Analysis of the cyp51 genes contribution to azole resistance in Aspergillus section Nigri with the CRISPR-Cas9 technique.

Cyp51 contribution to azole resistance has been broadly studied and characterized in Aspergillus fumigatus, whereas it remains poorly investigated in other clinically relevant species of the genus, such as those of section Nigri In this work, we aimed to analyze the impact of cyp51 genes (cyp51A and cyp51B) on the voriconazole (VRC) response and resistance of Aspergillus niger and Aspergillus tubingensis We generated CRISPR-Cas9 cyp51A and cyp51B knock-out mutants from strains with different genetic backgrounds and diverse patterns of azole susceptibility. Single gene deletions of cyp51 genes resulted in 2 to 16-fold decrease of the VRC Minimum Inhibitory Concentration (MIC) values, which were below the VRC Epidemiological Cutoff Value (ECV) established by the Clinical and Laboratory Standards Institute (CLSI) irrespective of their parental strains susceptibilities. Gene expression studies in the tested species confirmed that cyp51A participates more actively than cyp51B in the transcriptional response of azole stress. However, ergosterol quantification revealed that both enzymes comparably impact the total ergosterol content within the cell, as basal and VRC-induced changes to ergosterol content was similar in all cases. These data contribute to our understanding on Aspergillus azole resistance, especially in non-fumigatus species.

A new pleosporalean fungus isolated from superficial to deep human clinical specimens.

Human infections by pleosporalean fungi (class Dothideomycetes, phylum Ascomycota) are rarely reported. Because their identification is challenging using morphological characterization, several phylogenetic markers must be sequenced for an accurate identification and taxonomical placement of the isolates. Three isolates of clinical origin were phenotypically characterized, but due to the absence of relevant morphological traits, D1-D2 domains of the 28S nrRNA gene (LSU), the internal transcribed spacer region (ITS) of the nrRNA, and fragments of the RNA polymerase II subunit 2 (rpb2) and translation elongation factor 1-alpha (tef1) genes were sequenced to allow a phylogenetic analysis that would solve their phylogenetic placement. That analysis revealed that these isolates did not match any previously known pleosporalean genera, and they are proposed here as the new fungal genus, Gambiomyces. Unfortunately, the isolates remained sterile, which, consequently, made the morphological description of the reproductive structures impossible. Future studies should try to understand the behaviour of this fungus in nature as well as its characteristics as an opportunistic fungal pathogen. Molecular identification is becoming an essential tool for proper identification of Dothideomycetes of clinical origin. LAY ABSTRACT: We describe a new pleosporalen pathogenic fungus, Gambiomyces profunda, found in superficial to deep samples from a human patient. Because all strains remained sterile, the fungus was finally identified following a phylogenetic analysis by using four different molecular markers.

ERG11 Polymorphism in Voriconazole-Resistant Candida tropicalis: Weak Role of ERG11 Expression, Ergosterol Content, and Membrane Permeability.

Mutations in ERG11 were detected by gene sequencing and amino acid alignment in 18 Candida tropicalis strains with different degrees of sensitivity to voriconazole (VRC). ERG11 expression, sterol content, and membrane permeability were also evaluated. We report three missense mutations in ERG11 that resulted in resistance to VRC. The transcriptional levels of ERG11 as well as the ergosterol content and membrane permeability demonstrated no correlation to only a slight correlation with the obtained MIC values, but the data did suggest a tendency toward such a correlation.

Current knowledge on the etiology and epidemiology of Scopulariopsis infections.

