Wastewater represents the main reusable water source after being adequately sanitized by wastewater treatment plants (WWTPs). In this sense, only bacterial quality indicators are usually checked to this end, and human pathogenic viruses usually escape from both sanitization procedures and controls, posing a health risk on the use of effluent waters. In this study, we evaluated a protocol based on aluminum adsorption–precipitation to concentrate several human enteric viruses, including norovirus genogroup I (NoV GI), NoV GII, hepatitis A virus (HAV), astrovirus (HAstV), and rotavirus (RV), with limits of detection of 4.08, 4.64, 5.46 log genomic copies (gc)/L, 3.31, and 5.41 log PCR units (PCRU)/L, respectively. Furthermore, the method was applied in two independent laboratories to monitor the presence of NoV GI, NoV GII, and HAV in effluent and influent waters collected from five WWTPs at two different sampling dates. Concomitantly, a viability PMAxx-RT-qPCR was applied to all the samples to get information on the potential infectivity of both influent and effluent waters. The ranges of the titers in influent waters for NoV GI, NoV GII, RV, and HAstV were 4.80–7.56, 5.19–7.31 log gc/L, 5.41–6.52, and 4.59–7.33 log PCRU/L, respectively. In effluent waters, the titers ranged between 4.08 and 6.27, 4.64 and 6.08 log gc/L, < 5.51, and between 3.31 and 5.58 log PCRU/L. Moreover, the viral titers detected by viability RT-qPCR showed statistical differences with RT-qPCR alone, suggesting the potential viral infectivity of the samples despite some observed reductions. The proposed method could be applied in ill-equipped laboratories, due to the lack of a requirement for a specific apparatus (i.e., ultracentrifuge).
This is a preview of subscription content, access via your institution.
Buy single article
Instant access to the full article PDF.
Tax calculation will be finalised during checkout.
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
Tax calculation will be finalised during checkout.
Ashbolt, N. J. (2015). Microbial contamination of drinking water and human health from community water systems. Current Environmental Health Reports,2, 95–106. https://doi.org/10.1007/s40572-014-0037-5.
Aw, T. G., & Rose, J. B. (2012). Detection of pathogens in water: From phylochips to qPCR to pyrosequencing. Current Opinion in Biotechnology,23(3), 422–430. https://doi.org/10.1016/j.copbio.2011.11.016.
Barrios, M. E., Blanco Fernández, M. D., Cammarata, R. V., Torres, C., & Mbayed, V. A. (2018). Viral tools for detection of fecal contamination and microbial source tracking in wastewater from food industries and domestic sewage. Journal of Virological Methods,262, 79–88. https://doi.org/10.1016/j.jviromet.2018.10.002.
Becerra-Castro, C., Lopes, A. R., Vaz-Moreira, I., Silva, E. F., Manaia, C. M., & Nunes, O. C. (2015). Wastewater reuse in irrigation: A microbiological perspective on implications in soil fertility and human and environmental health. Environment International,75, 117–135. https://doi.org/10.1016/j.envint.2014.11.001.
Borgmästars, E., Jazi, M. M., Persson, S., Jansson, L., Rådström, P., Simonsson, M., et al. (2017). Improved detection of norovirus and hepatitis A virus in surface water by applying pre-PCR processing. Food and Environmental Virology,9(4), 395–405.
Carvajal, G., Roser, D. J., Sisson, S. A., Keegan, A., & Khan, S. J. (2017). Bayesian belief network modelling of chlorine disinfection for human pathogenic viruses in municipal wastewater. Water Research,109, 144–154. https://doi.org/10.1016/j.watres.2016.11.008.
Cashdollar, J. L., & Wymer, L. (2013). Methods for primary concentration of viruses from water samples: A review and meta-analysis of recent studies. Journal of Applied Microbiology,115(1), 1–11. https://doi.org/10.1111/jam.12143.
