Pilot survey of norovirus in Northern Italy: an example of surveillance of norovirus gastroenteritis.

In this study, we describe the results of virological investigations carried out on cases of gastroenteritis reported in different communities within a 2-year pilot surveillance programme (January 2012 to December 2013) in the autonomous province of Bolzano (Northern Italy). Among the 162 norovirus (NoV)-positive cases out of 702 cases investigated, 76 were grouped in nine suspected outbreaks, 37 were hospital-acquired and 49 were community-acquired sporadic cases. NoV infections were found in all age groups in outbreak and community-acquired cases, while the highest peak of hospital-acquired infections occurred in the elderly. Sequence analyses helped to identify suspected outbreaks both in the community and in hospital wards. Although GII.4 is the predominant genotype, sequence data confirmed that at least seven genotypes circulate causing sporadic cases. Findings in this study confirmed the relevance of NoV infections as a cause of outbreaks, and impact of NoV infections in community-acquired sporadic cases in adults that are rarely described because of a lack of reporting.

Serological survey of hepatitis E virus infection in farmed and pet rabbits in Italy.

The recent identification in rabbits of hepatitis E viruses (HEV) related to viruses infecting humans raises the question of the role of this species as possible HEV reservoir. A serological survey on rabbit HEV infection was conducted in Italy during 2013-2014, including both farmed and pet rabbits. We found an anti-HEV antibody seroprevalence of 3.40 % in 206 farmed rabbits (collected on 7 farms) and 6.56 % in 122 pets. RNA was extracted from IgG-positive sera and analyzed by HEV-specific real-time RT-PCR. None of the samples were positive, confirming that no viremia was present in the presence of IgG. Only one serum sample from a farmed rabbit was positive for IgM, but no HEV RNA was detected in it. Pet rabbit feces were also tested for HEV RNA, with negative results. This finding suggests that HEV is circulating in rabbits in Italy.

Epidemiology of rotavirus diarrhoea in Albania.

The aim of the study was to estimate the prevalence of rotavirus disease in children<5 years old in Tirana, Albania, and to monitor and characterize the rotavirus genotypes. Rotavirus was detected in 21% of samples, more frequently in children under 2 years of age, which accounted for 80.8% of all positive cases. Among all rotavirus-positive samples collected, G4P[8] was the most prevalent genotype (38%), followed by G1P[8] (36.6%). The use of safe and effective rotavirus vaccines for the prevention of severe diarrhoea and the reduction of treatment costs will be of great importance for Albania.

Rotavirus genotypes co-circulating in Europe between 2006 and 2009 as determined by EuroRotaNet, a pan-European collaborative strain surveillance network.

EuroRotaNet, a laboratory network, was established in order to determine the diversity of co-circulating rotavirus strains in Europe over three or more rotavirus seasons from 2006/2007 and currently includes 16 countries. This report highlights the tremendous diversity of rotavirus strains co-circulating in the European population during three years of surveillance since 2006/2007 and points to the possible origins of these strains including genetic reassortment and interspecies transmission. Furthermore, the ability of the network to identify strains circulating with an incidence of ≥1% allowed the identification of possible emerging strains such as G8 and G12 since the beginning of the study; analysis of recent data indicates their increased incidence. The introduction of universal rotavirus vaccination in at least two of the participating countries, and partial vaccine coverage in some others may provide data on diversity driven by vaccine introduction and possible strain replacement in Europe.

Rotavirus surveillance in europe, 2005-2008: web-enabled reporting and real-time analysis of genotyping and epidemiological data.

BACKGROUND: The first European rotavirus surveillance network, EuroRotaNet, comprising 16 laboratories in 15 European countries, has been established. METHODS: Fecal samples from gastroenteritis cases positive for group A rotavirus antigen were collected from multiple European countries from 2005 to mid-2008 and were subjected to G and P genotyping. Epidemiological data collected included age, sex, geographical location, setting, dates of onset and sample collection, and clinical symptoms. RESULTS: A total of 8879 rotavirus-positive samples were characterized: 2129 cases were from the 2005-2006 season, 4030 from the 2006-2007 season, and 2720 from the ongoing 2007-2008 season. A total of 30 different G and P type combinations of strains circulated in the region from 2005 through 2008. Of these strains, 90% had genotypes commonly associated with human infections-G1P[8], G2P[4], G3P[8], G4P[8], and G9P[8]-and 1.37% represented potential zoonotic introductions. G1P[8] remained the most prevalent genotype in Europe as a whole, but the incidence of infection with G1P[8] rotavirus strains was <50% overall, and all 3 seasons were characterized by a significant diversity of cocirculating strains. The peak incidence of rotavirus infection occurred from January through May, and 81% of case patients were aged <2.5 years. Conclusions. Data gathered through EuroRotaNet will provide valuable background information on the rotavirus strain diversity in Europe before the introduction of rotavirus vaccines, and the network will provide a robust method for surveillance during vaccine implementation.

