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Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) persistence in COVID-19 patients could play a key role in the emergence of variants of concern. The rapid intra-host evolution of SARS-CoV-2 may result in an increased transmissibility, immune and therapeutic escape which could be a direct consequence of COVID-19 epidemic currents. In this context, a longitudinal retrospective study on eight consecutive COVID-19 patients with persistent SARS-CoV-2 infection, from January 2022 to March 2023, was conducted. To characterize the intra- and inter-host viral evolution, whole genome sequencing and phylogenetic analysis were performed on nasopharyngeal samples collected at different time points. Phylogenetic reconstruction revealed an accelerated SARS-CoV-2 intra-host evolution and emergence of antigenically divergent variants. The Bayesian inference and principal coordinate analysis analysis showed a host-based genomic structuring among antigenically divergent variants, that might reflect the positive effect of containment practices, within the critical hospital area. All longitudinal antigenically divergent isolates shared a wide range of amino acidic (aa) changes, particularly in the Spike (S) glycoprotein, that increased viral transmissibility (K417N, S477N, N501Y and Q498R), enhanced infectivity (R346T, S373P, R408S, T478K, Q498R, Y505H, D614G, H655Y, N679K and P681H), caused host immune escape (S371L, S375F, T376A, K417N, and K444T/R) and displayed partial or complete resistance to treatments (G339D, R346K/T, S371F/L, S375F, T376A, D405N, N440K, G446S, N460K, E484A, F486V, Q493R, G496S and Q498R). These results suggest that multiple novel variants which emerge in the patient during persistent infection, might spread to another individual and continue to evolve. A pro-active genomic surveillance of persistent SARS-CoV-2 infected patients is recommended to identify genetically divergent lineages before their diffusion., (© 2024 Wiley Periodicals LLC.)

The SARS-CoV-2 pandemic has seriously affected the population in Turkey. Since the beginning, phylogenetic analysis has been necessary to monitor public health measures against COVID-19 disease. In any case, the analysis of spike (S) and nucleocapsid (N) gene mutations was crucial in determining their potential impact on viral spread. We screened S and N regions to detect usual and unusual substitutions, whilst also investigating the clusters among a patient cohort resident in Kahramanmaraş city, in a restricted time span. Sequences were obtained by Sanger methods and genotyped by the PANGO Lineage tool. Amino acid substitutions were annotated comparing newly generated sequences to the NC_045512.2 reference sequence. Clusters were defined using phylogenetic analysis with a 70% cut-off. All sequences were classified as Delta. Eight isolates carried unusual mutations on the S protein, some of them located in the S2 key domain. One isolate displayed the unusual L139S on the N protein, while few isolates carried the T24I and A359S N substitutions able to destabilize the protein. Phylogeny identified nine monophyletic clusters. This study provided additional information about SARS-CoV-2 epidemiology in Turkey, suggesting local transmission of infection in the city by several transmission routes, and highlighting the necessity to improve the power of sequencing worldwide.

Background: Monoclonal antibodies (mAbs) and antivirals have been approved for early therapy of coronavirus disease (COVID-19), however, in the real-life setting, there are difficulties to prescribe these therapies within few days from symptom onset as recommended, and effectiveness of combined use of these drugs have been hypothesised in most-at-risk patients (such as those immunocompromised) but data supporting this strategy are limited., Methods: We describe the real-life experience of SARS-CoV-2 antivirals and/or monoclonal antibodies (mAbs) and focus on the hospitalisation rate due to the progression of COVID-19. Clinical results obtained through our risk-stratification algorithm and benefits achieved through a strategic proximity territorial centre are provided. We also report a case series with an in-depth evaluation of SARS-CoV-2 genome in relationship with treatment strategy and clinical evolution of patients., Results: Two hundred eighty-eight patients were analysed; 94/288 (32.6%) patients were treated with mAb monotherapy, 171/288 (59.4%) patients were treated with antivirals, and 23/288 (8%) patients received both mAbs and one antiviral drug. Haematological malignancies were more frequent in patients treated with combination therapy than in the other groups (p = 0.0003). There was a substantial increase in the number of treated patients since the opening of the centre dedicated to early therapies for COVID-19. The provided disease-management and treatment appeared to be effective since 98.6% patients recovered without hospital admission. Moreover, combination therapy with mAbs and antivirals seemed successful because all patients admitted to the hospital for COVID-19 did not receive such therapies, while none of the most-at-risk patients treated with combination therapy were hospitalized or reported adverse events., Conclusions: A low rate of COVID-19 progression requiring hospital admission was observed in patients included in this study. The dedicated COVID-19 proximity territorial service appeared to strengthen the regional sanitary system, avoiding the overwhelming of other services. Importantly, our results also support early combination therapy: it is possible that this strategy reduces the emergence of escape mutants of SARS-CoV-2, thereby increasing efficacy of early treatment, especially in immunocompromised individuals., (© 2022. The Author(s).)

