FATAL cryptococcal meningitis in a child with hyper-immunoglobulin M syndrome, with an emphasis on the agent.

Following a fatal case of Cryptococcus neoformans meningitis in a child with X-linked hyper-immunoglobulin M syndrome (XHIGM), we evaluated the fungal isolate in an experimental infection in a mouse model with respect to microbiology, epidemiology, virulence and response to therapy. The minimum inhibitory concentrations for antifungals in the susceptibility test were 0.5mg/L for amphotericin B, 4.0mg/L for fluconazole and 0.12mg/L for voriconazole. Evaluation of pathogenicity by means of an experimental infection in BALB/c mice showed that fungus isolated from the blood and cerebrospinal fluid of the child was able to disseminate, reaching the spleen, lungs and brain, where it caused significant macroscopic alterations in the size and texture of each organ. Treatment of infected mice with amphotericin B reduced the fungal load in the spleen and lungs, but not in the brain.

Identification of chromoblastomycosis agents by PCR based reverse line blot (PCR-RLB) hybridization assay.

Chromoblastomycosis is one of the most prevalent implantation fungal infections caused by melanized fungi, affecting individuals with certain risk factors with high morbidity due to its recalcitrant nature. It is difficult to identify the etiological agents and thus a suitable reproductive molecular identification method applicable in developing countries has been investigated. We report the identification of four different fungal causative agents of chromoblastomycosis by reverse line blotting hybridization (RLB) based on biotin-labeled PCR products and amine labeled probes to hybridize. Sixty five reference strains, including type strains, i.e. Fonsecaea pedrosoi, F. monophora, F. nubica, and Phialophora verrucosa, obtained from the CBS-KNAW were included in this study. Internal transcribed spacer 1 (ITS1) regions of relevant species were aligned and adjusted using BIONUMERICS v. 4.61 in order to design four specific probes to identify informative nucleotide polymorphisms. The final identification of these species by RLB assay was concordant with ITS sequencing and showed 100% specificity with no cross hybridization, able to identify all tested strains. The time and cost were less compare to other routine identification methods such as sequencing. This assay allows sensitive and specific simultaneous detection and identification of a different fungal species.

Correction to: Rapid Identification of Seven Waterborne Exophiala Species by RCA DNA Padlock Probes.

The Editorial Office of Mycopathologia reports that several paragraphs of Najafzadeh et al. were transcribed with only minor edits from previously published material by Najafzadeh M.J.

Rapid Identification of Seven Waterborne Exophiala Species by RCA DNA Padlock Probes.

The black yeast genus Exophiala includes numerous potential opportunistic species that potentially cause systematic and disseminated infections in immunocompetent individuals. Species causing systemic disease have ability to grow at 37-40 °C, while others consistently lack thermotolerance and are involved in diseases of cold-blooded, waterborne vertebrates and occasionally invertebrates. We explain a fast and sensitive assay for recognition and identification of waterborne Exophiala species without sequencing. The ITS rDNA region of seven Exophiala species (E. equina, E. salmonis, E. opportunistica, E. pisciphila, E. aquamarina, E. angulospora and E. castellanii) along with the close relative Veronaea botryosa was sequenced and aligned for the design of specific padlock probes for the detection of characteristic single-nucleotide polymorphisms. The assay demonstrated to successfully amplify DNA of target fungi, allowing detection at the species level. Amplification products were visualized on 1% agarose gels to confirm specificity of probe-template binding. Amounts of reagents were reduced to prevent the generation of false positive results. The simplicity, tenderness, robustness and low expenses provide padlock probe assay (RCA) a definite place as a very practical method among isothermal approaches for DNA diagnostics.

Exploring the genomic diversity of black yeasts and relatives (Chaetothyriales, Ascomycota).

