Performance evaluation of gastrointestinal viral ELIte panel multiplex RT-PCR assay for the diagnosis of rotavirus, adenovirus and astrovirus infection.

Rotavirus, adenovirus, norovirus and astrovirus are considered to be among the major causes of sporadic cases and outbreaks of acute gastroenteritis globally. Rapid and accurate identification of enteric viruses is still a challenge for the clinical laboratory. Recently, several molecular platforms for the detection of viral enteric pathogens have become available. In this study, the diagnostic accuracy of InGenius Gastrointestinal Viral (GV) Elite Panel, a newly developed one-step multiplex real-time RT-PCR assay simultaneously detecting rotavirus, adenovirus and astrovirus, was evaluated retrospectively analyzing an archival collection of 128 stool samples of children hospitalized with acute gastroenteritis. The overall sensitivity and specificity for the GV assay was 100% and 96.2% for rotavirus, 96.9% and 100% for astrovirus, 100% and 100% for adenovirus, respectively. The InGenius GV assay showed a high concordance with the reference methods and was able to detect all tested genotypes of rotavirus (including G1, G3, G4, G9 and G12P[8] and G2P[4]), adenovirus and astrovirus (AstV-1 and 2). Studies of considerable sample size are required to determine robust Cycle threshold cut-off values to effectively correlate infection to disease. These preliminary results suggest that InGenius GV assay can be recommended as a valuable method for accurate diagnosis of epidemic and sporadic gastroenteritis.

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 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.

Introduction and prolonged circulation of G12 rotaviruses in Sicily.

Genotype G12 strains are now considered to be the sixth most prevalent human rotaviruses worldwide. In two Sicilian cities, Palermo and Messina, surveillance of rotavirus circulation performed since 1985 and 2009, respectively, did not detect G12 strains until 2012. From 2012 to 2014 rotavirus infection was detected in 29·7% of 1647 stool samples collected from children admitted for acute gastroenteritis to three Sicilian hospitals in Palermo, Messina and Ragusa. In 2012, G12P[8] was first detected in Palermo and then in Messina where it represented the second most frequent genotype (20% prevalence) after G1P[8]. Thereafter, G12 strains continued to circulate in Sicily, showing a marked prevalence in Ragusa (27·8%) in 2013 and in Palermo (21%) and Messina (16·6%) in 2014. All but one of the Sicilian G12 strains carried a P[8] VP4 genotype, whereas the single non-P[8] rotavirus strain was genotyped as G12P[9]. Phylogenetic analysis of the VP7 and VP4 sequences allowed distinction of several genetic lineages and separation of the G12P[8] strains into three cluster combinations. These findings indicate independent introductions of G12 rotavirus strains in Sicily in recent years.

Full-genome sequencing of a Hungarian canine G3P[3] Rotavirus A strain reveals high genetic relatedness with a historic Italian human strain.

A canine Rotavirus A strain was identified in the fecal specimen of a young dog during 2012 in Hungary. The strain RVA/Dog-wt/HUN/135/2012/G3P[3] shared complete genotype constellation (G3-P[3]-I3-R3-C3-M3-A15-N2-T3-E3-H6) and high genome sequence similarity (nt, 98.8 %) with a historic human strain, RVA/Human-tc/ITA/PA260-97/1997/G3P[3]. This study provides evidence for the canine origin of the unusual NSP1 genotype, A15, and reinforces the hypothesis of direct interspecies transmission of canine rotaviruses to humans.

Analysis of early strains of the norovirus pandemic variant GII.4 Sydney 2012 identifies mutations in adaptive sites of the capsid protein.

Global surveillance for norovirus identified in 2012 the emergence of a novel pandemic GII.4 variant, termed Sydney 2012. In Italy, the novel pandemic variant was identified as early as November 2011 but became predominant only in the winter season 2012-2013. Upon sequencing and comparison with strains of global origin, the early Sydney 2012 strains were found to differ from those spreading in 2012-2013 in the capsid (ORF2) putative epitopes B, C and D, segregating into a distinct phylogenetic clade. At least three residues (333, 340 and 393, in epitopes B, C and D, respectively) of the VP1 varied among Sydney 2012 strains of different clades. These findings suggest that the spread of the pandemic variant in Italy during the winter season 2012-2013 was due to the introduction of strains distinct from those circulating at low frequency in the former winter season and that similar strains were also circulating elsewhere worldwide.

