Evaluation of the impact of CSF prion RT-QuIC and amended criteria on the clinical diagnosis of Creutzfeldt-Jakob disease: a 10-year study in Italy.

BACKGROUND: The 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. METHODS: We 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. RESULTS: The 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). CONCLUSIONS: CSF RT-QuIC is highly sensitive and specific for diagnosing CJD in vitam. The Q-CM criteria provide a high diagnostic value for CJD.

Biochemical and Neuropathological Findings in a Creutzfeldt-Jakob Disease Patient with the Rare Val180Ile-129Val Haplotype in the Prion Protein Gene.

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.

Retrospective analysis of acute HBV infections occurred in 1978-79 and 1994-95 in North-East Italy: increasing prevalence of BCP/pre-core mutants in sub-genotype D3.

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.

Incidence of hepatitis E virus infection among blood donors in a high endemic area of Central Italy.

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.

Human hepatitis E virus circulation in Bulgaria: Deep Bayesian phylogenetic analysis for viral spread control in the country.

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 data sets of HEV virus was built for genotyping by the maximum likelihood method, for evolutionary rate estimated by Bayesian Markov Chain Monte Carlo approach, for demographic history investigation and for selective pressure analysis. The evolutionary rate for genotype 3e, was 351 × 10-3 substitution/site/year (95% highest posterior density [95% HPD]: 145 × 10 -3 -575 × 10 -3 ). The root of the time to the most recent common ancestor of the Bayesian maximum clade credibility tree of HEV 3e genotype corresponded to 1965 (HPD 95% 1949-1994). The Bulgarian sequences mainly clustered in the 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 64 statistically significant sites under negative selection. Molecular epidemiological surveillance by Bayesian phylogeny of HEV virus can contribute to trace the way of human infection after contact with swine source directly or heating meat improving public health control.

Hepatitis A outbreak affecting men who have sex with men (MSM) in South Italy.

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 A virus strains circulating during 1997-2015 in Campania, a Southern Italy region with periodic outbreaks.

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.

Evolutionary dynamics of HBV-D7 subgenotype in Tunisia.

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.

Cerebrospinal fluid real-time quaking-induced conversion is a robust and reliable test for sporadic creutzfeldt-jakob disease: An international study.

Real-time quaking-induced conversion (RT-QuIC) has been proposed as a sensitive diagnostic test for sporadic Creutzfeldt-Jakob disease; however, before this assay can be introduced into clinical practice, its reliability and reproducibility need to be demonstrated. Two international ring trials were undertaken in which a set of 25 cerebrospinal fluid samples were analyzed by a total of 11 different centers using a range of recombinant prion protein substrates and instrumentation. The results show almost complete concordance between the centers and demonstrate that RT-QuIC is a suitably reliable and robust technique for clinical practice. Ann Neurol 2016;80:160-165.

Hepatitis a virus genotypes and strains from an endemic area of Europe, Bulgaria 2012-2014.

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.

Naturally Occurring Surface Antigen Variants of Hepatitis B Virus in Tunisian Patients.

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.

Naturally occurring mutations associated with resistance to HCV NS5B polymerase and NS3 protease inhibitors in treatment-naïve patients with chronic hepatitis C.

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.

Migration pattern of hepatitis A virus genotype IA in North-Central Tunisia.

BACKGROUND: Hepatitis A virus (HAV) epidemiology in Tunisia has changed from high to intermediate endemicity in the last decades. However, several outbreaks continue to occur. The last reported sequences from Tunisian HAV strains date back to 2006. In order to provide an updated overview of the strains currently circulating in Tunisia, a large-scale molecular analysis of samples from hepatitis A cases was performed, the first in Tunisia. RESULTS: Biological samples were collected from patients with laboratory confirmed hepatitis A: 145 sera samples in Tunis, Monastir, Sousse and Kairouan from 2008 to 2013 and 45 stool samples in Mahdia in 2009. HAV isolates were characterised by nested RT-PCR (VP1/2A region) and sequencing. The sequences finally obtained from 81 samples showed 78 genotype IA and 3 genotype IB isolates. A Tunisian genotype IA sequence dataset, including both the 78 newly obtained IA sequences and 51 sequences retrieved from GenBank, was used for phylogenetic investigation, including analysis of migration pattern among six towns. Virus gene flow from Sfax and Monastir was directed to all other towns; in contrast, the gene flows from Sousse, Tunis, Mahdia and Kairouan were directed to three, two, one and no towns, respectively. CONCLUSIONS: Several different HAV strains co-circulate in Tunisia, but the predominant genotype still continues to be IA (78/81, 96% isolates). A complex gene flow (migration) of HAV genotype IA was observed, with Sfax and Monastir showing gene flows to all other investigated towns. This approach coupled to a wider sampling can prove useful to investigate the factors underlying the spread of HAV in Tunisia and, thus, to implement appropriate preventing measures.