The order Chaetothyriales (Pezizomycotina, Ascomycetes) harbours obligatorily melanised fungi and includes numerous etiologic agents of chromoblastomycosis, phaeohyphomycosis and other diseases of vertebrate hosts. Diseases range from mild cutaneous to fatal cerebral or disseminated infections and affect humans and cold-blooded animals globally. In addition, Chaetothyriales comprise species with aquatic, rock-inhabiting, ant-associated, and mycoparasitic life-styles, as well as species that tolerate toxic compounds, suggesting a high degree of versatile extremotolerance. To understand their biology and divergent niche occupation, we sequenced and annotated a set of 23 genomes of main the human opportunists within the Chaetothyriales as well as related environmental species. Our analyses included fungi with diverse life-styles, namely opportunistic pathogens and closely related saprobes, to identify genomic adaptations related to pathogenesis. Furthermore, ecological preferences of Chaetothyriales were analysed, in conjuncture with the order-level phylogeny based on conserved ribosomal genes. General characteristics, phylogenomic relationships, transposable elements, sex-related genes, protein family evolution, genes related to protein degradation (MEROPS), carbohydrate-active enzymes (CAZymes), melanin synthesis and secondary metabolism were investigated and compared between species. Genome assemblies varied from 25.81 Mb (Capronia coronata) to 43.03 Mb (Cladophialophora immunda). The bantiana-clade contained the highest number of predicted genes (12 817 on average) as well as larger genomes. We found a low content of mobile elements, with DNA transposons from Tc1/Mariner superfamily being the most abundant across analysed species. Additionally, we identified a reduction of carbohydrate degrading enzymes, specifically many of the Glycosyl Hydrolase (GH) class, while most of the Pectin Lyase (PL) genes were lost in etiological agents of chromoblastomycosis and phaeohyphomycosis. An expansion was found in protein degrading peptidase enzyme families S12 (serine-type D-Ala-D-Ala carboxypeptidases) and M38 (isoaspartyl dipeptidases). Based on genomic information, a wide range of abilities of melanin biosynthesis was revealed; genes related to metabolically distinct DHN, DOPA and pyomelanin pathways were identified. The MAT (MAting Type) locus and other sex-related genes were recognized in all 23 black fungi. Members of the asexual genera Fonsecaea and Cladophialophora appear to be heterothallic with a single copy of either MAT-1-1 or MAT-1-2 in each individual. All Capronia species are homothallic as both MAT1-1 and MAT1-2 genes were found in each single genome. The genomic synteny of the MAT-locus flanking genes (SLA2-APN2-COX13) is not conserved in black fungi as is commonly observed in Eurotiomycetes, indicating a unique genomic context for MAT in those species. The heterokaryon (het) genes expansion associated with the low selective pressure at the MAT-locus suggests that a parasexual cycle may play an important role in generating diversity among those fungi.

The role of melanin pathways in extremotolerance and virulence of Fonsecaea revealed by de novo assembly transcriptomics using illumina paired-end sequencing.

