Detection of Echovirus 11 lineage 1 in wastewater samples in Sicily.

We report the presence of Echovirus 11 (E11) in wastewater in Sicily (Southern Italy), since August 2022. Overall, the 5.4 % of sewage samples (7/130) collected in 2022 were positives for E11 and then the percentage of E11-positive sewage samples reached the value of 27.27(18/66) in the first semester of 2023. Phylogenetic analysis of VP1 sequences showed for most E11-positive samples (16/25: 64 %) close genetic correlation (98.4-99.4 % nucleotide identity) to E11 lineage 1 strains involved in recently reported severe neonatal infections.

Identification of a novel intra-genotype reassortant G1P[8] rotavirus in Italy, 2021.

OBJECTIVES: Rotaviruses G1P[8] are epidemiologically relevant and are targeted by vaccines. The introduction of vaccines has altered rotavirus epidemiology. Hospital-based surveillance conducted in Sicily, Italy, showed a progressive decline in rotavirus prevalence since 2014, along with an increasing vaccine coverage (63.8% in 2020), and a marked decrease in circulation of G1P[8] strains. Surprisingly in 2021, G1P[8] viruses accounted for 90.5% (19/21) of rotavirus infections. This study aimed to understand if the increased activity of G1P[8]'s was related to virus-related peculiarities. DESIGN: In 2021, 266 patients <15 years of age were hospitalized with acute gastroenteritis (AGE) and included in rotavirus surveillance. Viral proteins (VP7 and VP4) genotyping and sequence data were generated from all rotavirus-positive samples. The genetic makeup of G1P[8] rotaviruses was investigated by full-genome sequencing. RESULTS: Peculiar G1P[8] rotaviruses, with VP7 and VP4 belonging to novel sub-lineages, circulated in 2021, accounting for 76.2% (16/21) of all rotavirus infections. On full-genome analysis, the novel G1P[8] variant displayed an intra-genotype (Wa-like) reassortant constellation, involving G12 and G1 strains, into a unique arrangement never observed before. The novel G1P[8] variant showed peculiar amino acid substitutions in 8-1 and 8-3 epitopes of the VP4 with respect to the Rotarix strain. CONCLUSIONS: Prompt identification of virus variants circulating in the human population is pivotal to understanding epidemiological trends and assessing vaccine efficacy.

Biological Specimen Banking as a Time Capsule to Explore the Temporal Dynamics of Norovirus Epidemiology.

Norovirus is recognised as a major cause of epidemic and sporadic acute gastroenteritis (AGE) in all age groups. Information on the genetic diversity of the noroviruses circulating in the 1980s and 1990s, before the development and adoption of dedicated molecular assays, is limited compared with the last decades. Between 1986 and 2020, uninterrupted viral surveillance was conducted in symptomatic children hospitalized with AGE in Palermo, Italy, providing a unique time capsule for exploring the epidemiological and evolutionary dynamics of enteric viruses. A total of 8433 stool samples were tested using real-time RT-PCR. All samples were stored at -20 or -80 °C until processing. In this 35-year long time span, noroviruses of genogroup II (GII) were detected in 15.6% of AGE requiring hospitalization, whilst GI noroviruses were detected in 1.4% of AGE. Overall, the predominant norovirus capsid (Cap) genotype was GII.4 (60.8%), followed by GII.3 (13.3%) and GII.2 (12.4%). Temporal replacement of the GII.4 Cap variants associated with different polymerase (Pol) types were observed over the study period. The chronology of emergence and circulation of the different GII.4 variants were consistent with data available in the literature. Also, for GII.3 and GII.2 NoVs, the circulation of different lineages/strains, differing in either the Cap or Pol genes or in both, was observed. This long-term study revealed the ability of noroviruses to continuously and rapidly modify their genomic makeup and highlights the importance of surveillance activities in vaccine design.

Impact of Vaccination on Rotavirus Genotype Diversity: A Nearly Two-Decade-Long Epidemiological Study before and after Rotavirus Vaccine Introduction in Sicily, Italy.