Condit, R. C. (2013). Principles of virology. In D. M. Knipe & P. M. Howley (Eds.), Fields virology (pp. 21–25). Philadelphia: Wolters Klewer/Lippincott Williams & Wilkins.
Coudray-Meunier, C., Fraisse, A., Martin-Latil, S., Guillier, L., & Perelle, S. (2013). Discrimination of infectious hepatitis A virus and rotavirus by combining dyes and surfactants with RT-qPCR. BMC Microbiology,13(1), 216. https://doi.org/10.1186/1471-2180-13-216.
D’Ugo, E., Marcheggiani, S., Fioramonti, I., Giuseppetti, R., Spurio, R., Helmi, K., et al. (2016). Detection of human enteric viruses in freshwater from European countries. Food and Environmental Virology,8(3), 206–214.
da Silva, Le, Guyader, F. S., Le Saux, J. C., Pommepuy, M., Montgomery, M. A., & Elimelech, M. (2008). Norovirus removal and particle association in a waste stabilization pond. Environmental Science and Technology,42(24), 9151–9157. https://doi.org/10.1021/es802787v.
da Silva, Le, Saux, J.-C., Parnaudeau, S., Pommepuy, M., Elimelech, M., & Le Guyader, F. S. (2007). Evaluation of removal of noroviruses during wastewater treatment, using real-time reverse transcription-PCR: Different behaviors of genogroups I and II. Applied and Environmental Microbiology,73(24), 7891–7897. https://doi.org/10.1128/aem.01428-07.
Dias, E., Ebdon, J., & Taylor, H. (2019). Estimating the concentration of viral pathogens and indicator organisms in the final effluent of wastewater treatment processes using stochastic modelling. Microbial Risk Analysis,11, 47–56. https://doi.org/10.1016/j.mran.2018.08.003.
EPA. (2016). Drinking Water Contaminant Candidate List 4-Final. (2016-27667). Retrieved from https://www.federalregister.gov/documents/2016/11/17/2016-27667/drinking-water-contaminant-candidate-list-4-final.
Farkas, K., Marshall, M., Cooper, D., McDonald, J. E., Malham, S. K., Peters, D. E., et al. (2018). Seasonal and diurnal surveillance of treated and untreated wastewater for human enteric viruses. Environmental Science and Pollution Research,25(33), 33391–33401. https://doi.org/10.1007/s11356-018-3261-y.
Gerba, C. P., Betancourt, W. Q., & Kitajima, M. (2017). How much reduction of virus is needed for recycled water: A continuous changing need for assessment? Water Research,108, 25–31. https://doi.org/10.1016/j.watres.2016.11.020.
Gerba, C. P., Betancourt, W. Q., Kitajima, M., & Rock, C. M. (2018). Reducing uncertainty in estimating virus reduction by advanced water treatment processes. Water Research,133, 282–288. https://doi.org/10.1016/j.watres.2018.01.044.
Gyawali, P., & Hewitt, J. (2018). Detection of infectious noroviruses from wastewater and seawater using PEMAXTM treatment combined with RT-qPCR. Water,10(7), 841. https://doi.org/10.3390/w10070841.
Hamza, I. A., Jurzik, L., Überla, K., & Wilhelm, M. (2011). Methods to detect infectious human enteric viruses in environmental water samples. International Journal of Hygiene and Environmental Health,214, 424–436. https://doi.org/10.1016/j.ijheh.2011.07.014.
Haramoto, E., Fujino, S., & Otagiri, M. (2015). Distinct behaviors of infectious F-specific RNA coliphage genogroups at a wastewater treatment plant. Science of the Total Environment,520, 32–38. https://doi.org/10.1016/j.scitotenv.2015.03.034.
Haramoto, E., Katayama, H., Oguma, K., Yamashita, H., Tajima, A., Nakajima, H., et al. (2006). Seasonal profiles of human noroviruses and indicator bacteria in a wastewater treatment plant in Tokyo, Japan. Water Science and Technology,54(11–12), 301–308. https://doi.org/10.2166/wst.2006.888.
