The Laboratory Diagnosis of Malaria: A Focus on the Diagnostic Assays in Non-Endemic Areas.

Even if malaria is rare in Europe, it is a medical emergency and programs for its control should ensure both an early diagnosis and a prompt treatment within 24-48 h from the onset of the symptoms. The increasing number of imported malaria cases as well as the risk of the reintroduction of autochthonous cases encouraged laboratories in non-endemic countries to adopt diagnostic methods/algorithms. Microscopy remains the gold standard, but with limitations. Rapid diagnostic tests have greatly expanded the ability to diagnose malaria for rapid results due to simplicity and low cost, but they lack sensitivity and specificity. PCR-based assays provide more relevant information but need well-trained technicians. As reported in the World Health Organization Global Technical Strategy for Malaria 2016-2030, the development of point-of-care testing is important for the improvement of diagnosis with beneficial consequences for prompt/accurate treatment and for preventing the spread of the disease. Despite their limitations, diagnostic methods contribute to the decline of malaria mortality. Recently, evidence suggested that artificial intelligence could be utilized for assisting pathologists in malaria diagnosis.

Insights into the knowledge of complex diseases: Environmental infectious/toxic agents as potential etiopathogenetic factors of systemic sclerosis.

Systemic sclerosis (SSc) is a connective tissue disease secondary to three cardinal pathological features: immune-system alterations, diffuse microangiopathy, and fibrosis involving the skin and internal organs. The etiology of SSc remains quite obscure; it may encompass multiple host genetic and environmental -infectious/chemical-factors. The present review focused on the potential role of environmental agents in the etiopathogenesis of SSc based on epidemiological, clinical, and laboratory investigations previously published in the world literature. Among infectious agents, some viruses that may persist and reactivate in infected individuals, namely human cytomegalovirus (HCMV), human herpesvirus-6 (HHV-6), and parvovirus B19 (B19V), and retroviruses have been proposed as potential causative agents of SSc. These viruses share a number of biological activities and consequent pathological alterations, such as endothelial dysfunction and/or fibroblast activation. Moreover, the acute worsening of pre-existing interstitial lung involvement observed in SSc patients with symptomatic SARS-CoV-2 infection might suggest a potential role of this virus in the overall disease outcome. A variety of chemical/occupational agents might be regarded as putative etiological factors of SSc. In this setting, the SSc complicating silica dust exposure represents one of the most promising models of study. Considering the complexity of SSc pathogenesis, none of suggested causative factors may explain the appearance of the whole SSc; it is likely that the disease is the result of a multifactorial and multistep pathogenetic process. A variable combination of potential etiological factors may modulate the appearance of different clinical phenotypes detectable in individual scleroderma patients. The in-deep investigations on the SSc etiopathogenesis may provide useful insights in the broad field of human diseases characterized by diffuse microangiopathy or altered fibrogenesis.

Reduction trend of mcr-1 circulation in Emilia-Romagna Region, Italy.

This study aims to describe trends of mcr-positive Enterobacterales in humans based on laboratory surveillance with a defined catchment population. The data source is the Micro-RER surveillance system, established in Emilia-Romagna region (Italy), to monitor the trend of mcr resistance. Enterobacterales isolates from human clinical samples with minimum inhibitory concentration (MIC) ≥ 2 mg/L for colistin were sent to the study reference laboratory for the detection of mcr genes. Isolates prospectively collected in the period 2018-2020 were considered for the assessment of population rates and trends; further analyses were carried out for the evaluation of clonality and horizontal mcr gene transfer. Previous isolates from local laboratory collection were also described. In the period 2018-2020, 1164 isolates were sent to the reference laboratory, and 51 (4.4%) were confirmed as mcr-positive: 50 mcr-1 (42 Escherichia coli, 6 Klebsiella pneumoniae, 2 Salmonella enterica) and 1 mcr-4 (Enterobacter cloacae). The number of mcr-positive isolates dropped from 24 in the first half of 2018 to 3 in the whole of 2020 (trend p value < 0.001). Genomic analyses showed the predominant role of the horizontal transfer of mcr genes through plasmids or dissemination of transposable elements compared to clonal dissemination of mcr-positive microorganisms. The study results demonstrate a substantial decrease in the circulation of mcr-1 plasmid genes in Emilia-Romagna Region.

Impact of Human Cytomegalovirus and Human Herpesvirus 6 Infection on the Expression of Factors Associated with Cell Fibrosis and Apoptosis: Clues for Implication in Systemic Sclerosis Development.

