Azole-resistant Aspergillus Fumigatus Strains Research Articles

Prevalence of Azole-Resistant Aspergillus Section Fumigati Strains Isolated from Romanian Vineyard Soil Samples.

A total of 265 soil samples from various Romanian vineyards were screened for fungi resistant to azoles. Aspergillus section Fumigati isolates exhibited significant resistance to itraconazole and voriconazole, but no azole-resistant Aspergillus fumigatus strains were detected. Six percent of the samples were positive for Aspergillus section Fumigati strains, all of which were azole-resistant. The strains were mainly Aspergillus udagawae (93.75%) and Aspergillus lentulus (6.25%). The predominant azole-resistant Aspergillus species were Aspergillus section Nigri strains, which were found in 75 soil samples. This study highlights the importance of understanding fungal resistance in vineyard soils for both the agricultural and clinical sectors. The presence of resistant strains may affect vine health and wine production while also constituting a challenge in the selection of effective treatments against severe and potentially fatal fungal infections in humans, stressing the importance of species-specific antifungal resistance knowledge.

Inhibition of azole-resistant Aspergillus fumigatus biofilm at various formation stages by antifungal drugs, including olorofim.

Interest in aspergillosis has increased over the past decades. An increase in the incidence of azole-resistant Aspergillus fumigatus strains has been reported; therefore, the need for novel therapeutic approaches is urgent. The formation of biofilms contributes to pathogen resistance. We investigated the biofilm formation capabilities of azole-resistant A. fumigatus and analysed the susceptibility of biofilms at various developmental stages to three antifungal agents. Biofilm formation of 19 clinical A. fumigatus strains (3 azole-susceptible and 16 azole-resistant strains) was determined by crystal violet staining and by an XTT assay over a period of 48 h. We measured antibiofilm activity of voriconazole, amphotericin B and olorofim. These agents were added before adhesion, after adhesion, after germination and to mature fungal biofilm. Antibiofilm activity was assessed in an XTT assay and in confocal laser scan microscopy. Additionally, a growth-kinetic assay with planktonic A. fumigatus was performed. Each of the antifungal agents inhibited the metabolic activity of A. fumigatus biofilms when applied at early stages of biofilm formation. The mature biofilms were more resistant. Olorofim and voriconazole showed promising effects against A. fumigatus adhesion and germination, whereas the mature biofilm was not affected by treatment. In contrast, the biofilm of A. fumigatus showed amphotericin B susceptibility throughout the entire developmental process. The planktonic cells were susceptible to all three antifungal drug classes with an inhibition peak at 12 h after incubation. This is the first known study to demonstrate the antibiofilm activity of olorofim, voriconazole and amphotericin B against azole-resistant A. fumigatus.

First Investigative Study of Azole-Resistant Aspergillus fumigatus in the Environment in Burkina Faso.

Azole-resistant Aspergillus fumigatus (ARAF) strains have been reported on all continents, however, limited data exist on these strains in Africa, while several factors, mainly environmental ones, suggest their presence on this continent. This study aimed to assess the environmental prevalence of ARAF strains in Burkina Faso, a country situated in the West African region where data on ARAF is non-existent. In total, 120 environmental samples (soil) were collected and analyzed. Samples were screened for resistance using three azole-containing agar plates; one without azole antifungal (growth control) and two supplemented with either itraconazole (4 mg/L) or voriconazole (2 mg/L). The EUCAST susceptibility testing method was used to confirm the azole-resistant phenotype of A. fumigatus sensu-stricto isolates. Mutations in the cyp51A gene were determined by sequencing. Of the 120 samples, 51 positive samples showed growth of A. fumigatus isolates on control medium. One ARAF (2%; 1/51) isolate was found amongst A. fumigatus positive samples and harbored the F46Y/M172V/E427K cyp51A mutations. No TR34/L98H or TR46/Y121F/T289A mutations were observed. Our study described the first A. fumigatus isolate resistant to an azole antifungal in Burkina Faso.