Massive outbreak of viral gastroenteritis associated with consumption of municipal drinking water in a European capital city.

On 24 August 2008, an outbreak alert regarding cases of acute gastroenteritis in Podgorica triggered investigations to guide control measures. From 23 August to 7 September, 1699 cases were reported in Podgorica (population 136 000) and we estimated the total size of the outbreak to be 10 000-15 000 corresponding to an attack rate of approximately 10%. We conducted an age- and neighbourhood-matched case-control study, microbiologically analysed faecal and municipal water samples and assessed the water distribution system. All cases (83/83) and 90% (80/90) [corrected] of controls drank unboiled chlorinated municipal water [matched odds ratio (mOR) 11.2, 95% confidence interval (CI), 1.6-infinity]. Consumption of bottled water was inversely associated with illness (mOR 0.3, 95% CI 0.1-0.8). Analyses of faecal samples identified six norovirus genotypes (21/38 samples) and occasionally other viruses. Multiple defects in the water distribution system were noted. These results suggest that the outbreak was caused by faecally contaminated municipal water. It is unusual to have such a large outbreak in a European city especially when the municipal water supply is chlorinated. Therefore, it is important to establish effective multiple-barrier water-treatment systems whenever possible, but even with an established chlorinated supply, sustained vigilance is central to public health.

Analysis of integrated virological and epidemiological reports of norovirus outbreaks collected within the Foodborne Viruses in Europe network from 1 July 2001 to 30 June 2006.

The Foodborne Viruses in Europe network has developed integrated epidemiological and virological outbreak reporting with aggregation and sharing of data through a joint database. We analyzed data from reported outbreaks of norovirus (NoV)-caused gastroenteritis from 13 European countries (July 2001 to July 2006) for trends in time and indications of different epidemiology of genotypes and variants. Of the 13 countries participating in this surveillance network, 11 were capable of collecting integrated epidemiological and virological surveillance data and 10 countries reported outbreaks throughout the entire period. Large differences in the numbers and rates of reported outbreaks per country were observed, reflecting the differences in the focus and coverage of national surveillance systems. GII.4 strains predominated throughout the 5-year surveillance period, but the proportion of outbreaks associated with GII.4 rose remarkably during years in which NoV activity was particularly high. Spring and summer peaks indicated the emergence of genetically distinct variants within GII.4 across Europe and were followed by increased NoV activity during the 2002-2003 and 2004-2005 winter seasons. GII.4 viruses predominated in health care settings and in person-to-person transmission. The consecutive emergence of new GII.4 variants is highly indicative of immune-driven selection. Their predominance in health care settings suggests properties that facilitate transmission in settings with a high concentration of people such as higher virus loads in excreta or a higher incidence of vomiting. Understanding the mechanisms driving the changes in epidemiology and clinical impact of these rapidly evolving RNA viruses is essential to design effective intervention and prevention measures.

Molecular characterization of bovine rotavirus strains circulating in northern Italy, 2003-2005.

A total of 232 stools collected from calves with rotavirus infection in herds located in northern Italy from 2003 to 2005 was investigated. Determination of the rotavirus G and P types was carried out using nested RT-PCR. G6 was the most prevalent genotype, accounting for 78.5% of samples, G10 accounted for 9.9% of samples and viruses of G8 type were found in 4.7% of samples. In 3% of samples, viruses were not classified due to concomitant infection with more G type strains, whereas viruses in 3.9% of samples could not be characterized with any of the G-specific primers used in this study. Most common P types were P[11] and P[5], accounting for 65.1% and 25%, respectively. In 2.6% of cases, samples reacted with multiple P-specific primers; no P[1] serotype was identified. The G6P[11] combination was predominant throughout the study period, i.e. 52.5% in 2003, 50% in 2004 and 40% in 2005. The incidence of G6P[5] increased from 13.1% in 2003 to 27% in 2004 and 25.5% in 2005. The G10P[11] combination decreased markedly from 18% in 2003 to 2.6% in 2004, rising again to 7.3% in 2005. G8P[11] viruses were similarly present in 2003 (5%) and 2004 (4.3%), declining slightly in 2005 (1.8%).

