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Background: Human parechovirus (HPeV), like enteroviruses, usually causes mild self-limiting respiratory and gastrointestinal symptoms. In infants, HPeV can occasionally cause serious illnesses, including sepsis-like syndrome and encephalitis. In summer 2016, Public Health England (PHE) received increasing reports of severe HPeV infections nationally. We, therefore, reviewed all infants with confirmed HPeV across England during 2016., Methods: HPeV cases in infants aged <12 months reported to PHE during 2016 were followed up using a clinical questionnaire. Additional cases identified by clinicians completing the questionnaire were also included., Results: We identified 106 infants with confirmed HPeV infection during 2016. The disease peaked during early summer. Most infants (98/106, 92%) were aged <90 days, and 43% (46/106) were neonates. Fever was the most commonly reported symptom (92%) and signs of circulatory shock were present in 53%. Eighteen infants (18%) required paediatric intensive care admission. Most infants had normal or low C reactive protein concentrations (<10 mg/dL in 75%, <50 mg/dL in 98%). A lumbar puncture was performed in 98% of cases; 92% (33/36) of neonates and 93% (53/57) of older infants had normal white cell count in the cerebrospinal fluid (CSF). Nearly all reported cases (98%) were confirmed by CSF PCR. All infants survived, but five had ongoing seizures after hospital discharge., Conclusions: HPeV is an important cause of febrile illness in infants and can have severe clinical presentations. Early diagnosis may help reduce antimicrobial use, unnecessary investigations and prolonged hospitalisation. While prognosis remains favourable, some infants will develop long-term complications-paediatricians should ensure appropriate follow-up after discharge., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Background/objectives: Enteroviruses are the most common cause of aseptic or lymphocytic meningitis, particularly in children. With reports of unusually severe neurological disease in some patients infected with enterovirus D68 in North America, and a recent increase in the number of paediatric enterovirus meningitis cases presenting in this UK Midlands population, a retrospective regional surveillance study was performed., Study Design: Cerebrospinal fluid (CSF) samples received were tested using the polymerase chain reaction (PCR) for HSV-1/2, VZV, enteroviruses and parechoviruses. Enterovirus PCR positive CSF samples were sent for further serotyping. A phylogenetic tree was constructed of the echovirus 30 VP1 sequences, where sufficient sample remained for sequencing., Results: The number of enterovirus positive CSFs from each year were: 21 (2008), 7 (2011), 53 (2012), 58 (2013) and 31 (2014). Overall, 163 of the 170 serotyped enteroviruses belonged to the species B (echovirus 5, 6, 7, 9, 11, 13, 16, 17, 18, 21, 25, 30; coxsackie B1, B2, B3, B4, B5, A9), with only 7 belonging to species A (coxsackie A2, A6, A16 and enterovirus 71). Echovirus 30 was the predominant serotype overall, identified in 43 (25.3%) of samples, with a significantly higher proportion in the adult age group (37.3%) compared to the infant age group (12.3%). Phylogenetic analysis showed that these UK Midlands echovirus 30 VP1 sequences clustered most closely with those from Europe and China., Conclusion: This study showed a continued predominance of echovirus 30 as a cause of viral meningitis, particularly in adults, though more surveillance is needed., (Copyright © 2016 Elsevier B.V. All rights reserved.)

There have recently been significant changes in diagnostic practices for detecting enterovirus (EV) infections across England and Wales. Reports of laboratory-confirmed EV infections submitted by National Health Service (NHS) hospital laboratories to Public Health England (PHE) over a 12-year period (2000-2011) were analysed. Additionally, the PHE Virus Reference Department (VRD) electronic database containing molecular typing data from 2004 onwards was interrogated. Of the 13,901 reports, there was a decline from a peak of 2254 in 2001 to 589 in 2006, and then an increase year-on-year to 1634 in 2011. This increase coincided with increasing PCR-based laboratory diagnosis, which accounted for 36% of reported cases in 2000 and 92% in 2011. The estimated annual incidence in 2011 was 3.9/100,000 overall and 238/100,000 in those aged <3 months, who accounted for almost one-quarter of reported cases (n = 2993, 23%). During 2004-2011, 2770 strains were submitted for molecular typing to the VRD, who found no evidence for a predominance of any particular strain. Thus, the recent increase in reported cases closely reflects the increase in PCR testing by NHS hospitals, but is associated with a lower proportion of samples being submitted for molecular typing. The high EV rate in young infants merits further investigation to inform evidence-based management guidance., (© 2014 The Authors Clinical Microbiology and Infection © 2014 European Society of Clinical Microbiology and Infectious Diseases.)

