Search

BackgroundThe introduction of the prion Real-Time Quaking-Induced Conversion assay (RT-QuIC) has led to a revision of the diagnostic criteria for sporadic Creutzfeldt-Jakob disease (sCJD).Validation studies are needed for the amended criteria, especially for their diagnostic value in the clinical setting.MethodsWe studied 1250 patients with suspected CJD referred for diagnosis to two Italian reference centres between 2010 and 2020. Focusing on the first diagnostic assessment, we compared the diagnostic value of the old and the amended criteria and that of different combinations of clinical variables and biomarker results.ResultsThe studied cohort comprised 850 participants with CJD (297 definite sCJD, 151 genetic CJD, 402 probable sCJD) and 400 with non-CJD (61 with neuropathology). At first clinical evaluation, the sensitivity of the old criteria (76.8%) was significantly lower than that of the amended criteria (97.8%) in the definite CJD cohort with no difference between definite and probable sCJD cases. Specificity was ~94% for both criteria against the non-CJD cohort (82.0% against definite non-CJD group). Cerebrospinal fluid (CSF) RT-QuIC was highly sensitive (93.9%) and fully specific against definite non-CJD patients. Limiting the criteria to a positive RT-QuIC or/and the combination of a clinical course compatible with possible CJD with a positive MRI (Q-CM criteria) provided higher diagnostic accuracy than both the old and amended criteria, overcoming the suboptimal specificity of ancillary test results (ie, CSF protein 14-3-3).ConclusionsCSF RT-QuIC is highly sensitive and specific for diagnosing CJD in vitam. The Q-CM criteria provide a high diagnostic value for CJD.

Genetic Creutzfeldt–Jakob disease (gCJD) associated with the V180I mutation in the prion protein (PrP) gene (PRNP) in phase with residue 129M is the most frequent cause of gCJD in East Asia, whereas it is quite uncommon in Caucasians. We report on a gCJD patient with the rare V180I-129V haplotype, showing an unusually long duration of the disease and a characteristic pathological PrP (PrPSc) glycotype. Family members carrying the mutation were fully asymptomatic, as commonly observed with this mutation. Neuropathological examination showed a lesion pattern corresponding to that commonly reported in Japanese V180I cases with vacuolization and gliosis of the cerebral cortexes, olfactory areas, hippocampus and amygdala. PrP was deposited with a punctate, synaptic-like pattern in the cerebral cortex, amygdala and olfactory tract. Western blot analyses of proteinase-K-resistant PrP showed the characteristic two-banding pattern of V180I gCJD, composed of mono- and un-glycosylated isoforms. In line with reports on other V180I cases in the literature, Real-Time Quaking Induced Conversion (RT-QuIC) analyses did not demonstrate the presence of seeding activity in the cerebrospinal fluid and olfactory mucosa, suggesting that this haplotype also may result in a reduced seeding efficiency of the pathological PrP. Further studies are required to understand the origin, penetrance, disease phenotype and transmissibility of 180I-129V haplotype in Caucasians.

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.

Background At the end of the 1970s, in Italy more than 2% of the general population was HBsAg carrier. In the late ‘70s and late ‘80s, two remarkable events might have impacted on HBV strains transmitted in North-East Italy: (a) the increased HBV incidence due to parenteral drugs between 1978 and 1982; (b) the preventive anti-HIV educational campaign, started locally in 1985. Methods To address if those events impacted on circulating HBV variants, acute cases occurred in North-East Italy in 1978–79 (n = 50) and 1994–95 (n = 30) were retrospectively analysed. HBV sequences obtained from serum samples were subjected to phylogenetic analysis and search for BCP/pre-core and S mutations. Results HBV-D was the most prevalent genotype in both 1978–79 (43/50, 86%) and 1994–95 (24/30, 80.0%), with HBV-A in all but one remaining cases. Among HBV-D cases, sub-genotype HBV-D3 was the most prevalent (25/29, 86.2% in 1978–79; 13/16, 81.2% in 1994–95), with HBV-D1 and HBV-D2 in the remaining cases. All HBV-A cases were sub-genotype A2. Single and multiple BCP/pre-core mutations, responsible for HBeAg(−) hepatitis, were detected in 6/50 (12%) cases in 1978/79 vs. 12/30 (40.0%) in 1994/95 (p = 0.006). They were found exclusively in HBV-D; in the most abundant sub-genotype, HBV-D3, they were detected in 2/25 (8%) cases in 1978–79 vs. 6/13 (46%) in 1994–95 (p = 0.011). No vaccine escape S mutations were observed. The IDU risk factor was significantly more frequent in 1994–95 (8/30, 26.7%) than in 1978–79 (4/50, 8%) (p = 0.048). Conclusions The above mentioned epidemiological and public health events did not affect the proportion of genotypes and sub-genotypes that remained unchanged over 16 years. In contrast, the proportion of BCP/pre-core mutants increased more than three-fold, mostly in HBV-D3, a sub-genotype highly circulating in IDUs; drug abuse likely contributed to the spread of these mutants. The findings contribute to explain a previously described major change in HBV epidemiology in Italy: the proportion of HBeAg(−) cases in the carrier cohort changed from low in late 1970s, to high at the beginning of the 2000s. In addition to other recognized factors, the increased circulation of BCP/pre-core mutants likely represents a further factor that contributed to this change.

Background: On the analogy of the non-pathogenic microbiota found in oral cavity, skin and gastrointestinal tract, existence of blood microbiota was confirmed by DNA sequencing, but never deeply characterized. Hypothesis for the existence of dormant blood microbiota in healthy humans have been arisen and single species have been isolated. The aim of our study was to resuscitate and investigate the biodiversity of bacterial and fungal dormant blood microbiota in healthy individuals by blood culturing and NGS DNA sequencing. Results: Twenty eight blood samples of healthy individuals, seven for each blood type, were studied. Several culture media were tested. Blood microbiota resuscitation was performed in BHI broth supplemented with vitamin K 1 mg/ml, 2% sucrose, 0.25% sodium citrate and 0.2% yeastolate at 43?C for 72 h. All tested blood samples were culture positive, as confirmed by Gram staining and TEM. TEM images demonstrated well defined cell structures. Analysis for bacterial and eukaryotic species was performed by 16S rRNA and ITS2 targeted sequencing. The obtained sequences were clustered (≥97% identity) in Operational Taxonomic Units (OTUs). Among cultured and uncultured samples we identified OTUs similarity with 47 bacterial orders belonging to 15 phyla and 39 fungi orders blonging to 2 phyla. For the first time we demonstrated isolation and sequencing identification of fungal blood microbiota in healthy individuals. Blood-group differences were identified among the bacterial microbiome compositions. Conclusion: The dormant blood microbiome is innate of the healthy individuals. Interventional strategies to bind the host blood microbiome with the states of health and disease remain an unmet research goal.

