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Optimizing a real-time PCR assay for rapid detection of Candida auris in nasal and axillary/groin samples
Michael Malczynski1, Noor Dowllow1, Saba Rezaeian1
1Clinical Microbiology Laboratory, Department of Pathology, Northwestern Memorial Hospital, Chicago, IL, USA.
Abstract:
Introduction. Candida auris is an emerging fungal pathogen. The organism can cause invasive infections associated with high mortality, has been implicated in outbreaks in healthcare settings and is frequently resistant to multiple antifungal agents, making it a significant challenge to infection prevention and patient treatment.Aim. To implement a real-time PCR assay for detection of C. auris in patient surveillance samples collected with the Copan Liquid Amies elution swab (ESwab) collection and transport system.Methodology. We optimized a real-time PCR testing procedure based on the sample collection device used in our institution.Results . ESwab transport medium was strongly inhibitory to the real-time PCR. Removing the medium with centrifugation, followed by suspending the pellet in PBS-BSA buffer (concentration 1 %), sufficiently eliminated the inhibition. The manual sample preparation method, freeze-thaw followed by mechanical disruption, allowed the detection of C. auris at the lowest cell concentration.Conclusion . The optimized procedure was used to test 1414 patient surveillance samples. The real-time PCR detected all culture-positive samples with 100 % sensitivity and 100 % specificity.
Insights
A new real-time PCR assay effectively detects Candida auris in patient samples. This method overcomes inhibition from ESwab collection systems, ensuring high sensitivity and specificity for early detection and infection control.
Area of Science:
- Clinical Microbiology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Candida auris is a challenging emerging fungal pathogen causing invasive infections.
- High mortality rates and frequent antifungal resistance complicate C. auris infection control.
- Effective surveillance methods are crucial for preventing C. auris outbreaks in healthcare settings.
Purpose of the Study:
- To develop and implement a real-time PCR assay for detecting Candida auris.
- To adapt the assay for patient surveillance samples using the Copan Liquid Amies ESwab system.
- To optimize sample preparation to overcome inhibitory factors in the ESwab medium.
Main Methods:
- Optimization of a real-time PCR procedure tailored to the ESwab collection device.
- Investigated and mitigated inhibition caused by ESwab transport medium.
- Developed a manual sample preparation involving centrifugation, resuspension in PBS-BSA, freeze-thaw, and mechanical disruption.
Main Results:
- ESwab transport medium demonstrated significant inhibition of real-time PCR.
- Centrifugation and resuspension in PBS-BSA buffer effectively eliminated PCR inhibition.
- The optimized manual sample preparation enabled detection of C. auris at low concentrations.
- Tested on 1414 surveillance samples, the assay achieved 100% sensitivity and 100% specificity.
Conclusions:
- An optimized real-time PCR procedure effectively detects Candida auris in ESwab surveillance samples.
- The method successfully overcomes inhibitory components present in the ESwab transport medium.
- This validated assay provides a highly sensitive and specific tool for C. auris surveillance.