Systemic sclerosis (SSc) is a severe autoimmune disorder characterized by vasculopathy and multi-organ fibrosis; its etiology and pathogenesis are still largely unknown. Herpesvirus infections, particularly by human cytomegalovirus (HCMV) and human herpesvirus 6 (HHV-6), have been suggested among triggers of the disease based on virological and immunological observations. However, the direct impact of HCMV and/or HHV-6 infection on cell fibrosis and apoptosis at the cell microenvironment level has not yet been clarified. Thus, this study aimed to investigate the effects of HCMV and HHV-6 infection on the induction of pro-fibrosis or pro-apoptosis conditions in primary human dermal fibroblasts, one of the relevant SSc target cells. The analysis, performed by microarray in in vitro HCMV- or HHV-6-infected vs. uninfected cells, using specific panels for the detection of the main cellular factors associated with fibrosis or apoptosis, showed that both viruses significantly modified the expression of at least 30 pro-fibrotic and 20 pro-apoptotic factors. Notably, several recognized pro-fibrotic factors were highly induced, and most of them were reported to be involved in vivo in the multifactorial and multistep pathogenic process of SSc, thus suggesting a potential role of both HCMV and HHV-6.

Contribution of the FilmArray® Gastrointestinal Panel in the laboratory diagnosis of gastroenteritis in a cohort of children: a two-year prospective study.

This study represents a 2-year picture of the epidemiology of enteric pathogens in children suffering from gastroenteritis using the FilmArray® Gastrointestinal Panel (FA-GP), a multiplex molecular assay that allows to simultaneously detect a large panel of pathogens independently of the etiological suspicion and to evaluate its potential contribution to the diagnosis compared to the conventional methods. A total of 1716 stool samples, collected from children with clinical suspicion of bacterial and/or viral gastroenteritis attending the University Hospital of Parma, was submitted to the FA-GP and, when an adequate aliquot was available, to electron microscopy (n = 1163) for virus detection and to an enterovirus-targeting real-time PCR (n = 1703). Specimens with positive results for Salmonella, Yersinia enterocolitica, Vibrio, diarrheagenic Escherichia coli/Shigella, Campylobacter, Plesiomonas shigelloides and/or parasites by the FA-GP were also submitted to conventional diagnostic methods. The FA-GP gave positive results in 958 (55.8%) cases, 64.8% from inpatients: 647 (67.5%) contained a single agent and 311 (32.5%) multiple agents, for a total of 1374 pathogens. Enteropathogenic E. coli, rotavirus, norovirus, toxigenic Clostridioides difficile, and sapovirus were the most commonly detected pathogens. A total of 812 additional agents (344 of which as single pathogen) was detected by the FA-GP and not included in the clinical suspicion. The overall recovery rate of the conventional methods from stools that resulted positive by the FA-GP was 38.6% for bacteria, 50% and 84.2% for Giardia intestinalis and Cryptosporidium, respectively, and ranged from 3.7% to 64.6% for viruses, if excluding all electron microscopy-negative astroviruses. Enterovirus, an agent not targeted by the FA-GP, was revealed in 9.6% (164/1703) of the examined samples, and in 52 cases it was the only agent detected. The results of this study allowed to extend the range of detectable pathogens independently of the clinical suspicion, to detect co-infections in almost one third of children positive for at least one agent and to show that conventional methods would have missed more than half of the enteric agents detected by the FA-GP.

Mammalian Diaphanous-related formin-1 restricts early phases of influenza A/NWS/33 virus (H1N1) infection in LLC-MK2 cells by affecting cytoskeleton dynamics.

Viruses depend on cellular machinery to efficiently replicate. The host cytoskeleton is one of the first cellular systems hijacked by viruses in order to ensure their intracellular transport and promote the development of infection. Our previous results demonstrated that stable microfilaments and microtubules interfered with human influenza A/NWS/33 virus (H1N1) infection in semi-permissive LLC-MK2 cells. Although formins play a key role in cytoskeletal remodelling, few studies addressed a possible role of these proteins in development of viral infection. Here, we have demonstrated that mammalian Diaphanous-related formin-1 (mDia1) is involved in the control of cytoskeleton dynamics during human influenza A virus infection. First, by employing cytoskeleton-perturbing drugs, we evidenced a cross-talk occurring between microtubules and microfilaments that also has implications on the intracellular localization of mDia1. In influenza A/NWS/33 virus-infected LLC-MK2 cells, mDia1 showed a highly dynamic intracellular localization and partially co-localized with actin and tubulin. A depletion of mDia1 by RNA-mediated RNA interference was found to improve the outcome of influenza A/NWS/33 virus infection and to increase the dynamics of microfilament and microtubule networks in LLC-MK2 cells. Consistent with these findings, observations made in epithelial respiratory cells from paediatric patients with acute respiratory disease assessed that the expression of mDia1 is stimulated by influenza A virus but not by respiratory syncytial virus. Taken together, the obtained results suggest that mDia1 restricts the initiation of influenza A/NWS/33 virus infection in LLC-MK2 cells by counteracting cytoskeletal dynamics.