Scopulariopsis is a common fungus in the environment, characterized by its intrinsic resistance to the available antifungal drugs. Around 70 cases of infection by this fungus have been described in the literature. Pulmonary and disseminated infections are the most common and their treatment is difficult; therefore, very diverse approaches have been taken, with varied results. A successful outcome has been reported in only a few cases, generally attributed to a multitreatment strategy combining medical and surgical procedures that ultimately led to the resection of the infected tissue if possible, identification of the mould, and an aggressive long-term antifungal therapy. Although most of the infections are caused by Scopulariopsis brevicaulis, a few other species have also been linked to these cases, although molecular evidence has not been proven for all of them. On this basis, more knowledge on the epidemiology, presentation, diagnosis, treatment, and prognosis of these unusual infections would improve their management. This review aims to compile the current data on Scopulariopsis infections.

Expression of ERG11 and efflux pump genes CDR1, CDR2 and SNQ2 in voriconazole susceptible and resistant Candida glabrata strains.

Candida glabrata causes difficult to treat invasive candidiasis due to its antifungal resistance, mainly to azoles. The aim of the present work was to study the role of the genes ERG11, CDR1, CDR2, and SNQ2 on the resistance to voriconazole (VRC) in a set of C. glabrata strains with known in vitro and in vivo susceptibility to this drug. Eighteen clinical isolates of C. glabrata were exposed in vitro to VRC, and the expression of the cited genes was quantified by real time quantitative polymerase chain reaction (q-PCR). In addition, the ERG11 gene was amplified and sequenced to detect possible mutations. Ten synonymous mutations were found in 15 strains, two of them being reported for the first time; however, no amino acid changes were detected. ERG11 and CDR1 were the most expressed genes in all the strains tested, while the expression of CDR2 and SNQ2 was modest. Our results show that gene expression does not directly correlate with the VRC MIC. In addition, the expression profiles of ERG11 and efflux pump genes did not change consistently after exposure to VRC. Although individual analysis did not result in a clear correlation between MIC and gene expression, we did observe an increase in ERG11 and CDR1 expression in resistant strains. It is of interest that considering both in vitro and in vivo results, the slight increase in such gene expression correlates with the observed resistance to VRC.

Sarocladium and Acremonium infections: New faces of an old opportunistic fungus.

The genera Acremonium and Sarocladium comprise a high diversity of morphologically and genetically related fungi generally found in the environment, although a few species, mainly Sarocladium kiliense and Acremonium egyptiacum, can also be involved in many human infections. Clinical management of opportunistic infections caused by these fungi is very complex, since their correct identification is unreliable, and they generally show poor antifungal response. More than 300 clinical cases involving a broad range of Acremonium/Sarocladium infections have so far been published, and with this review we aim to compile and provide a detailed overview of the current knowledge on Acremonium/Sarocladium human infections in terms of presentation, diagnosis, treatments and prognoses. We also aim to summarise and discuss the data currently available on their antifungal susceptibility, emphasising the promising results obtained with voriconazole as well as their impact in terms of animal infections.

New Insights into the Cyp51 Contribution to Azole Resistance in Aspergillus Section Nigri.

Invasive aspergillosis (IA) is a severe condition mainly caused by Aspergillus fumigatus, although other species of the genus, such as section Nigri members, can also be involved. Voriconazole (VRC) is the recommended treatment for IA; however, the prevalence of azole-resistant Aspergillus isolates has alarmingly increased in recent years, and the underlying resistance mechanisms in non-fumigatus species remain unclear. We have determined the in vitro susceptibility of 36 strains from section Nigri to VRC, posaconazole (POS), and itraconazole (ITC), and we have explored the role of Cyp51A and Cyp51B, both targets of azoles, in azole resistance. The three drugs were highly active; POS displayed the best in vitro activity, while ITC and VRC showed MICs above the established epidemiological cutoff values in 9 and 16% of the strains, respectively. Furthermore, expression studies of cyp51A and cyp51B in control condition and after VRC exposure were performed in 14 strains with different VRC susceptibility. We found higher transcription of cyp51A, which was upregulated upon VRC exposure, but no correlation between MICs and cyp51 transcription levels was observed. In addition, cyp51A sequence analyses revealed nonsynonymous mutations present in both, wild-type and non-wild-type strains of A. niger and A. tubingensis Nevertheless, a few mutations were exclusively present in non-wild-type A. tubingensis strains. Altogether, our results suggest that azole resistance in section Nigri is not clearly explained by Cyp51A protein alteration or by cyp51 gene upregulation, which indicates that other mechanisms might be involved.