Melanisation has been considered to be an important virulence factor of Fonsecaea monophora. However, the biosynthetic mechanisms of melanisation remain unknown. We therefore used next generation sequencing technology to investigate the transcriptome and digital gene expression data, which are valuable resources to better understand the molecular and biological mechanisms regulating melanisation in F. monophora. We performed de novo transcriptome assembly and digital gene expression (DGE) profiling analyses of parent (CBS 122845) and albino (CBS 125194) strains using the Illumina RNA-seq system. A total of 17 352 annotated unigenes were found by BLAST search of NR, Swiss-Prot, Gene Ontology, Clusters of Orthologous Groups and Kyoto Encyclopedia of Genes and Genomes (KEGG) (E-value <1e‒5). A total of 2 283 unigenes were judged to be the differentially expressed between the two genotypes. We identified most of the genes coding for key enzymes involved in melanin biosynthesis pathways, including polyketide synthase (pks), multicopper oxidase (mco), laccase, tyrosinase and homogentisate 1,2-dioxygenase (hmgA). DEG analysis showed extensive down-regulation of key genes in the DHN pathway, while up-regulation was noted in the DOPA pathway of the albino mutant. The transcript levels of partial genes were confirmed by real time RT-PCR, while the crucial role of key enzymes was confirmed by either inhibitor or substrate tests in vitro. Meanwhile, numbers of genes involved in light sensing, cell wall synthesis, morphology and environmental stress were identified in the transcriptome of F. monophora. In addition, 3 353 SSRs (Simple Sequence Repeats) markers were identified from 21 600 consensus sequences. Blocking of the DNH pathway is the most likely reason of melanin deficiency in the albino strain, while the production of pheomelanin and pyomelanin were probably regulated by unknown transcription factors on upstream of both pathways. Most of genes involved in environmental tolerance to oxidants, irradiation and extreme temperatures were also assembled and annotated in transcriptomes of F. monophora. In addition, thousands of identified cSSR (combined SSR) markers will favour further genetic linkage studies. In conclusion, these data will contribute to understanding the regulation of melanin biosynthesis and help to improve the studies of pathogenicity of F. monophora.

Molecular characterisation and antifungal susceptibility of clinical Cryptococcus deuterogattii (AFLP6/VGII) isolates from Southern Brazil.

Cryptococcosis, caused by Cryptococcus gattii sensu lato, is an emerging disease that was initially found in (sub)tropical regions but recently expanded to temperate regions. Cryptococcus gattii s.l. infections are mostly encountered in healthy individuals, frequently affecting both lungs and the central nervous system (CNS). Usually, C. gattii s.l. is less susceptible to antifungal compounds than its counterpart, C. neoformans s.l. We studied 18 clinical C. gattii s.l. isolates with amplified fragment length polymorphism (AFLP) fingerprinting, mating-typing, multi-locus sequence typing (MLST) and antifungal susceptibility testing. All isolates were C. deuterogattii (genotype AFLP6/VGII), 14 were mating-type α and four were type a. Amphotericin B, itraconazole, voriconazole, posaconazole and isavuconazole showed high activity, with minimum inhibitory concentration (MIC) ranges of 0.063-0.25, 0.031-0.25, 0.031-0.25, 0.031-0.25 and <0.016-0.25 µg mL-1, respectively. Fluconazole and flucytosine had high geometric mean MICs of 2.07 and 3.7 µg mL-1, respectively. Most cases occurred in immunocompetent patients (n = 10; 55.6 %) and CNS involvement was the most common clinical presentation (n = 14; 77.8 %). Three patients (16.7 %) showed sequelae, hyperreflexia, dysarthria, diadochokinesia, anosmia and upper limb weakness. In conclusion, all infections were caused by C. deuterogattii (AFLP6/VGII) and the majority of patients were immunocompetent, with the CNS as the most affected site. All antifungal drugs had high in vitro activity against C. deuterogattii isolates, except fluconazole and flucytosine.

In vitro activities of eight antifungal drugs against 106 waterborne and cutaneous exophiala species.

The in vitro activities of eight antifungal drugs against 106 clinical and environmental isolates of waterborne and cutaneous Exophiala species were tested. The MICs and minimum effective concentrations for 90% of the strains tested (n = 106) were, in increasing order, as follows: posaconazole, 0.063 µg/ml; itraconazole, 0.25 µg/ml; micafungin, 1 µg/ml; voriconazole, 2 µg/ml; isavuconazole, 4 µg/ml; caspofungin, 8 µg/ml; amphotericin B, 16 µg/ml; fluconazole, 64 µg/ml.

Molecular identification of Penicillium marneffei using rolling circle amplification.

