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Interlaboratory Comparative Study to Detect Potentially Infectious Human Enteric Viruses in Influent and Effluent Waters

Abstract

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).

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References

  1. 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.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. 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.

    CAS  Article  PubMed  Google Scholar 

  3. 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.

    CAS  Article  PubMed  Google Scholar 

  4. 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.

    CAS  Article  PubMed  Google Scholar 

  5. 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.

    Article  Google Scholar 

  6. 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.

    CAS  Article  PubMed  Google Scholar 

  7. 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.

    CAS  Article  PubMed  Google Scholar 

  8. 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.

    Google Scholar 

  9. 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.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. 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.

    Article  Google Scholar 

  11. 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.

    CAS  Article  PubMed  Google Scholar 

  12. 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.

    Article  PubMed  PubMed Central  Google Scholar 

  13. 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.

    Article  Google Scholar 

  14. 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.

  15. 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.

    Article  PubMed  Google Scholar 

  16. 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.

    Article  PubMed  Google Scholar 

  17. 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.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. 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.

    CAS  Article  Google Scholar 

  19. 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.

    Article  PubMed  Google Scholar 

  20. 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.

    CAS  Article  PubMed  Google Scholar 

  21. 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.

    CAS  Article  PubMed  Google Scholar 

  22. 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.

    CAS  Article  PubMed  Google Scholar 

  23. 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.

    CAS  Article  PubMed  Google Scholar 

  24. 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.

    Article  Google Scholar 

  25. 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.

    Article  PubMed  Google Scholar 

  26. 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.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  27. 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.

    Google Scholar 

  28. 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.

    CAS  Article  PubMed  Google Scholar 

  29. 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.

    CAS  Article  PubMed  Google Scholar 

  30. 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.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. 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.

    CAS  Article  PubMed  Google Scholar 

  32. 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.

    CAS  Article  PubMed  Google Scholar 

  33. 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.

    CAS  Article  PubMed  Google Scholar 

  34. 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.

    CAS  Article  PubMed  Google Scholar 

  35. 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.

    CAS  Article  PubMed  Google Scholar 

  36. Ligges, U., & Mächler, M. (2003). Scatterplot3d—An R package for visualizing multivariate data. Journal of Statistical Software,8, 1–20.

    Article  Google Scholar 

  37. Limsawat, S., & Ohgaki, S. (1997). Fate of liberated viral RNA in wastewater determined by PCR. Applied and Environmental Microbiology,63(7), 2932–2933.

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 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.

    CAS  Article  PubMed  Google Scholar 

  39. 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.

    Article  PubMed  PubMed Central  Google Scholar 

  40. 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.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  41. National Research Council. (2012). Water reuse. Washington, DC: The National Academies Press.

    Google Scholar 

  42. 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.

    CAS  Article  PubMed  Google Scholar 

  43. 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.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  44. 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.

    CAS  Article  PubMed  Google Scholar 

  45. 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.

    CAS  Article  PubMed  Google Scholar 

  46. 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.

    CAS  Article  PubMed  Google Scholar 

  47. R Core Team. (2014). R: A language and environment for statistical computing. Vienna: R Core team.

    Google Scholar 

  48. 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.

    Article  PubMed  Google Scholar 

  49. 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.

    CAS  Article  PubMed  Google Scholar 

  50. 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.

    CAS  Article  PubMed  Google Scholar 

  51. 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.

    CAS  Article  PubMed  Google Scholar 

  52. 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.

    CAS  Article  Google Scholar 

  53. 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.

    CAS  Article  PubMed  Google Scholar 

  54. SEPA. (2008). Guidance to the Swedish Environmental Protection Agency’s regulation for environmental reports (Vägledning om Naturvårdsverkets föreskrifter om miljörapport).

  55. 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.

    CAS  Article  PubMed  Google Scholar 

  56. 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.

    Google Scholar 

  57. Standard Methods For the Examination of Water and Wastewater. (2011). In 9510 detection of enteric viruses: American Public Health Association.

  58. 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.

    Article  Google Scholar 

  59. 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.

    CAS  Article  PubMed  Google Scholar 

  60. WHO. (2017). Potable reuse: Guidance for producing safe drinking-water. Geneva: WHO.

    Google Scholar 

  61. 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.

    CAS  PubMed  Google Scholar 

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Acknowledgements

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).

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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

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Keywords

  • Foodborne viruses
  • Viability RT-qPCR
  • Sewage
  • Effluent water