Molecular epidemiology of invasive Group A Streptococcal infections before and after the COVID-19 pandemic in Switzerland (preprint)

Group A Streptococcus (GAS, aka Streptococcus pyogenes) poses a significant public health concern, causing a diverse spectrum of infections with high mortality rates. Following the COVID-19 pandemic, a resurgence of invasive GAS (iGAS) infections has been documented, necessitating efficient outbreak detection methods. Whole genome sequencing (WGS) serves as the gold standard for GAS molecular typing, albeit constrained by time and costs. This study aimed to characterize the postpandemic increased prevalence of iGAS on the molecular epidemiological level in order to assess whether new, more virulent variants have emerged, as well as to assess the performance of the rapid and cost-effective Fourier-transform infrared (FTIR) spectroscopy as an alternative to WGS for detecting and characterizing GAS transmission routes. A total of 66 iGAS strains isolated from nine Swiss hospitals during the COVID-19 post-pandemic increased GAS prevalence were evaluated and compared to 15 strains collected before and 12 during the COVID-19 pandemic. FT-IR measurements and WGS were conducted for network analysis. Demographic, clinical, and epidemiological data were collected. Skin and soft tissue infection was the most common diagnosis, followed by primary bacteremia and pneumonia. Viral co-infections were found in 25% of cases and were significantly associated with more severe disease requiring intensive care unit admission. WGS analysis did not reveal emerging GAS genetic distinct variants after the COVID-19 pandemic, indicating the absence of a pandemic-induced shift. FT-IR spectroscopy exhibited limitations in differentiating genetically distant GAS strains, yielding poor overlap with WGS-derived clusters. The emm1/ST28 gebotype was predominant in our cohort and was associated with five of the seven deaths recorded, in accordance with the molecular epidemiological data before the pandemic. Additionally, no notable shift in antibiotic susceptibility patterns was observed. Our data suggest that mainly non-pathogen related factors contributed to the recent increased prevalence of iGAS.

Herpesvirus Reactivations in Critically-Ill COVID-19 Patients with Autoantibodies Neutralizing Type I Interferons (preprint)

Autoantibodies neutralizing the antiviral action of type I interferons (IFNs) have been associated with pre-disposition to severe COVID-19. Here, we screened for such autoantibodies in 103 critically-ill COVID-19 patients in a tertiary intensive care unit in Switzerland. Eleven patients (10.7%), but no healthy donors, had neutralizing anti-IFN or anti-IFN/anti-IFN{omega} IgG in plasma/serum, but anti-IFN IgM or IgA was rare. One patient had non-neutralizing anti-IFN IgG. Strikingly, all patients with plasma anti-IFN IgG also had anti-IFN IgG in tracheobronchial secretions, identifying these autoantibodies at anatomical sites relevant for SARS-CoV-2 infection. Longitudinal analyses revealed patient heterogeneity in terms of increasing, decreasing, or stable anti-IFN IgG levels throughout the length of hospitalization. Notably, presence of anti-IFN autoantibodies in this critically-ill COVID-19 cohort appeared to predict herpesvirus (herpes simplex viruses types 1 and 2, HSV-1/-2; and/or cytomegalovirus, CMV) reactivations, which are linked to worse clinical outcomes. Indeed, all seven tested COVID-19 patients with anti-IFN IgG in our cohort (100%) suffered from one or more herpesvirus reactivations, and analysis revealed that these patients were substantially more likely to experience CMV reactivation than COVID-19 patients without anti-IFN autoantibodies, even when adjusting for systemic steroid treatment (odds ratio 7.28, 95%-CI 1.14-46.31, p=0.036). As the IFN system deficiency caused by neutralizing anti-IFN autoantibodies likely directly and indirectly exacerbates herpesvirus reactivations in critically-ill patients, early diagnosis of anti-IFN IgG could be rapidly used to inform risk-group stratification and treatment options.