Norovirus strains were detected in two patients and in environmental swabs from a pediatric primary immunodeficiency unit in London, United Kingdom, during an infection control incident in November and December 2007. Detailed analyses of the gene encoding the P2 domain demonstrated that the majority of the strains were not related to the patients and that the environmental contamination was most likely due to secondary transfer by the hands of staff or visitors.

In this study, we demonstrate that differences within the P2 domain of norovirus genogroup I (GI) strains can be used to segregate outbreaks which are unrelated, whereas complete conservation within this region allows tracking of strains that are part of a single outbreak and likely to have a common source.

Background: A retrospective study of the clinical, epidemiologic, and virologic features of norovirus gastroenteritis in 12 adult allogeneic hematopoietic stem cell transplant (HSCT) recipients., Methods: Norovirus infection was diagnosed by reverse-transcriptase polymerase chain reaction. Strains were genotyped by nucleic acid sequence of the most highly conserved region of the norovirus gene encoding the capsid S (shell) domain., Results: Ten of 12 patients presented with vomiting of short duration, but diarrhea was present in all. The median time from onset to norovirus diagnosis was 1 month (range, 0.25-6.0 months). Eleven patients were receiving immunosuppression when norovirus infection was diagnosed: 8 for graft-versus-host disease (GVHD) in an organ other than gut, 1 for previous gut GVHD, and 2 for presumed gut GVHD that proved to be norovirus gastroenteritis. Six patients required enteral or parenteral nutrition for severe weight loss. In 10 patients, diarrhea lasted a median of 3 months (range, 0.5-14 months) and virus was shed at a high level throughout. The remaining 2 patients died after 4 months of diarrhea (one died of unrelated complications, and the other died of malnutrition). The noroviruses found were GII (untyped), GII-3, GII-4, and GII-7 in 1, 1, 9, and 1 patients, respectively. Eleven of the 12 patients had acquired their infection in the community. Phylogenetic analysis of the GII-4 strains demonstrated that all differed., Conclusions: Noroviruses are a hitherto unsuspected cause of prolonged morbidity and mortality in adults after allogeneic HSCT. The use of reverse-transcriptase polymerase chain reaction to detect high viral load levels in feces distinguishes norovirus gastroenteritis from gut GVHD.

The aim of this study was to apply sequence analysis of a hyper variable region of the norovirus (NoV) genome in order to identify point source outbreaks associated with suspect food or water. The hyper-variable region of the gene encoding the P2 domain was chosen as small differences in sequence are likely to indicate virus from different sources whereas identical sequence may reveal transmission routes and the source of contamination. Strains with 100% similarity were considered as originating from a common source, whereas, strains with one or more mutations in the hyper variable region sequenced were regarded as representing unrelated transmission events. This study was able to identify a point source outbreak of a dominant strain, GII-4, on a cruise ship but also of a less common strain, GII-2, between two schools. Also identical GII-3 strains were demonstrated in food handlers amongst the same outbreak; however epidemiologically related outbreaks showed different GII-3 strains indicating multiple sources of contamination., (Copyright 2009 Wiley-Liss, Inc.)