Data quality of 5 years of central norovirus outbreak reporting in the European Network for food-borne viruses.

BACKGROUND: The food-borne viruses in Europe (FBVE) network database was established in 1999 to monitor trends in outbreaks of gastroenteritis due to noroviruses (NoVs), to identify major transmission routes of NoV infections within and between participating countries and to detect diffuse international food-borne outbreaks. METHODS: We reviewed the total of 9430 NoV outbreak reports from 13 countries with date of onset between 1 January 2002 and 1 January 2007 for representativeness, completeness and timeliness against these objectives. RESULTS: Rates of reporting ranged from a yearly average of 1.8 in 2003 to 11.6 in 2006. Completeness of reporting of an agreed minimum dataset improved over the years, both for epidemiological and virological data. For the 10 countries that provided integrated (epidemiological AND virological) reporting over the 5-year period, the completeness of the minimum dataset rose from 15% in 2003 to 48% in 2006. Two countries have not been able to combine both data types due to the structure of the national surveillance system (England and Wales and Germany). Timeliness of reporting (median days between the onset of an outbreak and the date of reporting to the FBVE database) differed greatly between countries, but gradually improved to 47 days in 2006. CONCLUSION: The outbreaks reported to the FBVE reflect the lack of standardization of surveillance systems across Europe, making direct comparison of data between countries difficult. However, trends in reported outbreaks per country, distribution of NoV genotypes, and detection of diffuse international outbreaks were used as background data in acute questions about NoV illness and the changing genotype distribution during the 5-year period, shown to be of added value. Integrated reporting is essential for these objectives, but could be limited to sentinel countries with surveillance systems that allow this integration. For successful intervention in case of diffuse international outbreaks, completeness and timeliness of reporting would need to be improved and expanded to countries that presently do not participate.

Norovirus genotypes involved in the outbreaks of gastroenteritis in Croatia during the winter season 2004--2005.

Seven outbreaks and four sporadic cases of the non-bacterial gastroenteritis caused by a norovirus (NoV) were detected in Croatia between November 2004 and February 2005. An enzyme immunoassay (EIA) and three different RT-PCRs for the viral polymerase (ORF1 RT-PCR) and genogroup I (GI) or II (GII) of capsid gene regions (GI-ORF2 RT-PCR; GII-ORF2 RT-PCR) were performed to detect NoV in 21 stool samples. To characterize NoVs, sequencing of the ORF1 region was performed on 12 RT-PCR positive samples, whereas the ORF2 region was sequenced for 5 cases. Four outbreaks were caused by the genotype GII.4 (Lordsdale) and one outbreak was caused by the genotype GI.1 (Norwalk). One of the outbreaks was characterized as potentially mixed GII.4 and GI.1 infection. In the monitored period, genotype GII.4 dominated as the cause of noroviral infections in adults.

Group A rotavirus genotypes in hospital-acquired gastroenteritis in Italy, 2012-14.

BACKGROUND: Group A rotaviruses (RVA) are the leading cause of acute gastroenteritis (AGE) in young (aged <5 years) children, causing ∼250,000 deaths worldwide, mostly in developing countries. Differences on nucleotide sequences of VP7 (G-type) and VP4 (P-type) genes are the basis for the binary RVA nomenclature. Although at least 32 G-types and 47 P-types of rotavirus are presently known, most RVA infections in humans worldwide are related to five major G/P combinations: G1P[8], G2P[4], G3P[8], G4P[8], and G9P[8]. AIM: To provide the hospitals of the Italian surveillance network with update information on RVA AGE. METHODS: During RVA gastroenteritis surveillance in Italy in 2012-14, a total of 2341 RVA-positive faecal samples were collected from children hospitalized with AGE, and RVA strains were genotyped following standard EuroRotaNet protocols. FINDINGS: Most strains analysed belonged to the five major human genotypes and 118 out of 2341 (5.0%) were reported to be hospital-acquired. Comparison of the distributions of the RVA genotypes circulating in the community or associated with nosocomial infections showed a different distribution of genotypes circulating inside the hospital wards, with respect to those observed in the community. G1P[8] and G9P[8] RVA strains were detected frequently, whereas G12P[8] caused a single large nosocomial outbreak. CONCLUSION: The information from this study will be useful to implement guidelines for preventing RVA AGE and optimizing the management of patients in hospital wards.