Noroviruses (NoV) are a major cause of gastroenteritis worldwide. Recently, a novel variant of NoV GII.17 (GII.P17_GII.17 NoV), termed Kawasaki 2014, has been increasingly reported in NoV outbreaks in Asia, and has also been described in Europe and North America. In this study, sewage samples were investigated to study the occurrence and genetic diversity of NoV genogroup II (GII) along a 6-year period. Moreover, the spread of GII.17 strains (first appearance and occurrence along time) was specifically assessed. A total of 122 sewage samples collected from 2011 to 2016 from four wastewater treatment plants in Rome (Italy) were initially tested using real-time RT-(q)PCR for GII NoV. Positive samples were subsequently subjected to genotypic characterization by RT-nested PCRs using broad-range primes targeting the region C of the capsid gene of GII NoV, and specific primers targeting the same region of GII.17 NoV. In total, eight different genotypes were detected with the broad-range assay: GII.1 (n = 6), GII.2 (n = 8), GII.3 (n = 3), GII.4 (n = 13), GII.6 (n = 3), GII.7 (n = 2), GII.13 (n = 2), and GII.17 (n = 3), with the latter two genotypes detected only in 2016. Specific amplification of GII.17 NoV was successful in 14 out of 110 positive samples, spanned over the years 2013-2016. The amplicons of the broad-range PCR, pooled per year, were further analyzed by next-generation sequencing (NGS) for a deeper analysis of the genotypes circulating in the study period. NGS confirmed the circulation of GII.17 NoV since 2013 and detected, beyond the eight genotypes identified by Sanger sequencing, three additional genotypes regarded as globally uncommon: GII.5, GII.16, and GII.21. This study provides evidence that GII.17 NoV Kawasaki has been circulating in the Italian population before its appearance and identification in clinical cases, and has become a major genotype in 2016. Our results confirm the usefulness of wastewater surveillance coupled with NGS to study the molecular epidemiology of NoV and to monitor the emergence of NoV strains.

Human adenoviruses (HAdVs) are of major public health importance and are associated with a variety of clinical manifestations, including gastroenteritis, respiratory, ocular and urinary tract infections. To study the occurrence, prevalence and diversity of HAdV species and types circulating in Italy, we conducted a large-scale molecular-epidemiological investigation, a yearlong monitoring of 22 wastewater treatment plants, covering 10 Italian regions, representative of northern, central, and southern Italy. A total of 141 raw sewage samples were collected from January to December 2013, and processed to detect and characterize by phylogenetic analysis a fragment of the hexon coding region of HAdVs. Nested PCR results showed the presence of HAdVs in 85 out of 141 samples (60% of samples). Fifty-nine samples were characterized by conventional Sanger sequencing as belonging to four HAdV species and four types: A (type 12, 5 samples), B (type 3, 8 samples), C (type 5, 1 sample) and F (type 41, 45 samples). The remaining 26 samples could not be characterized because of uninterpretable (mixed) electropherograms suggesting the presence of multiple species and/or types. Pools of characterized and uncharacterized PCR amplicons were further analyzed by next-generation sequencing (NGS). NGS results revealed a marked HAdV diversity with 16 additional types detected beyond the four types found by Sanger sequencing. Overall, 19 types were identified, belonging to HAdV species A-F: types 12 and 31 (species A), type 3 (species B), types 1, 2, and 5 (species C), types 9, 17, 24, 26, 37, 38, 42, 44, 48, and 70 (species D), type 4 (species E), and types 40 and 41(species F). An untypeable HAdV was also detected, showing similar percentages of identity with more than one prototype (types 15, 30, 56, and 59). Our findings documented the circulation of a wide variety of species and types in raw sewage, potentially able to affect other surface water environments and hence human health. Next-generation sequencing proved to be an effective strategy for HAdV genotyping in wastewater samples. It was able to detect a wide range of "less prevalent" types unidentified by conventional Sanger sequencing, confirming that studies based on conventional technologies may grossly underestimate the existence of some, possibly less common, types. Knowledge of the distribution of HAdV species and types would improve our understanding of waterborne HAdV-related health risks.

Purpose. In 2013/2014, Italy experienced one of the largest community-wide prolonged outbreaks of hepatitis A virus (HAV) throughout the country. The article provides a comprehensive description of the outbreak and the investigation carried out by a multidisciplinary National Task Force, in collaboration with regional and local public health authorities. Control strategies of food-borne HAV infection in both the human and food sectors are also described. Methodology. Enhanced human epidemiological and microbiological surveillance together with microbiological monitoring of HAV in food and trace-back investigation were conducted. Results. A total of 1803 HAV cases were identified from 1 January 2013 to 31 August 2014, in Italy. Sequencing was possible for 368 cases (20.4 %), mostly collected between 1 January 2013 and 28 February 2014, and 246 cases (66.8 %) harboured an HAV outbreak strain. Imported frozen berries contaminated with HAV were identified as the vehicle of the outbreak which also involved many other European countries in 2013 and 2014. Epidemiological evidence obtained through a case–control study was supported by the finding of a 100 % nucleotide similarity of the VP1/2A sequences of HAVs detected in human and food samples. Trace-back investigation revealed an extremely complex supplying network with no possibility for a point source potentially explaining the vast contamination of berries found in Italy. Conclusion. The investigation benefited from an excellent collaboration among different sectors who shared proactively the available information. Our findings highlight the importance of considering frozen berries among the highest risk factors for HAV.

