Here we review how evolving species concepts have been applied to understand yeast diversity. Initially, a phenotypic species concept was utilized taking into consideration morphological aspects of colonies and cells, and growth profiles. Later the biological species concept was added, which applied data from mating experiments. Biophysical measurements of DNA similarity between isolates were an early measure that became more broadly applied with the advent of sequencing technology, leading to a sequence-based species concept using comparisons of parts of the ribosomal DNA. At present phylogenetic species concepts that employ sequence data of rDNA and other genes are universally applied in fungal taxonomy, including yeasts, because various studies revealed a relatively good correlation between the biological species concept and sequence divergence. The application of genome information is becoming increasingly common, and we strongly recommend the use of complete, rather than draft genomes to improve our understanding of species and their genome and genetic dynamics. Complete genomes allow in-depth comparisons on the evolvability of genomes and, consequently, of the species to which they belong. Hybridization seems a relatively common phenomenon and has been observed in all major fungal lineages that contain yeasts. Note that hybrids may greatly differ in their post-hybridization development. Future in-depth studies, initially using some model species or complexes may shift the traditional species concept as isolated clusters of genetically compatible isolates to a cohesive speciation network in which such clusters are interconnected by genetic processes, such as hybridization.

For decades etiologic agents of cryptococcosis were named C. neoformans var. grubii, C. neoformans var. neoformans and C. gattii. Nowadays, based on the combination of various speciation criteria, including ecological, geographical, genetic, phenetic and phylogenetic characteristics, C. neoformans var. grubii and C. neoformans var. neoformans are recognized as C. neoformans and C. deneoformans, and C. gattii is divided into 5 species, i.e. C. gattii, C. bacillisporus, C. deuterogattii, C. tettragattii and C. decagattii. Most cases of cryptococcal meningitis are caused by C. neoformans, with most patients (>90%) occurring in Sub-Saharan Africa and the Asia-Pacific region. Using MLST analysis the global genetic diversity of C. neoformans showed that isolates from Africa had a high genetic diversity, while isolates from Asia, particular East-Southeast-South Asia, had a more limited genetic diversity. Population structure analysis of C. neoformans in Asia showed that each genotype (sequence type) had an unique distribution at each locale. In addition, microsatellite analysis showed that each genotype or microsatellite complex had a limited geographic occurrence. Ancestral reconstruction using Bayesian Binary Markov Chain Monte Carlo analysis confirmed that Africa is a possible place of origin of C. neoformans before spreading globally. In contrast, S-DIVA supported the “Continental drift” hypothesis that C. neoformans originated on Pangea before moving to the modern continents. In our study, 5-fluorocytosine-, fluconazole- and double (5-fluorocytosine-fluconazole and fluconazole-voriconazole)-resistant isolates of C. neoformans were found. It is common that 5-fluorocytosine and azole resistant isolates occur among clinical isolates. However, the presence of isolates that were intrinsically resistant to 5-fluorocytosine in Indonesia and Thailand is a concern when this antifungal drug will become available for therapy of cryptococcosis in these countries. Cryptococcus gattii was isolated for the first time from soil and debris samples collected from a hollow of a Castanopsis argyrophylla tree in Chiang Mai, Thailand. This isolate was typed as AFLP4/VGI and, hence, represented C. gattii sensu stricto. The isolation of members of the C. neoformans/C. gattii species complex is performed by culturing on selective and differential media, such as caffeic acid agar or Niger seed agar. These culture media induce melanization of yeasts of the C. neoformans/C. gattii species complex, resulting in brown colonies that differ from most other yeasts. Based on this melanization process of these yeasts, a novel Banana blossom agar was formulated for isolation of members of the C. neoformans/C. gattii species complex. This medium may find use in resource limited settings as banana trees occur widely in areas where many cryptococcosis patients live.