Evidence for recombination between pandemic GII.4 norovirus strains New Orleans 2009 and Sydney 2012.

During 2012, a novel pandemic GII.4 norovirus variant, Sydney 2012, emerged worldwide. A signature of the variant was a GII.Pe ORF1, in association with GII.4 Apeldoorn 2008-like ORF2-ORF3 genes. We report the detection of recombinant GII.4 Sydney 2012 strains, possessing the ORF1 gene of the former pandemic variant New Orleans 2009.

Nationwide surveillance study of human astrovirus infections in an Italian paediatric population.

The study investigated the genetic diversity of human astroviruses (HAstVs) detected in children hospitalized with gastroenteritis in Italy in 2008-2009. A total of 1321 faecal samples were collected in Parma (northern Italy), Bari (southern Italy), and Palermo (Sicily) and screened for the presence of HAstVs. RT-PCR amplification of a portion at the 5'-end of ORF2 allowed the detection of HAstVs in 3·95% of the patients. Four different genotypes (HAstV-1, HAstV-2, HAstV-4, HAstV-5) were found to be circulating during the study period, with HAstV-1 being the predominant type. Interestingly, a novel lineage, proposed as HAstV-2d, was found to have emerged in Parma in 2009. Investigating the genetic variability of HAstVs will be important for understanding the epidemiological trends and evolution of these viruses.

Genotypic Analysis of E. coli Strains Isolated from Patients with Cystitis and Pyelonephritis.

BACKGROUND: Urinary tract infection is the most common health problem affecting millions of people each year, mainly caused by a large genetically heterogeneous group of Escherichia coli called uropathogenic E. coli This study investigates the genotypic analysis of E. coli strains isolated from patients with cystitis and pyelonephritis. METHODS: During 2008-2009, 90 E. coli strains were analyzed, consisting of 48 isolates causing pyelonephritis in children and 42 isolates causing cystitis. Having identified the strains by standard methods, they were subtyped by pulsed field gel electrophoresis (PFGE) and their corresponding patterns were compared using dendrogram. RESULTS: Sixty five PFGE profiles were obtained from the genome of E. coli strains by this genotyping method. Thirty six and thirty three patterns were obtained for pyelonephritis and cystitis, respectively. Most strains exhi-bited twelve and thirteen bands and the patterns with eight or nineteen bands had the lowest rate. Genome sizes of the strains were between 1610-4170 kbp. CONCLUSION: With due attention to these results, genetic patterns showed that the strains had different clonalities and it could be suggested in some cases that the strains causing pyelonephritis or cystitis have common patterns and different diseases could be explained by different gene factors.

Genetic heterogeneity and recombination in human type 2 astroviruses.

Novel lineages of human astrovirus (HAstV) types 2, 2c, and 2d have been identified. Upon sequencing of the 3' end of the genome, the type 2c and 2d HAstVs were found to be open reading frame 1b (ORF1b)-ORF2 recombinant, with ORF1b being derived from type 3 and type 1 HAstVs, respectively. An ORF2 interlineage recombinant strain, 2c/2b, was also identified.

A feline rotavirus G3P[9] carries traces of multiple reassortment events and resembles rare human G3P[9] rotaviruses.

The full-length genome sequence of a feline G3P[9] rotavirus (RV) strain, BA222, identified from the intestinal content of an adult cat, was determined. Strain BA222 possessed a G3-P[9]-I2-R2-C2-M2-A3-N1-T3-E2-H3 genomic constellation, differing substantially from other feline RVs. Phylogenetic analyses of each genome segment revealed common origins with selected animal and zoonotic human RVs, notably with rare multi-reassortant human G3P[9] RVs (Ita/PAI58/96 and Ita/PAH136/96). Altogether, the findings suggest that feline RVs are genetically diverse and that human RVs may occasionally originate either directly or indirectly (via reassortment) from feline RVs.