BACKGROUND Evaluation of clinical performance of the anti-hepatitis C virus (HCV) rapid tests were carried out mostly in chronic hepatitis C patients and in individuals at high risk of HCV infection. METHODS The aim of this study was to evaluate the performance of OraQuick and Wantai rapid tests on archived serum samples from 1408 individuals (mean age 46, range 18-90; 65% female) recruited with a systematic sampling procedure during a general population survey. RESULTS The analysis of samples by Ortho HCV 3.0 ELISA and Cobas Taqman HCV RNA assays resulted in 69 anti-HCV antibody positive sera, including 42 HCV RNA positive (group 1) and 27 HCV RNA negative (group 2) samples. The performance of rapid tests was evaluated on the 69 anti-HCV positive (group 1+2) and 206 (OraQuick) and 198 (Wantai) anti-HCV negative sera, randomly selected from the 1339 anti-HCV negative samples. The OraQuick and Wantai rapid assays showed a sensitivity in group 1 of 92.9% and 90.5%, respectively. The sensitivity in group 2 was 40.7% and 51.9%, respectively. The anti-HCV antibodies signal/cutoff mean value was the only parameter that statistically differed between group 1 and group 2 individuals (P

From April to October 2017, 27 cases of Hepatitis A (HA), 22 male and 5 female, were reported in Cosenza (South Italy). The median age of cases was 32 years (range 3-49 years). Out of 21 male adults, 14 were identified as men who have sex with men (MSM). Phylogenetic analysis was conducted in 15 cases and revealed two distinct sequences of genotype IA linking to clusters recognised in MSM in other European countries in 2016; genotype IB was recognized in only 2 cases. The report confirms that HA is an emerging issue among MSM. As suggested by the WHO, in countries with low HAV circulation, vaccination programmes should be tailored on local epidemiological patterns to prevent outbreaks among high risk groups and eventual spill-over of the infection into the general population.

Hepatitis C virus subtype 1b (HCV1b) is still the most prevalent subtype worldwide, with massive expansion due to poor health care standards, such as blood transfusion and iatrogenic procedures. Despite safe and effective new direct antiviral agents (DAA), treatment success can depend on resistance-associated substitutions (RASs) carried in target genomic regions. Herein we investigated transmission clusters and RASs among isolates from HCV1b positive subjects in the Calabria Region. Forty-one NS5B and twenty-two NS5A sequences were obtained by Sanger sequencing. Phylogenetic analysis was performed using the maximum likelihood method and resistance substitutions were analyzed with the Geno2pheno tool. Phylogenetic analysis showed sixteen statistically supported clusters, with twelve containing Italian sequences mixed with foreign HCV1b isolates and four monophyletic clusters including only sequences from Calabria. Interestingly, HCV1b spread has been maintained by sporadic infections in geographically limited areas and by dental treatment or surgical intervention in the metropolitan area. The L159F NS5B RAS was found in 15 isolates and in particular 8/15 also showed the C316N substitution. The Y93H and L31M NS5A RASs were detected in three and one isolates, respectively. The A92T NS5A RAS was found in one isolate. Overall, frequencies of detected NS5B and NS5A RASs were 36.6% and 22.7%, respectively. For the eradication of infection, improved screening policies should be considered and the prevalence of natural RASs carried on viral strains.

Hepatitis B virus (HBV) is the main cause of diseases liver related infecting more than 200 milion persons worldwide. HBV infection shows high level of prevalence in South-East Europe and in Mediterranean basin. In Tunisia, a country with an intermediate level endemicity, HbsAg prevalence ranges from 2 to 5%. Most of the HBV isolates from Tunisia were classified as subgenotype D7 whose circulation is restricted to a specific area of North Africa including Maghreb region. In this paper, the phylogeny of HBV-D7 isolated from 38 Tunisian patients was investigated by analyzing the S gene region of HBV. A Bayesian coalescent-based framework was used to estimate the origin of the HBV-D7 in the country. The Tunisian D7 isolates were found to share a common ancestor whose origin was traced back to 1958. Population dynamics indicated that HBV-D7 epidemic in Tunisia grew exponentially from 1960s to 1990s. After that, the curve reached a plateau around the years 2000 likely due to the implementation of the infant vaccination program in 1996. Epidemiological data suggested that the exponential growth phase was likely sustained by intra-familial transmission events occurring during infancy. Further characterization of HBV-D7 isolates should be performed to evaluate, in the post-vaccination era, the emergence of new transmission routes, and to monitor the efficacy of the vaccination program. J. Med. Virol. 89:469-475, 2017. © 2016 Wiley Periodicals, Inc.

In Tunisia, the prevalence of naturally occurring surface (S) gene variants of hepatitis B virus (HBV) has not been determined. In the present study, the prevalence of these variants was examined in terms of the clinical and viral state in a series of 99 Tunisian patients with HBV infection. The S genes were amplified and directly sequenced. Genotype D was predominant (98%), 40.4% isolates belonged to subgenotypes D7 and 1 to subgenotype D2. The most common subtype was ayw2 (95.9%). In total, 60.6% of the studied strains harbored S mutations. Several novel mutation patterns were detected. Interestingly, the presence of S mutations was significantly correlated with the D7 subgenotype, low HBV DNA and advancing age (≥35 years), and tended to be higher in liver cirrhosis than in chronic infection. The global prevalence of the major hydrophilic region variants was 12.1%, with substitution S143L/T as the most frequent (4%). Only 33.9% of S substitutions produced amino acid changes in the polymerase gene. In conclusion, a high prevalence of naturally occurring HBsAg variants was observed among Tunisian HBV carriers. Natural viral variability in a geographical region and duration of infection are among the major factors associated with the occurrence of S mutations.