Molecular epidemiology and phylogenetic analysis of Hepatitis B virus in a group of migrants in Italy.

BACKGROUND: Hepatitis B virus infection (HBV) is widespread and it is considered a major health problem worldwide. The global distribution of HBV varies significantly between countries and between regions of the world. Among the many factors contributing to the changing epidemiology of viral hepatitis, the movement of people within and between countries is a potentially important one. In Italy, the number of migrant individuals has been increasing during the past 25 years. HBV genotype D has been found throughout the world, although its highest prevalence is in the Mediterranean area, the Middle East and southern Asia. We describe the molecular epidemiology of HBV in a chronically infected population of migrants (living in Italy), by using the phylogenetic analysis. METHODS: HBV-DNA was amplified and sequenced from 43 HBV chronically infected patients. Phylogenetic and evolutionary analysis were performed using both maximum Likelihood and Bayesian methods. RESULTS AND CONCLUSION: Of the 43 HBV S gene isolates from migrants, 25 (58.1 %) were classified as D genotype. Maximum Likelihood analysis showed an intermixing between Moldavian and foreigners sequences mostly respect to Italian ones. Italian sequences clustered mostly together in a main clade separately from all others. The estimation of the time of the tree's root gave a mean value of 17 years ago, suggesting the origin of the tree back to 1992 year. The skyline plot showed that the number of infections softly increased until the early 2005s, after which reached a plateau. Comparing phylogenetic data to the migrants date of arrival in Italy, it should be possible that migrants arrived in Italy yet infected from their country of origin. In conclusion, this is the first paper where phylogenetic analysis and genetic evolution has been used to characterize HBV sub genotypes D1 circulation in a selected and homogenous group of migrants coming from a restricted area of Balkans and to approximately define the period of infection besides the migration date.

Evolutionary dynamics of HBV-D1 genotype epidemic in Turkey.

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.

Molecular characterisation of human hepatitis E virus from Italy: comparative analysis of five reverse transcription-PCR assays.

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.

Surveillance of hepatitis A virus in urban sewages and comparison with cases notified in the course of an outbreak, Italy 2013.

BACKGROUND: Over the past 20 years, Hepatitis A notifications in Italy have been in decline. Since the beginning of 2013 however, Italy has been experiencing a foodborne hepatitis A outbreak caused by genotype IA, involving hundreds of cases. Consumption of frozen mixed berries was deemed the potential vehicle of infection.We aimed to investigate the spread of hepatitis A virus (HAV) in Italy through the monitoring of urban sewages collected at Wastewater Treatment Plants (WTPs) and a subsequent comparison of environmental surveillance data with data from the clinical surveillance performed during the epidemic. METHODS: The study covered 15 months, from July 2012 to September 2013, comprising the outbreak and the preceding six months. Environmental surveillance consisted of the analysis of urban sewage samples collected at 19 WTPs in seven of the Italian regions most affected by the epidemic. HAV isolates were detected and typed using a nested RT-PCR targeting the VP1/2A junction. Parallel clinical surveillance was performed by the sentinel surveillance system for acute viral hepatitis (SEIEVA) and by the ministerial Central Task Force on Hepatitis A, established with the purpose of determining the source of the outbreak and adopting appropriate outbreak control strategies. RESULTS: A total of 38/157 wastewater samples (24.2%) were positive for HAV, 16 collected in 2012 and 22 in 2013. Several HAV strains were detected, including the IA variant implicated in the outbreak and isolated from clinical cases over the same period. The vast majority of sequences belonged to genotype IB. Interestingly however, although these included variants related to strains that had been involved in past Italian epidemics, none were detected in recent clinical samples, probably due to underreporting or asymptomatic circulation. Conversely, a number of sequences were identified in clinical samples that were not found in wastewaters. CONCLUSIONS: The percentage of sewage samples detected as HAV-positive in this study are consistent with the classification of Italy as a country with low/intermediate endemicity. A combined environmental/clinical surveillance is able to provide a more complete picture of the spread of HAV and of the genotypes circulating in the population, allowing a better understanding of changes in disease trends.

Isolation and Characterisation of Human-Derived blaKPC-3-Producing Salmonella enterica Serovar Rissen in 2018.

