Acute gastroenteritis (AGE) is a very common and at times severe infection, especially in children. However, current techniques fail to determine a causal infectious agent in 30-70 percent of cases. Our goal is to investigate what currently undetected infectious agents are responsible for this diagnostic void. We have begun a prospective clinical study of 1500 children admitted to a children's hospital, half for AGE and half (the control group) for other reasons. All specimens are stored at -70 degrees C to form a well documented comprehensive archive. With half the enrolments achieved, and following exhaustive testing, fecal samples from nearly one quarter of those with AGE remain negative. By study end, this will represent 218 children, each with strictly assessed symptoms of AGE and some even with a history of transmission to close contacts. We hypothesize that these infections are being caused by agents, probably viruses, not yet associated with AGE. Having used specific diagnostic tests, including very sensitive PCR assays, we propose to now use non-specific amplification assays to detect possibly novel agents within these samples.
The specific aims are: 1. Develop the Nuclease pretreated Sequence Independent Single Primer Amplification (NSlSPA) assay for screening fecal extracts. We will optimize NSISPA to detect both RNA and DNA viruses in the presence of contaminating human, bacterial and food-derived nucleic acids. 2. Using NSISPA, screen samples from the 218 symptomatic children for the presence of novel viruses. We will examine for the presence of NSISPA amplification products, and compare the sequence homology of these products to either currently recognized viral sequences, or coding or control regions suggestive of viral origin. 3. Extend these NSISPA-obtained sequences to identify viral agents and permit construction of specific PCR assays for each agent. We will extend the NSlSPA-obtained sequences to encompass at least the majority of the genome, using PCR specific to the detected sequences, alternative non-specific amplification to obtain additional sequences from other locations on the genome, cloning and RACE techniques. 4. Assess importance of new viruses. Using the specific PCRs developed in (3) above; we will examine the complete specimen archive to determine prevalence, statistical association with AGE, and clinical and epidemiological features. Vaccine developments for rotavirus have the potential to eliminate most of the 3 million cases of AGE, and 800,000 deaths globally caused by this virus. Some therapies to treat AGE are virus specific. Understanding the presence and role of other viruses is the important first step in determining strategies to reduce or eliminate disease caused by them in the community.
| Arthur, Jane L; Higgins, Geoffrey D; Davidson, Geoffrey P et al. (2009) A novel bocavirus associated with acute gastroenteritis in Australian children. PLoS Pathog 5:e1000391 |
