Search

Inflammasomes are important sentinels of innate immune defence that are activated in response to diverse stimuli, including pathogen-associated molecular patterns (PAMPs)1. Activation of the inflammasome provides host defence against aspergillosis2,3, which is a major health concern for patients who are immunocompromised. However, the Aspergillus fumigatusPAMPs that are responsible for inflammasome activation are not known. Here we show that the polysaccharide galactosaminogalactan (GAG) of A. fumigatusis a PAMP that activates the NLRP3 inflammasome. The binding of GAG to ribosomal proteins inhibited cellular translation machinery, and thus activated the NLRP3 inflammasome. The galactosamine moiety bound to ribosomal proteins and blocked cellular translation, which triggered activation of the NLRP3 inflammasome. In mice, a GAG-deficient Aspergillusmutant (?gt4c) did not elicit protective activation of the inflammasome, and this strain exhibited enhanced virulence. Moreover, administration of GAG protected mice from colitis induced by dextran sulfate sodium in an inflammasome-dependent manner. Thus, ribosomes connect the sensing of this fungal PAMP to the activation of an innate immune response.

A steroid-immunosuppressed rat model of invasive pulmonary aspergillosis was use to examine the usefulness of galactomannan enzyme immunoassay (GM) and quantitative real time PCR (RT-PCR) in evaluating the association between response and exposure after a high dose of prophylactic posaconazole.

ABSTRACTIsogenic bar-coded strains of Aspergillus fumigatuscarrying the G54W or M220K mutation in Cyp51A were constructed. In vitro, the growth and conidiation capacities of the mutants were similar to those of the parental strain. Competition studies in the absence of azoles showed that there was no adverse fitness cost for the azole-resistant A. fumigatusstrains in vitroor in vivocompared to the parental strain.

ABSTRACTFungal infections have increased dramatically in the last 2 decades, and fighting infectious diseases requires innovative approaches such as the combination of two drugs acting on different targets or even targeting a salvage pathway of one of the drugs. The fungal cell wall biosynthesis is inhibited by the clinically used antifungal drug caspofungin. This antifungal activity has been found to be potentiated by humidimycin, a new natural product identified from the screening of a collection of 20,000 microbial extracts, which has no major effect when used alone. An analysis of transcriptomes and selected Aspergillus fumigatusmutants indicated that humidimycin affects the high osmolarity glycerol response pathway. By combining humidimycin and caspofungin, a strong increase in caspofungin efficacy was achieved, demonstrating that targeting different signaling pathways provides an excellent basis to develop novel anti-infective strategies.

ABSTRACTAspergillussection Fumigaticontains 12 clinically relevant species. Among these Aspergillusspecies, A. fumigatusis the most frequent agent of invasive aspergillosis, followed by A. lentulusand A. viridinutans. Genealogical concordance and mating experiments were performed to examine the relationship between phylogenetic distance and mating success in these three heterothallic species. Analyses of 19 isolates from section Fumigatirevealed the presence of three previously unrecognized species within the broadly circumscribed species A. viridinutans. A single mating type was found in the new species Aspergillus pseudofelisand Aspergillus pseudoviridinutans, but in Aspergillus parafelis, both mating types were present. Reciprocal interspecific pairings of all species in the study showed that the only successful crosses occurred with the MAT1-2isolates of both A. parafelisand A. pseudofelis. The MAT1-2isolate of A. parafeliswas fertile when paired with the MAT1-1isolates of A. fumigatus, A. viridinutans, A. felis, A. pseudoviridinutans, and A. wyomingensisbut was not fertile with the MAT1-1isolate of A. lentulus. The MAT1-2isolates of A. pseudofeliswere fertile when paired with the MAT1-1isolate of A. felisbut not with any of the other species. The general infertility in the interspecies crossings suggests that genetically unrelated species are also biologically incompatible, with the MAT1-2isolates of A. parafelisand A. pseudofelisbeing the exception. Our findings underscore the importance of genealogical concordance analysis for species circumscription, as well as for accurate species identification, since misidentification of morphologically similar pathogens with differences in innate drug resistance may be of grave consequences for disease management.

The incidence of fungal infections due to C. parapsilosisand closely related cryptic species (-psilosiscomplex) has increased in the last few years, but differences in virulence among these species have not been widely studied. Fifteen clinical isolates of C. parapsilosis, C. orthopsilosis, and C. metapsilosis, including the type strains, were used to evaluate their virulence in Galleria mellonellalarvae. Fluctuations in the hemocyte density and in the phagocytic activity were also tested. Differences in the median survival for these species were demonstrated at 37 °C (2.6 ± 1.02, 2.3 ± 0.92, and 4.53 ± 1.65 d for C. parapsilosis, C. orthopsilosis, and C. metapsilosis, respectively). Galleria mellonellahemocytes phagocytosed C. metapsilosisstrains more effectively than did for C. orthopsilosisand C. parapsilosis(P< 0.05). The phagocytosis rate was lower for C. parapsilosisthan for C. orthopsilosis(P< 0.05). The hemocyte density was increased in larvae infected with C. metapsilosiscompared with those infected with C. parapsilosisor C. orthopsilosis(P< 0.05). Moreover, in vitro studies of virulence factors such as pseudohyphae production and hydrolytic enzyme secretion showed that the capability of C. metapsilosisstrains to produce those virulence factors was reduced. Infections due to -psilosiscomplex species produced tissue damage in G. mellonellaand pseudohyphae could be also observed during infection with C. parapsilosis.

