Whole-Genome Sequencing of Newly Emerged Fungal Pathogen Aspergillus Lentulus and Its Azole Resistance Gene Prediction.

Aims: Aspergillus lentulus is an important newly recorded species in the A. fumigatus complex and its resistance to azole drugs and the high mortality rate of infected individuals have emerged as problems. Comprehensive understanding of the A. lentulus is limited due to lack of genome-wide fine mapping data. The aim of this study was to investigate the A. lentulus signature at the molecular level, analyze the genome-wide profile of this strain, and predict its possible genes that execute azole resistance. Methods: In this study, a whole-genome sequencing of a clinically isolated A. lentulus strain (named A. lentulus PWCAL1) was studied by PacBio Sequel sequencing platform. Azole resistance genes were predicted based on whole-genome sequencing data analysis, gene function annotation, comparative genomic analysis, and BLASTP alignment using the Mycology Antifungal Resistance Database to comprehensively understanding the genome-wide features, pathogenicity, and resistance mechanisms of A. lentulus. Results: The results of whole-genome sequencing demonstrated that the total length of A. lentulus PWCAL1 genome was 31255105 bp, the GC content was 49.24%, and 6883 coding genes were predicted. A total of 4565, 1824, and 6405 genes were annotated in the Gene Ontology, Clusters of Orthologous Groups, and Kyoto Encyclopedia of Genes and Genomes databases, respectively. In the Pathogen Host Interactions Database and the Database of Fungal Virulence Factors, 949 and 259 interacting virulence factors were identified, respectively, with the main virulence factor-mutant virulence phenotype, being enriched in reduced virulence. Comparative genomic analysis showed that there were 5456 consensus core genes in this strain and four closely related strains of A. fumigatus complex, which were mainly involved in human diseases, metabolism, organismal systems, etc. Among the three aligned A. lentulus strains, the number of unique genes of this bacterium was the highest with a number of 171, and these genes were mainly associated with carbohydrate metabolism and cell growth and death. Resistance gene prediction demonstrated that the A5653 gene of this bacterium had F46Y/N248T double point mutations on the CYP51A gene, but no tandem repeat mutations in the promoter region were detected. Furthermore, 12 genes belonging to the fungal multidrug resistance ATP-binding cassette (ABC) transporters were identified based on the complete genome sequence and phylogenetic analysis of A. lentulus, which belonged to the ALDp subfamily, the PDR subfamily (AtrB, CDR1, and CDR2), and the MDR subfamily (MDR1), respectively, and there were four genes that are annotated to the major facilitator superfamily multidrug transporter. Further phylogenetic tree classification of the ABC transporter subfamilies predicted in the nine selected A. fumigatus complex strains showed that these putative ABC proteins were divided into two main clusters, which belonged to the PDR (CDR1, CDR2, AtrB, and AtrF) and MDR subfamilies (MDR1, MDR2, and MDR3). The distribution of these ABC proteins varies among different species of the A. fumigatus complex. Conclusions: The main result obtained from this study for the whole genome of A. lentulus provide new insights into better understanding the biological characteristics, pathogenicity, and resistance mechanisms of this bacterium. In this study, two resistance mechanisms, which include CYP51A gene mutation and multidrug-resistant ABC transporter, were predicted in a single isolate. Based on the predicted CYP51A-F46Y/N248T site mutation combination, we speculate that the CYP51A gene of A. lentulus may be partially responsible for azole resistance. Based on the predicted ABC transporter family genes, we hypothesize that resistance to multiple azoles in A. lentulus is mediated, at least in part, by these ABC transporters with resistance.

Unveiling the roles of CaSDH8 in Candida albicans: Implications for virulence and azole resistance.

