Rapid and Accurate Diagnosis Based on Real-Time PCR Cycle Threshold Value for the Identification of Campylobacter jejuni, astA Gene-Positive Escherichia coli, and eae Gene-Positive E. coli

Abstract

We previously developed a multiplex real-time PCR assay (Rapid Foodborne Bacterial Screening 24 ver.5, [RFBS24 ver.5]) for simultaneous detection of 24 foodborne bacterial targets. Here, to overcome the discrepancy of the results from RFBS24 ver.5 and bacterial culture methods (BC), we analyzed 246 human clinical samples from 49 gastroenteritis outbreaks using RFBS24 ver.5 and evaluated the correlation between the cycle threshold (CT) value of RFBS24 ver.5 and the BC results. The results showed that the RFBS24 ver.5 was more sensitive than BC for Campylobacter jejuni and Escherichia coli harboring astA or eae, with positive predictive values (PPV) of 45.5-87.0% and a kappa coefficient (KC) of 0.60-0.92, respectively. The CTs were significantly different between BC-positive and -negative samples (p ＜ 0.01). All RFBS24 ver.5-positive samples were BC-positive under the lower confidence interval (CI) limit of 95% or 99% for the CT of the BC-negative samples. We set the 95% or 99% CI lower limit to the determination CT (d-CT) to discriminate for assured BC-positive results (d-CTs: 27.42-30.86), and subsequently the PPVs (94.7%-100.0%) and KCs (0.89-0.95) of the 3 targets were increased. Together, we concluded that the implication of a d-CT-based approach would be a valuable tool for rapid and accurate diagnoses using the RFBS24 ver.5 system.

Keywords: Campylobacter; bacterial pathogen; diarrheagenic E. coli; gastroenteritis outbreak; real-time PCR.