Novel Approach for Detection of Enteric Viruses To Enable Syndrome Surveillance of Acute Viral Gastroenteritis

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.

Improved Detection of Five Major Gastrointestinal Pathogens by Use of a Molecular Screening Approach

“…Previous studies also reported improvements in the detection rate of enteric pathogens by molecular assays (1,7,18,25,36,37,40). The reason why S. enterica was the only pathogen not detected more frequently by MSA than by culture (3.0%) may be the use of a sensitive selective enrichment broth.…”

“…However, broad application remains limited due to their assumed high costs, inhibition caused by fecal constituents (22), and the need for specialized laboratories. Due to the high throughput of stool screening and the number of possible enteric pathogens, implementation of a molecular approach which uses multiplexing of targets is mandatory (9,10,20,25,26,36,43,44,47). For the detection of enteric pathogens it has been proven feasible to use molec-ular methods with improved performance and turnaround time (TAT) (7,25,32 MATERIALS AND METHODS mPCR technical validation.…”

Epidemiology and Genotype Analysis of Emerging Sapovirus-Associated Infections across Europe

“…Outbreak surveillance has been operational in the Netherlands since 1994 and provides a systematic overview of all notified outbreaks (29). From 1 November 2007 to 1 January 2009, 478 GE outbreaks (3,233 samples) were reported to the surveillance program, and samples were tested for the presence of noroviruses, rotaviruses, enteric adenoviruses, astroviruses, and SaVs by a combination of multiplex RT-PCR assays (3,(28)(29)(30). Outbreaks were attributed to one of the pathogens mentioned above, depending on the diagnostic yield as a proportion of samples tested (3).…”

“…Detection of sapoviruses was preformed as described by Svraka et al (30). A multiplex RT-PCR that uses random primers and is sapovirus specific was used; it proved to be more sensitive than previously used sapovirus PCR assays, as described by Svraka et al (30). Sequencing of partial capsid genes of SaVs was performed using primers previously described by Yan et al (35).…”

“…Sequencing of partial capsid genes of SaVs was performed using primers previously described by Yan et al (35). The RT reaction step was slightly modified and performed using random primers and Superscript III (30). PCR amplification was performed as described by Svraka et al (30) and by adding 0.1 l forward and reverse primers (100 pmol/l) to the PCR mixture (Table 1) (35).…”