Molecular Methods for the Detection of Waterborne Pathogens - Waterborne Pathogens: Detection Methods and Applications - page 255

Although traditionally cell culture was the most widely used method for
the detection of viruses, it is now combined with PCR or immunological
testing to improve the sensitivity of detection. The use of molecular tech-
niques can contribute to more accurate assessment of the occurrence and
health impact of waterborne viruses by overcoming the need to culture
the viruses. However, currently none of the molecular tests are practical for
routine or large-scale monitoring; therefore, viral presence is often based on
the presence/absence of bacteria indicators such as E. coli , total coliforms,
Enterococcus , C. perfringens spores, and bacteriophages. 164 Although enteric
viruses cannot multiply outside of their host, they are able to survive lon-
ger in water than most intestinal bacteria, including indicator organisms. 164
Therefore, the absence of fecal bacteria does not ensure the corresponding
absence of the virus.
Detection of viruses in water samples is limited at various stages in the
traditional methodology. In samples where viral concentrations are high,
such as feces (may excrete up to 10 11 viral particles per gram of stool), elec-
tron microscopy, enzyme immunoassay, latex agglutination, or polyacryl-
amide gel electrophoresis can be used. However, in water samples where the
virus may only reach 1-10 particles per liter, these methods have not been
found to be sensitive enough. Therefore, sensitivity requires the concentra-
tion of large volumes of the sample to overcome infectious levels of viruses
that are often very low. This, in itself, can also cause detection problems
because it also concentrates contaminants that may interfere with subse-
quent testing. Filtration by absorption or size exclusion (ultrafiltration) have
both been used successfully, 165-167 although sometimes a second concen-
tration step is necessary to improve sensitivity. 128 Once concentrated, the
detection efficiency of current methods may vary from only 50-95%. 46,162
A cell culture of the viruses may still be the most definite test because it
can confirm both the presence and infectivity of the viruses; however, it
suffers from the lack of detection of slow-growing viruses, the occasional
inability to produce clear cytopathic effects, and a possible inability to dis-
tinguish between plaques produced by multiple viruses. 168,169 Although the
detection of adenoviruses, enteroviruses, astroviruses, and rotaviruses have
been achieved with appropriate cell lines, 46,128 it is not a realistic approach
for hepatitis A and norovirus due to assay complexity. 128 Furthermore, cell
cultures can give false positives due to contamination that causes cells to
change morphologically. 131
For viruses where antibodies are available, IMS can be used to concen-
trate the viruses from samples. In this method, antibody-coated paramagnetic