The COVID-19 pandemic has strengthened the interest in the biological mechanisms underlying the complex interplay between infectious agents and the human host. The spectrum of phenotypes associated with the SARS-CoV-2 infection, ranging from the absence of symptoms to severe systemic complications, raised the question as to what extent the variable response to coronaviruses (CoVs) is influenced by the variability of the hosts’ genetic background.To explore the current knowledge about this question, we designed a systematic review encompassing the scientific literature published from Jan. 2003 to June 2020, to include studies on the contemporary outbreaks caused by SARS-CoV-1, MERS-CoV and SARS-CoV-2 . Studies were eligible if human genetic variants were tested as predictors of clinical phenotypes.ACE2, CLEC4M (L-SIGN), MBL, MxA (n = 3), ACE, CD209, FCER2, OAS-1, TLR4, TNF-α (n = 2). Only variants in MBL and MxA were found as possibly implicated in CoV-associated phenotypes in at least two studies. The number of studies for each predictor was insufficient to conduct meta-analyses.An ad hoc protocol for the rapid review process was designed according to the PRISMA paradigm and registered at the PROSPERO database (ID: CRD42020180860). The systematic workflow provided 32 articles eligible for data abstraction  reporting data on 26 discovery cohorts. Most studies considered the definite clinical diagnosis as the primary outcome, variably coupled with other outcomes . Ten studies analysed HLA haplotypes (1 in patients with COVID-19) and did not provide consistent signals of association with disease-associated phenotypes. Out of 22 eligible articles that investigated candidate genes (2 as associated with COVID-19), the top-ranked genes in the number of studies were Studies collecting large cohorts from different ancestries are needed to further elucidate the role of host genetic variants in determining the response to CoVs infection. Rigorous design and robust statistical methods are warranted. Infectious diseases are known to accompany human evolution through a complex interaction between the host and the infection . InfectiThe global impact of tuberculosis and HIV infection, the interest in the understanding of the genetic background of infectious disease, coupled with the emerging molecular technologies, lead to an increase of investigations on the role of the human host genetics profile . Most caSeveral human-tropic coronaviruses (CoVs) constantly circulate in the human population and usually cause mild respiratory disease. Two of these RNA viruses—SARS-CoV-1 and MERS-CoV—had been discovered in 2002 and 2012, respectively, as the cause of severe acute respiratory syndromes. The former emerged in Guangdong province, China, and its spread in China is known as the SARS epidemic. The latter caused an epidemic that began in Saudi Arabia and was limited in the Middle East  .In December 2019, a new infectious respiratory disease emerged in Wuhan, Hubei province, China. The disease, termed coronavirus disease 19 (COVID-19), rapidly spread from China as a global devastating pandemic –9. At thAs the knowledge of the COVID-19 risk factors has progressed, age has become recognised as one of the predominant determinants of severe outcomes , 11, in The latest findings of seroprevalence in COVID-19 were allowed to estimate a higher prevalence of infection than previously inferred . This isSoon after the SARS outbreak in 2003, researchers tried to respond to the question as to whether germline genetic variants influence the occurrence of the clinical syndrome caused by CoVs as well as its variable outcome. At present, this question has begun a priority for human genomics.IL4 gene and additional genetic risk factors for tuberculosis. The study implemented a formal assessment of the risk of bias and concluded that almost 95% of eligible studies were affected by a strong risk of bias or confounding .After interim results, a domain was added (“CoV-associated Biomarkers”) which targeted specific genes and genomic biomarkers found during the preliminary inclusion phase; the domain was combined with the “CoV” dimension with “and”; the resulting records were checked for duplicates and screened, then added to the final list of included articles. Search strings are reported in Supplementary Table Records were filtered for language (English) and date of publication (Jan 2003 to Apr 2020). The publication year field was restricted to the period from 2003 onwards, to include all articles published after the SARS outbreak dated 2003. The distribution of relevant publications indexed in PubMed confirmed a sharp rise since 2003 and a massive increase in 2020 (not shown).Given the rapid evolution of the field, the literature search was replicated on June 17, in order to retrieve the latest articles and published versions of pre-print documents. To this purpose, PubMed and Scopus were interrogated.Each identified study was indexed and manually abstracted. A total of 37 fields were pre-set for data extraction. Findings for all individual genetic variants under investigation were recorded, whether significantly associated or not. Some studies provided more than one data point (corresponding to one variant—one outcome measure entry) and were consequently represented with more than one record. The consistency of results between studies was analysed by gene and by the individual genomic biomarker. The study protocol did not envisage clinical recommendations after data synthesis.Owing to the need for a rapid appraisal, the following modifications of the standard protocol for systematic reviews were applied, according to the current recommendations for rapid reviews , 26.Grey literature was not systematically searched. Only articles in English were included in the screening phase and considered as eligible. One reviewer completed the screening phase according to an expedited PICO-based method previously described  and applThe systematic literature search in PubMed, Scopus, medRxive and bioRxive provided 1567 unique records and additional 362 records found in the last update . After the screening phase and full-text inspection, 32 articles fulfilled the criteria and were eligible for data abstraction. The detailed flow of literature assessment according to the PRISMA statement is depicted in Supplementary Figure Considering all variants tested as a predictor and the different outcome measures modelled as a dependent variable, >500 data points were included. In fact, this count should be further multiplied by the models tested in each study, i.e. allele and genotype association, dominant, recessive, codominant, and by the number of tests. However, we abstracted for each variant the statistics showing the larger effect size or the most significant result, if applicable.The 32 articles included for data abstraction analysed 26 independent cohorts—i.e. 3 cohorts were described in 4, 3 and 2 studies, respectively Table .Table 1Consistently with the outbreaks experienced in the last decades and the period considered for inclusion, SARS infection was by far the most frequent endpoint. Therefore, all studies investigated patients of Asian ancestry—Chinese Han and Vietnamese—except the only study which focused on MERS in Saudi Arabia and one recent analysis on individuals of European ancestry based in the UK Biobank.Patient series were ascertained according to the current diagnostic criteria and recruited retrospectively. No randomisation procedure was found in eligible studies. The healthcare setting—i.e. whether in-patient or out-patient clinic, follow-up, etc.—was seldom reported.The control series resulted to be consistent for ancestry. No paired case-control enrolment was applied.The discovery samples were heterogeneous in size across studies, ranging from 44 to 323,570, while the patient series count was from 20 to 817  and 1 measured COVID-19 severity . Correction for multiple testing was not common in included studies, though hypothesis testing in genetic association studies implies multiple testing , MBL, MxA (3 studies); ACE, CD209, FCER2, OAS-1, TLR4, TNF-α (2 studies). Out of these, only MBL and MxA provided positive signal of association in at least two studies. The CCL5/RANTES gene was reported in one article . The following HLA haplotypes were found differentially distributed in patients with respect to the reference population: B*4601; B*0703; B*0301; Cw0801: DRB1*1202; Cw1502; DR0301; DRB1*11:01; C*07:29; B*15:27. None of these provided positive signals in more than one cohort , they could cover the SARS-CoV-1 outbreak, though with some limitations such as the exclusion of HLA haplotypes . A narraBy developing this comprehensive review, we recapitulated the body of knowledge about the influence of host germline genetic variants on the clinical phenotypes associated with CoVs infection. In total, we examined 32 articles that met the criteria and were eligible for data extraction.HLA haplotypes were examined in ten studies spanning the entire period from the SARS-CoV-1 outbreak in 2003 up to the SARS-CoV-2 pandemic in 2020. The remaining 22 studies tested the hypothesis of association with single-gene variants. A summary of the findings is reported below.N-acetylglucosamine sugar motifs frequently present on the microbial surfaces of bacteria, viruses and protozoa [MBL gene seem to have functional effects on MBL serum levels. The variant alleles causing low plasma concentrations of functional MBL were described as associated with an increased risk of developing infections [Two different studies reported an association between mannose-binding lectin (MBL) polymorphisms and susceptibility to SARS-CoV infection , 37. MBLprotozoa . After bprotozoa . Polymorfections . Since bIFN-γ gene in a Chinese cohort [Il1A and IL18 showed nominal association with nasopharyngeal shedding [TNF-α promoter region were found as associated with femoral head necrosis [Abnormalities in the production levels of several cytokines such as IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IFN-γ, TNF-α and TGF -β1 were detected in patients with SARS-CoV infection . Cytokine cohort . Moreoveshedding , and varnecrosis . None ofOAS1) and myxovirus resistance 1 (MxA) harbour genetic variants that might affect susceptibility to the SARS-CoV infection and progression [OAS1 genetic variants located in exon 3, exon 6 [MxA with CoVs-related phenotypes needs to be investigated in large cohorts by applying a robust study design.Type 1 interferon (IFN-a/b) plays an important role in the innate immune response against viral infections. Among the antiviral proteins induced by IFN1, 2'-5' oligoadenylate synthetase 1  gene, also known as CCL5 [RANTES codes for a chemokine responsible for the recruitment of eosinophils, lymphocytes, monocytes and basophils at the site of inflammation [RANTES expression and enhances NF-κB binding in vitro, thus suggesting its role in promoting inflammation [One article reported the association between SARS susceptibility and a genetic variant in the  as CCL5 . RANTES ammation . The stuammation . This waACE2 variants, as well as other factors implicated along the pathway of SARS-CoV2 infection. In fact, biochemical interaction studies and crystal structure analyses revealed that the SARS-CoV-2 spike protein has a strong binding affinity with the human ACE2 extracellular domain [ACE2 polymorphisms obtained from reference data with the prevalence of COVID-19 in different countries [After the spread of the COVID-19 pandemic, it has been speculated on the possible role of r domain , 69, 70,r domain , 71, 72,ountries , 74. TheIL4 and IL6, were not sufficiently investigated so far. One out of the 32 eligible studies included variants of both genes in a panel of 65 loci, with negative results [Although the role of the host immune system