Molecular characterization of emerging Echovirus 11 (E11) shed light on the recombinant origin of a variant associated with severe hepatitis in neonates.

Echovirus 11 (E11) has gained attention owing to its association with severe neonatal infections. Due to the limited data available, the World Health Organization (WHO) considers public health risk to the general population to be low. The present study investigated the genetic variation and molecular evolution of E11 genomes collected from May to December 2023. Whole genome sequencing (WGS) was performed for 16 E11 strains. Phylogenetic analysis on WG showed how all Italian strains belonged to genogroup D5, similarly to other E11 strains recently reported in France and Germany all together aggregated into separate clusters. A cluster-specific recombination pattern was also identified using phylogenetic analysis of different genome regions. Echovirus 6 was identified as the major recombinant virus in 3Cpro and 3Dpol regions. The molecular clock analysis revealed that the recombination event probably occurred in June 2018 (95% HPD interval: Jan 2016-Jan 2020). Shannon entropy analyses, within P1 region, showed how 11 amino acids exhibited relatively high entropy. Five of them were exposed on the canyon region which is responsible for receptor binding with the neonatal Fc receptor. The present study showed the recombinant origin of a new lineage of E11 associated with severe neonatal infections.

Epidemiology and molecular characteristics of respiratory syncytial virus (RSV) among italian community-dwelling adults, 2021/22 season.

BACKGROUND: Respiratory syncytial virus (RSV) is a leading cause of acute respiratory infections worldwide. While historically RSV research has been focused on children, data on RSV infection in adults are limited. The goal of this study was to establish the prevalence of RSV in community-dwelling Italian adults and analyze its genetic variability during the 2021/22 winter season. METHODS: In this cross-sectional study, a random sample of naso-/oropharyngeal specimens from symptomatic adults seeking for SARS-CoV-2 molecular testing between December 2021 and March 2022 were tested for RSV and other respiratory pathogens by means of reverse-transcription polymerase chain reaction. RSV-positive samples were further molecularly characterized by sequence analysis. RESULTS: Of 1,213 samples tested, 1.6% (95% CI: 0.9-2.4%) were positive for RSV and subgroups A (44.4%) and B (55.6%) were identified in similar proportions. The epidemic peak occurred in December 2021, when the RSV prevalence was as high as 4.6% (95% CI: 2.2-8.3%). The prevalence of RSV detection was similar (p = 0.64) to that of influenza virus (1.9%). All RSV A and B strains belonged to the ON1 and BA genotypes, respectively. Most (72.2%) RSV-positive samples were also positive for other pathogens being SARS-CoV-2, Streptococcus pneumoniae and rhinovirus the most frequent. RSV load was significantly higher among mono-detections than co-detections. CONCLUSION: During the 2021/22 winter season, characterized by the predominant circulation of SARS-CoV-2 and some non-pharmaceutical containment measures still in place, a substantial proportion of Italian adults tested positive for genetically diversified strains of both RSV subtypes. In view of the upcoming registration of vaccines, establishment of the National RSV surveillance system is urgently needed.

Heat-Treated Lysozyme Hydrochloride: A Study on Its Structural Modifications and Anti-SARS-CoV-2 Activity.

Lysozyme (E.C. 3.2.1.17), an about 14 kDa protein and pI 11, widely spread in nature, is present in humans mainly in milk, saliva, and intestinal mucus as a part of innate defense mechanisms. It is endowed with antimicrobial activity due to its action as an N-acetylmuramidase, cleaving the 1-4ß glycosidic linkage in the peptidoglycan layer of Gram-positive bacteria. This antimicrobial activity is exerted only against a limited number of Gram-negative bacteria. Different action mechanisms are proposed to explain its activity against Gram-negative bacteria, viruses, and fungi. The antiviral activity prompted the study of a possible application of lysozyme in the treatment of SARS-CoV-2 infections. Among the different sources of lysozyme, the chicken egg albumen was chosen, being the richest source of this protein (c-type lysozyme, 129 amino acids). Interestingly, the activity of lysozyme hydrochloride against SARS-CoV-2 was related to the heating (to about 100 °C) of this molecule. A chemical-physical characterization was required to investigate the possible modifications of native lysozyme hydrochloride by heat treatment. The FTIR analysis of the two preparations of lysozyme hydrochloride showed appreciable differences in the secondary structure of the two protein chains. HPLC and NMR analyses, as well as the enzymatic activity determination, did not show significant modifications.