Background: Nursing homes have represented important hotspots of viral spread during the initial wave of COVID-19 pandemics. The proximity of patients inside nursing homes allows investigate the dynamics of viral transmission, which may help understand SARS-Cov2 biology and spread., Methods: SARS-CoV-2 viral genomes obtained from 46 patients infected in an outbreak inside a nursing home in Calabria region (South Italy) were analyzed by Next Generation Sequencing. We also investigated the evolution of viral genomes in 8 patients for which multiple swabs were available. Phylogenetic analysis and haplotype reconstruction were carried out with IQ-TREE software and RegressHaplo tool, respectively., Results: All viral strains isolated from patients infected in the nursing home were classified as B.1 lineage, clade G. Overall, 14 major single nucleotide variations (SNVs) (frequency > 80%) and 12 minor SNVs (frequency comprised between 20% and 80%) were identified with reference to the Wuhan-H-1 sequence (NC_045512.2). All patients presented the same 6 major SNVs: D614G in the S gene; P4715L, ntC3037T (F924F) and S5398P in Orf1ab gene; ntC26681T (F53F) in the M gene; and ntC241T in the non-coding UTR region. However, haplotype reconstruction identified a founder haplotype (Hap A) in 36 patients carrying only the 6 common SNVs indicated above, and 10 other haplotypes (Hap BK) derived from Hap A in the remaining 10 patients. Notably, no significant association between a specific viral haplotype and clinical parameters was found., Conclusion: The predominant viral strain responsible for the infection in a nursing home in Calabria was the B.1 lineage (clade G). Viral genomes were classified into 11 haplotypes (Hap A in 36 patients, Hap BK in the remaining patients)., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The activity of the SARS-CoV-2 virus has not yet been studied in a post-mortem setting. The absence of these data has led to the prohibition of exposure of infected corpses during burial procedures. Our aim was to assess the virus's persistence and the possibility of transmission in the post-mortem phase including autopsy staff. The sample group included 29 patients who were admitted to our Covid-19 Centre who died during hospitalisation and the autopsy staff. All the swabs were subjected to a one-step real-time reverse transcription polymerase chain reaction with cycle threshold (Ct) values. Swab collection was performed at 2 h, 4 h, 6 h, 12 h, over 24 since death. The following were the analysis of patients' swabs: 10 cases were positive 2 h after death; 10 cases positive 4 h after death; 9 cases were found positive 6 h after death; 7 cases positive 12 h after death; 9 cases remained positive 24 h after death. The swabs performed on all the forensic pathologist staff on duty who performed the autopsies were negative. The choice to avoid rituals and the display of corpses before and at the burial procedures given appears cautiously valid due to the persistence of the SARS-CoV-2 virus in the post-mortem period. Although the caution in choosing whether or not to perform an autopsy on infected corpses is acceptable, not to perform autopsies is not biologically supported.

