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To assess influenza viruses (IVs) circulation and to evaluate A(H3N2) molecular evolution during the 2021-2022 season in Italy.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 PThe 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 PAfter 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.

This study adds insight regarding the occurrence and spread of SARS-CoV-2 Variants of Concern (VOCs) and Var-iants of Interest (VOIs) in Italy in October and November 2022, by testing urban wastewater collected through-out the country. A total of 332 wastewater samples were collected from 20 Italian Regions/Autonomous Provinces (APs) within the framework of national SARS-CoV-2 environmental surveillance. Of these, 164 were collected in the first week of October and 168 in the first week of November. A similar to 1600 bp fragment of the spike protein was sequenced by Sanger (for individual samples) and long-read nanopore sequencing (for pooled Region/AP samples).In October, mutations characteristic of Omicron BA.4/BA.5 were detected in the vast majority (91 %) of the samples amplified by Sanger sequencing. A fraction of these sequences (9 %) also displayed the R346T mutation. Despite the low prevalence documented in clinical cases at the time of sampling, amino acid substitutions characteristic of sublineages BQ.1 or BQ.1.1 were detected in 5 % of sequenced samples from four Regions/APs. A significantly higher variability of sequences and variants was documented in November 2022, when the rate of se-quences harbouring mutations of lineages BQ.1 and BQ1.1 increased to 43 %, and the number of Regions/APs positive for the new Omicron subvariant more than tripled (n = 13) compared to October. Moreover, an increase in the number of sequences with the mutation package BA.4/BA.5 + R346T (18 %), as well as the detection of variants never observed before in wastewater in Italy, such as BA.2.75 and XBB.1 (the latter in a Region where no clinical cases asso-ciated with this variant had ever been documented) was recorded.The results suggest that, as predicted by the ECDC, BQ.1/BQ.1.1 is rapidly becoming dominant in late 2022. Environ-mental surveillance proves to be a powerful tool for tracking the spread of SARS-CoV-2 variants/subvariants in the population.

Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) cause a high burden of disease, particularly in children and the elderly. With the aim to add knowledge on RSV and HMPV infections in Italy, a prospective, multicenter study was conducted by eight centers of the Working Group on Respiratory Virus Infections (GLIViRe), from December 2018-April 2019. Weekly distribution and patients' demographic and clinical data were compared in 1300 RSV and 222 HMPV-positive cases. Phylogenetic analysis of the G-glycoprotein coding region was performed to characterize circulating strains. RSV positivity ranged from 6.4% in outpatients of all ages to 31.7% in hospitalized children; HMPV positivity was 4-1.2% with no age-association. RSV season peaked in February and ended in mid-April: HMPV circulation was higher when RSV decreased in early spring. RSV was more frequent in infants, whereas HMPV infected comparatively more elderly adults; despite, their clinical course was similar. RSV-B cases were two-thirds of the total and had similar clinical severity compared to RSV-A. Phylogenetic analysis showed the circulation of RSV-A ON1 variants and the predominance of RSV-B genotype BA10. HMPV genotype A2c was the prevalent one and presented insertions of different lengths in G. This first multicenter Italian report on seasonality, age-specific distribution, and clinical presentation of RSV and HMPV demonstrated their substantial disease burden in young patients but also in the elderly. These data may provide the basis for a national respiratory virus surveillance network.

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

(1) Background: The surveillance of SARS-CoV-2 RNA in urban wastewaters allows one to monitor the presence of the virus in a population, including asymptomatic and symptomatic individuals, capturing the real circulation of this pathogen. The aim of this study was to evaluate the performance of different pre-analytical and analytical methods for identifying the presence of SARS-CoV-2 in untreated municipal wastewaters samples by conducting an inter-laboratory proficiency test. (2) Methods: three methods of concentration, namely, (A) Dextran and PEG-6000 two-phase separation, (B) PEG-8000 precipitation without a chloroform purification step and (C) PEG-8000 precipitation with a chloroform purification step were combined with three different protocols of RNA extraction by using commercial kits and were tested by using two primers/probe sets in three different master mixes. (3) Results: PEG-8000 precipitation without chloroform treatment showed the best performance in the SARS-CoV-2 recovery; no major differences were observed among the protocol of RNA extraction and the one-step real-time RT-PCR master mix kits. The highest analytic sensitivity was observed by using primers/probe sets targeting the N1/N3 fragments of SARS-CoV-2. (4) Conclusions: PEG-8000 precipitation in combination with real-time RT-PCR targeting the N gene (two fragments) was the best performing workflow for the detection of SARS-CoV-2 RNA in municipal wastewaters.

