Donor Cytomegalovirus Transmission Patterns in Solid Organ Transplant Recipients With Primary Infection.

BACKGROUND: The epidemiology of single versus multiple cytomegalovirus (CMV) strain transmission from donor (D+) to seronegative solid organ transplant (SOT) recipients (R-) is uncertain, as is whether "relapsing" recipient infection represents changing strain predominance when multiple strains are transmitted. Here we characterized CMV strain transmission patterns in D+/R- SOT recipients. METHODS: We studied pairs or groups of D+/R- SOT recipients who received organs from a common donor (group A) and recipients who experienced ≥2 waves of CMV DNAemia (group B). CMV in plasma was characterized by genotype-specific real-time PCR for genes gB and gH. RESULTS: Single concordant genotypes were identified in 12 of 18 recipient pairs/group sharing a common donor (group A); at least 6 of 18 (33%) donors transmitted > 1 strain. A single CMV strain was detected in 14 of 15 recipients in group B; only 1 recipient had coinfection. A shift in CMV strain predominance occurred after the first posttransplant year in at least 4 recipients with coinfection. CONCLUSIONS: Using a common donor approach, we confirmed that multiple CMV strain transmission from donors to R- SOT recipients is not uncommon. D+/R- SOT recipients with CMV coinfection can undergo changes in strain predominance in late waves of CMV DNAemia.

Changes in norovirus genotype diversity in gastroenteritis outbreaks in Alberta, Canada: 2012-2018.

BACKGROUND: The emergence of norovirus genotype GII.4 variants has been associated with gastroenteritis pandemics worldwide, prompting molecular surveillance for early detection of novel strains. In this study, we aimed to analyze the outbreak activity of norovirus and characterize the norovirus strains circulating in Alberta between July 2012 and February 2018. METHODS: Stool samples from gastroenteritis outbreaks in Alberta were tested for norovirus at the Provincial Laboratory for Public Health using a multiplex real time-RT PCR assay. The ORF1 and ORF2-genotypes of norovirus positive samples were assigned based on phylogenetic analyses of partial polymerase and capsid sequences, respectively. RESULTS: A total of 530 norovirus outbreaks were identified. During July 2012 and June 2017 there was a gradual decrease in the annual number of GII.4 outbreaks, however, outbreak numbers increased from June 2017-February 2018. Four novel strains emerged: GII.17 Kawasaki in July 2014-June 2015, GII.P16/GII.4 Sydney in July 2015-June 2016, GII.P16/GII.2 and GII.P4 New Orleans/GII.4 Sydney in July 2016-June 2017. GII.Pe/GII.4 Sydney was the single predominant strain responsible for the majority (over 50%) of all norovirus outbreaks up to June 2015. Between June 2017 and February 2018, GII.P16/GII.4 Sydney was the leading strain causing 63% of all norovirus outbreaks. CONCLUSIONS: GII.4 stands as the predominant capsid genotype causing a large majority of the norovirus outbreaks in early 2018. An increase in genotype diversity was observed in the last years, characterized by a high circulation of non-GII.4 strains and GII.4 recombinants.

Determination of the Biological Form of Human Cytomegalovirus DNA in the Plasma of Solid-Organ Transplant Recipients.

Background: Whether cytomegalovirus (CMV) DNA exists in plasma as virion-associated or free DNA is uncertain. Methods: An assay combining DNase I digestion and CMV quantitative polymerase chain reaction (DNase-CMV-qPCR) was developed to differentiate free naked DNA from virion DNA. One hundred three frozen and 10 fresh CMV DNA-positive plasma samples from solid-organ transplant recipients (SOTRs) were tested. Three sets of paired qPCR (P-qPCR) assays with amplicons of variable length were used to study CMV DNA fragmentation in 20 SOTR plasma samples, viral stocks (Towne, Merlin, AD169) and the first World Health Organization (WHO) international standard (IS) for CMV DNA. Results: In all plasma samples, 98.8%-100% of CMV DNA was free DNA; this was the only form in 93 of 103 (90.3%) frozen and all 10 fresh samples tested using DNase-CMV-qPCR. Low levels of virion CMV DNA were found in 10 of 103 (9.7%) samples with higher total DNA load. Cytomegalovirus DNA results were highly reproducible for 3 CMV virus stocks and WHO IS (P > .80), tested by three sets of paired q-PCR. However, for the 20 SOTR plasma samples, the smaller amplicon assay result was 2.6-fold, 3.4-fold, and 6.5-fold higher than the longer amplicion result (P < .001). Conclusions: Cytomegalovirus DNA in SOTR plasma is almost exclusively free DNA, highly fragmented, and not virion associated.