Longitudinal, virological, and serological assessment of hospitalized COVID-19 patients.

Here we described the virological and serological assessment of 23 COVID-19 patients hospitalized and followed up in Milan, Italy, during the first wave of COVID-19 pandemic. Nasopharyngeal (NPS), anal swabs, and blood samples were collected from 23 COVID-19 patients, at hospital admission, and periodically up to discharge, for a median time of 20 days (3-83 days). RNA was isolated and tested for SARS-CoV-2 by qRT-PCR; anti-SARS-CoV-2 IgM and IgG antibody titers were evaluated in serum samples by ELISA. SARS-CoV-2 genome was detected in the NPS swabs of the 23 patients, at the admission, and 8/19 (42.1%) were still positive at the discharge. Anal swabs were positive to SARS-CoV-2 RNA detection in 20/23 (86.9%) patients; 6/19 (31.6%) were still positive at discharge. The mean time of RNA negative conversion was 17 days (4-36 days) and 33 days (4-77 days), for NPS and anal swabs, respectively. SARS-CoV-2-RNA was detected in the blood of 6/23 (26.1%) patients. Thirteen/23 (56.5%) and 17/23 (73.9%) patients were seropositive for IgM and IgG, respectively, at the admission, and the median IgM and IgG levels significantly (p < 0.05) increased after 13 days. Although the limited cohort size, our report provides evidence that SARS-CoV-2 is shed through multiple routes, with important implications in healthcare settings.

Viral Agents and Systemic Levels of Inflammatory Cytokines in Vulnerable and Stable Atherosclerotic Carotid Plaques.

BACKGROUND: To investigate the presence of genetic material of viral agents and the serum level of inflammatory cytokines in patients submitted to carotid endarterectomy having vulnerable versus stable atherosclerotic plaques. METHODS: Data of patients consecutively submitted to carotid endarterectomy for a significant stenosis from July 2019 to December 2019 were prospectively collected. The genetic material of Epstein-Barr (EBV), CitoMegalo (CMV), Herpes Simplex (HSV), Varicella-Zoster (VZV) and Influenza (IV) Viruses was searched in the patient's plaques, both in the "mid" of the plaque and in an adjacent lateral portion of no-plaque area. The serum levels of TNF-α, IL-1ß, IL-6, IL10 and CCL5 were determined. The obtained results were then correlated to the histologic vulnerability of the removed carotid plaque. P values < 0.05 were considered statistically significant. RESULTS: Data of 50 patients were analyzed. A vulnerable plaque was found in 31 patients (62%). The genome of CMV, HSV, VZV and IV was not found in any of the vascular samples, while the EBV genome was found in the "mid" of 2 vulnerable plaques, but not in their respective control area. Eighty-two percent of patients who did not receive anti-IV vaccination (23/28) had vulnerable carotid plaque, compared with 36% of vaccinated patients (8/22, P = 0.001). Serum levels of TNF-α and IL-6 were higher in patients with a vulnerable plaque compared to patients with a stable plaque (73.6 ± 238.2 vs. 3.9 ± 13.1 pg/ml, P= 0.01, and 45.9 ± 103.6 vs. 10.1 ± 25.3 pg/ml, P= 0.01, respectively), independent of comorbidities, viral exposure or flu vaccination. CONCLUSIONS: The EBV genome was found in the "core" of 2 vulnerable carotid plaques, but not in their respective adjacent control. Influenza vaccination was associated with a lower incidence of carotid plaque vulnerability. Serum levels of TNF-α and IL-6 were higher in patients with a vulnerable plaque compared to patients with a stable plaque.