High prevalence of malaria in a non-endemic setting: comparison of diagnostic tools and patient outcome during a four-year survey (2013-2017).

BACKGROUND: Malaria is no longer endemic in Italy since 1970 when the World Health Organization declared Italy malaria-free, but it is now the most commonly imported disease. The aim of the study was to analyse the trend of imported malaria cases in Parma, Italy, during January 2013-June 2017, reporting also the treatment and the outcome of cases, exploring the comparison of the three diagnostic tests used for malaria diagnosis: microscopy, immunochromatographic assay (ICT) (BinaxNOW®) and Real-time PCR assays detecting Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale curtisi, Plasmodium ovale wallikeri, and Plasmodium knowlesi. RESULTS: Of the 288 patients with suspected malaria, 87 were positive by microscopy: 73 P. falciparum, 2 P. vivax, 8 P. ovale, 1 P. vivax/P. ovale, 1 P. malariae and 2 Plasmodium sp. All samples were positive by ICT except 6. Plasmodial DNA was revealed in the 87 cases and in 2 additional cases showing P. falciparum-specific bands by ICT, as follows: 75 P. falciparum, 2 P. vivax, 6 P. ovale curtisi, 3 P. ovale wallikeri, 1 P. malariae, and 2 mixed infections. 72 patients were foreigners and 17 Italians travelling for tourism or business. The majority of these patients presented with fever at blood collection and did not have chemoprophylaxis. No fatal cases were observed and the drug mostly used was quinine observing a negative blood smear or a parasitaemia < 0.001% after 48-72 h' therapy. CONCLUSIONS: The study shows an update and a thorough analysis of imported malaria cases in the area of Parma during 4.5 years from the point of view of the total case management, clinical and diagnostic. The prevalence of malaria in such area in the considered period was especially due to immigrants mostly from Africa. Molecular methods were more sensitive and specific than microscopy and ICT, both detecting additional cases of P. falciparum malaria missed by microscopy and correctly identifying the Plasmodium species of medical interest. The data reported in this study may stimulate the clinicians in non-endemic areas to suspect malaria also in cases, where the most typical symptoms are absent, and the parasitologists to confirm the results of microscopy, remaining the reference method, with molecular methods to avoid misdiagnosis.

Emergence of novel recombinant GII.P16_GII.2 and GII. P16_GII.4 Sydney 2012 norovirus strains in Italy, 2016/2017.

In the winter season 2014/15, the GII.P17_GII.17 norovirus strain Kawasaki 2014 emerged in Italy, cocirculating with pandemic GII.4 strains. In March 2016, molecular investigation identified novel GII.P16 recombinant noroviruses in children with gastroenteritis in Italy. In 43.10% of the genotyped noroviruses GII.P16 strains were identified: 12 were characterized as GII.2 and 13 as GII.4 Sydney 2012 capsid genotypes. The GII.P16 genotype became predominant in January- February 2017 along with an increase in norovirus activity. The capsid gene was characterized as GII.2 or GII.4 Sydney 2012 variant. The emergence of two different recombinant GII.P16 viruses, of which one harboring a pandemic GII.4 capsid sequence, suggests the potential for a future pandemic.

Norovirus GII.17 as Major Epidemic Strain in Italy, 2015-16.

In winter 2015-16, norovirus GII.17 Kawasaki 2014 emerged as a cause of sporadic gastroenteritis in children in Italy. Median patient age was higher for those with GII.17 than GII.4 infection (55 vs. 24 months), suggesting limited cross-protection for older children.

Analysis of the full genome of human group C rotaviruses reveals lineage diversification and reassortment.