ABSTRACTA voriconazole-resistant isolate of Aspergillus fumigatuswas recovered from an immunocompetent patient receiving long-term antifungal therapy for cerebral aspergillosis. A G448S amino acid substitution in the azole target (Cyp51A) was identified as the cause of the resistance phenotype. This article describes the first isolation of a voriconazole-resistant A. fumigatusisolate from an immunocompetent patient in Spain.

Natural products are an inexhaustible source for drug discovery. However, the validation and selection of primary screening assays are vital to guarantee a selection of extracts or molecules with relevant pharmacological action and worthy of following up. The assay must be rapid, simple, easy to implement, and produce quick results and preferably at a low cost. In this work, we developed and validated a colorimetric microtiter assay using the resazurin viability dye. The parameters of the resazurin method for high-throughput screening (HTS) using natural extracts against Aspergillus fumigatuswere optimized and set up. The extracts plus RPMI-1640 modified medium containing the spores and 0.002% resazurin were added per well. The fluorescence was read after 24 to 30 h of incubation. The resazurin proved to be as suitable as Alamar Blue for determining the minimal inhibitory concentration of different antifungals against A. fumigatusand effective to analyze fungicidal and fungistatic compounds. An HTS of 12 000 microbial extracts was carried out against two A. fumigatusstrains, and 2.7% of the extracts displayed antifungal activity. Our group has been the first to use this methodology for screening a collection of natural extracts to identify compounds with antifungal activity against the medically important human pathogen A. fumigatus.

ABSTRACTRecent studies have demonstrated that some morphologically atypical Aspergillus fumigatusstrains are different species belonging to the section Fumigati. Aspergillus lentulus, one of these sibling species, is increasingly reported in patients under corticosteroid treatment. MICs of most antifungals in clinical use are elevated against A. lentulus, and it shows primary resistance to azole drugs. Two A. lentuluscytochrome P450 14-α sterol demethylases, encoded by A. lentuluscyp51A (Alcyp51A) and Alcyp51Bgenes, were identified. Targeted cyp51Agene knockout in A. lentulusshowed that the intrinsic azole resistance of this species is cyp51Adependent. The Δcyp51Astrain was morphologically indistinguishable from the A. lentuluswild-type strain, retaining the ability to cause pulmonary disease in neutropenic mice. The heterologous expression of A. lentuluscyp51Awas performed in an A. fumigatuscyp51A-deficient strain, confirming that Cyp51A is responsible for the differences in A. lentulus-azole drug interaction.

Recent studies have demonstrated that some morphologically atypical Aspergillus fumigatus strains are different species belonging to the section FUMIGATI: Aspergillus lentulus, one of these sibling species, is increasingly reported in patients under corticosteroid treatment. MICs of most antifungals in clinical use are elevated against A. lentulus, and it shows primary resistance to azole drugs. Two A. lentulus cytochrome P450 14-α sterol demethylases, encoded by A. lentulus cyp51A (Alcyp51A) and Alcyp51B genes, were identified. Targeted cyp51A gene knockout in A. lentulus showed that the intrinsic azole resistance of this species is cyp51A dependent. The Δcyp51A strain was morphologically indistinguishable from the A. lentulus wild-type strain, retaining the ability to cause pulmonary disease in neutropenic mice. The heterologous expression of A. lentulus cyp51A was performed in an A. fumigatus cyp51A-deficient strain, confirming that Cyp51A is responsible for the differences in A. lentulus-azole drug interaction.

Respiratory tract colonization by molds in patients with cystic fibrosis (CF) were analyzed, with particular focus on the frequency, genotype, and underlying mechanism of azole resistance among Aspergillus fumigatus isolates. Clinical and demographic data were also analyzed. A total of 3,336 respiratory samples from 287 CF patients were collected during two 6-month periods in 2007 and 2009. Azole resistance was detected using an itraconazole screening agar (4 mg/liter) and the EUCAST method. cyp51A gene sequencing and microsatellite genotyping were performed for isolates from patients harboring azole-resistant A. fumigatus. Aspergillus spp. were present in 145 patients (51%), of whom 63 (22%) were persistently colonized. Twelve patients (4%) harbored other molds. Persistently colonized patients were older, provided more samples, and more often had a chronic bacterial infection. Six of 133 patients (4.5%) harbored azole-nonsusceptible or -resistant A. fumigatus isolates, and five of those six patients had isolates with Cyp51A alterations (M220K, tandem repeat [TR]/L98H, TR/L98H-S297T-F495I, M220I-V101F, and Y431C). All six patients were previously exposed to azoles. Genotyping revealed (i) microevolution for A. fumigatus isolates received consecutively over the 2-year period, (ii) susceptible and resistant isolates (not involving TR/L98H isolates) with identical or very closely related genotypes (two patients), and (iii) two related susceptible isolates and a third unrelated resistant isolate with a unique genotype and the TR/L98H resistance combination (one patient). Aspergilli were frequently found in Danish CF patients, with 4.5% of the A. fumigatus isolates being azole nonsusceptible or resistant. Genotyping suggested selection of resistance in the patient as well as resistance being achieved in the environment.

