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The coronavirus SARS-CoV-2 has highlighted the criticality of an accurate and rapid diagnosis in order to contain the spread of the virus. Knowledge of the viral structure and its genome is essential for diagnosis development. The virus is still quickly evolving and the global scenario could easily change. Thus, a greater range of diagnostic options is essential to face this threat to public health. In response to the global demand, there has been a rapid advancement in the understanding of current diagnostic methods. In fact, innovative approaches have emerged, leveraging the benefits of nanomedicine and microfluidic technologies. Although this development has been incredibly fast, several key areas require further investigation and optimization, such as sample collection and preparation, assay optimization and sensitivity, cost effectiveness, scalability device miniaturization, and portability and integration with smartphones. Addressing these gaps in the knowledge and these technological challenges will contribute to the development of reliable, sensitive, and user-friendly NAAT-based POCTs for the diagnosis of SARS-CoV-2 and other infectious diseases, facilitating rapid and effective patient management. This review aims to provide an overview of current SARS-CoV-2 detection methods based on nucleic acid detection tests (NAATs). Additionally, it explores promising approaches that combine nanomedicine and microfluidic devices with high sensitivity and relatively fast 'time to answer' for integration into point-of-care testing (POCT).

Coronavirus disease 2019 (COVID-19) is the greatest threat to global health at the present time, and considerable public and private effort is being devoted to fighting this recently emerged disease. Despite the undoubted advances in the development of vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, uncertainty remains about their future efficacy and the duration of the immunity induced. It is therefore prudent to continue designing and testing vaccines against this pathogen. In this article we computationally designed two candidate vaccines, one monopeptide and one multipeptide, using a technique involving optimizing lambda-superstrings, which was introduced and developed by our research group. We tested the monopeptide vaccine, thus establishing a proof of concept for the validity of the technique. We synthesized a peptide of 22 amino acids in length, corresponding to one of the candidate vaccines, and prepared a dendritic cell (DC) vaccine vector loaded with the 22 amino acids SARS-CoV-2 peptide (positions 50-71) contained in the NTD domain (DC-CoVPSA) of the Spike protein. Next, we tested the immunogenicity, the type of immune response elicited, and the cytokine profile induced by the vaccine, using a non-related bacterial peptide as negative control. Our results indicated that the CoVPSA peptide of the Spike protein elicits noticeable immunogenicity in vivo using a DC vaccine vector and remarkable cellular and humoral immune responses. This DC vaccine vector loaded with the NTD peptide of the Spike protein elicited a predominant Th1-Th17 cytokine profile, indicative of an effective anti-viral response. Finally, we performed a proof of concept experiment in humans that included the following groups: asymptomatic non-active COVID-19 patients, vaccinated volunteers, and control donors that tested negative for SARS-CoV-2. The positive control was the current receptor binding domain epitope of COVID-19 RNA-vaccines. We successfully developed a vaccine candidate technique involving optimizing lambda-superstrings and provided proof of concept in human subjects. We conclude that it is a valid method to decipher the best epitopes of the Spike protein of SARS-CoV-2 to prepare peptide-based vaccines for different vector platforms, including DC vaccines., (© 2022. The Author(s).)

The objective of this study was to analyse the mechanisms of resistance to carbapenems and other extended-spectrum-β-lactams and to determine the genetic relatedness of multidrug-resistant Enterobacterales (MDR-E) causing colonization or infection in solid-organ transplantation (SOT) recipients. Prospective cohort study in kidney (n = 142), liver (n = 98) or kidney/pancreas (n = 7) transplant recipients between 2014 and 2018 in seven Spanish hospitals. We included 531 MDR-E isolates from rectal swabs obtained before transplantation and weekly for 4-6 weeks after the procedure and 10 MDR-E from clinical samples related to an infection. Overall, 46.2% Escherichia coli, 35.3% Klebsiella pneumoniae, 6.5% Enterobacter cloacae, 6.3% Citrobacter freundii and 5.7% other species were isolated. The number of patients with MDR-E colonization post-transplantation (176; 71.3%) was 2.5-fold the number of patients colonized pre-transplantation (71; 28.7%). Extended-spectrum β-lactamases (ESBLs) and carbapenemases were detected in 78.0% and 21.1% of MDR-E isolates respectively. In nine of the 247 (3.6%) transplant patients, the microorganism causing an infection was the same strain previously cultured from surveillance rectal swabs. In our study we have observed a low rate of MDR-E infection in colonized patients 4-6 weeks post-transplantation. E. coli producing bla CTX-M-G1 and K. pneumoniae harbouring bla OXA-48 alone or with bla CTX-M-G1 were the most prevalent MDR-E colonization strains in SOT recipients.