Background Hepatitis E virus (HEV) genotype 3 has a worldwide distribution. The food-borne transmission of HEV associated with the consumption of products derived from domestic pig, wild boar has been reported in various countries. In this study the genetic diversity, evolutionary rates of HEV 3f, 3c among swine and wild boar in Italy were estimated. Methods Sampling was performed on a wild boar population living in an area located in Abruzzo region. The HEV RNA amplification was performed by real-time RT-PCR. Nested RT-PCR and sequencing of the ORF2 region were carried out by the Super Script III First-Strand Synthesis System. Sequencing of purified PCR products was carried out by the Genome Lab Dye Terminator Cycle Sequencing (DTCS) Quick Start Kit. The maximum likelihood trees were generated by using Phyml. The mean evolutionary rates and the dated trees were co-estimated by BEAST. Results The phylogenetic analysis showed that the HEV ORF2 isolates from Abruzzo region belonged to 3f subtype. The prevalent subtypes in Italy were those belonging to 3f and 3c. The estimated mean values of the HEV ORF2 capsid gene evolutionary rates were 1.915 × 10−2 substitutions/site/year (95% HPD: 1.64 × 10−3 – 3.97 × 10−2) and 2.81 × 10−2 substitutions/site/year (1.83 × 10−2 - 3.8 × 10−2) for 3f and 3c subtype datasets, respectively. The HEV 3f dated back to 1985 (1960–2000), whereas the 3c subtype entered in Italy during the year 2006 (2005–2006). The majority of the HEV 3f sequences collected from swine didn't appear intermixed, except in two cases. The HEV 3c population circulating in Italy remained segregated without significant transfer to swine. Conclusion Our study provide insight into the evolution, circulation of HEV 3f and 3c in Italy. Continued genomic surveillance of HEV in animal reservoir, as well as improving sanitary control measures are required., Bioinformatics; Microbiology; Epidemiology; Hepatitis E virus; Molecular evolution; Phylogeny; Animal reservoir; Genomic surveillance.

Sequence analysis of HPV16 isolates reveals the presence of genome variants with characteristic mutations. The HPV16 variants have different geographical distribution and diverge into four phylogenetic lineages (A, B, C and D) and 16 sub-lineages: A1, A2, A3 (previously known as European variants), A4 (Asian variant), B1, B2, B3, B4, C1, C2, C3, and C4 (African variants), D1 (North-American variant), D2, D3 (Asian-American variants) and D4. Population studies showed that infections with viruses belonging to specific HPV16 sublineages confer different risks of viral persistence and cancer. In this study, 39 HPV16-positive cervical smears from European women living in Calabria (Italy) were analyzed for the presence of HPV16 variants. Cervical DNA extracts were processed by PCR to amplify L1, the Long Control Region (LCR), E6 and E7, which were sequenced. The sequences were concatenated and the 3169 nucleotides long fragments were characterized by BLAST and phylogenetic analysis. A total of 96 Single Nucleotide Polymorphism (SNPs) were detected, 29 of which mapping in the L1, 45 in the LCR, 15 in the E6 and 7 in the E7. The most common SNP was the T350G (29/39 samples, 74.4%), causing the L83 V amino acid change in the E6. Most of the HPV16 isolates (89.7%) had 99% of nucleotide (nt) identity to members of the A1 and A2 sublineages, while 4 isolates had 99% nt identity to members of the B2, B4, C1 and D4 sublineages. In conclusion, viruses belonging to the A1, A2, B2, B4, C1 and D4 HPV16 sublineages were found to circulate in the Calabria region.

In Europe, autochthonous hepatitis E virus (HEV) infection is mainly a foodborne zoonosis, but it is also transmitted by blood transfusion. Despite the numerous prevalence surveys, only a few studies have investigated HEV incidence. We aimed to determine HEV incidence and risk factors among blood donors in a hyperendemic area in Central Italy. Of 296 blood donors who had tested HEV negative in two previous seroprevalence surveys in L'Aquila, 198 agreed to undergo at least another blood sampling for estimating HEV incidence nearly 2 years after the prevalence surveys. Ten newly acquired infections were detected, yielding an overall incidence of 2.1/100 person-years (95%CI: 1.0-3.9), with an estimated participant's cumulative probability of becoming HEV infected of 6.5% (95%CI: 3.5-12.0) at 4 years after enrolment. Seven newly infected blood donors were IgG positive only, two were IgM positive (one also IgG positive) and one was HEV RNA positive only, harbouring subtype 3c. Incident infection was most strongly associated with eating game meat, raw-dried pork liver sausage and raw-dried wild boar sausage. None of these exposures was statistically significant, even if eating raw-dried wild boar sausage approached significance (P = 0.06). The HEV incidence we found was considerable compared with other similar studies. The nearly significant association of incident infection with wild boar and other game meat consumption was in agreement with the 3c subtype isolation in the viremic donor. However, beyond eating habits, also other exposure sources are likely important in hyperendemic areas, where incidence and risk exposure studies need to be undertaken for effectively preventing HEV transmission.

Supplemental Digital Content is available in the text, The purpose of this study was to analyze sequences of hepatitis A virus (HAV) Ia and Ib genotypes from Bulgarian patients to investigate the molecular epidemiology of HAV genotype I during the years 2012 to 2014. Around 105 serum samples were collected by the Department of Virology of the National Center of Infectious and Parasitic Diseases in Bulgaria. The sequenced region encompassed the VP1/2A region of HAV genome. The sequences obtained from the samples were 103. For the phylogenetic analyses, 5 datasets were built to investigate the viral gene in/out flow among distinct HAV subpopulations in different geographic areas and to build a Bayesian dated tree, Bayesian phylogenetic and migration pattern analyses were performed. HAV Ib Bulgarian sequences mostly grouped into a single clade. This indicates that the Bulgarian epidemic is partially compartmentalized. It originated from a limited number of viruses and then spread through fecal-oral local transmission. HAV Ia Bulgarian sequences were intermixed with European sequences, suggesting that an Ia epidemic is not restricted to Bulgaria but can affect other European countries. The time-scaled phylogeny reconstruction showed the root of the tree dating in 2008 for genotype Ib and in 1999 for genotype Ia with a second epidemic entrance in 2003. The Bayesian skyline plot for genotype Ib showed a slow but continuous growth, sustained by fecal-oral route transmission. For genotype Ia, there was an exponential growth followed by a plateau, which suggests better infection control. Bidirectional viral flow for Ib genotype, involving different Bulgarian areas, was observed, whereas a unidirectional flow from Sofia to Ihtiman for genotype Ia was highlighted, suggesting the fecal-oral transmission route for Ia.

