title: Incidence of enteroviruses in Mamala Bay, Hawaii using cell culture and direct polymerase chain reaction methodologies.
abstract: The consequence of point and nonpoint pollution sources, discharged into marine waters, on public recreational beaches in Mamala Bay, Hawaii was evaluated using virus cell culture and direct reverse transcriptase-polymerase chain reaction (RT-PCR). Twelve sites, nine marine, two freshwater (one stream and one canal), and one sewage, were assessed either quarterly or monthly for 1 year to detect the presence of human enteric viruses. Water samples were concentrated from initial volumes of 400 L to final volumes of 30 mL using Filterite electronegative cartridge filters and a modified beef extract elution procedure. Cell culture was applied using the Buffalo Green Monkey kidney cell line to analyze samples for enteroviruses. Positive samples were also evaluated by RT-PCR, using enterovirus-specific primers. Levels of RT-PCR inhibition varied with each concentrated sample. Resin column purification increased PCR detection sensitivity by at least one order of magnitude in a variety of sewage outfall and recreational marine water samples but not in the freshwater canal samples. Using cell culture, viable enteroviruses were found in 50 and 17% of all outfall and canal samples, respectively. Samples were positive at beaches 8% of the time. These data illustrate the potential public health hazard associated with recreational waters. Using direct PCR, viruses were detected at the outfall but were not found in any beach or canal samples, in part, owing to substances that inhibit PCR. Therefore, conventional cell culture is the most effective means of detecting low levels of infectious enteroviruses in environmental waters, whereas direct RT-PCR is rendered less effective by inhibitory compounds and low equivalent reaction volumes.
authors.affiliation: Department of Soil, Water, and Environmental Science, University of Arizona, Tucson 85721, USA. reynolds@u.arizona.edu
authors.forename: K A
authors.initials: KA
authors.lastname: Reynolds
authors.forename: K
authors.initials: K
authors.lastname: Roll
authors.forename: R S
authors.initials: RS
authors.lastname: Fujioka
authors.forename: C P
authors.initials: CP
authors.lastname: Gerba
authors.forename: I L
authors.initials: IL
authors.lastname: Pepper
mesh_terms: D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D003588:Cytopathogenic Effect, Viral; D004770:Enterovirus / Q000302:isolation & purification*; D000882:Haplorhini; D006254:Hawaii; D006801:Humans; D007668:Kidney; D008137:Longitudinal Studies; D016133:Polymerase Chain Reaction; D014871:Water Microbiology