Candida albicans is the most common pathogen in systemic fungal diseases, exhibits a complex pathogenic mechanism, and is increasingly becoming drug tolerant. Therefore, it is particularly important to study the genes associated with virulence and resistance of C. albicans. Here, we identified a gene (orf19.1588) that encodes a conserved mitochondrial protein known as CaSDH8, upon deletion of CaSdh8, the deleted strain (Casdh8Δ/Δ) experienced impaired growth, hyphal development, and virulence. Casdh8Δ/Δ displayed a reduced capacity to utilize alternative carbon sources, along with detrimental alterations in reactive oxygen species (ROS), mitochondrial membrane potential (MMP) depolarization, and adenosine triphosphate (ATP) levels. Interestingly, Casdh8Δ/Δ demonstrated resistance to azole drugs, and under the influence of fluconazole, the cell membrane permeability and mitochondrial function of Casdh8Δ/Δ were less compromised than those of the wild type, indicating a reduction in the detrimental effects of fluconazole on Casdh8Δ/Δ. These findings highlight the significance of CaSDH8 as a crucial gene for the maintenance of cellular homoeostasis. Our study is the first to document the effects of the CaSDH8 gene on the virulence and azole resistance of C. albicans at both the molecular and animal levels, providing new clues and directions for the antifungal infection and the discovery of antifungal drug targets.

Routine laboratory screening for CYP51A-related azole resistance in Aspergillus fumigatus using Etest: concordance with gene sequencing.

We evaluated the combined performance of itraconazole and voriconazole Etest® gradient concentration strips for detecting A. fumigatus azole resistance associated with cyp51a mutations confirmed by gene sequencing. Among 118 Aspergillus fumigatus clinical isolates collected in a French center, 6 (5%) had azole resistance mutations, 5 of which were probably of environmental origin. Using recent method-dependent Epidemiological Cut-Off Values (ECVs) as thresholds, the combination's sensitivity and specificity were 100% [95% confidence interval 61-100] and 99% [95-100]. Our results support itraconazole and voriconazole Etest® combined use as a promising self-sufficient method for simple, efficient and reliable cyp51a-related azole resistant A fumigatus detection.

Azole resistance in Aspergillus fumigatus is mainly due to mutations in the cyp51a gene and is a challenge for laboratory detection and therapeutic management. We evaluated a combination of two Etest® strips as a simple screening method by evaluating its results in concordance with gene sequencing.

Detection in Orchards of Predominant Azole-Resistant Candida tropicalis Genotype Causing Human Candidemia, Taiwan.

Fluconazole-resistant clade 4 Candida tropicalis causing candidemia in humans has been detected in tropical/subtropical areas, including those in China, Singapore, and Australia. We analyzed 704 individual yeasts isolated from fruits, soil, water, and farmers at 80 orchards in Taiwan. The most common pathogenic yeast species among 251 isolates recovered from farmers were Candida albicans (14.7%) and C. parapsilosis (11.6%). In contrast, C. tropicalis (13.0%), C. palmioleophila (6.6%), and Pichia kudriavzevii (6.0%) were prevalent among 453 environmental isolates. Approximately 18.6% (11/59) of C. tropicalis from the environment were resistant to fluconazole, and 81.8% (9/11) of those belonged to the clade 4 genotype. C. tropicalis susceptibility to fluconazole correlated with susceptibilities to the agricultural azole fungicides, difenoconazole, tebuconazole, and triadimenol. Tandem gene duplications of mutated ERG11 contributed to azole resistance. Agriculture environments are a reservoir for azole-resistant C. tropicalis; discontinuing agricultural use of azoles might reduce emergence of azole-resistant Candida spp. strains in humans.

Long-read sequencing and de novo genome assembly data of Candida parapsilosis HMC1 and Rhodotorula mucilaginosa LBMH1012, two novel isolates with antifungal resistance signatures.

Candida parapsilosis and Rhodotorula mucilaginosa are opportunistic pathogens affecting mostly immunocompromised hosts. Both species have emerged as causes of invasive candidiasis and sepsis respectively. Here we present high-quality long-read genome assemblies for a strain of C. parapsilosis isolated from human breast milk, with multiple predicted signatures consistent with Candida Drug Resistance CDR1/CDR2 and Multi Drug Resistance MDR1-type genes, also for an environmental strain of R. mucilaginosa with multiresistance to azole antifungals. The genome sequencing was performed using the R9.4.1 flowcell with the MinION Mk1B sequencer (Oxford Nanopore Technologies, Oxford, UK). The draft genome of C. parapsilosis HMC1 was assembled from 85,745 long-reads and has 13,114,208 bp in length and comprises 10 contigs making it a highly contiguous assembly. The R. mucilaginosa LBMH1012 assembly has 23,636,156 bp in length and comprises 54 contigs. The genome completeness was estimated as 94.02 % and 91.40 % respectively using BUSCO. These data may be useful to explore the genetic diversity landscape in both species, infer potential causal genes for antifungal resistance and virulence, and represent an addition to the useful sequence space on emerging fungal pathogens.