Hospital Environment as a Source of Azole-Resistant Aspergillus fumigatus Strains with TR34/L98H and G448S Cyp51A Mutations.

Azole-resistant Aspergillus fumigatus is an emerging worldwide problem with increasing reports of therapy failure cases produced by resistant isolates. A case of azole-resistant A. fumigatus hospital colonization in a patient is reported here. Investigations of the hospital environment led to the recovery of A. fumigatus strains harboring the TR34/L98H and the G448S Cyp51A azole resistance mechanisms. Isolate genotyping showed that one strain from the environment was isogenic with the patient strains. These are the first environmental A. fumigatus azole resistant strains collected in a hospital in Spain; it supports the idea of the hospital environment as a source of dissemination and colonization/infection by azole resistant A. fumigatus in patients. The isolation of an azole-resistant strain from an azole-naïve patient is an interesting finding, suggesting that an effective analysis of clinical and environmental sources must be done to detect azole resistance in A. fumigatus. The emergence and spread of these resistance mechanisms in A. fumigatus is of major concern because it confers high resistance to voriconazole and is associated with treatment failure in patients with invasive aspergillosis.

Transition of triazole-resistant Aspergillus fumigatus isolates in a Japanese tertiary hospital and subsequent genetic analysis

ObjectiveTo evaluate the annual variation in the frequency of patient-acquired azole-resistant Aspergillus fumigatus (ARAf), and correlate it to the amount of oral triazole prescribed, in Nagasaki, Japan. MethodsA. fumigatus isolates from respiratory specimens collected in the Nagasaki University Hospital (NUH) between 1996 and 2017 were included in the study. The amount of oral triazole prescribed in NUH since 2001 was obtained from the medical ordering system. Mutations in cyp51A, hmg1, and erg6 genes of ARAf were also analysed. ResultsFrom a total of 240 ARAf strains, 12 (5%), 6 (2.5%), 15 (6.25%), and 3 (1.25%) strains were resistant to itraconazole (ITC), voriconazole (VRC), to either ITC or VRC, and both triazoles, respectively. The amount of prescribed VRC increased annually, and was three times as large as that of ITC in 2017. All eleven patients harbouring ITC-resistant strains had a history of prior ITC treatment, while only one of six patients harbouring VRC-resistant strains had a history of prior VRC treatment. cyp51A mutations were recorded in 10 strains; however, tandem repeat mutations of the promoter region of cyp51A were not observed. Several azole-resistant strains had non-cyp51A mutations. ConclusionsThe frequency of patient-acquired ARAf is not increasing in Nagasaki, Japan. Furthermore, the prevalence of VRC-induced ARAf was rare despite the remarkable increase in the amount of prescribed VRC. Mutations in genes other than cyp51A should also be considered when ARAf strains are obtained from patients treated with azole antifungals.

Anti-fungal activity of a novel triazole, PC1244, against emerging azole-resistant Aspergillus fumigatus and other species of Aspergillus.

ObjectivesThe growing emergence of azole-resistant Aspergillus fumigatus strains worldwide is a major concern for current systemic antifungal treatment. Here we report antifungal activities of a novel inhaled triazole, PC1244, against a collection of multi-azole-resistant A. fumigatus strains.MethodsMICs of PC1244 were determined for A. fumigatus carrying TR34/L98H (n = 81), TR46/Y121F/T289A (n = 24), M220 (n = 6), G54 (n = 11), TR53 (n = 1), TR463/Y121F/T289A (n = 2), G448S (n = 1), G432C (n = 1) and P216S (n = 1) resistance alleles originating from either India, the Netherlands or France. The effects of PC1244 were confirmed in an in vitro model of the human alveolus and in vivo in temporarily neutropenic, immunocompromised mice.ResultsPC1244 exhibited potent inhibition [geometric mean MIC (range), 1.0 mg/L (0.125 to >8 mg/L)] of growth of A. fumigatus strains carrying cyp51A gene mutations, showing much greater potency than voriconazole [15 mg/L (0.5 to >16 mg/L)], and an effect similar to those on other azole-susceptible Aspergillus spp. (Aspergillus flavus, Aspergillus terreus, Aspergillus tubingensis, Aspergillus nidulans, Aspergillus niger, Aspergillus nomius, Aspergillus tamarii) (0.18–1 mg/L). In TR34/L98H and TR46/Y121F/T289A A. fumigatus-infected in vitro human alveolus models, PC1244 achieved superior inhibition (IC50, 0.25 and 0.34 mg/L, respectively) compared with that of voriconazole (IC90, >3 mg/L and >10 mg/L, respectively). In vivo, once-daily intranasal administration of PC1244 (0.56–70 μg/mouse) to the A. fumigatus (AF91 with M220V)-infected mice reduced pulmonary fungal load and serum galactomannan more than intranasal posaconazole.ConclusionsPC1244 has the potential to become a novel topical treatment of azole-resistant pulmonary aspergillosis.

