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Quantitative PCR Effectively Quantifies Triazole-Susceptible and Triazole-Resistant Aspergillus fumigatus in Mixed
Agustin Resendiz-Sharpe1, Wannes Van Holm2, Rita Merckx3
1Department of Imaging and Pathology, Biomedical MRI, KU Leuven, 3000 Leuven, Belgium.
Abstract:
Increasing resistance to triazole antifungals in Aspergillus fumigatus is worrisome because of the associated high mortality of triazole-resistant A. fumigatus (TRAF) infections. While most studies have focused on single triazole-susceptible (WT) or TRAF infections, reports of TRAF cases developing mixed WT and TRAF infections have been described in several studies. However, the prevalence of mixed infections and their responses to current recommended therapies are unknown and could be inappropriate, leading to poor clinical outcomes. To address the urgent need for tools to diagnose, monitor disease development and therapy efficacies in mixed infection settings where quantification of WT versus TRAF is key, this study developed a novel qPCR assay to differentiate WT and TRAF harboring the cyp51A-TR34/L98H mutation. The proposed assay successfully quantified A. fumigatus and discriminated TRAF-TR34 in vitro and in vivo, which was achieved by increasing the yield of extracted DNA through improved homogenization and specific primers targeting the WT-sequence or TR34-insertion and a TaqMan-probe directed to A. fumigatus. The here-developed qPCR assay overcomes sensitivity issues of methodologies such as CFU counts, providing specific, reproducible, and reliable quantitative information to study and follow up the (interplay and individual) effects of mixed A. fumigatus infections on disease development and treatment responses.
Insights
A new qPCR assay can now quantify triazole-susceptible and triazole-resistant Aspergillus fumigatus (TRAF) in mixed infections. This tool is crucial for monitoring disease progression and treatment effectiveness in patients with TRAF infections.
Area of Science:
- Medical Mycology
- Infectious Diseases
- Molecular Diagnostics
Background:
- Increasing triazole antifungal resistance in Aspergillus fumigatus (TRAF) is a significant clinical concern due to high mortality rates.
- Mixed infections involving both susceptible (WT) and resistant (TRAF) Aspergillus fumigatus strains are increasingly reported.
- The prevalence and clinical impact of mixed infections, along with optimal therapeutic strategies, remain poorly understood.
Purpose of the Study:
- To develop a novel quantitative PCR (qPCR) assay for differentiating and quantifying WT and TRAF strains of Aspergillus fumigatus in mixed infections.
- To provide a reliable tool for monitoring disease development and assessing treatment efficacy in mixed Aspergillus fumigatus infections.
Main Methods:
- Development of a specific qPCR assay targeting the cyp51A-TR34/L98H mutation.
- Optimization of DNA extraction through improved homogenization techniques.
- Design of specific primers and a TaqMan probe for accurate quantification of WT and TRAF strains.
Main Results:
- The developed qPCR assay successfully quantified Aspergillus fumigatus and discriminated TRAF-TR34 strains both in vitro and in vivo.
- The assay demonstrated superior sensitivity compared to traditional methods like CFU counts.
- The assay provides specific, reproducible, and reliable quantitative data for mixed infections.
Conclusions:
- The novel qPCR assay offers a sensitive and specific method for diagnosing and monitoring mixed Aspergillus fumigatus infections.
- This tool will aid in understanding the interplay between WT and TRAF strains and their impact on disease progression and treatment outcomes.
- The assay addresses a critical need for quantitative tools in managing triazole-resistant Aspergillus fumigatus infections.
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