Blunted Fas signaling favors RIPK1-driven neutrophil necroptosis in critically ill COVID-19 patients (preprint)

Critically ill COVID-19 patients are characterized by a severely dysregulated cytokine profile and elevated neutrophil counts, which are thought to contribute to disease severity. However, to date it remains unclear how neutrophils contribute to pathophysiology during COVID-19. Here, we assessed the impact of the dysregulated cytokine profile on the tightly regulated cell death program of neutrophils. We show that in a subpopulation of neutrophils, canonical apoptosis was skewed towards rapidly occurring necroptosis. This phenotype was characterized by abrogated caspase-8 activity and increased RIPK1 levels, favoring execution of necroptosis via the RIPK1-RIPK3-MLKL axis, as further confirmed in COVID-19 biopsies. Moreover, reduction of sFas-L levels in COVID-19 patients and hence decreased signaling to Fas directly increased RIPK1 levels and correlated with disease severity. Our results suggest an important role for Fas signaling in the regulation of cell death program ambiguity via the ripoptosome in neutrophils during COVID-19 and a potential therapeutic target to curb inflammation and thus influence disease severity and outcome.

IgA autoantibodies target pulmonary surfactant in patients with severe COVID-19 (preprint)

Complications affecting the lung are hallmarks of severe coronavirus disease 2019 (COVID-19). While there is evidence for autoimmunity in severe COVID-19, the exact mechanisms remain unknown. Here, we established a prospective observational cohort to study lung specific autoantibodies (auto-Abs). Incubation of plasma from severe COVID-19 patients with healthy human lung tissue revealed the presence of IgA antibodies binding to surfactant-producing pneumocytes. Enzyme-linked immunosorbent assays (ELISA) and protein pull-downs using porcine surfactant confirmed the presence of auto-Abs binding to surfactant proteins in severe COVID-19 patients. Mass spectrometry and ELISAs with recombinant proteins identified IgA auto-Abs that target human surfactant proteins B and C. In line with these findings, lungs of deceased COVID-19 patients showed reduced pulmonary surfactant. Our data suggest that IgA-driven autoimmunity against surfactant may result in disease progression of COVID-19.

Differential effects of antiseptic mouth rinses on SARS-CoV-2 infectivity in vitro (preprint)

SARS-CoV-2 is detectable in saliva from asymptomatic individuals, suggesting the potential necessity for the use of mouth rinses to suppress viral load to reduce virus spread. Published studies on anti-SARS-CoV-2 activities of antiseptics determined by virus-induced cytotoxic effects cannot exclude antiseptic-associated cytotoxicity. Here, we determined the effect of commercially available mouth rinses and antiseptic povidone-iodine on the infectivity of pseudotyped SARS-CoV-2 virus. We first determined the effect of mouth rinses on cell viability to ensure that antiviral activity was not a consequence of mouth rinse-induced cytotoxicity. Colgate Peroxyl (hydrogen peroxide) exhibited the most cytotoxicity, followed by povidone-iodine-10% solution, chlorhexidine gluconate-0.12% (CHG), and Listerine (essential oils and alcohol). Analysis of the anti-viral activity of mouth rinses at non-cytotoxic concentrations showed that 1.5% (v/v) diluted CHG was a potent inhibitor when present in cells during infection, but the potency was reduced when CHG was removed after viral attachment, suggesting that the prolonged effect of mouth rinses on cells impacts the anti-viral activity. To minimalize mouth rinse-associated cytotoxicity, we pelleted treated-viruses to remove most of the mouth rinse prior to infection of cells. Colgate Peroxyl or povidone-iodine at 5% (v/v) completely blocked the viral infectivity. Listerine or CHG at 5% (v/v) had a moderate suppressive effect on the virus, and 50% (v/v) Listerine or CHG blocked the viral infectivity completely. Prolonged incubation of virus with mouth rinses was not required to block viral infectivity. Our results indicate that mouth rinses can significantly reduce virus infectivity, suggesting their potential use to reduce SARS-CoV-2 spread.

Neutrophil and monocyte dysfunctional effector response towards bacterial challenge in critically-ill COVID-19 patients (preprint)

COVID-19 displays diverse disease severities and symptoms. Elevated inflammation mediated by hypercytokinemia induces a detrimental dysregulation of immune cells. However, there is limited understanding of how SARS-CoV-2 pathogenesis impedes innate immune signaling and function against secondary bacterial infections. We assessed the influence of COVID-19 hypercytokinemia on the functional responses of neutrophils and monocytes upon bacterial challenges from acute and corresponding recovery COVID-19 ICU patients. We show that severe hypercytokinemia in COVID-19 patients correlated with bacterial superinfections. Neutrophils and monocytes from acute COVID-19 patients showed severely impaired microbicidal capacity, reflected by abrogated ROS and MPO production as well as reduced NETs upon bacterial challenges. We observed a distinct pattern of cell surface receptor expression on both neutrophils and monocytes leading to a suppressive autocrine and paracrine signaling during bacterial challenges. Our data provide insights into the innate immune status of COVID-19 patients mediated by their hypercytokinemia and its transient effect on immune dysregulation upon subsequent bacterial infections

