ADVANTAGES OF EVA GREEN REAL-TIME MPCR WITH CULTURE AND IMMUNOCHROMATOGRAPHIC METHODS FOR DIFFERENTIATING C. JEJUNI / COLI DIRECTLY FROM FECES

Objective: To develop and optimize a rapid molecular method for diagnosing campylobacteriosis directly from a clinical fecal sample and at the same time determining the most common causing agents - C. jejuni/coli. Materials and methods: 40 clinical fecal samples of hospitalized patients in the Pediatric Clinic of Infectious Diseases - Sofia were tested, the patients aged between 0 and 7 years and with diarrheal syndrome. The clinical samples were tested using a rapid immunochromatographic test (ICT) (CerTest Biotec). All positive samples were tested for confirmation by culturing in a microaerophilic atmosphere at 42 degrees C and subsequently the isolates were biochemically differentiated. The Eva Green real-time mPCR reaction of a direct fecal sample was conducted using the "IQ5TM Real-Time PCR System" apparatus. Results: Out of 40 clinical fecal samples which were ICT positive, 20 strains were isolated by culture - 18 of C. jejuni and 2 of C. coli. The Eva Green real-time mPCR reaction also reported 20 positive samples for Campylobacter - 18 out of which were of C. jejuni and 2 of C. coli. All other samples were negative for Campylobacter spp. The analytical sensitivity and specificity of the mPCR method were 100%. Discussion: We developed and optimized the Eva Green real-time mPCR for detection and species differentiation of C. jejuni/coli directly from a clinical fecal sample. This analysis ensures the faster and more reliable detection of bacterial cells when compared to the conventional culture methods using biochemical differentiation.