Development of a real-time PCR assay and comparison to CHROMagar(TM) STEC to screen for Shiga toxin-producing Escherichia coli in stool, Cape Town, South Africa

INTRODUCTION: Shiga toxin-producing Escherichia coli (STEC) is an emerging infectious pathogen which could lead to haemolytic uremic syndrome. Even though previous studies have compared the performance of CHROMagar(TM)STEC to real-time polymerase chain reaction (PCR) in Europe, no study has been done to assess its performance on African isolates. OBJECTIVES: This project aimed to validate and test an in-house-developed duplex real-time PCR and use it as a reference standard to determine the performance of CHROMagar(TM)STEC on African isolates from diarrhoeic stool samples. METHODS: This study evaluated STEC diagnostic technology on African isolates. An in-house-developed duplex real-time PCR assay for detection of stx(1) and stx(2) was validated and tested on diarrhoeic stool samples and then used as a reference standard to assess the performance of CHROMagar(TM)STEC. Real-time PCR was used to screen for stx in tryptic soy broth and the suspected STEC isolates, while conventional PCR was used to detect the other virulence genes possessed by the isolates. RESULTS: The real-time PCR limit of detection was 5.3 target copies/μL of broth. The mean melting temperature on melt-curve analysis for detection of stx(1) was 58.2 °C and for stx(2) was 65.3 °C. Of 226 specimens screened, real-time PCR detected stx in 14 specimens (6.2%, 95% confidence interval = 3.43% – 10.18%). The sensitivity, specificity, negative predictive value and positive predictive value of the CHROMagar(TM)STEC were 33.3%, 77.4%, 95.3% and 11.3%. CONCLUSIONS: The in-house developed real-time PCR assay is a sensitive and specific option for laboratory detection of STEC as compared to CHROMagar(TM)STEC in this setting.