Emerging Threats in Antifungal-Resistant Fungal Pathogens.

The use of antifungal drugs in the therapy of fungal diseases can lead to the development of antifungal resistance. Resistance has been described for virtually all antifungal agents in diverse pathogens, including Candida and Aspergillus species. The majority of resistance mechanisms have also been elucidated at the molecular level in these pathogens. Drug resistance genes and genome mutations have been identified. Therapeutic choices are limited for the control of fungal diseases, and it is tempting to combine several drugs to achieve better therapeutic efficacy. In the recent years, several novel resistance patterns have been observed, including antifungal resistance originating from environmental sources in Aspergillus fumigatus and the emergence of simultaneous resistance to different antifungal classes (multidrug resistance) in different Candida species. This review will summarize these current trends

Azole and fungicide resistance in clinical and environmental Aspergillus fumigatus isolates

Aspergillus fumigatus is a human pathogen but it is also a widespread filamentous fungus in the environment. A. fumigatus can therefore be exposed to antifungals used in medical and agricultural environments. Only the class of azoles is used in both of these environments (i.e., voriconazole and itraconazole in medicine; prochloraz, propiconazole or imazalil in agriculture). Exposure to azoles provides the potential for the development of resistance. Several clinical itraconazole-resistant isolates have been reported in A. fumigatus and their resistance mechanisms have been partially resolved. Since limited data exist on the susceptibility of A. fumigatus to both medical and agricultural antifungals, we undertook a drug susceptibility study including clinical (400) and agricultural (150) A. fumigatus isolates (Swiss origin). We tested azoles and also compounds of major antifungal classes used in agriculture (i.e., azoxystrobin, iprodione, benalaxyl or cyprodinil). The results showed that all A. fumigatus isolates were intrinsically resistant to iprodione, benalaxyl or cyprodinil (MIC90>32 µg.ml−1) and that azoxystrobin minimal inhibitory concentrations (MICs) showed a wide range (0.06 to 32 µg.ml−1). MIC ranges of azoles were compound-dependent. MIC90 for voriconazole, itraconazole, imazalil and prochloraz were within a range of 0.125 to 1 µg.ml−1 and similar between clinical and environmental isolates, whereas propiconazole was the least active compound (MIC90: 4-8 µg.ml−1). Ten clinical and 36 environmental isolates with high itraconazole MIC (≥2 µg.ml−1) were detected. In clinical isolates, no cross-resistance was observed between itraconazole and all others azoles tested. Several patterns of azole MICs for were, however, observed in the environmental isolates. Unexpectedly, a single environmental isolate was voriconazole-resistant (MIC of 16 µg.ml−1) but still susceptible to itraconazole (MIC of 2 µg.ml−1). Taken together, our results demonstrate the absence of susceptibility of A. fumigatus isolates to non-azole agricultural agents and that there is little impact of azole resistance in both clinical and environmental isolates. When detected, azole resistance was compound-specifi

Defining the frontiers between antifungal resistance, tolerance and the concept of persistence.

A restricted number of antifungal agents are available for the therapy of fungal diseases. With the introduction of epidemiological cut-off values for each agent in important fungal pathogens based on the distribution of minimal inhibitory concentration (MIC), the distinction between wild type and drug-resistant populations has been facilitated. Antifungal resistance has been described for all currently available antifungal agents in several pathogens and most of the associated resistance mechanisms have been deciphered at the molecular level. Clinical breakpoints for some agents have been proposed and can have predictive value for the success or failure of therapy. Tolerance to antifungals has been a much more ignored area. By definition, tolerance operates at antifungal concentrations above individual intrinsic inhibitory values. Important is that tolerance to antifungal agents favours the emergence of persister cells, which are able to survive antifungal therapy and can cause relapses. Here we will review the current knowledge on antifungal tolerance, its potential mechanisms and also evaluate the role of antifungal tolerance in the efficacy of drug treatments

Novel Approaches for Fungal Transcriptomics from Host Samples.