Re-emergence of enterovirus D68 in Europe after easing the COVID-19 lockdown, September 2021.

We report a rapid increase in enterovirus D68 (EV-D68) infections, with 139 cases reported from eight European countries between 31 July and 14 October 2021. This upsurge is in line with the seasonality of EV-D68 and was presumably stimulated by the widespread reopening after COVID-19 lockdown. Most cases were identified in September, but more are to be expected in the coming months. Reinforcement of clinical awareness, diagnostic capacities and surveillance of EV-D68 is urgently needed in Europe.

Respiratory syncytial virus in influenza-like illness cases: Epidemiology and molecular analyses of four consecutive winter seasons (2014-2015/2017-2018) in Lombardy (Northern Italy).

BACKGROUND: Besides seasonal influenza viruses (IV), several other pathogens-including respiratory syncytial virus (RSV)-are involved in clinically undistinguished influenza-like illnesses (ILIs). This study aimed at investigating the contribution of RSV in ILI cases in Lombardy (Northern Italy) during four consecutive winter seasons. MATERIALS AND METHODS: In the framework of influenza surveillance, respiratory samples from ILI outpatients were collected from 2014-2015 to 2017-2018 season. IV-negative swabs were included in the study and analyzed to detect and molecularly characterize RSV-A and RSV-B. RESULTS: A total of 12.9% (135/1047) of samples were positive to RSV that was mostly detected among children ≤5 years (51/183, 27.8%) and those aged 6 to 15 years (30/158, 18.9%), whereas elderly >65 years accounted for 12% of RSV cases (15/125). The median start of RSV epidemic was in the end of November, with a peak in mid-February and a width of nearly 4 months, almost overlapping seasonal influenza epidemic. RSV-A and RSV-B co-circulated in all considered seasons, with RSV-B predominating on RSV-A (63.6% vs 36.4%; P < .001). Most (85.2%) RSV-A belonged to genotype ON1 and the remaining to NA1. All RSV-B clustered within the BA genotype. CONCLUSIONS: In this study, RSV significantly contributed to ILI cases, especially among pediatric population (<15 years), although it was detected in all age groups. RSV-B predominated on RSV-A, and the most recent evolved genotypes (BA and ON1, respectively) circulated. Investigating the epidemiological and molecular characteristics of RSV in ILI cases can increase baseline epidemiological information before the introduction of RSV vaccination.

SARS-CoV-2 IgG/IgM Rapid Test as a Diagnostic Tool in Hospitalized Patients and Healthcare Workers, at a large Teaching Hospital in northern Italy, during the 2020 COVID-19 Pandemic.

We describe the outcome of a Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) IgG/IgM rapid test, and discuss the potential suitability of antibody testing. Retrospective single cohort study on patients with suspected Coronavirus Disease 2019 (COVID-19) and asymptomatic Healthcare Workers, enrolled from March to April 2020. Subjects had quantitative PCR (qPCR) test for detection of SARS-CoV-2 via nasal swab and serological testing using the COVID-19 IgG/ IgM Rapid Test (PRIMA Lab SA) immunochromatographic assay. Some subjects underwent chemiluminescence immunoassay (CLIA) after rapid test. The aim of the study was to analyse the proportion of those who developed a positive IgM/IgG response for SARS-CoV-2. The correspondence between the results from rapid testing and CLIA, when available, was evaluated. 97 subjects underwent qPCR for SARS-CoV-2 through nasal swab, which resulted positive in 40/43 (93.0%) of symptomatic patients, 2/40 (5%) of asymptomatic HCW, in no subjects with suspected COVID- 19 (clinical and radiological findings) then excluded by repeated nasal swabs and alternative diagnosis (COVID-19-negative patients, CNPs), and in 6/6 (100%) of patients with confirmed diagnosis and negative follow-up nasal swabs (COVID-19-recovered patients, CRPs). IgM resulted positive in 8/43 (18.6%) of symptomatic patients and in 1/6 (16.7%) of CRPs. IgG resulted positive in 36/43 (83.7%) of symptomatic patients, 2/40 (5%) of HCW, and in 1/8 (12.5%) and 6/6 (100%) of CNPs and CRPs, respectively. A comparison between an IgG/IgM Rapid Test and a following CLIA test showed consistency in negative results in 25/28 of HCW and 8/8 of CNPs tested. Our preliminary data support the role of IgG/IgM Rapid Test (PRIMA Lab SA) immunochromatographic assay as a point-of-care test that may complement molecular tests in the screening of SARS-CoV-2 carriers. The test may gain particular relevance in shortening the time needed to refer patients to a COVID or non-COVID Hospital area and to achieve diagnosis in patients with persistently negative nasal swabs.