Gene-specific transcriptional activation by the Aspergillus fumigatus AtrR factor requires a conserved C-terminal domain.

Treatment of fungal infections associated with the filamentous fungus Aspergillus fumigatus is becoming more problematic as this organism is developing resistance to the main chemotherapeutic drug at an increasing rate. Azole drugs represent the current standard-of-care in the treatment of aspergillosis with this drug class acting by inhibiting a key step in the biosynthesis of the fungal sterol ergosterol. Azole compounds block the activity of the lanosterol α-14 demethylase, encoded by the cyp51A gene. A common route of azole resistance involves an increase in transcription of cyp51A. This transcriptional increase requires the function of a Zn2Cys6 DNA-binding domain-containing transcription activator protein called AtrR. AtrR was identified through its action as a positive regulator of expression of an ATP-binding cassette transporter (abcC/cdr1B here called abcG1). Using both deletion and alanine scanning mutagenesis, we demonstrate that a conserved C-terminal domain in A. fumigatus is required for the expression of abcG1 but dispensable for cyp51A transcription. This domain is also found in several other fungal pathogen AtrR homologs consistent with a conserved gene-selective function of this protein segment being conserved. Using RNA sequencing (RNA-seq), we find that this gene-specific transcriptional defect extends to several other membrane transporter-encoding genes including a second ABC transporter locus. Our data reveal that AtrR uses at least two distinct mechanisms to induce gene expression and that normal susceptibility to azole drugs cannot be provided by maintenance of wild-type expression of the ergosterol biosynthetic pathway when ABC transporter expression is reduced. IMPORTANCE: Aspergillus fumigatus is the primary human filamentous fungal pathogen. The principal chemotherapeutic drug used to control infections associated with A. fumigatus is the azole compound. These drugs are well-tolerated and effective, but resistance is emerging at an alarming rate. Most resistance is associated with mutations that lead to overexpression of the azole target enzyme, lanosterol α-14 demethylase, encoded by the cyp51A gene. A key regulator of cyp51A gene expression is the transcription factor AtrR. Very little is known of the molecular mechanisms underlying the effect of AtrR on gene expression. Here, we use deletion and clustered amino acid substitution mutagenesis to map a region of AtrR that confers gene-specific activation on target genes of this transcription factor. This region is highly conserved across AtrR homologs from other pathogenic species arguing that its importance in transcriptional regulation is maintained across evolution.

Environmental Hot Spots and Resistance-Associated Application Practices for Azole-Resistant Aspergillus fumigatus, Denmark, 2020-2023.

Azole-resistant Aspergillus fumigatus (ARAf) fungi have been found inconsistently in the environment in Denmark since 2010. During 2018-2020, nationwide surveillance of clinical A. fumigatus fungi reported environmental TR34/L98H or TR46/Y121F/T289A resistance mutations in 3.6% of isolates, prompting environmental sampling for ARAf and azole fungicides and investigation for selection of ARAf in field and microcosmos experiments. ARAf was ubiquitous (20% of 366 samples; 16% TR34/L98H- and 4% TR46/Y121F/T289A-related mechanisms), constituting 4.2% of 4,538 A. fumigatus isolates. The highest proportions were in flower- and compost-related samples but were not correlated with azole-fungicide application concentrations. Genotyping showed clustering of tandem repeat-related ARAf and overlaps with clinical isolates in Denmark. A. fumigatus fungi grew poorly in the field experiment with no postapplication change in ARAf proportions. However, in microcosmos experiments, a sustained complete (tebuconazole) or partial (prothioconazole) inhibition against wild-type A. fumigatus but not ARAf indicated that, under some conditions, azole fungicides may favor growth of ARAf in soil.

Comparative Genomics of the First Resistant Candida auris Strain Isolated in Mexico: Phylogenomic and Pan-Genomic Analysis and Mutations Associated with Antifungal Resistance.

