Evaluation and limitations of the novel chemiluminescent enzyme immunoassay technique for measuring total tau protein in the cerebrospinal fluid in patients with human prion disease: a 10-year prospective study (2011–2020) (preprint)

Background: Recently, the investigation of cerebrospinal fluid (CSF) biomarkers for diagnosing human prion diseases (HPD) has garnered significant attention. Reproducibility and accuracy are paramount in biomarker research, particularly in the measurement of total tau (t-tau) protein, which is a crucial diagnostic marker. Given the global impact of the coronavirus disease pandemic, the frequency of measuring this protein using one of the world's fully automated assays, chemiluminescent enzyme immunoassay (CLEA), has increased. At present, the diagnosis and monitoring of neurological diseases mainly rely on traditional methods, but their accuracy and responsiveness are limited.there is a limited knowledge on the accuracy of CLEA in Tau measurements. We aimed to measure t-tau protein using CLEA and to elucidate its merits and limitations. Methods: We analysed CSF samples obtained from 91 patients with rapidly progressive dementia using ELISA and CLEA. Additionally, we used western blotting to detect the presence of 14-3-3 protein and employed real-time quaking-induced conversion (RT-QuIC) assays to analyse the same set of samples. Furthermore, we examined the correlation coefficient between ELISA and CLEA results in a subset of 30 samples. Moreover, using CLEA, we evaluated the diurnal reproducibility, storage stability, dilutability, and freeze-thaw effects in three selected samples. Results:Among the 91 patients, a total of 45 (22 men and 23 women) tested positive for HPD in the RT-QuIC assay. In contrast, all CSF samples from the remaining 46 patients without HPD (23 men and 23 women) tested negative in the RT-QuIC assay. Both ELISA and CLEA showed perfect sensitivity and specificity (100%) in measuring t-tau protein levels. Furthermore, there was a strong correlation coefficient (R² = 0.9363) between ELISA and CLEA results. However, despite its advantages, CLEA analysis exhibited instability for certain samples with t-tau protein levels exceeding 2,000 pg/mL, leading to low reproducibility during dilution analysis. Conclusions: Our findings indicate that CLEA outperforms ELISA in terms of diurnal reproducibility, storage stability, and freeze-thaw effects. However, ELISA demonstrated superior performance in the dilution assay. Therefore, it is imperative to develop innovative approaches for the dilution of biomarker samples for CLEA measurements during clinical trials.

Inflammatory proteins as strong predictors of death in COVID-19 patients with coexisting neurological diseases (preprint)

Background: Although many studies published so far on COVID-19 have examined its clinical prognosis, there is still no universal laboratory test that can assess the risk of a fatal outcome in patients with coexisting neurological diseases. Methods: The plasma concentrations of C-reactive protein (CRP), procalcitonin (PCT), lactate dehydrogenase (LDH), ferritin, interleukin 6 (IL-6), D-dimers, and highly sensitive troponin I (hsTnI) were determined in a group of 400 consecutive in-patients with COVID-19 and concomitant neurological comorbidities. Results: The median concentration levels of most of the inflammatory mediators/indicators, calculated for the whole group of patients, remained above the normal reference ranges, whereas the median concentrations of these substances were much higher in the sub-group of patients who died. Backward stepwise logistic regression confirmed the statistically significant predictors of death in a descending order of odds ratios as follows: LDH (3.8), ferritin (2.8), hsTnI (2.0), IL-6 (1.7), and age (1.01). A concentration of hsTnI > 64 ng/L appeared to constitute a strong predictor of an unfavorable prognosis. Patients who were treated with lopinavir and ritonavir, who required mechanical ventilation, and treatment with dexamethasone presented with significant increase in the concentrations of all the studied inflammatory proteins and increased odds ratio for death. Conclusion: High plasma concentrations of pro-inflammatory proteins in patients suffering from COVID-19 and concomitant neurological diseases were associated with a more serious clinical course and an increased risk of death. The presence of these substances is worth monitoring as a valuable indicator of the current clinical condition of COVID-19 patients.