The SARS-CoV-2 Omicron variant emerged in South Africa in November 2021, and has later been identified worldwide, raising serious concerns. A real-time RT-PCR assay was designed for the rapid screening of the Omicron variant, targeting characteristic mutations of the spike gene. The assay was used to test 737 sewage samples collected throughout Italy (19/21 Regions) between 11 November and 25 December 2021, with the aim of assessing the spread of the Omicron variant in the country. Positive samples were also tested with a real-time RT-PCR developed by the European Commission, Joint Research Centre (JRC), and through nested RT-PCR followed by Sanger sequencing. Overall, 115 samples tested positive for Omicron SARS-CoV-2 variant. The first occurrence was detected on 7 December, in Veneto, North Italy. Later on, the variant spread extremely fast in three weeks, with prevalence of positive wastewater samples rising from 1.0% (1/104 samples) in the week 5-11 December, to 17.5% (25/143 samples) in the week 12-18, to 65.9% (89/135 samples) in the week 19-25, in line with the increase in cases of infection with the Omicron variant observed during December in Italy. Similarly, the number of Regions/Autonomous Provinces in which the variant was detected increased from one in the first week, to 11 in the second, and to 17 in the last one. The presence of the Omicron variant was confirmed by the JRC real-time RT-PCR in 79.1% (91/115) of the positive samples, and by Sanger sequencing in 66% (64/97) of PCR amplicons. In conclusion, we designed an RT-qPCR assay capable to detect the Omicron variant, which can be successfully used for the purpose of wastewater-based epidemiology. We also described the history of the introduction and diffusion of the Omicron variant in the Italian population and territory, confirming the effectiveness of sewage monitoring as a powerful surveillance tool.

Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract illness in young children and can also cause influenza-like illness (ILI). Here we investigated the epidemiological features of RSV infection in pediatric ILI cases in Lombardy (a region in Northern Italy accounting nearly 10 million inhabitants) from 2014-2015 to 2020-2021 winter seasons.Data for this study were retrieved and statistically analyzed from the database of virological influenza surveillance of the regional reference laboratory for Lombardy within the Italian influenza surveillance network (InfluNet).RSV accounted for nearly 19% of pediatric ILI with a risk of infection nearly two-fold greater than that of individuals ≥15 years. RSV positivity rate increased to 28% considering 0-5 years old children. Although in children ≤5 years the risk of infection from influenza viruses resulted nearly two-fold higher than the risk of RSV infection, the age group 4-6 months and 7-12 months showed a five-fold greater risk of infection from RSV than from influenza. Children ≤5 years of age with pre-existing underlying health conditions had a nearly five-fold greater risk of getting RSV infection than otherwise healthy 0-5 years old children. RSV was identified in ILI cases15 years of age in all considered winter seasons except in the 2020-2021 season.Sentinel surveillance of ILI allowed us to identify groups at higher risk of RSV and influenza infection and to define the start, duration, timing, and intensity of the RSV and influenza community circulation. This surveillance approach can be implemented to assess the RSV circulation and impact in a real-time manner.

INTRODUCTION. Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract illness in young children and can also cause influenza-like illness (ILI). Here we investigated the epidemiological features of RSV infection in pediatric ILI cases in Lombardy (a region in Northern Italy accounting nearly 10-million inhabitants) from 2014-2015 to 2020-2021 winter seasons. MATERIAL AND METHODS. Data for this study were retrieved and statistically analyzed from the database of virological influenza surveillance of the regional reference laboratory for Lombardy within the Italian influenza surveillance network (InfluNet). RESULTS. RSV accounting for nearly 19% of pediatric ILI with a risk of infection nearly 2-fold greater than that of individuals ≥15 years. The RSV positivity rate increased to 28% considering 0-5 years old children. Although in children ≤5 years the risk of infection from influenza viruses resulted nearly 2-fold higher than the risk of RSV infection, the age group 4-6 months and 7-12 months showed 5-fold greater risk of infection from RSV than from influenza. Children ≤5 years of age with presence of one or more comorbidities had a nearly 5-fold greater risk of getting RSV infection than otherwise healthy 0-5 years old children. DISCUSSION. The use of the ILI sentinel surveillance allowed us to identify groups at higher risk of RSV and influenza infection and to define the start, duration, timing and intensity of the RSV and influenza community circulation, determining thresholds based on historical data. This surveillance approach can be implemented to assess the nearly real-time RSV circulation and impact.