in the development and clinical course of the severe respiratory syndrome caused by SARS-CoV1 and SARS-CoV-2 has been increasingly apparent, it is also worth to underline that some promising candidate genes, namely  results .IL4 as the single result that was significant in pooled analyses, and marginally significant in disease-specific meta-analyses. IL4 promotes both T-cell and B-cell differentiation and provides a balance between Th1 and Th2 response [In the systematic review and meta-analysis of studies addressing host genetic factors implicated in five common respiratory tract infectious diseases—i.e tuberculosis, influenza, respiratory syncytial virus (RSV) and SARS-CoV—published in 2015 , Patarčiresponse . Given iIL6 genetic variants should be considered as a potential determinant of the host response against CoVs [IL6R gene polymorphisms with the effectiveness of tocilizumab was found in rheumatoid arthritis [IL6R in CoV-related diseases.IL-6 is one of the pro-inflammatory cytokines whose levels appear to increase sharply in patients with COVID19 . In partnst CoVs . Based onst CoVs . The assrthritis  and was rthritis . ConversWe did not focus on pharmacogenetics of treatments against CoV infections, as a response to drugs is a distinct phenotype which deserves a specific focus. Preliminary data pointed to germline genetic variants influencing the efficacy of specific therapies administered in patients with COVID-19 . This liAs the involvement of immune response in the clinical course of CoV-related diseases had been apparent since the first severe cases with SARS occurred in 2003, several efforts have been made to identify susceptibility or resistance factors associated with the variability of HLA alleles in the human population. This review included ten studies that investigated the hypothesis of association with HLA. In summary, no HLA haplotype was found to be significantly associated with CoV-related phenotypes in more than one study. Moreover, most studies appear rather weak in terms of study design and robustness of statistics .Our findings are in line with the recent review by Sanchez-Mazas , who criIn general, the sample size of eligible studies was limited by the absolute number of cases available for clinical and genetic ascertainment, and supposedly by the logistic constraints of an outbreak. A few studies, however, used a very small case-control sample—i.e. <100 cases, see Tables Many nominally significant signals of association listed in tables would not survive to rigorous corrections for multiple testing  had not been ascertained.Studies on HLA used reference data from the general population as the control series  who were ascertained as either seronegative or mildly affected. The latter individuals could be reasonably classified as resistant—either to infection or severe manifestations—and could serve as matched controls with respect to selected cases. Our recognition found one study that implemented a procedure to enrol control individuals .ApoE ε4 homozygous individuals are more likely to be COVID-19 test positives compared with ε3 homozygotes, independently from dementia [To deploy this approach, clinical registries and linked biobanks are a helpful source of information on COVID-19 phenotype variability. The UK Biobank (UKB) has already provided pieces of evidence on COVID-19 , and onedementia .This systematic review and summary of findings should be weighted for some limitations.The rapid protocol may have limited the sensitivity of the literature search; thus, some relevant articles might have been missed. To reduce this risk, we performed an extensive hand-search of reference lists and updated the search in order to include the latest studies and unrefereed articles previously found.The present study also suffers from the inability to adjust significance and effect size for multiple testing and basic covariates, such as age, sex and comorbidity that were found to account for most of the variability in the clinical manifestations of CoV-associated diseases, particularly COVID-19 , 11. TheThe choice of rapid systematic review protocol implied also to renounce a formal quality assessment of eligible articles. Therefore, we included only accepted articles, relying on the ability of the peer-review process of rejecting papers that did not meet the minimal criteria for publication. Since no meta-analysis was possible on the set of extracted data, the lack of a structured assessment of the risk of bias did not affect the final synthesis.Somatic variants in the target tissues of CoVs were not comprised in the inclusion criteria of the present systematic search. The variable expression of virus receptors implies a complex network which controls expression level and processing in response to the virus, encompassing both coding and non-coding RNA molecules. The post-transcriptional regulation of the interaction between CoVs and the human host is a fascinating field of investigation—however, it was beyond the scope of our effort.We weighted these limitations against the utility of a synopsis which summarises a heterogeneous and promising line of research. We are also aware that the body of evidence on the host genetic determinants of CoV-related phenotypes is increasing—our synthesis provides a snapshot of the current situation but is far from being conclusive. Due to the short time-lapse from the discovery of COVID-19, the majority of studies that were appraised in the present review are related to the SARS outbreak caused by SARS-CoV-1. This finding underlines the importance of reliable genetic association studies targeting the human host response to SARS-CoV-2 infection. In a rapidly changing scenario which has a major impact on public health, such as the current pandemic, it is mandatory to maintain the knowledge with the most recent evidence and discuss the research priorities. In adherence to the recommendations recently reminded by the Cochrane’s initiative , the revhttps://www.ukbiobank.ac.uk/2020/04/covid/). In Finland, the COVID-19 Host Genetics Initiative intends to integrate data with major biobanks (https://www.covid19hg.org/). The Greek public initiative, COVID-19-GR, will genotype COVID-19 patients and their SARS-CoV-2 genome, linking data with the Greek COVID-19 registry . In the UK, the GenOMICC Consortium is working in the project ‘GenOMICC study on COVID-19 patients’ aimed at achieving whole genome sequencing of up to 20,000 patients who were severely affected by COVID-19 and 15,000 who had mild symptoms (https://genomicc.org/). In Iceland, the company deCODE Genetics has partnered with Iceland’s government to sequence the genomes of patients who were previously infected with COVID-19 (https://www.decode.com/). In the USA, researchers from Harvard’s Wyss Institute and the Personal Genome Project at Harvard University are launching a multi-omics project to compare genomes, microbiomes, viromes, and immune systems of individuals with extreme susceptibility to SARS-CoV-2 infection and individuals who have shown resistance (https://wyss.harvard.edu). In collaboration with Yale University, the Yale SARS-CoV-2 Genomic Surveillance Initiative is using a variety of tools, including viral sequencing, case surveillance, test development, and wastewater surveillance, in order to monitor the spreading of the virus (https://covidtrackerct.com/). International initiatives, such as the COVID Human Genetic Effort [www.covidhge.com), are having a pivotal role in facilitating the implementation of standardised phenotyping, collection of reliable clinical data, as well as biological samples, according to the best practices in adherence to shared ethical and legal standards. The GEFACOVID consortium, made up of 29 participating institutions, is focused on virus’ genetic polymorphisms and pathogenesis mechanisms as well as genetic, genomic, metabolomic, epidemiological and clinical data in order to identify biomarkers that confer susceptibility to virus infection and increase the risk of life-threatening complications (https://davincidtx.com/gefacovid-project/).The considerations outlined above highlights the need for consortia aimed at coordinating multidisciplinary efforts to elucidate the genetic determinants of COVID-19 , 93. To Registries and biobanks are invaluable sources of demographic and clinical assessments, including imaging and biomarkers. These large data sets may be used to explore different models of response to infection and generate hypotheses encompassing multiple genetic risk factors and interactions with other predictors. Promising hypotheses could be subsequently tested in clinical studies.Assuming that ethnicity does not merely reflect the genetic background, as it is a complex construct made of genetic inheritance, cultural heritage and shared social and behavioural profiles, ancestry is a mandatory ascertainment in any clinical data set. Most genetic association studies included in the present review were targeted on patients of Chinese Han ancestry. In a global pandemic, the genetic diversity of populations across the world implies the need for inclusive investigations recruiting participants from the different genealogical and geographical origin. We endorse the recommendations recently invoked  and follAfter the global spread of COVID-19, the knowledge about the interplay between CoVs and the human host has been growing at an impressive rate. However, as compared to the number of clinical studies that were delivered so far, we found a quite limited number of investigations focused on the effect of human genetic variability on clinical phenotypes.A handful of candidate genes were tested in more than one study, and no single variant was confirmed to be associated with the clinical outcome in independent cohorts. The paucity of studies converging on common genetic determinants hampered any quantitative synthesis. In the light of the present synopsis, the discovery of a single major gene which determines a substantial variability of the CoV-associated phenotype cannot be sensibly anticipated. Since all genetic association studies eligible for the present review were presumably prone to both type I and type II errors, their findings should be regarded as exploratory.Plenty of recent studies addressing the COVID-19 biological mechanisms and response to therapy might provide the rationale for novel candidate genes. Genetic association analyses warrant rigorous design and robust statistical methods, in order to test precise models of interaction among multiple genetic variants and between genetic variants and exposure. To this purpose, the shared effort of the human genomics community will enable the development of robust methodological approaches through a close multidisciplinary interchange, as well as the assembly of large data sets. Prompt dissemination of genetic studies—including negative findings—is helpful to focus on the rapidly evolving research questions.Genetic variants are non-modifiable risk factors and cannot be claimed to be directly translated into the clinic as actionable markers in the present emergency. However, once a panel of associated genetic biomarkers is established, genetic biomarkers could be included in multidimensional models with age, comorbidities, socioeconomic status, etc., to prioritise public health interventions, such as active surveillance for the vulnerable population strata.From the speculative perspective, although association studies do not prove mechanisms per se, evidence from genetic variants might be crucial to elucidate the biological pathways underlying the severe clinical presentation associated with CovS infection and to identify promising therapeutic targets.Additional file 1: Supplementary table 1. Search strings used to interrogate publication databases. Supplementary figure 1. PRISMA workflow.


