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<jats:sec id="abs1-1"><jats:title>Background</jats:title><jats:p>Every year approximately 5000–9000 patients are admitted to a hospital with diarrhoea, which in up to 90% of cases has a non-infectious cause. As a result, single rooms are ‘blocked’ by patients with non-infectious diarrhoea, while patients with infectious diarrhoea are still in open bays because of a lack of free side rooms. A rapid test for differentiating infectious from non-infectious diarrhoea could be very beneficial for patients.</jats:p></jats:sec><jats:sec id="abs1-2"><jats:title>Objective</jats:title><jats:p>To evaluate MassCode multiplex polymerase chain reaction (PCR) for the simultaneous diagnosis of multiple enteropathogens directly from stool, in terms of sensitivity/specificity to detect four common important enteropathogens:<jats:italic>Clostridium difficile</jats:italic>,<jats:italic>Campylobacter</jats:italic>spp.,<jats:italic>Salmonella</jats:italic>spp. and norovirus.</jats:p></jats:sec><jats:sec id="abs1-3"><jats:title>Design</jats:title><jats:p>A retrospective study of fixed numbers of samples positive for<jats:italic>C. difficile</jats:italic>(<jats:italic>n</jats:italic> = 200),<jats:italic>Campylobacter</jats:italic>spp. (<jats:italic>n</jats:italic> = 200),<jats:italic>Salmonella</jats:italic>spp. (<jats:italic>n</jats:italic> = 100) and norovirus (<jats:italic>n</jats:italic> = 200) plus samples negative for all these pathogens (<jats:italic>n</jats:italic> = 300). Samples were sourced from NHS microbiology laboratories in Oxford and Leeds where initial diagnostic testing was performed according to Public Health England methodology. Researchers carrying out MassCode assays were blind to this information. A questionnaire survey, examining current practice for infection control teams and microbiology laboratories managing infectious diarrhoea, was also carried out.</jats:p></jats:sec><jats:sec id="abs1-4"><jats:title>Setting</jats:title><jats:p>MassCode assays were carried out at Oxford University Hospitals NHS Trust. Further multiplex assays, carried out using Luminex, were run on the same set of samples at Leeds Teaching Hospitals NHS Trust. The questionnaire was completed by various NHS trusts.</jats:p></jats:sec><jats:sec id="abs1-5"><jats:title>Main outcome measures</jats:title><jats:p>Sensitivity and specificity to detect<jats:italic>C. difficile</jats:italic>,<jats:italic>Campylobacter</jats:italic>spp.,<jats:italic>Salmonella</jats:italic>spp., and norovirus.</jats:p></jats:sec><jats:sec id="abs1-6"><jats:title>Results</jats:title><jats:p>Nucleic acids were extracted from 948 clinical samples using an optimised protocol (200<jats:italic>Campylobacter</jats:italic>spp., 199<jats:italic>C. difficile</jats:italic>, 60<jats:italic>S. enterica</jats:italic>, 199 norovirus and 295 negative samples; some samples contained more than one pathogen). Using the MassCode assay, sensitivities for each organism compared with standard microbiological testing ranged from 43% to 94% and specificities from 95% to 98%, with particularly poor performance for<jats:italic>S. enterica</jats:italic>. Relatively large numbers of unexpected positives not confirmed with quantitative PCR were also observed, particularly for<jats:italic>S. enterica</jats:italic>,<jats:italic>Giardia lamblia</jats:italic>and<jats:italic>Cryptosporidium</jats:italic>spp. As the results indicated that<jats:italic>S. enterica</jats:italic>detection might provide generic challenges to other multiplex assays for gastrointestinal pathogens, the Luminex xTag<jats:sup>®</jats:sup>gastrointestinal assay was also run blinded on the same extracts (937/948 remaining) and on re-extracted samples (839/948 with sufficient material). For<jats:italic>Campylobacter</jats:italic>spp.,<jats:italic>C. difficile</jats:italic>and norovirus, high sensitivities (&gt; 92%) and specificities (&gt; 96%) were observed. For<jats:italic>S. enterica</jats:italic>, on the original MassCode/Oxford extracts, Luminex sensitivity compared with standard microbiological testing was 84% [95% confidence interval (CI) 73% to 93%], but this dropped to 46% on a fresh extract, very similar to MassCode, with a corresponding increase in specificity from 92% to 99%. Overall agreement on the per-sample diagnosis compared with combined microbiology plus PCR for the main four/all pathogens was 85.6%/64.7%, 87.0%/82.9% and 89.8%/86.8% for the MassCode assay, Luminex assay/MassCode extract and Luminex assay/fresh extract, respectively. Luminex assay results from fresh extracts implied that 5% of samples did not represent infectious diarrhoea, even though enteropathogens were genuinely present. Managing infectious diarrhoea was a significant burden for infection control teams (taking 21% of their time) and better diagnostics were identified as having major potential benefits for patients.</jats:p></jats:sec><jats:sec id="abs1-7"><jats:title>Conclusions</jats:title><jats:p>Overall, the Luminex xTag gastrointestinal panel showed similar or superior sensitivity and specificity to the MassCode assay. However, on fresh extracts, this test had low sensitivity to detect a key enteric pathogen,<jats:italic>S. enterica</jats:italic>; making it an unrealistic option for most microbiology laboratories. Extraction efficiency appears to be a major obstacle for nucleic acid-based tests for this organism, and possibly the whole Enterobacteriaceae family. To improve workflows in service microbiology laboratories, to reduce workload for infection control practitioners, and to improve outcomes for NHS patients, further research on deoxyribonucleic acid-based multiplex gastrointestinal diagnostics is urgently needed.</jats:p></jats:sec><jats:sec id="abs1-8"><jats:title>Funding</jats:title><jats:p>The Health Technology Assessment programme of the National Institute for Health Research.</jats:p></jats:sec>

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Journal article


Health Technology Assessment


National Institute for Health Research

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