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

An Italian 13-year-old boy immunosuppressed due to kidney transplant presented in November 2018 with acute flaccid paralysis with anterior horn cell involvement resembling the clinical, radiological, and laboratory features of poliomyelitis. Enterovirus was molecularly identified in cerebral spinal fluid and stool samples and the sequence analysis of the VP1 gene of enterovirus genome revealed the presence of Echovirus 30 both in CSF and in stool samples. Echovirus 30 is an emerging neurotropic virus able to cause outbreaks of aseptic meningitis and meningoencephalitis all over the world, but acute flaccid paralysis is not a classical manifestation. A 6-month follow-up revealed a poor outcome with severe motor deficits and only slight improvement in disability. Clinicians must be aware of the possible role of Echovirus 30 in acute flaccid paralysis and active surveillance should consider the possible influence of immunosuppression on the symptoms caused by the widening spectrum of enterovirus infections.

Enteroviruses (EVs) and Hepatitis A Viruses (HAVs) are human pathogens with a wide spectrum of clinical manifestations. The monitoring of sewage samples enables to monitor the EVs and HAVs in circulation among the general population and recognize possible outbreaks. This study focused on the molecular characterization and phylogenetic analysis of the EVs and HAVs identified in 33 sewage samples collected every 15 days at the influent of a wastewater treatment plant located in Northern Italy from March to October 2016. According to the results of the molecular characterization, the most frequently identified viruses were Echovirus 6 (E-6), E-11 and HAV-IA. The phylogenetic analyses indicated the rapid genetic evolution of E-6 and E-1; noteworthy, most E-11 strains clustered with a strain isolated from a clinical sample collected in the same geographical area over the same period by our laboratory. Most of the HAV strains detected clustered with epidemic HAV-IA strains identified during the European hepatitis A outbreak that occurred in 2016-2017 affecting men who have sex with men (MSM). The detection of environmental HAV strains before and at the beginning of its spread amongst humans demonstrated that this outbreak could have been predicted by monitoring sewage samples. Moreover, conducting a genetic comparison between the HAV and EV strains identified in sewage and clinical samples may improve knowledge of viral epidemiology. EV and HAV molecular environmental surveillance may prove useful for identifying viral circulation and for issuing early warning alerts on possible outbreaks among the human population.

Wastewater-based viral surveillance is a promising approach to monitor the circulation of SARS-CoV-2 in the general population. The aim of this study was to develop an analytical method to detect SARS-CoV-2 RNA in urban wastewater, to be implemented in the framework of a surveillance network in the Lombardy region (Northern Italy). This area was the first hotspot of COVID-19 in Europe. Composite 24h samples were collected weekly in eight cities from end-March to mid-June 2020 (first peak of the epidemic). The method developed and optimized, involved virus concentration, using PEG centrifugation, and one-step real-time RT-PCR for analysis. SARS-CoV-2 RNA was identified in 65 (61%) out of 107 samples, and the viral concentrations (up to 2.1 E +05 copies/L) were highest in March-April. By mid-June, wastewater samples tested negative in all the cities. Viral loads were used for inter-city comparison and Brembate, Ranica and Lodi had the highest. The pattern of decrease of SARS-CoV-2 in wastewater was closely comparable to the decline of active COVID-19 cases in the population, reflecting the effect of lock-down. Wastewater surveillance of SARS-CoV-2 can integrate ongoing virological surveillance of COVID-19, providing information from both symptomatic and asymptomatic individuals, and monitoring the effect of health interventions.

This paper outlines the role of Lombardy’s regional influenza reference laboratory (Northern Italy) in the surveillance of influenza-like illnesses (ILIs) in monitoring SARS-CoV-2 circulation by analyzing 631 consecutive nasopharyngeal swabs (NPSs) collected from ILI outpatients by sentinel physicians during the 2019–2020 season. The samples were tested by specific real-time RT-PCRs targeting SARS-CoV-2, influenza viruses, and RSVs. Results: Of these NPSs, 31% tested positive for influenza viruses, 10% for SARS-CoV-2, and 7% for RSV. No coinfections were detected. Influenza viruses and RSVs circulated throughout the surveillance period until the end of February (week 9-2020), when they suddenly ceased to circulate seven weeks earlier than during the previous five influenza seasons. After the first detection of SARS-CoV-2 in our ILI outpatients at the beginning of March (week 10-2020), SARS-CoV-2 remained the only virus identified throughout the surveillance period. Patients ≥ 65 years had a 3.2-fold greater risk of being infected with SARS-CoV-2, while school-age children (5–14 years) and children < 5 years proved to be the age groups most at risk of contracting influenza viruses and RSV, respectively. Our experience demonstrates that laboratory-based ILI surveillance networks are essential for identifying SARS-CoV-2 cases that would otherwise remain undetected, in order to stop their spread within our communities.