Prospective multicenter surveillance of clinically isolated Aspergillus species revealed azole-resistant Aspergillus fumigatus isolates with TR34/L98H mutation in the Kyoto and Shiga regions of Japan.

The prevalence of azole-resistant Aspergillus fumigatus (ARAF) in Japan is unclear. We aimed to investigate the epidemiology of clinically isolated Aspergillus species and the frequency of azole resistance in Aspergillus species, particularly Aspergillus fumigatus, in the Kyoto and Shiga regions of Japan. Strains of clinically isolated Aspergillus species were prospectively collected from nine acute care hospitals. Species identification was performed by DNA sequence analysis, and all strains were subjected to antifungal susceptibility testing. Sequencing of the Aspergillus cyp51A gene and promoter region and genotyping by short tandem repeats were performed for ARAF isolates. A total of 149 strains were collected, and 130 strains were included for the subsequent analysis after the exclusion of duplicate isolates. The most commonly isolated species was Aspergillus fumigatus, accounting for 43.1% (56 isolates) overall, and seven (12.7%) of 55 strains of A. fumigatus were azole-resistant. Azole-resistance of other Aspergillus species were also found that two (22.2%) of nine strains of A. tubingensis and two (28.6%) of seven strains of A. flavus were azole-resistant. DNA sequence analysis of the ARAF strains revealed that two carried the cyp51A TR34/L98H mutation, one carried G448S, one carried M220I, and three had no relevant mutations (wild type). Genotyping and phylogenetic analyses showed that the TR34/L98H strains were clustered with the strains from the Netherlands and France. These data suggest the emergence of ARAF with TR34/L98H in Japan, and continuous surveillance will be important to identify trends in resistance.

1639. Efficacy of Voriconazole Prophylaxis Followed by Therapeutic Liposomal Amphotericin B for the Treatment of Murine Pulmonary Aspergillosis Caused by Azole-Resistant Aspergillus fumigatus Strains

