Original Research

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

John B. Kalule, Karen H. Keddy, Anthony Smith, Mark P. Nicol, Lourens Robberts
African Journal of Laboratory Medicine | Vol 6, No 1 | a609 | DOI: https://doi.org/10.4102/ajlm.v6i1.609 | © 2017 John B. Kalule, Karen H. Keddy, Anthony Smith, Mark P. Nicol, Lourens Robberts | This work is licensed under CC Attribution 4.0
Submitted: 28 January 2017 | Published: 14 December 2017

About the author(s)

John B. Kalule, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa
Karen H. Keddy, Center for Enteric Disease Research Unit, National Institute for Communicable Diseases, Johannesburg, South Africa
Anthony Smith, Center for Enteric Disease Research Unit, National Institute for Communicable Diseases, Johannesburg, South Africa
Mark P. Nicol, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa
Lourens Robberts, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa


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Abstract

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 CHROMagarTMSTEC 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 CHROMagarTMSTEC 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 stx1 and stx2 was validated and tested on diarrhoeic stool samples and then used as a reference standard to assess the performance of CHROMagarTMSTEC. 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 stx1 was 58.2 °C and for stx2 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 CHROMagarTMSTEC 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 CHROMagarTMSTEC in this setting.


Keywords

Shiga toxin-producing E. coli; CHROMagarTMSTEC; Assay development

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Crossref Citations

1. Characterisation of STEC and other diarrheic E. coli isolated on CHROMagar™STEC at a tertiary referral hospital, Cape Town
John Bosco Kalule, Karen H. Keddy, Mark P. Nicol
BMC Microbiology  vol: 18  issue: 1  year: 2018  
doi: 10.1186/s12866-018-1195-7