Noemi Urone

Phylogenetic analysis of isolates from new cases of HBV infection in Southern Italy

The level of endemicity of hepatitis B virus (HBV) infections in Italy is low and genotype D infections predominant. New HBV strains may however be introduced as a result of movements of people from regions of high endemicity. The aim of the present study was to determine whether strains from new cases of acute hepatitis B detected in southern Italy were due to endemic or new HBV strains. We studied 34 isolates from patients with acute hepatitis B infection, and 35 from chronic hepatitis B patients. A phylogenetic analysis of preS/S region was done by comparing the sequences from the acute and chronic cases with references sequences. The study showed that 44% of strain from acute hepatitis B patients were of genotype A, 53% of genotype D, and 3% of genotype E. The molecular analysis of isolates from acute hepatitis B patients from Sicily showed a change in the local epidemiology of this infection, with an increase in HBV/A infections and a clustering effect for HBV D2, possibly correlated to immigration. The introduction of new genotypes , could have an effect on HBV-correlated diseases due to the different association between genotype, liver disease and response to antiviral therapy.

Characterization of measles virus strains circulating in Southern Italy (Palermo area, Sicily) between 2010 and 2011

Measles virus (MV) was classified in 24 genotypes that show a distinct geographic distribution. Genotypes contain multiple distinct lineages. In 2011 large outbreaks of measles occurred in Italy and in many European countries. Aims of this study are to analyze the intra-genotype variability and to follow the importation and the spread of new MV strains in Sicily. A fragment of 450 bps of MV C-terminal nucleoprotein was sequenced from sera of 73 Sicilian patients with symptomatic measles infections, occurred between 2010 and 2011. Five MV strains were D4 genotype and 68 were D8 genotype. The MV/D4 sequences were related to MV/D4-Enfield variant. Two lineages of MV/D8 genotypes, related to MV/D8-Villupuram variant and to a strain found in Birmingham in 2006 respectively, were identified. This is the first study that reports the co-circulation of different MV genotypes and lineages in Sicily suggesting multiple origins of the outbreak that occurred during 2010 and 2011 years.

HCV-1b intra-subtype variability: Impact on genetic barrier to protease inhibitors

Due to error-prone RNA polymerase and the lack of proofreading mechanisms, to the spread worldwide and probable long-term presence in human population, HCV showed a high degree of inter- and intra-subtype genetic variability. Protease inhibitors (PIs), a new class of drugs, have been designed specifically on the HCV genotype 1 NS3 protease three-dimensional structure. The viral genetic barrier limits the efficacy of PIs, and fourteen loci in the HCV NS3 gene are involved in resistance to PIs. A sensitive method (15UI/ml) for study the HCV genetic profile of 125 strains from patients naïve to PIs, was developed through the use of new degenerate primers for subtype 1b. We observed the presence of naturally resistance-associated variants in 14% of the HCV strains (V36L, F43S, T54S, I153V, R155Q, D168A/G). T54S was the most common mutation (4%) detected. We investigated, through minimal score (m.s.) calculating, how the HCV intra-subtype 1b variability modifies the genetic barrier to PIs. For >60% of strains a single transition (m.s. of 1) was required for selection of low to medium resistance mutations, while more than one transition/transversion (m.s. ⩾2.5) or one transition plus one transversion (m.s. ⩾3.5) was necessary for most of the high level PI-resistant-associated mutations, except for A156V, for which a single transition was sufficient (m.s. of 1). However, the presence at locus 36 of the amino acid polymorphism S36 in one case and the wild type V36 in 6 isolates, encoded by unusual GTA or GTG codons, might determined a higher probability of V36L/M mutations because of the reduction of the genetic barrier. Instead, the presence of the CGA and CGT codons in the 155(th) position increases the genetic barrier for R155M or R155Q/M. The large intra-subtype variability, suggests that a routine baseline resistance test must be used before PIs-treatment.

Molecular evolutionary analysis of type-1 human astroviruses identifies putative sites under selection pressure on the capsid protein

Human astroviruses (HAstV) are important enteric pathogens that can be classified into eight sero/genotypes (HAstV-1 to -8). Although the various HAstV types show global spread, type-1 strains tend to be predominant. Molecular analysis of the genomic region encoding the capsid protein (ORF2) has revealed discrete sequence variation, with different lineages within each HAstV type and at least three major lineages have been identified within HAstV-1. Longitudinal epidemiological surveillance has revealed temporal shift of the various HAstV-1 lineages. Metadata analysis of HAstV-1 sequences available in the databases also revealed temporal shifts of the circulation of HAstV-1 lineages, suggesting possible antigenic-related mechanisms of selection at the sub-genotype level. By comparison of HAstV-1 capsid sequences, lineage-defining residues under positive selection were identified. Structural analysis of HAstV-1 capsid allowed identifying at least six residues exposed on the virion surface. Two residues were located in the VP34 (shell region) whilst four residues were mapped in the VP25/27 (protruding region) of HAstV capsid protein, in proximity of the putative receptor binding S site. These findings suggest that mechanisms similar to those observed and/or hypothesized for other enteric viruses are also shaping the evolution of HAstVs, with intra-typic diversification being a possible mechanism to decrease the antigenic pressure to which these viruses are exposed.