Alzheimer’s disease (AD) is the most common form of dementia. An increasing body of evidence describes an elevated incidence of epilepsy in patients with AD, and many transgenic animal models of AD also exhibit seizures and susceptibility to epilepsy. However, the biological mechanisms that underlie the occurrence of seizure or increased susceptibility to seizures in AD is unknown. Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase that regulates various cellular signaling pathways, and plays a crucial role in the pathogenesis of AD. It has been suggested that GSK-3 might be a key factor that drives epileptogenesis in AD by interacting with the pathological hallmarks of AD, amyloid precursor protein (APP) and tau. Furthermore, seizures may also contribute to the progression of AD through GSK-3. In this way, GSK-3 might be involved in initiating a vicious cycle between AD and seizures. This review aims to summarise the possible role of GSK-3 in the link between AD and seizures. Understanding the role of GSK-3 in AD-associated seizures and epilepsy may help researchers develop new therapeutic approach that can manage seizure and epilepsy in AD patients as well as decelerate the progression of AD. Alzheimer’s disease (AD) is the most common form of dementia and is clinically characterised by cognitive impairment and progressive loss of memory. It has been estimated that one in every 85 individuals worldwide will be living with AD by 2050 , imposinRecent evidence has raised the possibility of a connection between AD and epilepsy, another common CNS disorder in the elderly that is characterised by an enduring predisposition to generate unprovoked seizures. Multiple studies have reported an increased incidence of unprovoked seizures in AD patients ,4,5. ForIn addition to the clinical evidence, animal models harbouring AD-associated mutations provide a useful insight on the possible causation and mechanisms underlying AD-associated epilepsy . There aGlycogen synthase kinase-3 (GSK-3) is a proline-rich serine/threonine kinase which plays a pivotal role in the pathogenesis of AD ,18,19. IIn this article, we will review the possible mechanisms of AD-related seizures, present hypothesised pathways of the role of GSK-3 in the development of seizures in AD, and propose future research to clarify the role of GSK-3 in this process. It is worth noting that multiple studies using AD animal models employed models that harbour familial AD mutations and, thus, the neuropathological process in the context of familial AD. However, that does not exclude patients with sporadic AD from the possibility of similar pathological changes.There is growing clinical evidence suggestive of an increased risk of epilepsy or epileptic seizures in AD. Higher incidence of unprovoked seizures in AD patients has been reported by numerous studies ,23,24,25In parallel to the clinical evidence, many transgenic mouse models carrying identified AD mutations also show unprovoked seizures or epileptiform EEG patterns. Although AD mouse models cannot fully recapitulate the pathological features of AD, they serve as controllable models of the disease with one or few genetic modifications that manifest AD-associated symptoms. The most well-studied models are mice that overexpress human mutant APP or specific APP cleavage products such as amyloid intracellular domain (AICD). The process of APP cleavage is shown in Aside from the potential immediate consequences of epileptic seizures, such as physical injuries, to AD patients, it is found that the occurrence of seizures might also be relevant to the acceleration of cognition impairment, which creates a complex bidirectional association between AD and seizures. The cognitive functions of patients with AD or other dementias may abruptly worsen after the first onset of seizure ,38, suggGSK-3 is a ubiquitously expressed kinase that is involved in various physiological and pathological processes. GSK-3 was originally found as a key kinase that phosphorylated glycogen synthase during glycogen accumulation, a fundamental process of energy storage . There aThe role of GSK-3 in the pathogenesis of AD has been discussed in recent reviews ,55,56,57In addition, studies showed that GSK-3 might be involved in AD-associated seizures through alternate pathways. Multiple studies have shown that GSK-3β can be associated with the activity of Fyn ,74,75, aAlthough the interaction between GSK-3 and tau has been widely studied, the mechanisms which initiate the dysregulation of GSK-3 activity potentially leading to subsequent seizures and epileptiform events in AD remain to be clarified. Multiple results from in vitro and in vivo experiments showed that GSK-3β might be activated by APP or its metabolites ,79,80,81Interestingly, some evidence showed that the AICD may also be pertinent to the occurrence of spontaneous seizures and increased susceptibility of induced seizures in animal models. It is reported that the cleavage of APP at the C-terminus may be related to neuronal network abnormality , while oSome other studies suggest that GSK-3 might also be involved in the production of AICD through the cleavage of APP ,89. PhieCollectively, accumulating evidence suggests that there might be a GSK-3-amyloid-tau triad that induces the development of seizures and epilepsy in AD. However, the specific interaction between GSK-3 and AICD remains to be clarified. Answering these questions might help us understand the pathophysiology of AD-associated epilepsy as well as develop new therapeutic approaches to manage seizures and epilepsy in AD patients.As previously discussed, the occurrence of seizures may accelerate the progress of AD. Some evidence showed that GSK-3 might also be involved in this process. It is clear that GSK-3 plays an important role in the pathogenesis of AD through the phosphorylation of tau ,91 and tBased on the current evidences, it may be hypothesized that GSK-3 contributes to the “vicious cycle” between seizure and AD. After being activated in AD, GSK-3 might induce the occurrence of epileptic seizures, which could further increase the activity of GSK-3 and establish a positive feedback mechanism. Therefore, regulation of GSK-3 activity might be a potential intervention for both AD and epileptic seizures.c-, a system that imports cystine and exports glutamate [There are also multiple studies that link GSK-3 to seizures in non-AD models. One study showed that GSK-3β is an essential regulator of the cystine/glutamate antiporter xlutamate . The dislutamate . Inhibitlutamate . Howeverlutamate . Furtherlutamate , which wThere is increasing evidence supporting the cooccurrence of AD and seizures. To improve the understanding of both fields and to develop novel therapeutic approaches targeting AD-associated seizures, there are many questions to be addressed. Firstly, GSK-3 activity in AD models has not been well studied. Although some studies have found that GSK-3 could be activated in transgenic human APP mouse models ,84, the Importantly, whether and at what time point GSK-3 is activated in AD models requires further study. To date, no study has reported the chronological analysis on the activity of GSK-3 in animal models carrying AD-relevant mutations. It is possible that GSK-3 activity is increased at an early age due to the gene mutation or the overexpression of genes, such as APP and AICD. The former is more likely to be relevant to human condition while the latter is limited to animal models, so carefully designed studies need to exclude simple overexpression of APP as a driving factor. In addition, a concern raised by Saito et al. suggested the possibility that some phenotypes in APP-overexpressing mice might come from membrane protein overexpression . FurtherSecond, the roles of GSK-3 in AD-associated seizure need to be clarified. Although GSK-3 has been recognised as one of the contributors of AD pathogenesis, whether GSK-3 causes or accelerates the development of epilepsy in AD has not been well studied. One study reported that genetically modified GSK-3 with increased activity decelerates kainate-induced epileptogenesis , which sLast but not least, the pathway that involves GSK-3 in AD-associated epileptic seizures requires further investigation. GSK-3 is involved in multiple cell signalling pathways , which sTo summarise, emerging evidence suggests that GSK-3 may play a crucial but complex role in the pathogenesis of epileptic seizures in AD. Unravelling this relationship could potentially open up new therapeutic strategies.