ABSTRACTRespiratory tract colonization by molds in patients with cystic fibrosis (CF) were analyzed, with particular focus on the frequency, genotype, and underlying mechanism of azole resistance among Aspergillus fumigatusisolates. Clinical and demographic data were also analyzed. A total of 3,336 respiratory samples from 287 CF patients were collected during two 6-month periods in 2007 and 2009. Azole resistance was detected using an itraconazole screening agar (4 mg/liter) and the EUCAST method. cyp51Agene sequencing and microsatellite genotyping were performed for isolates from patients harboring azole-resistant A. fumigatus. Aspergillusspp. were present in 145 patients (51%), of whom 63 (22%) were persistently colonized. Twelve patients (4%) harbored other molds. Persistently colonized patients were older, provided more samples, and more often had a chronic bacterial infection. Six of 133 patients (4.5%) harbored azole-nonsusceptible or -resistant A. fumigatusisolates, and five of those six patients had isolates with Cyp51A alterations (M220K, tandem repeat [TR]/L98H, TR/L98H-S297T-F495I, M220I-V101F, and Y431C). All six patients were previously exposed to azoles. Genotyping revealed (i) microevolution for A. fumigatusisolates received consecutively over the 2-year period, (ii) susceptible and resistant isolates (not involving TR/L98H isolates) with identical or very closely related genotypes (two patients), and (iii) two related susceptible isolates and a third unrelated resistant isolate with a unique genotype and the TR/L98H resistance combination (one patient). Aspergilli were frequently found in Danish CF patients, with 4.5% of the A. fumigatusisolates being azole nonsusceptible or resistant. Genotyping suggested selection of resistance in the patient as well as resistance being achieved in the environment.

This article identifies novel stress-protective proteins that belong to the family of intrinsically unstructured proteins, DprA and DprB, which are associated with the cytosol and the peroxisomes in Aspergillus fumigatus.

During a search for genes controlling conidial dormancy in Aspergillus fumigatus, two dehydrin-like genes, DprA and DprB, were identified. The deduced proteins had repeated stretches of 23 amino acids that contained a conserved dehydrin-like protein (DPR) motif. Disrupted DprAΔ mutants were hypersensitive to oxidative stress and to phagocytic killing, whereas DprBΔ mutants were impaired in osmotic and pH stress responses. However, no effect was observed on their pathogenicity in our experimental models of invasive aspergillosis. Molecular dissection of the signaling pathways acting upstream showed that expression of DprA was dependent on the stress-activated kinase SakA and the cyclic AMP-protein kinase A (cAMP-PKA) pathways, which activate the bZIP transcription factor AtfA, while expression of DprB was dependent on the SakA mitogen-activated protein kinase (MAPK) pathway, and the zinc finger transcription factor PacC. Fluorescent protein fusions showed that both proteins were associated with peroxisomes and the cytosol. Accordingly, DprA and DprB were important for peroxisome function. Our findings reveal a novel family of stress-protective proteins in A. fumigatus and, potentially, in filamentous ascomycetes.

ABSTRACTA single mechanism of azole resistance was shown to predominate in clinical and environmental Aspergillus fumigatusisolates from the Netherlands, and a link to the use of azoles in the environment was suggested. To explore the prevalence of azole-resistant A. fumigatusand other aspergilli in the environment in other European countries, we collected samples from the surroundings of hospitals in Copenhagen, Innsbruck, and Madrid, flowerbeds in an amusement park in Copenhagen, and compost bags purchased in Austria, Denmark, and Spain and screened for azole resistance using multidish agars with itraconazole, voriconazole, and posaconazole. EUCAST method E.DEF 9.1 was used to confirm azole resistance. The promoter and entire coding sequence of the cyp51Agene were sequenced to identify azole-resistant A. fumigatusisolates. A. fumigatuswas recovered in 144 out of 185 samples (77.8%). Four A. fumigatusisolates from four Danish soil samples displayed elevated azole MICs (8%), and all harbored the same TR/L98H mutation of cyp51A. One A. lentulusisolate with voriconazole MIC of 4 mg/liter was detected in Spain. No azole-resistant aspergilli were detected in compost. Finally, A. terreuswas present in seven samples from Austria. Multi-azole-resistant A. fumigatusis present in the environment in Denmark. The resistance mechanism is identical to that of environmental isolates in the Netherlands. No link to commercial compost could be detected. In Spain and Austria, only Aspergillusspecies with intrinsic resistance to either azoles or amphotericin B were found.

ABSTRACTSpecies identification using both phenotypic and molecular methods and antifungal susceptibility tests was carried out with 60 uncommon clinical yeasts. Our data show that phenotypic methods were insufficient for correct identification (only 25%) and that most of the wrongly identified strains showed a resistant antifungal profile.

ABSTRACTA phylogenetic analysis was performed for 34 Aspergillusstrains belonging to section Nigri. Molecular methods allowed for the correct classification into three different clades (A. niger, A. tubingensis, and A. foetidus). Correlation with in vitro itraconazole susceptibility distinguished the following three profiles: susceptible, resistant, and showing a paradoxical effect. A number of different species whose morphological features resemble those of A. nigershowed unusual MICs to itraconazole that have never been described for the Aspergillusgenus.

ABSTRACTA collection of 2,278 isolates belonging to 86 different fungal species was tested with micafungin and eight other drugs using the EUCAST procedures. Micafungin was active against species of Candidaand Aspergillus(even azole-resistant species) as well as Penicilliumspp., Scedosporium apiospermum, and Acremoniumspp. It was inactive for species of Basidiomycotaand Mucoralesand for multiresistant species such as those of Fusarium.