Neocucurbitaria keratinophila: An emerging opportunistic fungus causing superficial mycosis in Spain.

Although there have been few reports of opportunistic infections (superficial and systemic) caused by coelomycetous fungi, they are becoming more frequent. Neocucurbitaria keratinophila (formerly Pyrenochaeta keratinophila), characterized by producing pycnidial conidiomata and small hyaline conidia, seems to be an emergent opportunistic pathogen in Spain. Since this fungus was first reported from human keratitis, eight strains have been isolates from clinical cases in Spain. This is a retrospective study of these fungal strains, including phenotypic and molecular characterizations, and in vitro antifungal susceptibility assays. These clinical strains were identified by sequencing four phylogenetic markers such as the internal transcribed spacer region (ITS1-5.8S-ITS2) and fragments of the 28S nrRNA (LSU), beta-tubulin (tub2), and RNA polymerase II subunit 2 (rpb2) genes, and by morphological characterization. All the strains tested were susceptible to the majority of antifungals, being isavuconazole the only drug that showed a poor antifungal activity.

Novel Paranannizziopsis species in a Wagler's viper (Tropidolaemus wagleri), tentacled snakes (Erpeton tentaculatum), and a rhinoceros snake (Rhynchophis boulengeri) in a zoological collection.

We report several cases of fungal infections in snakes associated with a new species within the genus Paranannizziopsis. Three juvenile Wagler's vipers (Tropidolaemus wagleri) presented with skin abnormalities or ulcerative dermatitis, and two snakes died. Histologic examination of skin from the living viper revealed hyperplastic, hyperkeratotic, and crusting epidermitis with intralesional fungal elements. The terrestrial Wagler's vipers were housed in a room with fully aquatic tentacled snakes (Erpeton tentaculatum), among which there had been a history of intermittent skin lesions. Approximately 2 months after the biopsy of the viper, a skin sample was collected from one tentacled snake (TS1) with skin abnormalities and revealed a fungal infection with a similar histologic appearance. Fungal isolates were obtained via culture from the Wagler's viper and TS1 and revealed a novel species, Paranannizziopsis tardicrescens, based on phenotypic characterization and molecular analysis. P. tardicrescens was cultured and identified by DNA sequence analysis 8 months later from a dead tentacled snake in an exhibit in an adjacent hallway and 13 months later from a living rhinoceros snake (Rhynchophis boulengeri) with two focal skin lesions. Antifungal susceptibility testing on three of four cultured isolates demonstrated potent in vitro activity for terbinafine and voriconazole.

Two new species of Gloniopsis (Hysteriales, Ascomycota) from clinical specimens: Morphological and molecular characterisation.

BACKGROUND: The coelomycetes comprise a wide range of fungal species distributed in at least three different classes of the phylum Ascomycota. These are morphologically characterised by producing their conidia inside of fruiting bodies called pycnidia or acervuli, and only a reduced number of species are able to cause human infections. However, their identification in the clinical laboratory is often difficult, due to their few morphological features or because they remain sterile. MATERIALS AND METHODS: In the present study, three isolates of coelomycetes of clinical origin were phenotypically and molecularly studied, by sequencing the D1-D2 fragment of the 28S nuclear ribosomal RNA (nrRNA) (LSU), the internal transcribed spacer region (ITS1-5.8S-ITS2) and a fragment of the translation elongation factor 1-alpha (tef1) genes. RESULTS AND CONCLUSIONS: As result of the molecular analysis, the isolates were identified as belonging to the genus Gloniopsis (order Hysteriales, Dothideomycetes) but without the characteristics of any of the species described so far. Therefore, we propose the new species Gloniopsis percutanea and Gloniopsis pneumoniae. Furthermore, this study revealed that some isolates from clinical specimens identified previously as Rhytidhysteron spp. were misidentified, and considering the few studies in the order Hysteriales and the scarce number of sequences of phylogenetic markers, future revisions of this order should be performed to clarify their taxonomy and obtain a better identification from isolates involved in human mycoses.