Background. Enteroviruses (EVs) can cause infections and outbreaks of mild to severe diseases, such as central nervous system (CNS) and systemic infections. The contribution of EVs to acute CNS/systemic infections requiring hospitalization was assessed by analysing data extracted from virology laboratory database. Methods. Real-life data obtained from two molecular virology laboratories located in Northern Italy were retrieved from databases and analysed retrospectively. The queries used to extract the data were (i) requests for EV-RNA detection in clear cerebrospinal fluid (CSF) specimens collected from hospitalized patients with suspected acute CNS (including aseptic meningitis, encephalitis, and acute flaccid myelitis/paralysis) or systemic infections (sepsis-like illness or fever (≥ 38°C) of unknown origin), (ii) CSF samples collected from January 1st, 2015, to December 31st, 2017. Results. 582 requests of EV-RNA detection in CSF samples collected from as many patients of any age were recorded. EV-RNA was detected in 4.5% of the CSF samples; 92.3% of EV-positive cases were patients<15 years, 58.3% of whom were < 3 months. EVs circulated all-year-round, and the highest EV-positive rates were observed from May to August. The risk of EV infection and the relative illness ratio value among children<1−year−old were significantly higher than those observed for older patients. Conclusions. EV surveillance should be carried out for all pediatric patients<15 years and especially children less than 1 year of age with clinically suspected CNS infection/systemic infections. The implementation of a laboratory-based surveillance established for analysing the virological data provided by laboratories that routinely perform EV molecular testing may enable us to determine the impact of EVs that can cause infections requiring hospitalization.

Wastewater-based viral surveillance was proposed as a promising approach to monitor the circulation of SARS-CoV-2 in the general population. The aim of this study was to develop an analytical method to detect SARS-CoV-2 RNA in urban wastewater, and apply it to follow the trends of epidemic in the framework of a surveillance network in the Lombardy region (Northern Italy). This area was the first hotspot of COVID-19 in Europe and was severely affected. Composite 24 h samples were collected weekly in eight cities from end-March to mid-June 2020 (first peak of the pandemic). The method developed and optimized, involved virus concentration using PEG centrifugation, and one-step real-time RT-PCR for analysis. SARS-CoV-2 RNA was identified in 65 (61%) out of 107 samples, and the viral concentrations (up to 2.1 E + 05 copies/L) were highest in March-April. By mid-June, wastewater samples tested negative in all the cities corresponding to the very low number of cases recorded in the same period. Viral loads were calculated considering the wastewater daily flow rate and the population served by each wastewater treatment plant, and were used for inter- city comparison. The highest viral loads were found in Brembate, Ranica and Lodi corresponding to the hotspots of the first peak of pandemic. The pattern of decrease of SARS-CoV-2 in wastewater was closely comparable to the decline of active COVID-19 cases in the population, reflecting the effect of lock-down. This study tested wastewater surveillance of SARS-CoV-2 to follow the pandemic trends in one of most affected areas worldwide, demonstrating that it can integrate ongoing virological surveillance of COVID-19, providing information from both symptomatic and asymptomatic individuals, and monitoring the effect of health interventions., Graphical abstract Unlabelled Image

A previously healthy 6-year-old boy was admitted to hospital with hypotonia and hyposthenia of lower limbs. Electromyography and slow motor nerve conduction velocity test identified a lower limb acute motor axonal neuropathy. Brain and spinal cord magnetic resonance imaging demonstrated multifocal cortical gray matter lesions in both cerebral hemispheres consistent with gray matter acute disseminated encephalitis otherwise with viral/Mycoplasma pneumoniae encephalitis, and signs of involvement of anterior nerve roots of the cauda equina consistent with Guillain-Barré syndrome. The patient resulted negative to routinely bacterial and viral investigations but positive to human parechovirus that sequence analyses confirmed as type 6. Intravenous immunoglobulins and methylprednisolone treatment were administered but did not relieve the symptoms of Guillain-Barré syndrome. The disease improved gradually over the next 3-month follow-up with a complete remission of both central and peripheral nervous system symptoms.