Haramoto, E., Kitajima, M., Hata, A., Torrey, J. R., Masago, Y., Sano, D., et al. (2018). A review on recent progress in the detection methods and prevalence of human enteric viruses in water. Water Research,135, 168–186. https://doi.org/10.1016/j.watres.2018.02.004.
Hewitt, J., Leonard, M., Greening, G. E., & Lewis, G. D. (2011). Influence of wastewater treatment process and the population size on human virus profiles in wastewater. Water Research,45(18), 6267–6276. https://doi.org/10.1016/j.watres.2011.09.029.
Hill, V. R., Mull, B., Jothikumar, N., Ferdinand, K., & Vinjé, J. (2010). Detection of GI and GII noroviruses in ground water using ultrafiltration and TaqMan real-time RT-PCR. Food and Environmental Virology,2(4), 218–224.
Ikner, L. A., Gerba, C. P., & Bright, K. R. (2012). Concentration and recovery of viruses from water: A comprehensive review. Food and Environmental Virology,4(2), 41–67. https://doi.org/10.1007/s12560-012-9080-2.
Ikner, L. A., Soto-Beltran, M., & Bright, K. R. (2011). New method using a positively charged microporous filter and ultrafiltration for concentration of viruses from tap water. Applied and Environmental Microbiology,77(10), 3500–3506. https://doi.org/10.1128/AEM.02705-10.
ISO 15216–1:2017. (2017). Microbiology of food and animal feed—horizontal method for determination of Hepatitis A virus and norovirus in food using real-time RT-PCR—Part 1: Method for quantification. Geneva: Switzerland.
Katayama, H., Haramoto, E., Oguma, K., Yamashita, H., Tajima, A., Nakajima, H., et al. (2008). One-year monthly quantitative survey of noroviruses, enteroviruses, and adenoviruses in wastewater collected from six plants in Japan. Water Research,42(6–7), 1441–1448. https://doi.org/10.1016/j.watres.2007.10.029.
Kazama, S., Masago, Y., Tohma, K., Souma, N., Imagawa, T., Suzuki, A., et al. (2016). Temporal dynamics of norovirus determined through monitoring of municipal wastewater by pyrosequencing and virological surveillance of gastroenteritis cases. Water Research,92, 244–253. https://doi.org/10.1016/j.watres.2015.10.024.
Kazama, S., Miura, T., Masago, Y., Konta, Y., Tohma, K., Manaka, T., et al. (2017). Environmental surveillance of norovirus genogroups I and II for sensitive detection of epidemic variants. Applied and Environmental Microbiology,83(9), e03406-16. https://doi.org/10.1128/aem.03406-16.
Kim, K., Katayama, H., Kitajima, M., Tohya, Y., & Ohgaki, S. (2011). Development of a real-time RT-PCR assay combined with ethidium monoazide treatment for RNA viruses and its application to detect viral RNA after heat exposure. Water Science and Technology,63(3), 502–507. https://doi.org/10.2166/wst.2011.249.
Kingsley, D. H., Vincent, E. M., Meade, G. K., Watson, C. L., & Fan, X. (2014). Inactivation of human norovirus using chemical sanitizers. International Journal of Food Microbiology,171, 94–99. https://doi.org/10.1016/j.ijfoodmicro.2013.11.018.
Kitajima, M., Iker, B. C., Pepper, I. L., & Gerba, C. P. (2014). Relative abundance and treatment reduction of viruses during wastewater treatment processes—Identification of potential viral indicators. Science of the Total Environment,488–489, 290–296. https://doi.org/10.1016/j.scitotenv.2014.04.087.
Kundu, A., McBride, G., & Wuertz, S. (2013). Adenovirus-associated health risks for recreational activities in a multi-use coastal watershed based on site-specific quantitative microbial risk assessment. Water Research,47(16), 6309–6325. https://doi.org/10.1016/j.watres.2013.08.002.