Diagnosis of congenital CMV infection via DBS samples testing and neonatal hearing screening: an observational study in Italy.

BACKGROUND: Congenital Cytomegalovirus (cCMV) is the most common cause of non-genetic hearing loss in childhood. A newborn hearing screening program (NHSP) is currently running in Italy, but no universal cCMV nor statewide hearing-targeted CMV screening programs have been implemented yet. This observational monocentric study was aimed at estimating the rate of cCMV infections identified by CMV-DNA analysis on Dried Blood Spots (DBS) samples in deaf children identified via NHSP in Northern Italy in the period spanning from 2014 to 2018. METHODS: Children with a confirmed diagnosis of deafness and investigated for CMV-DNA by nucleic acid extraction and in-house polymerase-chain reaction (PCR) on stored newborns screening cards (DBS-test) were included in this study. Deafness was defined by a hearing threshold ≥20 decibel (dB HL) by Auditory Brainstem Responses (ABR); all investigated DBS samples were collected within 3 days of life. RESULTS: Overall, 82 children were included (median age: 3.4 months; lower-upper quartiles: 2-5.3 months; males: 60.9%). Most of them (70.7%) presented bilateral hearing loss with a symmetrical pattern in 79.3% of the cases. ABR thresholds were ≥ 70 dB HL (severe/profound deafness) in 46.5% of children. Among all tested children, 6.1% resulted positive for cCMV. The rate of severe/profound deafness was statistically higher in children with cCMV infection. CONCLUSIONS: The addition of DBS-test to the NHSP allowed the identification, in their first months of life, of a cCMV infection in 6.1% of children who had failed NHS. The introduction of a targeted CMV screening strategy could help clinicians in the differential diagnosis and in the babies' management. DBS samples can be considered a "universal newborns biobank": their storage site and duration should be the subject of political decision-making.

Emergence of divergent enterovirus (EV) D68 sub-clade D1 strains, northern Italy, September to October 2018.

Between September and October 2018, an enterovirus D68 (EV-D68) outbreak occurred in patients hospitalised with severe acute respiratory infection in northern Italy; 21 laboratory-confirmed cases were reported. Phylogenetic analysis revealed that 16/20 of the EV-D68 sequences belonged to a divergent group within the sub-clade D1. Since its upsurge, EV-D68 has undergone rapid evolution with the emergence of new viral variants, emphasising the need for molecular surveillance that include outpatients with respiratory illness.

Molecular Characterization of Influenza Strains in Patients Admitted to Intensive Care Units during the 2017-2018 Season.