Within the initiatives for poliomyelitis eradication by WHO, Italy activated an environmental surveillance (ES) in 2005. ES complements clinical Acute Flaccid Paralysis (AFP) surveillance for possible polio cases, detects poliovirus circulation in environmental sewage, and is used to monitor transmission in communities. In addition to polioviruses, the analyses comprised: (i) the monitoring of the presence of non-polio enteroviruses in sewage samples and (ii) the temporal and geographical distribution of the detected viruses. From 2009 to 2015, 2880 sewage samples were collected from eight cities participating in the surveillance. Overall, 1479 samples resulted positive for enteroviruses. No wild-type polioviruses were found, although four Sabin-like polioviruses were detected. The low degree of mutation found in the genomes of these four isolates suggests that these viruses have had a limited circulation in the population. All non-polio enteroviruses belonged to species B and the most frequent serotype was CV-B5, followed by CV-B4, E-11, E-6, E-7, CV-B3, and CV-B2. Variations in the frequency of different serotypes were also observed in different seasons and/or Italian areas. Environmental surveillance in Italy, as part of the 'WHO global polio eradication program', is a powerful tool to augment the polio surveillance and to investigate the silent circulation or the re-emergence of enteroviruses in the population.

Aim: This paper discusses the unexpected findings from the RotaNet-Italy hospital-based surveillance study carried out in northern Italy. Materials & methods: From September 2015 to August 2016, 51 rotavirus-A (RVA) positive fecal samples were collected from children aged less than 15 years, who were hospitalized for acute gastroenteritis in Lombardy, northern Italy. Results: Molecular characterization revealed the predominance of the uncommon G12P[8] RVA strain, which was detected in 49% of cases. Phylogenetic analysis showed that these G12 strains clustered into lineage 3. Conclusion: To our knowledge, this is the first study on the G12P[8] genotype’s introduction in northern Italy. Our findings emphasize the importance of the surveillance of RVA gastroenteritis with the aim of obtaining new insight into the unusual newly emerging RVA strains.

Background/aim: Autism spectrum disorders (ASD) are neurodevelopmental disorders without a definitive etiology in most cases. Environmental factors, such as viral infections, have been linked with anomalies in brain growth, neuronal development, and functional connectivity. Congenital cytomegalovirus (CMV) infection has been associated with the onset of ASD in several case reports. The aim of this study was to evaluate the prevalence of congenital CMV infection in children with ASD and in healthy controls. Patients and methods: The CMV genome was tested by polymerase chain reaction (PCR) on dried blood spots collected at birth from 82 children (38 with ASD and 44 controls). Results: The prevalence of congenital CMV infection was 5.3% (2/38) in cases and 0% (0/44) in controls (p=0.212). Conclusion: The infection rate was about 10-fold higher in patients with ASD than in the general Italian population at birth. For this reason, detection of CMV-DNA on dried blood spots could be considered in the work-up that is usually performed at ASD diagnosis to rule-out a secondary form. Given the potential prevention and treatment of CMV infection, this study could have intriguing consequences, at least for a group of patients with ASD. Keywords: Cytomegalovirus; congenital infection; nervous system.

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.

Aim: Several authors have hypothesized an association between congenital viral infections and the onset of autism spectrum disorders (ASD). We aimed to assess the prevalence of congenital varicella zoster virus (VZV) infection in patients with ASD. Patients & methods: Congenital infection by VZV was evaluated in a cohort of 38 children with ASD and in 44 healthy controls. PCR for VZV-DNA performed on dried blood spots collected at birth. Results & conclusion: No VZV infection was detected in both groups. With the limitation of the small sample size of this study, the results are not in favor of a role of VZV in the etiology of ASD.

