The global epidemiology of emerging Histoplasma species in recent years.

Histoplasmosis is a serious infectious disease in humans caused by Histoplasma spp. (Onygenales), whose natural reservoirs are thought to be soil enriched with bird and bat guano. The true global burden of histoplasmosis is underestimated and frequently the pulmonary manifestations are misdiagnosed as tuberculosis. Molecular data on epidemiology of Histoplasma are still scarce, even though there is increasing recognition of histoplasmosis in recent years in areas distant from the traditional endemic regions in the Americas. We used multi-locus sequence data from protein coding loci (ADP-ribosylation factor, H antigen precursor, and delta-9 fatty acid desaturase), DNA barcoding (ITS1/2+5.8s), AFLP markers and mating type analysis to determine the genetic diversity, population structure and recognise the existence of different phylogenetic species among 436 isolates of Histoplasma obtained globally. Our study describes new phylogenetic species and the molecular characteristics of Histoplasma lineages causing outbreaks with a high number of severe outcomes in Northeast Brazil between 2011 and 2015. Genetic diversity levels provide evidence for recombination, common ancestry and clustering of Brazilian isolates at different geographic scales with the emergence of LAm C, a new genotype assigned to a separate population cluster in Northeast Brazil that exhibited low diversity indicative of isolation. The global survey revealed that the high genetic variability among Brazilian isolates along with the presence of divergent cryptic species and/or genotypes may support the hypothesis of Brazil being the center of dispersion of Histoplasma in South America, possibly with the contribution of migratory hosts such as birds and bats. Outside Brazil, the predominant species depends on the region. We confirm that histoplasmosis has significantly broadened its area of occurrence, an important feature of emerging pathogens. From a practical point of view, our data point to the emergence of histoplasmosis caused by a plethora of genotypes, and will enable epidemiological analysis focused on understanding the processes that lead to histoplasmosis. Further, the description of this diversity opens avenues for comparative genomic studies, which will allow progress toward a consensus taxonomy, improve understanding of the presence of hybrids in natural populations of medically relevant fungi, test reproductive barriers and to explore the significance of this variation.

ESCMID and ECMM joint guidelines on diagnosis and management of hyalohyphomycosis: Fusarium spp., Scedosporium spp. and others.

Mycoses summarized in the hyalohyphomycosis group are heterogeneous, defined by the presence of hyaline (non-dematiaceous) hyphae. The number of organisms implicated in hyalohyphomycosis is increasing and the most clinically important species belong to the genera Fusarium, Scedosporium, Acremonium, Scopulariopsis, Purpureocillium and Paecilomyces. Severely immunocompromised patients are particularly vulnerable to infection, and clinical manifestations range from colonization to chronic localized lesions to acute invasive and/or disseminated diseases. Diagnosis usually requires isolation and identification of the infecting pathogen. A poor prognosis is associated with fusariosis and early therapy of localized disease is important to prevent progression to a more aggressive or disseminated infection. Therapy should include voriconazole and surgical debridement where possible or posaconazole as salvage treatment. Voriconazole represents the first-line treatment of infections due to members of the genus Scedosporium. For Acremonium spp., Scopulariopsis spp., Purpureocillium spp. and Paecilomyces spp. the optimal antifungal treatment has not been established. Management usually consists of surgery and antifungal treatment, depending on the clinical presentation.

ESCMID and ECMM joint clinical guidelines for the diagnosis and management of mucormycosis 2013.

These European Society for Clinical Microbiology and Infectious Diseases and European Confederation of Medical Mycology Joint Clinical Guidelines focus on the diagnosis and management of mucormycosis. Only a few of the numerous recommendations can be summarized here. To diagnose mucormycosis, direct microscopy preferably using optical brighteners, histopathology and culture are strongly recommended. Pathogen identification to species level by molecular methods and susceptibility testing are strongly recommended to establish epidemiological knowledge. The recommendation for guiding treatment based on MICs is supported only marginally. Imaging is strongly recommended to determine the extent of disease. To differentiate mucormycosis from aspergillosis in haematological malignancy and stem cell transplantation recipients, identification of the reverse halo sign on computed tomography is advised with moderate strength. For adults and children we strongly recommend surgical debridement in addition to immediate first-line antifungal treatment with liposomal or lipid-complex amphotericin B with a minimum dose of 5 mg/kg/day. Amphotericin B deoxycholate is better avoided because of severe adverse effects. For salvage treatment we strongly recommend posaconazole 4×200 mg/day. Reversal of predisposing conditions is strongly recommended, i.e. using granulocyte colony-stimulating factor in haematological patients with ongoing neutropenia, controlling hyperglycaemia and ketoacidosis in diabetic patients, and limiting glucocorticosteroids to the minimum dose required. We recommend against using deferasirox in haematological patients outside clinical trials, and marginally support a recommendation for deferasirox in diabetic patients. Hyperbaric oxygen is supported with marginal strength only. Finally, we strongly recommend continuing treatment until complete response demonstrated on imaging and permanent reversal of predisposing factors.

