The Application of New Molecular Methods in the Investigation of a Waterborne Outbreak of Norovirus in Denmark, 2012

In December 2012, an outbreak of acute gastrointestinal illness occurred in a geographical distinct area in Denmark covering 368 households. A combined microbiological, epidemiological and environmental investigation was initiated to understand the outbreak magnitude, pathogen(s) and vehicle in order to control the outbreak. Norovirus GII.4 New Orleans 2009 variant was detected in 15 of 17 individual stool samples from 14 households. Norovirus genomic material from water samples was detected and quantified and sequencing of longer parts of the viral capsid region (>1000 nt) were applied to patient and water samples. All five purposely selected water samples tested positive for norovirus GII in levels up to 1.8×10(4) genomic units per 200 ml. Identical norovirus sequences were found in all 5 sequenced stool samples and 1 sequenced water sample, a second sequenced water sample showed 1 nt (<0.1%) difference. In a cohort study, including 256 participants, cases were defined as residents of the area experiencing diarrhoea or vomiting onset on 12-14 December 2012. We found an attack rate of 51%. Being a case was associated with drinking tap-water on 12-13 December (relative risk = 6.0, 95%CI: 1.6-22) and a dose-response relation for the mean glasses of tap-water consumed was observed. Environmental investigations suggested contamination from a sewage pipe to the drinking water due to fall in pressure during water supply system renovations. The combined microbiological, epidemiological and environmental investigations strongly indicates the outbreak was caused by norovirus contamination of the water supply system

Attack rates (AR),relative risk (RR) and 95% confidence interval (CI) of diarrhoea and/or vomiting compared to the reference (Ref) on 12–14 December by tap-water consumption and mean daily tap-water consumption on 12–13 December, Kalundborg, Denmark.

<p>Attack rates (AR),relative risk (RR) and 95% confidence interval (CI) of diarrhoea and/or vomiting compared to the reference (Ref) on 12–14 December by tap-water consumption and mean daily tap-water consumption on 12–13 December, Kalundborg, Denmark.</p

Results of positive microbiological findings in water samples.

a<p>Estimations are figured with or without applying corrective factors derived by extraction efficiencies.</p>b<p>Below theoretical limit of quantification estimated at 100 genomic units/200 ml.</p><p>Results of positive microbiological findings in water samples.</p

Phylogenetic tree of NoV capsid gene sequences.

<p>Phylogenetic tree based on 11 nucleotide sequences of 1140 nt of the NoV capsid gene. All sequences are indicated by their GenBank Accession number. GenBank accession numbers used in this manuscript are from norovirus sequences from patient samples (KF798196, KF798197, KF798198 and KF798200) and water samples (KF798199 (W3) and KF798201 (W1)). The remaining norovirus sequences from patient samples used in the phylogenetic tree have been published previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105053#pone.0105053-Fonager1" target="_blank">[32]</a>. Legend: Circles and triangles: New Orleans 2009 variants and Squares: Sydney 2012 variants, Circles: belonging to the outbreak, Triangles: not belonging to the outbreak described here. Open triangle and square are the reference sequences for the New Orleans 2009 and Sydney 2012 variants respectively. Open circles:sequences obtained from water samples (KF798201,W1) and KF798199,W3) and closed circles are the sequences obtained from patient stool samples.</p