Global Spread of Norovirus GII.17 Kawasaki 308, 2014-2016.

Analysis of complete capsid sequences of the emerging norovirus GII.17 Kawasaki 308 from 13 countries demonstrated that they originated from a single haplotype since the initial emergence in China in late 2014. Global spread of a sublineage SL2 was identified. A new sublineage SL3 emerged in China in 2016.

Are We There Yet? Impact of the First International Standard for Cytomegalovirus DNA on the Harmonization of Results Reported on Plasma Samples.

BACKGROUND: Interassay harmonization of cytomegalovirus (CMV) DNA measurement is important for infection management. Uncertainty exists regarding the result harmonization achievable in patient plasma samples using quantitative polymerase chain reaction (qPCR) assays with calibrators now traceable to the First World Health Organization International Standard (IS) for CMV DNA. METHOD: Serial dilutions of the IS and a blinded panel of 40 genotyped CMV DNA-positive pooled plasma samples and 10 negative plasma samples were tested by 6 laboratories using 10 qPCR assays calibrated to the IS. Each clinical sample was constructed using plasma from a single unique transplant recipient. RESULTS: The variance for individual CMV DNA-positive samples was greater for clinical samples (median, 1.50 [range, 1.22-2.82] log10 IU/mL) than for IS dilutions (median, 0.94 [range, 0.69-1.35] log10 IU/mL) (P < .001); 58.9% of all clinical sample results and 93.6% of IS dilution results fell within ±0.5 log10 IU/mL of the mean viral load of each sample. Result variability was not impacted by either genotype or quantitative levels of CMV DNA. Testing procedure differences can significantly influence results, even when analyte-specific reagents are identical. For clinical samples, all assays demonstrated result bias (P < .008). Assays with amplicon sizes ≤86 bp had significantly higher results compared to assays with larger amplicon sizes (≥105 bp) (P < .001). CONCLUSIONS: The variability in CMV DNA results reported on individual samples has been reduced by the IS, but ongoing clinically relevant variability persists, preventing meaningful interassay result comparison.

A next generation sequencing-based method to study the intra-host genetic diversity of norovirus in patients with acute and chronic infection.

BACKGROUND: Immunocompromised individuals with chronic norovirus (NoV) infection and elderly patients are hypothesized to be reservoirs where NoV might accumulate mutations and evolve into pandemic strains. Next generation sequencing (NGS) methods can monitor the intra-host diversity of NoV and its evolution but low abundance of viral RNA results in sub-optimal efficiency. In this study, we: 1) established a next generation sequencing-based method for NoV using bacterial rRNA depletion as a viral RNA enrichment strategy, and 2) measured the intra-host genetic diversity of NoV in specimens of patients with acute NoV infection (n = 4) and in longitudinal specimens of an immunocompromised patient with chronic NoV infection (n = 2). RESULTS: A single Illumina MiSeq dataset resulted in near full-length genome sequences for 5 out of 6 multiplexed samples. Experimental depletion of bacterial rRNA in stool RNA provided up to 1.9 % of NoV reads. The intra-host viral population in patients with acute NoV infection was homogenous and no single nucleotide variants (SNVs) were detected. In contrast, the NoV population from the immunocompromised patient was highly diverse and accumulated SNVs over time (51 SNVs in the first sample and 122 SNVs in the second sample collected 4 months later). The percentages of SNVs causing non-synonymous mutations were 27.5 % and 20.5 % for the first and second samples, respectively. The majority of non-synonymous mutations occurred, in increasing order of frequency, in p22, the major capsid (VP1) and minor capsid (VP2) genes. CONCLUSIONS: The results provide data useful for the selection and improvement of NoV RNA enrichment strategies for NGS. Whole genome analysis using next generation sequencing confirmed that the within-host population of NoV in an immunocompromised individual with chronic NoV infection was more diverse compared to that in individuals with acute infection. We also observed an accumulation of non-synonymous mutations at the minor capsid gene that has not been reported in previous studies and might have a role in NoV adaptation.

Enhanced enteric virus detection in sporadic gastroenteritis using a multi-target real-time PCR panel: a one-year study.

