Diagnosis of SARS-CoV-2 during the Pandemic by Multiplex RT-rPCR hCoV Test: Future Perspectives.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has rapidly become a significant threat to public health. However, among the Coronaviridae family members, there are other viruses that can also cause infections in humans. Among these, severe acute respiratory syndrome (SARS-CoV) and Middle East respiratory syndrome (MERS-CoV) have posed significant threats to human health in the past. Other human pathogenic coronaviruses have been identified, and they are known to cause respiratory diseases with manifestations ranging from mild to severe. In this study, we evaluated the performance of a multiplex RT-rPCR specific to seven human pathogenic coronaviruses in mainly detecting SARS-CoV-2 directly from nasopharyngeal swabs obtained from suspected COVID-19 infected patients, while simultaneously detecting different human pathogenic coronaviruses in case these were also present. We tested 1195 clinical samples suspected of COVID-19 infection. The assay identified that 69% of the samples tested positive for SARS-CoV-2 (1195), which was confirmed using another SARS-CoV-2 RT-PCR kit available in our laboratory. None of these clinical samples were positive for SARS-CoV, MERS-CoV or HCoV. This means that during the endemic phase of COVID-19, infection with other human pathogenic coronaviruses, even the common cold coronavirus (HCoV), is very uncommon. Our study also confirmed that the multiplex RT-rPCR is a sensitive assay for detecting SARS-CoV-2 regardless of differences among the variants. This multiplex RT-rPCR is also time- and cost-saving and very easy to apply in the diagnostic laboratory due to its simple procedure and its stability in storage after preparation. These features make the assay a valuable approach in screening procedures for the rapid detection of SARS-CoV-2 and other human pathogenic coronaviruses that could affect public health.

SARS-CoV-2 Gamma and Delta Variants of Concern Might Undermine Neutralizing Activity Generated in Response to BNT162b2 mRNA Vaccination.

The Delta variant raised concern regarding its ability to evade SARS-CoV-2 vaccines. We evaluated a serum neutralizing response of 172 Italian healthcare workers, three months after complete Comirnaty (BNT162b2 mRNA, BioNTech-Pfizer) vaccination, testing their sera against viral isolates of Alpha, Gamma and Delta variants, including 36 subjects with a previous SARS-CoV-2 infection. We assessed whether IgG anti-spike TRIM levels and serum neutralizing activity by seroneutralization assay were associated. Concerning Gamma variant, a two-fold reduction in neutralizing titres compared to the Alpha variant was observed, while a four-fold reduction of Delta virus compared to Alpha was found. A gender difference was observed in neutralizing titres only for the Gamma variant. The serum samples of 36 previously infected SARS-CoV-2 individuals neutralized Alpha, Gamma and Delta variants, demonstrating respectively a nearly three-fold and a five-fold reduction in neutralizing titres compared to Alpha variant. IgG anti-spike TRIM levels were positively correlated with serum neutralizing titres against the three variants. The Comirnaty vaccine provides sustained neutralizing antibody activity towards the Alpha variant, but it is less effective against Gamma and even less against Delta variants.

Defend as You Can, React Quickly: The Effects of the COVID-19 Shock on a Large Fishery of the Mediterranean Sea

This paper presents an analysis of the effect of SARS-CoV-2 coronavirus pandemic and related restrictive measures on the activity of the Italian fleet of trawlers, which represents one of the most important fisheries in the Mediterranean Sea. We integrated multiple sources of information including: 1) fleet activity data from Vessel Monitoring System, the most important satellite-based tracking device;2) vessel-specific landing data disaggregated by species;3) market and economic drivers affecting the effort variation during the lockdown and in the related fishing strategies;4) monthly landings of demersal species in the main Italian harbours. These data sources are combined to: 1) assess the absolute and relative changes of trawling effort in the geographical sub-areas surrounding the Italian coasts;2) integrate and compare these changes with the market and economic drivers in order to explain the observed changes in fishing effort and strategy;3) analyse the changes of the fishing effort on the Landing-per-unit-effort (LPUE) in order to further understand the strategy adopted by fishers during this crisis and to infer the potential consequence for the different stocks. The results provide an overview of the effects of the “COVID-19 shock”, in terms of fishing activity and socio-economic drivers, demonstrating that the consequences of the pandemic have been very varied. Although the COVID-19 shock has caused a marked overall reduction in activity in the first semester of 2020, in some cases the strategies adopted by fishermen and the commercial network linked to their activity have significantly reduced the impact of the emergency and taken back catch and effort to levels similar to those of previous years. These results could provide insights for management measures based on fleet reduction and temporal stops.