The aims of this study were to examine the extent of gastroenteric virus contamination in a pediatric primary immunodeficiency (PPI) ward and a general pediatric ward over a winter season and to determine whether changes to hospital infection control interventions would have an impact on environmental contamination levels within pediatric units. Environmental swabs were collected weekly from 11 sites in both wards from 15 December 2005 to 3 March 2006 and examined for the presence of norovirus (NoV), astrovirus, and rotavirus (RV) by reverse transcriptase PCR. Viruses were detected in 17% and 19% of swabs from both wards. Virus contamination for NoV and RV decreased from 20% to 6% and 15% to 10% of swabs, respectively, in the PPI ward from the 2004 study by Gallimore et al. (C. I. Gallimore, C. Taylor, A. R. Gennery, A. J. Cant, A. Galloway, M. Iturriza-Gomara, and J. J. Gray, J. Clin. Microbiol. 44:395-399, 2006). Overall, changes to cleaning protocols were deemed to have reduced the level of environmental contamination with gastroenteric viruses, but contamination still occurred due to a breakdown in infection control procedures indicated by contamination in areas frequented by parents but used only occasionally by staff.

Background: Noroviruses (NoVs) are associated with outbreaks of diarrhoeal illness in hospitals, nursing and residential homes and other institutional settings. NoV strains exhibit wide genetic diversity, and different virus genogroups and genotypes co-circulate in any geographical region at the same time, although most outbreaks of gastroenteritis are predominantly associated with genogroup II. The reverse transcription-polymerase chain reaction (RT-PCR) is the gold standard for detecting NoVs in clinical samples., Objectives: This study evaluates commercialised Loopamp kits for detecting NoV GI and NoV GII in faecal samples collected from patients with gastroenteritis and compares the results with those obtained using real-time RT-PCR with NoV genogroup sequence-specific detection., Study Design: Five hundred and ten faecal samples collected from patients with gastroenteritis were evaluated for the presence of NoV using the gold-standard real-time RT-PCRs and the Loopamp assays., Results: The Loopamp Norovirus GI and GII detection kits performed well compared to genogroup-specific real-time RT-PCR. Although the sensitivity of detection of GI strains (83.3%) was less than that for GII strains (97.4%), this will have little impact on the laboratory diagnosis of NoV, since GII strains are associated with the majority of outbreaks examined., Conclusions: The Loopamp GII detection kit is a sensitive method for detecting all the commonly circulating GII-4 strains included in the evaluation panel.

Tracking the spread of noroviruses during outbreaks of gastroenteritis is hampered by the lack of sequence diversity in those regions of the genome chosen for virus detection and characterization. Sequence analysis of regions of the genes encoding the RNA-dependent RNA polymerase and the S domain of the capsid does not provide sufficient discrimination between genotypically related strains of different outbreaks. However, analysis of sequences derived from the region encoding the P2 domain showed 100% similarity among strains from the same outbreak and <100% similarity among strains of different outbreaks. The prolonged nature of some hospital outbreaks, links between hospitals, and the introduction of multiple strains of a single genotype associated with an outbreak aboard a cruise ship were determined using this method. This provides a powerful tool for tracking outbreak strains and the subsequent analysis and validation of interventions in a background of multiple introductions of virus strains of the same genotype or genetic cluster.

Background: Human noroviruses are a highly diverse group of viruses classified into three of the five currently recognised Norovirus genogroups, and contain numerous genotypes or genetic clusters. Noroviruses are the major aetiological agent of endemic gastroenteritis in all age groups, as well as the cause of periodic epidemic gastroenteritis. The noroviruses most commonly associated with outbreaks of gastroenteritis are genogroup II genotype 4 (GII-4) strains. The relationship between genotypes of noroviruses with their phenotypes and antigenic profile remains poorly understood through an inability to culture these viruses and the lack of a suitable animal model., Methodology/principal Findings: Here we describe a study of the diversity of amino acid sequences of the highly variable P2 region in the major capsid protein, VP1, of the GII-4 human noroviruses strains using sequence analysis and homology modelling techniques., Conclusions/significance: Our data identifies two sites in this region, which show significant amino acid substitutions associated with the appearance of variant strains responsible for epidemics with major public health impact. Homology modelling studies revealed the exposed nature of these sites on the capsid surface, providing supportive structural data that these two sites are likely to be associated with putative variant-specific epitopes. Furthermore, the patterns in the evolution of these viruses at these sites suggests that noroviruses follow a neutral network pattern of evolution.