La Rosa, G., Iaconelli, M., Pourshaban, M., & Muscillo, M. (2010). Detection and molecular characterization of noroviruses from five sewage treatment plants in central Italy. Water Research,44(6), 1777–1784. https://doi.org/10.1016/j.watres.2009.11.055.
Ligges, U., & Mächler, M. (2003). Scatterplot3d—An R package for visualizing multivariate data. Journal of Statistical Software,8, 1–20.
Limsawat, S., & Ohgaki, S. (1997). Fate of liberated viral RNA in wastewater determined by PCR. Applied and Environmental Microbiology,63(7), 2932–2933.
López-Gálvez, F., Randazzo, W., Vásquez, A., Sánchez, G., Tombini Decol, L., Aznar, R., et al. (2018). Irrigating lettuce with wastewater effluent: Does disinfection with chlorine dioxide inactivate viruses? Journal of Environmental Quality,47(5), 1139–1145. https://doi.org/10.2134/jeq2017.12.0485.
Miura, T., Lhomme, S., Le Saux, J. C., Le Mehaute, P., Guillois, Y., Couturier, E., et al. (2016). Detection of Hepatitis E virus in sewage after an outbreak on a French Island. Food and Environmental Virology,8(3), 194–1999. https://doi.org/10.1007/s12560-016-9241-9.
Montazeri, N., Goettert, D., Achberger, E. C., Johnson, C. N., Prinyawiwatkul, W., & Janes, M. E. (2015). Pathogenic enteric viruses and microbial indicators during secondary treatment of municipal wastewater. Applied and Environmental Microbiology,81(18), 6436–6445. https://doi.org/10.1128/aem.01218-15.
National Research Council. (2012). Water reuse. Washington, DC: The National Academies Press.
Nordgren, J., Matussek, A., Mattsson, A., Svensson, L., & Lindgren, P. E. (2009). Prevalence of norovirus and factors influencing virus concentrations during one year in a full-scale wastewater treatment plant. Water Research,43(4), 1117–1125. https://doi.org/10.1016/j.watres.2008.11.053.
Parshionikar, S., Laseke, I., & Fout, G. S. (2010). Use of propidium monoazide in reverse transcriptase PCR to distinguish between infectious and noninfectious enteric viruses in water samples. Applied and Environmental Microbiology,76(13), 4318–4326. https://doi.org/10.1128/aem.02800-09.
Prevost, B., Goulet, M., Lucas, F. S., Joyeux, M., Moulin, L., & Wurtzer, S. (2016). Viral persistence in surface and drinking water: Suitability of PCR pre-treatment with intercalating dyes. Water Research,91, 68–76. https://doi.org/10.1016/j.watres.2015.12.049.
Prevost, B., Lucas, F. S., Goncalves, A., Richard, F., Moulin, L., & Wurtzer, S. (2015). Large scale survey of enteric viruses in river and waste water underlines the health status of the local population. Environment International,79, 42–50. https://doi.org/10.1016/j.envint.2015.03.004.
Qiu, Y., Lee, B. E., Neumann, N., Ashbolt, N., Craik, S., Maal-Bared, R., et al. (2015). Assessment of human virus removal during municipal wastewater treatment in Edmonton, Canada. Journal of Applied Microbiology,119(6), 1729–1739. https://doi.org/10.1111/jam.12971.
R Core Team. (2014). R: A language and environment for statistical computing. Vienna: R Core team.
Randazzo, W., Khezri, M., Ollivier, J., Le Guyader, F. S., Rodríguez-Díaz, J., Aznar, R., et al. (2018a). Optimization of PMAxx pretreatment to distinguish between human norovirus with intact and altered capsids in shellfish and sewage samples. International Journal of Food Microbiology,266, 1–7. https://doi.org/10.1016/j.ijfoodmicro.2017.11.011.
