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. 2016 Jan 1;62(1):1-10.
doi: 10.1093/cid/civ747. Epub 2015 Oct 26.

Epidemiologic, Virologic, and Host Genetic Factors of Norovirus Outbreaks in Long-term Care Facilities

Veronica P Costantini  1 , Emilie M Cooper  1 , Hope L Hardaker  2 , Lore E Lee  2 , Marieke Bierhoff  3 , Christianne Biggs  2 , Paul R Cieslak  2 , Aron J Hall  1 , Jan Vinjé  1
Affiliations

Epidemiologic, Virologic, and Host Genetic Factors of Norovirus Outbreaks in Long-term Care Facilities

Veronica P Costantini et al. Clin Infect Dis. .

Abstract

Background: In the Unites States, long-term care facilities (LTCFs) are the most common setting for norovirus outbreaks. These outbreaks provide a unique opportunity to better characterize the viral and host characteristics of norovirus disease.

Methods: We enrolled 43 LTCFs prospectively to study the epidemiology, virology, and genetic host factors of naturally occurring norovirus outbreaks. Acute and convalescent stool, serum, and saliva samples from cases, exposed and nonexposed controls were collected. Norovirus infection was confirmed using quantitative polymerase chain reaction testing of stool samples or 4-fold increase in serum antibody titers. The presence of histo-blood group antigens (secretor, ABO, and Lewis type) was determined in saliva.

Results: Sixty-two cases, 34 exposed controls, and 18 nonexposed controls from 10 norovirus outbreaks were enrolled. Forty-six percent of acute, 27% of convalescent case, and 11% of control stool samples tested norovirus positive. Outbreak genotypes were GII.4 (Den Haag, n = 3; New Orleans, n = 4; and Sydney, n = 2) and GI.1 (n = 1). Viral load in GII.4 Sydney outbreaks was significantly higher than in outbreaks caused by other genotypes; cases and controls shed similar amounts of virus. Forty-seven percent of cases shed virus for ≥ 21 days. Symptomatic infections with GII.4 Den Haag and GII.4 New Orleans were detected among nonsecretor individuals.

Conclusions: Almost half of all symptomatic individuals shed virus for at least 21 days. Viral load was highest in GII.4 viruses that most recently emerged; these viruses also infect the nonsecretor population. These findings will help to guide development of targeted prevention and control measures in the elderly.

Keywords: long-term care facilities; norovirus; secretor status; shedding.

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Conflict of interest statement

Potential conflicts of interest. P. R. C., L. E. L., and H. L. H. report grants and nonfinancial support from the CDC Foundation. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