Candida auris is an emerging multidrug-resistant and opportunistic pathogenic yeast. Whole-genome sequencing analysis has defined five major clades, each from a distinct geographic region. The current study aimed to examine the genome of the C. auris 20-1498 strain, which is the first isolate of this fungus identified in Mexico. Based on whole-genome sequencing, the draft genome was found to contain 70 contigs. It had a total genome size of 12.86 Mbp, an N50 value of 1.6 Mbp, and an average guanine-cytosine (GC) content of 45.5%. Genome annotation revealed a total of 5432 genes encoding 5515 proteins. According to the genomic analysis, the C. auris 20-1498 strain belongs to clade IV (containing strains endemic to South America). Of the two genes (ERG11 and FKS1) associated with drug resistance in C. auris, a mutation was detected in K143R, a gene located in a mutation hotspot of ERG11 (lanosterol 14-α-demethylase), an antifungal drug target. The focus on whole-genome sequencing and the identification of mutations linked to the drug resistance of fungi could lead to the discovery of new therapeutic targets and new antifungal compounds.

Azole Resistance in Veterinary Clinical Aspergillus fumigatus Isolates in the Netherlands.

Aspergillus fumigatus is a saprophytic fungal pathogen that causes opportunistic infections in animals and humans. Azole resistance has been reported globally in human A. fumigatus isolates, but the prevalence of resistance in isolates from animals is largely unknown. A retrospective resistance surveillance study was performed using a collection of clinical A. fumigatus isolates from various animal species collected between 2015 and 2020. Agar-based azole resistance screening of all isolates was followed by in vitro antifungal susceptibility testing and cyp51A gene sequencing of the azole-resistant isolates. Over the 5 year period 16 (11.3%) of 142 A. fumigatus culture-positive animals harbored an azole-resistant isolate. Resistant isolates were found in birds (15%; 2/13), cats (21%; 6/28), dogs (8%; 6/75) and free-ranging harbor porpoise (33%; 2/6). Azole-resistance was cyp51A mediated in all isolates: 81.3% (T-67G/)TR34/L98H, 12.5% TR46/Y121F/T289A. In one azole-resistant A. fumigatus isolate a combination of C(-70)T/F46Y/C(intron7)T/C(intron66)T/M172V/E427K single-nucleotide polymorphisms in the cyp51A gene was found. Of the animals with an azole-resistant isolate and known azole exposure status 71.4% (10/14) were azole naive. Azole resistance in A. fumigatus isolates from animals in the Netherlands is present and predominantly cyp51A TR-mediated, supporting an environmental route of resistance selection. Our data supports the need to include veterinary isolates in resistance surveillance programs. Veterinarians should consider azole resistance as a reason for therapy failure when treating aspergillosis and consider resistance testing of relevant isolates.

Azole resistance screening in Aspergillus fumigatus sensu stricto using the azole-containing agar method (EUCAST E.Def 10.2): conidial suspension filtration and inoculum adjustment before inoculum preparation may not be needed.

Azole resistance screening in Aspergillus fumigatus sensu stricto can be routinely carried out by using azole-containing agar plates (E.Def 10.2 procedure); however, conidial suspension filtering and inoculum adjustment before inoculum preparation are time-consuming. We evaluated whether skipping the filtration and inoculum adjustment steps negatively influenced the performance of the E.Def 10.2 procedure. A. fumigatus sensu stricto isolates (n = 98), previously classified as azole susceptible or azole resistant (E.Def 9.4 method), were studied. Azole-resistant isolates had either the wild-type cyp51A gene sequence (n = 1) or the following cyp51A gene substitutions: TR34-L98H (n = 41), G54R (n = 5), TR46-Y121F-T289A (n = 1), or G448S (n = 1). In-house azole-containing agar plates were prepared according to the EUCAST E.Def 10.2 procedure. Conidial suspensions obtained by adding distilled water (Tween 20 0.1%) were either filtered and the inocula adjusted to 0.5 McFarland or left unfiltered and unadjusted. Agreements between the agar screening methods using inocula prepared by each procedure were high for itraconazole (99%), voriconazole (100%), and posaconazole (94.9%). Sensitivity and specificity (considering the susceptibility category as per the microdilution E.Def 9.4 method as the gold standard) of E.Def 10.2 were 100% to rule in or rule out resistance when unfiltered and unadjusted suspensions were used; the resistance phenotype of isolates harboring the TR34-L98H, G54R, or TR46-Y121F-T289A substitutions was correctly detected. Unfiltered and unadjusted conidial suspensions do not negatively influence the performance of the E.Def 10.2 method when screening for azole resistance in A. fumigatus sensu stricto. IMPORTANCE: Azole resistance screening in Aspergillus fumigatus sensu stricto can be routinely carried out by using azole-containing plates (E.Def 10.2 procedure); however, conidial suspension filtering and inoculum adjustment before inoculation of plates are time-consuming. We, here, showed that unfiltered and unadjusted conidial suspensions do not negatively influence the performance of the E.Def 10.2 method when screening for azole resistance in A. fumigatus sensu stricto.