Candida albicans adaptation to the host requires a profound reprogramming of the fungal transcriptome as compared to in vitro laboratory conditions. A detailed knowledge of the C. albicans transcriptome during the infection process is necessary in order to understand which of the fungal genes are important for host adaptation. Such genes could be thought of as potential targets for antifungal therapy. The acquisition of the C. albicans transcriptome is, however, technically challenging due to the low proportion of fungal RNA in host tissues. Two emerging technologies were used recently to circumvent this problem. One consists of the detection of low abundance fungal RNA using capture and reporter gene probes which is followed by emission and quantification of resulting fluorescent signals (nanoString). The other is based first on the capture of fungal RNA by short biotinylated oligonucleotide baits covering the C. albicans ORFome permitting fungal RNA purification. Next, the enriched fungal RNA is amplified and subjected to RNA sequencing (RNA-seq). Here we detail these two transcriptome approaches and discuss their advantages and limitations and future perspectives in microbial transcriptomics from host material

Signaling Pathways Governing the Caspofungin Paradoxical Effect in Aspergillus fumigatus.

Aspergillus fumigatus is responsible for a wide range of diseases affecting several million people worldwide. Currently, a few families of antifungals are available to fight aspergillosis, and we are facing a worrisome increase in resistance to azoles, the drugs used for both first-line treatment and prophylaxis of invasive aspergillosis. In this context, some of the latest antifungals, i.e., echinocandins, have gained attention. Even though acquired resistance to echinocandins is yet uncommon in A. fumigatus clinical isolates, some strains exhibit another characteristic that relies on their capacity to grow at suprainhibitory echinocandin concentrations in vitro This intriguing phenomenon, especially observed with caspofungin and now referred to as the caspofungin paradoxical effect (CPE), relies on molecular mechanisms that were hitherto little understood. Here, we discuss the recent key findings of Valero and colleagues published in mBio (C. Valero, A. C. Colabardini, J. Chiaratto, L. Pardeshi, et al., mBio 11:e00816-20, 2020, https://doi.org/10.1128/mBio.00816-20) that will allow a better understanding of the complex regulatory pathway involved in governing the response of A. fumigatus to caspofungin

DAMA detection claim is still compatible with all other DM searches

We show that the annual modulation signal observed by DAMA can be reconciled with all other negative results from dark matter searches with a conventional halo model for particle masses around 5 to 9 GeV. We also show which particular dark matter stream could produce the DAMA signal.Comment: Talk given at TAUP2005, Sept. 10-14 2005, Zaragoza (Spain). 3 pages, 4 figure

Red-Shifted Firefly Luciferase Optimized for Candida albicans In vivo Bioluminescence Imaging.

Candida albicans is a major fungal pathogen causing life-threatening diseases in immuno-compromised patients. The efficacy of current drugs to combat C. albicans infections is limited, as these infections have a 40-60% mortality rate. There is a real need for novel therapeutic approaches, but such advances require a detailed knowledge of C. albicans and its in vivo pathogenesis. Additionally, any novel antifungal drugs against C. albicans infections will need to be tested for their in vivo efficacy over time. Fungal pathogenesis and drug-mediated resolution studies can both be evaluated using non-invasive in vivo imaging technologies. In the work presented here, we used a codon-optimized firefly luciferase reporter system for detecting C. albicans in mice. We adapted the firefly luciferase in order to improve its maximum emission intensity in the red light range (600-700 nm) as well as to improve its thermostability in mice. All non-invasive in vivo imaging of experimental animals was performed with a multimodal imaging system able to detect luminescent reporters and capture both reflectance and X-ray images. The modified firefly luciferase expressed in C. albicans (Mut2) was found to significantly increase the sensitivity of bioluminescence imaging (BLI) in systemic infections as compared to unmodified luciferase (Mut0). The same modified bioluminescence reporter system was used in an oropharyngeal candidiasis model. In both animal models, fungal loads could be correlated to the intensity of emitted light. Antifungal treatment efficacies were also evaluated on the basis of BLI signal intensity. In conclusion, BLI with a red-shifted firefly luciferase was found to be a powerful tool for testing the fate of C. albicans in various mice infection models

How Yeast Antifungal Resistance Gene Analysis Is Essential to Validate Antifungal Susceptibility Testing Systems.

