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Monkeypox virus (MPXV), a member of the family Poxviridae, causes monkeypox, a viral zoonosis detected in north Africa in the 1970s (1). MPXV can be transmitted between humans through contact [...]

Human noroviruses (HNoVs) are a leading cause of acute gastroenteritis worldwide, with significant public health implications. In this study, wastewater-based epidemiology (WBE) was used to monitor the circulation and genetic diversity of HNoVs in Rome over an eight-year period (2017–2024). A total of 337 wastewater samples were analyzed using RT-nested PCR and next-generation sequencing (NGS) to identify genogroups GI and GII and their respective genotypes. The results showed that GII had higher detection rates (66.5%) compared to GI (50.7%), with significant variation between years. Detection rates peaked in 2019 before declining sharply in 2020, coinciding with the COVID-19 pandemic and rebounding after the pandemic in 2023. A total of 24 genotypes were identified (8 GI and 17 GII), including persistent variants GII.2, GII.3 and GII.4 and emerging genotypes such as GII.8, GII.10 and GII.14. Only two GII.4 variants, Sydney_2016 and Sydney_2012, were detected in the study. These results demonstrate the utility of WBE in tracking HNoVs circulation, identifying genotype diversity and capturing shifts in transmission dynamics. WBE provides a cost-effective and comprehensive tool for public health surveillance, particularly in regions with limited clinical surveillance. Sustained investment in WBE is crucial for advancing our understanding of HNoVs epidemiology and its long-term trends. [ABSTRACT FROM AUTHOR]

Abstract Background Microbiological indicators are commonly used in the assessment of public health risks associated with fecal contamination of freshwater ecosystems. Sediments are a reservoir of microorganisms, and can thus provide information on past pollution events, not obtainable through the testing of surface water. Moreover, pathogens present in sediment may represent future threats to human health. Clostridium perfringens, a typical colonizer of sediments, has been suggested as an alternative indicator of fecal pollution. In order to be suitable for such purpose, the microorganism should be widely distributed in contaminated environments. The objective of this study was thus to determine the composition of the anaerobic community in sediment samples of the lower Tiber basin, in central Italy, through a combined approach involving granulometric analysis of sediment samples, as well as a microbiological and molecular (16S rRNA) analysis of strains. Results Granulometry showed a similar, clayey sediment composition, in most sampling sites. The microbiological method, employing, an adaptation of the standard method, proved to be effective in isolating anaerobic bacteria from the environmental matrix for the purpose of genetic analysis. Eighty-three strains of bacteria were isolated and the partial 16S rRNA gene sequenced. While biochemical analysis detected only C. perfringens strains, phylogenetic analysis indicated the presence of three clusters: C. perfringens, C. bifermentans and B. cereus, comprising eight taxa. C. perfringens, the commonest in almost all sediment sampling sites, was present in all sites, and in both seasons (seasonal sampling was carried out only along the Tiber and Aniene rivers). None of the described genetic profiles showed complete similarity with GenBank sequences. Conclusion The study underlines the value of C. perfringens as an alternative microbial indicator of fecal contamination in river sediments. This is supported by the bacterium's presence in all sampling sites, and in both seasons, coupled with its detectability using commercial diagnostic kits. The study also illustrates the presence of an anaerobic community of considerable biodiversity in the lower Tiber basin, with C. perfringens as its main component. The 16S rRNA analysis, while confirming the phylogenetic relationships among isolated species, also showed haplotype patterns different from those present in the NCBI database.

The occurrence of hepatitis E virus (HEV) through the molecular screening of raw sewage samples collected from urban wastewater treatment plants (WPTs) in different parts of Italy is examined. The results have shown that HEV strains are more widespread than previously thought.

Background Hepatitis E (HEV) is an important public-health concern as a major cause of enterically transmitted hepatitis worldwide. In industrialised countries it is considered rare, and largely confined to travellers returning from endemic areas. However, autochthonous (locally acquired) HEV infection is also emerging in these regions. The infection is caused by different genotypes, depending on whether it is travel-related or autochthonous. Conventional RT-PCR followed by sequencing of PCR products can identify HEV genotype and, depending on the region, the subtype, thus helping in defining the origin of infection and tracing the source of contamination. Methods We re-analysed a collection of serum samples previously confirmed as hepatitis E positive by anti-HEV IgM and IgG assays as well as by Real-Time PCR, with the aim to compare the performances of five different broad range RT-PCR assays that could be provided for molecular characterisation of HEV. This approach is certainly valuable to investigate the molecular epidemiology of acute hepatitis E in countries where co-circulation of different genotypes occurs, like Italy. Results Samples were analyzed by five assays targeting the ORF1, ORF2, and ORF2/3 regions. The sensitivity of these assays varied significantly, depending on the target region. Only 46% of samples tested positive by nested PCR; moreover, no single method was able to detect all positive samples. Most sequences originated from patients who had travelled to endemic areas (genotype 1), while the minority originated from Italian patients with no travel history (genotype 3). Conclusion Broad range methods for molecular characterization of HEV still need to be improved to detect all circulating strains. [ABSTRACT FROM AUTHOR]

Noroviruses cause acute viral gastroenteritis worldwide. They are classified in five genogroups, of which GI, GII, and GIV infect humans. Little information is available on the prevalence and clinical effects of GIV noroviruses. We conducted a large-scale molecular-epidemiological investigation, a yearlong monitoring of 11 wastewater treatment plants throughout Italy, with the aim of studying the circulation of GIV NoV, as well as its genetic diversity. Eight percent of samples tested positive, and sequence analysis showed a considerable degree of genetic variability. These results imply the need for further studies to elucidate the role of this virus as a gastroenteritis-causing pathogen. [ABSTRACT FROM AUTHOR]