In this study, we describe a Salmonella enterica serovar (S.) Rissen strain with a reduced susceptibility to meropenem, isolated from a urinary infection in an 89-year-old woman in 2018 during activity surveillance in Italy (Enter-Net Italia). The genomic characteristics, pathogenicity, and antimicrobial resistance mechanisms were investigated via a genomic approach. Antimicrobial susceptibility testing revealed a "susceptible, increased exposure" phenotype to meropenem in the S. Rissen strain (4_29_19). Whole-genome sequencing (WGS) was performed using both the NovaSeq 6000 S4 PE150 XP platform (Illumina, San Diego, CA, USA) and MinION (Oxford Nanopore). The S. Rissen 4_29_19 strain harboured two plasmids: a pKpQIL-like plasmid carrying the blaKPC-3 resistance gene in a Tn4401a transposon (pKPC_4_29_19), and a ColE-like plasmid (p4_4_29_19) without resistance genes, highly prevalent among Enterobacterales. Comparative analysis revealed that the pKPC_4_29_19 plasmid was highly related to the pKpQIL reference plasmid (GU595196), with 57% coverage and 99.96% identity, but lacking a region of about 30 kb, involving the FIIK2 replicon region and the entire transfer locus, causing the loss of its ability to conjugate. To our knowledge, this is the first time that a pKpQIL-like plasmid, carrying blaKPC-3, highly diffused in Klebsiella pneumoniae strains, has been identified in a Salmonella strain in our country. The acquisition of blaKPC genes by Salmonella spp. is extremely rare, and is reported only sporadically. In zoonotic bacteria isolated from humans, the presence of a carbapenem resistance gene carried by mobile genetic elements, usually described in healthcare-associated infection bacteria, represents an important concern for public health.

Molecular Characterization and Cluster Analysis of SARS-CoV-2 Viral Isolates in Kahramanmaras City, Turkey: The Delta VOC Wave within One Month.

The SARS-CoV-2 pandemic has seriously affected the population in Turkey. Since the beginning, phylogenetic analysis has been necessary to monitor public health measures against COVID-19 disease. In any case, the analysis of spike (S) and nucleocapsid (N) gene mutations was crucial in determining their potential impact on viral spread. We screened S and N regions to detect usual and unusual substitutions, whilst also investigating the clusters among a patient cohort resident in Kahramanmaras city, in a restricted time span. Sequences were obtained by Sanger methods and genotyped by the PANGO Lineage tool. Amino acid substitutions were annotated comparing newly generated sequences to the NC_045512.2 reference sequence. Clusters were defined using phylogenetic analysis with a 70% cut-off. All sequences were classified as Delta. Eight isolates carried unusual mutations on the S protein, some of them located in the S2 key domain. One isolate displayed the unusual L139S on the N protein, while few isolates carried the T24I and A359S N substitutions able to destabilize the protein. Phylogeny identified nine monophyletic clusters. This study provided additional information about SARS-CoV-2 epidemiology in Turkey, suggesting local transmission of infection in the city by several transmission routes, and highlighting the necessity to improve the power of sequencing worldwide.

Characterization of SARS-CoV-2 Variants in Military and Civilian Personnel of an Air Force Airport during Three Pandemic Waves in Italy.

We investigated SARS-CoV-2 variants circulating, from November 2020 to March 2022, among military and civilian personnel at an Air Force airport in Italy in order to classify viral isolates in a potential hotspot for virus spread. Positive samples were subjected to Next-Generation Sequencing (NGS) of the whole viral genome and Sanger sequencing of the spike coding region. Phylogenetic analysis classified viral isolates and traced their evolutionary relationships. Clusters were identified using 70% cut-off. Sequencing methods yielded comparable results in terms of variant classification. In 2020 and 2021, we identified several variants, including B.1.258 (4/67), B.1.177 (9/67), Alpha (B.1.1.7, 9/67), Gamma (P.1.1, 4/67), and Delta (4/67). In 2022, only Omicron and its sub-lineage variants were observed (37/67). SARS-CoV-2 isolates were screened to detect naturally occurring resistance in genomic regions, the target of new therapies, comparing them to the Wuhan Hu-1 reference strain. Interestingly, 2/30 non-Omicron isolates carried the G15S 3CLpro substitution responsible for reduced susceptibility to protease inhibitors. On the other hand, Omicron isolates carried unusual substitutions A1803V, D1809N, and A949T on PLpro, and the D216N on 3CLpro. Finally, the P323L substitution on RdRp coding regions was not associated with the mutational pattern related to polymerase inhibitor resistance. This study highlights the importance of continuous genomic surveillance to monitor SARS-CoV-2 evolution in the general population, as well as in restricted communities.