During the prevaccine era, rotavirus (RV) was the major cause of pediatric hospitalizations and emergency department visits for acute gastroenteritis (GE) in Canada.1–7 Among Quebec children aged less than 5 years, approximately 1 in 66 to 1 in 85 visited an emergency department for RV annually, and between 1 in 135 and 1 in 200 were hospitalized for RV GE.7 In November 2011, the Province of Quebec implemented a publicly funded, routine childhood RV vaccination program with the exclusive-use of monovalent Rotarix (GlaxoSmithKline Biologicals Rixensart, Belgium) vaccine.
After the implementation of childhood RV vaccination in other countries, the burden of pediatric RV infection declined significantly,8–18 and changes in circulating RV genotypes were initially observed.18–21 In the US and Finland,22–24 declines in RV have led to norovirus (NV) replacing RV in relative importance as the leading cause of pediatric medically attended GE, with 1 in 278 US children hospitalized and 1 in 14 requiring emergency care for NV infection by age 5 years.23 Comparison of RV and NV clinical features in the prevaccination era reveal potential differences in the clinical severity of RV and NV disease,25–31 with symptoms of pediatric RV GE, on average, more clinically severe than NV GE. Development of a vaccine to protect against NV GE is currently underway.32,33
To date, the relative burden of RV and NV pediatric GE in Quebec in the postvaccine era has not yet been established, and comparison of RV and NV GE among a pediatric population is useful to inform childhood NV vaccination recommendations, should a vaccine become available in the future. The objectives of our prospective study were to examine the pediatric burden of RV and NV over time after the Quebec RV vaccination program and compare clinical characteristics of RV and NV cases.
MATERIALS AND METHODS
Prospective, active surveillance for acute RV GE among children aged 8 weeks to less than 3 years was conducted at 3 teaching hospitals in Quebec: The Montreal Children’s Hospital and Centre Hospitalier Universitaire Sainte-Justine, located in Montreal, and Centre Hospitalier Universitaire de Sherbrooke, located in Sherbrooke. The active surveillance protocol was approved by Research Ethics Boards at each hospital.
Patient Recruitment and Eligibility
Patients were eligible for inclusion if hospitalized or seeking emergency care for acute GE at a study location and their parent or legal guardian consented to be contacted for research purposes. Acute GE was defined as either (i) diarrhea (liquid stools for >12 hours with ≥3 stools in a 24-hour period), (ii) vomiting (≥1 episode in a 24-hour period) or (iii) an emergency department diagnosis of diarrhea, vomiting or gastroenteritis, where symptom onset (for participants meeting any criteria) occurred ≤7 days of hospital presentation. Patients were excluded from study participation if RV vaccination was contraindicated, in accordance with the Quebec Immunization Protocol.34
Written consent to participate in active surveillance was obtained from a parent or legal guardian of all participants.
Participant demographics, medical information, vaccination history and history of present illness were systematically collected via phone interview with the child’s caretaker and review of medical records. Vaccination history, including vaccine type and date, was collected in reference to the participant’s immunization card. Symptoms were ascertained as of the time of interview and included fever and duration of febrile illness; diarrhea, duration of diarrheal illness, and the maximum number of stools produced in a 24-hour period at the height of diarrheal illness; and vomiting, duration of vomiting illness and the maximum number of vomiting episodes in a 24-hour period at the height of vomiting illness. Medical records were used to ascertain information regarding hospitalization, a clinical history of dehydration, seizures, hematemesis, hematochezia, prematurity and presence of underlying conditions. Prematurity was defined as gestational age of less than 37 weeks at birth; underlying conditions were defined as documentation of any of the following underlying disorders: cardiovascular, respiratory, nonmalignant hematologic, neurological, developmental, genitourinary, renal, gastrointestinal, hepatic, endocrine, nutritional, metabolic, inherited immunodeficiency, bone, joint, connective tissue or severe skin disorders.
Stool samples from participants were collected ≤14 days of onset from specimens collected at home by the participant’s parent/guardian or from stool retrieved during routine emergency or hospital care.
All stool specimens were stored at −80°C until viral testing. RV enzyme immunoassay (EIA) testing was first performed on all specimens via the commercially available, Premier Rotaclone (Meridian Bioscience, Inc., Cincinnati, OH) kit. RV-positives were additionally confirmed via real-time reverse-transcriptase polymerase chain reactions (RT-PCR) testing, in consideration of the high specificity but lower sensitivity performance characteristics of the Premier Rotaclone EIA kit.35 In case of discordant results between RV RT-PCR and EIA, RV RT-PCR results were used. Only EIA RV-negative or RT-PCR RV-negative specimens (following confirmatory RV RT-PCR testing for specimens RV-positive by EIA) were additionally tested for NV and sapovirus (SV) via RT-PCR.
