Detection of Enteric Viruses in Surface (Source) Water and Groundwater in Alberta, Canada

Transcription

1 Detection of Enteric Viruses in Surface (Source) Water and Groundwater in Alberta, Canada XL Pang 1,2, BE Lee 2, D Mooney 3, S Gabos 2, S Craik 4, R Zurawell 5, and N Neumann 1,2 1 Provincial Public Health Laboratory, Alberta, 2 University of Alberta, 3 Alberta Health, Alberta, 4 EPCOR, Edmonton, Canada, 5 Alberta Environment and Sustainable Resource Development, Alberta, Canada

2 Background-why interest? Because the viruses are: Extremely contagious Diverse transmission pathways High human gastroenteritis burden The candidates -The US EPA Likely underestimated A novel panel assay is available

3 Objectives To determine the prevalence and level of selected viruses in surface/source water and groundwater in Alberta To provide the baseline data of the viruses in surface/source water and groundwater for evidence-based review/development of the guideline for drinking water safety

4 Human viruses targets Enteric viruses Human caliciviruses (Norovirus & Sapovirus): +ssrna viruses, non-cultivable Rotavirus: dsrna virus, cultivable but grows extremely slowly Reovirus: dsrna virus, grown in BGM and MA104 cell lines Astrovirus: +ssrna virus, not grown in ordinary cells Human adenoviruses: dsdna virus, grown well in BGM and AM104 cell lines Enteroviruses Human coxsackievirus: +ssrna viruses, grown in the BGM cell line Human echoviruses: +ssrna viruses, grown in the BGM cell line Polyomavirus JC virus and BK virus: double-stranded circular DNA viruses. JC virus is a human virus causing latent and chronic infections. Recently JC virus was found in 98% to 100% of sewage samples and was proposed to be an indicator of human fecal contamination (Bofill-Mas et all 2006)

5 Sample preparation and detection Step 1-Viral concentration: Filtration Elution Flocculation Step 2-Viral detection: Real time PCR for quantification of the viruses Cell culture for identification of infectious viral particles Integration of cell culture and PCR (ICC-PCR)

6 Concentration of viruses 1-2 hrs Filtration NanoCeram laminated filter Capacity: L Virus Concentration 0.5-1hr Elution 1.0 L of 1.5% BE, ph hrs Flocculation 15 ml concentrated viral specimen

7 WSD for on-field filtration of large volume water sample The NanoCeram VS2.5-5 cartridge

8 Instruments used for concentration of viruses

9 Detection and quantification ABI 7500 Concentrated Specimen Extraction of viral nucleic acid Amplification and detection Hours CT value Standard Curve for TCID 50 IU/ml log10 coxsackievirus Cycle Number

10 Viral cell culture Concentrated Specimen Identifying of infectious viruses CPE positive - detection of infectious viruses CPE negative - no infectious viruses

11 Integrated viral culture with PCR (ICC-PCR) To enhance the sensitivity for infectious virus To confirm the presence of specific viruses

12 Detection of viruses in surface/source water in Alberta 20 liters of water was collected from each site monthly (Jun 2012 May 2013) A total of 216 surface/source water samples were collected from 18 locations across the province in this study

13 Results-Detection of viruses Overall, 91% (197/216) samples tested positive for one or more viruses Virus Positive (%) Range of positive % in the 18 sampling sites Rotavirus 158 (88%) (%) Adenovirus 87 (48%) (%) Sapovirus 60 (33%) 0-70 (%) Norovirus 57 (32%) 0-70 (%) Astrovirus 56 (31%) 0-90 (%) JC virus 52 (29%) 0-90 (%) Enterovirus 27 (15%) 0-50 (%) Cell ulture 21 (12%) 0-40 (%)

14 Results-Quantitation of viruses Copies/Llog10 NoV RotaV SapV AstroV EnteroV AdV JCV

15 Results-Seasonality-1 No. sample Monthy

16 Results-Seasonality-2 No. Sample Monthy

17 Groundwater collection 500 liters of well-water was filtrated through viral capture filter cartridge at each well biweekly from Jun to Oct, 2013 in Bonneville area in Northern Alberta. A total of 54 samples were processed from 5 wells in liters of well-water was filtrated through viral capture filter cartridge at each well biweekly from Jun to Sept, 2014 in rural areas surrounding Edmonton. A total of 46 samples were processed from 8 wells in 2014.

18 Summary of the viruses in groundwater Tested samples and wells in 2013: Viruses (at least one in the testing panel) were detected in 6 out of 54 samples and the positive rate was 11% of all samples tested in 5 wells. The viruses were frequently detectable in 2 wells (#1 and #5). Tested samples and wells in 2014: Viruses (at least one in the testing panel) were detected in 10 out of 46 samples and the positive rate was 22% of all samples. The viruses were detectable in 4 of the 8 wells (#2, #3, #5 and #6) and the positive rate was 50% of the wells tested. Cell culture was negative for all samples tested. Of samples with virus, rotavirus was the most commonly found followed by adenovirus. Sapovirus and astrovirus co-existed with rotavirus at various frequencies in No norovirus, enterovirus and JC virus was found in samples tested in 2013 & 2014.

19 Detection of viruses from groundwater Well Bonneville area (Jun to Oct 2013) Rural area of Edmonton (Jun to Sep 2014) Sample # PCR Positive (%) Culture positive Sample # PCR Positive (%) Culture positive (19%) (17%) (14%) (25%) (40%) (100%) Total 54 6 (11%) (22%) 0

20 Conclusions Our new method of integrated real-time PCR with viral culture was established with good sensitivity, specificity, rapid detection, excellent precision and is cost effectiveness for rapid and quantitative measurement of enteric viruses and other viruses in various water matrices. Viruses were widely distributed in surface/source and groundwater water systems for recreation and drinking use in Alberta. The presence of viruses in aquatic environment under ambient temperature represents a reservoir for potential transmission and risk for gastroenteritis. Further studies on the association of enteric viruses in surface water/groundwater and gastroenteritis is needed to assess the risks to public health.

21 Acknowledgement Min Cao for her technical Support All staff of ProvLab environmental laboratory for helping with sample transportation Jessica Popadynetz for providing groundwater information This was a collaborative study with Alberta Health, Alberta Health Services, University of Alberta and Alberta Environment and Sustainable Resource and Development, Long Term River Network, Alberta Environment and EPCOR The Project was funded by Alberta Health under the Framework of Alberta Water for Life Strategies