Is Italian population protected from Poliovirus? Results of a seroprevalence survey in Florence, Italy

ABSTRACT Objectives: Periodical assessments of population susceptibility to polioviruses (PV) is essential for evaluating population protection and planning appropriate vaccination strategies. The aim of the current work was to assess serological protective titers against all three polioviruses in the general population of Florence. Methods: A convenience sample of 328 sera, collected in 2009 in Florence (Central Italy) was analyzed. Samples were considered protective if neutralizing antibodies were detected at dilutions ≥1:8, according to the WHO protocols. Results: The immune coverage was 75.3%, 69.2% and 46% for PV1, PV2 and PV3, respectively. The protective titers of neutralizing antibodies were generally higher in children up to 14 years of age, with 74.4% (PV1), 75.6% (PV2) and 56.7% (PV3) of seroprevalence. From the age of 11 years, most of the study subjects were seronegative for PV3. Conclusions: In a polio-free country with strong migration pressures, such as Italy, our results bring clear support to the recent recommendation of Italian health authorities to introduce a fifth dose of IPV vaccine in adolescence all over the country.

Waterborne Norovirus outbreak at a seaside resort likely originating from municipal water distribution system failure

In May 2016 a Norovirus (NoV) gastroenteritis outbreak involved a high school class visiting a seaside resort near Taormina (Mascali, Sicily). Twenty-four students and a teacher were affected and 17 of them showed symptoms on the second day of the journey, while the others got ill within the following 2 days. Symptoms included vomiting, diarrhoea and fever, and 12 students required hospitalisation. Stool samples tested positive for NoV genome by Real-Time polymerase chain reaction assay in all 25 symptomatic subjects. The GII.P2/GII.2 NoV genotype was linked to the outbreak by ORF1/ORF2 sequence analysis. The epidemiological features of the outbreak were consistent with food/waterborne followed by person-to-person and/or vomit transmission. Food consumed at a shared lunch on the first day of the trip was associated to illness and drinking un-bottled tap water was also considered as a risk factor. The analysis of water samples revealed the presence of bacterial indicators of faecal contamination in the water used in the resort as well as in other areas of the municipal water network, linking the NoV gastroenteritis outbreak to tap water pollution from sewage leakage. From a single water sample, an amplicon whose sequence corresponded to the capsid genotype recovered from patients could be obtained.

Performance evaluation of a newly developed molecular assay for the accurate diagnosis of gastroenteritis associated with norovirus of genogroup II

The performance of a newly proposed fully automated cassette-based sample-to-results solution for norovirus (NoV) detection, InGenius Norovirus ELITe MGB®, was evaluated. A total of 120 selected archival stool samples from children hospitalized for acute gastroenteritis were used to compare the results to a reference real-time RT-PCR. The InGenius NoV assay showed optimal diagnostic accuracy (sensitivity, 100%; specificity, 95.7%) and was able to correctly detect the entire wide panel of epidemiologically relevant genotypes tested. These preliminary results suggest that the InGenius NoV assay can be recommended as a valuable method for accurate diagnosis of NoV GII infection in epidemic and sporadic gastroenteritis.

Performance analysis of two immunochromatographic assays for the diagnosis of rotavirus infection

Group A rotaviruses (RVAs) are the primary cause of acute gastroenteritis (AGE) in young children worldwide. Several commercial tests including latex agglutination, enzyme-linked assays (ELISA) and immunochromatographic tests (ICT) have been developed for the diagnosis of RVA infection. In the present study, the performance of two commercially available one-step chromatographic immunoassays, CerTest Rotavirus+Adenovirus (Biotec S.L, Zaragoza, Spain) and Vikia Rota-Adeno (bioMerieux SA, Lyon, France) were retrospectively evaluated using Real-time PCR as reference test. Re-testing by Real-time PCR of 2096 stool samples of children hospitalized with AGE previously screened by ICTs (1467 by CerTest and 629 by Vikia) allowed to calculate higher sensitivity for Vikia (94% vs 85% of CerTest) and higher specificity for CerTest (93% vs 89% of Vikia). Accordingly, higher Positive Predictive Values (87% vs 78%) and Positive Likelihood Ratios (12.32 vs 8.8) were found for CerTest and lower Negative Predictive Values (91% vs 97%) and Negative Likelihood Ratios (0.16 vs 0.06) for Vikia. However, both CerTest and Vikia showed a substantial agreement (κ=0.79) with the Real-time PCR. A correlation between false negative results by ICTs and high Cycle Threshold values of Real-time PCR, indicative of low viral load, was observed. False positive results by the two ICT assays were not related to Norovirus, Adenovirus or Astrovirus infections, therefore the risk of cross-reactions was excluded. Both CerTest and VIKIA were able to detect the wide range of RVA genotypes circulating over the study period (including G1P[8], G2P[4], G3, G4, G9 and G12P[8]). The results of the present study showed a satisfactory efficacy of the two diagnostic tests analyzed.