Group C rotaviruses (RVC) are enteric pathogens of humans and animals. Whole-genome sequences are available only for few RVCs, leaving gaps in our knowledge about their genetic diversity. We determined the full-length genome sequence of two human RVCs (PR2593/2004 and PR713/2012), detected in Italy from hospital-based surveillance for rotavirus infection in 2004 and 2012. In the 11 RNA genomic segments, the two Italian RVCs segregated within separate intra-genotypic lineages showed variation ranging from 1.9 % (VP6) to 15.9 % (VP3) at the nucleotide level. Comprehensive analysis of human RVC sequences available in the databases allowed us to reveal the existence of at least two major genome configurations, defined as type I and type II. Human RVCs of type I were all associated with the M3 VP3 genotype, including the Italian strain PR2593/2004. Conversely, human RVCs of type II were all associated with the M2 VP3 genotype, including the Italian strain PR713/2012. Reassortant RVC strains between these major genome configurations were identified. Although only a few full-genome sequences of human RVCs, mostly of Asian origin, are available, the analysis of human RVC sequences retrieved from the databases indicates that at least two intra-genotypic RVC lineages circulate in European countries. Gathering more sequence data is necessary to develop a standardized genotype and intra-genotypic lineage classification system useful for epidemiological investigations and avoiding confusion in the literature.

Human cytomegalovirus reactivation from latency: validation of a "switch" model in vitro.

BACKGROUND: Human cytomegalovirus (HCMV) is an opportunistic pathogen leading to severe and even fatal diseases in 'at-risk' categories of individuals upon primary infection or the symptomatic reactivation of the endogenous virus. The mechanisms which make the virus able to reactivate from latency are still matter of intense study. However, the very low number of peripheral blood monocytes (an important latent virus reservoir) harbouring HCMV DNA makes it very difficult to obtain adequate viral quantities to use in such studies. Thus, the aim of the present study was to demonstrate the usefulness of human THP-1 monocytes, mostly employed as HCMV latent or lytic infection system, as a reactivation model. METHODS: THP-1 monocytes were infected with HCMV TB40E strain (latency model) at multiplicities of infection (MOI) of 0.5, 0.25 or 0.125. After infection, THP-1 aliquots were differentiated into macrophages (reactivation model). Infections were carried out for 30 h, 4, 6 and 7 days. Viral DNA evaluation was performed with viable and UV-inactivated virus by q-Real-Time PCR. RNA extracted from latency and reactivation models at 7 days post-infection (p.i.) was subjected to RT-PCR to analyse viral latency and lytic transcripts. To perform viral progeny analysis and titration, the culture medium from infected THP-1 latency and reactivation models (7 days p.i.) was used to infect human fibroblasts; it was also checked for the presence of exosomes. For viral progeny analysis experiments, the Towne strain was also used. RESULTS: Our results showed that, while comparable TB40E DNA amounts were present in both latent and reactivation models at 30 h p.i., gradually increased quantities of viral DNA were only evident in the latter model at 4, 6, 7 days p.i.. The completion of the lytic cycle upon reactivation was also proved by the presence of HCMV lytic transcripts and an infectious viral yield at 7 days p.i. CONCLUSIONS: Our data demonstrate the effectiveness of THP-1 cells as a "switch" model for studying the mechanisms that regulate HCMV reactivation from latency. This system is able to provide adequate quantities of cells harbouring latent/reactivated virus, thereby overcoming the intrinsic difficulties connected to the ex vivo system.

MALDI-TOF mass spectrometry as a potential tool for Trichomonas vaginalis identification.

BACKGROUND: Trichomonas vaginalis is a flagellated protozoan causing trichomoniasis, a sexually transmitted human infection, with around 276.4 million new cases estimated by World Health Organization. Culture is the gold standard method for the diagnosis of T. vaginalis infection. Recently, immunochromatographic assays as well as PCR assays for the detection of T. vaginalis antigen or DNA, respectively, have been also available. Although the well-known genome sequence of T. vaginalis has made possible the application of proteomic studies, few data are available about the overall proteomic expression profiling of T. vaginalis. The aim of this study was to investigate the potential application of MALDI-TOF MS as a new tool for the identification of T. vaginalis. METHODS: Twenty-one isolates were analysed by MALDI-TOF MS after the creation of a Main Spectrum Profile (MSP) from a T. vaginalis reference strain (G3) and its subsequent supplementation in the Bruker Daltonics database, not including any profile of protozoa. This was achieved after the development of a new identification method created by modifying the range setting (6-10 kDa) for the MALDI-TOF MS analysis in order to exclude the overlapping of peaks derived from the culture media used in this study. RESULTS: Two MSP reference spectra were created in 2 different range: 3-15 kDa (standard range setting) and 6-10 kDa (new range setting). Both MSP spectra were deposited in the MALDI BioTyper database for further identification of additional T. vaginalis strains. All the 21 strains analysed in this study were correctly identified by using the new identification method. CONCLUSIONS: In this study it was demonstrated that changes in the MALDI-TOF MS standard parameters usually used to identify bacteria and fungi allowed the identification of the protozoan T. vaginalis. This study shows the usefulness of MALDI-TOF MS in the reliable identification of microorganism grown on complex liquid media such as the protozoan T. vaginalis, on the basis of the proteic profile and not on the basis of single markers, by using a "new range setting" different from that developed for bacteria and fungi.