BackgroundAntifungal treatment for pulmonary aspergillosis is more difficult if the fungal strain causing the infection is azole resistant. To investigate this problem, we used a murine model of pulmonary aspergillosis caused by azole-resistant Aspergillus fumigatus strains V29 and V45, and compared treatment with voriconazole (Vr, oral 40 mg/kg, bid) or liposomal amphotericin B (L-AmB, 5 mg/kg, IV) used alone or in combination.MethodsMice (n = 14/gp) were immunosuppressed with 24 mg/kg triamcinolone acetonide, IP, d-3, d-1, and d+1 relative to fungal challenge (d0). For 2 groups, Vr was given prophylactically (proph) d-3, d-2, d-1 followed by L-AmB or buffer, d+1, d+2, and d+3. The other groups were given Vr, L-AmB, Vr+L-AmB, or buffer d+1, d+2, and d+3. On d0, mice were given 1.3 to 1.6 × 107A. fumigatus spores intranasally (Vr MIC = 64 μg/mL, V29; Vr MIC = 8 μg/mL, V45). On d+3, lungs were collected from 7 mice/gp and fungal burden determined by plating for colony forming units; 7 mice/gp were then monitored for morbidity to d+21.ResultsOptimum treatment was observed when Vr was given proph, followed by L-AmB post-challenge (Vr/L-AmB), with better survival (100%) for both fungal strains vs. buffer or Vr post-challenge (P ≤ 0.04); for V29, significantly better survival was also seen with Vr/L-AmB vs. L-AmB or Vr+L-AmB post-challenge (P ≤ 0.01). For strain V45, lung fungal burden was significantly lower for Vr/L-AmB versus all other treatments (P ≤ 0.04), while for strain V29, fungal burden was lower for the Vf/L-AmB and L-AmB post-challenge groups versus the other groups, but the differences were not significant. Notably, although the lung fungal burden with Vr proph and Vr postchallenge were both similar to the buffer control, Vr proph yielded significantly better survival than Vr post-challenge (P ≤ 0.001).ConclusionThese preclinical observations demonstrate that combining L-AmB with Vr for the postchallenge treatment of pulmonary aspergillosis caused by azole-resistant strains is not an effective therapeutic option. However, the results do show that Vr proph, but not Vr postchallenge, can have some limited antifungal activity, and can significantly enhance the antifungal effects of post-challenge L-AmB. This regimen could be considered in areas where there is a high incidence of azole-resistant A. fumigatus.Disclosures All authors: No reported disclosures.

Prevalence of azole-resistant Aspergillus fumigatus in the environment of Thailand.

Occurrence of azole-resistant Aspergillus fumigatus (ARAF) in the environment is an emerging problem worldwide, likely impacting on patient treatment. Several resistance mutations are thought to have initially arisen through triazole-based fungicide use in agriculture and subsequently being propagated in a similar manner. Here we investigated the prevalence of ARAF in the environment of Thailand and characterized their susceptibility profiles toward clinically used azole compounds along with underlying resistance mutations. Three hundred and eight soil samples were collected and analyzed, out of which 3.25% (n = 10) were positive for ARAF. All isolates obtained were resistant to itraconazole (MIC ≥ 8μg/ml), two showed additional increased MIC values toward posaconazole (MIC = 0.5μg/ml), and one other toward voriconazole (MIC = 2μg/ml). Sequencing of the respective cyp51A genes revealed that eight of the isolates carried the TR34/L98H allele and those two with elevated MIC values to posaconazole the G54R substitution. Although a clear correlation between the use of triazole-based fungicides and isolation of ARAF strains from agricultural lands could not be established for Thailand, but this study clearly demonstrates the spread of globally observed ARAF strains to the environment of South East Asia.

Whole Genome Sequencing of Azole-Resistant Aspergillus Fumigatus Strains from Hematopoietic Stem Cell Recipients Identifies Candidate Molecular Targets Potentially Implicated in Novel Resistance Mediating Mechanisms - First Results