Noroviruses (NoVs) are a major cause of acute non-bacterial gastroenteritis in all age groups. They efficiently circulate in both clinical and environmental contexts. In this study, real-time RT-PCR methods based on TaqMan probe technology were used to enumerate human noroviruses (genogroups I and II) in clinical samples, and in estuarine, seawater and sewage water samples, with the aim of obtaining quantitative information on the level of viral contamination. This was achieved through a quantitative analysis of the highly conserved region between ORF1 and ORF2, using genogroup-specific assays. RNA standards used to construct calibration curves for the two genogroups were generated by in vitro transcription from recombinant pCR4TOPO vectors containing a partial sequence coding for RdRp polymerase. Sewages were found to contain from 6.8 × 102 to 6.7 × 104 genome copies (GC) per millilitre; seawater samples contained from 7.6 × 101 to 2.4 × 103 per 10 l. As for clinical samples, the concentrations of NoVs per gram of stool varied, ranging from 6.1 × 103 to 1.4 × 108. Real-time PCR is an easy to use, sensitive and specific tool able to generate quantitative data, and could prove useful in both environmental and clinical settings. [ABSTRACT FROM AUTHOR]

The genetic diversity of Hepatitis A Virus (HAV) circulating in the Campania Region in years 2015–2018 was investigated through the monitoring of sentinel bivalve shellfish and water matrices. Overall, 463 water samples (71 sewage samples, 353 coastal discharge waters, and 39 seawaters samples), and 746 bivalve shellfish samples were analyzed. Positivity for HAV was detected in 20/71 sewage samples, 14/353 coastal discharge waters, 5/39 seawaters, and 102/746 bivalve shellfish. Sixty-one of the positive samples were successfully sequenced and were characterized as genotype IA (n = 50) and IB (n = 11). The prevalent strain circulating in 2015 in both bivalves and waters was the IA strain responsible for the outbreak occurring around the same time in the Naples area. This variant was no longer identified in subsequent years (2017–2018) when, instead, appeared two of the IA variants of the multistate outbreak affecting men who have sex with men (MSM), VRD_521_2016, and RIVM-HAV16–090, with the former prevailing in both shellfish and water environments. HAV IB isolates were detected over the years in shellfish and in water matrices, but not in clinical samples, suggesting that this genotype had been circulating silently. An integrated surveillance system (environment/food/clinical cases) can be a useful tool to monitor changes in viral variants in the population, as well as an early warning system. [ABSTRACT FROM AUTHOR]

Increasing numbers of hepatitis E cases are being reported in several European countries, including Italy, but the burden of hepatitis E virus (HEV) infection is largely unknown in the latter. To gain a better understanding of HEV epidemiology at national level in Italy, we piloted a strengthened and integrated human (epidemiological and virological) and environmental HEV surveillance system between 2012 and 2016. Over the 5-year period, 169 confirmed hepatitis E cases were identified, with a national annual incidence of 0.72 cases per 1,000,000. Of 65 HEV-RNA positive samples of sufficient quality for molecular analysis, 66% were genotype HEV3, 32% HEV1 and 1% HEV4. The most frequent risk factor reported by all HEV3 infected cases, was the consumption of undercooked pork and sausage. For the environmental surveillance, 679 urban sewage samples were collected from 53 wastewater treatment plants and HEV-RNA was detected in 38/679 of the samples. Among these, 25 (66%) were genotype HEV3 and the remaining were HEV1. We demonstrate that autochthonous transmission and environmental circulation of genotype HEV3 is adding to travel-related HEV transmission in Italy. We recommend the 'One Health' approach to integrated surveillance, and to include HEV-related messages within health information campaigns focussing on food security.

The prevalence of enteric viruses in wastewater, the efficacy of wastewater treatments in eliminating such viruses, and potential health risks from their release into the environment or by recycling of treated wastewaters, are very important issues in environmental microbiology. In this study we performed a quantitative TaqMan real-time PCR (polymerase chain reaction) analysis of enteric viruses on samples of influents and effluents from 5 wastewater treatment plants in and around Rome. Three epidemiologically important, waterborne enteric viruses were analyzed: adenoviruses, enteroviruses and noroviruses (GI and GII) and compared to classical bacterial indicators of fecal contamination. The concentration of adenoviruses was the highest, in both raw and treated waters. Mean values in influents were ranked as follows: adenovirus > norovirus GI > norovirus GII > enterovirus. In effluents, the ranking was: adenovirus > norovirus GI > enterovirus > norovirus GII. Removal efficiencies ranged from 35% (enterovirus) to 78% (norovirus GI), while removal efficiency for bacterial indicators was up to 99%. Since molecular quantification does not necessarily indicate an actual threat to human health, we proceeded to evaluate the infectivity of enterovirus particles in treated effluents through integrated cell culture and real-time PCR. Infectivity assays detected live virions in treated water, pointing to potential public health risks through the release of these viruses into the environment. A better understanding of viral presence and resistance to sewage purification processes have the potential of contributing to the effective management of risks linked to the recycling of treated wastewater, and its discharge into the environment.

There is little information on the epidemiology of Human Adenoviruses (HAdVs) in Italy. In this study, 103 HAdV isolates, collected by the A. Gemelli Hospital (Catholic University Medical School of Rome, Italy) between 1987 and 2005, were genotyped by sequencing and phylogenetic analysis on a partial hexon gene region. Nine different serotypes belonging to all six HAdV species were identified. Serotype 2 was the most frequent (53.4%), followed by serotype 1 (15.53%) and serotype 41 (9.7%). Partial-hexon-based identification was confirmed as an effective tool for studying the molecular epidemiology of HAdVs.