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.

Vitamin D: a steroid hormone with progesterone-like activity.

OBJECTIVE: This review is aimed at demonstrating the progesterone-like activity exerted by the active form of vitamin D, or calcitriol (1,25(OH)2D). To achieve this outcome, we compared the effects in vivo and in vitro exerted by progesterone and vitamin D, with a special focus on the female reproductive system and pregnancy. MATERIALS AND METHODS: This is a literature review of the most important articles published in English on vitamin D as a hormone, mainly found by MEDLINE. Furthermore, a section of our review contains some unpublished data, concerning the analysis in silico of the similarities between the steric structure of progesterone and calcitriol, based on the availability of the experimental structures of progesterone and vitamin D3 receptors in complex with their physiological ligands in the RCSB Protein Data Bank. RESULTS: Vitamin D was shown to exert many physiological activities during the very early stages of gestation in perfect synchrony with progesterone. Both the molecules mutually help and reinforce the activity exerted by each one. A little bit later than progesterone is released, vitamin D secretion rises, but only if pregnancy occurs. Calcitriol contributes to prepare the endometrium to be receptive. Moreover, it supports the implantation process and the course of pregnancy through different but similar pathways to those used by progesterone, giving rise to a significant synergy of action. It is increasingly evident that vitamin D gives an essential support from the luteal phase onwards. CONCLUSIONS: Based on the evidence displayed in this review we may define appropriately vitamin D as a steroid hormone with progesterone-like activity.

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.

Predictors of Hepatitis B Surface Antigen Titers two decades after vaccination in a cohort of students and post-graduates of the Medical School at the University of Palermo, Italy.

Introduction and objective. The introduction of a vaccine against hepatitis B virus (HBV) for newborn babies in Italy in 1991, extended to 12-year-old children for the first 12 years of application, has been a major achievement in terms of the prevention of HBV infection. The objective of this study was to analyse the long-term immunogenicity and effectiveness of HBV vaccination among healthcare students with different working seniorities. Materials and method. A cross-sectional observational study of undergraduate and postgraduate students attending the Medical School of the University of Palermo was conducted from January 2014 - July 2016. HBV serum markers were performed with commercial chemiluminescence assays. Categorical variables were analyzed using the chi-square test (Mantel-Haenszel), whereas means were compared by using the Student's t test. Adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were also calculated by a multivariable logistic regression, using a model constructed to examine predictors of anti-HBs titer above 10 mIU/mL, assumed as protective. Results. Of the 2,114 subjects evaluated - all vaccinated at infancy or at the age of 12 years and were HBsAg/anti-HBc negative - 806 (38.1%) had an anti-HBs titre <10 IU/L. The latter were younger, more likely to be attending a healthcare profession school (i.e., nursing and midwifery), than a medical postgraduate level school, and more likely to have been vaccinated in infancy (p <0.001, 95% CI 2.63-5.26, adjusted OR 3.70). Conclusion. The results of the study suggest that assessment of HBV serum markers in workers potentially exposed to hospital infections is useful for identifying small numbers of unvaccinated subjects, or vaccinated subjects with low antibody titre, all of whom should be referred to a booster series of vaccinations.

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.

N-acetylcysteine as powerful molecule to destroy bacterial biofilms. A systematic review.

OBJECTIVE: Biofilms are microbial communities consisting of bacteria, extremely capable to self-reproduce on biological surfaces, causing infections. Frequently, these biofilms are resistant to classical antibacterial treatments and host immune response. Thus, new adjuvant molecules are mandatory in clinical practice. N-acetylcysteine (NAC), a precursor to the antioxidant glutathione, has been investigated for its effectiveness both in inhibiting biofilm formation and in destroying developed biofilms. The aim of our study was to conduct a systematic literature review of clinical trials involving NAC as adjuvant treatment to eradicate pre-formed mature biofilms and to inhibit new biofilm production. MATERIALS AND METHODS: A careful analysis of the Medline was conducted and eight studies were selected according to the following criteria: site of infection, kind of bacteria, design of the research, dose of the treatment, administration, biological effects and results. We fixed an arbitrary scale of scores from 0 (lowest score) to 5 (highest score) for each criterion and a threshold value of 3. RESULTS: The studies analyzed, with score over 3, suggested a potential role for NAC as adjuvant molecule in the treatment of bacterial biofilms, with an excellent safety and efficacy profile. NAC, in combination with different antibiotics, significantly promoted their permeability to the deepest layers of the biofilm, overcoming the problem of the resistance to the classic antibacterial therapeutic approach. CONCLUSIONS: Overall, these results are encouraging to a more widespread clinical use of NAC, as adjuvant therapy for microbial infections followed by biofilm settle, which may occur in several body districts, such as the vaginal cavity.

Peripheral neuropathy in obstetrics: efficacy and safety of α-lipoic acid supplementation.

OBJECTIVE: Neuropathic pain during pregnancy is a common condition due to the physical changes and compression around pregnancy and childbirth that make pregnant women more prone to develop several medical conditions such as carpal tunnel syndrome, sciatica, meralgia paraesthetica and other nerve entrapment syndromes. Most of the treatments usually performed to counteract neuropathic pain are contraindicated in pregnancy so that, the management of these highly invalidating conditions remains an issue in the clinical practice. We aimed to review the efficacy and safety of alpha lipoic acid supplementation in the treatment of neuropathic pain. DISCUSSION: Lipoic acid is a co-factor essential in the regulation of mitochondrial energy. It has been demonstrated that lipoic acid supplementation is involved in several biochemical processes and actions, exerting important antioxidant and anti-inflammatory activity and significantly improving pain and paraesthesia in patients with sciatica, carpal tunnel syndrome and diabetic neuropathy. CONCLUSIONS: Efficacy of lipoic acid is combined with a high safety profile, making this molecule a novel candidate for the management of several diseases. Data reported so far are promising and dietary supplementation with lipoic acid seems a useful tool to contrast neuropathic pain during pregnancy.

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.

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.

Genetic heterogeneity of porcine enteric caliciviruses identified from diarrhoeic piglets.

Enteric caliciviruses (noroviruses and sapoviruses) are responsible for the majority of non-bacterial gastroenteritis in humans of all age groups. Analysis of the polymerase and capsid genes has provided evidence for a huge genetic diversity, but the understanding of their ecology is limited. In this study, we investigated the presence of porcine enteric caliciviruses in the faeces of piglets with diarrhoea. A total of 209 samples from 118 herds were analysed and calicivirus RNA was detected by RT-PCR in 68 sample (32.5%) and in 46 herds (38.9%), alone or in mixed infection with group A and C rotaviruses. Sequence and phylogenetic analysis of the calicivirus-positive samples characterized the majority as genogroup III (GGIII) sapoviruses. Unclassified caliciviruses, distantly related to the representatives of the other sapovirus genogroups, were identified in five herds, while one outbreak was associated with a porcine sapovirus related genetically to human GGII and GGIV sapovirus strains. By converse, norovirus strains were not detected. Altogether, these data suggest the epidemiological relevance of porcine enteric caliciviruses and suggest a role in the etiology of piglets diarrhoea.

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.

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.