Mapping the Global Spread of T. indotineae: An Update on Antifungal Resistance, Mutations, and Strategies for Effective Management.

INTRODUCTION: The global spread of Trichophyton indotineae presents a pressing challenge in dermatophytosis management. This systematic review explores the current landscape of T. indotineae infections, emphasizing resistance patterns, susceptibility testing, mutational analysis, and management strategies. METHODS: A literature search was conducted in November 2023 using Embase, PubMed, Scopus, and Web of Science databases. Inclusion criteria covered clinical trials, observational studies, case series, or case reports with T. indotineae diagnosis through molecular methods. Reports on resistance mechanisms, antifungal susceptibility testing, and management were used for data extraction. RESULTS AND DISCUSSION: A total of 1148 articles were identified through the systematic search process, with 45 meeting the inclusion criteria. The global spread of T. indotineae is evident, with cases reported in numerous new countries in 2023. Tentative epidemiological cut-off values (ECOFFs) suggested by several groups provide insights into the likelihood of clinical resistance. The presence of specific mutations, particularly Phe397Leu, correlate with higher minimum inhibitory concentrations (MICs), indicating potential clinical resistance. Azole resistance has also been reported and investigated in T. indotineae, and is a growing concern. Nevertheless, itraconazole continues to be an alternative therapy. Recommendations for management include oral or combination therapies and individualized approaches based on mutational analysis and susceptibility testing. CONCLUSION: Trichophyton indotineae poses a complex clinical scenario, necessitating enhanced surveillance, improved diagnostics, and cautious antifungal use. The absence of established clinical breakpoints for dermatophytes underscores the need for further research in this challenging field.

Investigation of Azole Resistance Involving cyp51A and cyp51B Genes in Clinical Aspergillus flavus Isolates.

This study aimed to investigate azole resistance mechanisms in Aspergillus flavus, which involve cyp51A and cyp51B genes. Real-time Reverse Transcriptase qPCR method was applied to determine the overexpression of cyp51A and cyp51B genes for 34 A. flavus isolates. PCR sequencing of these two genes was used to detect the presence of gene mutations. Susceptibility test found sensitivity to voriconazole (VOR) in all strains. 14.7% and 8.8% of isolates were resistant to itraconazole (IT) and posaconazole (POS), respectively, with a cross-resistance in 5.8%. For the double resistant isolates (IT/POS), the expression of cyp51A was up to 17-fold higher. PCR sequencing showed the presence of 2 mutations in cyp51A: a synonymous point mutation (P61P) in eight isolates, which did not affect the structure of CYP51A protein, and another non synonymous mutation (G206L) for only the TN-33 strain (cross IT/POS resistance) causing an amino acid change in the protein sequence. However, we noted in cyp51B the presence of the only non-synonymous mutation (L177G) causing a change in amino acids in the protein sequence for the TN-31 strain, which exhibits IT/POS cross-resistance. A short single intron of 67 bp was identified in the cyp51A gene, whereas three short introns of 54, 53, and 160 bp were identified in the cyp51B gene. According to the models provided by PatchDock software, the presence of non-synonymous mutations did not affect the interaction of CYP51A and CYP51B proteins with antifungals. In our study, the overexpression of the cyp51A and cyp51B genes is the primary mechanism responsible for resistance in A. flavus collection. Nevertheless, other resistance mechanisms can be involved.

The TAC1 Gene in Candida albicans: Structure, Function, and Role in Azole Resistance: A Mini-Review.