IntroductionInvasive aspergillosis (IA) represents a critical complication that frequently emerges during induction chemotherapy for acute leukemia or after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and is associated with high mortality rates. During the last years, routine administration of antimold prophylaxis based on triazole regimens provided a major advance in controlling fungal infections in patients at high risk for IA. However, several studies recently reported azole resistant fungal strains, which are detectable in patients at risk with rapidly increasing frequency. While mutations in the 14α-sterol demethylase (cyp51A) gene have been implicated in mediating azole resistance, in approximately 50% of resistant patient isolates the underlying genetic cause is unknown (van der Linden, Emerging Infectious Diseases, 2015). In view of a rising prevalence of azole resistance in the high risk clinical setting we performed whole genome sequencing (WGS) of azole-resistant cyp51A-wildtype Aspergillus fumigatus (Afu) specimen, the main effector of IA, with the aim to identify novel genetic alterations leading to azole resistance.MethodsAfu clinical strains were isolated from six different patients with IA undergoing intensive chemotherapy for acute leukemia or after allo-HSCT. Phenotypic resistance screening confirmed azole resistance in all samples. Moreover, Sanger sequencing revealed characteristic resistance associated cyp51A mutations ("TR46/Y121F/T289A") in four of the six specimen, while two samples harbored cyp51A wildtype sequences. High molecular weight genomic DNA (gDNA) was isolated using a modified DNeasy Plant Mini Kit protocol (Qiagen). WGS sequencing libraries were prepared utilizing the transposome fragmentation based Nextera XT DNA Library Prep Kit (Illumina) and sequenced on a MiSeq next generation sequencing (NGS) system (Illumina) with V2 sequencing chemistry (2x250bp). Raw paired reads were mapped to the NCBI Af293 (ASM265v1) reference genome using BWA followed by calibration with GATK. Subsequently, genomic variants were identified by GATK Haplotypecaller module and ANNOVAR software was used for functional variant annotation.ResultsWGS of four cyp51A mutated and two cyp51A wildtype samples resulted in an average genomic coverage of 26x (range 21.2x - 30.1x) allowing robust detection of genomic sequence variants. Of note, the presence or absence of cyp51A "TR46/Y121F/T289A" mutational genotype was reliably validated by WGS data using both our variant calling pipeline and manual screening of mapped reads with the IGV browser. In an attempt to screen for novel, previously undocumented candidate genes that are mechanistically involved in azole resistance we first identified all mutations that are exclusively present in the cyp51A wildtype samples and undetectable in the cyp51A mutated cohort. Next, we excluded all variants that have previously been reported as being polymorphic in WGS data from 22 different Afu strains sequenced by the J. Craig Venter Institute. Using this strategy we identified a core candidate cluster of 112 nucleotide variants in 62 genes that were exclusively found in the cyp51A wildtype group and predicted to have an impact on the amino acid sequence of the corresponding translational products. In particular, exemplary identified mutations affected genes coding for glycosyl hydrolase family 31 proteinimplicated in carbohydrate metabolic processes and 3-oxo-5-alpha-steroid 4-dehydrogenase family protein, which is, similar to cyp51A, involved in steroid metabolism.ConclusionThe rapid increase in detection of triazole resistant fungal strains in clinical contexts represents a critical problem, particularly for immunocompromised patients at high risk for IA, and highlights the need to explore the underlying molecular causes. Pending further validation in a larger cohort, our NGS-based approach for sequencing the genomes of azole resistant Afu strains identified several candidate genomic alterations that might pinpoint alternative pathways to acquisition of resistance, independent of the previously described cyp51A mutations. Ultimately, our approach for discovery of previously undescribed resistance pathways in fungal strains might aid in exploration of novel therapeutic targets and identify suitable biomarkers for effective clinical and epidemiological surveillance. DisclosuresBuchheidt:Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Other: travel grant, Research Funding, Speakers Bureau; Astellas: Other: travel grant, Speakers Bureau; Merck Sharp & Dohme: Other: travel grant, Speakers Bureau.

Résistance aux antifongiques azolés d’origine environnementale : quelles alternatives pour l’avenir ?

Azole resistant Aspergillus fumigatus strains are increasingly reported in many countries. One resistance mechanism is attributed to the use of azole fungicides in environment. Two mutations, TR34/L98H and TR46/Y121F/T289A, on the cyp51A gene, have been described. Results of 40publications about azole resistant strain detections, with TR34/L98H and TR46/Y121F/T289A mutations, in clinical and/or environmental samples, are presented in this review. These cases, observed in many countries, suggest spreading phenomenon. Measures to moderate fungicides treatments and/or alternative treatments in environment should be established to preserve the effectiveness of azole antifungal therapy for at-risk patients.

Aspergillus fumigatus harbouring the sole Y121F mutation shows decreased susceptibility to voriconazole but maintained susceptibility to itraconazole and posaconazole.