Species of Aspergillus section Aspergillus from clinical samples in the United States.

The diversity of Aspergillus species in clinical samples is continuously increasing. Species under the former name Eurotium, currently accommodated in section Aspergillus of the genus Aspergillus, are xerophilic fungi widely found in the human environment and able to grow on substrates with low water activity. However, their prevalence in the clinical setting is poorly known. We have studied the presence of these species in a set of clinical samples from the United States using a multilocus sequence analysis based on the internal transcribed spacer (ITS) region of the rRNA, and fragments of the genes ß-tubulin (BenA), calmodulin (CaM), and polymerase II second largest subunit (RPB2). A total of 25 isolates were studied and identified as follows: A. montevidensis (44%), A. chevalieri (36%), A. pseudoglaucus (8%), and A. costiformis (4%). A new species Aspergillus microperforatus is also proposed, which represented 8% of the isolates studied and is characterized by uniseriate conidial heads, subglobose to pyriform vesicles, rough conidia, globose to subglobose cleistothecia, and lenticular and smooth ascospores. The in vitro antifungal activity of eight clinically available antifungals was also determined against these isolates, with the echinocandins and posaconazole having the most potent activity.

DNA sequencing to clarify the taxonomical conundrum of the clinical coelomycetes.

The taxonomy of the fungi that produce human infections and that develop asexual fruiting bodies in culture has become very complex. Recent molecular studies have produced dramatic changes in their classification. Currently, the coelomycetes traditionally included in Sphaeropsidales and Melanconiales are in fact distributed across at least three different classes of the Phylum Ascomycota. Approximately 1000 genera and 7000 species have been grouped in the classes Dothideomycetes, Leotiomycetes and Sordariomycetes and their proper identification can only be made by analysing their DNA sequences and comparing them with those corresponding to type strains available in the adequate databases. To facilitate this task for scientists and clinicians involved in the study of these complex, and every day more numerous taxa, we have updated the knowledge about the taxonomy of the commonest coelomycetes of clinical interest with the aim of improving their identification and antifungal treatment.

Cryptic Aspergillus from clinical samples in the USA and description of a new species in section Flavipedes.

BACKGROUND: In the last few decades there has been an emergence of cryptic Aspergillus as agents of human infections due to the increase in immunocompromised population and to the improvement of identification tools. METHODS: Continuing our study on Aspergillus isolates from clinical origin deposited in a mycological reference centre in the United States, we selected 37 isolates belonging to less common sections of the genus, to study their species diversity and detect cryptic species by using a polyphasic approach. RESULTS: From this set of isolates, a total of 16 species were identified; the most frequent being A. calidoustus (48.6%, section Usti), A. terreus (13.5%, section Terrei), and A. nidulans (5.7%, section Nidulantes). The remaining isolates corresponded to 13 species of rare or cryptic Aspergillus, i.e. A. europaeus (section Cremei); A. iizukae, A. micronesiensis, A. spelaeus (section Flavipedes); A. pachycristatus, A. quadrilineatus, A. spinulosporus, A. unguis (section Nidulantes); A. alabamensis, A. carneus, A. hortai (section Terrei), A. granulosus (section Usti); and the new species A. suttoniae (section Flavipedes), which is described here. CONCLUSIONS: Correct identification of cryptic species is crucial to reveal new potential pathogens, to gather accurate epidemiological data and to choose an appropriate treatment.

Coelomycetous Fungi in the Clinical Setting: Morphological Convergence and Cryptic Diversity.

