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Abstract Background Persistent digestive disorders account for considerable disease burden in the tropics. Despite advances in understanding acute gastrointestinal infections, important issues concerning epidemiology, diagnosis, treatment and control of most persistent digestive symptomatologies remain to be elucidated. Helminths and intestinal protozoa are considered to play major roles, but the full extent of the aetiologic spectrum is still unclear. We provide an overview of pathogens causing digestive disorders in the tropics and evaluate available reference tests. Methods We searched the literature to identify pathogens that might give rise to persistent diarrhoea, chronic abdominal pain and/or blood in the stool. We reviewed existing laboratory diagnostic methods for each pathogen and stratified them by (i) microscopy; (ii) culture techniques; (iii) immunological tests; and (iv) molecular methods. Pathogen-specific reference tests providing highest diagnostic accuracy are described in greater detail. Results Over 30 pathogens may cause persistent digestive disorders. Bacteria, viruses and parasites are important aetiologic agents of acute and long-lasting symptomatologies. An integrated approach, consisting of stool culture, microscopy and/or specific immunological techniques for toxin, antigen and antibody detection, is required for accurate diagnosis of bacteria and parasites. Molecular techniques are essential for sensitive diagnosis of many viruses, bacteria and intestinal protozoa, and are increasingly utilised as adjuncts for helminth identification. Conclusions Diagnosis of the broad spectrum of intestinal pathogens is often cumbersome. There is a need for rapid diagnostic tests that are simple and affordable for resource-constrained settings, so that the management of patients suffering from persistent digestive disorders can be improved.