Viral gastroenteritis causes significant mortality and morbidity worldwide. Identifying the etiology of viral gastroenteritis is a challenge as most enteric viruses (EVs) are non-culturable. This study is to develop an EV testing panel using real-time PCR (EVPrtPCR) to simultaneously detect rotavirus, norovirus, sapovirus, astrovirus, and enteric adenovirus in stool samples. EVPrtPCR using universal amplification conditions was run in a single instrument run. EVPrtPCR was used to test 2,486 sporadic gastroenteritis samples submitted for EV testing using electron microscopy (EM) between July 2008 and July 2009. Retesting spiked negative stool samples and Salmon DNA as internal control were used to evaluate inhibition. EVPrtPCR detected viruses in significantly more samples: 748 (34%) as compared to 94 (3.8%) by EM. EM did not detect any norovirus, sapovirus, and mixed infection, and detected only 39% of rotavirus and 38.2% of enteric adenovirus positive samples. Four samples that tested positive for rotavirus and two for adenovirus and for small-round-structured viruses by EM were negative by EVPrtPCR. Norovirus was the most common virus detected (17.6%) with 92.4% as genogroup II, followed by rotavirus (6.8%), sapovirus (4.2%), astrovirus (2.0%), and enteric adenovirus (1.4%) with 9% samples positive for mixed infection. Overall, EV identification followed a U-shaped age distribution; positive samples were more common in children ≤5 years old and adults >60 years old. Norovirus, sapovirus and astrovirus showed winter predominance and rotavirus peaked in the spring. No inhibition was observed. Molecular technology significantly enhanced the identification of EV causing sporadic gastroenteritis in Alberta.

Emergence of a new norovirus GII.4 variant and changes in the historical biennial pattern of norovirus outbreak activity in Alberta, Canada, from 2008 to 2013.

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.

Wastewater surveillance monitoring of SARS-CoV-2 variants of concern and dynamics of transmission and community burden of COVID-19.

Wastewater-based surveillance is a valuable approach for monitoring COVID-19 at community level. Monitoring SARS-CoV-2 variants of concern (VOC) in wastewater has become increasingly relevant when clinical testing capacity and case-based surveillance are limited. In this study, we ascertained the turnover of six VOC in Alberta wastewater from May 2020 to May 2022. Wastewater samples from nine wastewater treatment plants across Alberta were analysed using VOC-specific RT-qPCR assays. The performance of the RT-qPCR assays in identifying VOC in wastewater was evaluated against next generation sequencing. The relative abundance of each VOC in wastewater was compared to positivity rate in COVID-19 testing. VOC-specific RT-qPCR assays performed comparatively well against next generation sequencing; concordance rates ranged from 89% to 98% for detection of Alpha, Beta, Gamma, Omicron BA.1 and Omicron BA.2, with a slightly lower rate of 85% for Delta (p < 0.01). Elevated relative abundance of Alpha, Delta, Omicron BA.1 and BA.2 were each associated with increased COVID-19 positivity rate. Alpha, Delta and Omicron BA.2 reached 90% relative abundance in wastewater within 80, 111 and 62 days after their initial detection, respectively. Omicron BA.1 increased more rapidly, reaching a 90% relative abundance in wastewater after 35 days. Our results from VOC surveillance in wastewater correspond with clinical observations that Omicron is the VOC with highest disease burden over the shortest period in Alberta to date. The findings suggest that changes in relative abundance of a VOC in wastewater can be used as a supplementary indicator to track and perhaps predict COVID-19 burden in a population.

Differences in Illness Severity among Circulating Norovirus Genotypes in a Large Pediatric Cohort with Acute Gastroenteritis.

Norovirus is a major pathogen identified in children with acute gastroenteritis (AGE), little is known about the strain's diversity and their clinical severity. Stool and/or rectal swabs were collected from children ≤18 years of age recruited at emergency departments (ED), and a provincial nursing advice phone line due to AGE symptoms in the province of Alberta, Canada between December 2014 and August 2018. Specimens were tested using a reverse transcription real time PCR and genotyped by Sanger sequencing. The Modified Vesikari Scale score (MVS) was used to evaluate the disease severity. The objectives are to identify the Genogroup and Genotype distribution and to compare illness severity between the GI and GII genogroups and to complete further analyses comparing the GII genotypes identified. GII.4 was the genotype most commonly identified. Children with GII.4 had higher MVS scores (12.0 (10.0, 14.0; p = 0.002)) and more prolonged diarrheal (5 days (3.0, 7.8)) and vomiting (3.2 days (1.7, 5.3; p < 0.001)) durations compared to other non GII.4 strains. The predominant strain varied by year with GII.4 Sydney[P31] predominant in 2014/15, GII.4 Sydney[P16] in 2015/16 and 2017/18, and GII.3[P12] in 2016/17. Genogroup II norovirus strains predominated in children with AGE with variance between years; clinical severity associated with different strains varied with episodes being most severe among GII.4 infected children.