ABSTRACTAntifungal susceptibility testing of molds has been standardized in Europe and in the United States. Aspergillus fumigatusstrains with resistance to azole drugs have recently been detected and the underlying molecular mechanisms of resistance characterized. Three hundred and ninety-three isolates, including 32 itraconazole-resistant strains, were used to define wild-type populations, epidemiological cutoffs, and cross-resistance between azole drugs. The epidemiological cutoff for itraconazole, voriconazole, and ravuconazole for the wild-type populations of A. fumigatuswas ≤1 mg/liter. For posaconazole, the epidemiological cutoff was ≤0.25 mg/liter. Up till now, isolates susceptible to itraconazole have not yet displayed resistance to other azole drugs. Cross-resistance between azole drugs depends on specific mutations in cyp51A. Thus, a substitution of glycine in position 54 of Cyp51A confers cross-resistance between itraconazole and posaconazole. A substitution of methionine at position 220 or a duplication in tandem of a 34-bp fragment in the cyp51Apromoter combined with a substitution of leucine at position 98 for histidine confers cross-resistance to all azole drugs tested. The results obtained in this study will help to develop clinical breakpoints for azole drugs and A. fumigatus.

This study analyzed 28 Aspergillus strains belonging to the section Fumigati that were isolated from clinical samples in Spain. All isolates sporulated slowly and were unable to grow at 48°C. Phylogenetic analysis based on sequencing of partial sequences of the β-tubulin and rodlet A genes was used to classify the 28 strains into six different clades (Neosartorya hiratsukae, Neosartorya pseudofischeri, Aspergillus viridinutans, Aspergillus lentulus, Aspergillus fumigatiaffinis, and Aspergillus fumisynnematus). Antifungal susceptibility testing showed heterogeneous patterns and grouped the strains together by species. Most A. lentulus and A. fumigatiaffinis isolates showed high MICs of amphotericin B (geometric mean [GM] MICs, ≥4.5 µg/ml), itraconazole (GM MICs, ≥6 µg/ml), voriconazole (GM MICs, ≥3 µg/ml), and ravuconazole (GM MICs, ≥3 µg/ml); N pseudofischeri and A. viridinutans showed high MICs of itraconazole (GM MICs, ≥8 µg/ml), voriconazole (GM MICs, ≥3.33 µg/ml), and ravuconazole (GM MICs, ≥2 µg/ml); and N. hiratsukae and A. fumisynnematus were susceptible to all the antifungals tested. In conclusion, a number of different species whose morphological features resemble those of Aspergillus fumigatus could succeed in producing invasive infections in the susceptible host. In addition, some of them showed high MICs for most of the antifungals available for the treatment of patients infected with these organisms. The epidemiology and clinical relevance of these species should therefore be addressed.

ABSTRACTThis study analyzed 28 Aspergillusstrains belonging to the section Fumigatithat were isolated from clinical samples in Spain. All isolates sporulated slowly and were unable to grow at 48°C. Phylogenetic analysis based on sequencing of partial sequences of the β-tubulin and rodlet A genes was used to classify the 28 strains into six different clades (Neosartorya hiratsukae, Neosartorya pseudofischeri, Aspergillus viridinutans, Aspergillus lentulus, Aspergillus fumigatiaffinis, and Aspergillus fumisynnematus). Antifungal susceptibility testing showed heterogeneous patterns and grouped the strains together by species. Most A. lentulusand A. fumigatiaffinisisolates showed high MICs of amphotericin B (geometric mean [GM] MICs, ≥4.5 μg/ml), itraconazole (GM MICs, ≥6 μg/ml), voriconazole (GM MICs, ≥3 μg/ml), and ravuconazole (GM MICs, ≥3 μg/ml); N pseudofischeriand A. viridinutansshowed high MICs of itraconazole (GM MICs, ≥8 μg/ml), voriconazole (GM MICs, ≥3.33 μg/ml), and ravuconazole (GM MICs, ≥2 μg/ml); and N. hiratsukaeand A. fumisynnematuswere susceptible to all the antifungals tested. In conclusion, a number of different species whose morphological features resemble those of Aspergillus fumigatuscould succeed in producing invasive infections in the susceptible host. In addition, some of them showed high MICs for most of the antifungals available for the treatment of patients infected with these organisms. The epidemiology and clinical relevance of these species should therefore be addressed.

ABSTRACTTriazole resistance in Aspergillus fumigatusis an uncommon but rising phenomenon. Susceptibility testing is rarely performed and can take 48 h or longer, which is an impediment to effective therapy. Molecular diagnostic probing of well-defined resistance mechanisms, which serve as surrogate markers, provides an alternative approach to rapidly (within hours) and efficiently identify resistant strains. The mechanisms of triazole resistance in A. fumigatusare limited to amino acid substitutions in the drug target Cyp51A and include amino acid substitutions at the positions Gly 54, Gly 138, Met 220, and Leu 98, coupled with a tandem repetition in the gene promoter. We report the development of a real-time PCR assay utilizing molecular beacons to assess triazole resistance markers in A. fumigatus. When combined in a multiplex platform, the assay provides a comprehensive evaluation of drug resistance in A. fumigatus.

ABSTRACTActivities of 35 combinations of antifungal agents against Scedosporiumspp. were analyzed by a checkerboard microdilution design and the summation of fractional concentration index. An average indifferent effect was detected apart from combinations of azole agents and echinocandins against Scedosporium apiospermum. Antagonism was absent for all antifungal combinations against both species.