Aims: Patients with acute coronary syndrome (ACS) often arrive in the catheterization (cath) lab directly from the field or an emergency department without an accurate triage for Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.Although in the pandemic period the treatment in the cath laboratory of high-risk ACS should not be delayed because the operators wear special protection systems, the subsequent risk of contagion in a non-Covid coronary care unit could be high in the case of patients positive for SARS-CoV-2., Methods: We tested the possibility of a fast-track protocol in 51 consecutive patients (mean age 65 ± 12 years) transferred from spokes centres or from the field to our HUB centre and admitted to our coronary care unit (CCU). Once the patient had arrived in the cath lab, the nasopharyngeal swab was performed. The real-time PCR to extract RNA for SARS-CoV-2 detection was performed with an automated rapid molecular Xpert Xpress test. Meanwhile, coronary angiography or percutaneous coronary intervention was performed if necessary., Results: In this fast-track protocol, the time to perform nasopharyngeal swab was 11 ± 11 min; time spent to transport nasopharyngeal swab to the laboratory was 29 ± 20 min; time to detect viral nucleic acid was 68 ± 16 min. The overall time from the execution of nasopharyngeal swab to the result was 109 ± 26 min. The results were immediately put into the hospital computer system and made readily available. Depending on the test result, patients were then transferred to the regular CCU or Covid area., Conclusion: This study demonstrates that 0-1.5 h fast-track triage for coronavirus disease 2019 (COVID 19) is feasible in patients with ACS. The execution of nasopharyngeal swab in the cath lab and its analysis with a rapid molecular test allows rapid stratification of SARS-CoV-2 infection.

In a convenience sample of 93 patients treated with monoclonal antibodies (moAbs) against SARS-CoV-2, the interleukin-62/lymphocyte count ratio (IL-62/LC) was able to predict clinical worsening both in early stages of COVID-19 and in oxygen-requiring patients. Moreover, we analysed 18 most at-risk patients with asymptomatic or mild disease treated with both moAbs and antiviral treatment and found that only 2 had clinical progression, while patients with a similar risk were reported to have an unfavourable outcome in most cases from recent data. In only one of our 18 patients, clinical progression was attributable to COVID-19, and in the other cases, clinical progression was observed despite IL-62/LC being above the risk cut-off. In conclusion, IL-62/LC may be a valuable method to identify patients requiring more aggressive treatments both in earlier and later stages of the disease; however, most at-risk patients can be protected from clinical worsening by combining moAbs and antivirals, even if levels of the IL-62/LC biomarker are lower than the risk cut-off. [ABSTRACT FROM AUTHOR]

We investigated SARS-CoV-2 variants circulating, from November 2020 to March 2022, among military and civilian personnel at an Air Force airport in Italy in order to classify viral isolates in a potential hotspot for virus spread. Positive samples were subjected to Next-Generation Sequencing (NGS) of the whole viral genome and Sanger sequencing of the spike coding region. Phylogenetic analysis classified viral isolates and traced their evolutionary relationships. Clusters were identified using 70% cut-off. Sequencing methods yielded comparable results in terms of variant classification. In 2020 and 2021, we identified several variants, including B.1.258 (4/67), B.1.177 (9/67), Alpha (B.1.1.7, 9/67), Gamma (P.1.1, 4/67), and Delta (4/67). In 2022, only Omicron and its sub-lineage variants were observed (37/67). SARS-CoV-2 isolates were screened to detect naturally occurring resistance in genomic regions, the target of new therapies, comparing them to the Wuhan Hu-1 reference strain. Interestingly, 2/30 non-Omicron isolates carried the G15S 3CLpro substitution responsible for reduced susceptibility to protease inhibitors. On the other hand, Omicron isolates carried unusual substitutions A1803V, D1809N, and A949T on PLpro, and the D216N on 3CLpro. Finally, the P323L substitution on RdRp coding regions was not associated with the mutational pattern related to polymerase inhibitor resistance. This study highlights the importance of continuous genomic surveillance to monitor SARS-CoV-2 evolution in the general population, as well as in restricted communities. [ABSTRACT FROM AUTHOR]