Objective: It has been known that VNI molecular type of Cryptococcus neoformans/C. gattii is strongly associated with HIV patients. However, this paradigm has recently been challenged because of the high prevalence of VNI molecular type among non-HIV patients with cryptococcosis in East Asia. The purpose of this study was to answer the question: “Among cryptococcosis in Asia, is there an association between the genotype and the patient’s HIV status?” Methods: Using a systematic review and meta-analysis study design, we included all relevant published data, which were any type of study designs, mainly studied clinical Cryptococcus neoformans/C. gattii strains isolated in Asia and had available molecular typing data. The primary study variables were Cryptococcus neoformans molecular type (VNI/non-VNI or ST5/non-ST5) and the HIV status of the patients at the time of diagnosis. We used a randomeffects meta-analysis model to estimate the prevalence of HIV infection. Results: Sixteen retrospective descriptive studies during 2005 – 2018 (1,584 isolates) were included. Most of the cryptococcosis cases in East Asian countries were in non-HIV patients (72.4-81.8%), which differed from non-East Asian countries (2.6-28.3% associated with non-HIV patients). In East Asia, the HIV prevalence among VNI and ST5 infected patients ranged from 7.5% - 46.7% with the pooled prevalence of 19.8% (95% CI, 12.2% - 30.4%) and 5.3% - 52.4% with the pooled prevalence of 19.9% (95% CI, 6.9% - 45.3%), respectively. In non-East Asia, the HIV prevalence among VNI and ST5 infected patients ranged from 48.3% - 98.8% with the pooled prevalence of 81.9% (95% CI, 73.3% - 88.2%) and 52.3% - 88.0% with the pooled prevalence of 74.9% (95% CI, 40.7% - 92.8%), respectively. Statistical heterogeneity was high in both analyses with the I2 of 79-89% in all analyses. Conclusion: Our results confirmed the low prevalence of HIV prevalence among VNI and ST5 strains in East Asian countries. The emergence of high virulence genotype causing disease in non-HIV patient is highly unlikely, because the VNI and ST5 were associated with HIV patients in other Asian countries. It can be hypothesized that the low HIV prevalence among VNI and ST5 strains in East Asian is due to the high susceptibility to cryptococcosis of people living in this region. This requires further investigation.

SummaryIsolation of representatives of the Cryptococcus neoformans/Cryptococcus gattii species complex can be made using dopamine containing media, such as Niger seed agar and l‐DOPA agar. Here, we describe an alternative medium that uses banana flowers. Banana is a dopamine containing fruit and is widely available in tropical and subtropical countries that have high numbers of cryptococcosis patients. This banana blossom‐based agar is useful for the enrichment of isolates of the C. neoformans/C. gattii species complex from environmental and clinical materials. The banana blossom agar (BABA) with and without creatinine can differentiate between the melanin forming isolates of the C. neoformans/C. gattii species complex from other yeasts that do not form melanin.

Cryptococcosis is a major fungal disease caused by members of the Cryptococcus gattii and Cryptococcus neoformans species complexes. After more than 15 years of molecular genetic and phenotypic studies and much debate, a proposal for a taxonomic revision was made. The two varieties within C. neoformans were raised to species level, and the same was done for five genotypes within C. gattii. In a recent perspective (K. J. Kwon-Chung et al., mSphere 2:e00357-16, 2017, https://doi.org/10.1128/mSphere.00357-16), it was argued that this taxonomic proposal was premature and without consensus in the community. Although the authors of the perspective recognized the existence of genetic diversity, they preferred the use of the informal nomenclature "C. neoformans species complex" and "C. gattii species complex." Here we highlight the advantage of recognizing these seven species, as ignoring these species will impede deciphering further biologically and clinically relevant differences between them, which may in turn delay future clinical advances.

Knowledge of the environmental prevalence of members of the Ctyptococcus neoformans/Cryptococcus gattii species complex is important, since cryptococcal infection is acquired from the environment. We determined whether trees located in two South African recreational areas harboured pathogenic cryptococci and compared the isolates to clinical isolates obtained from Western Cape hospitals with molecular typing techniques. The majority of isolates originating from trees in a public park in Cape Town (PPCT) were C. gattii sensu stricto, followed by C neoformans sensu stricto genotype AFLP1/VNI. The PPCT trees might be a source of infection, since all genotype AFLP1/VNI isolates from these trees and one clinical isolate belonged to the same sequence type (ST), i.e. ST23. Recombination and basidiospore production might be occurring in PPCT trees that contained C gattii s.s. isolates belonging to both mating types. The presence of C gattii s.s. in PPCT trees might therefore pose a risk to human health. (C) 2017 Elsevier Ltd and British Mycological Society. All rights reserved.

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.