SARS-CoV-2 infects neurons, astrocytes, choroid plexus epithelial cells and pericytes of the human central nervous system (preprint)

SARS-CoV-2, the coronavirus responsible for the COVID-19 pandemic, is associated with a range of neurological manifestations including haemorrhage, thrombosis and ischaemic necrosis and encephalitits. However, the mechanism by which this occurs is unclear. Neurological disease associated with SARS-CoV-2 infection has been proposed to occur following direct infection of the central nervous system and/or indirect sequelae as a result of peripheral inflammation. We profiled ACE2 and TMPRSS2 in brain tissue from five healthy human donors, and observed expression of these proteins in astrocytes, neurons and choroid plexus epithelium within frontal cortex and medulla. Primary human astrocytes, neurons and choroid plexus epithelial cells supported productive SARS-CoV-2 infection in an ACE2- dependent manner. Infected cells supported the full viral lifecycle, releasing infectious virus particles. In contrast, primary brain microvascular endothelial cells, pericytes and microglia were refractory to SARS-CoV-2 infection. These data support a model whereby SARS-CoV-2 is neurotropic, and this may in part explain the neurological sequelae of infection. ImportanceA subset of patients with COVID-19 develop neurological symptoms, but the underlying cause is poorly understood. We observed that cells within normal human brain express the SARS-CoV-2 entry factors ACE-2 and TMPRRS2, with expression mainly observed within astrocytes, neurons and choroid plexus epithelium. Primary human astrocytes, neurons and choroid plexus epithelial cells cultured in vitro supported the full SARS-CoV-2 life cycle with a range of SARS-CoV-2 variants. This study demonstrates that cells of the human central nervous system express SARS-CoV-2 entry factors in vivo and support viral infection in vitro, thus supporting a model where neurological symptoms seen in some COVID-19 patients may be as a result of direct viral infection of the central nervous system. Furthermore, these data highlight the importance of investigating the ability of therapeutics to clear virus from this potential reservoir of infection.

Genetic Risk Factors for Severe and Fatigue Dominant Long COVID and Commonalities with ME/CFS Identified by Combinatorial Analysis (preprint)

Background Long COVID is a debilitating chronic condition that has affected over 100 million people globally. It is characterized by a diverse array of symptoms, including fatigue, cognitive dysfunction and respiratory problems. Studies have so far largely failed to identify genetic associations, the mechanisms behind the disease, or any common pathophysiology with other conditions such as ME/CFS that present with similar symptoms. Methods We used a combinatorial analysis approach to identify combinations of genetic variants significantly associated with the development of long COVID and to examine the biological mechanisms underpinning its various symptoms. We compared two subpopulations of long COVID patients from Sano Genetics' Long COVID GOLD study cohort, focusing on patients with severe or fatigue dominant phenotypes. We evaluated the genetic signatures previously identified in an ME/CFS population against this long COVID population to understand similarities with other fatigue disorders that may be triggered by a prior viral infection. Finally, we also compared the output of this long COVID analysis against known genetic associations in other chronic diseases, including a range of metabolic and neurological disorders, to understand the overlap of pathophysiological mechanisms. Results Combinatorial analysis identified 73 genes that were highly associated with at least one of the long COVID populations included in this analysis. Of these, 9 genes have prior associations with acute COVID-19, and 14 were differentially expressed in a transcriptomic analysis of long COVID patients. A pathway enrichment analysis revealed that the biological pathways most significantly associated with the 73 long COVID genes were mainly aligned with neurological and cardiometabolic diseases. Expanded genotype analysis suggests that specific SNX9 genotypes are a significant contributor to the risk of or protection against severe long COVID infection, but that the gene-disease relationship is context dependent and mediated by interactions with KLF15 and RYR3. Comparison of the genes uniquely associated with the Severe and Fatigue Dominant long COVID patients revealed significant differences between the pathways enriched in each subgroup. The genes unique to Severe long COVID patients were associated with immune pathways such as myeloid differentiation and macrophage foam cells. Genes unique to the Fatigue Dominant subgroup were enriched in metabolic pathways such as MAPK/JNK signaling. We also identified overlap in the genes associated with Fatigue Dominant long COVID and ME/CFS, including several involved in circadian rhythm regulation and insulin regulation. Overall, 39 SNPs associated in this study with long COVID can be linked to 9 genes identified in a recent combinatorial analysis of ME/CFS patient from UK Biobank. Among the 73 genes associated with long COVID, 42 are potentially tractable for novel drug discovery approaches, with 13 of these already targeted by drugs in clinical development pipelines. From this analysis for example, we identified TLR4 antagonists as repurposing candidates with potential to protect against long term cognitive impairment pathology caused by SARS-CoV-2. We are currently evaluating the repurposing potential of these drug targets for use in treating long COVID and/or ME/CFS. Conclusion This study demonstrates the power of combinatorial analytics for stratifying heterogeneous populations in complex diseases that do not have simple monogenic etiologies. These results build upon the genetic findings from combinatorial analyses of severe acute COVID-19 patients and an ME/CFS population and we expect that access to additional independent, larger patient datasets will further improve the disease insights and validate potential treatment options in long COVID.

