Abstract

Acute gastroenteritis is one of the most common diseases worldwide, with viruses, particularly noroviruses, being the leading cause in developed countries. In The Netherlands, systematic surveillance of gastroenteritis outbreaks of suspected viral etiology was established by the National Institute for Public Health and the Environment in 1994. Since 2002, the total number of outbreaks reported has been increasing, and with that comes the need for sensitive assays that can be performed quickly. In addition, the diagnostic demand changed so that now the proportion of samples from hospitals is higher and there is a need for patient-based test results. In order to target the diagnosis of acute gastroenteritis, we reviewed our data on outbreaks of gastroenteritis and the prevalence of individual viruses to provide a priority list of viruses for which samples should be evaluated. Random primers were used to replace the separate specific primers for each virus used in the reverse transcription steps. The individual PCR assays were replaced by multiplex PCR assays. We employed a two-step method in which in the first step we screened for the most common causes of viral gastroenteritis, noroviruses of genogroup II and rotaviruses of group A, with equine arteritis virus used as the internal control. Subsequently, in the second step, two parallel PCR assays were developed for the detection of noroviruses of genogroup I and equine arteritis virus in one run and adenoviruses, sapoviruses, and astroviruses in the other run. The specificities of the assays were calculated to be 92.5% for the assay for noroviruses of genogroup I and 100% for the assays for all other viruses, the detection limits were equal for all viruses, and the turnaround time was reduced to 1 day compared to the at least 3 days required for the methods used previously. This approach allows the targeted, rapid, and cost-effective elucidation of the causes of acute gastroenteritis outbreaks.