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Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR
Published on: February 1, 2014
Clinical validation of loop-mediated isothermal amplification for the detection of Escherichia coli sequence type
Gisele Peirano1,2, L Ricardo Castellanos1, Yasufumi Matsumura3
1Cummings School of Medicine, University of Calgary, Calgary, Alberta, Canada.
Abstract:
The dissemination of Escherichia coli multidrug-resistant (MDR) STc131 is related to its persistence in the human gastrointestinal tract as efficient gut colonizers. Infection and prevention measures are the cornerstones for preventing STc131 spread. Oral decolonization therapies that target ST131 are being developed. There are no rapid methods available to identify STc131 in human specimens. A loop-mediated isothermal amplification (LAMP) assay (named LAMP-ST131) was developed for the detection of STc131 on well-characterized E. coli isolates and then compared to culture and PCR for urines and stool swabs. With E. coli isolates (n = 720), LAMP-ST131 had a sensitivity (sens) of 100% [95% confidence interval (C.I.) = 98.1-100%)] and a specificity (spec) of 98.9% (95% C.I. = 97.5-99.5%). On urines (n = 550), LAMP-ST131 had a sens of 97.6% (95% C.I. = 89.68-94.33%) and a spec of 92.3% (95% C.I. = 87.68-99.88%), while on stool swabs (n = 278), LAMP-ST131 had a sens of 100% (95% C.I. = 88.7-100%) and a spec of 83.9% (95% C.I. = 78.8-87.9%). LAMP-ST131 detected 10 (urines) and 100 (stool swabs) gene copies/μL. LAMP-ST131 accurately identified STc131 within E. coli isolates and human specimens. The implementation of LAMP-ST131 will aid genomic surveys, enable the rapid implementation of effective infection prevention measures, and identify patients suitable for ST131 decolonization therapies. Such approaches will curb the spread of STc131 and decrease incidence rates of global MDR E. coli infections.
Importance:
We developed an accurate non-culture-based loop-mediated isothermal amplification (LAMP) methodology for the detection of (sequence type) STc131 among Escherichia coli isolates and human specimens. The use of LAMP-ST131 for global genomic surveillance studies and to identify patients that are suitable for ST131 decolonization therapies will be important for decreasing multidrug-resistant E. coli infections across the globe.
Insights
A new loop-mediated isothermal amplification (LAMP) assay accurately detects multidrug-resistant Escherichia coli STc131. This rapid method aids infection control and identifies patients for decolonization therapies, curbing global infections.
Area of Science:
- Microbiology
- Infectious Diseases
- Molecular Diagnostics
Background:
- Multidrug-resistant (MDR) Escherichia coli STc131 is a significant global health threat due to its efficient colonization of the human gut.
- Current infection control strategies and the development of targeted decolonization therapies are hampered by the lack of rapid identification methods for STc131.
Purpose of the Study:
- To develop and validate a rapid, non-culture-based diagnostic assay for the specific detection of MDR Escherichia coli STc131.
- To assess the performance of the developed assay in identifying STc131 in both laboratory isolates and clinical specimens.
Main Methods:
- A loop-mediated isothermal amplification (LAMP) assay, designated LAMP-ST131, was designed for the detection of STc131.
- The LAMP-ST131 assay was evaluated using a collection of well-characterized E. coli isolates (n=720).
- The assay's performance was further assessed using clinical samples, including urine specimens (n=550) and stool swabs (n=278), and compared against traditional culture and PCR methods.
Main Results:
- LAMP-ST131 demonstrated high accuracy in identifying STc131 in E. coli isolates, with a sensitivity of 100% and specificity of 98.9%.
- In clinical specimens, the assay showed a sensitivity of 97.6% for urine and 100% for stool swabs, with specificities of 92.3% and 83.9%, respectively.
- The assay is capable of detecting as few as 10 gene copies/μL in urine and 100 gene copies/μL in stool swabs, indicating high sensitivity.
Conclusions:
- The developed LAMP-ST131 assay provides a rapid and accurate method for identifying MDR Escherichia coli STc131 in clinical settings.
- Implementation of LAMP-ST131 can significantly enhance genomic surveillance, facilitate timely infection control interventions, and enable patient selection for decolonization therapies.
- This diagnostic tool has the potential to reduce the global burden of MDR E. coli infections by curbing STc131 dissemination.
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