New breakthrough offers quick and precise detection of low levels of enteric viruses

Waterborne microbial contaminants, once thought to be under control, are attracting renewed attention. An awareness of the presence of previously undetected microbial contaminants in drinking water is increasing. By developing an improved methodology for testing for viruses in water, a University of Arizona microbiologist is contributing to this expanded awareness Kelly A. Reynolds, University of Arizona Department of Soil, Water and Environmental Science, developed a method capable of quickly and precisely detecting low levels of enteric viruses in large volumes of water concentrates, thus overcoming limitations of previous testing strategies.

Her method is considered a major breakthrough in detecting viruses in drinking water. Viruses are not easily detected. Often present in water in very low numbers, viruses still can pose a health risk since it takes only one virus in a water system to infect a person with a waterborne disease. (Bacteria are different. Some require only 10, but usually the infectious dose is closer to 1,000.) A challenge in testing for viruses therefore is to be able to detect very low levels of viruses in very large water volumes.

Since human viruses generally occur in very low numbers in the environment, water samples need to be concentrated before analysis. The conventional methods for virus detection that are then applied rely on animal cell cultures. Water sample concentrates are added to culture flasks containing monkey or human cells that support virus growth. The cells are then observed for periods of a few days to weeks to detect signs of cell destruction indicating virus growth. The advantage of cell culture is that it detects only infectious strains of viruses and can test large sample volumes, after concentration. Cell culture, however, can require long periods of time. Some strains of enteric viruses may need two weeks of growth for preliminary results, with confirmed results possibly requiring as long as three weeks or more. Also some strains of viruses, although growing in cells, do not show any visual signs of cell destruction and therefore go undetected.

Examples of such viruses, called noncytopathogenic viruses, are certain strains of rotavirus and hepatitis A. The limitations of cell culture have prompted scientists to turn to molecular detection methods to routinely monitor for viruses. The distinct nucleic acid sequences of different organisms can be differentiated at the genetic level, and molecular methods can detect the presence of a pathogen's genetic material (RNA or DNA). The most commonly used molecular method, the polymerase chain reaction (PCR) can quickly detect enteric viruses, with only 24 hours needed for definitive results. In many respects, PCR is more effective than conventional cell culture and has proven to be a rapid, sensitive, specific and inexpensive method for detecting viruses.

Molecular methods, however, also have shortcomings. Their detection sensitivity often is decreased by inhibitory compounds often present in environmental concentrates. False negatives can result. Also, PCR does not distinguish between noninfectious and infectious virus particles, thus complicating interpreting a PCR positive result and its implication to public health. It is within this context i.e., in response to the limitations of both cell cultures and molecular methods that Reynolds developed the integrated cell culture/PCR method to routinely monitor for infectious enteric viruses. ICC/PCR retains many of the advantages of both conventional cell culture and molecular methods but without their limitations.

After adding sample concentrates to cell culture flasks, Reynolds applies PCR on the cell culture medium. By applying PCR to the medium, lengthy incubation times are unnecessary because PCR is capable of detecting low levels of virus growth in the cell culture. If PCR were not used, results would be delayed until visual signs of cell destruction become apparent. Further, by integrating the molecular method with cell culture, PCR results are more reliable. No confusion exists about whether a PCR-detected virus is infectious or not since only infectious viruses develop in the cell culture. All viruses detected by ICC/PCR then are infectious and results are available in 24-48 hours, compared to days or weeks required by cell culture alone.

Also, ICC/PCR overcomes the effect of PCR inhibitory compounds that otherwise could lead to false negative results and is able to detect noncytopathogenic viruses e.g., certain strains of rotavirus and hepatitis A that grow in cells without visual signs of cell destruction. With improved, viable virus detection sensitivity and reduced assay times, ICC/PCR is the future for effective environmental virus monitoring. Even with samples that are suitable for direct PCR amplification monitoring, having low inhibitory compounds and sufficiently high levels of target organisms, subsequent use of ICC/ PCR would aid evaluation of the viable nature of the target, with minimal cost and time involvement.

The implications of the ICC/PCR method will gain importance as water quality testing increasingly includes more frequent monitoring of viruses. Also the method will serve to evaluate the effectiveness of various water treatment and disinfection methods concerned with removing or inactivating human enteric viruses.