Retrospective Study Evaluating Seroprevalence of Hepatitis E Virus in Blood Donors and in Swine Veterinarians in Italy (2004).

Hepatitis E is an emerging viral disease in developed countries, with sporadic cases occasionally linked to the consumption of raw or undercooked pork, wild boar or deer meat. Cases due to transfusion or transplantation have also been reported. In developed countries, hepatitis E is considered a zoonosis and pig is the main reservoir. In the last few years, several studies conducted in Europe reported variable seroprevalence rates among the general population, ranging between 0.26% and 52.5%. A higher seroprevalence was described among workers who come in contact with pigs. The aim of this retrospective study was to evaluate the seroprevalence of anti-HEV IgG and IgM antibodies in blood donors (170) and in pig veterinarians (83). Archival sera were collected in Italy in 2004. The observed seroprevalence was 9.64% and 8.82% in veterinarians and blood donors, respectively. Overall, only three sera from blood donors were positive for IgM, but no HEV-RNA was detected.

Presence of Hepatitis E Virus in a RED Deer (Cervus elaphus) Population in Central Italy.

Hepatitis E is an acute human disease caused by the hepatitis E virus (HEV). In addition to humans, HEV has been detected in several animal species and is recognized as a zoonotic pathogen. Pigs, wild boar and deer can be reservoir. In this study, we evaluated HEV prevalence in a free-living red deer (Cervus elaphus) population in central Italy by detecting virus-specific antibodies and RNA in sera. A total of 35 of 251 red deer sera were positive for anti-HEV IgG. HEV RNA was detected in 10 of 91 sera examined. Two genomic fragments targeted by diagnostic PCRs in the capsid region were sequenced, both matching with genotype 3 HEV. Overall results confirmed the occurrence of HEV infection in deer also in Italy.

Oral immunoglobulins for treatment of acute rotaviral gastroenteritis.

OBJECTIVE: Preliminary evidence has been reported on the antirotavirus effect of human serum immunoglobulin administered orally. The aim was to see whether such treatment might be effective in rotavirus acute gastroenteritis. METHODS: A prospective, double-blind, placebo-controlled study was performed. Ninety-eight children admitted with acute gastroenteritis were enrolled and randomly assigned to groups A (treated) and B (control). Children in group A received a single oral dose of 300 mg/kg body weight of human serum immunoglobulin. Parameters of efficacy were clinical condition, frequency and consistency of stools, duration of diarrhea, duration of viral excretion, and length of hospital stay. Antirotaviral activity was determined in the immunoglobulin preparation by a specific neutralization assay. RESULTS: Seventy-one of the 98 children enrolled had rotaviral gastroenteritis; 36 belonged to group A. Children who received immunoglobulin had significantly faster clinical improvement of clinical condition and stool pattern than control children. Mean total duration of rotaviral diarrhea was 76 hours in group A and 131 in group B (P < .01). Viral excretion lasted 114 and 180 hours, respectively (P < .01). Hospital stay was significantly reduced in children in group A. Neutralizing antibodies against rotavirus were detected in the immunoglobulin preparation. CONCLUSION: Oral administration of immunoglobulin is associated with a faster recovery from acute gastroenteritis and should be given to children hospitalized with this illness.

Measles epidemiology in Italy.

In preparation for a mass vaccination programme, the immune status with regard to measles was determined in over 8300 unvaccinated children aged 0-13 years, residing in eight Italian cities with different socioeconomic situations and geographical locations. The age corresponding to the 50% prevalence of immunes appeared to be intermediate (2.9-5.5 years) between that reported for industrialized (6-7 years) and developing countries (1-2 years). The 50% prevalence of natural immunity was reached at an earlier age in southern cities in which poorer socioeconomic and hygienic conditions prevailed; the earlier occurrence of measles in these areas was confirmed by a more detailed serological study of children in the first 24 months of life. For children aged 2-13 years, serological results showed that the history of measles reported by parents on questionnaires gave high positive predictive values (over 85%). Our seroepidemiological study shows that, on the basis of the ages of 25 and 75% prevalence of immunes, the target population for a mass immunization programme in Italy can be assumed to be aged from 12 months to 7 years. However, special attention should be given to the poorest areas, especially in southern Italy, where measles occurs earlier and can be particularly severe.