COVID-19 Infection in Children and Infants: Current Status on Therapies and Vaccines.

Since the beginning in December 2019, the SARS-CoV-2 outbreak appeared to affect mostly the adult population, sparing the vast majority of children who only showed mild symptoms. The purpose of this investigation is to assess the status on the mechanisms that give children and infants this variation in epidemiology compared to the adult population and its impact on therapies and vaccines that are aimed towards them. A literature review, including in vitro studies, reviews, published guidelines and clinical trials was performed. Clinical trials concerned topics that allowed a descriptive synthesis to be produced. Four underlying mechanisms were found that may play a key role in providing COVID-19 protection in babies. No guidelines are available yet for therapy due to insufficient data; support therapy remains the most used. Only two vaccines are approved by the World Health Organization to be used in children from 12 years of age, and there are currently no efficacy or safety data for children below the age of 12 years. The COVID-19 clinical frame infection is milder in children and adolescents. This section of the population can act as vectors and reservoirs and play a key role in the transmission of the infection; therefore, vaccines are paramount. More evidence is required to guide safely the vaccination campaign.

Emerging Mutations Potentially Related to SARS-CoV-2 Immune Escape: The Case of a Long-Term Patient.

SARS-CoV-2 isolates from long-term COVID-19 patients play a significant role in understanding the mechanisms of infection and virus persistence. This study describes a SARS-CoV-2 isolate from a 53-year-old woman from Apulia (Italy), who was COVID-19 positive for approximately four months. In this paper we aimed to investigate any potential correlation between genetic mutations and clinical features of this case of infection. The viral isolate was assigned to lineage B.1.177.51 through whole-genome sequencing (WGS) and harbored a novel set of mutations on the Spike protein (V143D, del144/145 and E484K); furthermore, seroneutralization assays showed impaired response of the surveyed strain to BNT162b2 (Comirnaty) Pfizer/BioNTech vaccine-induced (average reduction of 70%) and convalescent sera (average reduction of 19.04%), when compared to VOC P.1. This study highlights the importance of genomic surveillance for the management of the COVID-19 pandemic, the relevance of monitoring of emerging SARS-CoV-2 mutations in all lineages, and the necessity of testing the response of emerging variants to available therapies and vaccines.

Laniakea@ReCaS: exploring the potential of customisable Galaxy on-demand instances as a cloud-based service.

BACKGROUND: Improving the availability and usability of data and analytical tools is a critical precondition for further advancing modern biological and biomedical research. For instance, one of the many ramifications of the COVID-19 global pandemic has been to make even more evident the importance of having bioinformatics tools and data readily actionable by researchers through convenient access points and supported by adequate IT infrastructures. One of the most successful efforts in improving the availability and usability of bioinformatics tools and data is represented by the Galaxy workflow manager and its thriving community. In 2020 we introduced Laniakea, a software platform conceived to streamline the configuration and deployment of "on-demand" Galaxy instances over the cloud. By facilitating the set-up and configuration of Galaxy web servers, Laniakea provides researchers with a powerful and highly customisable platform for executing complex bioinformatics analyses. The system can be accessed through a dedicated and user-friendly web interface that allows the Galaxy web server's initial configuration and deployment. RESULTS: "Laniakea@ReCaS", the first instance of a Laniakea-based service, is managed by ELIXIR-IT and was officially launched in February 2020, after about one year of development and testing that involved several users. Researchers can request access to Laniakea@ReCaS through an open-ended call for use-cases. Ten project proposals have been accepted since then, totalling 18 Galaxy on-demand virtual servers that employ ~ 100 CPUs, ~ 250 GB of RAM and ~ 5 TB of storage and serve several different communities and purposes. Herein, we present eight use cases demonstrating the versatility of the platform. CONCLUSIONS: During this first year of activity, the Laniakea-based service emerged as a flexible platform that facilitated the rapid development of bioinformatics tools, the efficient delivery of training activities, and the provision of public bioinformatics services in different settings, including food safety and clinical research. Laniakea@ReCaS provides a proof of concept of how enabling access to appropriate, reliable IT resources and ready-to-use bioinformatics tools can considerably streamline researchers' work.

Accurate detection and quantification of SARS-CoV-2 genomic and subgenomic mRNAs by ddPCR and meta-transcriptomics analysis.