A Systematic Review of Diffusion Microstructure Imaging (Dmi): Current and Future Applications in Neurology Research (preprint)

Background: Diffusion microstructure imaging (DMI) is a novel diffusion magnetic resonance imaging (MRI) technique that provides rich estimates of microscopic tissue properties, such as axon morphologies and fiber configurations. DMI has potential applications in neurology, where various diseases and disorders affect the brain tissue's microstructure and connectivity.Objectives: To investigate the current and future applications of DMI in neurology, covering various diseases and disorders such as brain tumors and metastases, Parkinson's syndromes, COVID-19-related neurological symptoms, temporal lobe epilepsy, and acute ischemic stroke.Methods: The PRISMA 2020 statement was followed. Four electronic databases were searched from inception to May the 5th 2023. Two reviewers independently screened, selected, and extracted data from the eligible studies.Results: Seven studies were included in the review. The studies showed that DMI can differentiate between various neurological diseases or disorders based on alterations in brain tissue microstructure and connectivity. The studies also showed that DMI can be superior to conventional diffusion imaging techniques, such as diffusion tensor imaging (DTI) and neurite orientation dispersion and density imaging (NODDI), in detecting subtle differences between pathological conditions.Conclusions: DMI is a powerful diffusion imaging technique that can provide rich estimates of microscopic tissue properties and differentiate between various neurological diseases or disorders. However, more research is needed to compare DMI with other imaging modalities or clinical measures and to evaluate longitudinal changes or treatment effects using DMI in neurological diseases or disorders.

Rethinking immunologic risk: a retrospective cohort study of severe SARS-CoV-2 infections in individuals with congenital immunodeficiencies (preprint)

Background. Debates on the allocation of medical resources during the COVID-19 pandemic revealed the need for a better understanding of immunologic risk. Studies highlighted variable clinical outcomes of SARS-CoV-2 infections in individuals with defects in both adaptive and innate immunity, suggesting additional contributions from other factors. Notably, none of these studies controlled for variables linked with social determinants of health. Objective. To determine the contributions of determinants of health to risk of hospitalization for SARS-CoV-2 infection among individuals with inborn errors of immunodeficiencies. Methods. This is a retrospective, single-center cohort study of 166 individuals with inborn errors of immunity, aged two months through 69 years, who developed SARS-CoV-2 infections from March 1, 2020 through March 31, 2022. Risks of hospitalization was assessed using a multivariable logistic regression analysis. Results. The risk of SARS-CoV-2-related hospitalization was associated with underrepresented racial and ethnic populations (odds ratio [OR] 5.29; confidence interval [CI], 1.76-17.0), a diagnosis of any genetically-defined immunodeficiency (OR 4.62; CI, 1.60-14.8), use of B cell depleting therapy within one year of infection (OR 6.1; CI, 1.05-38.5), obesity (OR 3.74; CI, 1.17-12.5), and neurologic disease (OR 5.38; CI, 1.61-17.8). COVID-19 vaccination was associated with reduced hospitalization risk (OR 0.52; CI, 0.31-0.81). Defective T cell function, immune-mediated organ dysfunction, and social vulnerability were not associated with increased risk of hospitalization after controlling for covariates. Conclusions. The associations between race, ethnicity, and obesity with increased risk of hospitalization for SARS-CoV-2 infection indicate the importance of variables linked with social determinants of health as immunologic risk factors for individuals with inborn errors of immunity.

Prevalence of SARS-CoV-2 antibodies and associated factors in the adult population of Belgium: a general population cohort study between March 2021 and April 2022 (preprint)

Background This cohort study assessed seroprevalence trends of SARS-CoV-2 antibodies in the general Belgian population between March 2021 and April 2022, and explored factors associated with seropositivity among the vaccinated and unvaccinated population. Seroreversion and its potential determinants were also examined. Methods A random sample of the adult population in Belgium was invited to provide a saliva sample and to complete a survey questionnaire. Participants were followed up twice for a new saliva sample and updated information. Antibodies were assessed with a semi-quantitative measure of anti-RBD (Receptor Binding Domain) IgG ELISA. Seven time periods were defined for estimating SARS-CoV-2 antibody prevalence using post stratification weights to match the population distribution. Seroreversion was defined as passing from a positive to a negative antibody test from one data collection point to the next. Potential determinants of seropositivity were assessed through hierarchical multiple logistic regressions. Results In total 6,178 valid observations were obtained from 2,768 individuals. SARS-CoV-2 antibody prevalence increased from 25.1% in the beginning of the study period to 92.3% in the end. Among the vaccinated population, factors significantly associated with a higher seropositivity were being younger, having a bachelor diploma, living with others, having had a vaccine in the last 3 months and having received a nucleic-acid vaccine or a combination. Lower seropositivity rates were observed among vaccinated people with a neurological disease and transplant patients. Factors significantly associated with a higher seropositivity rate among the unvaccinated population were having non-O blood type and being non-smoker. Among fully vaccinated people the seroreversion rate was much lower (0.3%) among those who had received their latest vaccine in the last 3 months compared to those who had received their latest vaccine more than 3 months ago (2.7%). Conclusions The rapid increase in antibody seropositivity in the general adult population in Belgium during the study period was driven by the vaccination campaign which ran at full speed during this period. Factors associated with higher and lower seropositivity were identified among the vaccinated and unvaccinated people.