Background/aim Autism spectrum disorders (ASD) are neurodevelopmental disorders without a definitive etiology in most cases. Environmental factors, such as viral infections, have been linked with anomalies in brain growth, neuronal development, and functional connectivity. Congenital cytomegalovirus (CMV) infection has been associated with the onset of ASD in several case reports. The aim of this study was to evaluate the prevalence of congenital CMV infection in children with ASD and in healthy controls. Patients and methods The CMV genome was tested by polymerase chain reaction (PCR) on dried blood spots collected at birth from 82 children (38 with ASD and 44 controls). Results The prevalence of congenital CMV infection was 5.3% (2/38) in cases and 0% (0/44) in controls (p=0.212). Conclusion The infection rate was about 10-fold higher in patients with ASD than in the general Italian population at birth. For this reason, detection of CMV-DNA on dried blood spots could be considered in the work-up that is usually performed at ASD diagnosis to rule-out a secondary form. Given the potential prevention and treatment of CMV infection, this study could have intriguing consequences, at least for a group of patients with ASD.

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.

JC virus (JCV) is a widespread member of the Polyomaviridae family. Following primary infection, which occurs asymptomatically during childhood, JCV establishes latency in the host. JCV seroprevalence can reach 80 % in healthy adults, but the age of viral exposure has not been yet characterized. This study was conducted to define JCV seroprevalence in Italian infants and to estimate the date of primary infection. A JCV viral protein 1 (VP1)-GST fusion protein was used in conjunction with a homemade indirect enzyme-linked immunosorbent assay (ELISA) to test for the presence of IgG antibodies to JCV in 981 serum samples collected from 644 Italian infants of different ages (1 day to 3 years old) and in 102 breast milk samples. IgM antibody presence was also evaluated in longitudinally collected samples from 17 selected children. JCV antibody prevalence and normalized optical density (nOD) were calculated. For the longitudinal analysis, generalized estimating equation techniques and spline functions were used to estimate the possible non-linear effects of time on antibody production kinetics. JCV IgG was detected in 71.8 % of the sera. Prevalence increased over time from 46.1 % (1 month old) to 80.7 % (12 months old), 85.9 % (24 months old), and 85.5 % (36 months old). As determined by nOD, the longitudinal analysis of serum IgG amounts in children of this study (ages 1 day to 3 years old) illustrated IgG kinetic changes with statistically significant trends (p = 0.001). One-month-old children were largely negative for JCV IgM (82.4 %), and 58.8 % of children produced JCV IgM within the second and sixth months of life. JCV IgG was detected in 27.3 % of breast milk samples. JCV primary infection likely occurs before 6 months of age, and a sizeable percentage of Italian infants will become JCV seropositive within 2 years of age. This study can be used to determine the optimal age for potential future JCV vaccination in infants.

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.

Group A rotaviruses (RVA) are the leading cause of acute gastroenteritis in young (5 years of age) children, causing up to 450.000 deaths worldwide, mostly in developing countries. VP7 (G-type) and VP4 (P-type) genotypes are the basis for the binary RVA classification. Although at least 27 G-types and 37 P-types of rotavirus are presently known, most RVA infections in humans worldwide are associated with the five major G/P combinations G1P[8], G2P[4], G3P[8], G4P[8] and G9P[8]. During RVA gastroenteritis surveillance in Italy, a total of 1112 samples collected from children hospitalized with acute gastroenteritis in 2013 were RVA positive and were genotyped following standardized protocols from the EuroRotaNet. Most strains analyzed belonged to the five major human genotypes. Among these common strains, 22 G4P[8] RVA strains from different Italian regions were subjected to nucleotide sequencing of their VP4, VP6, VP7 and NSP4 genes to investigate their evolution. The phylogenetic analysis showed that the Italian strains belonged to lineage G4-I for VP7 and to lineage P[8]-III for VP4, in line with the modern G4P[8] RVA strains detected in children worldwide. The phylogenetic trees revealed high degrees of nucleotide identity between the RVA strains involved in this study and G4P[8] strains detected previously in Europe, Asia and Africa, but also demonstrated at least three separate evolution clusters within the same lineage. Based on the amino acid sequences deduced for their hypervariable regions, both the VP7 and VP8* proteins of the Italian G4P[8] RVA strains presented amino acid substitutions near known neutralizing epitopes.