This study aimed at assessing the frequency and the distribution of influenza virus types/subtypes in 172 laboratory-confirmed influenza-positive patients admitted to intensive care units (ICUs) during the 2017-2018 season in the Lombardy region (Northern Italy), and to investigate the presence of molecular pathogenicity markers. A total of 102/172 (59.3%) patients had influenza A infections (83 A/H1N1pdm09, 2 H3N2 and 17 were untyped), while the remaining 70/172 (40.7%) patients had influenza B infections. The 222G/N mutation in the hemagglutinin gene was identified in 33.3% (3/9) of A/H1N1pdm09 strains detected in the lower respiratory tract (LRT) samples and was also associated with more severe infections, whereas no peculiar mutations were observed for influenza B strains. A single-point evolution was observed in site 222 of A/H1N1pdm09 viruses, which might advantage viral evolution by favouring virus binding and replication in the lungs. Data from 17 paired upper respiratory tract (URT) and LRT samples showed that viral load in LRT samples was mostly higher than that detected in URT samples. Of note, influenza viruses were undetectable in 35% of paired URT samples. In conclusion, LRT samples appear to provide more accurate clinical information than URT samples, thus ensuring correct diagnosis and appropriate treatment of patients with severe respiratory infections requiring ICU admission.

Hepatitis E Virus Genotype 3 Genomes from RNA-Positive but Serologically Negative Plasma Donors Have CUG as the Start Codon for ORF3.

Hepatitis E virus (HEV) is a pathogen that causes hepatitis worldwide. Molecular studies have identified HEV RNA in blood products although its significance is not understood. This study was undertaken to characterize HEV genomes in asymptomatic plasma donors from Sweden and Germany lacking anti-HEV. Complete open reading frames (ORFs) were obtained from HEV strains in 5 out of 18 plasma donors who tested positive for HEV RNA. All strains had CUG as the start codon of ORF3, while 147 GenBank strains all had AUG as the start codon (p < 0.0001). This substitution was found in both interrelated and unrelated strains belonging to different phylogenetic clades. The HEV strains from the seronegative plasma donors had no other substitution in common, which may be why the CUG substitution seems to explain the seronegativity.

Epidemiology and molecular characterization of influenza viruses, human parechoviruses and enteroviruses in children up to 5 years with influenza-like illness in Northern Italy during seven consecutive winter seasons (2010-2017).

Besides the influenza virus (IV), several other viruses are responsible for influenza-like illness (ILI). Although human parechoviruses (HPeVs) and enteroviruses (EVs) may impact on ILI, limited data on their epidemiological characteristics are available. During seven consecutive winter seasons (from 2010-2011 to 2016-2017), within the framework of an influenza surveillance system (InfluNet), 593 respiratory swabs were collected from children ≤5 years of age with ILIs. Molecular detection showed that 58.3 % of swabs were positive for at least one of the viruses under study: 46 % for IV, 13 % for EV and 5.4 % for HPeV. A single virus was identified in 51.3 % of samples while more than one virus was detected in 7 % of the samples. The risk of contracting IV was higher than the risk associated with EV, which in turn was higher than the risk of contracting HPeV. The risk of developing an IV infection was twofold greater in children >3 years than in those ≤3 years, who had higher risk of EV/HPeV infection. The frequency of EV/HPeV-positive swabs increased significantly during the 2016-2017 winter season compared to the previous six seasons. Sixteen EV genotypes were identified belonging to species A and B. HPeV-1 was the most frequently detected genotype, followed by -6 and -3. In this study, IV was mainly responsible for ILI, however EV and HPeV were also involved and particularly affected children ≤3 years of age. Influenza surveillance samples could provide us with valuable insight into the epidemiological features of viruses involved in ILI.

Acute Hepatitis B After the Implementation of Universal Vaccination in Italy: Results From 22 Years of Surveillance (1993-2014).

