Innate host defenses against <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>

Cryptococcus neoformans, the predominant etiological agent of cryptococcosis, can cause life-threatening infections of the central nervous system in immunocompromised and immunocompetent individuals. Cryptococcal meningoencephalitis is the most common disseminated fungal infection in AIDS patients, and remains the third most common invasive fungal infection among organ transplant recipients. The administration of highly active antiretroviral therapy (HAART) has resulted in a decrease in the number of cases of AIDS-related cryptococcosis in developed countries, but in developing countries where HAART is not readily available, Cryptococcus is still a major concern. Therefore, there is an urgent need for the development of novel therapies and/or vaccines to combat cryptococcosis. Understanding the protective immune responses against Cryptococcus is critical for development of vaccines and immunotherapies to combat cryptococcosis. Consequently, this review focuses on our current knowledge of protective immune responses to C. neoformans, with an emphasis on innate immune responses.     

Cryptococcosis in HIV-negative Patients with Renal Dialysis: A Retrospective Analysis of Pooled Cases

Cryptococcosis is a lethal fungal infection mainly caused by Cryptococcus neoformans/C. gattii species. Currently, our understanding of cryptococcosis episodes in HIV-negative patients during renal dialysis remains scarce and fragmented. Here, we performed an analysis of pooled cases to systemically summarize the epidemiology and clinical characteristics of cryptococcosis among HIV-negative patients with renal dialysis. Using pooled data from our hospital and studies identified in four medical databases, 18 cases were identified and analyzed. The median duration time of renal dialysis for peritoneal renal dialysis and hemodialysis cases was 8 months and 36 months, respectively. Several non-neoformans/gattii species were identified among the renal dialysis recipients with cryptococcosis, particularly Cryptococcus laurentii and Cryptococcus albidus, which share similar clinical manifestations as those caused by C. neoformans and C. gattii. Our analyses suggest that physicians should consider the possibility of the occurrence of cryptococcosis among renal dialysis recipients even when cryptococcal antigen test result is negative. The timely removal of the catheter is crucial for peritoneal dialysis patients with cryptococcosis. In addition, there is a need for optimized antifungal treatment strategy in renal dialysis recipients with cryptococcal infections.     

Time-Kill Curves Studies with Amphotericin B Against <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>/<Emphasis Type="Italic">C</Emphasis>. <Emphasis Type="Italic">gattii</Emphasis> Species Complex Clinical Isolates

Purpose of Review

We reviewed data on amphotericin B (AmB) tolerance among Cryptococcus neoformans/C. gattii species complex clinical isolates and present our results of large recent study on this issue.

Recent Findings

The standard method to detect antifungal susceptibility is based on MIC (minimal inhibitory concentration) determination; however, there is no interpretative clinical breakpoints defined for antifungal agents against Cryptococcus species, and to date, there is no correlation of MIC and clinical response. The time-kill curves (TKC) methodology seems to provide some correlation with outcome and it could identify distinct profiles of AmB-fungicidal activity.

Summary

Our group analyzed 83 human isolates from cryptococcosis cases. The isolates were tested by TKC and showed up 8.3% of tolerance to AmB. Importantly, the AmB-MIC was low for all isolates, including tolerant ones. Our findings are similar to other authors, due the ability of TKC to identify distinct AmB-fungicidal activity and detecting low susceptible isolates.

     

Equine Pulmonary Cryptococcosis: A Comparative Literature Review and Evaluation of Fluconazole Monotherapy

Cryptococcus is the most common fungal respiratory pathogen in Australian horses, manifesting primarily as pulmonary granulomas. Disease severity at presentation is dependent on the athletic use of the horse. The diagnosis and estimation of disease severity are centred around clinical findings, cytological evaluation of respiratory tract secretions, diagnostic imaging, and antigen titre testing. Both the lateral flow assay and the latex cryptococcal antigen titre are used, and important similarities and differences between species are discussed. Cryptococcus gattii occurs with greater frequency than Cryptococcus neoformans in equine pulmonic cryptococcosis and can be successfully treated with enteral fluconazole monotherapy, with disease severity determining treatment length.     