Surveillance of human astrovirus circulation in Italy 2002-2005: emergence of lineage 2c strains.

By screening faecal samples collected over four consecutive years (2002-2005) from hospitalized children with diarrhoea in Palermo, Italy, astroviruses (HAstVs) were detected in 3.95% of the patients. The predominant type circulating was HAstV-1 but, in 2002, only HAstV-2 and -4 were identified. Interestingly, the HAstVs-2 detected appeared to be consistently different in 5' end of their open reading frame 2 from the previously described subtypes. These novel type 2 strains were included in a new 2c lineage based on the phylogenetic analysis and the presence of nine peculiar substitutions.

Chemical composition and antibacterial potential of Artemisia arborescens L. essential oil.

This study was undertaken to characterize the essential oil (EO) of Artemisia arborescens growing wild in Sicily. EO, extracted by steam distillation, was examined for its chemical composition and for its capability to inhibit some food-borne pathogen bacteria. A total of 43 compounds (13 monoterpene hydrocarbons, 14 oxygenated monoterpenes, 10 sesquiterpene hydrocarbons, three oxygenated sesquiterpenes and less amount of other three compounds), which account 93.73% of the total oil, were identified by gas chromatography and gas chromatography-mass spectrometry. Oxygenated monoterpenes (57.32%) constituted the main fraction, with ß-thujone as the main compound (45.04%), followed by the sesquiterpene hydrocarbon chamazulene (22.71%). Undiluted EO showed a large inhibition spectrum against strains of Listeria monocytogenes (34 out of 44), whilst it was ineffective against enterobacteria and salmonellas. The minimum inhibition concentration (MIC) was evaluated for the two most sensitive strains (L. monocytogenes 186 and 7BO) at two cellular concentrations (10(6) and 10(7) CFU ml(-1)). The lowest MIC (0.625 µl ml(-1), dilution of oil with acetone) was found for strain L. monocytogenes 186 at 10(6) CFU ml(-1).

Rare AU-1-like G3P[9] human rotaviruses with a Kun-like NSP4 gene detected in children with diarrhea in Italy.

Three G3P[9] rotaviruses, detected in children hospitalized with gastroenteritis in Palermo, Italy, were found to be genetically related to strains of either human or feline origin in the VP7, VP4, and VP6 genes. In contrast, in the NSP4 gene the viruses resembled G2P[4] human strains, suggesting a reassortment between AU-1-like and Kun-like strains.

Tonometers and infectious risk: myth or reality? Efficacy of different disinfection regimens on tonometer tips.

PURPOSE: To evaluate the adequacy of common disinfection regimens for disposable tonometer tips and assess if disinfection of reusable prisms or the use of disposable tips is preferable. METHODS: We used disposable tonometer tips, using the same material and tip diameter of standard Goldmann tonometer prism. Strains of Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilisand Candida albicanswere tested according to the European standard guidelines for disinfectants test. Antimicrobial effectiveness of the following disinfection practices has been assessed: dry wipe, Minuten wipes (Alpro), soaking in 3% hydrogen peroxide, 0.5% benzalkonium chloride, and 0.5% Pantasept for 1, 5, and 15 min. All tests have been performed three times and all conditions tested in duplicate. RESULTS: Dry wiping and 1 min soak in 3% hydrogen peroxide were ineffective on all microrganisms. Minuten wipes, 1 min soak in 0.5% benzalkonium chloride or 3% hydrogen peroxide were ineffective on B. subtilis. 0.5% Pantasept soak was effective in 1 min for all microrganisms tested, whereas 3% hydrogen peroxide and 0.5% benzalkonium chloride soaks were effective when performed for at least 5 min. B. subtiliswas the most resistant organism to disinfectant regimes at 1 min time. CONCLUSIONS: Results of our study demonstrate a relative disinfection efficacy for the different evaluated regimens, provided that correct exposure times are adopted for the chosen disinfectants, a condition difficult to ensure in a busy clinic setting. We conclude that disposable prism tonometry provides a safe alternative to Goldmann tonometry.