BACKGROUND: Hepatitis B vaccination has proven to be very safe and highly effective. This study assessed the proportion of successfully vaccinated individuals among cases with acute hepatitis B, the proportion of preventable cases if individuals were vaccinated as recommended, and the reasons for failures. METHODS: We analyzed data reported to the Italian Surveillance System for Acute Viral Hepatitis from 1993 to 2014. RESULTS: A total of 362 of 11 311 (3.2%) cases with acute hepatitis B were vaccinated. Of the 277 cases for whom immunization data were available, 50 (18%) received a complete vaccination course according to the correct schedule and before exposure to hepatitis B virus. Molecular characterization of 17 of these cases showed that 6 were infected with S-gene mutants. Among the 10 949 unvaccinated cases, 213 (1.9%) escaped mandatory vaccination and 2821 (25.8%) were not vaccinated despite being at increased risk of infection. Among the latter, the most common risk factors were cohabitation with hepatitis B surface antigen (HBsAg) carriers, intravenous drug use, and homosexual/bisexual practices. Thirty-seven percent of the unvaccinated households with HBsAg carriers were aware of their risk. Lack of trust in the vaccination, negative attitude, and inaccurate beliefs followed by lack of or poor communication and low perceived severity of the disease were the most frequent reasons for vaccine hesitancy. CONCLUSIONS: Development of acute disease in successfully vaccinated individuals is a rare event. Further efforts are needed to enhance the vaccine coverage rate in individuals at increased risk of infection.

Sequence analysis of respiratory syncytial virus cases reveals a novel subgroup -B strain circulating in north-central Italy after pandemic restrictions.

BACKGROUND: Following the pandemic restrictions, the epidemiology of respiratory syncytial virus (RSV) has changed, leading to intense hospitalization peaks. OBJECTIVES: This study, conducted at multiple sites in Italy, aimed to describe the temporal dynamics of two post-COVID-19 RSV epidemics. Additionally, the circulating RSV-A and -B lineages were characterized and compared to those found in 2018 and 2019. STUDY DESIGN: Respiratory specimens and data were collected from RSV-positive patients, both inpatients, and outpatients, of all ages at three sites in north-central Italy. To analyze these samples, roughly one-sixth were sequenced in the attachment glycoprotein G gene and subjected to phylogenetic and mutational analyses, including pre-pandemic sequences from north-central Italy. RESULTS: The first post-pandemic surge of RSV cases was quite intense, occurring from October 2021 to early January 2022. The subsequent RSV epidemic (from November 2022 to early March 2023) also had a high impact, characterized by a rise in elderly patient cases. Post-pandemic cases of RSV-A were caused by various strains present in Italy prior to COVID-19. In contrast, a distinct RSV-B lineage, which was concurrently spreading in other countries, was identified as the main cause of the surge in 2022-2023 but remained undetected in Italy before the pandemic. CONCLUSIONS: This study describes the temporal dynamics of post-pandemic RSV subgroups and uncovers a lineage of RSV-B with high genetic divergence that may have increased the impact of decreased population immunity.

Increased circulation of echovirus 11 in the general population and hospital patients as elicited by the non-polio enterovirus laboratory-based sentinel surveillance in northern Italy, 2023.

OBJECTIVES: Following the alert of echovirus 11 (E-11) infection in neonates in EU/EEA Member States, we conducted an investigation of E-11 circulation by gathering data from community and hospital surveillance of enterovirus (EV) in northern Italy from 01 August 2021 to 30 June 2023. METHODS: Virological results of EVs were obtained from the regional sentinel surveillance database for influenza-like illness (ILI) in outpatients, and from the laboratory database of ten hospitals for inpatients with either respiratory or neurological symptoms. Molecular characterization of EVs was performed by sequence analysis of the VP1 gene. RESULTS: In our ILI series, the rate of EV-positive specimens showed an upward trend from the end of May 2023, culminating at the end of June, coinciding with an increase in EV-positive hospital cases. The E-11 identified belonged to the D5 genogroup and the majority (83%) were closely associated with the novel E-11 variant, first identified in severe neonatal infections in France since 2022. E-11 was identified sporadically in community cases until February 2023, when it was also found in hospitalized cases with a range of clinical manifestations. All E-11 cases were children, with 14 out of 24 cases identified through hospital surveillance. Of these cases, 60% were neonates, and 71% had severe clinical manifestations. CONCLUSION: Baseline epidemiological data collected since 2021 through EV laboratory-based surveillance have rapidly tracked the E-11 variant since November 2022, alongside its transmission during the late spring of 2023.