Penicillium marneffei is the aetiological agent of a severe systemic disease in immunocompromised hosts in Southeast Asia. In the present study, we evaluated an identification method based on rolling circle amplification (RCA) enabling rapid and specific detection of single nucleotide differences. Three padlock probes were designed on the basis of the internal transcribed spacers 1 and 2 (ITS) of the rRNA operon. One of these (PmPL1) allowed specific amplification of P. marneffei DNA within one working day using a newly conceived protocol, while no cross-reactivity was observed with other fungi including related biverticillate penicillia. Amplification products can be detected by electrophoresis on agarose gel. The method provides a powerful tool for a rapid specific identification of P. marneffei in the clinical laboratory and has potential for ecological studies.

Rapid identification of fungal pathogens by rolling circle amplification using Fonsecaea as a model.

We aimed to describe a rapid and sensitive assay for identification of pathogenic fungi without sequencing. The method of rolling circle amplification (RCA) is presented with species of Fonsecaea, agents of human chromoblastomycosis, as a model. The internal transcribed spacer (ITS) rDNA region of 103 Fonsecaea strains was sequenced and aligned in view of designing three specific padlock probes to be used for the detection of single nucleotide polymorphisms in three Fonsecaea species. The 38 strains included for testing the specificity of RCA comprised 17 isolates of Fonsecaea pedrosoi, 13 of Fonsecaea monophora and eight of Fonsecaea nubica. The assay successfully amplified DNA of the target fungi at the level of species, while no cross reactivity was observed. The amplification product was visualised on a 1% agarose gel to verify the specificity of probe-template binding. Amounts of reagents were minimised to avoid the generation of false-positive results. The simplicity, sensitivity, robustness and low costs provide RCA a distinct position among isothermal techniques for DNA diagnostics as a very practical identification method.

Fonsecaea multimorphosa sp. nov, a new species of Chaetothyriales isolated from a feline cerebral abscess.

A novel fungal species is described originating from the left occipital lobe of the cerebrum of an 18-month-old spayed female cat in Australia. Neurological disorder of the animal became apparent by circling movements and uncoordinated behaviour. Sequencing of the SSU rRNA gene reveals this strain as belonging to the genus Fonsecaea in Chaetothyriales. This order includes many black yeasts and relatives known as etiologic agents of disease in humans and animals, including several neurotropic species. Novelty of the species was corroborated by morphology and by multilocus sequencing of the ribosomal internal transcribed spacers (ITS) and partial sequences of the ß-tubulin (BT2) and translation elongation factor (TEF1) genes. The strain is very similar to several strains recovered by a selective isolation technique from the natural environment in Brazil.

Waterborne Exophiala species causing disease in cold-blooded animals.

The majority of mesophilic waterborne species of the black yeast genus Exophiala (Chaetothyriales) belong to a single clade judging from SSU rDNA data. Most taxa are also found to cause cutaneous or disseminated infections in cold-blooded, water animals, occasionally reaching epidemic proportions. Hosts are mainly fish, frogs, toads, turtles or crabs, all sharing smooth, moist or mucous skins and waterborne or amphibian lifestyles; occasionally superficial infections in humans are noted. Cold-blooded animals with strictly terrestrial life styles, such as reptiles and birds are missing. It is concluded that animals with moist skins, i.e. those being waterborne and those possessing sweat glands, are more susceptible to black yeast infection. Melanin and the ability to assimilate alkylbenzenes are purported general virulence factors. Thermotolerance influences the choice of host. Exophiala species in ocean water mostly have maximum growth temperatures below 30 °C, whereas those able to grow until 33(-36) °C are found in shallow waters and occasionally on humans. Tissue responses vary with the phylogenetic position of the host, the lower animals showing poor granulome formation. Species circumscriptions have been determined by multilocus analyses involving partial ITS, TEF1, BT2 and ACT1.

Some biomolecules and a partially O-acetylated exo-galactomannan containing ß-Galf units from pathogenic Exophiala jeanselmei, having a pronounced immunogenic response.

