Clinical Utility of Nested Multiplex RT-PCR for Group F Adenovirus, Rotavirus and Norwalk-like Viruses in Acute Viral Gastroenteritis in Children and Adults

Background: The diagnosis of viral gastroenteritis can be carried out by non-molecular techniques such as electron microscopy (EM), enzyme-immunoassay and latex agglutination tests and various molecular techniques. Normally molecular detection requires the use of three separate protocols to detect the three main causes of viral gastroenteritis, adenoviruses, rotaviruses and norwalk-like viruses (NLV) which have different types of nucleic acid. The development of a sensitive and specific assay which could detect these targets would have major advantages for the clinical virology laboratory.

Objectives: The aims of the present study were to develop a sensitive and specific multiplex molecular assay and to apply it to the detection of viral agents in clinical cases of acute gastroenteritis.

Study Design: The multiplex assay was designed using Access RT-PCR (Promega). Primers were researched and selected for their specificity and broad range detection of the viral agents across the various genotypes of group A rotaviruses, NLV and group F adenoviruses.

Results: From September 2000 to August 2001 we tested 1945 clinical specimens. Rotavirus infections were detected in 190 with an age range from 12 days to 8 years old. Group F adenovirus was detected in 96 patients ranging from 15 days to 10 years old. A further single case of group F adenovirus was detected in an adult of 75 years old. NLVs were detected in 132 patients. There were 55 infections in children less than 7 years old. In 10 different outbreaks involving 130 adult patients there were 57 NLV positives. Sporadic NLV infection was detected in 11 of 600 adult patients. There were 4 patients with dual infections.

Conclusions: The assay detailed here has proved an invaluable tool for the investigation of acute gastroenteritis in specimens from patients of all ages. We found it convenient to use a single mastermix with a single protocol to test all specimens from patients of all ages. NLV in children is often overlooked and/or under reported, particularly where less sensitive assays such as EM are being employed for diagnosis.