Human infections by coelomycetous fungi are becoming more frequent and range from superficial to systemic dissemination. Traumatic implantation of contaminated plant material is the most common cause. The typical morphological feature of these fungi is the production of asexual spores (conidia) within fruiting bodies called conidiomata. This study aimed to determine the distribution of the coelomycetes in clinical samples by a phenotypic and molecular study of a large set of isolates received from a U.S. reference mycological institution and by obtaining the in vitro antifungal susceptibility pattern of nine antifungals against a selected group of isolates. A total of 230 isolates were identified by sequencing the D1 and D2 domains of the large subunit (LSU) nuclear ribosomal RNA (nrRNA) gene and by morphological characterization. Eleven orders of the phylum Ascomycota were identified: Pleosporales (the largest group; 66.1%), Botryosphaeriales (19.57%), Glomerellales (4.35%), Diaporthales (3.48%), Xylariales (2.17%), Hysteriales and Valsariales (0.87%), and Capnodiales, Helotiales, Hypocreales and Magnaporthales (0.43% each). The most prevalent species were Neoscytalidium dimidiatum, Paraconiothyrium spp., Phoma herbarum, Didymella heteroderae, and Epicoccum sorghinum The most common anatomical site of isolation was superficial tissue (66.5%), followed by the respiratory tract (17.4%). Most of the isolates tested were susceptible to the majority of antifungals, and only flucytosine showed poor antifungal activity.

Voriconazole MICs are predictive for the outcome of experimental disseminated scedosporiosis.

Background: Scedosporiosis is associated with a mortality rate of up to 90% in patients suffering from disseminated infections. Recommended first-line treatment is voriconazole, but epidemiological cut-off values and clinical breakpoints have not been determined. Objectives: To correlate voriconazole treatment response in mice suffering from disseminated scedosporiosis with MIC values determined using CLSI broth microdilution, Etest (bioMérieux) and disc diffusion. Methods: Voriconazole MICs for 31 Scedosporium apiospermum strains were determined using CLSI broth microdilution, Etest and disc diffusion. Groups of mice were challenged intravenously with 1 out of 16 S. apiospermum strains (voriconazole CLSI broth microdilution MIC range: 0.125-8.0 mg/L) and treated with 40 mg/kg voriconazole orally by gavage once daily. Efficacy of voriconazole was evaluated by a statistically significant ( P < 0.05) reduction in fungal burden in brain. Results: A categorical agreement of 90.4% was reached for CLSI broth microdilution and disc diffusion and of 93.6% for CLSI broth microdilution and Etest. Correlation of CLSI MICs and in vivo outcome was good, as mice challenged with strains with an MIC ≤2 mg/L responded to voriconazole therapy in 92.3% and those challenged with strains with an MIC ≥4 mg/L responded to voriconazole therapy in 33.3%. Conclusions: CLSI broth microdilution and Etest deliver comparable results that enable a prediction of in vivo outcome. Our results suggest that voriconazole is able to reduce fungal burden in the brain of 92.3% of all mice challenged with strains with voriconazole CLSI MICs ≤2 mg/L, while mice challenged with strains with CLSI MICs ≥4 mg/L showed limited response to voriconazole treatment.

Synergistic effect of anidulafungin combined with posaconazole in experimental aspergillosis.

Clinical and experimental data have shown discrepancies on the efficacy of combinations between triazoles and echinocandins. In this study, anidulafungin plus posaconazole have shown efficacy against a murine systemic infection by three strains of Aspergillus fumigatus. The combination increased mice survival and reduced burden in the kidneys over the corresponding monotherapies and voriconazole. Clearance of kidneys was observed in 62% to 100% of animals (strain dependant). We observed good in vitro- in vivo correlation when a cutoff < 1 was indicative of synergy. Our results showed that the combination could be a therapeutical option, especially against infections refractory to the first line therapy.