Hepatitis E virus (HEV) infection in Bulgaria is endemic, as demonstrated by the seroprevalence of antibody against the virus in the general population and by the high prevalence of clinical cases registered. In this study, a deep Bayesian phylogenetic analysis has been performed to provide information on the genetic diversity and the spread of HEV genotypes in Bulgaria. Three different dataset of HEV virus were built for genotyping by the maximum likelihood method, for evolutionary rate estimate by Bayesian Markov Chain Monte Carlo (MCMC) approach, for demographic history investigation and for selective pressure analysis. The evolutionary rate for genotype 3e, was 3,51 x10-3 substitution/site/year (95% HPD: 1,45 x10-3 - 5,75 x10-3 ). The root of the tMRCA of the Bayesian Maximum clade credibility (MCC) tree of HEV 3e genotype corresponded to 1965 (HPD 95% 1949 - 1994). The Bulgarian sequences mainly clustered in a main clade (clade A). The monophyletic clade included all Bulgarian genotype 3e sequences. The demographic history showed a slight growth from 1995 to 2000, followed by a sort of bottleneck in 2010s, a peak in 2011 and a new growth to 2015. Selection pressure analysis did not show sites under positive pressure but Sixty-four statistically significant sites under negative selection. Molecular epidemiological surveillance by Bayesian phylogeny of HEV virus can contributes to trace the way of human infection after contact with swine source directly or heating meat improving public health control. This article is protected by copyright. All rights reserved.

Several studies have reported the detection of hepatitis A (HAV) and E (HEV) virus in sewage waters, indicating a possibility of contamination of aquatic environments. The objective of the present study was to assess the occurrence of HAV and HEV in different water environments, following the route of contamination from raw sewage through treated effluent to the surface waters receiving wastewater discharges . Bivalve molluscan shellfish samples were also analyzed, as sentinel of marine pollution. Samples were tested by RT-PCR nested type in the VP1/2A junction for HAV, and in the ORF1 and ORF2 regions for HEV. Hepatitis A RNA was detected in 12 water samples: 7/21 (33.3%) raw sewage samples, 3/21 (14.3%) treated sewage samples, and 2/27 (7.4%) river water samples. Five sequences were classified as genotype IA, while the remaining 7 sequences belonged to genotype IB. In bivalves, HAV was detected in 13/56 samples (23.2%), 12 genotype IB and one genotype IA. Whether the presence of HAV in the matrices tested indicates the potential for waterborne and foodborne transmission is unknown, since infectivity of the virus was not demonstrated. HEV was detected in one raw sewage sample and in one river sample, both belonging to genotype 3. Sequences were similar to sequences detected previously in Italy in patients with autochthonous HEV (no travel history) and in animals (swine). To our knowledge, this is the first detection of HEV in river waters in Italy, suggesting that surface water can be a potential source for exposure .

Background Hepatitis A virus (HAV) infection is endemic in Eastern European and Balkan region countries. In 2012, Bulgaria showed the highest rate (67.13 cases per 100,000) in Europe. Nevertheless, HAV genotypes and strains circulating in this country have never been described. The present study reports the molecular characterization of HAV from 105 patients from Bulgaria. Methods Anti-HAV IgM positive serum samples collected in 2012–2014 from different towns and villages in Bulgaria were analysed by nested RT-PCR, sequencing of the VP1/2A region and phylogenetic analysis; the results were analysed together with patient and geographical data. Results Phylogenetic analysis revealed two main sequence groups corresponding to the IA (78/105, 74%) and IB (27/105, 26%) sub-genotypes. In the IA group, a major and a minor cluster were observed (62 and 16 sequences, respectively). Most sequences from the major cluster (44/62, 71%) belonged to either of two strains, termed "strain 1" and "strain 2", differing only for a single specific nucleotide; the remaining sequences (18/62, 29%) showed few (1 to 4) nucleotide variations respect to strain 1 and 2. Strain 2 is identical to the strain previously responsible for an outbreak in the Czech Republic in 2008 and a large multi-country European outbreak caused by contaminated mixed frozen berries in 2013. Most sequences of the IA minor cluster and the IB group were detected in large/medium centers (LMCs). Overall, sequences from the IA major cluster were more frequent in small centers (SCs), but strain 1 and strain 2 showed an opposite relative frequency in SCs and LMCs (strain 1 more frequent in SCs, strain 2 in LMCs). Conclusions Genotype IA predominated in Bulgaria in 2012–2014 and phylogenetic analysis identified a major cluster of highly related or identical IA sequences, representing 59% of the analysed cases; these isolates were mostly detected in SCs, in which HAV shows higher endemicity than in LMCs. The distribution of viral sequences suggests the existence of some differences between the transmission routes in SCs and LMCs. Molecular characterization of an increased number of isolates from Bulgaria, regularly collected over time, will be useful to explore specific transmission routes and plan appropriate preventing measures.

Carbapenems resistant Enterobacteriaceae infections are increasing worldwide representing an emerging public health problem. The application of phylogenetic and phylodynamic analyses to bacterial whole genome sequencing (WGS) data have become essential in the epidemiological surveillance of multi-drug resistant nosocomial pathogens. Between January 2012 and February 2013, twenty-one multi-drug resistant K. pneumoniae strains, were collected from patients hospitalized among different wards of the University Hospital Campus Bio-Medico. Epidemiological contact tracing of patients and Bayesian phylogenetic analysis of bacterial WGS data were used to investigate the evolution and spatial dispersion of K. pneumoniae in support of hospital infection control. The epidemic curve of incident K. pneumoniae cases showed a bimodal distribution of cases with two peaks separated by 46 days between November 2012 and January 2013. The time-scaled phylogeny suggested that K. pneumoniae strains isolated during the study period may have been introduced into the hospital setting as early as 2007. Moreover, the phylogeny showed two different epidemic introductions in 2008 and 2009. Bayesian genomic epidemiology is a powerful tool that promises to improve the surveillance and control of multi-drug resistant pathogens in an effort to develop effective infection prevention in healthcare settings or constant strains reintroduction.