Wastewater-based epidemiology revealed in advance the increase of enterovirus circulation during the Covid-19 pandemic.

Wastewater-based epidemiology (WBE) was conducted to track Enteroviruses (EVs) circulation in the Milan metropolitan area (Northern Italy) during Covid-19 pandemic (March 2020-December 2022). 202 composite 24-hour wastewater samples (WWSs) were collected weekly from March 24, 2020, to December 29, 2022 at the inlet of two wastewater treatment plants (WWTP) in Milan (1.5 million inhabitants). EV-RNA was quantified and molecular characterization of non-polio EVs (NPEV) was performed by Sanger sequence analysis. Data from WWS were matched with virological data collected in the framework of Influenza-Like Illness (ILI) surveillance in the same place and time. EV-RNA was identified in 88.2 % of WWSs. The peak in EVs circulation was observed in late August 2020 (upon conclusion of the first national lockdown), in late August 2021, and in mid-April 2022. EV-RNA concentration in WWS (normalized as copies/d/1000 people) at peak of circulation presented a yearly increase (2020: 2.47 × 1010; 2021: 6.81 × 1010; 2022: 2.14 × 1011). This trend overlapped with trend in EV-positivity rate in ILI cases, expanded from 21.7 % in 2021 to 55.6 % in 2022. EV trends in WWS preceded clinical sample detections in 2021 and 2022 by eight and five weeks, respectively, acting as an early warning of outbreak. Although sequencing of EV-positive WWSs revealed the presence of multiple EV strains, typing remained inconclusive. Molecular characterization of EVs in clinical samples revealed the co-circulation of several genotypes: EV-A accounted for 60 % of EVs, EV-B for 16.7 %, EV-D68 for 23.3 %. EVs were circulating in Milan metropolitan area between March 2020 and December 2022. The epidemiological trends unfolded the progressive accumulation of EV transmission in the population after removal of Covid-19 restrictions. The increased circulation of EVs in 2021-2022 was identified at least 35 days in advance compared to the analysis of clinical data. The inconclusive results of Sanger sequencing lookout for improvement and innovative molecular approaches to deepen track EVs.

A Flexible Regression Modeling Approach Applied to Observational Laboratory Virological Data Suggests That SARS-CoV-2 Load in Upper Respiratory Tract Samples Changes with COVID-19 Epidemiology.

(1) Background. Exploring the evolution of SARS-CoV-2 load and clearance from the upper respiratory tract samples is important to improving COVID-19 control. Data were collected retrospectively from a laboratory dataset on SARS-CoV-2 load quantified in leftover nasal pharyngeal swabs (NPSs) collected from symptomatic/asymptomatic individuals who tested positive to SARS-CoV-2 RNA detection in the framework of testing activities for diagnostic/screening purpose during the 2020 and 2021 winter epidemic waves. (2) Methods. A Statistical approach (quantile regression and survival models for interval-censored data), novel for this kind of data, was applied. We included in the analysis SARS-CoV-2-positive adults >18 years old for whom at least two serial NPSs were collected. A total of 262 SARS-CoV-2-positive individuals and 784 NPSs were included: 193 (593 NPSs) during the 2020 winter wave (before COVID-19 vaccine introduction) and 69 (191 NPSs) during the 2021 winter wave (all COVID-19 vaccinated). We estimated the trend of the median value, as well as the 25th and 75th centiles of the viral load, from the index episode (i.e., first SARS-CoV-2-positive test) until the sixth week (2020 wave) and the third week (2021 wave). Interval censoring methods were used to evaluate the time to SARS-CoV-2 clearance (defined as Ct < 35). (3) Results. At the index episode, the median value of viral load in the 2021 winter wave was 6.25 log copies/mL (95% CI: 5.50-6.70), and the median value in the 2020 winter wave was 5.42 log copies/mL (95% CI: 4.95-5.90). In contrast, 14 days after the index episode, the median value of viral load was 3.40 log copies/mL (95% CI: 3.26-3.54) for individuals during the 2020 winter wave and 2.93 Log copies/mL (95% CI: 2.80-3.19) for those of the 2021 winter wave. A significant difference in viral load shapes was observed among age classes (p = 0.0302) and between symptomatic and asymptomatic participants (p = 0.0187) for the first wave only; the median viral load value is higher at the day of episode index for the youngest (18-39 years) as compared to the older (40-64 years and >64 years) individuals. In the 2021 epidemic, the estimated proportion of individuals who can be considered infectious (Ct < 35) was approximately half that of the 2020 wave. (4) Conclusions. In case of the emergence of new SARS-CoV-2 variants, the application of these statistical methods to the analysis of virological laboratory data may provide evidence with which to inform and promptly support public health decision-makers in the modification of COVID-19 control measures.

