Quantitative PCR Effectively Quantifies Triazole-Susceptible and Triazole-Resistant Aspergillus fumigatus in Mixed Infections

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.