Under the ongoing COVID-19 prevention and control measures in China, increasing the laboratory severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid testing capacity has become the top priority. Since the COVID-19 outbreak in Xinfadi market in Beijing in June 2020, large-scale screening of key populations has been carried out, challenging the nucleic acid testing capabilities of hospital laboratories. Therefore, within 48 hours, Peking University People’s Hospital (PKUPH) transformed a non-nucleic acid testing laboratory into a SARS-CoV-2 nucleic acid testing laboratory. Based on the original structure of the building, we adapted measures to local conditions, sorted out a new testing process, and quickly started testing for COVID-19. The nucleic acid testing process has been optimized, including quality control, personal operating specifications, and the timeliness of the release of LIS results to form closed-loop management. This high-throughput COVID-19 testing optimization process provides a reference model for other countries that are fighting the epidemic. The spread of coronavirus disease COVID-19) worldwide has caused considerable economic losses and has affected people’s daily lives 9 worldwi. HoweverOn June 20, 2020, Peking University People’s Hospital (PKUPH) urgently reconstructed an out-of-use laboratory on another campus as a SARS-CoV-2 nucleic acid testing laboratory to increase the volume of nucleic acid testing. To perform the required nucleic acid tests, the laboratory required modifications included physical partitioning of staff and equipment, alterations to airflow direction and laboratory air pressure, and re-planning of cleaning directions. The modifications were completed within 48 hours, including hydropower transformation; fresh air commissioning; installation of equipment furniture, and a laboratory information system (LIS); and preparation of experimental reagents and various protective materials. From June 24 to July 12, 2020, the laboratory performed a total of 41,897 nucleic acid tests for SARS-CoV-2, with a maximum of 4,030 tests in a single day. The average test turn-around time (TAT) was 7 hours from initial patient swab. Here, we summarize the aspects of laboratory reconstruction and testing process optimization to provide a multi-department cooperation model for COVID-19 testing and screening in large cities like Beijing. Our experience will provide a basis for increasing nucleic acid testing capacity in the continued fight against the COVID-19 pandemic.The layout of the SARS-CoV-2 nucleic acid testing laboratory has an important impact on the testing workflow and on laboratory biosafety . Figure To satisfy the urgent need of actively screening a large number of samples in high-risk areas in Beijing, our laboratory has optimized the number of tests performed in a given day. The following laboratory plan is determined based on a daily output of 4000 tests. Taking 93 clinical samples as a batch, 44 batches of PCR detection (4000/93 ≈ 44) are required. With 5 nucleic acid extractors and 10 PCR machines in the laboratory, 4.5 rounds of PCR tests are therefore required. The time required for each batch of nucleic acid extraction is 25 minutes. Each batch of PCR detection takes approximately 2 hours; therefore, it takes 8–10 hours to complete the 4000 tests. Prior to nucleic acid testing, the hospital requires all identifying information from the different communities in advance and generates sample barcodes in batches. Furthermore, during the release of test results, a closed-loop informatization is created, whereby the hospital health information system (HIS) releases the results to socialized public platforms, including the WeChat, Beijing Jiankangbao, and Peking University People’s Hospital (PKUPH) applications, via cell phones. Patients and government authorities will receive nucleic acid test results as soon as possible, especially positive test results.COVID-19 laboratory personnel must be qualified for PCR testing and interpretation. The SARS-CoV-2 Nucleic Acid Testing Laboratory maintained a streamlined operation with 7 staff members . One staff member each is assigned to the sample registration, reagent preparation, and amplification areas, and these three personnel can cooperate according to specific scenarios. The nucleic acid extraction area is equipped with four staff members. Of these personnel, three are responsible for adding samples to the lysate plate, and one is responsible for the operation of the nucleic acid extractor and adding templates to the system. When the number of specimens was relatively large (i.e. >3000 tests per day), the entire group made all possible efforts to keep more than 8–10 PCR machines in operation simultaneously. When a large quantity of samples require tests results quickly, the staff members in the nucleic acid extraction area enter the scanning area first.In the reagent preparation area, the total sample quantity should be estimated as accurately as possible, and enough 96-well reaction plates should be prepared at one time to avoid a delay due to repeated preparation of reagents. The scanned and numbered specimens are then transferred to the nucleic acid extraction area through the transfer window. Sample addition should always be performed in a biosafety cabinet. A high throughput extractor was used with 96 samples per batch. To this end, the operator’s outer gloves must be taken off within the safety cabinet after disinfection. Once the sample nucleic acids have been added to the corresponding reaction tube or 96-well reaction plate, they should be covered with film and sealed within the PCR reaction tube or reaction plate. In the amplification area, a dedicated staff member removed the prepared PCR test tube, centrifuged it, set the amplification parameters according to the kit instructions, and analyzed the results. Due to the technical characteristics of the sensitivity and limitations of testing, two staff members reviewed the test results comprehensively. The analysis results are recorded in real time, and the codes of the specimens that require re-examination are recorded on the re-examination record sheet. After the results are released from the laboratory, the hospital’s HIS platform quickly releases the results on the public application platforms of PKUPH, WeChat, and Beijing Jiankangbao within the same day. Those who have been tested can also check their test reports through a mobile phone application.The quality control of nucleic acid testing is reflected in all elements, including the pre-analysis, analysis and post-analysis stage. Specimen collection personnel are trained according to guidelines on the collection of oropharyngeal and nasopharyngeal swabs during large-sample screening . LaboratThe rapid and successful control of the recent COVID-19 epidemic in Beijing was mainly attributable to the strengthening of laboratory SARS-CoV-2 nucleic acid testing capacity. Optimization of workflow of the whole testing process and shortening the time spent for each step are key to managing the large testing volumes in an infectious disease epidemic.Click here for additional data file.


The COVID-19 coronavirus pandemic has critically struck the world economy and healthcare systems. The highly contagious virus spreads rapidly and can result in potentially life-threatening acute respiratory distress. The current established test for diagnosing COVID-19 is using the RT-PCR laboratory test. However, the test requires specialized laboratories and testing kits. Recent reports also showed high false-negative rates. Experts recognize the urgent need to develop a rapid point of care diagnostic tests. Ultrasonography is a widely established safe diagnostic imaging test for detecting various lung abnormalities. Recent publications from China and Italy provided limited evidence on its usefulness for diagnosing COVID-19 in emergency departments earlier than RT-PCR. Ultrasound is sensitive to pleural and subpleural abnormalities, which suggests a great potential diagnostic role given the predilection for COVID-19 in peripheral subpleural regions.This paper reviews the current evidence and discusses the problems with specificity and scoring. It is an extremely contagious virus, which could potentially cause acute respiratory distress and potentially organ failure in advanced stages.2,3 In contrast, alternative diagnostic tests such as chest CT has not been recommended by international and local guidelines due to several reasons, including poor specificity and radiation risks.4 Lung ultrasonography (LU) is an established technique for evaluating lung pathologies. It is sensitive to abnormalities in the pleura and subpleural spaces. The initial evidence on COVID-19 suggesting peripheral lung involvement at the terminal alveoli, which alludes a significant role for LU in diagnosis and management. This paper presents an up-to-date review of the LU evidence in COVID-19 and discusses the pitfalls and future perspectives.COVID-19 is diagnosed by reverse-transcription polymerase chain reaction (RT-PCR), which was criticized after reports of poor diagnostic sensitivity.5–16 The publications were divided between simple case-studies, letters of opinion and commentaries discussing the potential usefulness of LU. Despite a few useful descriptions of how LU can be included in the COVID-19 diagnostic workup and management, they present inconclusive results and apparent lack of thoroughness. The paragraphs below discuss the overall findings.There have been several publications describing the role of LU in COVID-19.6 was of a female heath practitioner in her sixties with confirmed COVID-19 that developed a cough and sore throat followed by dyspnea. LU demonstrated multiple B-lines, consolidation and thickened pleural line. Both of these cases can be considered moderate in terms of severity (both febrile with low oxygen saturation). The third, and most interesting, case study14 was of pregnant females with positive lung ultrasound findings suggesting COVID-19 which had an early negative PCR result. The findings included bilateral thick B-lines in posterior lung regions, which correlated perfectly with the CT results. She later tested positive on RT-PCR. This report demonstrates the early detection capability of LU in cases with false-negative RT-PCR results. It also further supports Ai et al.3 recommendations regarding the importance of lung imaging considering the high sensitivity prior to positive RT-PCR results.The first case report was of a 57-year-old male presenting to the ER with fever, cough, general weakness, and headache. The LU findings showed bilateral irregular pleural line with minimal consolidations and thick multiple B-lines in the anterior and posterior hemithorax. As a control case, a suspected patient (later confirmed negative) with typical COVID-19 symptoms exhibited normal lung pleura with A-lines on LU. Notably, the scan was performed by two operators using a handheld device, one scanning and the other holding the tablet (freezing and storing images). This practice was recommended by them to mitigate the risks of infection. The second case study11 Despite not being peer-reviewed, their report described lesions located bilaterally in the posterior inferior lung regions. They were characterized by fixed confluent B-lines , diffused B-lines (white lung sign), irregular and thickened pleural line, and consolidation with air bronchograms. Additionally, they reported localized pleural effusion in a few cases (exact number not mentioned), which is seldom found in viral pneumonia. They highlighted that these are characteristic findings different from bacterial pneumonia or pulmonary edema, where