Quantitative PCR Effectively Quantifies Triazole-Susceptible and Triazole-Resistant <i>Aspergillus fumigatus</i> in Mixed Infections

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

Exploring the oxidative mechanisms of bitumen after laboratory short- and long-term ageing

Understanding the fundamental mechanisms of oxidative ageing in bitumen is considered of paramount importance in order to take steps towards durable binders able to tackle distresses related to this phenomenon which deteriorates the asphalt performance. This paper focuses on the identification of the intermediate and final oxygenated products after short- and long-term laboratory ageing simulated with rolling thin-film oven testing (RTFOT) and pressurised ageing vessel (PAV) respectively. Three binders were investigated in this study, two originated from the same wax-free crude source, while the third was obtained from a different source, containing natural wax, and followed a different manufacturing process. Fourier-Transform Infrared (FTIR) spectroscopy demonstrated a clear increase of the sulfoxide and carbonyl functional groups upon ageing for all the binders independently of origin, manufacturing or performance. Electron Paramagnetic Resonance (EPR) spectroscopy showed an increase of the organic carbon-centred radicals after short-term ageing (RTFOT), whereas after PAV these radicals remained constant in the two wax-free binders originating from the same crude source, and even decreased for the third, waxy binder. Proton Nuclear Magnetic Resonance (1H NMR) spectroscopy reported differences in the relative distribution of protons between the binders in the unaged state, and similar minor changes after both ageing steps regardless of the binder's crude source and distillation. The results of Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS) revealed that SOx- and (OH)x-containing compounds are produced after the sequentially occurring short- and long-term ageing in both wax-free bitumens, whereas an almost constant behaviour of aliphatics after PAV ageing can be seen for the same bitumens. Finally, the strengths and weaknesses of each of these experimental techniques were reviewed and compared versus the obtained results and possible ageing mechanisms.Green Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public.Pavement Engineerin