In Italy, the incidence of hepatitis A has progressively declined over the last 30 years, though not homogeneously throughout the country. In Campania, Southern Italy, high annual incidence rates have been reported and several periodic outbreaks have occurred. To investigate the phylogenetic and epidemiologic relationships among HAV strains circulating in Campania over the period 1997-2015, 87 hepatitis A cases were investigated. The most frequent risk factor was the consumption of raw/undercooked shellfish (75/87, 86.2%). During 1997-2002 most viral strains were subtype IA (16/23, 70%); the phylogenetic pattern suggests that the incidence peaks observed in 2000-2001 had likely been caused by multiple strains. During a large 2004 outbreak, almost all viral variants were subtype IB (38/41, 93%); most of them (22/38, 58%) were recognized to be one of two main strains (differing for just a single nucleotide), the remaining sequences were strictly related variants. In 2014/2015, only IA strains were observed; two phylogenetically related but distinct strains were responsible, respectively, for a small cluster in 2014 and an outbreak in 2015. In each outbreak, several strains unrelated to those responsible for most cases were detected in a minority of patients, documenting a background of sporadic cases occurring even in the course of outbreaks; some of them proved to be identical to strains detected 11-14 years previously. Overall, the data suggest that several related and unrelated HAV strains have endemically circulated over the last 15 years in Campania, with some strains gaining epidemic transmission likely because of a local combination of multiple factors, including inadequate waste water purification and dietary habits.

The original version of this article unfortunately contained a mistake. The presentation of Table 1 was incorrect. The corrected table is given below. The original article has been corrected.

Hepatitis B virus (HBV), is the leading cause of liver diseases infecting an estimated 240 million persons worldwide. The HBV prevalence rates are variables between different countries, with an high level of endemicity in the south-eastern part of Europe. Seven main HBV-D subgenotypes have been described until now (D1–D7). Turkey, seems to have played an important role in the penetration of HBV-D1 in the Mediterranean area. The importance of Turkey in the European epidemiology of HBV is also suggested by the observation that the highest spread of HBV infection in the Continent are reported in Turkey with Romania, Bulgaria, Greece, Albania and some southern regions of Italy. In this paper the molecular epidemiology and the epidemiological history of HBV-D in Turkey was studied, by characterizing 34 new Turkish isolates and performing a phylogeographic reconstruction. By using a phylodynamic and phylogeographic Bayesian approach, the analysis suggested that HBV-D1 originated in Turkey about in the early 1940s. The large prevalence of D1 in comparison to the other subgenotypes in Turkey confirms the importance of this Country as epidemiological reservoir of HBV-D1 dispersion. The phylogeny suggests that after each initial introduction of the virus in a specific population, separate transmission clusters have been evolving along independent phylogenetic lineages. Better characterization and continuous monitoring of such groups are going to be crucial to understand in detail the epidemiology of HBV-D1 subgenotype in Turkey and to assess the efficacy of prevention, vaccination and therapy in controlling the epidemic. J. Med. Virol. 86:109–116, 2014. © 2013 Wiley Periodicals, Inc.

Gerstmann-Straussler-Scheinker syndrome (GSS) is a dominantly inherited disorder belonging to the group of transmissible human spongiform encephalopathies or prion diseases. Several families affected by GSS with patients carrying mutations in the prion protein gene have been described worldwide. We report clinical, genealogical, neuropathology and molecular study results from two members of the first Argentine kindred affected by GSS. Both family members presented a frontotemporal-like syndrome, one with and the other without ataxia, with different lesions on neuropathology. A Pro to Leu point mutation at codon 102 (P102L) of the prion protein gene was detected in one of the subjects studied. The pathogenic basis of phenotypic variability observed in this family remains unclear, but resembles that observed in other P102L GSS patients from the same family.

Hepatitis A virus is a widely occurring disease, with different prevalence rates between countries in the North and West and those in the South and East. In Italy endemicity is low/medium, but not homogeneously distributed: in the northern/central regions a large hepatitis A outbreak due to genotype IA, related to the consumption of contaminated mixed frozen berries, occurred between 2013 and 2014, whereas in southern Italian regions recurrent outbreaks of hepatitis A, due to the IB genotype, still result from consumption of raw seafood. In 2014 an uncommon genotype IA strain was isolated from five patients (2 adults and 3 children) with hepatitis A, living in the surroundings of Naples (Campania) who did not have any of the most common risk factors for hepatitis A in Italy, such as consumption of raw shellfish or frozen berries, or travel to endemic countries. Moreover, based on the analysis of viral sequences obtained, this strain differed from several others in the national database, which had been recently isolated during Italian outbreaks. This case report reinforces the need to implement both information campaigns about the prevention of hepatitis A and vaccination programmes in childhood; in addition, it would be suitable to sequence strains routinely not only during large outbreaks of hepatitis A in order to obtain a more detailed national database of HAV strains circulating in Italy.

Hepatitis E virus (HEV) is an emergent causative agent of acute hepatitis, transmitted by fecal-oral route. Infection with HEV is a global cause for morbidity and mortality throughout the world: it mainly causes large outbreaks in endemic areas and sporadic autochthonous cases in industrialized countries where HEV infections seem to be an emergent zoonotic disease. Infection of porcine livestock and its relationship with the human cases have been demonstrated. The present study describes an investigation on the prevalence and diversity of HEV in pig slurry in Italy. Slurry samples (24) were collected from ten farms located in North Italy during 2015 and analyzed for HEV, using four broad-range nested PCR assays targeting ORF1 (MTase), ORF2 (capsid) genes, and ORF2/3 regions. Overall, 18 samples (75%) were positive for HEV RNA, and characterized as genotype 3. Nine samples could be subtyped by ORF2 sequencing: Eight belonged to subtype 3f, while one sequence could not be characterized by blast analysis and phylogenetic analysis and may actually represent a new subtype. Furthermore, similarity of 99% was found between 3f Italian HEV sequences of human and swine origins. Real-Time PCR assay was also performed, in order to obtain quantitative data on positive samples. Two swine slurry samples were positive, containing 600 and 1000 UI per mL of sewage. The results of this study show that HEV strains belonging to zoonotic genotype 3 are widely present in swine excreta, and have high degree of identity with strains detected in autochthonous HEV cases. Improving swine farming operations safety and increasing operators' awareness of the zoonotic potential connected with the handling of swine effluents turn out to be key points in order to reduce the environmental and sanitary problem represented by the possible dissemination of HEV to water bodies.