We have compared the activities of posaconazole and other currently available antifungal agents against a collection of 3,378 clinical isolates of yeasts and filamentous fungi. A total of 1,997 clinical isolates of Candida spp., 359 of other yeast species, 697 strains of Aspergillus spp., and 325 nondermatophyte non-Aspergillus spp. were included. The average geometric means of the MICs of agents that were tested against Candida spp. were 0.23 µg/ml for amphotericin B, 0.29 µg/ml for flucytosine, 0.97 µg/ml for fluconazole, 0.07 µg/ml for itraconazole, 0.04 µg/ml for voriconazole, 0.15 µg/ml for caspofungin, and 0.03 µg/ml for posaconazole. Voriconazole and posaconazole were active in vitro against the majority of isolates, with resistance to fluconazole and itraconazole, and against Cryptococcus neoformans and other Basidiomycota yeasts. Posaconazole was the most active of antifungal agents tested against Aspergillus spp., with an average geometric mean of 0.10 µg/ml. It was active against Paecilomyces spp., Penicillium spp., Scedosporium apiospermum, and some black fungi, such as Alternaria spp. Multiresistant filamentous fungi, such as Scedosporium prolificans, Scopulariopsis brevicaulis, and Fusarium solani, were also resistant to voriconazole, caspofungin, and posaconazole. Amphotericin B and posaconazole were found to be active against most of the Mucorales strains tested. Posaconazole and currently available antifungal agents exhibit a potent activity in vitro against the majority of pathogenic fungal species.

ABSTRACTWe have compared the activities of posaconazole and other currently available antifungal agents against a collection of 3,378 clinical isolates of yeasts and filamentous fungi. A total of 1,997 clinical isolates of Candidaspp., 359 of other yeast species, 697 strains of Aspergillusspp., and 325 nondermatophyte non-Aspergillusspp. were included. The average geometric means of the MICs of agents that were tested against Candidaspp. were 0.23 μg/ml for amphotericin B, 0.29 μg/ml for flucytosine, 0.97 μg/ml for fluconazole, 0.07 μg/ml for itraconazole, 0.04 μg/ml for voriconazole, 0.15 μg/ml for caspofungin, and 0.03 μg/ml for posaconazole. Voriconazole and posaconazole were active in vitro against the majority of isolates, with resistance to fluconazole and itraconazole, and against Cryptococcus neoformansand other Basidiomycotayeasts. Posaconazole was the most active of antifungal agents tested against Aspergillusspp., with an average geometric mean of 0.10 μg/ml. It was active against Paecilomycesspp., Penicilliumspp., Scedosporium apiospermum, and some black fungi, such as Alternariaspp. Multiresistant filamentous fungi, such as Scedosporium prolificans, Scopulariopsis brevicaulis, and Fusarium solani, were also resistant to voriconazole, caspofungin, and posaconazole. Amphotericin B and posaconazole were found to be active against most of the Mucoralesstrains tested. Posaconazole and currently available antifungal agents exhibit a potent activity in vitro against the majority of pathogenic fungal species.

Two erg3 genes encoding C-5 sterol desaturase enzymes (Erg3A and Erg3B) in Aspergillus fumigatus were characterized with respect to their nucleotide sequences and null mutant phenotypes. Targeted disruption of the erg3A and erg3B genes and a double gene knockout, erg3A– erg3B–, showed that they are not essential for A. fumigatus viability. Mutant phenotypes clearly showed that Erg3B is a C-5 sterol desaturase, but no apparent role for Erg3A in A. fumigatus ergosterol biosynthesis was found. Susceptibility to amphotericin B, itraconazole, fluconazole, voriconazole, and ketoconazole was not altered in isolates in which erg3A and erg3B were knocked out alone and in combination.

ABSTRACTTwo erg3genes encoding C-5 sterol desaturase enzymes (Erg3A and Erg3B) in Aspergillus fumigatuswere characterized with respect to their nucleotide sequences and null mutant phenotypes. Targeted disruption of the erg3Aand erg3Bgenes and a double gene knockout, erg3A−erg3B−, showed that they are not essential for A. fumigatusviability. Mutant phenotypes clearly showed that Erg3B is a C-5 sterol desaturase, but no apparent role for Erg3A in A. fumigatusergosterol biosynthesis was found. Susceptibility to amphotericin B, itraconazole, fluconazole, voriconazole, and ketoconazole was not altered in isolates in which erg3Aand erg3Bwere knocked out alone and in combination.

ABSTRACTThe physiological patterns, the sequence polymorphisms of the internal transcriber spacer (ITS), and intergenic spacer regions (IGS) of the rRNA genes and the antifungal susceptibility profile were evaluated for their ability to identify Trichosporonspp. and their specificity for the identification of 49 clinical isolates of Trichosporonspp. Morphological and biochemical methodologies were unable to differentiate among the Trichosporonspecies. ITS sequencing was also unable to differentiate several species. However, IGS1 sequencing unambiguously identified all Trichosporonisolates. Following the results of DNA-based identification, Trichosporon asahiiwas the species most frequently isolated from deep sites (15 of 25 strains; 60%). In the main, other Trichosporonspecies were recovered from cutaneous samples. The majority of T. asahii, T. faecale, and T. coremiiformeclinical isolates exhibited resistance in vitro to amphotericin B, with geometric mean (GM) MICs >4 μg/ml. The other species of Trichosporondid not show high MICs of amphotericin B, and GM MICs were <1 μg/ml. Azole agents were active in vitro against the majority of clinical strains. The most potent compound in vitro was voriconazole, with a GM MIC ≤0.14 μg/ml. The sequencing of IGS correctly identified Trichosporonisolates; however, this technique is not available in many clinical laboratories, and strains should be dispatched to reference centers where these complex methods are available. Therefore, it seems to be more practical to perform antifungal susceptibility testing of all isolates belonging to Trichosporonspp., since correct identification could take several weeks, delaying the indication of an antifungal agent which exhibits activity against the infectious strain.