Candidiasis is a common fungal infection caused by Candida species, with Candida albicans being the most prevalent. Resistance to azole drugs, commonly used to treat Candida infections, poses a significant challenge. Transcriptional activator candidate 1 (TAC1) gene has emerged as a key player in regulating drug resistance in C. albicans. This review explores the structure and function of the TAC1 gene and its role in azole resistance. This gene encodes a transcription factor that controls the expression of genes involved in drug resistance, such as efflux pump genes (CDR1, CDR2, and MDR1) and ERG11. Mutations in TAC1 can increase these genes' expression and confer resistance to azoles. Various TAC1 gene mutations, mostly gain-of-function mutations, have been identified, which upregulate CDR1 and CDR2 expression, resulting in azole resistance. Understanding the mechanisms of azole resistance mediated by the TAC1 gene is crucial for the strategies in the effective antifungal development pipeline.

Detection of pan-azole resistant Aspergillus fumigatus in horticulture and a composting facility in Belgium.

Azole resistance in Aspergillus fumigatus (ARAf) is becoming a worldwide health threat due to increasing occurrence in the environment. However, environmental surveillance programs are not commonly in place and are lacking in Belgium. Since no data on the occurrence of ARAf and the presence of hotspots for the selection of azole resistance is available in Belgium, a first study on the prevalence of ARAf in the environment was conducted. A total of 232 air and compost or soil samples were taken from two composting facilities, and from horticultural and agricultural crops. The azole susceptibility pattern was determined using the EUCAST method (E. Def. 9.4), and the cyp51A gene and its promotor region were sequenced in A. fumigatus isolates with phenotypic azole resistance. Six pan-azole-resistant A. fumigatus isolates were identified, originating from compost and horticultural crops. Four isolates carried the TR34/L98H mutation, and one isolate carried the TR46/Y121F/T289A mutation. However, we did not observe any ARAf isolates from agricultural crops. In conclusion, this study reported the first TR34/L98H and TR46/Y121F/T289A mutation isolated from a composting facility and horticulture in Belgium. The implementation of standardization in environmental surveillance of A. fumigatus on a European level would be beneficial in order to identify hotspots.

The ubiquitous fungus Aspergillus fumigatus can cause serious invasive diseases in humans. Due to the extensive use of environmental azoles, an increase of clinical infections with azole-resistant A. fumigatus is seen. This pilot study aimed to estimate the prevalence of azole-resistant A. fumigatus in environmental reservoirs in Belgium.

Antifungal activity of nisin against clinical isolates of azole-resistant Candida tropicalis.

The rapid emergence of invasive infections caused by azole-resistant Candida tropicalis has become a public health concern, and there is an urgent need for alternative treatment strategies. Studies have demonstrated the antibacterial effects of nisin, a well-known peptide naturally produced by Lactococcus lactis subsp. lactis. However, there is scant information about the antifungal effect of nisin against C. tropicalis. The present study aims to investigate the in vitro antifungal activity of nisin against clinical isolates of azole-resistant C. tropicalis strains, as well as its inhibitory effect on biofilm formation. A total of 35 C. tropicalis strains isolated from patients with invasive fungal infections were divided into the azole-resistant group and the azole-sensitive group, containing 21 and 14 strains, respectively. The relative expression levels of the ERG11 and UPC2 genes in the azole-resistant group were higher than those in the azole-sensitive group (p < 0.0001), while no significant differences were observed in the expression levels of the MDR1 and CDR1 genes. The minimum inhibitory concentration of nisin against C. tropicalis ranged from 2 to 8 µg/mL. Nisin treatment inhibited the growth of azole-resistant C. tropicalis, with over a four-fold reduction in OD600 nm values observed at the 8-h time point, while it promoted the transition of C. tropicalis from the spore phase to the hyphal phase, as observed on cryo-scanning electron microscopy. The results of biofilm quantification using crystal violet staining indicated a significant decrease in OD570 nm values in the nisin-treated group compared to the controls (p < 0.0001). Among the 21 azole-resistant C. tropicalis strains, the biofilm formation was inhibited in 17 strains (17/21, 81%), and more than 85% inhibition of biofilm formation was observed in the representative strains. With regard to the molecular mechanisms, the expression of the BCR1 and UPC2 genes in the azole-resistant strains was down-regulated on nisin treatment (p < 0.05). In conclusion, we demonstrated, for the first time, that nisin has antifungal activity and significant anti-biofilm activity against clinical isolates of azole-resistant C. tropicalis strains. Based on the findings, nisin could be a promising alternative antifungal agent for combating azole-resistant C. tropicalis infections.