B-lines are mobile and focal. We argue otherwise as explained below.Several publications included descriptive case series. Huang et al were the first to publish a case series on 20 patients of non-critical patients in the emergency department.9 They reported the same findings of Huang et al except for finding focal B-lines. An Italian group of researchers published a letter on 12 patients presenting to the ER.10 In agreement with the above reports, they found diffused B-lines in all patients and posterior consolidation in three patients only. They also mentioned a good correlation between CT and LU.In the emergency settings, Peng et al published a brief letter explaining the ultrasonographic features of COVID-19 in 20 cases.12 proposed an unvalidated protocol to perform LU in COVID-19 patients. They recommended scanning patients in a sitting position (when possible) using convex or linear transducers operating low mechanical index settings with a single focus point on the pleura. However, their recommended 14-zone scanning protocol is cumbersome and has not been described before. It extends the 12-zone protocol by adding two extra zones at the back of the chest. In our experience, the lung is mostly obscured at the back by the scapula, and the 12-zone is a sufficient protocol to thoroughly scan the lung even in ICU settings where the scanning time should be short. It was also the employed protocol in multiple publications5,9,11,13 and endorsed by multiple societies.17All reports above omitted reporting the severity of the cases. This is vital information as it can be postulated that LU features vary depending on the disease development. Additionally, none of the case series examined the usefulness of handheld ultrasound devices. With regard to LU scanning techniques, there is a clear discrepancy in the number of regions screened. Some studies opted for the limited four-zone testing, while others employed the 12- and 14-zone protocols. Soldati et al13 They noted the application of LU helped in decreasing the rates of chest x-rays and CT scans, which contributed to making care delivery and management more efficient. They also described a total scanning time of less than 5 min, which we believe underestimates the actual whole time especially with dressing, positioning and documentation. Despite the promising results of accuracy, efficiency, safety, reproducibility, and point of care use of LU, it has several drawbacks. For example, LU is limited to visualizing the pleural surface and cannot detect deep lung lesions. However, this may not be an issue considering the predilection of COVID-19 in peripheral subpleural regions. A second limitation is a need for experienced users to operate the ultrasound device. Practitioners should also be aware that LU, like CT, does not exclude COVID-19 in subjects with no pulmonary complications.Vetrugno et al described quite positively their Italian experience with LU in COVID-19.The LU features of COVID-19 include irregular pleural line, confluent B-lines, and consolidations in more severe cases. Two studies described pleural effusion, which is seldom found in viral pneumonia. The problem is that none of these signs are pathognomonic for COVID-19 as they merely describe the density state of the lung surface. We should be cautious when calling these signs characteristics amidst hospitals flooding with COVID-19 as the pre-test likelihood for positive findings in suspected COVID-19 is currently high.18,19 This feature corresponds to acute ground glass alteration which are prevalent in COVID-19 pneumonia. It may be a specific sign especially when imaged in young patients with no history of lung diseases. Nevertheless, the described features bring some hope. They can be considered significantly different to signs of pulmonary fibrosis and interstitial pneumonia caused by other viruses where a diffused B-lines with no spared areas.Few researchers argue the presence of a typical LU sign coined as the ‘light beam artifact’ characterized by a vertical echoic broadband-like artifact, similar to the white lung sign, which comes in and out of the frame with respiration.12 proposed a scoring criterion for LU images. It scores each zone in the 12-zone LU protocol from 0  to 3 (severe) based on the LU lung involvement patterns relating to pleural lines, vertical artifacts and consolidations. This scoring system was also recently proposed by Manivel et al20 but with minor modifications in each category definition. This semiquantitative scoring system is a promising attempt to standardize the reporting of COVID-19 scans. However, it was developed based on the sole observations of the research team of Soldati et al.12 Hence, the criterion and content validity must be established as well as the inter- and intra-reader reproducibility.There is currently no validated image scoring system for diagnosing COVID-19 on LU. Soldati et alIt can be difficult to appreciate or count the number of B-lines especially as minute changes can significantly influence the image. Hence, we recommend obtaining the best possible image for each zone then ‘freezing’ to count the lines for scoring. A best practice approach is to acquire a clip and still images of each zone for post scanning documentation, inter-reader deliberation and medicolegal purposes.To date, all results provided positive outcomes on the usefulness of LU in COVID-19. However, the presented evidence thus far is considered weak. None of the studies included a control group or reported their findings longitudinally. Moreover, the sample sizes were extremely small, impeding results generalization. Several international societies, such as the British Medical Ultrasound Society, the intensive care society, and the WFUMB are calling for more research on LU in COVID 19. Future research should focus on validating the LU signs and patterns based on the proposed scoring system to suggest optimum cut-off scores.e.g., symptoms, decision to supplement oxygen). For instance, it can be used to check lung aeration before and after non-invasive ventilation. This may indicate that there is more hope for LU in COVID-19 as a bed-side-management tool for confirmed cases, potentially saving them from re-exposure to ionizing radiation. However, in screening tests, it is better to have a high sensitivity than high specificity. As a false-negative case infected with COVID-19 can be discharged and mingle back in society, eventually infecting numerous new cases.In our opinion, LU in COVID-19 can have elevated rates of false-positives due to the overlapping features with other viral pneumonia. LU findings should never be interpreted without consideration of the clinical context (The novel coronavirus unprecedented healthcare crisis indisputably calls for point of care screening solutions that can deliver rapid and sensitive results. The previous and recent evidence suggests that LU could be the called-for solution to diagnose positive cases and grade their severity. Modern handheld ultrasound devices can be particularly useful considering their relatively cheap prices and ease of disinfection without significant compromises on quality. The future potential for a radiation-free and relatively cheap bed-side-tool carries hope.


Our results highlight the general importance of the SARS-CoV-2 structural proteins in immunological control in early infection spread looking at a global census in various countries and taking case fatality rate into account. Other factors such as health system and control measures become more important after the early spread. Our study should encourage further studies on MHCII alleles as potential risk factors in COVID-19 including assessment of local populations and specific allele distributions.We observed substantial differences in predicted Major Histocompatibility Complex II (MHCII) epitope presentation of SARS-CoV-2 proteins for different populations but only minor differences in predicted MHCI epitope presentation. A comparison of this predicted epitope MHC-coverage revealed for the early phase of infection spread (till day 15 after reaching 128 observed infection cases) highly significant negative correlations with the case fatality rate. Specifically, this was observed in different populations for MHC class II presentation of the viral spike protein ( Since early 2020, SARS-CoV-2, which causes COVID-19, developed into a global pandemic. Many potential approaches to treatment and prophylaxis have been developed over a relatively short period. Currently, 238 vaccine candidates are in pre-clinical and clinical development  and six are at the same time already used worldwide . However, most of the treatment agents show disputable efficacy or side effects ,5. HowevThe spread of viral diseases is influenced by several factors, such as the movements of the human population, social behavior, virus mutations and population immunity. Therefore, to understand the country-specific responses elicited by the MHCI and MHCII repertoires, population data on the spread of COVID-19 infection and the calculated lethality should be correlated with predictions of the immunological repertoire in a particular population.To address this issue, our analysis considers: (i) infection spread and pattern of COVID-19-patient growth curves in different countries; (ii) focus on naïve population and natural immunity, defined for the time period from disease-free population till day 15 after reaching 128 infected cases in the population and (iii) country-specific human host immune response according to epitope response reaction frequencies separated for T- and B-cells (major eliciting MHCII epitopes mapped on SARS-CoV-2 surface epitopes).In this paper, we establish and present several correlations between pandemic spread in different countries and the immune response to epitope-representation in immunogenic SARS-CoV-2 proteins. Full data of a broad analysis including different parameter choices are given see also , includiProtein annotation and protein–protein interactions with the human host did provide some hints on SARS-CoV-2 infection biology and treatment strategies. However, there are some lacunas in the methods and in the analyses provided to date. Therefore, we decided to take the analysis one decisive step further to in silico predictions of potential linear B-cell epitopes. These analyses were performed with several online tools to rule out bias from individual prediction algorithms. The number of predicted epitopes significantly varied between the analyzed proteins.For the analysis of B-cell epitopes, three distinct methods were used. The methods include ABCpre, BepiPred and IEDB. These methods work on discrete principles which help to explore various aspects of epitopes. Therefore the results are in varied combinations which can be further processed and interpreted. For example, the ABCpred method predicted a high number of epitopes with the default parameters. However, compared to BepiPred, IEDB analysis revealed unique epitopes which are neither predicted by BepiPred nor ABCpred. The BepiPred is one of the most popular tools for linear B-cell analysis. We compared the epitopes predicted in this study with experimentally verified epitopes, and most of them also matched . All theThe non-structural protein ORF1ab was evaluated as the most antigenic protein. However, due to its nature and length, it is controversial to consider ORF1ab as a vaccine candidate. On the other hand, the ORF1ab encodes viral enzymes crucial for viral replication, such as viral protease . The predictions by IEDB are overlapping 7-mers starting at 782 AA.To compare our findings with already experimentally validated epitopes, we selected epitope patients . Interes280NENGTITDA288) and IEDB (1229MVTIMLCCMTS1239).Several studies have recently been published on the bioinformatics prediction of B-cell epitope prediction in SARS-CoV-2 proteins. A summary of these articles is summarized in Noorimotlagh et al. 2020 . In the Also, we found several similarities between already validated epitopes of previous SARS-CoV and novel SARS-CoV-2 epitopes, however, the amino acid sequences of these viruses are not identical, and thus several differences in predicted epitopes were observed . A similIdentification of T-cell epitopes was performed using the analysis resource TepiTool (IEDB). We included the 27 most frequent alleles for the MHCI and the 26 most frequent alleles of MHC class II. Interestingly, multiple alleles of each MHCI and MHCII bind the same epitopes or overlap. For example, we found 16 epitopes in OFR10, of which only two (YINVFAFPF and MGYINVFAF) bind exclusively MHCI. The number of epitopes predicted in silico for each structural protein is given in Each of the viral proteins was further analyzed by the VaxiJen server  alleles is crucial for understanding the spread of disease, and the pathogenicity of viruses, bacteria and parasites. MHC molecules are highly polymorphic and provide information on how the patients will respond to an antigen . AllelicThe population coverage analysis was performed by testing in silico predicted epitopes and their recognizing HLA alleles using the IEDB Population coverage analysis tool . For anaWe observed significant differences in the coverage of MHC alleles by SARS-CoV-2 proteins for different populations. Similar results were observed for MHCII alleles . The higAn important factor in understanding the prevalence of SARS-CoV-2 is the analysis of different ethnicities. MHC molecules are highly polymorphic and their frequencies vary in different ethnical groups . These d7) COVID-19 patients were observed in each country. An exception to this common pattern can be seen in South Korea with a much lower slope of growth of identified cases. We speculate that this is due to better protection from the beginning of the pandemic because wearing a protective mask is widespread in the Korean population even before the pandemic. Countries such as Colombia, South Africa, Italy, but also Belgium and Mexico, have much higher disease burden  over time due to an underestimation of cases at the beginning of an epidemic. To analyze whether we see the same trend with COVID-19, we plotted the CFR against time . Surprishttps://nextstrain.org/ncov/global; 4992 genomes sampled December 2019 till the end of September 2020 according to Nextstrain database [p-value: 0.00053) and the envelope protein , but not for other proteins or MHCI coverage.We correlated the overall predicted MHC-presentation of the four structural SARS-CoV-2-proteins for different populations with the case fatality rate (CFR) observed in different countries at day 15 since 128 patients . We chosdatabase —accessedp-value: 0.1 level , since it is probably the important target for neutralizing antibodies and it is, therefore, a remarkable observation that predicted MHCII-coverage of epitopes of this protein reveals the strongest correlation with the observed CFR in different countries.The quality of MHCII presentation by the T-cell is an important prerequisite for T-cell-dependent antibody production. Besides the envelope protein and membrane protein, the SARS-CoV-2 surface (spike) protein also shows a clear correlation but only below the The correlations in this study investigate differences in the adaptive immune response to SARS-CoV-2 envelope proteins in different populations. Several observed strong correlations suggest that these may play a role in the observed clear variation in fatality rates in different countries. According to these observations, strong MHC-restricted responses directed at the membrane protein and the envelope protein would be important to achieve better control over SARS-CoV-2-infection and are implied to lower CFR. The correlation of MHCII-presentation of the predicted epitopes of membrane, envelope and spike protein with a low CFR observed in different populations supports also the importance of these proteins as vaccine and potential therapeutic candidates.As a major conclusion of our study, we see that overall the strong epitope binders in T-cells are underrepresented in the Italian population as compared to the German population. However, the current data are not strong enough to identify particular alleles that definitely drive the differences. What we did study and deliver in our work are the differences in adaptive immune response regarding T-cell and B-cell epitope presentation averaging on the global population, not looking at individual populations but covering most of the prevalent alleles in most of the world’s major geographic regions. We primarily investigated MHC-restricted responses, which comprise both, humoral and cell-mediated reactions.Doubtless, many other factors may also contribute to country-specific differences: for instance in some Asian countries such as in Japan may be connected to cultural habits such as many people wearing a face mask even before the pandemic. Nevertheless, for viral diseases such as H7N9 influenza A virus, analysis of T-cell immunity in human populations applying refined immune-informatics provided also here valuable insights such as clear ethnic differences ,25 and aRegarding responses in specific subpopulations, the panel and the predictive power are not strong enough. For example, alleles associated with the DR14 and DR16 serological families, or various DR4 splits such as DR0404 or DR0407 (which will have different binding patterns than DR0401) that may be prevalent in the “negative” populations with reasonably high frequency, but not in the “positive” populations, are not represented in our prediction analysis. Associations of infectious diseases with the HLA DR4 type of the host have been found among distinct populations, racial or ethnic groups. In Mexico, a greater frequency of HLA-DR4 antigens was found in patients with Chagas’ disease with an increased frequency of HLA-DR16 antigens compared to asymptomatic patients or healthy controls . ParticuAnother important fact is the impact of COVID-19 on different races and ethnicities, which was noticed in several studies ,29,30. Ap-value: 0.00053) and for the envelope protein , and still clear for the viral spike protein . However, we cannot, of course, rule out that a completely different factor such as health system and non-HLA immunological factors could bear out an even higher correlation. However, for the latter, we did all reasonable efforts to account for this by (i) purposely averaging on the world population so that local trends do not lead to biases , (ii) monitoring overall immune response by looking at case fatality rate and (iii) by avoiding multiple other comparisons and hypothesis as then the statistical correlation has to be corrected and is weakened by multiple testing. In addition, the obviously higher correlation for MHCII compared to MHCI may indicate that CD4 cells play a more crucial role in fighting against the SARS-CoV-2 infection for the human immune system, in particular for severe COVID-19 patients. This may lead us to reconsider the strategy and development of novel therapeutics. An exception is a nucleocapsid, though highly expressed, this is unlikely able to offer sufficient neutralizing antibody response on its own.There is also another important point to discuss: could it be that other factors  have an equally high correlation or even higher? Well, firstly we only observe a correlation, not a molecular proof. To claim molecular proof, we would need to consider various additional factors: rare antigens, highly represented antigens in local populations, and importantly, direct molecular assays of the immune response. Instead, we focused here only on the correlation of MHC epitopes. However, this correlation is surprisingly strong for the membrane protein . Accession numbers, annotations, PDB identifiers and the length of analyzed proteins are given in Protein sequences of SARS-CoV-2 were screened for linear continuous B-cell epitopes using three tools:http://www.cbs.dtu.dk/services/BepiPred, accessed date: 5 February 2020 [BepiPred Linear Epitope Prediction tool (ver. 2) (ary 2020 ) was usehttps://www.iedb.org, accessed date: 5 February 2020).IEDB Database  used thehttp://crdd.osdd.net/raghava/abcpred, accessed date: 5 February 2020) [The parameters for the ABCpred Tool (ry 2020) ) were ushttp://tools.iedb.org/tepitool; accessed date: 10 February 2020 [T-cell epitopes prediction was performed using TepiTool (ary 2020 ). To preary 2020 .The distribution of COVID-19 recognizing MHCI and MHCII alleles was assessed using the IEDB population coverage tool . Here, wThe analysis was implemented in R by acquiring data from the nCov2019 library  and the During the outbreak, the proportion of death from COVID-19 (CFR) is observed for different countries at 15 days since 128 cases were reported. Accordingly, antigen presentation is collected as a percentage of MHCI and MHCII epitopes. The linear correlation is calculated using the “ggpubr” package in R, and a Pearson method is applied to run the test. We hence assume that the population data are normally distributed.We hypothesize that countries with poor case fatality rates are associated with poor immune response. We predict immunity by monitoring well-represented epitopes, whether these are already sufficient to derive a good correlation with the observed immunity, which is given inversely correlating parameter case fatality rate. We observe that this is really the case in the initial phase of infection. Our analysis combines multiple approaches to investigate the pandemic of SARS-CoV-2 in different countries. There are striking differences predicted for natural population immunity in different populations. Differences in the severity of COVID-19 disease observed in different appear to be related to differences in the potential of different populations to present SARS-CoV-2 epitopes on MHCII in a naïve population . Geographical areas with a high incidence of virus and disease burden such as Italy and Iran have a low epitope-binding MHCII-repertoire compared to countries with less severe disease progression such as Germany, Austria, Sweden, Norway and South Korea. We emphasize here that these are only correlations but these were confirmed using well-established epitope prediction tools that include experimental validation data and predict successfully also established SARS-CoV-2 and other viral epitopes. Moreover, the case fatality rate is a direct indicator of whether the immune response in the population is sufficient, including other epitopes or other immune factors. Our results highlight the importance of the SARS-CoV-2 structural proteins as a target to gain MHCII restricted control over the infection, e.g., by vaccine strategies and should encourage further studies probing the molecular immunology of SARS-CoV-2 further and examining MHCII alleles as potential individual risk factors in COVID-19. Future studies should focus on specific geographic regions as well as other factors taking over after the initial phase (later than day 15 after reaching 128 observed cases) such as health system, potential strain variation effects and, most importantly, different control measures. The evidence presented here suggests that epitopes predicted to provide broad coverage worldwide are also likely to provide broad coverage in specific populations and correlated strongly and inversely with a case fatality rate in the early phase of infection spread.