The pathogenic fungus Exophiala jeanselmei (Ej4) was grown in submerged MM medium, glucose being consumed after six days with maximum biomass and EPS production. Cells were extracted with CHCl3-MeOH (2:1, v/v) yielding a product containing 10% lipid, with high levels of unsaturated C(18:1) (43.6%) and C(18:2) (21.0%), 2D-TLC showed the presence of PE (17.7%), PS (11.6%), PC (35.8%), PI (1.2%) and lyso-phospholipids, LPE (10.7%), LPC (2.0%), PA (10.4%), cardiolipin (10.5%) and glucosyl-ceramide. Analysis of EPS-1 (120 kDa) showed a galactomanan, containing a main chain of Manp-(1→2) (24.2%), substituted by side chains containing terminal Galf (16.8%) and Manp (3.5%) and acetyl groups attached at O-6 of terminal Galf. An immune response against antigens was obtained using Balb/C mice. Anti-EPS-1 antibodies recognized purified fraction containing cellular walls very titer and higher than 1:20,000 for EPS. The studied biomolecules showed biotechnological potential and point to important perspectives in diagnosis of fungi and immunomodulatory products.

Environmental isolation of black yeast-like fungi involved in human infection.

The present study focuses on potential agents of chromoblastomycosis and other endemic diseases in the state of Paraná, Southern Brazil. Using a highly selective protocol for chaetothyrialean black yeasts and relatives, environmental samples from the living area of symptomatic patients were analysed. Additional strains were isolated from creosote-treated wood and hydrocarbon-polluted environments, as such polluted sites have been supposed to enhance black yeast prevalence. Isolates showed morphologies compatible with the traditional etiological agents of chromoblastomycosis, e.g. Fonsecaea pedrosoi and Phialophora verrucosa, and of agents of subcutaneous or systemic infections like Cladophialophora bantiana and Exophiala jeanselmei. Some agents of mild disease were indeed encountered. However, molecular analysis proved that most environmental strains differed from known etiologic agents of pronounced disease syndromes: they belonged to the same order, but mostly were undescribed species. Agents of chromoblastomycosis and systemic disease thus far are prevalent on the human host. The hydrocarbon-polluted environments yielded yet another spectrum of chaetothyrialean fungi. These observations are of great relevance because they allow us to distinguish between categories of opportunists, indicating possible differences in pathogenicity and virulence.

Selective factors involved in oil flotation isolation of black yeasts from the environment.

The oil flotation isolation technique has been successfully applied to recover chaetothyrialean black yeasts and relatives from the environment. The selective mechanisms playing a role in isolation are unknown. The fungi concerned are supposed to occupy specialized microniches in nature, taking advantage of (1) oligotrophism. Mineral oil as a main selective agent may be based on (2) hydrophobicity or on (3) assimilation. All three hypotheses are tested in this paper. Results show that cell wall hydrophobicity is unlikely to be a selective factor. Incubation under poor nutrient conditions provides competitive advantage for black yeasts, especially for Exophiala strains, which are subsequently enriched by mineral oil which enhances growth in this group of fungi. Incubation under mineral media and mineral oil can be used as selective factor.

Molecular ecology and pathogenic potential of Fonsecaea species.

The genus Fonsecaea is revised on the basis of ribosomal DNA internal transcribed spacer (ITS) sequence data. Two species are recognized, F. pedrosoi and the new defined F. monophora. The distinction between these species does not correspond with the classical distinction of F. pedrosoi and F. compacta. The latter appears to be no more than a morphological variant. Both species recognized in this study are agents of human chromoblastomycosis; however, in F. pedrosoi a strict association with this disease is noted, while F. monophora is a more general opportunist. Subspecific randomly amplified polymorphic DNA (RAPD) typing revealed a high degree of strain diversity, although clonal reproduction is also likely to occur. Most strains with Fonsecaea-like morphology isolated from environments to which symptomatic human patients were exposed were found to be more closely related to species of Cladophialophora than to Fonsecaea.