Cytoskeletal changes induced in HEp-2 cells by the cytotoxic necrotizing factor of Escherichia coli.

The effect of the cytotoxic necrotizing factor of Escherichia coli on HEp-2 cells was studied by fluorescence and scanning electron microscopy. This cytotoxin, known for inducing the formation of giant multinucleated cells in several cell lines, caused changes in actin and tubulin organization. The presence of membrane ruffles at the cell border and of numerous thick bundles of actin crossing the cell body, suggests that the factor promotes cell spreading; this probably interferes with cytokinesis, ultimately leading to the formation of very large flattened multinucleated cells. Moreover, the nuclear segmentation observed in treated cells seems to be associated with a rearrangement of actin in the perinuclear region and with the presence of tubulin bundles in proximity to nuclear clefts. Although the primary target is still unknown, these findings suggest that the cytoskeleton is affected accounting for the multinucleation process induced by the factor.

Rotavirus infection among children with diarrhoea in Italy.

Despite the absence of a nationwide surveillance system for rotavirus infection, relevant information concerning the epidemiology of this pathogen in Italy can be obtained from hospital-based studies carried out since the early 1980s on patients with acute diarrhoea. A review of more than 50 papers and congress proceedings published in both international and national literature indicates that rotavirus is the most important cause of diarrhoea in Italy among young children requiring hospitalization, with a prevalence ranging from approximately 20% to 40% in different studies. Infection is predominant among children aged 6-24 months, although cases are also common in younger children and in children 2-3 y of age. Despite differences among studies in geographical area, years and age group under investigation, an increase in rotavirus cases is consistently reported in the winter months, with a peak in February through April. Although a few studies have been conducted in non-hospitalized patients, rotavirus infection is significantly less frequent among outpatients with enteritis than among inpatients. Most circulating rotavirus strains typed from 1981 to 1992 belong to serotype 1 and, to a lesser extent, 4. However, untypable rotavirus strains have been found in these years, with prevalences up to 27%, suggesting a possible spread of non-serotype 1 through 4 strains.

Molecular characterization of human G8P[4] rotavirus strains in Italy: proposal of a more complete subclassification of the G8 genotype in three major lineages.

In 2011, two children with acute rotavirus gastroenteritis were hospitalized in Sardinia, Italy. Two RVA strains with G8P[4] genotype were detected in their stools, and were named SS56/2011 and SS65/2011. The aim of the study was to characterize these two rare strains, collected within a national RVA gastroenteritis surveillance program. Eight of the 11 RVA genes were sequenced and phylogenetic analysis performed. VP7 amino acid sequence was also analyzed. Sequencing of genes encoding the VP4, VP6, VP7, and NSP1-5 proteins classified both strains as G8-P[4]-I2-A2-N2-T2-E2-H2, not detected previously in Italy. Phylogenetic analysis revealed that most genes of Italian RVA strains were closely similar to typical DS-1 like strains circulating worldwide, whereas the VP7 gene was strictly related to G8 strains firstly reported in Africa. This finding of G8P[4] RVA strains with a DS-1 like genomic constellation also in a southern European country further confirms the wide circulation of this uncommon genotype in the world. Comparison of the deduced amino acid sequence of the VP7 capsid protein of the Italian G8P[4] RVA strains with sequences reported previously suggests that the G8 genotype should be divided into three major lineages.

Genetic diversity of G9P[8] rotavirus strains circulating in Italy in 2007 and 2010 as determined by whole genome sequencing.

This study reports the molecular characterization of G9P[8] rotavirus strains from children with acute diarrhea identified in different cities of Italy, in 2007 and 2010. Seventeen samples exhibited a G9P[8] genotype by RT-PCR and semi-nested PCR. Preliminary sequence analysis of the VP7 and VP8(*) encoding genes revealed nucleotide identities ranging between 96% and 100%. Full genome sequencing of four G9P[8] strains selected in different cities or years showed that the investigated Italian strains possessed a complete Wa-like genotype constellation. However, phylogenetic analyses assigned strains to different clusters reflecting point mutations and possibly earlier reassortment between Wa-like RVA strains. Deduced amino acid sequence of the VP7 and VP4 genes for the G9P[8] strains revealed at least five substitutions in relevant antigenic sites of both proteins.