Impact of SARS-CoV-2 infection and vaccination on migraine symptoms during the COVID-19 pandemic (preprint)

Introduction Migraine is one of the most disabling neurological diseases and is aggravated by anxiety, stress, and sleep dysfunction. Recent studies have shown worsening of migraine associated with the COVID-19 infection and the pandemic more broadly. COVID-19 vaccination has also been associated with symptomatic headache, with high frequency among migraineurs. We aim to assess the impact of COVID-19 diagnosis and vaccine administration on migraine, during the SARS-CoV-2 pandemic. Methods An online questionnaire was sent to migraine patients followed in a neurology outpatient clinic. The survey inquired about migraine symptoms and treatment changes, as well as sleep changes, anxiety and depressive symptoms during the pandemic, after a COVID-19 diagnosis and/or vaccination. Results Out of the 185 patients included, 108 (58.4%) reported a change in headache pattern (42% before a COVID-19 diagnosis) and 72.2% needed to escalate treatment during the pandemic, regardless of infection. Migraine worsening was associated with higher rates of persistent symptomatic headache. A change in sleep pattern was more frequently reported in migraineurs after COVID-19 and in the group with worsening migraine. Abnormal levels of anxiety and depression were high among patients with worsening migraine, irrespective of COVID-19 diagnosis. After vaccination, 49 (27.1%) described a change in headache pattern, with worsening pattern in 29 (16%), 48% up to 3 weeks. Conclusion Worsening of migraine was more likely associated to psychosocial factors related to the pandemic rather than COVID-19 diagnosis per se. COVID-19 vaccination might have acted as an additional but less relevant and temporary trigger for migraine worsening.

Neurological manifestations of Multisystem Inflammatory Syndrome in children (MIS-C) associated with SARS-CoV-2 infection: A clinico-demographic profile with short-term outcome in patients admitted to a tertiary care hospital in Eastern India. (preprint)

Background: In April 2020, a novel syndrome termed multisystem inflammatory syndrome in children (MIS-C), related to SARS-CoV-2 infection was first described. This syndrome has a wide spectrum of systemic involvement, has shown to include the nervous system as well. Our study aims to obtain a baseline clinical and demographic profile of varied neurological manifestations of MIS-C. It also aims to delineate a profile of short-term outcome in these patients with regards to residual neurological sequelae. Methods: A single-centre, retrospective, observational and hospital based study for 5 months was conducted among patients in the age group from 1 month to 12 years, with MIS-C with co-existing neurological symptoms. The subjects who had pre-existing neurological diseases were excluded from the study. A total of 34 subjects were collected. Post-discharge, each patient was followed up for a period of 1 month. The residual neurological deficits, if any, at discharge and follow-up. Results: The neurological complication found were Acute Symptomatic Seizure (29.4%), Aseptic Meningitis (23.5%), Encephalitis (11.8%), Guillain-Barré Syndrome (11.8%), Miller-Fisher Syndrome (2.9%), ADEM (8.8%), Autoimmune Encephalitis (8.8%) and Acute Haemorrhaegic Stroke (2.9%). 11.8% expired and 50% required P.I.C.U admissions. 23.5% had residual neurological deficit at discharge and 8.8% at follow-up after 1 month of discharge. Conclusions: In spite of great variability in manifestations, prognosis is favourable if early aggressive treatment is initiated.

Innate immune response to SARS-1 CoV-2 infection contributes to neuronal damage in human iPSC-derived peripheral neurons (preprint)

Severe acute respiratory coronavirus 2 (SARS-CoV-2) infection causes neurological disease in some patients suggesting that infection can affect both the peripheral and central nervous system (PNS and CNS, respectively). It is not clear whether the outcome of SARS-CoV-2 infection of PNS and CNS neurons is similar, and which are the key factors that cause neurological disease: SARS-CoV-2 infection or the subsequent immune response. Here, we addressed these questions by infecting human induced-pluripotent stem cell-derived CNS and PNS neurons with the beta strain of SARS-CoV-2. Our results show that SARS-CoV-2 infects PNS neurons more efficiently than CNS neurons, despite lower expression levels of angiotensin converting enzyme 2. Infected PNS neurons produced interferon lambda 1, several interferon stimulated genes and proinflammatory cytokines. They also displayed neurodegenerative-like alterations, as indicated by increased levels of sterile alpha and Toll/interleukin receptor motif-containing protein 1, amyloid precursor protein and alpha-synuclein and lower levels of nicotinamide mononucleotide adenylyltransferase 2 and beta-III-tubulin. Interestingly, blockade of the Janus kinase and signal transducer and activator of transcription pathway by Ruxolitinib did not increase SARS-CoV-2 infection, but reduced neurodegeneration, suggesting that an exacerbated neuronal innate immune response contributes to pathogenesis in the PNS.