For RT-PCR testing, nucleic acids were extracted from precleared stool samples using the NucliSENS easyMag automated extractor and reagents (bioMérieux, Inc., Marcy-l'Étoile, France). RV RT-PCR was conducted according to protocols outlined by Gentsch et al,36 with an assay targeting serotype-conserved regions of the VP4 coding segment, using CON 3 and CON 2 primers. Real-time RT-PCR assays used for genogroup I and genogroup II norovirus were performed according to Kageyama et al37 and Kojima et al38 protocols, respectively. Sapovirus detection was detected using the RT-PCR assay described by Oka et al.39
From the total number of study participants, we estimated the proportion that tested positive for RV, NV, SV, coinfected with NV and SV (ie, tested positive for NV and SV simultaneously) and those with GE of an unknown etiology (GEUE), defined as patients who tested negative for RV, NV and SV. Proportions were calculated for (i) the entire study period, (ii) 2 complete years: June 2012 to May 2014 and (iii) the years, June 2012 to May 2013 and June 2013 to May 2014 separately, for comparison. June was chosen as a starting month for annual estimates in accordance with previous research examining annual RV activity in Quebec.1 The SAS MULTINOM module40 was used to estimate 95% confidence intervals (CI) for each multinomial proportion and differences and 95% CI between the prevalence of NV-positive and RV-positive specimens during each time period.
Demographic and clinical features of participants were stratified by etiology. Participants’ age (in months) and the duration from onset to stool collection (in days) were compared by etiology to participants with RV by estimation of mean differences and 95% CI via the Welch–Satterthwaite method41; participants’ sex, history of an underlying medical condition and history of prematurity were compared with that of participants with RV by estimation of prevalence differences and Wilson 95% CI.42–44
Risk and absolute differences of disease severity outcomes were estimated to compare the exposures of NV and GEUE versus RV (referent) after adjustment for continuous patient age in months, centered at participants’ mean age. For these analyses, risk differences and 95% CI for the binary outcomes of fever, diarrhea, vomiting, dehydration and hospitalization were estimated using binomial regression with an identity link function; absolute differences and 95% CI were estimated via generalized linear regression to examine associations with the outcomes: durations of fever, diarrhea, and vomiting and the maximum number of diarrheal and vomiting episodes experienced in a 24-hour period. Sensitivity analyses were conducted excluding RV-positive participants who had received RV vaccination to examine any changes in results.
All analyses were conducted using SAS version 9.3 statistical software (SAS Institute, Inc., Cary, NC).
From February 1, 2012 through May 31, 2014, 734 patients enrolled in active RV GE surveillance were eligible for study inclusion; of these, 705 (96.0%) participants with complete laboratory information were included in analyses. All acute GE participants met the study definitions for diarrhea or vomiting; no participant had an emergency department diagnosis of diarrhea, vomiting or gastroenteritis, only. On average, 25.2 (95% CI: 20.6–29.8) participants were recruited monthly (range: 9–49). Mean age of participants was 17.1 (95% CI: 16.4–17.7) months, 52.4% (95% CI: 48.7–56.1%) were male. Overall, 14.4% (95% CI: 12.0–17.2%) of participants were hospitalized, and the risk of hospitalization did not change when analyses were stratified by study year. Mean duration between symptom onset and specimen collection dates was 6.1 (95% CI: 5.8–6.3) days.
Acute GE by Etiology
Among active surveillance participants, 149 tested positive for RV by EIA; of these, 144 (96.6%) were also positive for RV by RT-PCR. Results of RT-PCR testing by etiology are presented in Figure 1. During the entire study period, 20.4% (95% CI: 16.5–24.3%) of participants tested RV-positive, 25.5% (95% CI: 21.3–29.8%) tested NV-positive, with a difference of 5.1% (95% CI: 0.1–10.1%). NV/SV coinfection was found in 0.9% (95% CI: 0.0–1.7%) of specimens. When analyses were limited to 2 full years, RV represented 16.3% (95% CI: 12.2–20.4%) of acute GE cases and NV 24.5% (95% CI: 19.8–29.3%), with a difference of 8.2% (95%: 2.9–13.6%). Stratified by study year, RV and NV represented 23.3% (95% CI: 17.2–29.4%) and 22.4% (95% CI: 16.4–28.3%) of acute GE cases, respectively, with a difference of −0.9% (−8.3 to 6.5%) during June 2012 to May 2013, and 6.3% (95% CI: 2.1–10.4%) and 27.7% (95% CI: 20.0–35.4%), respectively, with a difference of 21.4% (95% CI: 14.3–28.5%) during June 2013 to May 2014.
Participant demographic and other characteristics are compared by etiology in Table 1. On average, stool of RV-positive patients was collected after symptom onset 1.3 (95% CI: 0.7–1.9), 1.1 (95% CI: 0.2–2.0) and 2.1 (95% CI: 1.6–2.6) days earlier than NV-positive, SV-positive and GEUE patients, respectively. RV-positive patients were on average, older than NV-positive, SV-positive and GEUE patients, by a mean difference of 7.0 (95% CI: 5.3–8.8), 6.4 (95% CI: 4.0–8.9) and 7.2 (95% CI: 5.5–8.8) months, respectively.