Diversity of human rotaviruses detected in Sicily, Italy, over a 5-year period (2001-2005).

Rotavirus infection was detected in 39.9% of 1030 children hospitalized with gastroenteritis in Palermo, Italy, in the period 2001-2005. Rotavirus strains belonging to G1, G4 and G9 types were continually detected, with G1 being the most common type in 2001, 2002 and 2004. A G4 epidemic occurred in 2003, while G9 was predominant in 2005. G2 strains displayed a low prevalence, except in 2003. G3 rotaviruses accounted for 2.7-17% of the gastroenteritis episodes in 2002-2005. The P-type of a subset of 166 strains confirmed the circulation of the usual G/P combinations, but single G1P[6], G9P[9] and G6P[9] strains were also found.

Viral gastroenteritis in children hospitalised in Sicily, Italy.

The aim of the present study was to describe the epidemiologic and clinical characteristics of acute viral gastroenteritis in hospitalised Italian children. A total of 215 stool specimens were collected from January to December 2003 from patients hospitalised in Palermo for acute diarrhoea. Samples were tested for group A rotavirus, astrovirus, adenovirus, norovirus, enteropathogenic bacteria, and parasites. Rotaviruses, mostly belonging to types G1-G4, were detected in 25.1% of samples, astrovirus in 7%, adenovirus in 6%, norovirus in 18.6%, and bacterial agents in 17.2%. No parasitic infections were diagnosed. Mixed infections represented 9.8% of all cases. The mean and median ages of children with rotavirus gastroenteritis were lower than those of children with other viruses (p = 0.029), with the highest median ages being found in astrovirus-infected patients. Vomiting and dehydration were more frequent among patients with viral infection (p < 0.01), and the severity score was significantly higher for children infected with astrovirus or group A rotavirus (p = 0.008). Rotavirus was the leading cause of prolonged hospitalisation (p = 0.005). In conclusion, viruses were confirmed in Italy as the most common cause of severe enteric illness in childhood, with rotavirus types G1-G4, which correspond to those included in the rotavirus vaccines being developed, playing the main role. Routine testing should be introduced for noroviruses, since they seem to represent an important cause of sporadic paediatric gastroenteritis.

Genetic variability among serotype G4 Italian human rotaviruses.

A total of 254 serotype GH rotavirus strains were detected in Palermo, Italy, from 1985 to 2003. Out of 38 serotype G4 strains selected for genetic analysis, 14 were recognized by genotyping as type G9. Strains confirmed to belong to the G4 type showed temporal patterns of genetic evolution in their VP7 and VP4 gene sequences, and the latest Italian G4 strains were distantly related to the reference vaccinal ST3 strain.

Molecular epidemiology of astrovirus infection in Italian children with gastroenteritis.

Abstract A 1-year study involving 157 gastroenteritis samples was conducted to investigate the role of human astrovirus (HAstV) as a cause of gastroenteritis in Italian children aged < 2 years. The overall incidence of HAstV was 3.1%. Most cases occurred between March and May, and four of the five isolates were of the HAstV-1 type, the other being HAstV-3. Analysis of genetic variability showed that the three HAstV-1 isolates collected in 2000 clustered together, but separately from the 1999 isolate. The results indicated that HAstV should be considered as a potential diarrhoeal pathogen in Italian children.

Molecular characterization of the genotype G9 human rotavirus strains recovered in Palermo, Italy, during the winter of 1999-2000.

Among the known human rotavirus serotypes, types G1-G4 are ubiquitous and account for >80% of global human rotavirus strains. Since 1994 an increase in reports of G9 serotype isolates has been observed in both developed and developing countries. In the winter season of 1999-2000 we detected the appearance of G9 rotavirus in Palermo, Italy, accounting for 19% of all rotaviruses circulating in our geographical area. Some of these Italian G9 isolates have been submitted to partial sequencing of their VP7 gene. All of them showed complete nucleotide identity suggesting a clonal origin. The Italian VP7 sequences were found to be very closely related to that of other G9 strains recently isolated in Europe, America, Africa and Asia. Our results confirm that G9 strains circulating worldwide since 1994 are closely related genetically in their VP7 genes.