Experiences of parents of children with rare neurogenetic conditions during the COVID-19 pandemic: an interpretative phenomenological analysis. (preprint)

BackgroundThe Coronavirus disease 2019 (COVID-19) pandemic has impacted parental and child mental health and wellbeing in the UK. This study aimed to explore the experiences of parents of children with rare neurological and neurodevelopmental conditions with a known or suspected genetic cause (neurogenetic) across the first year of the pandemic in the UK. MethodsSemi-structured interviews were conducted with 11 parents of children with rare neurogenetic conditions. Parents were recruited via opportunity sampling from the CoIN Study, a longitudinal quantitative study exploring the impact of the pandemic on the mental health and wellbeing of families with rare neurogenetic conditions. Interviews were analysed using Interpretative Phenomenological Analysis.ResultsFour main themes were identified: (1) “A varied impact on child wellbeing: from detrimental to ‘no big drama’”; (2) “Parental mental health and wellbeing: impact, changes and coping”; (3) “The world had shut its doors and that was that’: care and social services during the pandemic”; and (4) “Time and luck: abstract concepts central to parents understanding of their pandemic experience”. The majority of parents described experiencing an exacerbation of pre-pandemic challenges due to increased uncertainty and a lack of support, with a minority reporting minimal or positive effects of the pandemic on family wellbeing.ConclusionsThese findings offer a unique insight into the experiences parents of children with rare neurogenetic conditions across the first year of the pandemic in the UK. They highlight that the experiences of parents are not pandemic-specific, and will continue to be highly relevant in a non-pandemic context. Future support should to be tailored to the needs of families identified in this study and implemented across diverse future scenarios to promote coping and positive wellbeing.

COVID-19 encephalitis as a differential diagnosis of a Cyclosporine related Posterior Leukoencephalopathy Syndrome (PRES) (preprint)

PRES is a rare neurological disease possibly associated with the use of calcineurin inhibitors like cyclosporine A. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel coronavirus, is responsible for the outbreak of coronavirus disease 19 (COVID-19) and can cause neurological manifestations. We describe a case of CSA-related PRES whose diagnosis was difficult due to concurrent infection with SARS-CoV-2. The 16-year-old patient was known to have corticosteroid-resistant nephrotic syndrome secondary to minimal change disease. CSA was therefore introduced and on the fifth day of treatment, he presented with seizures followed by fever. Biological and MRI data were in favor of SARS-CoV-2 encephalitis. Relief of immunosuppression by discontinuation of CSA was decided and the patient was put on anticonvulsants. After being declared cured of COVID-19, which was without other clinical signs, the CSA was reintroduced but the patient presented with seizures the next day. This allowed us to rectify the diagnosis and relate the seizures to a CSA-related PRES. We concluded that infection with SARS-CoV-2 could be a differential diagnosis of a PRES related to anticalcineurins.

SIRT-1 connects autophagy and release of virus-containing vesicles during picornavirus infection (preprint)

ABSTRACT Enterovirus D68 is a re-emerging enterovirus which causes acute respiratory illness in infants. EV-D68 infection has recently been associated with Acute Flaccid Myelitis, a severe polio-like neurological disease that causes limb weakness and loss of muscle tone in infants. There is currently no FDA-approved drug or prophylactic vaccine against EV-D68. Here, we investigated the role of the histone deacetylase, SIRT-1, in autophagy and EV-D68 infection. We show that SIRT-1 plays an important role in both autophagy and EV-D68 infection. siRNA-mediated knockdown of the cellular protein blocks basal and stress-induced autophagy and reduces EV-D68 extracellular viral titers. The proviral activity of SIRT-1 does not require deacetylase activity, since transient expression of both wild-type and deacetylase-inactive SIRT-1 mutant plasmids increased EV-D68 release. In non-lytic conditions, EV-D68 is primarily released in extracellular vesicles, and SIRT-1 is required for this process. Knockdown of SIRT-1 further impedes EV-D68 release in the autophagy-deficient ATG-7 knockout cells. Knockdown of SIRT-1 also decreases titers of poliovirus (PV) and SARS-CoV-2, but not Coxsackievirus-B3 (CVB3). CVB3 is the only tested virus that fails to induce SIRT-1 translocation to the cytosol. Our data suggest a correlation between SIRT-1 translocation during viral infection and extracellular vesicle-mediated non-lytic release of infectious viral particles. SIGNIFICANCE Picornaviruses, including EV-D68, constitute a significant cause of human disease. EV-D68 infection generally causes mild respiratory tract infection in infants but has recently been implicated in a severe polio-like neurological disease, AFM. Given the lack of prophylactic vaccines or antivirals against EV-D68, identifying host factors that modulate EV-D68 infection is crucial. Here, we show that SIRT-1 regulates autophagy and EV-D68 infection. Knockdown of SIRT-1 blocked autophagy and impeded the non-lytic release of EV-D68 in extracellular vesicles. We also show that SIRT-1 modulates the release of SARS-CoV-2 and poliovirus but not Coxsackievirus-B3 virus. Our data suggest that many RNA viruses require SIRT-1 for egress and that targeting SIRT-1 could constitute a broad-spectrum antiviral strategy.