Seasonality of RV, NV and SV is examined in Figure 2. During the study period, the relative prevalence of RV peaked in the springtime months, with the highest RV prevalence detected in May 2012 (51%) followed by April 2013 (44%). When analyses were stratified by year, RV cases were detected in 11 of 12 months (91.7%) from June 2012 to May 2013, and only 7 of 12 months (58.3%) from June 2013 to May 2014. NV cases occurred throughout the entire study period with the exception of 1 month (July 2013). NV prevalence tended to peak in winter, with the maximum peak in prevalence detected in February 2014 (67%). SV activity represented <25% of all GE cases per month in all but 1 month of the study period, with the maximum peak prevalence (32%) occurring in November 2013.
Figure 3 displays the risk of GE symptoms by etiology after adjustment for centered age. In comparison with RV patients of the same age, NV patients experienced on average a 33.4% (95% CI: 23.4–43.5%), 9.5% (95% CI: 0.8–18.2%) and 14.5% (95% CI: 4.2–24.8%) lower risk of fever, dehydration and hospitalization, respectively. Although no differences were detected between RV and NV patients with regard to risk of diarrhea, NV patients reported a mean of 2.2 (95% CI: 0.9–3.5) less episodes of diarrhea in a 24-hour period at the height of illness than RV patients (Fig. 4). GEUE patients had an 11.5% (95% CI: 2.9–20.0%), 9.5% (95% CI: 1.1–17.9%), 6.2% (95% CI: 1.4–11.0%) and 14.2% (95% CI: 8.2–20.2%) lower risk of fever, dehydration, diarrhea and vomiting, respectively, in comparison with RV-positive patients of the same age. GEUE patients also reported an average of 1.6 (95% CI: 0.5–2.8) less episodes of diarrhea and 3.4 (95% CI: 2.0–4.9) less episodes of vomiting than RV patients in a 24-hour period. No differences were detected by etiology in the durations of fever, diarrhea or vomiting symptoms. Risk and absolute differences did not significantly or meaningfully differ in sensitivity analyses excluding 9 RV-positive patients who had a history of vaccination with RV-vaccine.
In addition to these symptoms, 1 (0.7%) RV-positive patient experienced seizures, and 4 (1.2%) GEUE patients had hematochezia; no patients experienced hematemesis.
In this prospective, active surveillance study, we examined changes over time in the relative burden of RV and NV illness after the implementation of a population-based RV vaccination program. With more than 2 years of postimplementation data, we found that the relative prevalence of RV infection among acute GE cases hospitalized or seeking emergency care declined over time, and that NV infection accounted for a higher proportion of acute GE cases among children less than 3 years of age hospitalized or seeking emergency care. Notably, RV GE illness was, on average, more clinically severe in comparison with NV GE illness among participants of the same age.
An apparent reduction in the relative prevalence of RV-associated GE is not surprising, given the effectiveness of the Rotarix vaccine and the success of childhood RV vaccination programs in reducing the burden of pediatric RV GE in the US and elsewhere.9–11,17,18,45–54 Provincial ≥2-dose RV vaccination coverage estimates among children aged 1 year were 14% and 86% vaccination coverage during the years 2012 and 2014, respectively.55 Even in areas with suboptimal RV vaccination coverage, modest declines in both RV illness and all-cause diarrheal illness have been reported.10,55,56
Similar to other studies comparing the severity of RV and NV disease,25–27,57–59 we found that illness among RV-positive patients in our sample tended to be more clinically severe than NV illness based on the risk of several GE symptoms. We found that RV-positive patients were more likely to be febrile, dehydrated, hospitalized and report a greater frequency of diarrheal episodes at the height of illness in comparison with NV-positive patients of the same age. RV patients were also more likely to experience fever, dehydration, vomiting and diarrhea than GEUE patients of the same age.
Our analyses should be considered with regard to several limitations. First, our study only includes data from 3 active surveillance study locations; thus, our results may not be generalizable to the entire Quebec population. Second, we were only able to examine prevalence differences relative to the burden of other pediatric GE emergency department visits and hospitalizations. Despite this limitation, relative differences are meaningful, provided that acute GE caused by alternate etiologies has not changed over time. Third, the time between symptom onset and stool specimen collection for RV-positive participants was on average, approximately 1–2 days earlier than participants with NV, SV or GEUE. Although earlier stool collection for RV-positive participants may be because of stool collection from a higher proportion of RV patients at the emergency care encounter due to a greater clinical disease severity, we cannot rule out the possibility that later collection times may have impacted viral agent detection, particularly in cases where no viral agent was detected. Finally, another limitation is that we were unable to examine RV coinfection; because coinfection may also be predictive of more severe clinical disease,27,60,61 it is possible that our estimates of clinical severity may change if coinfection was more likely to occur among pediatric RV cases.
In conclusion, more than 2.5 years after the implementation of RV vaccination in Quebec, we found that NV was more prevalent than RV infections among acute GE cases hospitalized or seeking emergency care in Quebec children aged less than 3 years. Although NV has replaced RV disease in relative importance in Quebec, GE symptoms of RV illness were on average more clinically severe than NV disease among children of the same age.
We would like to thank Thomas Lemaître, Suzanne DeRome, and Léna Coïc for their help in recruiting patients on various sites, as well as the MUHC Vaccine Study Centre personnel.
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