Hepatitis B virus (HBV) is the main cause of diseases liver related infecting more than 200 milion persons worldwide. HBV infection shows high level of prevalence in South-East Europe and in Mediterranean basin. In Tunisia, a country with an intermediate level endemicity, HbsAg prevalence ranges from 2 to 5%. Most of the HBV isolates from Tunisia were classified as subgenotype D7 whose circulation is restricted to a specific area of North Africa including Maghreb region. In this paper, the phylogeny of HBV-D7 isolated from 38 Tunisian patients was investigated by analyzing the S gene region of HBV. A Bayesian coalescent-based framework was used to estimate the origin of the HBV-D7 in the country. The Tunisian D7 isolates were found to share a common ancestor whose origin was traced back to 1958. Population dynamics indicated that HBV-D7 epidemic in Tunisia grew exponentially from 1960s to 1990s. After that, the curve reached a plateau around the years 2000 likely due to the implementation of the infant vaccination program in 1996. Epidemiological data suggested that the exponential growth phase was likely sustained by intra-familial transmission events occurring during infancy. Further characterization of HBV-D7 isolates should be performed to evaluate, in the post-vaccination era, the emergence of new transmission routes, and to monitor the efficacy of the vaccination program. J. Med. Virol. 89:469-475, 2017. © 2016 Wiley Periodicals, Inc.

Hepatitis C Virus (HCV) genotype 4 predominates in Middle East and Central Africa countries. Recently, it has become also prevalent in Southern European countries where it is thought to have been introduced through immigration and the movement of intravenous drug users. In Italy, the prevalence of genotype 4 is particularly high (4.5%) in Southern regions, such as Calabria, and reaches values of 8.4% in specific areas where there appears to be endemic circulation of this genotype. In the present study, the phylogeny of HCV subtype 4d isolated from 19 Italian patients in Calabria was investigated by analysing a fragment of the NS5B viral genomic region. A Bayesian coalescent-based framework was used to estimate origin and spread of the HCV 4d in this area. The mean evolutionary rate HCV 4d NS5B sequences was estimated using a dataset of sequences sampled at known times and a relaxed clock constant model that best fitted the data. By using a Bayesian coalescent method, the Italian 4d isolates collected in Calabria were found to share a common ancestor with reference 4d isolates whose origin was traced back to 1940s. The genotype 4d epidemic in Southern Italy was maintained in a steady non-expanding phase until the late 1970s after that it grew exponentially up to 1990s probably sustained by the vast increase of unsafe blood transfusions and the spread of illicit intravenous drug users. J. Med. Virol. 84:1613–1619, 2012. © 2012 Wiley Periodicals, Inc.

Worldwide, 12.5% of the more than 170 million people infected by hepatitis C virus (HCV), live in Eastern Mediterranean countries. In Turkey, the prevalence of HCV infection ranges from 0.3% to 0.4% of the general population. We investigated the distribution of HCV subtype 1b in Turkey by analysing the NS5b viral genomic region, using a Bayesian coalescent-based framework and phylogeographical analysis to estimate the origin of the HCV 1b subtype epidemic and the genetic diversification of the virus in Turkey. The dataset consisted of 24 NS5b sequences obtained from patients chronically infected with HCV subtype 1b admitted to the different health districts of Ankara hospital plus the reference sequences for phylogenetic analysis. An independent dataset including the same 342-nt NS5b fragment from all over the world (203 sequences) was used to calibrate the evolutionary rate. Using the relaxed clock model, we estimated a mean evolutionary rate of 0.84 × 10 −3 sub/site/year (95% highest posterior density interval HPD 0.16–1.5 × 10 −3 ). The results of the phylogeographical analysis suggested that the HCV epidemic probably originated in Greece during the first decade of 1900 and, a few years later (in the 1920s or 1930s), successfully spread to neighboring countries such as Turkey and Cyprus. The clustering of the majority of the Turkish strains in a single monophyletic group suggests the subsequent segregated circulation of the virus in the country during the years 1940–1999, which was probably due to unsafe medical parenteral procedures, with drug addiction playing a relatively negligible role. The Bayesian skyline plot (BSP) showed a growth in the number of effective infections between the 1940s and the 1990s, when the curve reached a plateau that still remains today, suggesting a partial success of improved transfusional policies. A coalescent-based approach to population dynamics can improve our understanding of the origin and spread of epidemics in a limited geographical area.

Virus-like particles (VLPs) are excellent tools for vaccines against pathogens and tumors. They can accommodate foreign polypeptides whose incorporation efficiency and immunogenicity however decrease strongly with the increase of their size. We recently described the CD8(+) T cell immune response against a small foreign antigen (i.e., the 98 amino acid long human papilloma virus E7 protein) incorporated in human immunodeficiency virus (HIV)-1 based VLPs as product of fusion with an HIV-1 Nef mutant (Nef(mut)). Here, we extended our previous investigations by testing the antigenic/immunogenic properties of Nef(mut)-based VLPs incorporating much larger heterologous products, i.e., human hepatitis C virus (HCV) NS3 and influenza virus NP proteins, which are composed of 630 and 498 amino acids, respectively. We observed a remarkable cross-presentation of HCV NS3 in dendritic cells challenged with Nef(mut)-NS3 VLPs, as detected using a NS3 specific CD8(+) T cell clone as well as PBMCs from HCV infected patients. On the other hand, when injected in mice, Nef(mut)-NP VLPs elicited strong anti-NP CD8(+) T cell and CTL immune responses. In addition, we revealed the ability of Nef(mut) incorporated in VLPs to activate and mature primary human immature dendritic cells (iDCs). This phenomenon correlated with the activation of Src tyrosine kinase-related intracellular signaling, and can be transmitted from VLP-challenged to bystander iDCs. Overall, these results prove that Nef(mut)-based VLPs represent a rather flexible platform for the design of innovative CD8(+) T cell vaccines.