SARS-CoV-2 replication requires the synthesis of a set of structural proteins expressed through discontinuous transcription of ten subgenomic mRNAs (sgmRNAs). Here, we have fine-tuned droplet digital PCR (ddPCR) assays to accurately detect and quantify SARS-CoV-2 genomic ORF1ab and sgmRNAs for the nucleocapsid (N) and spike (S) proteins. We analyzed 166 RNA samples from anonymized SARS-CoV-2 positive subjects and we observed a recurrent and characteristic pattern of sgmRNAs expression in relation to the total viral RNA content. Additionally, expression profiles of sgmRNAs, as determined by meta-transcriptomics sequencing of a subset of 110 RNA samples, were highly correlated with those obtained by ddPCR. By providing a comprehensive and dynamic snapshot of the levels of SARS-CoV-2 sgmRNAs in infected individuals, our results may contribute a better understanding of the dynamics of transcription and expression of the genome of SARS-CoV-2 and facilitate the development of more accurate molecular diagnostic tools for the stratification of COVID-19 patients.

Tracking the international spread of SARS-CoV-2 lineages B.1.1.7 and B.1.351/501Y-V2 with grinch.

Late in 2020, two genetically-distinct clusters of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with mutations of biological concern were reported, one in the United Kingdom and one in South Africa. Using a combination of data from routine surveillance, genomic sequencing and international travel we track the international dispersal of lineages B.1.1.7 and B.1.351 (variant 501Y-V2). We account for potential biases in genomic surveillance efforts by including passenger volumes from location of where the lineage was first reported, London and South Africa respectively. Using the software tool grinch (global report investigating novel coronavirus haplotypes), we track the international spread of lineages of concern with automated daily reports, Further, we have built a custom tracking website (cov-lineages.org/global_report.html) which hosts this daily report and will continue to include novel SARS-CoV-2 lineages of concern as they are detected.

COVID-19 Infection in Children, Infants and Pregnant Subjects: An Overview of Recent Insights and Therapies.

BACKGROUND: The SARS-CoV-2 pandemic has involved a severe increase of cases worldwide in a wide range of populations. The aim of the present investigation was to evaluate recent insights about COVID-19 infection in children, infants and pregnant subjects. METHODS: a literature overview was performed including clinical trials, in vitro studies, reviews and published guidelines regarding the present paper topic. A descriptive synthesis was performed to evaluate recent insights and the effectiveness of therapies for SARS-CoV-2 infection in children, infants and pregnant subjects. RESULTS: Insufficient data are available regarding the relationship between COVID-19 and the clinical risk of spontaneous abortion and premature foetus death. A decrease in the incidence of COVID-19 could be correlated to a minor expression of ACE2 in childrens' lungs. At present, a modulation of the dose-effect posology for children and infants is necessary. CONCLUSIONS: Pregnant vertical transmission has been hypothesised for SARS-CoV-2 infection. Vaccines are necessary to achieve mass immunity for children and also pregnant subjects.

Next generation sequencing of SARS-CoV-2 genomes: challenges, applications and opportunities.

Various next generation sequencing (NGS) based strategies have been successfully used in the recent past for tracing origins and understanding the evolution of infectious agents, investigating the spread and transmission chains of outbreaks, as well as facilitating the development of effective and rapid molecular diagnostic tests and contributing to the hunt for treatments and vaccines. The ongoing COVID-19 pandemic poses one of the greatest global threats in modern history and has already caused severe social and economic costs. The development of efficient and rapid sequencing methods to reconstruct the genomic sequence of SARS-CoV-2, the etiological agent of COVID-19, has been fundamental for the design of diagnostic molecular tests and to devise effective measures and strategies to mitigate the diffusion of the pandemic. Diverse approaches and sequencing methods can, as testified by the number of available sequences, be applied to SARS-CoV-2 genomes. However, each technology and sequencing approach has its own advantages and limitations. In the current review, we will provide a brief, but hopefully comprehensive, account of currently available platforms and methodological approaches for the sequencing of SARS-CoV-2 genomes. We also present an outline of current repositories and databases that provide access to SARS-CoV-2 genomic data and associated metadata. Finally, we offer general advice and guidelines for the appropriate sharing and deposition of SARS-CoV-2 data and metadata, and suggest that more efficient and standardized integration of current and future SARS-CoV-2-related data would greatly facilitate the struggle against this new pathogen. We hope that our 'vademecum' for the production and handling of SARS-CoV-2-related sequencing data, will contribute to this objective.

Symptomatic SARS-CoV-2 Reinfection in a Healthy Healthcare Worker in Italy Confirmed by Whole-Genome Sequencing.