IntroductionEnterovirus A71 (EV-A71) is an emerging pathogen that causes a wide range of disorders including severe neurological manifestations. In the past 20 years, this virus has been associated with large outbreaks of hand, foot and mouth disease with neurological complications in the Asia-Pacific region, while in Europe mainly sporadic cases have been reported. In spring 2016, however, an EV-A71 outbreak associated with severe neurological cases was reported in Catalonia and spread further to other Spanish regions.AimOur objective was to investigate the epidemiology and clinical characteristics of the outbreak.MethodsWe carried out a retrospective study which included 233 EV-A71-positive samples collected during 2016 from hospitalised patients. We analysed the clinical manifestations associated with EV-A71 infections and performed phylogenetic analyses of the 3'-VP1 and 3Dpol regions from all Spanish strains and a set of EV-A71 from other countries.ResultsMost EV-A71 infections were reported in children (mean age: 2.6 years) and the highest incidence was between May and July 2016 (83%). Most isolates (218/233) were classified as subgenogroup C1 and 217 of them were grouped in one cluster phylogenetically related to a new recombinant variant strain associated with severe neurological diseases in Germany and France in 2015 and 2016. Moreover, we found a clear association of EV-A71-C1 infection with severe neurological disorders, brainstem encephalitis being the most commonly reported.ConclusionAn emerging recombinant variant of EV-A71-C1 was responsible for the large outbreak in 2016 in Spain that was associated with many severe neurological cases.

Background: Influenza A viruses are characterised by their rapid evolution, and the appearance of point mutations in the viral hemagglutinin (HA) domain causes seasonal epidemics. The A(H3N2) virus has higher mutation rate than the A(H1N1) virus. The aim of this study was to reconstruct the evolutionary dynamics of the A(H3N2) viruses circulating in Italy between 2004 and 2012 in the light of the forces driving viral evolution., Methods: Phylodinamic analyses were made using a Bayesian method, and codon-specific positive selection acting on the HA coding sequence was evaluated., Results: Global and local phylogenetic analyses showed that the Italian strains collected between 2004 and 2012 grouped into five significant Italian clades that included viral sequences circulating in different epidemic seasons. The time of the most recent common ancestor (tMRCA) of the tree root was between May and December 2003. The tMRCA estimates of the major clades suggest that the origin of a new viral strain precedes the effective circulation of the strain in the Italian population by 6-31 months, thus supporting a central role of global migration in seeding the epidemics in Italy. The study of selection pressure showed that four codons were under positive selection, three of which were located in antigenic sites. Analysis of population dynamics showed the alternation of periods of exponential growth followed by a decrease in the effective number of infections corresponding to epidemic and inter-epidemic seasons., Conclusions: Our analyses suggest that a complex interaction between the immune status of the population, migrations, and a few selective sweeps drive the influenza A(H3N2) virus evolution. Our findings suggest the possibility of the year-round survival of local strains even in temperate zones, a hypothesis that warrants further investigation.

Enterovirus 68 (EV-D68) was associated with mild to severe respiratory infections. In the last 4 years, circulation of different EV-D68 strains has been documented worldwide. In this study, the phylogenetic characterization of nine EV-D68 strains identified in patients in the 2010-2012 period and 12 additional EV-D68 Italian strains previously identified in 2008 in Italy was described. From January 2010 to December 2012, a total of 889 respiratory specimens from 588 patients stayed or visited at the Fondazione IRCCS Policlinico San Matteo were positive for HRV or HEV. Extracted nucleic acids were amplified by one-step RT-PCR with primer specific for VP1 region of EV-D68 and purified positive PCR products were directly sequenced. Overall, 9/3736 (0.24%) patients were EV-D68 positive. Of these, 7/9 (77.8%) were pediatric and two (22.2%) were adults. Five out of seven (71.4%) pediatric patients had lower respiratory tract infection with oxygen saturation <94%. Four cases were detected from August through October 2010, while five other cases from September through December 2012. The Italian EV-D68 strains in 2008 belonged to clade A (n = 5) and clade C (n = 7). In 2010 all the Italian strains belonged to clade A (n = 4) and in 2012, four Italian strains belonged to clade B and one to clade A. In conclusion, we provide additional evidence supporting a role of EV-D68 in severe respiratory infection in pediatric patients. In addition, all the three EV-D68 clades circulating worldwide were identified in Italy in a 5-year period of time., (© 2013 Wiley Periodicals, Inc.)