Short nucleotide sequence inserts within the signal (s) and mid (m) regions of the vacuolating cytotoxin gene (vacA) of Helicobacter pylori provide the basis for defining the allelic forms widely used for strain typing and as markers for toxin functionality and severity of interactions with host gastric epithelial cells. Here 484 signal region and 411 mid-region sequences (new and from public databases) from 32 countries were analysed to determine the effect of geographical location on insert diversity, which is currently undefined. Short (27 bp) inserts of 52 mol% G+C from 201 sequences (98 %) of the s2 allelic family encoded a highly conserved nine amino acid sequence irrespective of geographical origin. The longer (75 bp) mid-region insert of 38 mol% G+C in 255 sequences of the m2 allelic family was more diverse and represented by 23 peptide variants, with one predominant sequence (MRI type 4) representing 62 % of inserts. Mid-region inserts were widespread throughout European/North American (Western) sequences in the dataset whereas a lower insert frequency was a geographical feature of East Asian sequences. Each insert was preceded by an associated conserved motif that provided a marker of the insertion sites within vacA, and facilitated identification of the Chinese m2b genotype. It is concluded that the observed sequence conservation supports the continued global use of vacA genotyping, and that inserts could have a functional significance in the mature protein, particularly the s2 form of the toxin, as the same combination of signal and mid-region insert type and preinsert motif was highly conserved.

This study describes a method used to determine the diversity of NoVs co-circulating in the community that consisted of the analysis of a limited number of strains collected from outbreaks occurring at different times of the NoV season. The diversity of twenty NoV strains collected from outbreaks occurring at the beginning of each NoV season (September) was compared to the diversity found in the middle (December) and at the end of the season (March). The method was validated through the characterisation of greater numbers of strains at times when novel genotypes or variants were detected. A total of 864 strains from outbreaks of gastroenteritis from the 2003/04, 2004/05 and 2005/06 seasons were genotyped, with the majority of outbreaks occurring in the UK. There was a greater diversity of NoV genotypes at the beginning of two of the three seasons, 2003/04 and 2005/06, when compared to strains circulating at the end of the seasons, and GII-4 NoV strains predominated (>90%) at the end of each season. Data from this study also identified the co-circulation and differentiation of three major GII-4 variants (v2, v3, and v4). Detailed analysis of a larger number of strains throughout each season confirmed that variants emerged, became the predominant circulating strain and were ultimately replaced with another variant selected from a pool of variants. By June 2006, GII-4 v4 (Hu/NoV/Rhyl440/2005/UK) emerged as the predominant GII-4 strain, usurping the previous GII-4 v3 strain [Hu/NoV/Hunter284E/040/AU] to become the commonest co-circulating strain, in the UK in 2006.

Phenotypic and phylogenetic studies were performed on four Campylobacter-like organisms recovered from three seals and a porpoise. Comparative 16S rRNA gene sequencing studies demonstrated that the organisms represent a hitherto unknown subline within the genus Campylobacter, associated with a subcluster containing Campylobacter jejuni, Campylobacter coli and Campylobacter lari. DNA-DNA hybridization studies confirmed that the bacteria belonged to a single species, for which the name Campylobacter insulaenigrae sp. nov. is proposed. The type strain of Campylobacter insulaenigrae sp. nov. is NCTC 12927(T) (=CCUG 48653(T)).

Objective: Surveillance data on Helicobacter pylori antibiotic susceptibilities in Wales are limited, despite resistance being a key factor in treatment failure. A single-centre survey was undertaken over 3 years to determine local antibiotic resistance rates on isolates from dyspeptic patients in Bangor, Gwynedd (North Wales)., Methods: Susceptibilities were determined for 363 isolates by disc diffusion and the Etest. Isolates were also genotyped (cagA presence and vacA allelic types)., Results: Overall in vitro resistance rates were 24% for metronidazole and 7% for clarithromycin, with 4% resistant to both antibiotics. Resistant strains typically had high MICs of >256 mg/L. Tetracycline resistance was identified in only one isolate whereas no isolates showed resistance to amoxicillin. There was a two-fold increase in resistance over the study period. No gender and age associations with resistance were detected. Resistant and susceptible isolates were genotypically diverse with respect to cagA/vacA type but the vacA s1m2 form was a feature of all clarithromycin-resistant isolates compared with 56% of the susceptible isolates., Conclusion: Although the overall antibiotic resistance rates of H. pylori from North Wales were low compared with many other regions in Europe, continued surveillance, particularly of high-level resistance (MIC >256 mg/L), is recommended to monitor the effects of the 'test and treat' strategy for H. pylori eradication.