Case Series: Report of the First Two Human Indigenous Cases of <Emphasis Type="Italic">Cryptococcus gattii</Emphasis> Infection in Eastern Canada

We report the two first cases of human C. gattii meningoencephalitis acquired on the Canadian east coast, from the province of Quebec. Unlike C. neoformans, C. gattii is not known to have an established ecological niche on the North American east coast. C. gattii has recently been responsible for major outbreaks in British Columbia, Canada, and in the American pacific northwest. However, no human cases acquired in other Canadian provinces have been reported to our knowledge. The source of acquisition remains unclear for both patients but since neither had traveled outside of the province of Quebec, we discuss the possibilities of environmental and animal-associated acquisition, as well as the possible established endemicity in new areas. These cases add to the growing reported human and animal cases in areas previously not thought to be endemic for C. gattii.     

<Emphasis Type="Italic">Cryptococcus gattii</Emphasis> Infection Presenting as an Aggressive Lung Mass

Cryptococcus gattii is an endemic fungus predominantly isolated in the tropical and subtropical regions, causing predominantly pulmonary disease with a predilection for the central nervous system. Herein, we report a case of rapidly progressing C. gattii pneumonia in an immune-deficient but virologically suppressed host with underlying human immunodeficiency viral (HIV) infection, exhibiting various fungal morphologies from bronchoalveolar lavage (BAL) cytological specimens. A 51-year-old Chinese male with known HIV disease was admitted to the Singapore General Hospital for evaluation of functional decline, febrile episodes, and a left hilar mass on chest radiograph. Computed tomography (CT) showed consolidation in the apical segment of the left lower lobe. He underwent bronchoscopy and BAL. Positron emission tomography-computed tomography done 10 days after the initial CT showed approximate doubling of the pulmonary lesion. Cytological examination of the fluid revealed yeasts of varying sizes. Subsequent fungal culture from BAL fluid grew C. gattii 10 days later.     

Antifungal Susceptibility Testing of <Emphasis Type="Italic">Candida</Emphasis> and <Emphasis Type="Italic">Cryptococcus</Emphasis> Species and Mechanisms of Resistance: Implications for Clinical Laboratories

Purpose of Review

Resistance to antifungal drugs amongst Candida species is a growing concern, and azole resistance may be emerging in Cryptococcus species. This review provides a contemporary perspective, relevant to the clinical mycology laboratory, of antifungal susceptibility testing of these fungi, focussing on the challenges of phenotypic and genotypic methodologies to detect drug resistance.

Recent Findings

Standardised CLSI and EUCAST broth microdilution (BMD) susceptibility testing methods are the benchmark to determine clinical breakpoints (CBPs) and/or epidemiological cut-off values (ECVs) MICs for Candida and Cryptococcus spp. Commercial methods may be used but caution is required when employing BMD CBPs/ECVs to interpret results. Species-specific CBPs/ECVs for Candida spp. generally correlate well with predicting likelihood of therapeutic failure or of presence of a drug resistance mechanism with the exception of the echinocandins where the presence of specific FKS gene mutations and not the MIC correlates most accurately with clinical outcome. The relationship of presence of one or more mechanisms of azole resistance and drug MICs is uncertain. Next generation sequencing technology is offering insights into the relationships between susceptibility results obtained by phenotypic and genotypic methods. For Cryptococcus spp., CBPs are not established but species- and genetic type-specific EVCs are useful for guiding therapy where clinically indicated. Isolates of genotype VGII appear to exhibit the highest MICs.

Summary

Antifungal susceptibility testing of yeasts is important to detect drug resistance. For Candida spp., MICs have clinical utility for the azoles but detecting echinocandin resistance by genotypic methods is preferred. For Cryptococcus spp., ECVs are useful in guiding therapy.