This study aimed to translate simulation-based dietitian nutritionisteducation to clinical competency attainment in a group of practicingregistered dietitian nutritionists (RDNs). Using a standardized instrumentto measure performance on a newly-required clinical skill, thenutrition-focused physical exam (NFPE), competence was measured both beforeand after a simulation-based education (SBE) session.Eighteen practicing RDNs were recruited by their employer, Spectrum Health.Following a pre-briefing session, participants completed an initial10-minute encounter, performing NFPE on a standardized patient (SP). Next,participants completed a 90-minute SBE training session on skills within theNFPE, including hands-on practice and role play, followed by a post-trainingSP encounter. Video recordings of the SP encounters were scored to assesscompetence in 7 skill areas within the NFPE. Scores were analyzed forparticipants’ initial competence and change in competence.The proportions of participants with initial competence ranged from 0% to 44%across the 7 skill areas assessed. The only competency where participantsinitially scored in the “meets expectations” range was“approach to the patient.” When raw competence scores wereassessed for changes from pre- to post-SBE training, the paired t-testindicated significant increases in all 7 competency areas following thesimulation-based training (P<0.001).This study showed the effectiveness of a SBE training program for increasingcompetence scores of practicing RDNs on a defined clinical skill. Simulation-based education (SBE) in professional training programs has expandedin recent years ,2. ThefThe NFPE has been established as a key tool of dietetics practitioners for theidentification of malnutrition and micronutrient imbalances in adult andpediatric patients . As of 2This study examined a group of practicing RDNs to identify changes in competencescores for clinical skills after participating in a 90-minute SBE session.Further, this study obtained a snapshot of existing competence levels ofpracticing RDNs for the NFPE, a required clinical skill. The research questionswere as follows: first, what is the current rate of competence in the NFPE skillamong practicing clinical RDNs?; second, are years of clinical experience,frequency of performing NFPE, and the education level of RDNs related with theirinitial competence scores in the NFPE?; third, does simulation-based trainingincrease practicing clinicians’ competence in specific skills ?; and fourth, what variables  are associated with the greatest gains in competence frombefore to after simulation-based training?Ethical approval was obtained from the Grand Valley State University HumanSubjects Institutional Review Board as exempt research (#18-220-H).https://www.strobe-statement.org/.This pre-and post-intervention comparison study included RDNs employed atSpectrum Health. This article was described according to the STROBEstatement, available from: This study took place in a university simulation center setting, specifically theSP suite. This suite consisted of outpatient exam rooms outfitted with a videocamera system that enabled the recording of patient examinations conducted in arealistic environment. Specifically, the SPs were adults employed by thesimulation center and trained to portray a variety of simulated patient cases.Training and data collection occurred in 2 groups, held in January and February2020. The same researchers conducted the SBE training and scored thevideo-recorded SP encounters for both groups.The RDNs were selected for training by their employer, as part of a professionaldevelopment activity offering, and selection was not based on level ofexperience, area of practice, or level of interest. A total of 18 RDNsparticipated in the study in the 2 groups .Initially, all RDN participants were asked to complete a survey on their level ofeducation, time since dietetic registration, and frequency of performing NFPE inthe course of their typical practice. The remaining variables analyzed in thisstudy were performance scores on each of the 7 competencies that make up theoverall NFPE skill.The competency scores were awarded by a team of 3 researchers, among whom useof the standardized assessment tool had previously been tested forsatisfactory inter-rater reliability. The pre-training data were obtainedfrom an assessment of video-recorded SP encounters conducted prior to the90-minute SBE training. Initially, participants were led in a 10-minutepre-briefing activity where SP case information was shared. As part of thepre-briefing, participants were informed that the simulation center is asafe space for practice and development of skills, a component of thepre-briefing that has been discussed by other researchers as a key componentof SBE . FollowiThe SBE education methods utilized in the 90-minute training provided toparticipants included: pre-briefing and de-briefing, role playing, andpracticing skills on both SPs and peers. This session was taught andfacilitated by the 3 researchers, all faculty members from thegraduate-level dietetics program at the sponsoring university. The trainingincluded an evidence-based presentation of skills required for competence inNFPE, as determined by guidelines established by the Academy of Nutritionand Dietetics ,11. TradAfter completing the training, the participants performed a second 10-minuteNFPE encounter with a different SP. Following the second encounter, allparticipants met back at the classroom for a 20-minute de-briefing sessionto discuss what went well, what was difficult, and the connections betweenNFPE findings and the given case information for each SP.NFPE skills were assessed by reviewing video recordings of the SP encounters.Recordings were made accessible to the researchers through an encryptedwebsite for the purpose of assigning a competence score. The instrument usedto assess competence from the video encounters was developed based onevidence-based practice guidelines for clinical competence assessment andusing the guidelines of the Academy of Nutrition and Dietetics forNutrition-Focused Physical Exam ,11. The To reduce the potential for confirmation bias in scoring by the researchers, theraters were blinded to whether the participant videos they scored were from thepre- or post-training time points. The 2 patient cases portrayed and the skillsperformed in both the pre- and post-training encounters were identical; eachparticipant saw one case pre-training and the other case post-training, but thesame 2 cases were used in both sessions. Additionally, videos were marked with acode number (not names) for participants, and researchers did not have access tothe code list linking data from the survey of experience level and area ofpractice until after all videos were scored.http://www.gpower.hhu.de/)was used to estimate the sample size for the paired t-test. The a prioriG*Power test indicated that the required sample size was 16 based on a2-tailed test, an effect size of 0.9, alpha error of 0.05, and a power of 0.9.The actual power was 0.915.A total of 18 RDNs participated in this study. Each RDN completed both a pre- anda post-training SP encounter in which they performed the NFPE skill. TheG*Power tool  included: first, descriptive statisticsof the subject population; second, the percent of RDNs competent at the initialtime point; third, the change in competence scores following remediationtraining, with mean competence scores compared using the paired t-test; andfourth, analysis of variance (ANOVA) to determine whether the analyzed variables wereassociated with significant differences in mean competence scores from theinitial assessment to the post-training re-assessment. For analyses, scores wereconsidered both on the 9-point scale and categorically, in 3 levels. Categorieswere considered to better indicate the clinically relevant groups of“below expectations” (scores of 1–3), “meetsexpectations” (scores of 4–6), and “exceedsexpectations” (scores of 7–9). For a binary analysis of initialcompetence, scores within the category of “meets” or“exceeds” expectations were considered as“competent.” These categorical outcome analyses were included inorder to identify differences in NFPE skills that would be relevant andmeaningful in the professional health care setting.The subjects included 18 RDNs employed at Spectrum Health, a private healthsystem in the Midwest region of the United States. All 18 participants attendedthe 90-minute training and completed both pre- and post-training NFPEassessments. The raw data file is available in The mean number of years in practice as an RDN for the participants was 9.6 years(standard deviation=7.7). Most participants  hadearned a bachelor’s degree as their highest earned degree. The largestcategory reported for the average number of times performing NFPE in a week was15 or more times per week , followed by 8–14 times. The complete results of the survey are shown in When competence as a binary variable was assessed for each item, thepercentage of RDNs competent at the initial time-point ranged from 0% to 44% . The only competency where participants scored in the“meets expectations” range initially was competency 1. The initial rates and scores for eachcompetency are shown in One-way ANOVA of the initial competence rates by years in practice, frequencyof performing NFPE, and highest degree completed detected no significantdifferences in any of the competency areas.When competence scores on the 9-point scale were assessed for changes frompre- to post-SBE training, significant increases in competence scores wereobserved in all 7 competency areas. The greatest increase in competencescores among the RDN participants occurred in competency 6 ,with a mean increase of 3.72 points , followed bycompetency 7 . The smallest increase from before to after trainingoccurred in competency 1 . Further investigation into the significance ofdifferences between mean scores at the pre-training and post-training timepoints using the paired t-test revealed significant increases in all 7competency areas following the simulation-based training (P<0.001). Interms of the binary distinction between “meets expectations”and “below expectations,” across all 7 competencies, onaverage 15 out of the 18 participants (83.3%) scored as competent followingthe SBE training. Complete results of the competency score change and thepaired t-test are shown in Relationship between changes in competence level and variables—yearsof experience as an RDN, number of times per week performing NFPE, andhighest degree completed—were examined using 1-way ANOVA. Nosignificant relationships were found between the change in mean score frompre- to post-training and any groupings by experience and education.Based on the competence change results , it appeLimitations of this study include the fact that the researchers who developed thecompetency assessment tool also rated participants at the pre-/post-trainingtime points. Although steps were taken to reduce confirmation bias, such asblinding researchers to whether videos were from a pre- or post-training timepoint, it is possible that subconscious rating inflation could have occurred dueto the investment of the raters in the study.The competencies with the lowest initial scores  were the same competencies with thegreatest increase in scores after the simulation-based training. These areas areadvanced-level skills that are not always included in basic instruction onperformance of the NFPE ,13.ThisAlthough the largest group of participants  indicated on theexperience survey that they performed NFPE on average 15 or more times per week,the competence scores were initially low across all 7 skill areas. This may haveoriginated from the simulation lab setting, which was new to all of theparticipants, or from participants’ unfamiliarity with some of theinstrumentation provided for use .The results of this study serve multiple purposes, for different stakeholders.The results will serve clinical managers and educators in the field of dieteticsby providing data on competence levels of practicing RDNs in the NFPE skill.Additionally, the findings will serve to move the field of health professionseducation forward by translating SBE to competence gains of practicingclinicians, as opposed to assessments of learner-perceived traits, as previousstudies have done ,14.This study demonstrates a method of quantifying the changes in clinical skillcompetence that occurred as a result of SBE training. Within the field ofdietetics, other clinical skills that could be tested using SBE include feedingtube placement, such as guided nasogastric tube placement at the bedside. Futurework should seek to explore potential differences in the effectiveness of SBE atcompetency-building in clinicians with different levels of experience andeducation, as well as in health professions students.


By the year 2035, the older adult population is expected to expand to 78 million in the United States. Advancing technology has made aging in place a more accessible possibility; however, understanding what is preventing this population from adopting the advancing devices remains to be a challenge as the presence of a digital divide continues to exist. A 34-question survey adapted from the Technology Proficiency Self-Assessment Questionnaire, and the National Technology Readiness survey was administered to 101 participants over the age of 50 across five local senior centers. The average age range was 70-79 and most were female (79.2%), white (69%), and owned or had access to technology such as a computer or cell phone (93%). Examples of findings include 86% felt technology limited human interaction and 69% felt the use of technology could lead to security risk and a breach of privacy, while 79% felt technology could improve their quality of life. Results found 60-69-year-olds were significantly more likely (p<.05) to have or use technology versus 80-89-year-olds. Correlation between perception and intent to use technology among older adults was positive with a coefficient value of .59(p<.01). Showing a relationship between perceptions and behavioral intentions to use technology, specifically in 60-69-year-olds. This study found access to technology  was not a driving factor of usage among the older population attending a senior center. To increase understanding further exploration of perceptions and intentions to use technology is warranted in the older adult population.


