustic burn of the airways. In this study, we designed and developed a breathing system capable of safely delivering high concentrations of NO. Methods We developed a gas delivery system that utilizes standard respiratory circuit connectors, a reservoir bag, and a scavenging chamber containing calcium hydroxide (Ca(OH)2) (Fig. 1). The system was tested using a bench testing lung and a mechanical ventilator. The NO concentration was measured by a NO analyzer connected to the inspiratory limb via a sampling line. NO2 levels were simultaneously evaluated by the Cavity Attenuated Phase Shift (CAPS) NO2 monitor using the same sampling port. To assess the efficacy of the Ca(OH)2 scavenger in reducing the inspiratory levels of NO2, we used a range of target NO concentrations (50, 150, and 250 ppm), two different levels of FiO2 (0.21 and 0.40) and measured NO2 levels with and without the scavenger. We administered high-dose NO with our system to healthy adult volunteers as part of a trial conducted at MGH. Each administration lasted for 15 minutes. Peripheral oxygen saturation (SpO2) and methemoglobin (MetHb) were continuously monitored with a pulse co-oximeter. Results Using our delivery system, we were able to reach NO concentration up 250 ppm with an Inspired oxygen fraction (FiO2) between 0.21 and 0.4. The scavenger reduced the inhaled NO2 concentration to an average of 0.9 ppm (CI -1.58, -0.22;p=0.01). At 150 ppm of inhaled NO, the NO2 concentration was maintained below 1.2 ppm with FiO2 from 0.21 to 0.40. Our data suggest that the scavenger can efficiently reduce NO2 in the circuit for NO delivery. We administered NO to 4 adult volunteers. The total number of NO administrations was 30. The average concentration of inspired NO was 163.9±10.1 ppm with NO2 levels of 0.75±0.08 ppm. During the administration methemoglobin levels increased from a baseline value of 0.97±0.6% to 2.17±0.43%. The subjects did not experience any adverse events. Conclusions We built a NO delivery system that provides a safe alternative to a ventilator-based system to give high dose NO to spontaneously breathing patients.

Titration of mechanical ventilation in supine compared to prone position reveals different respiratory mechanics behavior in COVID19 patients

INTRODUCTION: The prone position and protective lung ventilation are the only interventions to improve survival in Acute Respiratory Distress Syndrome (ARDS) patients. Due to early reports during the COVID-pandemic showing dramatic improvements in oxygenation, the use of prone position has been broadly adopted in intubated patients around the globe. However, it remains unclear on whether titration of ventilation should be reassessed when the patient is repositioned. Therefore, the objective of this study was to characterize the response of respiratory mechanics in supine and prone positions during a decremental end-expiratory positive pressure trial in COVID-19 related ARDS patients. METHODS: This is a retrospective analysis of patients with COVID-19 related ARDS under invasive mechanical ventilation in supine and prone positions. The study was approved by the Investigational Review Board at the Massachusetts General Hospital and by the Ethics and Research Committee at Heart Institute (InCor) from the University of São Paulo. Prone position was recommended based on hypoxia, measured as PaO2/FIO2 ratio (< 150 mmHg). Patients were sedated, and under volume-controlled ventilation (5-6 mL/Kg PBW). Airway pressure, flow, esophageal pressure and electrical impedance tomography (EIT) were recorded. A decremental PEEP trial was performed on supine and prone position. RESULTS: We included 10 patients with COVID-19 related ARDS. Median age was 62 years (range, 35-72), 5 patients (50%) were female, and BMI was 35 (range, 27-46). After 24 hours of intubation, median PaO2/FIO2 was 174 mmHg (IQR, 166-192), PEEP was 10 cmH2O (IQR, 10-14.5), and static compliance of respiratory system (CRS) was 28.5 mL/cmH2O (IQR, 24.2-35.7). The time interval between intubation and the supine-prone assessment was 7 days (IQR, 5-10). During the supine/prone assessment, a variety of CRS responses were observed among patients (Figure 1). Overall, the highest CRS was 44 mL/cmH2O (IQR, 29-57) in supine and 52 mL/cmH2O (IQR, 39-67) in prone position. At the highest CRS, from supine to prone position: lung compliance (CL) increased by 15 mL/cmH2O (IQR, 13-31), suggesting lung recruitment, and chest wall compliance (CCW) was reduced by 28 ml/cmH2O (IQR, 14-48) indicating external compression of the chest;and end-expiratory transpulmonary pressure (PLend-exp) increased from-3.4 cmH2O (IQR,-4.6 to-2.5) to 0.4 cmH2O (IQR, 0.1-3.0) suggesting decreased pleural pressure. CONCLUSION: Patients with COVID-19 related ARDS assessed in supine and prone positions revels a variety response to prone position on CRS during decremental PEEP trial, suggesting the necessity to reassess the PEEP when the patient is repositioned. (Table Presented).