Multicenter comparison of two norovirus ORF2-based genotyping protocols.

Point source norovirus outbreaks can be difficult to track due to high background levels of the virus in the environment and the limited strain variation in some genotyping regions. However, rapid and accurate source identification can limit the spread of a foodborne outbreak and reduce the number of cases. Harmonization of genotyping assays is critical for enabling the rapid exchange of sequence data nationally and internationally. Several regions of the genome have been proposed for this purpose, but no consensus has been reached. In the present study, two standardized genotyping protocols (region C and region D) were evaluated by nine laboratories in Canada and the United States, using a coded panel of 96 fecal specimens representing 22 different norovirus genotypes. Overall, region C typing had a success rate of 78% compared to 52% for region D; however, region D provides greater nucleotide sequence diversity for identifying new GII.4 variant strains. Significant differences in the genotyping success rate were observed among the nine participating laboratories (10% to 100%) and among the different genotypes (6% to 100%). For several genogroup II strains, reduced region D amplification correlated directly with mismatches between primer sequences and the template. Based on overall performance, we recommend the region C protocol for routine genotyping of noroviruses, while the region D protocol may be useful for identifying new GII.4 variants. Standardized genotyping protocols will enable rapid exchange of outbreak and sequence data through electronic norovirus surveillance networks.

Prospective monitoring of BK polyomavirus infection early posttransplantation in nonrenal solid organ transplant recipients.

BACKGROUND: BK virus-associated nephropathy is an important cause of renal dysfunction in renal transplant recipients. Renal dysfunction after nonrenal solid organ transplantation (NRSOT) is common; however, the impact of BK virus remains uncertain. METHODS: Sixty (7 heart, 25 liver, and 28 lung) NRSOT recipients were enrolled in this single center prospective longitudinal study. Urine and plasma were collected for detection of BK viral load using a real-time quantitative polymerase chain reaction assay at transplantation and at 3, 6, and 9 months posttransplantation. Demographic and clinical data including serum creatinine and immunosuppressive therapy were also collected. RESULTS: BK viruria was detected in 16 of 193 (8.3%) samples corresponding to 9 of 60 (15%) subjects. The median BK viral load was 1.12 x 10 (range, 1.1 x 10-2.66 x 10) copies per milliliter. No viremia was detected. In seven of nine, viruria occurred by 3 months posttransplantation. At 9 months of posttransplantation, the median Modification of Diet in Renal Disease-estimated glomerular filtration rate in those with BK viruria on at least one sample was similar to those without viruria (58.0 [IQR 43.1-60.7] mL/min/1.73 m vs. 61.4 [IQR 50.6-74.4] mL/min/1.73 m; P=0.39). CONCLUSIONS: Although BK infection was common in this NRSOT population, BK viremia was not observed and there was no association between BK viruria and renal dysfunction. Our data suggest that routine surveillance for BK virus early posttransplantation in NRSOT may not be warranted but should be further examined in a larger multicenter trial.

Chronic norovirus and adenovirus infection in a solid organ transplant recipient.

A 10-month-old boy developed chronic diarrhea 2 months after a combined liver, pancreas, and small bowel transplant. Norovirus and adenovirus were detected in multiple stool specimens during a 114-day period. Enteric viral infectious should be considered in solid organ transplant recipients with chronic diarrhea.

The use of unprocessed urine samples for detecting and monitoring BK viruses in renal transplant recipients by a quantitative real-time PCR assay.

Results of quantitative BK viral load using real-time quantitative PCR (rt-QPCR) were compared using two types of samples, extracted urine DNA and unprocessed urine. An excellent correlation was observed in quantitative viral load between unprocessed urine and extracted urine DNA samples. (R2=0.96, p<0.001). Compared to extracted urine DNA when a small sample volume of unprocessed urine was used (2 microl per PCR reaction), 100% concordance is detection of BKV DNA was observed in 124 samples (106 positive and 18 negative) collected from renal transplant recipient (RTR). There was no significant difference in the quantitative BK viral load (log10 copies/ml) detected in extracted urine DNA (median=7.82) compared to unprocessed urine (median=7.17). Urine pH in the range of 5.2-7.1 and specimen freezing had no effect on the rt-QPCR reaction. The partial inhibition of the rt-QPCR reaction observed when 5 microl sample volume of unprocessed urine was used was markedly reduced at a sample volume of 2 microl. Using unprocessed urine for rt-QPCR detection of BK viral load is cost-saving while maintaining the sensitivity and accuracy associated with the use of extracted urine DNA, making a clinical BKV surveillance strategy in RTR based on urinary sample screening using rt-QPCR as the first line test more feasible.