It is well-known that Aspergillus terreus harbors higher MIC for amphotericin B and in the past couple of years the body of evidence showing azole-resistant Aspergillus spp. is increasing.Clinicians are treating more high-risk populations, and prophylactic therapy in clinical practice has a growing impact influencing the epidemiological data.In Candida disease there seems to be shift from Candida albicans to non-albicans Candida species.In ICUs, there is evidence that patients with non-albicans infection have a nearly seven-fold mortality compared with those with C. albicans.The global epidemiology is variable, with C. glabrata most common in North America and northern Europe (16%-24%), C. tropicalis most frequent in southeast Asia and South America (20%-35%), and C. parapsilosis seen most in Spain, South America, Turkey, and Australia (20%-32%).In invasive Aspergillus disease voriconazole is highly recommended by many scientific societies including ESCMID.The new azole isavuconazole received high recommendations as well, followed by a weaker recommendation for liposomal amphotericn B due to its higher incidence of nephrotoxicity.Since diagnostic procedures remain challenging, antifungal prophylaxis appears a worthy alternative.From an infectious diseases standpoint, prophylaxis is always second best to targeted treatment, however posaconazole prophylaxis has been shown to provide a better chance of survival for patients in AML induction/remission and allogeneic HSCT with GVHD.All three available echinocandins are strongly recommended for targeted initial treatment of candidaemia, while liposomal amphotericin B and voriconazole are supported with moderate, and fluconazole with marginal strength by ESCMID.Further studies demonstrated that delayed start of caspofungin treatment delivers a worse outcome.Acquired resistance appears rare, however there is ongoing discussion whether echinocandins are appropriate for treating Candida parapsilosis.

Phylogenetic analysis of 11 genetic loci and results from many genotyping studies revealed significant genetic diversity with the pathogenic Cryptococcus gattii/Cryptococcus neoformans species complex. Genealogical concordance, coalescence-based, and species tree approaches supported the presence of distinct and concordant lineages within the complex. Consequently, we propose to recognize the current C. neoformans var. grubii and C. neoformans var. neoformans as separate species, and five species within C. gattii. The type strain of C. neoformans CBS132 represents a serotype AD hybrid and is replaced. The newly delimited species differ in aspects of pathogenicity, prevalence for patient groups, as well as biochemical and physiological aspects, such as susceptibility to antifungals. MALDI-TOF mass spectrometry readily distinguishes the newly recognized species.

ABSTRACT An interlaboratory study using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) to determine the identification of clinically important yeasts ( n = 35) was performed at 11 clinical centers, one company, and one reference center using the Bruker Daltonics MALDI Biotyper system. The optimal cutoff for the MALDI-TOF MS score was investigated using receiver operating characteristic (ROC) curve analyses. The percentages of correct identifications were compared for different sample preparation methods and different databases. Logistic regression analysis was performed to analyze the association between the number of spectra in the database and the percentage of strains that were correctly identified. A total of 5,460 MALDI-TOF MS results were obtained. Using all results, the area under the ROC curve was 0.95 (95% confidence interval [CI], 0.94 to 0.96). With a sensitivity of 0.84 and a specificity of 0.97, a cutoff value of 1.7 was considered optimal. The overall percentage of correct identifications (formic acid-ethanol extraction method, score ≥ 1.7) was 61.5% when the commercial Bruker Daltonics database (BDAL) was used, and it increased to 86.8% by using an extended BDAL supplemented with a Centraalbureau voor Schimmelcultures (CBS)-KNAW Fungal Biodiversity Centre in-house database (BDAL+CBS in-house). A greater number of main spectra (MSP) in the database was associated with a higher percentage of correct identifications (odds ratio [OR], 1.10; 95% CI, 1.05 to 1.15; P < 0.01). The results from the direct transfer method ranged from 0% to 82.9% correct identifications, with the results of the top four centers ranging from 71.4% to 82.9% correct identifications. This study supports the use of a cutoff value of 1.7 for the identification of yeasts using MALDI-TOF MS. The inclusion of enough isolates of the same species in the database can enhance the proportion of correctly identified strains. Further optimization of the preparation methods, especially of the direct transfer method, may contribute to improved diagnosis of yeast-related infections.