An updated comprehensive systematic review of Cladophialophora bantiana and analysis of epidemiology, clinical characteristics, and outcome of cerebral cases.

Cladophialophora bantiana is a phaeoid fungus that only rarely has been isolated from sources other than the human brain. It has a particular tropism for the central nervous system (CNS). We have integrated and updated large-scale data related to several aspects of C. Bantiana and reviewed all the available reports on its cerebral infections, focusing on their geographical distribution, infection routes, immune status of infected individuals, type and location of infections, clinical manifestations and treatment and outcome, briefly looking over the spectrum of other disease entities associated with C. bantiana, that is, extra-cerebral and animal infections and on the environmental sources of this fungus. Among the agents of phaeohyphomycosis, a term used to describe an infection caused by a dark pigmented fungus, C. bantiana has some significant specific features. A total of 120 case reports were identified with a significantly higher percentage of healthy subjects than immune-debilitated patients (58.3% vs. 41.7%). Infections due to C. bantiana occur worldwide. The main clinical manifestations are brain abscess (97.5%), coinfection of brain tissue and meninges (14.2%) and meningitis alone (2.5%). Among immunocompetent patients, cerebral infection occurred in the absence of pulmonary lesions. The mortality rate is 65.0% regardless of the patient's immune status. The therapeutic options used include surgery or antifungals alone, and the combination of both, in most cases the fatal outcome being rapid after admission. Since the fungus is a true pathogen, laboratory workers should be made aware that BioSafety Level-3 precautions might be necessary.

Combined antifungal therapy against systemic murine infections by rare Cryptococcus species.

Cryptococcus albidus and Cryptococcus laurentii are uncommon species of this genus that in recent decades have increasingly caused opportunistic infections in humans, mainly in immunocompromised patients; the best therapy for such infection being unknown. Using a murine model of systemic infection by these fungi, we have evaluated the efficacy of amphotericin B (AMB) at 0.8 mg/kg, administered intravenously, fluconazole (FLC) or voriconazole (VRC), both administered orally, at 25 mg/kg and the combination of AMB plus VRC against three C. albidus and two C. laurentii strains. All the treatments significantly reduced the fungal burden in all the organs studied. The combination showed a synergistic effect in the reduction in fungal load, working better than both monotherapies. The histopathological study confirmed the efficacy of the treatments.

Four new species of Talaromyces from clinical sources.

The genus Talaromyces constitutes an important group of molds with species that are mainly found in soil, indoor environments and food products. Traditionally, it has been considered, together with Eupenicillium, the teleomorphic state of Penicillium. However, the taxonomy of these fungi has changed considerably, and Talaromyces currently includes sexually and asexually reproducing species. In a previous study of the occurrence of penicillium-like fungi from clinical samples in the USA, we used the combined phylogeny of the internal transcribed spacer (ITS) region of the rDNA and ß-tubulin (BenA) gene to identify 31 isolates of Talaromyces, 85 of Penicillium and two of Rasamsonia. However, seven isolates of Talaromyces were assigned to the corresponding sections but not to any particular species. In this study, we have resolved the taxonomy of these isolates through a multilocus sequence analysis of the ITS, fragments of the BenA, calmodulin (CaM), and RNA polymerase II second largest subunit (RPB2) genes, and a detailed phenotypic study. As a result, four new species are described and illustrated, ie Talaromyces alveolaris, T. georgiensis, T. minnesotensis and T. rapidus.

New Species Spiromastigoides albida from a Lung Biopsy.

The new species Spiromastigoides albida (Onygenales, Eurotiomycetes, Ascomycota), from a lung biopsy in USA, is proposed and described based on morphological data and the analysis of rRNA, and fragments of actin and ß-tubulin gene sequences. This species is characterized by white colonies and a malbranchea-like asexual morph with profusely branching curved conidiophores forming sporodochia-like structures. Moreover, new combinations for Gymnoascus alatosporus, and for some new species recently described under the generic name Spiromastix, are provided.