     

Identification and characterization of yeasts isolated from sedimentary rocks of Union Glacier at the Antarctica

The study of the yeasts that inhabit cold environments, such as Antarctica, is an active field of investigation oriented toward understanding their ecological roles in these ecosystems. In a great part, the interest in cold-adapted yeasts is due to several industrial and biotechnological applications that have been described for them. The aim of this work was to isolate and identify yeasts from sedimentary rock samples collected at the Union Glacier, Antarctica. Furthermore, the yeasts were physiologically characterized, including the production of metabolites of biotechnological interest. The yeasts isolated that were identified at the molecular level belonged to genera Collophora (1 isolate), Cryptococcus (2 isolates), Sporidiobolus (4 isolates), Sporobolomyces (1 isolate) and Torrubiella (2 isolates). The majority of yeasts were basidiomycetous and psychrotolerant. By cross-test assays for anti-yeast activity, it was determined that Collophora sp., Sporidiobolus salmonicolor, and Sporobolomyces roseus secreted a protein factor that kills Sporidiobolus metaroseus. The colored yeasts Sp. salmonicolor, Sp. metaroseus and Collophora sp. produced several carotenoid pigments that were identified as 2,3 dihydroxy-γ-carotene, -carotene, 4-ketotorulene, torulene β-cryptoxanthin and spirilloxanthin. Concerning analysis of mycosporines, these metabolites were only found in the yeasts Torrubiella sp. and Cryptococcus sp. T11-10-1. Furthermore, the yeasts were evaluated for the production of extracellular hydrolytic activities. Of the twelve activities analyzed, alkaline phosphatase, invertase, gelatinase, cellulase, amylase, and protease enzyme activities were detected. The yeasts Cryptococcus sp. T11-10-1 and Sporidiobolus metaroseus showed the highest number of different enzyme activities.     

Isolation of <Emphasis Type="Italic">Cryptococcus gattii</Emphasis> from a <Emphasis Type="Italic">Castanopsis argyrophylla</Emphasis> tree hollow (Mai-Kaw), Chiang Mai,Thailand

The pathogenic yeast Cryptococcus gattii was isolated from a tree hollow of a Castanopsis argyrophylla King ex Hook.f. (Fagaceae) in Chiang Mai, Thailand. Molecular characterization with amplified fragment length polymorphism analysis and multi-locus sequence typing showed that this isolate belonged to genotype AFLP4/VGI representing C. gattii sensu stricto. Subsequent comparison of the environmental isolate with those from clinical samples from Thailand showed that they grouped closely together in a single cluster.     

Recent Taxonomic Developments with <Emphasis Type="Italic">Candida</Emphasis> and Other Opportunistic Yeasts

Increases in susceptible patient populations and advances in identification methods have resulted in the continued recognition of novel yeasts as agents of human infection. Most of these agents are members of the well-recognized genera Candida, Cryptococcus, Trichosporon, and Rhodotorula. Some of these agents are “cryptic species,” members of species complexes, and may not be detectable using classical carbohydrate assimilation-based methods of yeast identification. Such species require DNA- or MALDI-based methods for correct identification, although sporadic isolates may not routinely require delineation to the individual species level. The coming end of the fungal taxonomy rules requiring separate names for sexual and asexual forms of the same fungus will hopefully allow greater clarity, as names for medically important yeast can now be based on the needs of the medical mycology community and the common goal of better communication between laboratory and clinician.     

Molecular genetic polymorphism of soil yeasts of the genus <Emphasis Type="Italic">Williopsis</Emphasis> from Taiwan Island

Comparative molecular genetic study of Williopsis yeasts isolated in different world regions reveals some peculiarities of species content in Taiwan. Some Williopsis yeasts may represent novel species. In Taiwan, four of the five known Williopsis species are documented: W. saturnus, W. suaveolens, W. mrakii, and W. subsufficiens. The W. saturnus yeasts predominate in Taiwanese soils, while W. suaveolens is more frequently isolated in Europe.     

Yeasts in <Emphasis Type="Italic">Hevea brasiliensis</Emphasis> latex

Yeast abundance and species diversity in the latex of rubber tree Hevea brasiliensis (Willd. ex Juss.) Müll. Arg., on its green leaves, and in soil below the plant were studied. The yeasts present in the fresh latex in numbers of up to 5.5 log(CFU/g) were almost exclusively represented by the species Candida heveicola. This species was previously isolated from Hevea latex in China. In the course of natural modification of the latex (turned from liquid to solid form), yeast diversity increased, while yeast abundance decreased. The yeasts in thickened and solidified latex were represented by typical epiphytic and ubiquitous species: Kodamea ohmeri, Debaryomyces hansenii, Rhodotorula mucilaginosa, and synanthropic species Candida parapsilosis and Cutaneotrichosporon arboriformis. The role of yeasts in latex modification at the initial stages of succession and their probable role in development of antifungal activity in the latex are discussed.     