ABSTRACTThe influence of several test variables on susceptibility testing of Aspergillusspp. was assessed. A collection of 28 clinical isolates was tested against amphotericin B, itraconazole, voriconazole, and terbinafine. Inoculum size (104CFU/ml versus 105CFU/ml) and glucose supplementation (0.2% versus 2%) did not have significant effects on antifungal susceptibility testing results and higher inoculum size and glucose concentration did not falsely elevate MICs. In addition, antifungal susceptibility testing procedure with an inoculum size of 105CFU/ml distinctly differentiated amphotericin B or itraconazole-resistant Aspergillusstrains in vivo from the susceptible ones. Time of incubation significantly affected the final values of MICs, showing major increases (two to six twofold dilutions, P< 0.01 by analysis of variance) between MIC readings at 24 and 48 h, but no differences were observed between antifungal susceptibility testing results obtained at 48 h and at 72 h. Significantly higher MICs were uniformly associated with higher concentrations of Tween (P< 0.01), used as a dispersing agent in the preparation of inoculum suspensions. The geometric mean MICs showed increases of between 1.5- and 10-fold when the Tween concentration varied from 0.1% (the geometric means for amphotericin B, itraconazole, voriconazole, and terbinafine were 1.29, 0.69, 1.06, and 0.64 µg/ml, respectively) to 5% (the geometric means for amphotericin B, itraconazole, voriconazole, and terbinafine were 1.97, 5.79, 1.60, and 4.66 µg/ml, respectively). The inhibitory effect of Tween was clearly increased with inoculum sizes of 105CFU/ml and was particularly dramatic for itraconazole, terbinafine, and Aspergillus terreus. The inoculum effect was not observed when the Tween concentration was below 0.5% (P> 0.01).

ABSTRACTInteractions in vitro between amphotericin B, itraconazole, voriconazole, and caspofungin against itraconazole-resistant Aspergillus fumigatusclinical strains were determined. Differential results were obtained depending on the criteria (MIC or minimal effective concentration) used. Caspofungin and voriconazole exhibited the most potent interactions, with synergy against at least 50% of isolates, and the average fractional concentration index was 0.38. Antagonism was not found for any combination.

ABSTRACTThe activities of ravuconazole and four other antifungal agents were tested against a collection of 1,796 clinical yeast isolates, including fluconazole-susceptible and -resistant strains. Ravuconazole was active against the majority of fluconazole-resistant isolates; but for 102 of 562 (18%) resistant isolates, mainly Candida tropicalis, Candida glabrata, and Cryptococcus neoformans, ravuconazole MICs were ≥1 μg/ml.

ABSTRACTSeven cases of disseminated infection due to Dipodascus capitatusare reported. Infections occurred in a hematological unit of a tertiary hospital during a period of 5 years. Five cases were refractory to antifungal therapy. Antifungal susceptibility testing of seven isolates was performed, and strains were typed by PCR fingerprinting with the core sequence of phage M13 and by random amplification of polymorphic DNA with two primers, Ap12h and W-80A. A very short range of MICs of each antifungal agent was observed. The MICs of amphotericin B ranged between 0.50 and 2 µg/ml. Strains were susceptible in vitro to flucytosine and susceptible (dose-dependent) to fluconazole and itraconazole. Voriconazole exhibited an activity in vitro comparable to that of itraconazole. Typing techniques allowed seven additional isolates of D. capitatusneither geographically nor temporally related to be classified into two different genomic patterns. The genomic type of the seven strains from the hematological unit was identical regardless of typing technique utilized. It would indicate that the seven cases of disseminated infection could be related epidemiologically.

ABSTRACTFive sequential Cryptococcus neoformansisolates recovered from an AIDS patient with recurrent meningitis were analyzed. Four isolates were fluconazole susceptible, while the fifth isolate developed fluconazole resistance. Analysis of the 14-α lanosterol demethylase gene (ERG11) showed a point mutation in the resistant strain responsible for the amino acid substitution G484S.

ABSTRACTThe aim of the present study was to identify retrospectively trends in the species distributions and the susceptibility patterns of Aspergillusspecies causing fungal infections in Spanish medical centers from 2000 to 2002. The susceptibilities of 338 isolates to amphotericin B, itraconazole, and voriconazole were tested. Aspergillus fumigatuswas the most common species (54.7%), followed by Aspergillus terreus(14.8%) and Aspergillus flavus(13.9%). Non-A. fumigatusspecies were encountered in 45.3% of the samples studied. The majority of Aspergillusisolates were obtained from respiratory tract specimens, followed by ear and skin samples. The geometric mean (GM) MIC of amphotericin B was 0.56 μg/ml, and the amphotericin B MIC was >2 μg/ml for 16 isolates (4.7%). Nine of them were A. terreus. The GM MIC of itraconazole was 0.37, and the itraconazole MIC was >4 μg/ml for 12 (3.5%) isolates. The voriconazole MICs were also high for 8 of the 12 strains for which itraconazole MICs were high (voriconazole MIC range, 2 to 8 μg/ml).