Changes in COVID-19-related mortality across key demographic and clinical subgroups: an observational cohort study using the OpenSAFELY platform on 18 million adults in England (preprint)

Objectives To quantify in absolute and relative terms how population-level COVID-19 death rates have changed in demographic and clinical subgroups. Design Retrospective cohort study on behalf of NHS England. Setting Linked primary care and death registry data from the OpenSAFELY-TPP platform, covering the first three pandemic waves in England (wave 1: March 23 to May 30, 2020; wave 2: September 7, 2020 to April 24, 2021; and wave 3, delta: May 28 to December 14, 2021). Participants In total, 18.7, 18.8, and 18.7 million adults were included for waves 1, 2, and 3 respectively. Main outcome measures COVID-19-related mortality based on linked death registry records. Results The crude rate of COVID-19-related death per 1,000 person-years decreased from 4.48 in wave 1 (95%CI 4.41;4.55), to 2.70 in wave 2 (95%CI 2.67;2.73), to 0.64 in wave 3 (95%CI 0.63;0.66). The death rate decreased by 90% between waves 1 and 3 in patients aged 80+, but by only 20% in patients aged 18-39. This higher proportional reduction in death rates was also seen for other groups, such as neurological disease, learning disability and severe mental illness. Conversely, death rates in transplant recipients stayed constant across successive waves at 10 per 1,000 person-years. There was also only a small decrease in death rates between waves in people with kidney disease, haematological malignancies or conditions associated with immunosuppression. Consequently, the relative hazard of COVID-19-related death decreased over time for some variables (e.g. age), remained similar for some (e.g. sex, ethnicity), and increased for others (e.g. transplant). Conclusions COVID-19 death rates decreased over the first three pandemic waves. An especially large decrease was seen in older age groups and people with neurological disease, learning disability or severe mental illness. Some demographic inequalities in death rates persisted over time. Groups more likely to experience impaired vaccine effectiveness did not see the same benefit in COVID-19 mortality reduction.

Nitazoxanide is a potent inhibitor of human seasonal coronaviruses acting at postentry level: effect on viral spike glycoprotein (preprint)

Coronaviridae is recognized as one of the most rapidly evolving virus family as a consequence of the high genomic nucleotide substitution rates and recombination. The family comprises a large number of enveloped, positive-sense single-stranded RNA viruses, causing an array of diseases of varying severity in animals and humans. To date, seven human coronaviruses (HCoV) have been identified, namely HCoV-229E, HCoV-NL63, HCoV-OC43 and HCoV-HKU1, which are globally circulating in the human population (seasonal HCoV, sHCoV), and the highly pathogenic SARS-CoV, MERS-CoV and SARS-CoV-2. Seasonal HCoV are estimated to contribute to 15-30% of common cold cases in humans; although diseases are generally self-limiting, sHCoV can sometimes cause severe lower respiratory infections, as well as enteric and neurological diseases. No specific treatment is presently available for sHCoV infections. Herein we show that the anti-infective drug nitazoxanide has a potent antiviral activity against three human endemic coronaviruses, the Alpha-coronaviruses HCoV-229E and HCoV-NL63, and the Beta-coronavirus HCoV-OC43 in cell culture with IC50 ranging between 0.05 and 0.15 g/ml, and high selectivity indexes. We found that nitazoxanide does not affect HCoV adsorption, entry or uncoating, but acts at postentry level and interferes with the spike glycoprotein maturation, hampering its terminal glycosylation at an endoglycosidase H-sensitive stage. Altogether the results indicate that nitazoxanide, due to its broad-spectrum anti-coronavirus activity, may represent a readily available useful tool in the treatment of seasonal coronavirus infections.

