The value of molecular methods in rapid diagnosis of campylobacter jejuni and the other etiologic agents in acute diarrhea

Thermophilic Campylobacters are one of the most significant causes of diarrhea especially in children aged 0-2 in developing countries. Microbiological diagnosis of Campylobacter infections which are among limited indications for antibiotics, is important for decreasing the mortality rate and establishing an effective treatment plan against infections in childhood diarrhea with high mortality. The conventional culture based methods for isolation and identification of thermophilic Campylobacter species in patients?s stool specimens are highly difficult, time-consuming, expensive, low sensitive and low specific. For these reasons, in last decade, nucleic acid amplification based methods which are more sensitive and specific than conventional based culture methods, have been using for diagnosis of Campylobacters in clinical specimens.The aim of this study was to determine the sensitivity of selective culture methods and PCR-RFLP based on 23S rDNA for detecting and identifying Campylobacter jejuni and the other thermophilic Campylobacter species in patients with acute diarrhea.In this study, a total of 434 faecal samples obtained from patients with acute diarrhea over a fifteen-month period from June 2006 to October 2007 were investigated by culture and PCR-RFLP.The isolation of Campylobacters from faecal samples was carried out on Skirrow selective agar (SSA), modified Campylobacter blood free selective agar (mCCDA) and CCDA with CAT supplement medium.Extraction of DNA was performed with Qia-Gen stool extraction kit from faecal samples and with freeze-boil method from colonies growth on selective medium. A fragment of 491 bp of the 23s rDNA gene was amplified for identification of thermotolerant Campylobacters and amplicons were digested with restriction enzymes Alu I and Tsp 509I. The restriction fragments were separated by gel electrophoresis giving a specific banding pattern for each one of Camphylobacter species.As a result, 22 (5,1 %) stool specimens (females 13(59 %); males 9(41 %)) were found Campylobacter positive in at least one of these method 16(3,7 %)specimen were positive on mCCDA and CCDA with CAT supplement medium and 13(3 %) specimen on SSA medium. 19(4,5 %) stool specimen were found PCR positive for thermophilic Campylobacter species.The Hippurate hydrolysis test results showed that of 17 isolates growth on selective medium, 13 (76,5 %) were identified as C.jejuni as the most frequent species. 14 (82,4 %) isolates were identified as C.jejuni by PCR-RFLP