The recent emergence and rapid global spread of coronavirus disease 2019 (COVID-19) is leading to public health crises worldwide. Alcohol consumption and cigarette smoking (CS) are two known risk factors in many diseases including respiratory infections.We performed a multi-center study in the four largest hospitals designated for COVID-19 patients in Wuhan. There are totally 1547 patients diagnosed with COVID-19 enrolled in the study, alcohol consumption and CS history were evaluated among these patients. The epidemiology, laboratory findings and outcomes of patients contracted COVID-19 were further studied.Our findings indicated that COVID-19 patients with a history of CS tend to have more severe outcomes than non-smoking patients. However, alcohol consumption did not reveal significant effects on neither development of severe illness nor death rates in COVID-19 patients.CS is a risk factor for developing severe illness and increasing mortality during the SARS-CoV-2 infection. We believe that our findings will provide a better understanding on the effects of alcohol intake and CS exposure in COVID-19 patients. The recent emergence and rapid global spread of Severe Acute Respiratory Syndrome (SARS) Coronavirus 2 (SARS-CoV-2) and the resulting coronavirus disease 2019 (COVID-19) is associated with more than 12,077,210 cases and more than 550,327 deaths worldwide as of July 9, 2020 (COVID-19 Map from Johns Hopkins Coronavirus Resource Center). Considering the high rate of SARS-CoV-2 transmission and that there is no known cure for this disease at present, identifying vulnerable populations will be crucial for taking measures to protect those who are at increased risk of infection or of severe disease from COVID-19 .Alcohol consumption and cigarette smoking (CS) are two known risk factors in many diseases including respiratory infections . ChronicOur multi-center study was performed in the four largest hospitals designated for COVID-19 patients in Wuhan. All hospitalized patients were confirmed COVID-19 positive according to the interim guidance from the World Health Organization and National Health Commission of China , from FeClinical course, laboratory findings and outcomes were obtained and reviewed. The medical records were analyzed by three investigators independently. Information included demographic characteristics, symptoms, signs, underlying comorbid conditions and treatments were collected from the medical records. Laboratory data on the day of admission were collected. Smoking history was defined according to the WHO guidelines, including smoking for more than 6 months, or smoking more than 5 or more cigarettes per day ; alcoholU test, x2 test, or Student’s t-test to compare differences between two different groups where appropriate. Univariable and multivariable logistic regression analyses were used to identify any association between serious illness and smoking history. Univariable and multivariable cox regression analyses were used to identify any association between death and smoking history. Variables with a P-value less than 0.05 at univariable analysis were subjected to multivariable analysis, and odds ratios with 95% confidence intervals (CIs) were reported. The areas under the receiver operating characteristic curve were used to evaluate the discriminative abilities of the disease severity status. P-values less than 0.05 were considered to indicate statistical significance. Statistical analyses were performed by using statistical software (SPSS version 26.0).Categorical variables were described as frequency rates and percentages. Continuous variables were presented as mean ± standard deviation (SD) or mean, and interquartile range (IQR) as appropriately. We used the Mann–Whitney A total of 1547 subjects, including 802 male patients (51.8%) and 745 female patients (48.2%), were enrolled in the study. According to past medical history, the most common concurrent diseases were hypertension (27.34%), diabetes (12.93%) and cardiovascular disease (10.92%) . Among 1p < 0.001). There was no statistically significant difference in age, BMI, comorbidities and symptoms between the smoking group and the non-smoking group. However, in terms of biochemical indicators, the counts of lymphocyte and thrombocyte of the smoking group were lower than that of the non-smoking group, while C-reactive protein and AST were higher than that of the non-smoking group. The difference was statistically significant . There was no statistically significant difference in age and BMI. Concerning comorbidities and symptoms upon admission, COVID-19 patients with and without alcohol consumption were similar except for a higher proportion of diabetes . In terms of laboratory indicators, the counts of lymphocyte from the alcohol group were lower than that of the non-alcohol group, while ALT was higher than that of the non-alcohol group. The difference was statistically significant , indicating that the risk of severe illness in patients with previous CS history was 1.91 times higher than that in non-smokers.Within the same population, two groups were divided according to whether they had a history of alcohol consumption. The proportion of males in the alcohol group was higher than that in the non-alcohol group , indicating that the risk of death in smokers was 1.825 times higher than that in non-smokers. Also, lymphocyte count, C-reactive protein, and ALT were risk factors for death. However, alcohol consumption did not show a significant effect on death rates of COVID-19 patients in both univariable and multivariable analyses (However, in both univariable and multivariable analyses, alcohol consumption did not reveal any significant effect on developing severe illness in COVID-19 patients . Among tanalyses . In thisAlcohol is the most commonly abused drug in the world. It affects almost every organ of our body. At this time, patients with a history of alcohol consumption did not develop more severe outcomes compared to patients without. That might be because the time we collect our data is still in the early stage of the pandemic. With the SARS-CoV-2 rapidly spreading, governments across the world have issued stay-at-home and face covering orders, which resulted in trillions of people being isolated from each other for long periods of time. Combining with the stress of rising unemployment, excessive use of alcohol consumption becomes a public health crisis . SeveralIn conclusion, CS is a risk factor for developing severe illness and increasing mortality during the SARS-CoV-2 infection. We believe that our findings will provide a better understanding on the effects of alcohol intake and CS exposure in COVID-19 patients.ML designed the research. MD and ZC were in charge of data collection and manuscript writing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Many public officials are calling for increased testing for the 2019 novel coronavirus disease (COVID-19), and some governments have taken extraordinary measures to increase the availability of testing. However, little has been published about the sensitivity and specificity of the reverse transcriptase-polymerase chain reaction (RT-PCR) nasopharyngeal swabs that are commonly used for testing. This narrative review evaluates the literature regarding the accuracy of these tests, and makes recommendations based on this literature. In brief, a negative RT-PCR nasopharyngeal swab test is insufficient to rule out COVID-19. Thus, over-reliance on the results of the test may be dangerous, and the push for widespread testing may be overstated. A novel coronavirus disease COVID-19), caused by SARS-CoV-2, has rapidly spread throughout many countries including the United States since its discovery in December 2019.9, causedAt this time, no peer-reviewed publications have reported a sensitivity and specificity of RT-PCR tests for COVID-19. One non-peer reviewed publication reports that, based on 87 Chinese patients who were ultimately diagnosed with COVID-19, pharyngeal RT-PCR tests have a sensitivity and specificity of 78.2% and 98.8%, respectively.First, a study by Wang et al took various types of specimens from 205 patients with confirmed COVID-19 and tested them with RT-PCR. Of 398 pharyngeal swabs, they found only 126 (32%) were positive. They took just eight nasal swabs, and found five (63%) were positive.9Along the same lines, Winichakoon et al published a letter to the editor in which they described a case of a COVID-19 patient who had a nasopharygeal/oropharyngeal RT-PCR swab that was negative for COVID-19, but RT-PCR of BAL fluid was positive.12Next, in a case series described by Xie et al, five patients from the Hunan province of China had ground-glass opacities on chest computed tomography (CT) that were suggestive of COVID-19, but initial pharyngeal RT-PCR tests were negative. Repeat RT-PCR swabs ended up being positive.14Other studies have also demonstrated that initial RT-PCR tests may be negative and then become positive with repeated tests. For example, Wu et al studied the clinical course of 80 patients from the Jiangsu Province who were ultimately diagnosed with COVID-19. Nine of those 80 patients (11.3%) had two negative RT-PCR nasal or oral swabs before their third swabs came back positive.16The sensitivity and specificity of nasopharyngeal swabs using RT-PCR for the diagnosis of COVID-19 cannot be precisely determined with the published data to this point. However, the available in vitro data along with minimal clinical data suggest that the test has very high specificity. On the other hand, the sensitivity is moderate (perhaps between 63–78%). Among the various ways of performing RT-PCR, pharyngeal swabs seem to have lowest sensitivity; nasal swabs may be a bit more sensitive than pharyngeal swabs. RT-PCR analysis of BAL fluid seems to be the most accurate means of virologic confirmation, but BAL fluid can only reasonably be collected on the sickest cohort of patients. For patients with moderate to severe COVID-19 symptoms, identifying characteristic findings on CT imaging of the chest may be more sensitive than RT-PCR testing.Given these findings, when a patient has a high pretest probability for COVID-19, a negative test does not rule out the disease. Consequently, policies that assume a high accuracy of RT-PCR testing are perilous. For example, employers should not use a negative test result to decide when someone should return to work. Meanwhile, the perceived need for increased testing propagated by the popular mediaIncreased testing could be beneficial in areas of the world where there are very few cases of COVID-19. Aggressive early testing could allow for early identification of cases to allow for early targeted isolation and social distancing measures. However, in cities where COVID-19 is already widespread, the testing of large numbers of individuals with mild illness will have minimal effect on treatment but will require massive resources. There is epidemiological benefit to testing, but in cities already being devastated by COVID-19, the numbers of hospitalizations and mortalities associated with it can be used as indicators of disease impact. Reduced testing of patients with mild disease could save testing materials so that sicker patients and healthcare professionals will have access to testing. Additionally, a large amount of personal protective equipment could be saved by not attempting to test the many thousands and perhaps what will be millions of mild COVID-19 cases.While the exact sensitivity and specificity of RT-PCR tests for COVID-19 are not known, it appears that a positive test is highly suggestive of true COVID-19, but a negative test does not rule out the disease. Patients and providers in epidemic areas should assume they have the disease if they have the signs and symptoms of the disease even if their test was negative. The push for increased testing in areas that already have widespread COVID-19 may be overstated, as the benefits of large-scale use of a moderate sensitivity test are minimal.