Nearly three years into the pandemic, SARS-CoV-2 infections are occurring in vaccinated and naturally infected populations. While humoral and cellular responses in COVID-19 are being characterized, novel immune biomarkers also being identified. Recently, an increase in angiotensin-converting enzyme 2 expressing (aka, ACE2 positive) circulating exosomes (ExoACE2) were identified in the plasma of COVID-19 patients (El-Shennawy et al.). In this pilot study, we describe a method to characterize the exosome-associated microRNA (exo-miRNA) signature in ACE2-positive and ACE2-negative exosomal populations (non-ExoACE2). We performed a sorting protocol using the recombinant biotin-conjugated SARS CoV-2 spike protein containing the receptor binding domain (RBD) on plasma samples from six patients. Following purification, exo-miRNA were characterized for ACE2-positive and ACE2-negative exosome subpopulations by RT-PCR. We identified differential expression of several miRNA. Specifically let-7g-5p and hsa-miR-4454+miR-7975 were upregulated, while hsa-miR-208a-3p and has-miR-323-3p were downregulated in ExoACE2 vs. non-ExoACE2. The SARS CoV-2 spike-protein guided exosome isolation permits isolation of ExoACE2 exosomes. Such purification facilitates detailed characterization of potential biomarkers (e.g. exo-miRNA) for COVID-19 patients. This method could be used for future studies to further the understanding mechanisms of host response against SARS CoV-2. [ABSTRACT FROM AUTHOR]

We investigated the evolution of SARS-CoV-2 spread in Calabria, Southern Italy, in 2022. A total of 272 RNA isolates from nasopharyngeal swabs of individuals infected with SARS-CoV-2 were sequenced by whole genome sequencing (N = 172) and/or Sanger sequencing (N = 100). Analysis of diffusion of Omicron variants in Calabria revealed the prevalence of 10 different sub-lineages (recombinant BA.1/BA.2, BA.1, BA.1.1, BA.2, BA.2.9, BA.2.10, BA.2.12.1, BA.4, BA.5, BE.1). We observed that Omicron spread in Calabria presented a similar trend as in Italy, with some notable exceptions: BA.1 disappeared in April in Calabria but not in the rest of Italy; recombinant BA.1/BA.2 showed higher frequency in Calabria (13%) than in the rest of Italy (0.02%); BA.2.9, BA.4 and BA.5 emerged in Calabria later than in other Italian regions. In addition, Calabria Omicron presented 16 non-canonical mutations in the S protein and 151 non-canonical mutations in non-structural proteins. Most non-canonical mutations in the S protein occurred mainly in BA.5 whereas non-canonical mutations in non-structural or accessory proteins (ORF1ab, ORF3a, ORF8 and N) were identified in BA.2 and BA.5 sub-lineages. In conclusion, the data reported here underscore the importance of monitoring the entire SARS-CoV-2 genome. [ABSTRACT FROM AUTHOR]

Early recognition and prompt management are crucial for improving survival in COVID-19 patients, and after 2 years of the pandemic, many efforts have been made to obtain an early diagnosis. A key factor is the use of fast microbiological techniques, considering also that COVID-19 patients may show no peculiar signs and symptoms that may differentiate COVID-19 from other infective or non-infective diseases. These techniques were developed to promptly identify SARS-CoV-2 infection and to prevent viral spread and transmission. However, recent data about clinical, radiological and laboratory features of COVID-19 at time of hospitalization could help physicians in early suspicion of SARS-CoV-2 infection and distinguishing it from other etiologies. The knowledge of clinical features and microbiological techniques will be crucial in the next years when the endemic circulation of SARS-CoV-2 will be probably associated with clusters of infection. In this review we provide a state of the art about new advances in microbiological and clinical findings of SARS-CoV-2 infection in hospitalized patients with a focus on pulmonary and extrapulmonary characteristics, including the role of gut microbiota. [ABSTRACT FROM AUTHOR]