Fungicidal activity of cellobiose lipids from culture broth of yeast <Emphasis Type="Italic">Cryptococcus humicola</Emphasis> and <Emphasis Type="Italic">Pseudozyma fusiformata</Emphasis>

Cellobiose lipids of yeast fungi Cryptococcus huminola and Pseudozyma fusiformata have similar fungicidal activities against different yeast, including pathogenic Cryptococcus and Candida species. Basidiomycetic yeast reveals maximum sensitivity to these preparations; e.g., cells of cryptococcus Filobasidiella neoformans almost completely die after 30-min incubation in a glycolipid solution at a concentration of 0.02 mg/ml. The same effect toward ascomycetous yeast, including pathogenic Candida species, is achieved only at five to eight times higher concentrations of glycolipids. The cellobiose lipid from P. fusiformata, which, unlike glycolipid from Cr. humicola, has hydroxycaproic acid residue as O-subtituent of cellobiose and additional 15-hydroxy group in aglycone, inhibits the growth of the studied mycelial fungi more efficiently than the cellobiose lipid from Cr. humicola.     

The yeast <Emphasis Type="Italic">Candida railenensis</Emphasis> in the fruits of English oak (<Emphasis Type="Italic">Quercus robur</Emphasis> L.)

The cotyledons of whole intact acorns were shown to contain yeasts; their number increased sharply before acorn germination. The yeasts in the cotyledons are mainly represented by one species, Candida railenensis, with the number in the germinating cotyledons reaching 10⁷ CFU/g. After germination or exocarp destruction, the cotyledons were colonized by the usual epiphytic and litter yeasts Cryptococcus albidus, Rhodotorula glutinis, and Cystofilobasidium capitatum.     

<Emphasis Type="Italic">Malassezia</Emphasis> spp. in Acoustic Meatus of Bats (<Emphasis Type="Italic">Molossus molossus</Emphasis>) of the Amazon Region,Brazil

The yeasts of the Malassezia genus are opportunistic microorganisms and can cause human and animal infections. They are commonly isolated from the skin and auricular canal of mammalians, mainly dogs and cats. The present study was aimed to isolate Malassezia spp. from the acoustic meatus of bats (Molossus molossus) in the Montenegro region, “Rondônia”, Brazil. From a total of 30 bats studied Malassezia spp. were isolated in 24 (80%) animals, the breakdown by species being as follows (one Malassezia sp. per bat, N = 24): 15 (62.5%) M. pachydermatis, 5 (20.8%) M. furfur, 3 (12.5%) M. globosa and 1 (4.2%) M. sympodialis. This study establishes a new host and anatomic place for Malassezia spp., as it presents the first report ever of the isolation of this genus of yeasts in the acoustic meatus of bats.     

The Mycotoxin Zearalenone Hinders <Emphasis Type="Italic">Candida albicans</Emphasis> Biofilm Formation and Hyphal Morphogenesis

Yeast–mold mycobiota inhabit several natural ecosystems, in which symbiotic relationships drive strategic pathoadaptation. Mycotoxins are metabolites produced by diverse mycotoxigenic fungi as a defense against yeasts, though at times yeasts secrete enzymes that degrade, detoxify, or bio-transform mycotoxins. The present study is focused on the in vitro inhibitory effects of zearalenone (ZEN), a F2 mycotoxin produced by several Fusarium and Gibberella species, on different microbial strains. ZEN exhibited no effect on the planktonic growth or biofilms of several Gram positive and negative bacteria at the tested concentrations. Remarkably, Candida albicans biofilm formation and hyphal morphogenesis were significantly inhibited when treated with 100 µg/mL of ZEN. Likewise, ZEN proficiently disrupted pre-formed C. albicans biofilms without disturbing planktonic cells. Furthermore, these inhibitions were confirmed by crystal violet staining and XTT reduction assays and by confocal and scanning electron microscopy. In an in vivo model, ZEN significantly suppressed C. albicans infection in the nematode Caenorhabditis elegans. The study reports the in vitro antibiofilm efficacy of ZEN against C. albicans strains, and suggests mycotoxigenic fungi participate in asymmetric competitive interactions, such as, amensalism or antibiosis, rather than commensal interactions with C. albicans, whereby mycotoxins secreted by fungi destroy C. albicans biofilms.     