Severe COVID-19 is associated with elevated serum IgA and antiphospholipid IgA-antibodies (preprint)

BackgroundWhile the pathogenesis of coronavirus disease 2019 (COVID-19) is becoming increasingly clear, there is little data on IgA response, the first line of bronchial immune defense. ObjectiveTo determine, whether COVID-19 is associated with a vigorous total IgA response and whether IgA autoantibodies are associated with complications of severe illness. Since thrombotic events are frequent in severe COVID-19 and resemble hypercoagulation of antiphospholipid syndrome (APS), our approach focused on antiphospholipid antibodies (aPL). Materials and methodsIn this retrospective cohort study we compared clinical data and aPL from 64 patients with COVID-19 from three independent centers (two in Switzerland, one in Liechtenstein). Samples were collected from April 9, 2020 to May 1, 2020. Total IgA and aPL were measured with FDA-approved commercially available clinical diagnostic kits. ResultsClinical records of the 64 patients with COVID-19 were reviewed and divided into a cohort with mild illness (mCOVID, n=26 [41%]), a discovery cohort with severe illness (sdCOVD, n=14 [22%]) and a confirmation cohort with severe illness (scCOVID, n=24 [38%]). Severe illness was significantly associated with increased total IgA (sdCOVID, P=0.01; scCOVID, P<0.001). Total IgG levels were similar in both cohorts. Among aPL, both cohorts with severe illness significantly correlated with elevated anti-Cardiolipin IgA (sdCOVID and scCOVID, P<0.001), anti-Cardiolipin IgM (sdCOVID, P=0.003; scCOVID, P<0.001), and anti-Beta2 Glycoprotein-1 IgA (sdCOVID and scCOVID, P<0.001). Systemic lupus erythematosus was excluded from all patients as a potential confounder of APS. ConclusionsHigher total IgA and IgA-aPL were consistently associated with severe illness. These novel data strongly suggest that a vigorous antiviral IgA-response triggered in the bronchial mucosa induces systemic autoimmunity.

Bacterial but no SARS-CoV-2 contamination after terminal disinfection of tertiary care intensive care units treating COVID-19 patients (preprint)

Background: In intensive care units (ICUs) treating patients with Coronavirus disease 2019 (COVID-19) invasive ventilation poses a high risk for aerosol and droplet formation. Surface contamination of severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) or bacteria can result in nosocomial transmission.Methods: Two tertiary care COVID-19 intensive care units treating 53 patients for 870 patient days were sampled after terminal cleaning and preparation for regular use to treat non-COVID-19 patients.Results: A total of 176 swabs were sampled of defined locations covering both ICUs. No SARS-CoV-2 ribonucleic acid (RNA) was detected. Gram-negative bacterial contamination was mainly linked to sinks and siphons. Skin flora was isolated from most swabbed areas and Enterococcus faecium was detected on two keyboards.Conclusions: After basic cleaning with standard disinfection measures no remaining SARS-CoV-2 RNA was detected. Bacterial contamination was low and mainly localised in sinks and siphons.

Timing COVID-19 - Synchronization of longitudinal patient data to the underlying disease progression using CRP as a temporal marker (preprint)

Advances in medical technology and IT infrastructure have led to increased availability of continuous patient data that allows to investigate the longitudinal progression of novel and known diseases in unprecedented detail. However, to accurately describe any underlying pathophysiology with longitudinal data, the individual patient trajectories have to be synchronized based on temporal markers. In this study, we use longitudinal data from 28 critically ill ICU COVID-19 patients to compare the commonly used alignment markers "onset of symptoms", "hospital admission" and "ICU admission" with a novel objective method based on the peak value of inflammatory marker C-reactive protein (CRP). By applying our CRP-based method to align the progression of neutrophils and lymphocytes, we were able to define a pathophysiological window that allowed further mortality risk stratification in our COVID-19 patient cohort. Our data highlights that proper synchronization of patient data to the underlying pathophysiology is crucial to differentiate severity subgroups and to allow reliable interpatient comparisons.