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.

Autor: Kalule JB; Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa., Keddy KH; Center for Enteric Disease Research Unit, National Institute for Communicable Diseases, Johannesburg, South Africa., Smith A; Center for Enteric Disease Research Unit, National Institute for Communicable Diseases, Johannesburg, South Africa., Nicol MP; Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa., Robberts L; Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa.
Jazyk: angličtina
Zdroj: African journal of laboratory medicine [Afr J Lab Med] 2017 Dec 14; Vol. 6 (1), pp. 609. Date of Electronic Publication: 2017 Dec 14 (Print Publication: 2017).
DOI: 10.4102/ajlm.v6i1.609
Abstrakt: 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.
Competing Interests: The authors declare that there are no financial or personal relationships that may have had influence in the writing of this article.
Databáze: MEDLINE
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