This study describes a case of SARS-CoV-2 reinfection confirmed by whole-genome sequencing in a healthy physician who had been working in a COVID-19 hospital in Italy since the beginning of the pandemic. Nasopharyngeal swabs were obtained from the patient at each presentation as part of routine surveillance. Nucleic acid amplification testing was performed on the two samples to confirm SARS-CoV-2 infection, and serological tests were used to detect SARS-CoV-2 IgG antibodies. Comparative genome analysis with whole-genome sequencing was performed on nasopharyngeal swabs collected during the two episodes of COVID-19. The first COVID-19 episode was in March 2020, and the second was in January 2021. Both SARS-CoV-2 infections presented with mild symptoms, and seroconversion for SARS-CoV-2 IgG was documented. Genomic analysis showed that the viral genome from the first infection belonged to the lineage B.1.1.74, while that from the second infection to the lineage B.1.177. Epidemiological, clinical, serological, and genomic analyses confirmed that the second episode of SARS-CoV-2 infection in the healthcare worker met the qualifications for "best evidence" for reinfection. Further studies are urgently needed to assess the frequency of such a worrisome occurrence, particularly in the light of the recent diffusion of SARS-CoV-2 variants of concern.

Investigation of an outbreak of symptomatic SARS-CoV-2 VOC 202012/01-lineage B.1.1.7 infection in healthcare workers, Italy.

OBJECTIVES: In December 2020, Italy began a national immunization campaign using the BNT162b2 coronavirus disease 2019 (COVID-19) mRNA vaccine, prioritizing healthcare workers (HCWs). Immune serum from vaccinated subjects seems (largely) to retain titres of neutralizing antibodies, even against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) VOC 202012/01-lineage B.1.1.7. Here, we describe an outbreak of SARS-CoV-2 lineage B.1.1.7 infection in three HCWs in a hospital setting; two of the HCWs were fully vaccinated (i.e. had received two doses). METHODS: Two physicians and one nurse working on the same shift on 20th February 2021 were involved in the outbreak. Real-time PCR, antigen tests, and serological tests for the IgG anti-spike protein of SARS-CoV-2 were performed, along with whole-genome sequencing (WGS). RESULTS: SARS-CoV-2 infection was confirmed in all three HCWs; all presented with mild symptoms of COVID-19. The two physicians were fully vaccinated with BNT162b2 vaccine, with the second dose administered 1 month before symptom onset. Both had high titres of IgG anti-spike antibodies at the time of diagnosis. WGS confirmed that all virus strains were VOC 202012/01-lineage B.1.1.7, suggesting a common source of exposure. Epidemiological investigation revealed that the suspected source was a SARS-CoV-2-positive patient who required endotracheal intubation due to severe COVID-19. All procedures were carried out using a full suite of personal protective equipment (PPE). CONCLUSIONS: This mini-outbreak highlights some important issues about the efficacy of vaccines against transmission of SARS-CoV-2 variants, the high risk of exposure among HCWs, and the need for optimized implementation of PPE in hospitals. The wide circulation of VOC 202012/01 in Europe and Italy highlights the need to improve surveillance and genetic sequencing.

Rapid Spread of the SARS-CoV-2 Variant of Concern 202012/01 in Southern Italy (December 2020-March 2021).

Epidemiological and virological studies have revealed that SARS-CoV-2 variants of concern (VOCs) are emerging globally, including in Europe. The aim of this study was to evaluate the spread of B.1.1.7-lineage SARS-CoV-2 in southern Italy from December 2020-March 2021 through the detection of the S gene target failure (SGTF), which could be considered a robust proxy of VOC B.1.1.7. SGTF was assessed on 3075 samples from week 52/2020 to week 10/2021. A subset of positive samples identified in the Apulia region during the study period was subjected to whole-genome sequencing (WGS). A descriptive and statistical analysis of the demographic and clinical characteristics of cases according to SGTF status was performed. Overall, 20.2% of samples showed SGTF; 155 strains were confirmed as VOC 202012/01 by WGS. The proportion of SGTF-positive samples rapidly increased over time, reaching 69.2% in week 10/2021. SGTF-positive cases were more likely to be symptomatic and to result in hospitalization (p < 0.0001). Despite the implementation of large-scale non-pharmaceutical interventions (NPIs), such as the closure of schools and local lockdowns, a rapid spread of VOC 202012/01 was observed in southern Italy. Strengthened NPIs and rapid vaccine deployment, first among priority groups and then among the general population, are crucial both to contain the spread of VOC 202012/01 and to flatten the curve of the third wave.