In this study, we report on the results of SARS-CoV-2 surveillance performed in an area of Southern Italy for 12 months (from March 2021 to February 2022). To this study, we have sequenced RNA from 609 isolates. We have identified circulating VOCs by Sanger sequencing of the S gene and defined their genotypes by whole-genome NGS sequencing of 157 representative isolates. Our results indicated that B.1 and Alpha were the only circulating lineages in Calabria in March 2021; while Alpha remained the most common variant between April 2021 and May 2021 (90 and 73%, respectively), we observed a concomitant decrease in B.1 cases and appearance of Gamma cases (6 and 21%, respectively); C.36.3 and Delta appeared in June 2021 (6 and 3%, respectively); Delta became dominant in July 2021 while Alpha continued to reduce (46 and 48%, respectively). In August 2021, Delta became the only circulating variant until the end of December 2021. As of January 2022, Omicron emerged and took over Delta (72 and 28%, respectively). No patient carrying Beta, Iota, Mu, or Eta variants was identified in this survey. Among the genomes identified in this study, some were distributed all over Europe (B1_S477N, Alpha_L5F, Delta_T95, Delta_G181V, and Delta_A222V), some were distributed in the majority of Italian regions (B1_S477N, B1_Q675H, Delta_T95I and Delta_A222V), and some were present mainly in Calabria (B1_S477N_T29I, B1_S477N_T29I_E484Q, Alpha_A67S, Alpha_A701S, and Alpha_T724I). Prediction analysis of the effects of mutations on the immune response (i.e., binding to class I MHC and/or recognition of T cells) indicated that T29I in B.1 variant; A701S in Alpha variant; and T19R in Delta variant were predicted to impair binding to class I MHC whereas the mutations A67S identified in Alpha; E484K identified in Gamma; and E156G and ΔF157/R158 identified in Delta were predicted to impair recognition by T cells. In conclusion, we report on the results of SARS-CoV-2 surveillance in Regione Calabria in the period between March 2021 and February 2022, identified variants that were enriched mainly in Calabria, and predicted the effects of identified mutations on host immune response. [ABSTRACT FROM AUTHOR]

The purpose of this review is to address some of the latest aspects regarding molecular features, pathogenic mechanisms, and immune system response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), based on recent publications in this field from March 2020 to May 2021. Interpretation keys for periodic re-emergence of coronavirus infections and other lethal viral pandemics are suggested. Antibody-dependent enhancement (ADE) and other potential mechanisms of immune system deception are put forward. Therefore, vaccine development must take into account ADE and other unwanted side effects of immune-based medical intervention. Features reported in our review will allow both clinicians and basic science researchers to take home ideas to improve their knowledge about SARS-CoV-2. [ABSTRACT FROM AUTHOR]

A multidisciplinary approach appears to be fundamental for the treatment of critically ill patients with COVID-19, improving clinical outcomes, even in the most severe cases. Such severe cases are advisable to be collegially discussed between intensivists, surgeons, infectious disease, and other physicians potentially involved. [ABSTRACT FROM AUTHOR]

Background: In December 2019, a new coronavirus (named severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) spread from China, causing a pandemic in a very short time. The main clinical presentation of SARS-CoV-2 infection (COVID-19, coronavirus disease-2019) is pneumonia, but several cardiovascular complications may also occur (e.g., acute coronary syndromes, pulmonary embolism, stroke, arrhythmias, heart failure and cardiogenic shock). Direct or indirect mechanisms induced by SARS-CoV-2 could be implicated in the pathogenesis of these events.Case Presentation: We report herein the third case of COVID-19 autoimmune haemolytic anaemia (AIHA) reported so far, which occurredwithout any other possible explanations in a Caucasian patient. The patient also suffered from ST-elevation myocardial injury.Conclusions: Both complications occurred quite late after COVID-19 diagnosis and were probably precipitated by systemic inflammation, as indicated by a significant delayed increase in inflammatory markers, including interleukin-6 (IL-6). [ABSTRACT FROM AUTHOR]