The public health impact of the emergence of new norovirus (NoV) strains is uncertain. A biennial pattern of alternating quiescent and epidemic levels of NoV outbreak activity associated with the emergence of new GII.4 variants was observed in Alberta, Canada, between July 2000 and June 2008. In this study, NoV genogroup I (GI) and GII strains isolated from 710 outbreak specimens in Alberta between July 2008 and January 2013 were characterized to update historical data. The seasonality and annual variation in NoV outbreak burden were analyzed over a 10-year period (July 2002 to June 2012). We found that GII.4-2006b had persisted as the predominant variant over three observation periods (July 2006 to June 2009) during which the biennial NoV outbreak pattern continued. The emergence of GII.4-2010 (winter 2009) was not associated with increased outbreak activity, and outbreak activity between July 2009 and June 2012 when GII.4-2010 predominated (67.5 to 97.7%) did not follow a biennial pattern. GII.4-2012 first emerged in Alberta in September 2011 and became predominant in observation period July 2012 to June 2013. NoV GI, relatively rare in past years, had a higher activity level (37.3%) as represented by GI.6 and GI.7 in the winter of 2012 to 2013. A higher proportion of GI outbreaks occurred in non-health care facility settings compared to GII. Our study suggests that factors other than new variants emergence contribute to the levels of NoV outbreak activity in Alberta.
This study describes the epidemiology and circulating strains of sapovirus associated with gastroenteritis outbreaks in Alberta, Canada, from 2004 to 2007. Sapovirus was an important cause of gastroenteritis outbreaks, accounting for 43 (17.6%) of 244 outbreaks in which all samples tested were negative for norovirus. All 4 human sapovirus genotypes, GI, GII, GIV, and GV, were found in samples during these outbreaks. The greatest amount of sapovirus-associated outbreak activity occurred in 2007, after the emergence of genotype GIV in December 2006. The majority of sapovirus-associated outbreaks in Alberta during this period (27 [62.8%] of 43) occurred in hospitals, community long-term care facilities, and senior lodges. Adults>65 years of age were the age group most commonly affected.
Six-hundred and twenty-six stool specimens collected from children with diarrhea over a 12-month period were tested for rotavirus using a real time reverse transcription-polymerase chain reaction (RT-PCR) assay, a conventional nested PCR assay and by electron microscopy (EM). A fragment of 87 bp in a highly-conserved region of non-structural protein 3 (NSP3) in rotavirus genome was amplified by a single-step RT-PCR protocol in a closed-tube system. Rotavirus was detected in 123 samples (20%) with the real time RT-PCR assay, 113 samples (18%) with the nested-PCR assay, and 79 samples (13%) with EM. Using serial diluted nucleic acid extract, we compared the sensitivity of real time RT-PCR with conventional RT-PCR and conventional nested PCR assays. Real time RT-PCR was two to four logs more sensitive than the conventional assays. The reaction time required for the RT-PCR assay is about half the time required for the conventional nested-PCR. The real time RT-PCR assay is both simple and rapid with advantages including enhanced sensitivity and a lower risk for cross-contamination making it a useful tool for the detection of rotavirus in various situations including sporadic gastroenteritis, outbreaks, and environmental investigations. G(1) was the predominant type (89%), followed by G(2) (10%), and G(4) (1%). No rotavirus of G(3), G(8), and G(9) types were found. The peak season for rotavirus infection was January to May in northern Alberta.
Conventional reverse transcription-polymerase chain reaction (Con RT-PCR) assay to detect norovirus is a complex multi-step procedure that requires gel electrophoresis as well as hybridization or sequencing to confirm the results.
To develop and evaluate a multiplex real time RT-PCR (Mrt RT-PCR) assay that detect and quantify norovirus GI and GII with a single amplification and detection step.
The primers and TaqMan probes for the Mrt RT-PCR were selected from the ORF1-ORF2 junction region. A total of 97 stools from 41 gastroenteritis outbreaks and 726 stools from children with sporadic diarrhoea were used for this study.
For the 97 outbreak samples, norovirus were detected in 61 of the 69 previously tested positive and 11 of the 28 previously tested negative samples. Eight samples that tested positive for GII by Con RT-PCR but negative by the Mrt RT-PCR also tested negative by a Light Cycler RT-PCR assay. Eighty-two GII and two GI were detected in the 726 sporadic samples. Random primers were more sensitive than specific primers in the cDNA synthesis. The two-step assay using the random primers in RT reaction was 100 times more sensitive than the one-step assay. The Mrt RT-PCR had the same sensitivity as that using two real time RT-PCR for separate detection of GI and GII. A wide dynamic range was obtained with the two-step assay, detecting from 3000 to 3x10(11) of copies RNA/g stool. Very good precision was observed with no cross-reaction with other enteric viruses. The new assay is able to detect both GI and GII in one reaction and brings a cost reduction of approximately 40% compared to separate reactions for GI and GII.
The assay has good precision, sensitivity and specificity and is cost-effective as a routine diagnostic test.