Background:

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.

Objective:

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: Clostridium difficile, Campylobacter spp., Salmonella spp. and norovirus.

Design:

A retrospective study of fixed numbers of samples positive for C. difficile (n = 200), Campylobacter spp. (n = 200), Salmonella spp. (n = 100) and norovirus (n = 200) plus samples negative for all these pathogens (n = 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.

Setting:

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.

Main outcome measures:

Sensitivity and specificity to detect C. difficile, Campylobacter spp., Salmonella spp., and norovirus.

Results:

Nucleic acids were extracted from 948 clinical samples using an optimised protocol (200 Campylobacter spp., 199 C. difficile, 60 S. enterica, 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 S. enterica. Relatively large numbers of unexpected positives not confirmed with quantitative PCR were also observed, particularly for S. enterica, Giardia lamblia and Cryptosporidium spp. As the results indicated that S. enterica detection might provide generic challenges to other multiplex assays for gastrointestinal pathogens, the Luminex xTag^® gastrointestinal assay was also run blinded on the same extracts (937/948 remaining) and on re-extracted samples (839/948 with sufficient material). For Campylobacter spp., C. difficile and norovirus, high sensitivities (> 92%) and specificities (> 96%) were observed. For S. enterica, 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.

Conclusions:

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, S. enterica; 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.

Funding:

The Health Technology Assessment programme of the National Institute for Health Research.

Article history

The research reported in this issue of the journal was funded by the HTA programme as project number 08/13/35. The contractual start date was in April 2010. The draft report began editorial review in September 2013 and was accepted for publication in January 2014. The authors have been wholly responsible for all data collection, analysis and interpretation, and for writing up their work. The HTA editors and publisher have tried to ensure the accuracy of the authors’ report and would like to thank the reviewers for their constructive comments on the draft document. However, they do not accept liability for damages or losses arising from material published in this report.

Declared competing interests of authors

The institution of DWC and TEAP received per-case funding from Optimer Pharmaceuticals to support fidaxomicin trial patient expenses for a trial in Clostridium difficile infection. DWC and TEAP also received honoraria from Optimer Pharmaceuticals for participation in additional meetings related to investigative planning for fidaxomicin. MHW has received consulting fees from Actelion, Astellas, AstraZeneca, Cerexa, Cubist, Durata, Merck, Nabriva, Novacta, Novartis, Optimer, Paratek, Pfizer, Roche, Sanofi Pasteur, Summit, Synthetic Biologics, The Medicines Company and VHsquared; lecture fees from Abbott, Astellas, AstraZeneca and Pfizer; grant support from Abbott, Actelion, Astellas, bioMérieux, Cubist, Da Volterra, The European Tissue Symposium, Merck and Summit; and a lecture fee from Alere paid to his department. The Medicines Company and VH Squared; lecture fees from Abbott, Astellas, AstraZeneca and Pfizer; grant support from Abbott, Actelion, Astellas, bioMerieux, Cubist, Da Volterra, The European Tissue Symposium, Merck and Summit; and a lecture fee from Alere paid to his department. Unrelated to C. difficile or any gastrointestinal pathogen, DWC has received research funding from BioMérieux for Staphylococcus aureus bacterial genome wide association studies, from Pfizer for Streptococcus pneumoniae surveillance, and from Microsoft Azure for cloud-based access for bacterial genomic analysis. The institution where ASW holds a part-time post (not involved or acknowledged in this study) has received funding from Gilead Sciences and ViiV Healthcare/GlaxoSmithKline for additional assays and/or analyses within clinical trials in human immunodeficiency virus (HIV) infection; from Gilead Sciences for her lecturing on educational workshops, and from Janssen and Janssen (formerly Tibotec) for her Data and Safety Monitoring Board membership. No other author has a conflict of interest.