The Role of Olorofim in the Treatment of Filamentous Fungal Infections: A Review of In Vitro and In Vivo Studies.

Invasive fungal infections have recently been recognized by the WHO as a major health, epidemiological, and economic issue. Their high mortality rates and the emergence of drug resistance have driven the development of new molecules, including olorofim, an antifungal belonging to a new family of compounds, the orotomides. A review was conducted on the PubMed database and the ClinicalTrials.gov website to summarize the microbiological profile of olorofim and its role in the treatment of filamentous fungal infections. Twenty-four articles were included from the search and divided into two groups: an "in vitro" group focusing on minimum inhibitory concentration (MIC) results for various fungi and an "in vivo" group evaluating the pharmacokinetics (PK), pharmacodynamics (PD), efficacy, and tolerability of olorofim in animal models of fungal infection and in humans. Olorofim demonstrated in vitro and in vivo activity against numerous filamentous fungi, including azole-resistant Aspergillus fumigatus, various dermatophytes, and endemic and dimorphic fungi. in vitro results showed higher MICs for certain Fusarium species and dematiaceous fungi Alternaria alternata and Exophiala dermatitidis; further in vivo studies are needed. Published PK-PD data in humans are limited. The results of the ongoing phase III clinical trial are eagerly awaited to evaluate olorofim's clinical impact.

Assessment of Portuguese fitness centers: Bridging the knowledge gap on harmful microbial contamination with focus on fungi.

The lack of knowledge regarding the extent of microbial contamination in Portuguese fitness centers (FC) puts attendees and athletes at risk for bioaerosol exposure. This study intends to characterize microbial contamination in Portuguese FC by passive sampling methods: electrostatic dust collectors (EDC) (N = 39), settled dust (N = 8), vacuum filters (N = 8), and used cleaning mops (N = 12). The obtained extracts were plated in selective culture media for fungi and bacteria. Filters, EDC, and mop samples' extracts were also screened for antifungal resistance and used for the molecular detection of the selected Aspergillus sections. The detection of mycotoxins was conducted using a high-performance liquid chromatograph (HPLC) system and to determine the cytotoxicity of microbial contaminants recovered by passive sampling, HepG2 (human liver carcinoma) and A549 (human alveolar epithelial) cells were employed. The results reinforce the use of passive sampling methods to identify the most critical areas and identify environmental factors that influence microbial contamination, namely having a swimming pool. The cardio fitness area presented the highest median value of total bacteria (TSA: 9.69 × 102 CFU m-2.day-1) and Gram-negative bacteria (VRBA: 1.23 CFU m-2.day-1), while for fungi it was the open space area, with 1.86 × 101 CFU m-2.day-1. Aspergillus sp. was present in EDC and in filters used to collect settled dust. Reduced azole susceptibility was observed in filters and EDC (on ICZ and VCZ), and in mops (on ICZ). Fumonisin B2 was the only mycotoxin detected and it was present in all sampling matrixes except settled dust. High and moderate cytotoxicity was obtained, suggesting that A549 cells were more sensitive to samples' contaminants. The observed widespread of critical toxigenic fungal species with clinical relevance, such as Aspergillus section Fumigati, as well as Fumonisin B2 emphasizes the importance of frequent and effective cleaning procedures while using shared mops appeared as a vehicle of cross-contamination.

Triazole resistance in Aspergillus fumigatus isolated from a tomato production environment exposed to propiconazole.