A cluster of Enterovirus 71 subgenogroup C2 in a nursery school, Italy, 2014.

During October 2014, enterovirus (EV) RNA was detected in the stools of four children attending the same class in a nursery school, and hospitalized with mild febrile and vomiting disease in Parma, Italy. Upon sequencing, the viruses were characterized as EV71 subgenogroup C2. Phylogenetic analysis of the four EV71 C2 viruses allowed the distinction of a diverging lineage within subgenogroup C2, containing the Italian EV71 C2 strains and viruses detected in France in 2013. The identification of an outbreak of EV71 C2 in Italy extended information on the geographic diffusion and clinical relevance of these viruses in Europe.

Diagnostic performances of antigen detection compared to conventional and nucleic acid detection of Entamoeba histolytica in a non-endemic setting.

This study evaluated the immunochromatographic (IC) assay "TECHLAB(®) E. HISTOLYTICA QUIK CHEK™" analysing 36 faecal samples and 7 cultured strains. This assay was compared to the methods performed in our laboratory for the diagnosis of amoebiasis. The IC assay revealed a detection limit of 103 trophozoites/g faeces and no cross-reactivity with other parasites and failed to detect E. histolytica antigen in frozen faeces. In our laboratory located in a non-endemic setting this assay could not replace the methods currently used for the diagnosis of amoebiasis.

Genetic diversity in three bovine-like human G8P[14] and G10P[14] rotaviruses suggests independent interspecies transmission events.

The group A rotavirus (RVA) P[14] genotype has been detected sporadically in humans and is thought to be acquired through zoonotic transmission. The present study describes the full-length genome analysis of two G8P[14] and one G10P[14] human RVAs detected in Italy. The strains possessed the typical bovine-like I2-R2-C2-M2-A3/A11-N2-T6-E2-H3 genotype constellation. All the segments of the two G8P[14] RVAs were most closely related to bovine(-like) strains but were relatively distant to each other, suggesting two independent interspecies transmission events. Likewise, the G10P[14] RVA gene segments were most similar to bovine(-like) RVAs but distinct from the G8 strains. The history of these strains probably involved the interspecies transmission of these viruses to humans from an as-yet-unidentified animal host, without evidence of reassortment events involving human RVAs. These results reinforce the potential of animal viruses to cross the host-species barrier, causing disease and increased viral genetic diversity in humans.

Epidemiological dynamics of norovirus GII.4 variant New Orleans 2009.

Norovirus (NoV) is one of the major causes of diarrhoeal disease with epidemic, outbreak and sporadic patterns in humans of all ages worldwide. NoVs of genotype GII.4 cause nearly 80-90 % of all NoV infections in humans. Periodically, some GII.4 strains become predominant, generating major pandemic variants. Retrospective analysis of the GII.4 NoV strains detected in Italy between 2007 and 2013 indicated that the pandemic variant New Orleans 2009 emerged in Italy in the late 2009, became predominant in 2010-2011 and continued to circulate in a sporadic fashion until April 2013. Upon phylogenetic analysis based on the small diagnostic regions A and C, the late New Orleans 2009 NoVs circulating during 2011-2013 appeared to be genetically different from the early New Orleans 2009 strains that circulated in 2010. For a selection of strains, a 3.2 kb genome portion at the 3' end was sequenced. In the partial ORF1 and in the full-length ORF2 and ORF3, the 2011-2013 New Orleans NoVs comprised at least three distinct genetic subclusters. By comparison with sequences retrieved from the databases, these subclusters were also found to circulate globally, suggesting that the local circulation reflected repeated introductions of different strains, rather than local selection of novel viruses. Phylogenetic subclustering did not correlate with changes in residues located in predicted putative capsid epitopes, although several changes affected the P2 domain in epitopes A, C, D and E.

