Els van de Vijver

40 METHODS Design This was an international multicenter, delayed-type cross-sectional diagnostic accuracy study with a paired design.(11) Previously undiagnosed children and teenagers presenting with persistent diarrhoea for more than 4 weeks or chronic or recurrent abdominal pain were screened with the calprotectin stool test (existing test) and with the calgranulin-C test (new test). Confirmation of the target condition (IBD) was based on endoscopy with biopsies (reference standard) or clinical follow-up (alternative reference standard). The study was registered before recruitment of the first participant, and the study protocol has been published in BMJ Open.(12) Patients Patients were recruited from sixteen secondary and three tertiary level hospitals in the Netherlands and Belgium. They were eligible when aged between 6 and 17 years. The flow of patients from the first hospital visit to the choice of the reference test was described comprehensively in our published study protocol.(12) In brief, during the first hospital visit baseline characteristics, date of birth, presence of major and minor red flag signs and symptoms for IBD and use of non-steroidal anti-inflammatory drugs were entered on the study website (www.cacatustudie.eu) . A stool specimen was collected at home and sent to the hospital laboratory of the coordinating study center, where it was immediately tested for calprotectin and colon pathogens (including Shigatoxin-producing Escherichia coli, Salmonella, Shigella, Enteroinvasive Escherichia coli, Campylobacter , and Giardia lamblia ) with a real-time multiplex PCR technique. Residual faeces was stored at −80°C for calgranulin- C batch testing at a later stage. Assays Stool calprotectin concentrations (μg/g) were measured with the fCAL ELISA (BÜHLMANN Laboratories AG, Schönenbuch, Switzerland) and stool calgranulin-C concentrations (μg/g) with the commercially available Inflamark ELISA (CisBio Bioassays, Codolet, France), both on a Dynex DS2 Automated ELISA System (Alpha Labs, Easleigh, UK) in the same laboratory. The extraction and measuring technique of calgranulin-C was previously described in detail.(13) In discordant pairs (i.e. increased calprotectin and normal calgranulin-C, or vice versa) we did a posthoc analysis of potential viral causes (adeno, entero, astro, rota, noro, parecho and Chapter 3