The emergence of azole-resistant Aspergillus fumigatus (ARAf) across the world is an important public health concern. We sought to determine if propiconazole, a demethylase inhibitor (DMI) fungicide, exerted a selective pressure for ARAf in a tomato production environment following multiple exposures to the fungicide. A tomato field trial was established in 2019 and propiconazole was applied weekly until harvest. Soil, leaf, and fruit (when present) samples were collected at baseline and after each propiconazole application. A. fumigatus isolates (n, 178) were recovered and 173 were tested for susceptibility to itraconazole, posaconazole, voriconazole, and propiconazole in accordance with CLSI M38 guidelines. All the isolates were susceptible to medical triazoles and the propiconazole MIC ranged from 0.25 to 8 mg/L. A linear regression model was fitted that showed no longitudinal increment in the log2-fold azole MIC of the isolates collected after each propiconazole exposure compared to the baseline isolates. AsperGenius real-time multiplex assay ruled out TR34/L98H and TR46/Y121F/T289A cyp51A resistance markers in these isolates. Sequencing of a subset of isolates (n, 46) demonstrated widespread presence of F46Y/M172V/E427K and F46Y/M172V/N248T/D255E/E427K cyp51A mutations previously associated with reduced susceptibility to triazoles. IMPORTANCE: The agricultural use of azole fungicides to control plant diseases has been implicated as a major contributor to ARAf infections in humans. Our study did not reveal imposition of selection pressure for ARAf in a vegetable production system. However, more surveillance studies for ARAf in food crop production and other environments are warranted in understanding this public and One Health issue.

COVID-19 associated candidemia: From a shift in fungal epidemiology to a rise in azole drug resistance.

Our understanding of fungal epidemiology and the burden of antifungal drug resistance in COVID-19-associated candidemia (CAC) patients is limited. Therefore, we conducted a retrospective multicenter study in Iran to explore clinical and microbiological profiles of CAC patients. Yeast isolated from blood, were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and subjected to antifungal susceptibility testing (AFST) using the broth microdilution method M27-A3 protocol. A total of 0.6% of the COVID-19 patients acquired CAC (43/6174). Fluconazole was the most widely used antifungal, and 37% of patients were not treated. Contrary to historic candidemia patients, Candida albicans and C. tropicalis were the most common species. In vitro resistance was high and only noted for azoles; 50%, 20%, and 13.6% of patients were infected with azole-non-susceptible (ANS) C. tropicalis, C. parapsilosis, and C. albicans isolates, respectively. ERG11 mutations conferring azole resistance were detected for C. parapsilosis isolates (Y132F), recovered from an azole-naïve patient. Our study revealed an unprecedented rise in ANS Candida isolates, including the first C. parapsilosis isolate carrying Y132F, among CAC patients in Iran, which potentially threatens the efficacy of fluconazole, the most widely used drug in our centers. Considering the high mortality rate and 37% of untreated CAC cases, our study underscores the importance of infection control strategies and antifungal stewardship to minimize the emergence of ANS Candida isolates during COVID-19.

Distribution of Aspergillus species and prevalence of azole resistance in clinical and environmental samples from a Spanish hospital during a three-year study period.

BACKGROUND: Surveillance studies are crucial for updating trends in Aspergillus species and antifungal susceptibility information. OBJECTIVES: Determine the Aspergillus species distribution and azole resistance prevalence during this 3-year prospective surveillance study in a Spanish hospital. MATERIALS AND METHODS: Three hundred thirty-five Aspergillus spp. clinical and environmental isolates were collected during a 3-year study. All isolates were screened for azole resistance using an agar-based screening method and resistance was confirmed by EUCAST antifungal susceptibility testing. The azole resistance mechanism was confirmed by sequencing the cyp51A gene and its promoter. All Aspergillus fumigatus strains were genotyped using TRESPERG analysis. RESULTS: Aspergillus fumigatus was the predominant species recovered with a total of 174 strains (51.94%). The rest of Aspergillus spp. were less frequent: Aspergillus niger (14.93%), Aspergillus terreus (9.55%), Aspergillus flavus (8.36%), Aspergillus nidulans (5.37%) and Aspergillus lentulus (3.28%), among other Aspergillus species (6.57%). TRESPERG analysis showed 99 different genotypes, with 72.73% of the strains being represented as a single genotype. Some genotypes were common among clinical and environmental A. fumigatus azole-susceptible strains, even when isolated months apart. We describe the occurrence of two azole-resistant A. fumigatus strains, one clinical and another environmental, that were genotypically different and did not share genotypes with any of the azole-susceptible strains. CONCLUSIONS: Aspergillus fumigatus strains showed a very diverse population although several genotypes were shared among clinical and environmental strains. The isolation of azole-resistant strains from both settings suggest that an efficient analysis of clinical and environmental sources must be done to detect azole resistance in A. fumigatus.