On the lookout for influenza viruses in Italy during the 2021-2022 season: Along came A(H3N2) viruses with a new phylogenetic makeup of their hemagglutinin.

AIMS: To assess influenza viruses (IVs) circulation and to evaluate A(H3N2) molecular evolution during the 2021-2022 season in Italy. MATERIALS AND METHODS: 12,393 respiratory specimens (nasopharyngeal swabs or broncho-alveolar lavages) collected from in/outpatients with influenza illness in the period spanning from January 1, 2022 (week 2022-01) to May 31, 2022 (week 2022-22) were analysed to identify IV genome and were molecularly characterized by 12 laboratories throughout Italy. A(H3N2) evolution was studied by conducting an in-depth phylogenetic analysis of the hemagglutinin (HA) gene sequences. The predicted vaccine efficacy (pVE) of vaccine strain against circulating A(H3N2) viruses was estimated using the sequence-based Pepitope model. RESULTS: The overall IV-positive rate was 7.2% (894/12,393), all were type A IVs. Almost all influenza A viruses (846/894; 94.6%) were H3N2 that circulated in Italy with a clear epidemic trend, with 10% positivity rate threshold crossed for six consecutive weeks from week 2022-11 to week 2022-16. According to the phylogenetic analysis of a subset of A(H3N2) strains (n=161), the study HA sequences were distributed into five different genetic clusters, all of them belonging to the clade 3C.2a, sub-clade 3C.2a1 and the genetic subgroup 3C.2a1b.2a.2. The selective pressure analysis of A(H3N2) sequences showed evidence of diversifying selection particularly in the amino acid position 156. The comparison between the predicted amino acid sequence of the 2021-2022 vaccine strain (A/Cambodia/e0826360/2020) and the study strains revealed 65 mutations in 59 HA amino acid positions, including the substitution H156S and Y159N in antigenic site B, within major antigenic sites adjacent to the receptor-binding site, suggesting the presence of drifted strains. According to the sequence-based Pepitope model, antigenic site B was the dominant antigenic site and the p(VE) against circulating A(H3N2) viruses was estimated to be -28.9%. DISCUSSION AND CONCLUSION: After a long period of very low IV activity since public health control measures have been introduced to face COVID-19 pandemic, along came A(H3N2) with a new phylogenetic makeup. Although the delayed 2021-2022 influenza season in Italy was characterized by a significant reduction of the width of the epidemic curve and in the intensity of the influenza activity compared to historical data, a marked genetic diversity of the HA of circulating A(H3N2) strains was observed. The identification of the H156S and Y159N substitutions within the main antigenic sites of most HA sequences also suggested the circulation of drifted variants with respect to the 2021-2022 vaccine strain. Molecular surveillance plays a critical role in the influenza surveillance architecture and it has to be strengthened also at local level to timely assess vaccine effectiveness and detect novel strains with potential impact on public health.