Whole genome sequencing and de novo assembly identifies Sydney-like variant noroviruses and recombinants during the winter 2012/2013 outbreak in England

Background: Norovirus is the commonest cause of epidemic gastroenteritis among people of all ages. Outbreaks frequently occur in hospitals and the community, costing the UK an estimated £110 m per annum. An evolutionary explanation for periodic increases in norovirus cases, despite some host-specific post immunity is currently limited to the identification of obvious recombinants. Our understanding could be significantly enhanced by full length genome sequences for large numbers of intensively sampled viruses, which would also assist control and vaccine design. Our objective is to develop rapid, high-throughput, end-to-end methods yielding complete norovirus genome sequences. We apply these methods to recent English outbreaks, placing them in the wider context of the international norovirus epidemic of winter 2012. Method. Norovirus sequences were generated from 28 unique clinical samples by Illumina RNA sequencing (RNA-Seq) of total faecal RNA. A range of de novo sequence assemblers were attempted. The best assembler was identified by validation against three replicate samples and two norovirus qPCR negative samples, together with an additional 20 sequences determined by PCR and fractional capillary sequencing. Phylogenetic methods were used to reconstruct evolutionary relationships from the whole genome sequences. Results: Full length norovirus genomes were generated from 23/28 samples. 5/28 partial norovirus genomes were associated with low viral copy numbers. The de novo assembled sequences differed from sequences determined by capillary sequencing by <0.003%. Intra-host nucleotide sequence diversity was rare, but detectable by mapping short sequence reads onto its de novo assembled consensus. Genomes similar to the Sydney 2012 strain caused 78% (18/23) of cases, consistent with its previously documented association with the winter 2012 global outbreak. Interestingly, phylogenetic analysis and recombination detection analysis of the consensus sequences identified two related viruses as recombinants, containing sequences in prior circulation to Sydney 2012 in open reading frame (ORF) 2. Conclusion: Our approach facilitates the rapid determination of complete norovirus genomes. This method provides high resolution of full norovirus genomes which, when coupled with detailed epidemiology, may improve the understanding of evolution and control of this important healthcare-associated pathogen

Cross-sectional study of the prevalence, causes and management of hospital-onset diarrhoea.

BACKGROUND:The National Health Service in England advises hospitals collect data on hospital-onset diarrhoea (HOD). Contemporaneous data on HOD are lacking. AIM:To investigate prevalence, aetiology and management of HOD on medical, surgical and elderly-care wards. METHODS:A cross-sectional study in a volunteer sample of UK hospitals, which collected data on one winter and one summer day in 2016. Patients admitted ≥72 hours were screened for HOD (definition: ≥2 episodes of Bristol Stool Type 5-7 the day before the study, with diarrhoea-onset >48 hours after admission). Data on HOD aetiology and management were collected prospectively. FINDINGS:Data were collected on 141 wards in 32 hospitals (16 acute, 16 teaching). Point-prevalence of HOD was 4.5% (230/5142 patients; 95% CI 3.9-5.0%). Teaching hospital HOD prevalence (5.9%, 95% CI 5.1-6.9%) was twice that of acute hospitals (2.8%, 95% CI 2.1-3.5%; odds ratio 2.2, 95% CI 1.7-3.0). At least one potential cause was identified in 222/230 patients (97%): 107 (47%) had a relevant underlying condition, 125 (54%) were taking antimicrobials, and 195 (85%) other medication known to cause diarrhoea. 9/75 tested patients were Clostridium difficile toxin positive (4%). 80 (35%) patients had a documented medical assessment of the diarrhoea. Documentation of HOD in medical notes correlated with testing for C. difficile (78% of those tested versus 38% not tested, p<0.001). 144 (63%) patients were not isolated following diarrhoea onset. CONCLUSION:HOD is a prevalent symptom affecting thousands of patients across the UK health system each day. Most patients have multiple potential causes of HOD, mainly iatrogenic, but only a third had medical assessment. Most were not tested for C. difficile and were not isolated