Digital Triage Tool Using Artificial Intelligence and Patient History for Detecting Selected Neurological Diseases and Sensing the Bottleneck between Symptoms, Diagnosis, and Therapy (preprint)

During the COVID-19 pandemic, individuals with symptoms other than cough or fever have refrained from seeking medical advice. However, a delay in treatment might lead to serious consequences. At the same time, digital health initiatives have emerged to overcome this bottleneck of healthcare. Herein, we report the results of a multi-center initiative using a combination of patient history and artificial intelligence (AI) to identify individuals with rare neuromuscular diseases. First, a questionnaire with 46 items was developed by interviewing patients with muscular dystrophies, amyotrophic lateral sclerosis, Morbus Pompe, neuropathies, and myasthenia gravis. Second, patients with proven neurological diseases answered the questionnaire. Third, a combination of classifiers (artificial neural network, support vector, and random forest) was trained and, finally, the system was challenged with new questionnaires. Users with an abnormal questionnaire pattern received a unique code for data privacy and contact details for a neurologist for further advice. The neurologists confirmed or refuted the AI-based diagnosis. The questionnaire was accessed 3122 times, leading to 853 unique codes. Only for a few patients the computer-based diagnoses and the confirmed final diagnoses were reported to us. However, for these few patients, the genetic testing and high CK levels finally ended their long-lasting diagnostic odyssey.

Rare neurogenetic conditions and mental health of families during COVID-19 (preprint)

Background: The impact of the COVID-19 pandemic on the wellbeing of parents and children in the general population has been well-documented. This study investigated wellbeing in parents of and children with rare neurogenetic conditions, who may have been at greater risk of negative impact on mental health and behavioural challenges during the first UK lockdown. Method: An online survey data was completed by parents of children with a rare neurogenetic condition between May and July 2020 (CoIN sample; N=123) and compared to responses from parents of children in the wider population (Co-SPACE sample; N=2121). Measures of wellbeing included the 21-item Depression, Anxiety and Stress Scale for parents and the Strength and Difficulties Questionnaire for child behaviour. Results: Parent anxiety was significantly higher in CoIN (MedianAnx = 4) than Co-SPACE (MedianAnx = 2). Parent-rated internalising, externalising and impact of child behavioural difficulties were also significantly higher in CoIN (MedianInt = 9.5; MedianExt = 11, MedianImp = 8) than Co-SPACE (MedianInt = 6; MedianExt = 7, MedianImp = 1). Only group differences in child behaviour and impact remained significant when matching for demographic factors and were also larger than previously reported pre-pandemic differences. Discussion: Families of children with rare neurogenetic conditions reported poorer wellbeing during the first lockdown compared to the wider population, affecting both parents and children. This likely reflects pre-existing complex needs, which should be prioritised during future national crises. Investigation of changes in wellbeing in this population over the course of the pandemic is warranted.

mRNA-COVID19 vaccination can be considered safe and tolerable for frail patients (preprint)

Background Frail patients are considered at relevant risk of complications due to COVID-19 infection and, for this reason, are prioritized candidates for vaccination. As these patients were originally not included in the registration trials, fear related to vaccine side-effects and disease worsening was one of the reasons for vaccine hesitancy. Herein we report the safety profile of the prospective, multicenter, national VAX4FRAIL study (NCT04848493) to evaluate vaccines in a large trans-disease cohort of patients with solid or hematological malignancies, neurological and rheumatological diseases. Methods Between March 3rd and September 2nd, 2021, 566 patients were evaluable for safety endpoint: 105 received the mRNA-1273 vaccine and 461 the BNT162b2 vaccine. Frail patients were defined per protocol as patients under treatment with hematological malignancies (131), solid tumors (191), immune-rheumatological diseases (86), and neurological diseases (158), including multiple sclerosis and generalized myasthenia. The impact of the vaccination on the health status of patients was assessed through a questionnaire focused on the first week after each vaccine dose. Results The most frequently reported moderate-severe adverse events were pain at the injection site (60.3% after the first dose, 55.4% after the second), fatigue (30.1% - 41.7%), bone pain (27.4% - 27.2%) and headache (11.8% - 18.9%). Risk factors associated with the occurrence of severe symptoms after vaccine administration were identified through a multivariate logistic regression analysis: age was associated with severe fever presentation (younger patients vs. middle-aged vs. older ones), females presented a higher probability of severe pain at the injection site, fatigue, headache, and bone pain; the mRNA-1237 vaccine was associated with a higher probability of severe pain at the injection site and fever. After the first dose, patients presenting a severe symptom were at a relevant risk of recurrence of the same severe symptom after the second one. Overall, 11 patients (1.9%) after the first dose and 7 (1.2%) after the second one required to postpone or suspend the disease-specific treatment. Finally, 2 fatal events occurred among our 566 patients. These two events were considered unrelated to the vaccine. Conclusions Our study reports that mRNA-COVID-19 vaccination is safe also in frail patients as expected side effects were manageable and had a minimum impact on patient care path.