ABSTRACTA two-laboratory study was performed to evaluate the correlation between the NCCLS M27-A and EUCAST microdilution procedures for antifungal testing of Candidaspp. A panel of 109 bloodstream isolates was tested against amphotericin B, flucytosine, fluconazole, and itraconazole. Overall, the agreement was 92% and the intraclass correlation coefficient was 0.90 (P< 0.05).

ABSTRACTRecently, the methodology that will serve as a basis of the standard for antifungal susceptibility testing of fermentative yeasts of the European Committee on Antibiotic Susceptibility Testing has been described. This procedure employs a spectrophotometric method for both inoculum adjustment and endpoint determination. However, the utilization of a spectrophotometer requires studies for standardization. The present work analyzes the following parameters: (i) accuracy of inoculum preparation, (ii) correlation between optical density and CFU per milliliter, (iii) influence of the wavelength on the endpoint determination, and (iv) influence of the dimethyl sulfoxide concentration on the growth kinetics. The main results can be summarized as follows: (i) inoculum preparation following the methodology recommended by the National Committee for Clinical Laboratory Standards is an exact procedure; (ii) the relationship between optical density and CFU per milliliter is linear (coefficient of determination, r2= 0.84); (iii) MICs obtained by means of spectrophotometric readings at different wavelengths are identical (for amphotericin B, an intraclass correlation coefficient of 0.98 was obtained; for fluconazole, the intraclass correlation coefficient was 1); and (iv) a 2% concentration of dimethyl sulfoxide produces a significantly slower and lower growth curve of Candidaspp. than other concentrations.

ABSTRACTA major limitation of the National Committee for Clinical Laboratory Standards M27-A methodology is reliable detection of amphotericin B (AMB) resistance. The results obtained by using Iso-Sensitest, a synthetic medium, to detect AMB resistance were analyzed and compared with those obtained with RPMI and antibiotic medium 3 (AM3). The ability to detect AMB resistance with RPMI is not enhanced by using a higher inoculum, glucose supplementation at a final concentration of 20 g/liter, spectrophotometric reading, or 24 h of incubation time. Testing using AM3 and an inoculum of 103CFU/ml detects resistance. Identification of resistant isolates is not improved by glucose supplementation, changes in reading method, or changes in incubation time. However, the use of AM3 as assay medium and an inoculum of 105CFU/ml did not allow detection of AMB resistance. Testing using Iso-Sensitest medium appears to be similar to AM3 in detecting resistance. The most pronounced discrimination is achieved by testing in Iso-Sensitest supplemented with glucose and spectrophotometric reading after 24 h of incubation. The reproducibility of MIC testing was greatest for Iso-Sensitest-based procedures. Use of Iso-Sensitest produces both highly reproducible MICs and reliable identification of AMB-resistant Candidaisolates.

ABSTRACTThe influences of inoculum size and glucose supplementation on the growth kinetics of 60 Candidaspp. clinical isolates (Candida albicans, Candida tropicalis, Candida parapsilosis, Candida glabrata, Candida krusei, and Candida lusitaniae[10 isolates each]) are assessed. The combined influence of growth and reading method (visual or spectrophotometric) on the determination of the MICs of amphotericin B, flucytosine, fluconazole, itraconazole, ketoconazole, and voriconazole is also analyzed, and the MICs are compared with those determined by the National Committee for Clinical Laboratory Standards standard microdilution method (NCCLS document M27-A). Glucose supplementation and inoculum size had a significant influence on the growth cycles of these yeasts, and a statistically significant denser growth (optical density at 540 nm) was seen for both incubation periods, 24 and 48 h (P< 0.01). A longer exponential phase and shorter lag phase were also observed. The A540values at 24 h of incubation with medium containing glucose and an inoculum of 105CFU/ml were >0.4 U for all species, with the exception of that for C. parapsilosis(A540= 0.26 ± 0.025). The MICs at 24 h determined by testing with 2% glucose and an inoculum of 105CFU/ml showed the strongest agreement (96.83%) with MICs determined by the reference method. MICs were not falsely elevated, and good correlation indexes were obtained. The reproducibility of results with this medium-inoculum combination was high (intraclass correlation coefficient, 0.955). The best agreement and reproducibility of results for spectrophotometric readings were achieved with endpoints of 50% growth inhibition for flucytosine and azoles and 95% for amphotericin B. Supplementation of test media with glucose and an inoculum size of 105CFU/ml yielded a reproducible technique that shows elevated agreement with the reference procedures and a shorter incubation period for obtaining reliable MIC determinations. The spectrophotometric method offers an advantage over the visual method by providing a more objective and automated MIC determination.

ABSTRACTWe report the simultaneous isolation of one Aspergillus flavusstrain from the aortic prosthesis of a heart surgery patient and another two isolates recovered from a dual-reservoir cooler-heater used in the operating room where this patient was operated on. Genetic typing of these three isolates by randomly amplified polymorphic DNA (RAPD) revealed identical genotypes. Eight unrelated control strains of A. flavushad eight different genotypes. These results clearly indicated the nosocomial origin of theA. flavusstrain isolated from the patient. We suggest that the RAPD technique is a rapid and reliable tool to ascertain the epidemiology of infections caused by A. flavus.