Sicily was the first Italian region to introduce rotavirus (RV) vaccination with the monovalent G1P[8] vaccine Rotarix® in May 2012. In this study, the seasonal distribution and molecular characterization of RV strains detected over 19 years were compared to understand the effect of Rotarix® on the evolutionary dynamics of human RVs. A total of 7846 stool samples collected from children < 5 years of age, hospitalized with acute gastroenteritis, were tested for RV detection and genotyping. Since 2013, vaccine coverage has progressively increased, while the RV prevalence decreased from 36.1% to 13.3% with a loss of seasonality. The local distribution of RV genotypes changed over the time possibly due to vaccine introduction, with a drastic reduction in G1P[8] strains replaced by common and novel emerging RV strains, such as equine-like G3P[8] in the 2018−2019 season. Comparison of VP7 and VP4 amino acid (aa) sequences with the cognate genes of Rotarix® and RotaTeq® vaccine strains showed specific aa changes in the antigenic epitopes of VP7 and of the VP8* portion of VP4 of the Italian RV strains. Molecular epidemiological surveillance data are required to monitor the emergence of novel RV strains and ascertain if these strains may affect the efficacy of RV vaccines.

Assessment of SARS-CoV-2 RNA shedding in semen of 36 males with symptomatic, asymptomatic, and convalescent infection during the first and second wave of COVID-19 pandemic in Italy.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) receptor, angiotensin-converting enzyme 2 (ACE2), has been identified in the human testis, but the risk of transmission of SARS-CoV-2 through sexual intercourse still needs to be defined. The goal of our study was to determine if SARS-CoV-2 is detectable in the semen of patients suffering or recovering from coronavirus disease-19 (COVID-19), still testing positive at nasopharyngeal swabs but showing mild or no symptoms at the time of sampling. Detection of SARS-CoV-2 RNA in semen was performed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR targeting open reading frame (ORF) 1ab. Medical history of the enrolled patients was taken, including COVID-19-correlated symptoms, both at the time of diagnosis and at the time of interview. Results of real-time RT-PCR and nested PCR in semen showed no evidence of SARS-CoV-2 RNA in the 36 patients suffering or recovering from COVID-19 but still positive in a nasopharyngeal swab, from over 116 patients enrolled in the study. SARS-CoV-2 detection and persistence in semen would have an impact on both clinical practice and public health strategies, but our results would suggest that SARS-CoV-2 is not present in the semen of men recovering from COVID-19.

Clinical and Epidemiologic Features of Viral Gastroenteritis in Hospitalized Children: An 11-Year Surveillance in Palermo (Sicily).

In order to acquire information regarding viral agents and epidemiologic features of severe paediatric Viral Acute Gastroenteritis (VAGE) across multiple seasons in the pre-rotavirus-vaccine era, the epidemiologic characteristics of VAGE were investigated among paediatric patients hospitalized in a major Sicilian paediatric hospital from 2003 to 2013. Overall, 4725 children were observed and 2355 (49.8%) were diagnosed with a viral infection: 1448 (30.6%) were found positive to rotavirus, 645 (13.7%) to norovirus, 216 (4.6%) to adenovirus, and 46 (0.97%) to astrovirus. Viral infections showed different patterns of hospitalization in terms of age at risk (younger for rotavirus and adenovirus infections), seasonality (increased risk in winter for rotavirus and norovirus), trend over time (reduced risk in 2011-2013 for norovirus and rotavirus) and major diagnostic categories (digestive diseases more frequent in adenovirus and astrovirus but not in norovirus). This study increases general knowledge of VAGE epidemiology and contributes to suggest some a priori diagnostic criteria that could help clinicians to identify and treat viral agents responsible for gastroenteritis in hospital settings.

Evaluation of the diagnostic performances of two commercially available assays for the detection of enteric adenovirus antigens.

