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Rapid Tetra-Primer Amplification Refractory Mutation System-Polymerase Chain Reaction Protocol for Detection of Y132F
Sevgi Öztürk1,2, Kübra Çam1,2, Gizem Babuccu1,2
1Koc University Is Bank Center for Infectious Disease (KUISCID), Istanbul, Turkey.
Abstract:
There is an emerging fluconazole resistance in Candida parapsilosis in recent years. The leading mechanism causing azole resistance in C. parapsilosis is the Y132F codon alteration in the ERG11 gene which encodes the target enzyme of azole drugs. In this study, we evaluated the sensitivity, compatibility, and specificity of a novel tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) method for rapid detection of the Y132F mutation in fluconazole nonsusceptible C. parapsilosis. Antifungal susceptibility tests for detection of fluconazole resistance were performed by broth microdilution according to the CLSI guidelines. All susceptible and nonsusceptible C. parapsilosis isolates were analyzed for ERG11 mutations with Sanger sequencing. T-ARMS-PCR was fully concordant with the Sanger sequencing (100% of sensitivity and specificity) for detection of Y132F mutations. T-ARMS-PCR method could be a rapid, simple, accurate, and economical assay in the early detection of the most common cause of fluconazole resistance in C. parapsilosis isolates. In routine laboratories with high C. parapsilosis isolation rates, performing the T-ARMS-PCR for early detection of the most common reason of fluconazole resistance in C. parapsilosis, could be a life-saving approach for directing antifungal therapy before obtaining the definitive antifungal susceptibility tests results.
Insights
Emerging fluconazole resistance in Candida parapsilosis is often due to the Y132F mutation. A new tetra-primer amplification refractory mutation system-PCR (T-ARMS-PCR) accurately detects this mutation rapidly.
Area of Science:
- Medical Mycology
- Molecular Diagnostics
- Antimicrobial Resistance
Background:
- Increasing fluconazole resistance in *Candida parapsilosis* poses a significant clinical challenge.
- The Y132F mutation in the *ERG11* gene is the primary mechanism driving azole resistance in *C. parapsilosis*.
- Accurate and rapid detection of this mutation is crucial for guiding antifungal therapy.
Purpose of the Study:
- To evaluate the sensitivity, compatibility, and specificity of a novel tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) method.
- To assess the utility of T-ARMS-PCR for the rapid detection of the Y132F mutation in fluconazole-nonsusceptible *C. parapsilosis*.
- To establish T-ARMS-PCR as a reliable diagnostic tool for *C. parapsilosis* resistance.
Main Methods:
- Antifungal susceptibility testing using broth microdilution (CLSI guidelines).
- Sanger sequencing of the *ERG11* gene to identify mutations.
- Validation of the novel T-ARMS-PCR assay against Sanger sequencing results.
Main Results:
- The T-ARMS-PCR method demonstrated 100% concordance with Sanger sequencing.
- The assay achieved 100% sensitivity and specificity in detecting the Y132F mutation.
- T-ARMS-PCR proved to be a rapid, simple, accurate, and economical detection method.
Conclusions:
- T-ARMS-PCR is a highly effective tool for the early detection of the Y132F mutation, the main cause of fluconazole resistance in *C. parapsilosis*.
- This rapid diagnostic approach can aid clinicians in making timely therapeutic decisions, potentially improving patient outcomes.
- Implementing T-ARMS-PCR in clinical laboratories can facilitate prompt identification of resistance mechanisms, guiding effective antifungal treatment strategies.