TheAspergillus fumigatus chsE(AfchsE) gene was isolated from anA. fumigatusDNA library on the basis of hybridization to a segment ofSaccharomyces cerevisiae CHS3(ScCHS3). The amino acid sequence derived from AfchsEis 28% identical with ScCHS3 and 80% identical with the product ofAspergillus nidulans chsD(AnchsD). A mutant strain constructed by disruption of AfchsEhas reduced levels of mycelial chitin, periodic swellings along the length of hyphae, and a block in conidiation that can be partially restored by growth in osmotic stabilizer. This phenotype is different from that reported for an AnchsDmutant, in which germinating conidia and hyphal tips undergo lysis and the colonial growth rate is significantly reduced. Despite the defects associated with the AfchsE−strain, its virulence was not significantly reduced when compared with the wild-type parental strain in a mouse model of pulmonary aspergillosis.

Two approaches were used to isolate fragments of chitin synthase genes from the opportunistic human pathogen Aspergillus fumigatus. Firstly, regions of amino acid conservation in chitin synthases of Saccharomyces cerevisiae were used to design degenerate primers for amplification of portions of related genes, and secondly, a segment of the S. cerevisiae CSD2 gene was used to screen an A. fumigatus ? genomic DNA library. The polymerase chain reaction (PCR)-based approach led to the identification of five different genes, designated chsA, chsB, chsC, chsD and chsE. chsA, chsB, and chsC fall into Classes I, II and III of the ‘zymogen type’ chitin synthases, respectively. The chsD fragment has approximately 35% amino acid sequence identity to both the zymogen type genes and the non-zymogen type CSD2 gene. chsF appears to be a homologue of CSD2, being 80% identical to CSD2 over 100 amino acids. An unexpected finding was the isolation by heterologous hybridization of another gene (chsE), which also has strong sequence similarity (54% identity at the amino acid level over the same region as chsF) to CSD2. Reverse transcriptase-PCR was used to show that each gene is expressed during hyphal growth in submerged cultures.

ABSTRACTIn vitro susceptibility profiles of 58 Paecilomycesclinical isolates are reported. Amphotericin B, itraconazole, and echinocandins showed poor activity against Paecilomyces lilacinus, while the new triazoles were active against it. Paecilomyces variotiiexhibited a different susceptibility pattern, being susceptible to most antifungal agents apart from voriconazole and ravuconazole.

A Correction to this paper has been published: https://doi.org/10.1038/s41586-020-03088-5

ABSTRACTA collection of Aspergillus fumigatusisolates was used to check if MICs can be read at 24 h. At 24 h, the geometric mean MIC of itraconazole for resistant isolates was determined to be 5.11 mg/liter, but the MIC was read as 16 mg/liter at 48 h. At 24 h, MICs for 51.5% of resistant strains were determined to be ≤2 mg/liter. MICs must be obtained at 48 h.

ABSTRACTThe activities of 10 combinations of antifungal agents against 25 clinical isolates of Scopulariopsis brevicauliswere tested by the checkerboard technique. An average indifferent effect was detected for all combinations. Synergy was observed for some isolates and combinations, particularly with posaconazole-terbinafine (68% of strains), amphotericin B-caspofungin (60%), and posaconazole-caspofungin (48%).

ABSTRACTThe in vitro activities of ravuconazole against 575 clinical strains of Aspergillusspp. and 348 nondermatophyte non-Aspergillusspp. were analyzed. Ravuconazole was active against Aspergillusspp., other hyaline filamentous fungi, black molds, and some Mucorales. Species such as Scedosporium prolificans, Fusariumspp., and Scopulariopsisspp. were resistant to the triazole.

ABSTRACTThe antifungal susceptibility results for 32 clinical isolates of Scopulariopsis brevicaulisare presented. Flucytosine and itraconazole were inactive in vitro, and MICs of amphotericin B, voriconazole, and terbinafine for all isolates were high, with geometric means of 13, 25.8, and 14.4 μg/ml, respectively.

ABSTRACTThe in vitro activity of the azasordarin GW 471558 was compared with those of amphotericin B, flucytosine, itraconazole, and ketoconazole against 177 clinical isolates of Candidaspp. GW 471558 showed potent activity against Candida albicans, Candida glabrata, and Candida tropicalis, even against isolates with decreased susceptibility to azoles. Candida krusei, Candida parapsilosis, Candida lusitaniae,and Candida guilliermondiiare resistant to GW 471558 in vitro (MICs, >128 μg/ml).

ABSTRACTTwo methods of inoculum preparation for filamentous fungi were compared: counting with a hematocytometer and spectrophotometric adjustment. One hundred eighty-two filamentous fungi pathogenic for humans were used. Colony counts were done for all inoculum preparations. The agreement between the hematocytometer counts and the colony counts (CFU per milliliter) was 97.2%. The reproducibility between the hematocytometer counts and the colony counts by means of an intraclass correlation coefficient was 0.70. Pearson's correlation index for hematocytometer counts versus colony counts was 0.56, whereas that for optical density versus colony counts was 0.008. Both methods can be used for inoculum size adjustment. However, the use of the spectrophotometric method requires that each species be standardized separately.

Antifungal resistance is one of the major causes of the increasing mortality rates for fungal infections, especially for those caused by Aspergillusspp. A surveillance program was established in 2014 in the Spanish National Center for Microbiology for tracking resistance in the most prevalent Aspergillusspecies. A total of 273 samples were included in the study and were initially classified as susceptible or resistant according to EUCAST breakpoints.