Human migrations and geographical separation over long periods may have resulted in ecologically distinct populations of Helicobacter pylori infecting individuals in different continents. This study used nucleotide sequence analysis with the aim of defining population-specific genomic motifs in isolates from East Asian and African dyspeptic patients. Sequences of internal fragments (542-627 bp) of three housekeeping genes (ureI, ahpC and atpA) were analysed for 85 isolates from individuals in Japan and China (30 isolates), Nigeria and South Africa (14 isolates), the United Kingdom (32 isolates), and nine miscellaneous reference strains. Phylogenetic analyses showed a high degree of intra-set relatedness amongst sequences from the Japanese and Nigerian isolates, with each robustly segregated as distinct lineages irrespective of cagA presence and vacA allelic type. All strains had unique combined sequence types except for identical paired (antrum/corpus) isolates. Population-specific polymorphisms were identified within each gene which were combined to provide unique motifs defining the Japanese and Nigerian regional populations. The alleles were present at variable frequencies in UK and South African isolates. The findings provide unique evidence of positive selection for conserved nucleotide sites linked to the geographical separation in Japan of a strain subpopulation for which we propose the designation H. pylori geovar 'orientalis'.

Helicobacter pylori, a Gram-negative bacterium, is a causal agent of peptic ulcers and is estimated to infect the gastric mucosa of at least half of the world's population. As primary infections are acquired mainly by household contact, studies on family clusters provide a model for investigating transmission and the natural history of initial infection. Here, sequence typing exploiting genetic variation in core fragments of three key housekeeping loci (ureI, atpA and ahpC) was used to determine clonal descent amongst isolates of ten members of four families in Northern Ireland and a family with three generations in central England. Phylogenetic analysis of each locus for 73 strains of H. pylori from 11 countries indicated high background intraspecific diversity, apart from identical paired isolates from five unrelated patients and strains with identical sequence types (STs) detected in adult members of two families. In several families carrying strains with different STs, evidence of residual clonal descent was detected at one or two loci by comparison of nucleotide and amino acid sequences. Pathogenicity-associated genotypes were heterogeneous with respect to ST and amino acid type. Analysis of these three housekeeping genes provides unique evidence for precise tracing of clonal descent in isolates of H. pylori in family groups.

The Helicobacter pylori vacuolating cytotoxin is a putative pathogenicity factor encoded by vacA, a mosaic gene with a global distribution. The vacA type prevalence and diversity of H. pylori isolated from antral gastric biopsies of 360 dyspeptic patients in mid-Essex, and of 79 patients from other locations, were investigated in order to test for links with disease severity. Mid (m)-region genotyping and subtyping by vacA HaeIII RFLP (restriction fragment length polymorphism) analysis showed that the m1 and m2 alleles were diverse, with 191 different subtypes. Variation in 44% of strains was accounted for by ten subtypes of which subtype v-1 represented a conserved core (33%) of the m1 form. Prevalence rates for combined mid and signal (s)-region genotypes were 40% for s1/m1, 46% for s1/m2, and 11% for s2/m2. Overall, vacA genotyping provided high typability and discrimination, but no specific RFLP markers could reliably predict a clinically significant presentation due to an H. pylori infection.