Background Group A rotaviruses (RVA) are the leading cause of acute gastroenteritis (AGE) in young (aged

Background: Polyomavirus (PyV) infection is common, ranging from 60% to 100% depending on the virus. The urinary excretion rates of JC virus (JCV) and BK virus (BKV) have been extensively studied, but less is known about the more recently discovered PyVs. Objectives: To investigate the urinary excretion of Merkel cell PyV (MCPyV), which is associated with Merkel cell carcinoma (MCC), in kidney transplant recipients and healthy subjects, as well as those of lymphotropic polyomavirus (LPV), which was isolated from the B-cells of African green monkeys but has also been found in human blood, JCV and BKV. Study design: Urine samples were collected from 62 adult (ATP) and 46 pediatric (PTP) kidney transplant recipients and from 67 adult (AHC) and 40 pediatric (PHC) healthy controls. DNA was isolated and analyzed using real-time PCR (Q-PCR) to determine the viral loads of MCPyV, LPV, JCV and BKV. Results: MCPyV DNA was more frequently detected (p

Aims Human Enteroviruses (HEVs) infections have a significant impact on public health, being implicated in outbreaks of meningitis, encephalitis, hand-foot-mouth disease and other acute and chronic manifestation. In the strategic plan for poliomyelitis eradication, the environmental surveillance of poliovirus (PV) has been identified by the World Health Organization (WHO) as an activity that can complement the surveillance of polio. Having wastewater samples available for PV surveillance allows us to study nonpolio enteroviruses (NPEVs) circulating in the study population, which are widely spread. Methods and Results This study was carried out according to the WHO guidelines for environmental surveillance of PV and analysed the circulation of PV and NPEVs through the isolation of viruses in cell cultures in Milan area; from 2006 to 2010, 321 wastewater samples were collected, regularly over time, at the inlet of three diverse waste water treatment plants (WWTPs). Culturable HEVs were isolated in 80% of sewage samples: all isolates belonged to the HEV-B group and those circulating more intensely were CVB5 and Echo 6, while CVB4 was the predominant serotype found in 2010. In this study, two type 2 PVs were isolated, both characterized as Sabin like. Conclusion Environmental monitoring of HEVs in Milan has proved to be an interesting tool to investigate the circulation and distribution of viruses. Significance and Impact of the Study The detection of PV and other NPEV could be predictive of possible re-emergence of these viruses with an impact on public health. NPEV monitoring could also be a powerful public health tool to investigate the possible role of NPEV in different clinical manifestations.

Although in the last years poliovirus (PV) transmission has been reported at the lowest levels ever recorded, the spread of virus from endemic countries endures; the high levels of immigration flows across the Mediterranean Sea jeopardize Italy for PV reintroduction. The World Health Organization (WHO) strategic plan for global poliomyelitis (polio) eradication indicates the nationwide surveillance of Acute Flaccid Paralysis (AFP) as the gold standard for detecting cases of polio. In addition, the Environmental Surveillance (ES), seeking the presence of PV and Non-Polio Enterovirus (NPEV) in sewage, is recognized as a powerful tool to confirm PV circulation in absence of AFP cases, especially in polio-free countries. Here we report the results of AFP surveillance (AFPS) and ES in Lombardy (Northern Italy) from 2012 to 2015. Forty-eight AFP cases were identified during the study period. No AFP case was caused by PV infection. NPEVs were identified in 6.3% (3/48) of AFP cases. The annual AFP incidence rate was 0.87/100'000 children15 y in 2012, 1.42/100'000 in 2013, 1.02/100'000 in 2014, and 0.47/100'000 in 2015; according to WHO indicators, the sensitivity of AFPS was adequate in 2013 and 2014. Completeness of case investigation raised progressively during the study period to achieve the WHO standards in 2014 (92.3%) and 2015 (100%). Completeness of follow-up increased from 72.7% in 2012 to 100% in 2014. In the framework of the ES conducted in Milan, 268 wastewater samples were collected from 2012 to 2015 and no PVs were isolated. In contrast, NPEVs were detected in 65.3% (175/268) of samples. All NPEVs characterized belonged to enterovirus species B: echovirus type 11, 6 and 3 were the most frequently detected viruses, representing 29.1% (41/141), 20.6% (29/141) and 9.2% (13/141) of genotyped NPEVs, respectively. Keeping strong and encouraging both AFPS and ES is crucial to ensure that PV will not return unnoticed in Italy - as well as in other polio-free countries - and, as a final point, to achieve the global polio eradication goal.

We report 2 neonates with human parechoviruses type 3 encephalitis. Both newborns presented with fever, irritability and seizures. Cerebrospinal fluid analyses were normal, but magnetic resonance imaging revealed white matter damage, suggesting human parechoviruse infection. Human parechoviruses type 3-RNA was detected in cerebrospinal fluid samples and in blood, stool, urine and respiratory samples, indicating the dissemination of the virus.