Figures

Figure 1
Figure 1
Flow diagram of the study experimental design. From 2009 through 2013, 43 long-term care facilities were enrolled, and 10 norovirus outbreaks that qualified for inclusion in the study were reported. A total of 62 cases, 34 exposed controls, and 18 nonexposed controls were enrolled based on the presence of clinical symptoms and exposure to a case. aThe immune responses will be described in another article.
Figure 2
Figure 2
Duration of illness, virus shedding, and severity score in cases reported during the study. A, Illness duration data from 49 cases were based on questionnaire responses. Graph represents median + interquartile range (IQR) for each age group. B, The severity of norovirus disease was assessed for all cases (n = 62) on the basis of a score system; scores ranged from 0 to 17, with higher scores indicating more severe disease (Supplementary Table 1). Graph represents median + IQR for each age group. C, Illness duration, virus shedding duration, and severity score were obtained from 29 cases from which both acute and convalescent stool samples were collected. Boxes represent 25th percentile, median, and 75th percentile, and the whiskers show the 10th–90th percentile for illness duration (white boxes), virus shedding duration (gray boxes), and severity score (stripe boxes).
Figure 3
Figure 3
Phylogenetic analysis of (A) norovirus GI strains (outbreak F) and (B) norovirus GII strains (outbreaks A–E and G–J) based on amino acid sequences of the P2 domain of major capsid protein. Samples are listed by outbreak ID, followed by participant ID and collection time (Ac, acute; Cv, convalescent). Reference sequences are labeled by their name. The evolutionary history was inferred by using the maximum likelihood method based on the JTT matrix-based model. The tree with the highest log likelihood is shown. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JDT model and then selecting the topology with superior log likelihood value [24]. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Evolutionary analyses were conducted in MEGA5 [21]. All sequences from cases, except where indicated. GenBank accession numbers of reference sequences are as follows: GI.1 Norwalk (M87661), GI.2 Southampton (L07418), GI.3 Desert Shield (U04469), GI.3b Stavanger (AF145709), GI.4 Chiba (AB042808), GI.5 Musgrove (AJ277614), GI.6 Hesse (AJ277615), GI.7 Winchester (AJ277609), GI.8 Boxer (AF538679), GI.9 Vancouver 730 (HQ637267), GII.4 Bristol (X76716), GII.4 Richmond (GU4453253), GII.4 US95/96 (AJ0048642), GII.4 Farmington Hills 2002 US (AY4856423), GII.4 Hunter 2004 AUS (AY8830962), GII.4 Yerseke 2006a NLD (EF1269632), GII.4 Den Haag 2006b NLD (EF1269652), GII.4 Apeldoorn_2007 (AB445395), GII.4 New Orleans 2009 (GU445325), and GII.4 Sydney 2012 (JX459908). Abbreviations: ExC, exposed control; non-ExC, nonexposed control.
Figure 4
Figure 4
Viral shedding during norovirus outbreaks reported during the study. Box plots represent log10 viral shedding (median and 25th and 75th quartiles [box], 10th and 90th percentiles [whiskers]). Data were stratified for cases/exposed controls/nonexposed controls, during acute (day 0–8) or convalescent (day 17–32) time with the same genotype. Only positive samples are included; therefore, lack of value indicates negative samples or sample not collected (Table 3). Classification was based on virus genotype detected in stool sample. Viral shedding was log10 transform and analysis of variance 1-way followed by Tukey multiple comparison test was applied to compare all mean values (P < .05). Abbreviations: Ac, acute; Cv, convalescent; ExC, exposed control; non-exC, nonexposed control.
Figure 5
Figure 5
Shedding duration among cases. Acute (day 0–8) and convalescent stools sample (day 17–32) were collected from 35 cases. A, Norovirus was detected in 16/35 convalescent samples. Data represent mean viral load (copies/g stool) plus standard deviation. Short shedding convalescent samples were negative for norovirus. B, Proportion of positive samples stratified by norovirus concentration in acute stool samples. The survival distributions were significantly different (log rank test, χ2 = 12.45, P < .05). Cases with an initial shedding higher than 1010 copies per gram stool were significantly more likely to continue shedding virus for 3–4 weeks (P < .001).
Figure 6
Figure 6
Binding of norovirus virus-like particle (VLP) to saliva samples collected during the study. Saliva samples were assayed for their ability to bind outbreak-specific norovirus VLPs. A, Norovirus VLP (0.5 µg/mL) binding to saliva samples collected from secretors (n = 103) and nonsecretors (n = 11) during norovirus outbreaks reported during the study. Triangles represent corrected optical density (OD) (450 nm) from individual saliva samples stratified for secretor status and outbreak-specific norovirus genotype. All cases/exposed controls/nonexposed controls were included. Line represents median + interquartile range. Saliva from secretors and nonsecretors were able to bind GII.4 Den Haag, but not GII.4 New Orleans or GII.4 Sydney VLPs. B, Saliva samples from nonsecretors (negative on A) were assayed for their ability to bind increasing amounts of genotype-specific norovirus VLPs (0.5, 1.0, 5.0 µg/mL). Significant differences were observed after incubation of 1.0 µg/mL or 5.0 µg/mL with saliva samples from Lea+b− (Lewis positive) (P < .0001), but not from Lea−b− (Lewis negative). Abbreviations: Ca, case; ExC, exposed control; non-ExC, nonexposed control.
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