INDUSTRY MATTERS
Rapid Candida auris Screening and Echinocandins Resistance Detection Through an Automated High- Throughput Extraction and Real-time PCR Solution
By Macy Veling , PhD , Principal Application Scientist , Revvity Health Sciences , Inc ; Christodoulos Filippis , PhD , Institute for Experimental Immunology , affiliated to EUROIMMUN Medizinische Labordiagnostika AG ; Peishan Xie , Application Scientist , Revvity Health Sciences , Inc ; Kathleen Franck , Institute for Experimental Immunology , affiliated to EUROIMMUN Medizinische Labordiagnostika AG ; Melanie Harder , PhD , Institute for Experimental Immunology , affiliated to EUROIMMUN Medizinische Labordiagnostika AG ; Ulf Steller , PhD , Institute for Experimental Immunology , affiliated to EUROIMMUN Medizinische Labordiagnostika AG ; and Yanhong Tong , PhD , Sr . R & D Manager , Revvity Health Sciences , Inc .
All authors are employees of Revvity or EUROIMMUN , companies that manufacture diagnostic tests and instruments . They did not benefit from any potential or actual financial gain as a result of the work .
Figure 1 . Sensitivity confirmation for the Candida auris Detection Real-time PCR Reagents workflow ( total ~ 3 hours )
A potentially deadly fungal infection by Candida auris including the multidrugresistant strains is spreading globally at an alarming rate . Such infection not only can cause severe illness and spread easily among patients in healthcare facilities but also can lead to failure of treatment due to resistance , making C . auris a high-priority pathogen to be monitored . Unlike traditional culturing methods , which could take days for species and drug resistance identification , real-time PCR provides a rapid solution for C . auris screening and potential drug resistance detection . Here , we present an automated extraction workflow together with two real-time PCR assays : the Candida auris Detection Real-time PCR Reagents ( herein screening assay ) with dUTP / UNG carryover prevention system integrated for qualitative C . auris screening and the EURORealTime C . auris ECH-R ( herein resistance assay ) for both C . auris and echinocandins resistance detection . These solutions are suitable with samples collected from human skin swabs , environmental surface swabs or laboratory cultures .
Methods
The chemagic TM Pathogen NA Kit H96 ( Revvity , CMG-1033-G ) is designed for fungal pathogen extraction with the chemagic TM 360 extraction system ( max . 96-reactions / run ). Both the screening ( Revvity , DXMDX-RGT-1001 ) and the resistance ( EUROIMMUN , MP 2866-0125 or MP 2866-0100 ) assays utilize sequencespecific oligonucleotides to amplify the partial genetic region of the internal transcribed spacer ( ITS ) 1 & 2 for C . auris identification . 1 Primer / probe sets to detect internal control ( IC ; only screening assay ) and endogenous human gene ( RNase P ) are included for extraction and PCR process monitoring as well as for skin swab sample quality check . In the resistance assay , additional primer / probes to detect three highly-echinocandinsresistance-associated mutations ( S639F / Y / P ) on the FKS1 gene HS1 region ( FKS ) are included . 2 – 4 An extra probe for validity purposes monitors amplification of the FKS on all strains .
Analytical sensitivity was evaluated for both PCR assays by spiking C . auris positive wild-type culture samples
( various concentrations ) and human gDNA into the Eswab collection / transport system ( BD , 220245 ), followed by extraction and one of the PCR assays . Plasmid constructs containing the relevant C . auris gene sections were spiked into PCR for S639F / Y / P sensitivity evaluation . Specificity of the oligonucleotide sequences of both PCR tests was analyzed in silico using BLASTn and publicly available C . auris genome sequences plus non-C . auris microorganisms ( either belonging to the Candida family or commonly found in skin infections ). 5 , 6 Furthermore , a wet-lab study with gDNA from eight closely related Candida species was evaluated .
Results
The analytical sensitivity for the entire workflow with culture showed 100 % C . auris detection at a concentration as low as 600 cfu / mL for both PCR assays , and 100 % FKS detection at 1800 cfu / mL for the resistance assay ( Figure 1 – 2 ). For S639F , S639P and S639Y detection , sensitivity using synthetic plasmids showed 100 % detection at 30 copies / reaction ( Figure 2 ).
12 LAB MATTERS Summer 2024
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