Phospholipase activity is important in bacterial pathogenicity and could contribute to the pathogenic role of Helicobacter pylori by degradation of the gastric mucus, and in maintaining long-term colonisation. Our aim was to determine the degree of variation in the phospholipase A gene (pldA) of H. pylori from different geographic locations, and to investigate links between pldA genotype and clinical disease severity, as well as with variation in cagA status and vacA genotypes. PCR-restriction fragment length polymorphism (RFLP) analysis with MboI and HaeIII was used to study 124 isolates from 10 countries that included the two genome-sequenced strains (26695 and J99), as well as Tx30a and NCTC 11637 (type strain). The 925-bp pldA fragment was amplified with a frequency of 90%. The presence of pldA was confirmed in the other strains using an alternative forward primer. Isolates were distinguished by PCR-RFLP analysis with 10 MboI and four HaeIII restriction patterns that combined to give 25 distinct pldA RFLP types. The pldA M2H2 strain genotype was most common (20%) in the UK but similar strains came from several other countries. Microdiversity was evident in pldA sequences of strains representing different RFLP types, and five M2H2 strains each had a distinct pldA sequence type. Intragenic variation was independent of gastric disease severity as well as strain cagA status and vacA genotype, with the exception of eight geographically diverse strains all with the pldA M4H3/cagA+/vacA s1m1 genotype predominantly from peptic ulcer patients. The study indicated a spectrum of genotypic variants and was supportive of a pldA function in H. pylori colonisation and persistence rather than in chronicity of infection.

Helicobacter pullorum was first isolated from the faeces and carcasses of poultry and has been associated with human gastroenteritis. The aim of this study was to examine interstrain genetic diversity within H. pullorum. Two fingerprinting techniques were used: amplified fragment length polymorphism (AFLP) and pulsed field gel electrophoretic (PFGE) analysis. The 20 strains examined were from four countries and comprised 13 human isolates and seven poultry isolates. Their identity was confirmed by a species-specific PCR assay. The human and poultry isolates had distinct genotypes and most strains showed a high degree of genetic diversity. Genotyping also indicated a clonal origin for two strains from the same poultry flock, and established a close relatedness between three chicken carcass isolates from a processing plant. It is concluded that these two genotyping techniques will provide a useful basis for future epidemiological investigations of H. pullorum in poultry, and may provide a link with its possible causal role in human gastrointestinal infections.

Helicobacter pylori urease subunit genes in 383 isolates from 10 countries were investigated by PCR-restriction fragment length polymorphism (HaeIII) analysis. Eighty-two different ureAB profiles were documented by reference to known sequences. Variation among 51% of strains was accounted for by 10 predominant patterns, which provided a unique framework for categorizing isolates with geographically diverse origins.

Amplified-fragment length polymorphism (AFLP) analysis is the name given to a genotypic technique in which adapter oligonucleotides are ligated to restriction enzyme fragments and then used as target sites for primers in a PCR amplification process. The amplified fragments are electrophoretically separated to give strain-specific band profiles. We have developed a single-enzyme approach that did not require costly equipment or reagents for the fingerprinting of strains of Helicobacter pylori. The method was assessed with 46 isolates of H. pylori from 28 patients, and the results were compared with those from other genotypic tests. The AFLP profiles derived from HindIII fragments differentiated strains of H. pylori from unrelated individuals and confirmed the common origin of strains in some family members. AFLP analysis was also applied to investigate persistent infection following antibiotic therapy. Overall, the modified technique was relatively rapid and technically simple yet gave reproducible and discriminatory results. AFLP analysis samples variation throughout the genome and is a valuable addition to the existing genotypic fingerprinting methods for H. pylori.

Streptococcus pyogenes strains were genotyped by a combination of molecular methods for high- resolution epidemiologic studies of disease outbreaks. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the emm gene is reported. Alone or in conjunction with other molecular techniques (16S ribotyping, pulsed-field gel electrophoresis, and detection of exotoxin genes), PCR-RFLP could differentiate outbreak-related strains from contemporaneous background strains of the same M serotype. Three outbreaks were studied: pharyngitis in a boarding school (serotype M5), cross-infection in a hospital burn unit (serotype M76), and severe invasive disease in two elderly care homes (serotype R28). It was possible, for example, to identify within serotype R28 a clone with particular potential for invasive disease. In all cases, the four molecular methods yielded complementary results that were hierarchically related. Strains could be assigned to the outbreak or the background in a precise, reproducible, and rapid manner.