Multiplex real time RT-PCR for the detection and quantitation of norovirus genogroups I and II in patients with acute gastroenteritis.

BACKGROUND: 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. OBJECTIVE: 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. STUDY DESIGN: 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. RESULTS: 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. CONCLUSIONS: The assay has good precision, sensitivity and specificity and is cost-effective as a routine diagnostic test.

Rapid genotyping of human rotavirus using SYBR green real-time reverse transcription-polymerase chain reaction with melting curve analysis.

AIM: To develop a real-time reverse transcription-polymerase chain reaction (RT-PCR) assay to genotype rotavirus (G and P) in Alberta from January 2012 to June 2013. METHODS: We developed and validated a different approach to perform rotavirus G and P genotyping using a two-step SYBR green RT-PCR (rt-gPCR) by selecting genotype-specific primers of published conventional RT nested PCR (cnRT-PCR) assay and optimizing the amplification conditions. cDNA was first synthesized from total RNA with SuperScript™ II reverse transcriptase kit followed by amplication step using monoplex SYBR green real-time PCR. After the PCR reaction, melting curve analysis was used to determine specific genotype. Sixteen samples previously genotyped using cnRT-PCR were tested using the new assay and the genotyping results were compared as sensitivity analysis. Assay specificity was evaluated by testing other gastroenteritis viruses with the new assay. The amplicon size of each available genotype was determined by gel-electrophoresis and DNA sequences were obtained using Sanger-sequencing method. After validation and optimization, the new assay was used to genotype 122 pediatric clinical stool samples previously tested positive for rotavirus using electron microscopy between January 2012 and June 2013. RESULTS: The new rt-gPCR assay was validated and optimized. The assay detected G1 to G4, G9, G12 and P[4] and P[8] that were available as positive controls in our laboratory. A single and clear peak of melting curve was generated for each of specific G and P genotypes with a Tm ranging from 80 °C to 82 °C. The sensitivity of rt-gPCR was comparable to cnRT-PCR with 100% correlation of the 16 samples with known G and P genotypes. No cross reaction was found with other gastroenteritis viruses. Using the new rt-gPCR assay, genotypes were obtained for 121 of the 122 pediatric clinical samples tested positive for rotavirus: G1P[8] (42.6%), G2P[4] (4.9%), G3P[8] (10.7%), G9P[8] (10.7%), G9P[4] (6.6%), G12P[8] (23.0%), and unknown GP[8] (0.8%). For the first time, G12 rotavirus strains were found in Alberta and G12 was the second most common genotype during the study period. Gel electrophoresis of all the genotypes showed expected amplicon size for each genotype. The sequence data of the two G12 samples along with other genotypes were blasted in NCBI BLAST or analyzed with Rota C Genotyping tool (http://rotac.regatools.be/). All genotyping results were confirmed to be correct. CONCLUSION: rt-gPCR is a useful tool for the genotyping and characterization of rotavirus. Monitoring of rotavirus genotypes is important for the identification of emerging strains and ongoing evaluation of rotavirus vaccination programs.

Detection and analysis of recombination in GII.4 norovirus strains causing gastroenteritis outbreaks in Alberta.

Recombination is an important mechanism generating genetic diversity in norovirus (NoV) that occurs commonly at the NoV polymerase-capsid (ORF1/2) junction. The genotyping method based on partial ORF2 sequences currently used to characterize circulating NoV strains in gastroenteritis outbreaks in Alberta cannot detect such recombination events and provides only limited information on NoV genetic evolution. The objective of this study was to determine whether any NoV GII.4 strains causing outbreaks in Alberta are recombinants. Twenty stool samples collected during outbreaks occurring between July 2004 and January 2012 were selected to include the GII.4 variants Farmington Hills 2002, Hunter 2004, Yerseke 2006a, Den Haag 2006b, Apeldoorn 2007, New Orleans 2009, and Sydney 2012 based on previous NoV ORF2-genotyping results. Near full-length NoV genome sequences were obtained, aligned with reference sequences from GenBank and analyzed with RDPv4.13. Two sequences corresponding to Apeldoorn 2007, and Sydney 2012 were identified as recombinants with breakpoints near the ORF1/2 junction and putative parental strains as previously reported. We also identified, for the first time, a non-recombinant sequence resembling the ORF2-3 parent of the recombinant cluster Sydney 2012 responsible for the most recent pandemic. Our results confirmed the presence of recombinant NoV GII.4 strains in Alberta, and highlight the importance of including additional genomic regions in surveillance studies to trace the evolution of pandemic NoV GII.4 strains.