The performance of 2 antigenic commercial assays for enteric adenovirus (AdV) infection, bioNexia Rota-Adeno ImmunoChromatographic Tests (ICT) and LIAISON® Adenovirus ChemiLuminescence Immuno Assays (CLIA), was evaluated on 321 stools from children hospitalized for acute gastroenteritis in Palermo, Italy, using a Real time-PCR (Rt-PCR) as reference method. The CLIA showed higher sensitivity (77% vs 60%), accuracy (94.4 vs 90.9) and concordance (k: 0.81 vs 0.67) with respect to ICT, despite equivalent specificity (98.8%). Using the Ct values of the Rt-PCR as a proxy of the fecal viral load, similar Ct values (mean 9.32 vs 9.89) were observed among the true positive samples, whilst a significant difference (P < 0.05) was observed in false negative samples of CLIA (mean Ct 25.68) and ICT (mean Ct 19.87). Cross-reactivity with other enteric viruses was not observed. These results indicate that both the assays tested are suitable for diagnosis of AdV gastroenteritis.

Recombinant GII.P16 genotype challenges RT-PCR-based typing in region A of norovirus genome.

OBJECTIVES: In latest years GII.4[P16] and GII.2[P16] noroviruses have become predominant in some temporal/geographical settings. In parallel with the emergence of the GII.P16 polymerase type, norovirus surveillance activity in Italy experienced increasing difficulties in generating sequence data on the RNA polymerase genomic region A, using the widely adopted JV12A/JV13B primer set. Two sets of modified primers (Deg1 and Deg2) were tested in order to improve amplification and typing of the polymerase gene. METHODS: Amplification and typing performance of region A primers was assessed in RT-PCR on 452 GII norovirus positive samples obtained from 2194 stool samples collected in 2016-2019 from children hospitalized with acute gastroenteritis. RESULTS: The use of Deg1 increased the rate of samples types in region A from 49.5% to 81.4% and from 21.9% to 69.7% in 2016 and 2017, respectively. The rate of Deg1 typed samples remained high in 2018 (90.1%), but sharply decreased to 11.8% in 2019. The second primers set, Deg2, was able to increase to 64.9% the rate of 2019 samples typed in region A, while typing efficiently 73.2%, 69%, and 86.4% of samples collected in 2016, 2017 and 2018, respectively. CONCLUSIONS: The plasticity of norovirus genomes requires continuous updates of the primers used for strain characterization.

Assessing the burden of viral co-infections in acute gastroenteritis in children: An eleven-year-long investigation.

BACKGROUND: Acute gastroenteritis is an important cause of childhood morbidity and mortality worldwide. A number of pathogens are responsible for human acute gastroenteritis. The recent introduction of syndromic assays for the diagnosis of enteric infections, including a wide panel of enteric pathogens, has unveiled the frequency of mixed infections. This study was carried out to assess the burden of viral co-infections and the genetic diversity of the viruses detected in children hospitalized with acute gastroenteritis in Italy. METHODS: A total of 4161 stool samples collected from diarrheic children over 11 years, from January 2008 to December 2018, were investigated for the presence of four enteric viruses, i.e. group A rotavirus, norovirus, astrovirus and adenovirus. The samples were initially screened by either molecular or immunochromatographic assays and subsequently confirmed by Real-time PCR and sequence analyses. RESULTS: At least one viral agent was detected in 48.6 %of specimens. Rotavirus was the most prevalent virus (24.7 %) followed by norovirus (19.6 %), adenovirus (5.3 %) and astrovirus (3%). Co-infections were detected in 8.3 % of virus-positive patients, with common viral combination being rotavirus with norovirus (70.6 % of co-infections) or with astrovirus (9.6 %). A variety of viral genotypes was detected in co-infections and in single infections. Using Real-time PCR cycle thresholds as a proxy measure of fecal viral load, rotavirus was generally detected at higher levels in co-infected patients. CONCLUSIONS: Combining and deciphering measurable indicators of viral load and epidemiological information could be useful for an accurate interpretation of viral co-infections.