Combined genetic variants of human cytomegalovirus envelope glycoproteins as congenital infection markers.

BACKGROUND: Human cytomegalovirus (HCMV) is still considered to be the main viral cause of birth defects and long-term neurological and sensory sequelae following congenital infection. Several Authors sustain a key role of HCMV envelope glycoproteins, such as gB, gN and gO - mainly involved in cell targeting, viral penetration and spread - as putative virulence factors. The genes coding for these glycoproteins possess hypervariable regions, resulting in a number of genetic variants in circulating clinical strains. Considering that the genetic polymorphisms underlying the specific differences between gB, gN and gO genotypes can influence the ability of HCMV to preferentially target specific host cells, it is very likely that they play an important role in defining HCMV infection outcome. In the present study, we analysed HCMV gB, gN and gO gene polymorphisms in viral strains isolated from paediatric patients with congenital or post-natal infection, to investigate whether specific genetic variants may be associated with congenital infection. METHODS: The restriction fragment polymorphisms of genes coding for HCMV gB (UL55), gN (UL73) and gO (UL74) were investigated by analysing viral DNA extracted from 40 urine samples of as many paediatric patients with congenital or post-natal HCMV infection. Randomly selected samples were subjected to DNA sequencing and phylogenetic analysis. Statistical analysis was performed using Fisher's exact test to assess the significance of single and combined glycoprotein genotypes frequency distribution. Statistical significance was considered at a P <0.05. RESULTS: While gB genomic variants were quite homogeneously represented in both paediatric groups, the gN4 genotype significantly prevailed in congenitally infected children (89.5 %) vs post-natally infected children (47.6 %), with a predominance of the gN4c variant (47.4 %). A similar trend was observed for gO3 (52.6 % vs 19 %). Concerning genotypes association, a statistically significant (P = 0.037) gN4-gO3 combination was found specifically in the congenitally infected group. CONCLUSIONS: The results indicate that the gN4 (mostly the gN4c variant) and gO3 combined genotypes could provide useful markers of congenital infection and represent suitable candidate molecules for prophylactic vaccine preparations.

Proteasomes raise the microtubule dynamics in influenza A (H1N1) virus-infected LLC-MK2 cells.

The dynamics of microtubule networks are known to have an impact on replication of influenza A virus in some cellular models. Here we present evidence suggesting that at late stages of LLC-MK2 cell infection by influenza A (H1N1) virus the ubiquitin-proteasome protein degradation system participates in destabilization of microtubules, and favours virus replication. Chemical inhibition of proteasome activity partially suppresses influenza A virus replication, while stimulation of proteasome activity favours influenza A virus replication. Conversely, in another cellular model, A549 cells, inhibitors and activators of proteasomes have a small effect on influenza A virus replication. These data suggest that influenza A virus might take selective advantage of proteasome functions in order to set up a favourable cytoskeletal "environment" for its replication and spread. Furthermore, the relationship between influenza virus and the host cell is likely to depend on both the cellular model and the virus strain.

Identification of the novel Kawasaki 2014 GII.17 human norovirus strain in Italy, 2015.

Surveillance of noroviruses in Italy identified the novel GII.17 human norovirus strain, Kawasaki 2014, in February 2015. This novel strain emerged as a major cause of gastroenteritis in Asia during 2014/15, replacing the pandemic GII.4 norovirus strain Sydney 2012, but being reported only sporadically elsewhere. This novel strain is undergoing fast diversification and continuous monitoring is important to understand the evolution of noroviruses and to implement the future strategies on norovirus vaccines.

Evidence of VP7 and VP4 intra-lineage diversification in G4P[8] Italian human rotaviruses.

Intragenotypic heterogeneity of co-circulating rotaviruses is remarkable. Sequence and phylogenetic analyses of the rotavirus VP7 and VP4 genes were performed on selected human G4P[8] strains identified in Parma, Northern Italy, during 2004-2005 and 2008-2012. All the strains clustered into lineages Ic (VP7) and P[8]-III (VP4) in different subclusters with a nucleotide sequence variation up to 4 %. VP7 and VP4 amino acid sequences of the Italian rotaviruses showed multiple changes with the corresponding reference strains as well as with vaccine viruses in the neutralizing epitopes. There is concern that the progressive intra-lineage diversification in the VP7 and VP4 through the accumulation of point mutations and amino acid differences between vaccine strains and currently circulating rotaviruses could generate, over the years, vaccine-resistant variants.