Evaluation of methodology for detection of human adenoviruses in wastewater, drinking water, stream water and recreational waters

Rapid diagnosis of human adenovirus (HAdV) infections was achieved by PCR in the recent years. However, conventional PCR has the risk of carry-over contamination due to open handling with its products, and results are only qualitative. Therefore, a quantitative "real-time" PCR with consensus primer and probe (dual fluorescence labelled, "TaqMan") sequences for a conserved region of the hexon gene was designed and evaluated. Real-time PCR detected all 51 HAdV prototypes. Sensitivity of the assay was

Environmental virology began with efforts to detect poliovirus in sewage and water more than 50 years ago. Since that time, cell-culture methods useful for detection of enteroviruses have been replaced by molecular biology techniques for detection of pathogens (hepatitis A and E viruses, caliciviruses, rotaviruses, and astroviruses) that do not grow in cell culture or grow with great difficulty. Amplification of viral nucleic acid using the polymerase chain reaction (PCR) is the current preferred method. PCR or RT-PCR (to detect RNA viral genomes) is rapid, sensitive, specific, and quantitative. Method shortcomings include potential inhibition by substances in some environmental samples and an inability of test results to distinguish between infectious and noninfectious virus. Current questions involving use of PCR/RT-PCR tests for public health purposes include: What is the public health significance of a positive test, and should direct tests for viruses replace current public health-monitoring programs?

A nested-PCR method was used to detect the occurrence of human adenovirus in coastal waters of Southern California. Twenty- to forty-liter water samples were collected from 12 beach locations from Malibu to the border of Mexico between February and March 1999. All sampling sites were located at mouths of major rivers and creeks. Two ultrafiltration concentration methods, tangential flow filtration (TFF) and vortex flow filtration (VFF), were compared using six environmental samples. Human adenoviruses were detected in 4 of the 12 samples tested after nucleic acid extraction of VFF concentrates. The most probable number of adenoviral genomes ranged from 880 to 7,500 per liter of water. Coliphages were detected at all sites, with the concentration varying from 5.3 to 3332 PFU/liter of water. F-specific coliphages were found at 5 of the 12 sites, with the concentration ranging from 5.5 to 300 PFU/liter. The presence of human adenovirus was not significantly correlated with the concentration of coliphage (r = 0.32) but was significantly correlated (r = 0.99) with F-specific coliphage. The bacterial indicators (total coliforms, fecal coliforms, and enterococci) were found to exceed California recreational water quality daily limits at 5 of the 12 sites. However, this excess of bacterial indicators did not correlate with the presence of human adenoviruses in coastal waters. The results of this study call for both a reevaluation of our current recreational water quality standards to reflect the viral quality of recreational waters and monitoring of recreational waters for human viruses on a regular basis.