The third study of infectious intestinal disease in the community microbiological methods.

IntroductionInfectious intestinal disease (IID) is a significant public health concern, with an estimated 17 million cases occurring annually in the UK. However, only a small proportion of cases in the community present to primary health care, and those that do may not be tested for the causative pathogen. The third study of IID in the community (IID3 study) will provide updated estimates of the incidence and aetiology of IID in the UK. This protocol describes the laboratory methods employed by the IID3 study in primary diagnostic and reference laboratories. Methods and analysisStool specimens obtained from symptomatic subjects enrolled in two cohorts recruited prospectively through Primary Care in the UK are tested for 21 pathogens using PCR-based methods, in two separate workflows at Liverpool Clinical Laboratories. The first workflow comprises EntericBio DX, Viral and Clostridium difficile assays (Serosep, Crawley, UK); and the second a panel of assays for detection of Clostridium perfringens, Aeromonas spp., Cyclospora cayetanensis and diarrhoeagenic Escherichia coli based on 3base(R) Technology (Genetic Signatures, Newtown, Australia). Specimens testing positive for bacterial pathogens are subjected to culture. Specimens positive for, and isolates of Salmonella, Campylobacter, Yersinia, Vibrio and Shigella species and diarrhoeagenic E. coli are referred to the Gastrointestinal Bacteria Reference Unit, UK Health Security Agency (UKHSA) for whole genome sequencing and antimicrobial susceptibility testing. The parasites Cryptosporidium, Giardia and Cyclospora are referred for genotyping to the national Cryptosporidium Reference Unit, Public Health Wales. Norovirus positive faecal specimens are referred for Sanger Sequencing-based genotyping to the Enteric Virus Unit, UKHSA. Ethics and disseminationFavourable ethical opinion was granted on the 10th of August 2022 by the East Midlands - Nottingham 1 Research Ethics Committee (IRAS ID 314268, REC Reference 22/EM/0130). The results will be disseminated to key stakeholders including the Food Standards Agency, participating general practices and to the academic community through publications and conference presentations. A real-time breakdown of pathogens identified in faecal samples could be viewed openly on the study Dashboard throughout the surveillance period. Article SummaryO_ST_ABSStrengths and limitations of this studyC_ST_ABS- A total of 5439 stool samples underwent molecular testing for a total of 21 organisms at the Liverpool Clinical Laboratories (LCL), with detailed pathogen characterisation at national reference laboratories. - A commercial Genetic Signatures assay was evaluated in a routine diagnostic setting using commercially-provided pathogen controls, reference isolates and clinical samples, enabling the inclusion of Aeromonas and ETEC PCR targets into the diagnostic panel. - Real-time referrals of PCR-reactive samples were made to UK Health Security Agency (UKHSA) regardless of bacterial culture results, and to Public Health Wales (PHW). - Norovirus PCR-reactive samples were referred to the Enteric Virus Unit at UKHSA for genotyping ahead of Norovirus becoming a notifiable infection on the 6th of April 2025, adding valuable information on Norovirus genotypes circulating in the general population. - A Biobank has been created for residual faecal specimens, forming a rich resource for future studies. - A major challenge was the lack of a harmonised UK-wide digital referral and reporting system, resulting in an inflated manual reporting workload for staff at LCL.