BACKGROUND:Infections caused by Malassezia yeasts are most likely underdiagnosed, because fatty acid supplementation is needed for growth. Rapid identification of Malassezia species is essential for appropriate treatment of Malassezia-related skin infections, fungaemia and nosocomial outbreaks in neonates, children and adults and can be life-saving for those patients. Ma-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been reported to be a rapid and reliable diagnostic tool to identify clinically important yeasts, but so far no data have been reported on identification of Malassezia isolates with this technique.OBJECTIVES:To create an extensive database of main mass spectra (MSPs) that will allow quick identification of Malassezia species by MALDI-TOF MS.METHODS:An in-house library of 113 MSPs was created from 48 reference strains from the CBS-KNAW yeast collection. The in-house library was challenged with two test sets of Malassezia strains, namely 165 reference strains from the CBS collection and 338 isolates collected in Greece, Italy, Sweden and Thailand.RESULTS:MALDI-TOF MS allowed correct identification of all 14 Malassezia spp. MALDI-TOF MS results were concordant with those of sequence analyses of the internal transcribed spacers (ITS1/ITS2) and the D1/D2 domains of the large subunit of the ribosomal DNA.CONCLUSIONS:Implementation of the MALDI-TOF MS system as a routine identification tool will contribute to correct identification of Malassezia yeasts with minimal effort and in a short turnaround time, which is especially important for the rapid identification of Malassezia in skin diseases and nosocomial outbreaks.

We had determined that 190 Cryptococcus neoformans isolates (104 clinical and 86 environmental isolates from northern Thailand) were all susceptible to amphotericin B, fluconazole, itraconazole and ketoconazole. Of 14 selected isolates, one clinical isolate (CN4969) exhibited heterogeneity in fluconazole resistance, which produced two subpopulations of the fluconazole-resistant (CN4969HR) and -susceptible (CN4969S) subpopulations. To clarify the molecular mechanisms of fluconazole resistance, the expression of drug resistance genes was determined by using reverse transcription-polymerase chain reaction (RT-PCR). Both the ERG11 and MDR1 transcript levels showed at least a 2-fold increase in CN4969HR isolate compared to the fluconazole-susceptible isolates (H99, CN4901 and CN4969S), whereas the expression level of theAFR1 gene in CN4969HR was equivalent to that of isolates H99, CN4901 or CN4969S. Collectively, this study demonstrates the existing of the fluconazole heteroresistant population among clinical isolates of C. neoformans in northern part of Thailand and molecular mechanism of fluconazole-susceptible and resistant isolates. Key words: Heteroresistance, fluconazole, C. neoformans, Thailand.

ABSTRACT Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) was used for an extensive identification study of arthroconidial yeasts, using 85 reference strains from the CBS-KNAW yeast collection and 134 clinical isolates collected from medical centers in Qatar, Greece, and Romania. The test set included 72 strains of ascomycetous yeasts (Galactomyces, Geotrichum, Saprochaete, and Magnusiomyces spp.) and 147 strains of basidiomycetous yeasts (Trichosporon and Guehomyces spp.). With minimal preparation time, MALDI-TOF MS proved to be an excellent diagnostic tool that provided reliable identification of most (98%) of the tested strains to the species level, with good discriminatory power. The majority of strains were correctly identified at the species level with good scores (>2.0) and seven of the tested strains with log score values between 1.7 and 2.0. The MALDI-TOF MS results obtained were consistent with validated internal transcribed spacer (ITS) and/or large subunit (LSU) ribosomal DNA sequencing results. Expanding the mass spectrum database by increasing the number of reference strains for closely related species, including those of nonclinical origin, should enhance the usefulness of MALDI-TOF MS-based diagnostic analysis of these arthroconidial fungi in medical and other laboratories.

Cryptococcosis is an important fungal disease in Asia with an estimated 140,000 new infections annually the majority of which occurs in patients suffering from HIV/AIDS. Cryptococcus neoformans variety grubii (serotype A) is the major causative agent of this disease. In the present study, multilocus sequence typing (MLST) using the ISHAM MLST consensus scheme for the C. neoformans/C. gattii species complex was used to analyse nucleotide polymorphisms among 476 isolates of this pathogen obtained from 8 Asian countries. Population genetic analysis showed that the Asian C. neoformans var. grubii population shows limited genetic diversity and demonstrates a largely clonal mode of reproduction when compared with the global MLST dataset. HIV-status, sequence types and geography were found to be confounded. However, a correlation between sequence types and isolates from HIV-negative patients was observed among the Asian isolates. Observations of high gene flow between the Middle Eastern and the Southeastern Asian populations suggest that immigrant workers in the Middle East were originally infected in Southeastern Asia.