Pre-analytical and analytical procedures for the detection of enteric viruses and enterovirus in water samples.

Practical pre-analytical and analytical procedures were developed and validated for detection of enteric viruses in three water matrices. Both RNA viruses (norovirus, coxsackievirus, echovirus, and rotavirus) and DNA virus (adenovirus 41) were included in the study. The NanoCeram 90mm laminated disc with electropositive filter and procedures of filtration, elution and flocculation were utilized to concentrate known amount of viruses in different water matrices. Real time quantitative PCR was used to evaluate the recovery of virus and cell culture to assess viral infectivity. There was no PCR inhibition using various concentrations and pH of beef extract eluting buffer. A good recovery of the viruses spiked in 10L of deionized water was achieved for serial dilutions of coxsackievirus (41-67%), echovirus (22-90%), norovirus (23-44%) and rotavirus (24-46%). Relatively lower recovery was observed for adenovirus 41 (24-35%). There was no significant difference in viral recovery from deionized, tap and river water samples. The infectivity of recovered adenovirus, coxsackievirus and echovirus was demonstrated using in vitro cell culture. The pre-analytical and analytic procedures attained consistent recovery of RNA and DNA viruses both as infectious viral particles and viral genome, provided effective removal of inhibitory substances, achieved reliable reproducibility, and were relatively inexpensive for monitoring viruses in water.

Influence of novel norovirus GII.4 variants on gastroenteritis outbreak dynamics in Alberta and the Northern Territories, Canada between 2000 and 2008.

BACKGROUND: Norovirus GII.4 is the predominant genotype circulating worldwide over the last decade causing 80% of all norovirus outbreaks with new GII.4 variants reported in parallel with periodic epidemic waves of norovirus outbreaks. The circulating new GII.4 variants and the epidemiology of norovirus outbreaks in Alberta, Canada have not been described. Our hypothesis is that the periodic epidemic norovirus outbreak activity in Alberta was driven by new GII.4 variants evolving by genetic drift. METHODOLOGY/PRINCIPAL FINDINGS: The Alberta Provincial Public Health Laboratory performed norovirus testing using RT-PCR for suspected norovirus outbreaks in the province and the northern Territories between 2000 and 2008. At least one norovirus strain from 707 out of 1,057 (66.9%) confirmed norovirus outbreaks were successfully sequenced. Phylogenetic analysis was performed using BioNumerics and 617 (91.1%) outbreaks were characterized as caused by GII.4 with 598 assigned as novel variants including: GII.4-1996, GII.4-2002, GII.4-2004, GII.4-2006a, GII.4-2006b, GII.4-2008a and GII.4-2008b. Defining July to June of the following year as the yearly observation period, there was clear biannual pattern of low and high outbreak activity in Alberta. Within this biannual pattern, high outbreak activity followed the emergence of novel GII.4 variants. The two variants that emerged in 2006 had wider geographic distribution and resulted in higher outbreak activity compared to other variants. The outbreak settings were analyzed. Community-based group residence was the most common for both GII.4 variants and non-GII.4 variants. GII.4 variants were more commonly associated with outbreaks in acute care hospitals while outbreaks associated non-GII.4 variants were more commonly seen in school and community social events settings (p<0.01). CONCLUSIONS/SIGNIFICANCE: The emergence of new norovirus GII.4 variants resulted in an increased norovirus outbreak activity in the following season in a unique biannual pattern in Alberta over an eight year period. The association between antigenic drift of GII.4 strains and epidemic norovirus outbreak activity could be due to changes in host immunity, viral receptor binding efficiency or virulence factors in the new variants. Early detection of novel GII.4 variants provides vital information that could be used to forecast the norovirus outbreak burden, enhance public health preparedness and allocate appropriate resources for outbreak management.

Epidemiology and genotype analysis of sapovirus associated with gastroenteritis outbreaks in Alberta, Canada: 2004-2007.

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.