Voriconazole, itraconazole and posaconazole are members of the azole family and widely used for the treatment of aspergillosis. They act by inhibiting the activity of the fungal Cyp51A enzyme. The emergence of environmental azole-resistant Aspergillus fumigatus strains raises major concerns for human health. Recently, a new cyp51A-mediated resistance mechanism (namely TR46/Y121F/T289A) was described in clinical samples and patient-frequented environmental sites. In an azole-naive patient, we isolated an A. fumigatus strain that was not susceptible to voriconazole but was susceptible to itraconazole and posaconazole. A molecular analysis indicated a single Y121F substitution without the TR46 or T289A alterations, which to our knowledge has never been reported. Structure modelling and molecular dynamics offered an explanation for the resistance profile consistent with the structural differences between the three azoles. Taken together, these observations suggest an original mechanism conferring resistance to azoles mediated by cyp51A of environmental origin. This uncommon susceptibility pattern might represent a 'missing link' between the wild-type A. fumigatus and the fully azole-resistant strain harbouring the TR46/Y121F/T289A mutations.

Correction: Emergence of Azole-Resistant Aspergillus fumigatus Strains due to Agricultural Azole Use Creates an Increasing Threat to Human Health

Correction: Emergence of Azole-Resistant Aspergillus fumigatus Strains due to Agricultural Azole Use Creates an Increasing Threat to Human Health

Emergence of Azole-Resistant Aspergillus fumigatus Strains due to Agricultural Azole Use Creates an Increasing Threat to Human Health

Aspergillus fumigatus, a ubiquitously distributed opportunistic pathogen, is the global leading cause of aspergillosis and causes one of the highest numbers of deaths among patients with fungal infections [1]. Invasive aspergillosis is the most severe manifestation with an overall annual incidence up to 10% in immunosuppressed patients, whereas chronic pulmonary aspergillosis affects about 3 million, primarily immunocompetent, individuals each year [2]. Three triazole antifungals, namely itraconazole, voriconazole, and posaconazole, are recommended first-line drugs in the treatment and prophylaxis of aspergillosis [3]. However, azole resistance in A. fumigatus isolates is increasingly reported with variable prevalence in Europe, the United States, South America, China, Japan, Iran, and India [4]–[9]. For example, about 10% of strains of A. fumigatus from the Netherlands are itraconazole resistant, and in the United Kingdom, the frequency increased from 0%–5% during 2002–2004 to 17%–20% in 2007–2009 [10]–[13]. In the ARTEMIS global surveillance program involving 62 medical centers, 5.8% of A. fumigatus strains showed elevated MICs to one or more triazoles [5]. Similarly, the prospective SCARE (Surveillance Collaboration on Aspergillus Resistance in Europe) study involving 22 medical centers in 19 countries identified an overall prevalence of 3.4% azole resistance. Azole-resistant A. fumigatus (ARAF) ranged from 0% to 26% among the 22 centres and was detected in 11 (57.9%) of the 19 participating European countries [4 and P.E. Verweij, personal communication]. Interestingly, almost half (48.9%) of the ARAF isolates from the SCARE network in European countries were resistant to multiple azoles and harbored the TR34/L98H mutation in the cyp51A gene [4 and P.E. Verweij, personal communication]. Indeed, multi-azole resistance in A. fumigatus due to the TR34/L98H mutations has become an emerging problem in both Europe and Asia and has been associated with high rates of treatment failures [12]–[14]. Azole antifungal drugs inhibit the ergosterol biosynthesis pathway, specifically the cytochrome p450 sterol 14-α-demethylase encoded by the cyp51A gene, which leads to depletion of ergosterol and accumulation of toxic sterols. The majority of ARAF isolates contain alterations in the target enzyme and the mutated target showed reduced or no binding to the drugs [15]. While most mutations in ARAF isolates were single nucleotide substitutions in the target gene (cyp51A), mutations at other genes such as the cdr1B have also been reported. For example, in the United Kingdom the frequency of ARAF isolates without cyp51A mutations has been reported to be more than 50% [16].