The antifungal susceptibility testing (AFST) of yeast pathogen alerts clinicians about the potential emergence of resistance. In this study, we compared two commercial microdilution AFST methods: Sensititre YeastOne read visually (YO) and MICRONAUT-AM read visually (MN) or spectrophotometrically (MNV), interpreted with Clinical and Laboratory Standards Institute and European Committee on Antimicrobial Susceptibility Testing criteria, respectively. Overall, 97 strains from 19 yeast species were measured for nine antifungal drugs including a total of 873 observations. First, the minimal inhibitory concentration (MIC) was compared between YO and MNV, and between MNV and MN, either directly or by assigning them to five susceptibility categories. Those categories were based on the number of MIC dilutions around the breakpoint or epidemiological cut-off reference values (ECOFFs or ECVs). Second, YO and MNV methods were evaluated for their ability to detect the elevation of MICs due to mutation in antifungal resistance genes, thanks to pairs or triplets of isogenic strains isolated from a single patient along a treatment previously analyzed for antifungal resistance gene mutations. Reproducibility measurement was evaluated, thanks to three quality control (QC) strains. YO and MNV direct MIC comparisons obtained a global agreement of 67%. Performing susceptibility category comparisons, only 22% and 49% of the MICs could be assigned to categories using breakpoints and ECOFFs/ECVs, respectively, and 40% could not be assigned due to the lack of criteria in both consortia. The YO and MN susceptibility categories gave accuracies as low as 50%, revealing the difficulty to implement this method of comparison. In contrast, using the antifungal resistance gene sequences as a gold standard, we demonstrated that both methods (YO and MN) were equally able to detect the acquisition of resistance in the Candida strains, even if MN showed a global lower MIC elevation than YO. Finally, no major differences in reproducibility were observed between the three AFST methods. This study demonstrates the valuable use of both commercial microdilution AFST methods to detect antifungal resistance due to point mutations in antifungal resistance genes. We highlighted the difficulty to conduct conclusive analyses without antifungal gene sequence data as a gold standard. Indeed, MIC comparisons taking into account the consortia criteria of interpretation remain difficult even after the effort of harmonization

Comparative Genomics of Two Sequential <i>Candida glabrata</i> Clinical Isolates.

&lt;i&gt;Candida glabrata&lt;/i&gt; is an important fungal pathogen which develops rapid antifungal resistance in treated patients. It is known that azole treatments lead to antifungal resistance in this fungal species and that multidrug efflux transporters are involved in this process. Specific mutations in the transcriptional regulator &lt;i&gt;PDR1&lt;/i&gt; result in upregulation of the transporters. In addition, we showed that the &lt;i&gt;PDR1&lt;/i&gt; mutations can contribute to enhance virulence in animal models. In this study, we were interested to compare genomes of two specific &lt;i&gt;C. glabrata&lt;/i&gt; -related isolates, one of which was azole susceptible (DSY562) while the other was azole resistant (DSY565). DSY565 contained a &lt;i&gt;PDR1&lt;/i&gt; mutation (L280F) and was isolated after a time-lapse of 50 d of azole therapy. We expected that genome comparisons between both isolates could reveal additional mutations reflecting host adaptation or even additional resistance mechanisms. The PacBio technology used here yielded 14 major contigs (sizes 0.18-1.6 Mb) and mitochondrial genomes from both DSY562 and DSY565 isolates that were highly similar to each other. Comparisons of the clinical genomes with the published CBS138 genome indicated important genome rearrangements, but not between the clinical strains. Among the unique features, several retrotransposons were identified in the genomes of the investigated clinical isolates. DSY562 and DSY565 each contained a large set of adhesin-like genes (101 and 107, respectively), which exceed by far the number of reported adhesins (63) in the CBS138 genome. Comparison between DSY562 and DSY565 yielded 17 nonsynonymous SNPs (among which the was the expected &lt;i&gt;PDR1&lt;/i&gt; mutation) as well as small size indels in coding regions (11) but mainly in adhesin-like genes. The genomes contained a DNA mismatch repair allele of &lt;i&gt;MSH2&lt;/i&gt; known to be involved in the so-called hyper-mutator phenotype of this yeast species and the number of accumulated mutations between both clinical isolates is consistent with the presence of a &lt;i&gt;MSH2&lt;/i&gt; defect. In conclusion, this study is the first to compare genomes of &lt;i&gt;C. glabrata&lt;/i&gt; sequential clinical isolates using the PacBio technology as an approach. The genomes of these isolates taken in the same patient at two different time points exhibited limited variations, even if submitted to the host pressure