SARS-CoV-2 Infection of Microglia Elicits Pro-inflammatory Activation and Apoptotic Cell Death (preprint)

Accumulating evidence suggests that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes various neurological symptoms in coronavirus disease 2019 (COVID-19) patients. The most dominant immune cells in the brain are microglia. Yet, the relationship between neurological manifestations, neuroinflammation, and host immune response of microglia to SARS-CoV-2 has not been well characterized. Here, we report that SARS-CoV-2 can directly infect human microglia, eliciting M1-like pro-inflammatory responses, followed by cytopathic effects. Specifically, SARS-CoV-2 infected human microglial clone 3 (HMC3), leading to inflammatory activation and cell death. RNA-seq analysis also revealed that ER stress and immune responses were induced in the early and apoptotic processes in the late phase of viral infection. SARS-CoV-2-infected HMC3 showed the M1 phenotype and produced pro-inflammatory cytokines such as interleukin (IL)-1{beta}, IL-6, and tumour necrosis factor (TNF-), but not the anti-inflammatory cytokine IL-10. After this pro-inflammatory activation, SARS-CoV-2 infection promoted both intrinsic and extrinsic death receptor-mediated apoptosis in HMC3. Using K18-hACE2 transgenic mice, murine microglia were also infected by intranasal inoculation of SARS-CoV-2. This infection induced the acute production of pro-inflammatory microglial IL- 6 and TNF- and provoked a chronic loss of microglia. Our findings suggest that microglia are potential mediators of SARS-CoV-2-induced neurological problems and, consequently, can be targets of therapeutic strategies against neurological diseases in COVID-19 patients.

Comorbidities Associated with In-Hospital Mortality in Adult Patients with COVID-19 in Lima, Peru: A Retrospective Cohort Study (preprint)

Background: We aimed to determine the comorbidities associated with in-hospital mortality in different age groups of hospitalized patients in Lima and Callao. Methods: In this retrospective cohort study, we analyzed data from hospitalized COVID-19 patients reported to the National Epidemiological Surveillance System (NotiWeb) of the Peruvian Ministry of Health from March 5, to October 31, 2020. Multiple logistic regression was used to determine the associated comorbidities with in-hospital mortality in the different age groups according to the Peruvian Ministry of Health life stage classification: youth (18-29 years), adults (30-59 years) and older adults (≥60 years). Findings: Among, 45639 patients, 2084 youth, 21206 adults, and 22349 older adults were included. Older adults had a higher mortality rate (59.9%) compared to adults (24.7%), and youth (6.7%) (Log-rank test: p<0.001). In young patients, kidney disease (OR, 7.94; 95% CI, 2.38-26.50), chronic neurological disease (OR, 10.38; 95% CI, 2.31–46.57), and cancer (OR, 16.60; 95% CI, 3.75–73.53) were associated comorbidities with in-hospital mortality. In adults, obesity (OR 1.69, 95% CI, 1.49–1.92), kidney disease (OR, 2.91; 95% CI, 2.23–3.81), chronic neurological disease (OR, 1.67; 95% CI, 1.05–2.66), immunodeficiencies (OR, 2.55; 95% CI, 1.53–4.23) and cancer (OR, 2.93; 95% CI, 2.12–4.04) were associated comorbidities. In older patients, obesity (OR, 1.47; 95% CI=1.27 - 1.71), kidney disease (OR, 1.30; 95% CI=1.08 – 1.55), chronic neurological disease (OR, 1.37; 95% CI=1.06 – 1.77), and cancer (OR, 1.49; 95% CI, 1.13–1.74), chronic lung disease (OR, 1.29; 95% CI=1.06–1.57) and liver disease (OR, 1.55; 95% CI, 1.08–2.21) were associated comorbidities. Interpretation: Common and different associated comorbidities with COVID-19 in-hospital mortality may exist among age groups. Independently of the age, individuals with kidney, chronic neurologic, and cancer would be a high risk of died. The risk of in-hospital mortality associated with comorbidities may be higher in 18-29 patients compare to older.Funding: This research team is supported by the regular budget of National Center for Epidemiology, Disease Prevention and Control (CDC Peru). The funders had no role in this work and decision to submit for publication.Declaration of Interest: All authors work at the National Center for Epidemiology, Prevention and Disease Control of the Peruvian Ministry of Health (CDC-MINSA). However, the views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the CDC Peru, Peruvian Ministry of Health, nor the Peruvian Government. No other disclosures are reported.Ethical Approval: No ethical approval was required as the study analyzed only secondary anonymous surveillance data.