The ability to detect HIV-2 and to discriminate between HIV-1 and HIV-2 infections was evaluated in 46 serum samples from Guinea-Bissau (GB) and Guinea-Conakry (GC) using serological tests and commercial (HIV-1) and in-house (HIV-2) real-time PCR assays. Samples were first identified as HIV-2 positive by Genie I/II assay in GB and GC. HIV positivity was detected in 44 of 46 samples by all screening and confirmatory assays. A diagnostic strategy based on Inno-LIA and HIV-1/2 RNA detection assays allowed accurate discrimination between HIV-1 and HIV-2 in 84% of single infections and confirmed 32% of double infections. In samples with double reactivity in the Inno-LIA test and no detection of both genomes, cross-reactivity likely hampered the identification of true double infections. In conclusion, the implementation of a diagnostic strategy, based on multiple specific serological tests and highly sensitive quantitative PCR assays, is recommended to ensure accurate HIV-2 diagnosis and appropriate therapy for individuals from areas in which the virus is endemic.

Background In industrialized areas of the world, including Europe, Hepatitis E Virus (HEV) is considered an emerging pathogen. In fact, autochthonous cases caused by HEV genotype 3 (HEV-3) are increasingly reported. Several studies described the human HEV-3 subtypes and strains circulating in West Europe countries; in contrast, very little is known about the HEV strains responsible for acute hepatitis E in countries of East Europe/Balkans, such as Bulgaria. Methods and findings Anti-HEV IgM positive serum samples (n = 103) from acute hepatitis cases (2013-2015) from all over Bulgaria were analysed for HEV RNA by Real-Time PCR. Viremia was detected in 90/103 samples. A fragment of the viral genome (ORF-2 region) was amplified by nested PCR from 76/90 viremic samples, leading to a sequence in 64 of them. Genotyping by phylogenetic analysis with standard reference sequences showed HEV-1 in 1/64 cases, HEV-3 in 63/64. Subtyping of HEV-3 sequences showed 3e (39/63, 62%), 3f (n = 15/63, 24%) and 3c (n = 8/63, 13%) subtypes; in one case the sequence subtype was uncertain and classified as 3hi. In the phylogenetic tree, most 3e sequences grouped in two well distinct clusters (A and B), each one with very low intragroup genetic distances. In contrast, 3f and 3c were interspersed with reference sequences and showed lower tendency to cluster and/or higher intragroup distances. Geographically, while 3f and 3c were scattered throughout the country, 3e was restricted to the South-West area, with most cases in two towns about 40 kilometres apart from each other. Conclusions Most acute hepatitis E cases in Bulgaria are caused by HEV-3, subtypes 3e, 3f and 3c. Circulation of 3e appears quite different from 3f and 3c, with 3e restricted to the South-West area while 3f and 3c diffused over the country. The factors underlying the observed molecular and geographical differences remain to be investigated.

Microbiological indicators such as Escherichia coli and Enterococci are commonly used to detect fecal pollution and presence of other pathogens, in the monitoring programs of sanitary surveillance of freshwater ecosystems. Clostridium perfringens (spores) is suggested as an alternative fecal indicator, it provides evidence of historical fecal contamination. This work aims at assessing the genetic variability of the anaerobic microbial community in river sediments and its relation to different fecal pollution sources. This can be obtained through a combined approach involving microbiological and molecular (16S rRNA) tools of isolated bacteria. The study was conducted on the river systems Foglia, Tevere, Astura, and Sitzerri located in three Italian regions. The frequencies of observed haplotypes proved to be remarkably different in the four sampling areas. The phylogenetic relationships among the species obtained from the comparison of the different genetic sequences made it possible to identify six homoge...

Hepatitis C virus (HCV) proteins are known to interfere at several levels with both innate and adaptive responses of the host. A key target in these effects is the interferon (IFN) signaling pathway. While the effects of nonstructural proteins are well established, the role of structural proteins remains controversial. We investigated the effect of HCV structural proteins on the expression of interferon regulatory factor 1 (IRF-1), a secondary transcription factor of the IFN system responsible for inducing several key antiviral and immunomodulatory genes. We found substantial inhibition of IRF-1 expression in cells expressing the entire HCV replicon. Suppression of IRF-1 synthesis was mainly mediated by the core structural protein and occurred at the transcriptional level. The core protein in turn exerted a transcriptional repression of several interferon-stimulated genes, targets of IRF-1, including interleukin-15 (IL-15), IL-12, and low-molecular-mass polypeptide 2. These data recapitulate in a unifying mechanism, i.e., repression of IRF-1 expression, many previously described pathogenetic effects of HCV core protein and suggest that HCV core-induced IRF-1 repression may play a pivotal role in establishing persistent infection by dampening an effective immune response.

Background The detection of baseline resistance mutations to new direct-acting antivirals (DAAs) in HCV chronically infected treatment-naïve patients could be important for their management and outcome prevision. In this study, we investigated the presence of mutations, which have been previously reported to be associated with resistance to DAAs in HCV polymerase (NS5B) and HCV protease (NS3) regions, in sera of treatment-naïve patients. Findings HCV RNA from 152 naïve patients (84 % Italian and 16 % immigrants from various countries) infected with different HCV genotypes (21,1a; 21, 1b; 2, 2a; 60, 2c; 22, 3a; 25, 4d and 1, 4k) was evaluated for sequence analysis. Amplification and sequencing of fragments in the NS5B (nt 8256–8640) and NS3 (nt 3420–3960) regions of HCV genome were carried out for 152 and 28 patients, respectively. The polymorphism C316N/H in NS5B region, associated with resistance to sofosbuvir, was detected in 9 of the 21 (43 %) analysed sequences from genotype 1b-infected patients. Naturally occurring mutations V36L, and M175L in the NS3 protease region were observed in 100 % of patients infected with subtype 2c and 4. Conclusion A relevant proportion of treatment naïve genotype 1b infected patients evaluated in this study harboured N316 polymorphism and might poorly respond to sofosbuvir treatment. As sofosbuvir has been approved for treatment of HCV chronic infection in USA and Europe including Italy, pre-treatment testing for N316 polymorphism on genotype 1b naïve patients should be considered for this drug.