The English case-control Infectious Intestinal Disease Study (1993-1996) failed to detect an enteric pathogen or toxin in 49% of cases of gastroenteritis. In the present study, polymerase chain reaction (PCR) assays were applied to DNA and cDNA generated from 4,627 faecal samples from cases and controls archived during the original study for the detection of norovirus, rotavirus, sapovirus, Campylobacter spp., Salmonella spp., enteroaggregative Escherichia coli, Cryptosporidium spp., and Giardia spp. The percentage of archived samples from cases and from controls in which at least one agent (or toxin) was detected increased from 53% in the original study to 75% and from 19 to 42%, respectively, after the application of PCR assays. Among cases, the following percentages of enteric pathogens were detected: norovirus 36%, rotavirus A 31%, sapovirus 4%, Salmonella spp. 6%, Campylobacter jejuni 13%, Campylobacter coli 2%, other Campylobacter spp. 8%, enteroaggregative E. coli 6%, Giardia spp. 2%, and Cryptosporidium spp. 2%. The present study provides additional insight into the aetiology of infectious intestinal disease in England and highlights the occurrence of viral infections in cases as well as in asymptomatic individuals. Other notable findings include the frequent presence of Campylobacter spp. other than C. jejuni or C. coli, the high frequency of multiple agents in 41% of cases and in 13% of controls, and the variation in the aetiology and rate of infection found for different age groups. The results demonstrate the greater sensitivity of PCR-based methods compared to current conventional methods.

A blinded trial in two different laboratories was performed to compare the detection of selected enteric pathogens in 92 unselected faecal samples collected from patients with community-acquired diarrhoea by conventional and PCR-based techniques. Conventional techniques detected a single potential etiological agent in 15% of the samples, whereas results of PCR detected evidence of at least one agent in 41% of the samples. Overall, the detection rates for the different pathogens were as follows: adenovirus serogroup F, 1%; Campylobacter spp., 7.6%; Salmonella spp., 4%; enteroaggregative Escherichia coli, 9.8%; enteropathogenic E. coli, 6.5%; enterotoxigenic Clostridium perfringens, 3%; Cryptosporidium spp., 13%; and Giardia spp., 11%. Results for the detection of Salmonella spp., Campylobacter spp. and C. perfringens were similar by both techniques, whereas Cryptosporidium and Giardia spp. were detected 22 times more often by PCR than by conventional microscopy. It was not possible to compare the results for detection of enteroaggregative E. coli and enteropathogenic E. coli since these were only investigated by PCR. The results of this small study clearly demonstrate the advantages of PCR-based methods compared to conventional techniques for the detection of gastrointestinal pathogens.

Fecal samples were collected from cases and controls as part of the Infectious Intestinal Disease (IID) study in England and were stored as frozen suspensions for 8 to 12 years. The purpose of this study was to apply PCR-based procedures to assess the stability of pathogen-specific nucleic acid sequences present in this archive. Samples from which Cryptosporidium, Giardia, Salmonella, Campylobacter, enteroaggregative Escherichia coli (EAggEC), enterotoxigenic Clostridium perfringens, rotaviruses, noroviruses, or sapoviruses had been previously detected during the IID study using conventional methods were selected from the archive. A generic nucleic acid extraction method to recover RNA or DNA was used. Complementary DNA was generated from RNA by reverse transcription with random priming. Block-based and real-time PCR assays were used to amplify and detect gene fragments from each of these pathogens. The percentage reconfirmation of target was as follows: Giardia duodenalis 68%, Cryptosporidium 96%, Campylobacter 98%, Salmonella 98%, enterotoxigenic C perfringens 34%, EAggEC 93.3%, rotavirus 95%, norovirus 73%, and sapovirus 85%. This study has shown that nucleic acid can be extracted and specific sequences amplified and detected from archived fecal samples. The IID archive therefore represents a valuable resource for further studies, especially the investigation of the samples from which no pathogens had previously been detected.