Up-Regulation of Antimicrobial Peptides Gallerimycin and Galiomicin in <Emphasis Type="Italic">Galleria mellonella</Emphasis> Infected with <Emphasis Type="Italic">Candida</Emphasis> Yeasts Displaying Different Virulence Traits

Galleria mellonella has been described as a cheap and an easy-to-reproduce model for the study of fungal infections. We hypothesized that yeasts with higher virulence potential decrease survival and significantly trigger an immune response in G. mellonella through the regulation of innate immunity-related genes encoding antimicrobial peptides (AMPs) such as gallerimycin and galiomicin. Candida albicans SC5314 and Candida dubliniensis CBS 7987, selected because of their different virulence potential, were used for a killing assay followed by the determination of gene expression using qPCR. In vivo results confirmed a significantly (p?=?0.0321) lower pathogenicity for C. dubliniensis than for C. albicans. Accordingly, the induction of C. dubliniensis AMPs was lower at all the selected time points post-infection (1 h, 24 h, 48 h). Moreover, we observed an extremely high regulation of the galiomicin gene compared to the gallerimycin one, suggesting a different role of the tested AMPs in protecting G. mellonella from candidiasis.     

Identification of functional <Emphasis Type="Italic">flavonol synthase</Emphasis> genes from fragrant wild cyclamen (<Emphasis Type="Italic">Cyclamen purpurascens</Emphasis>)

Cyclamen purpurascens is considered suitable for horticultural breeding of cyclamens because it has an attractive fragrance that is not found in other wild species. To improve the commercial value of cyclamen flowers, this fragrance has been introduced into ornamental cultivars. However, variation in flower color is somewhat limited in these cultivars, and therefore understanding the genetic networks of flower coloration in C. purpurascens is required. We previously isolated DNA fragments of anthocyanin biosynthetic genes from C. purpurascens, broadening our understanding of the biosynthetic pathway of flavonols, which are co-pigments in flower coloration. In this study, we isolated complete open reading frames of flavonol synthase genes from C. purpurascens (CpurFLS1 and CpurFLS2) and analyzed the in planta functions of the genes by molecular complementation assay using the fls mutant of Arabidopsis thaliana. Expression patterns in several organs of C. purpurascens were also determined. The results strongly suggest that the CpurFLS genes participate in flavonol synthesis. We discuss the involvement of these two FLSs in flower coloration in C. purpurascens.     

Novel <Emphasis Type="Italic">Collophorina</Emphasis> and <Emphasis Type="Italic">Coniochaeta</Emphasis> species from <Emphasis Type="Italic">Euphorbia polycaulis</Emphasis>, an endemic plant in Iran

During a study on the biodiversity of yeasts and yeast-like ascomycetes from wild plants in Iran, four strains of yeast-like filamentous fungi were isolated from a healthy plant of Euphorbia polycaulis in the Qom Province, Iran (IR. of). All four strains formed small hyaline one-celled conidia from integrated conidiogenous cells directly on hyphae and sometimes on discrete phialides, as well as by microcyclic conidiation. Two strains additionally produced conidia in conidiomata that open by rupture. The internal transcribed spacer (ITS) sequences suggested the placement of these strains in the genera Collophorina (Leotiomycetes) and Coniochaeta (Sordariomycetes), respectively. Blast search results on NCBI GenBank and phylogenetic analyses of ITS, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the translation elongation factor 1α (EF-1α) sequences, and the nuclear large subunit ribosomal gene (LSU), partial actin (ACT), and β-tubulin (TUB) sequences, respectively, revealed the isolates to belong to three new species, that are described here as Collophorina euphorbiae, Coniochaeta iranica, and C. euphorbiae. All three species are characterised by morphological, physiological, and molecular data.