RAPID IDENTIFICATION OF THE ETIOLOGICAL FACTORS CAUSING DIARRHEAL DISEASES.

The main objective of this investigation was to develop and pilot a real-time Polymerase Chain Reaction (rt-PCR) diagnostic system for rapid and simultaneous identification of pathogens with a particular emphasis on diarrheal disease diagnostics. The diarrheal diseases were selected as a target for the pilot because they constitute a primary public health priority in Georgia and worldwide. The product developed by our research team "Neo_PCR_Diagnostics" represents an original system for the identification of pathogens associated with gastrointestinal tract infections and diarrhea. The advantages of the proposed technology over existing conventional methods include the ability of simultaneous identification of multiple pathogens and the detection of pathogenic agents directly from the fecal samples. For the evaluation of the new diagnostic system, stool samples were collected at collaborating hospitals and clinics and analyzed by real-time PCR using the Neo_PCR_Diagnostic system. The selection of the pathogens for detection was based on their epidemiological and clinical importance. The following bacterial pathogens were targets for detection: Salmonella spp., Campylobacter spp., Shigella spp., Clostridium difficile (Toxin A/B), Escherichia coli (ETEC, STEC and O157), Yersinia enterocolitica and Vibrio cholerae. The following viral pathogens were studied: adenoviruses, rotaviruses and noroviruses. Genetic material (DNA) of the following parasites were targets in our study: Giardia lamblia, Entamoeba histolitica and Cryptosporidium spp. We also compared the results obtained by our molecular technology with the conventional methods - bacterial culture (for bacterial growth) and ELISA (for bacterial toxins). For viral and parasitic pathogens, comparison tests were performed with immunochromatographic assays for direct detection of antigens in the stool samples or with the data obtained by use of home-made end-point PCR (where available). Advantages of the proposed technology over existing conventional technologies include the ability of simultaneous identification of diarrheal infections by multiple pathogens. The proposed test system allows the detection of pathogenic agents directly from the fecal samples and can be completed within one working day. In general, the spectrum of pathogens detected by our approach was wider than those detected by the conventional methods used in the clinical setting, taking into consideration the list of pathogenic agents requisitioned by physicians within the framework of the routine clinical visit. Given these promising results, Neo_PCR_Diagnostics test performance and accuracy may be sufficient for use in molecular microbiological diagnostics in clinical and/or research settings.