title: Evaluation of methods used to concentrate and detect hepatitis A virus in water samples.
abstract: Two adsorption-elution concentration methods, both involving negatively charged membranes, were evaluated in order to monitor hepatitis A virus (HAV) contamination in tap, river, mineral and coastal water samples: elution with urea-arginine phosphate buffer/reconcentration with magnesium chloride (method 1); and sodium hydroxide elution/reconcentration with a commercial concentrator (method 2). Nested (qualitative) reverse transcriptase PCR (RT-PCR) and real-time (quantitative) RT-PCR were used to detect and quantify HAV RNA in concentrated water samples. For concentrating HAV, method 1 was found to be the most suitable for tap water and method 2 most suitable for mineral water. HAV inoculated experimentally was detected in river water samples by both methods and in coastal water samples by neither method. The detection limits were 6 x 10(9) g equiv./ml for qualitative PCR and 60 g equiv./ml for quantitative PCR. In a field application study, HAV was detected in 20% of river and tap water samples but not in coastal or mineral water samples. River water samples contained subgenotype IA, and tap water samples contained subgenotype IB. It is concluded that, although influencing qualitative PCR, the concentration method does not affect quantitative PCR, which could therefore be used for all types of water samples. Both techniques are recommended for detecting HAV in environmental water samples.
authors.affiliation: Department of Virology, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Av. Brazil 4365, 21045-900 Rio de Janeiro, RJ, Brazil. lvillar@ioc.fiocruz.br
authors.forename: Livia Melo
authors.initials: LM
authors.lastname: Villar
authors.forename: Vanessa Salete
authors.initials: VS
authors.lastname: de Paula
authors.forename: Leonardo
authors.initials: L
authors.lastname: Diniz-Mendes
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Lampe
authors.forename: Ana Maria Coimbra
authors.initials: AM
authors.lastname: Gaspar
mesh_terms: D005618:Fresh Water / Q000821:virology; D005838:Genotype; D030041:Hepatitis A virus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D008900:Mineral Waters / Q000821:virology; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012623:Seawater / Q000821:virology; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D055672:Static Electricity; D014773:Virology / Q000379:methods*; D014871:Water Microbiology
chemical_list: D008900:Mineral Waters; D012367:RNA, Viral