Genomic Surveillance of Circulating SARS-CoV-2 in South East Italy: A One-Year Retrospective Genetic Study.

In order to provide insights into the evolutionary and epidemiological viral dynamics during the current COVID-19 pandemic in South Eastern Italy, a total of 298 genomes of SARS-CoV-2 strains collected in the Apulia and Basilicata regions, between March 2020 and January 2021, were sequenced. The genomic analysis performed on the draft genomes allowed us to assign the genetic clades and lineages of belonging to each sample and provide an overview of the main circulating viral variants. Our data showed the spread in Apulia and Basilicata of SARS-CoV-2 variants which have emerged during the second wave of infections and are being currently monitored worldwide for their increased transmission rate and their possible impact on vaccines and therapies. These results emphasize the importance of genome sequencing for the epidemiological surveillance of the new SARS-CoV-2 variants' spread.

VOC 202012/01 Variant Is Effectively Neutralized by Antibodies Produced by Patients Infected before Its Diffusion in Italy.

The coronavirus disease 2019 (Covid-19) pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and presents a global health emergency that needs urgent intervention. Viruses constantly change through mutation, and new variants of a virus are expected to occur over time. In the United Kingdom (UK), a new variant called B.1.1.7 has emerged with an unusually large number of mutations. The aim of this study is to evaluate the level of protection of sera from 12 patients infected and later healed in Apulia Region (Italy) with Covid-19 between March and November 2020, when the English variant was not circulating in this territory yet, against the new VOC 202012/01 variant by seroneutralization assay. The sera of patients had already been tested before, using a virus belonging to the lineage B.1 and showed an antibody neutralizing titer ranging between 1:160 and 1:320. All the 12 sera donors confirmed the same titers of neutralizing antibodies obtained with a strain belonging to the lineage B.1.1.7 (VOC 202012/01). These data indicate that antibodies produced in subjects infected with variants of Sars-CoV-2 strain before the appearance of the English one, seem to have a neutralizing power also against this variant.

Genome Sequence of a SARS-CoV-2 VUI 202012/01 Strain Identified from a Patient Returning from London, England, to the Apulia Region of Italy.

The coding-complete sequence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was obtained from a sample from a 25-year-old female returning to the Apulia region of Italy from England. The characterized strain showed all of the spike protein mutations defining SARS-CoV-2 VUI 202012/01, as well as other mutations in the spike protein and in other genomic regions.

Real Time PCR and Culture-Based Virus Isolation Test in Clinically Recovered Patients: Is the Subject Still Infectious for SARS-CoV2?

BACKGROUND: The highly variable manifestation of the COVID-19 disease, from completely asymptomatic to fatal, is both a clinical and a public health issue. The criteria for discharge of hospitalized patients have been based so far on the negative result of Real-Time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) tests, but the persistence of viral fragments may exceed that of the integral virus by weeks. The aim of our study was to verify the clearance of the virus at viral culture in patients hospitalized for COVID-19 that have clinically recovered but are still positive on nasopharyngeal swab. METHODS: The study was conducted in hospitalized patients with positive RT-PCR on nasopharyngeal swab. Patients included were from asymptomatic to severe cases and performed nasopharyngeal control swabbing on day 14 for asymptomatic patient or at least three days after remission of symptoms. RT-PCR positive specimens were sent to a biosafety level 3 laboratory for viral culture. RESULTS: We performed a combined analysis of RT-PCR and a highly sensitive in vitro culture from 84 samples of hospitalized patients. The average age was 46 ± 20.29, and 40.5% of the subjects had radiologically confirmed pneumonia, with average PaO2 of 72.35 ± 12.12and P/F ratio of 315 ± 83.15. Ct values for the N gene were lower in the first swab than in the control one (p < 0.001). The samples from 83 patients were negative at viral culture, and RT-PCR on the respective supernatants always confirmed the absence of viral growth. CONCLUSIONS: Our preliminary results demonstrate that patients clinically recovered for at least three days show the viral clearance at viral culture, and presumably they continued to not be contagious.

Sars-CoV-2 isolation from a 10-day-old newborn in Italy: A case report.

This report describes the evolution of COVID-19 in a 10 day-old-baby. The mother developed the disease immediately after childbirth and therefore a vertical transmission can be excluded. The isolation of the virus in cell culture with a cytopathic effect already visible after 48 h, indicates that the viral load of the newborn was quite high, but not serious course of the disease was observed. This paper wants to highlight the possible role of newborns and children in the spread of the disease.