Randazzo, W., López-Gálvez, F., Allende, A., Aznar, R., & Sánchez, G. (2016). Evaluation of viability PCR performance for assessing norovirus infectivity in fresh-cut vegetables and irrigation water. International Journal of Food Microbiology,229, 1–6. https://doi.org/10.1016/j.ijfoodmicro.2016.04.010.
Randazzo, W., Piqueras, J., Rodríguez-Díaz, J., Aznar, R., & Sánchez, G. (2018b). Improving efficiency of viability-qPCR for selective detection of infectious HAV in food and water samples. Journal of Applied Microbiology,124(4), 958–964. https://doi.org/10.1111/jam.13519.
Rodríguez-Díaz, J., Querales, L., Caraballo, L., Vizzi, E., Liprandi, F., Takiff, H., et al. (2009). Detection and characterization of waterborne gastroenteritis viruses in urban sewage and sewage-polluted river waters in Caracas, Venezuela. Applied and Environmental Microbiology,75(2), 387–394. https://doi.org/10.1128/AEM.02045-08.
Sano, D., Amarasiri, M., Hata, A., Watanabe, T., & Katayama, H. (2016). Risk management of viral infectious diseases in wastewater reclamation and reuse: Review. Environment International,91, 220–229. https://doi.org/10.1016/j.envint.2016.03.001.
Schmitz, B. W., Kitajima, M., Campillo, M. E., Gerba, C. P., & Pepper, I. L. (2016). Virus reduction during advanced Bardenpho and conventional wastewater treatment processes. Environmental Science and Technology,50(17), 9524–9532. https://doi.org/10.1021/acs.est.6b01384.
SEPA. (2008). Guidance to the Swedish Environmental Protection Agency’s regulation for environmental reports (Vägledning om Naturvårdsverkets föreskrifter om miljörapport).
Simmons, F. J., & Xagoraraki, I. (2011). Release of infectious human enteric viruses by full-scale wastewater utilities. Water Research,45(12), 3590–3598. https://doi.org/10.1016/j.watres.2011.04.001.
Sinclair, R. G., Choi, C. Y., Riley, M. R., & Gerba, C. P. (2008). Chapter 9—Pathogen surveillance through monitoring of sewer systems. In A. I. Laskin, S. Sariaslani, & G. M. Gadd (Eds.), Advances in applied microbiology (Vol. 65, pp. 249–269). New York: Academic Press.
Standard Methods For the Examination of Water and Wastewater. (2011). In 9510 detection of enteric viruses: American Public Health Association.
Van Abel, N., Schoen, M. E., Kissel, J. C., & Meschke, J. S. (2017). Comparison of risk predicted by multiple norovirus dose-response models and implications for quantitative microbial risk assessment. Risk Analalysis,37(2), 245–264. https://doi.org/10.1111/risa.12616.
Verbyla, M. E., & Mihelcic, J. R. (2015). A review of virus removal in wastewater treatment pond systems. Water Research,71, 107–124. https://doi.org/10.1016/j.watres.2014.12.031.
WHO. (2017). Potable reuse: Guidance for producing safe drinking-water. Geneva: WHO.
Wilrich, C., & Wilrich, P. T. (2009). Estimation of the pod function and the LOD of a qualitative microbiological measurement method. Journal of AOAC International,92(6), 1763–1772.
This study was supported by the “VIRIDIANA” Project AGL2017-82909 (AEI/FEDER, UE) funded by Spanish Ministry of Science, Innovation and Universities; the APOTI Grant (APOTIP/2018/007) from the Generalitat Valenciana; and the CSIC internal Project 201770I088. W. Randazzo was supported by a postdoctoral fellowship from Generalitat Valenciana (APOSTD/2018/150).
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
About this article
Cite this article
Randazzo, W., Piqueras, J., Evtoski, Z. et al. Interlaboratory Comparative Study to Detect Potentially Infectious Human Enteric Viruses in Influent and Effluent Waters. Food Environ Virol 11, 350–363 (2019). https://doi.org/10.1007/s12560-019-09392-2
- Foodborne viruses
- Viability RT-qPCR
- Effluent water