Chromoblastomycosis as an endemic disease in temperate Europe: first confirmed case and review of the literature.

This study reports a case of a 56-year-old white male, retired coal-miner, diagnosed with chromoblastomycosis lasting 20 years. The infection site was the burnt skin of the back. For many years the patient had not undertaken any treatment believing that the lesion had been a burn scar. A gradual increase in lesion size prompted the patient to start therapy. The diagnosis was made by histopathological examination and mycological culture. Identification of the causative agent at the species level was achieved by sequence analysis of the internal transcribed spacer (ITS) region and D1/D2 domains of the 26S rDNA. To our knowledge, this is the first documented case of chromoblastomycosis caused by Fonsecaea monophora in temperate Europe, outside the endemic area for the disease. This finding is highly significant for understanding the routes of infection of chromoblastomycosis and radically revises the traditional view of the natural ecology of the etiological agents of the disease.

Diversity of Bipolaris species in clinical samples in the United States and their antifungal susceptibility profiles.

A set of 104 isolates from human clinical samples from the United States, morphologically compatible with Bipolaris, were morphologically and molecularly identified through the sequence analysis of the internal transcribed space (ITS) region of the nuclear ribosomal DNA (rDNA). The predominant species was Bipolaris spicifera (67.3%), followed by B. hawaiiensis (18.2%), B. cynodontis (8.6%), B. micropus (2.9%), B. australiensis (2%), and B. setariae (1%). Bipolaris cynodontis, B. micropus, and B. setariae represent new records from clinical samples. The most common anatomical sites where isolates were recovered were the nasal region (30.7%), skin (19.2%), lungs (14.4%), and eyes (12.5%). The antifungal susceptibilities of 5 species of Bipolaris to 9 drugs are provided. With the exception of fluconazole and flucytosine, the antifungals tested showed good activity.

A virulent genotype of Microsporum canis is responsible for the majority of human infections.

The zoophilic dermatophyte species Microsporum canis belongs to the Arthroderma otae complex and is known to mate with tester strains of that teleomorph species, at least in the laboratory. Human infections are likely to be acquired from the fur of cats, dogs and horses. Epidemiological studies to reveal sources and routes of infection have been hampered by a lack of polymorphic molecular markers. Human cases mainly concern moderately inflammatory tinea corporis and tinea capitis, but, as cases of highly inflammatory ringworm are also observed, the question arises as to whether all lineages of M. canis are equally virulent to humans. In this study, two microsatellite markers were developed and used to analyse a global set of 101 M. canis strains to reveal patterns of genetic variation and dispersal. Using a Bayesian and a distance approach for structuring the M. canis samples, three populations could be distinguished, with evidence of recombination in one of them (III). This population contained 44 % of the animal isolates and only 9 % of the human strains. Population I, with strictly clonal reproduction (comprising a single multilocus genotype), contained 74 % of the global collection of strains from humans, but only 23 % of the animal strains. From these findings, it was concluded that population differentiation in M. canis is not allopatric, but rather is due to the emergence of a (virulent) genotype that has a high potential to infect the human host. Adaptation of genotypes resulting in a particular clinical manifestation was not evident. Furthermore, isolates from horses did not show a monophyletic clustering.

Survival of a neglected case of brain abscess caused by Cladophialophora bantiana.

Cladophialophora bantiana is an uncommon fungus related to the black yeasts which causes, if untreated, mostly fatal cerebral infections in immunosuppressed and competent patients. We report a case of a patient who survived a recurrent cerebral abscess caused by C. bantiana despite delayed and apparently inappropriate therapy.

Dermatophytes: recognizing species of clonal fungi.

Now that molecular data have forever changed our perspective on the anthropophilic and zoophilic dermatophyte species, the concepts of these species needs re-evaluation. In this paper, main concepts (morphological, biological (BSC), phylogenetic and genealogical concordance phylogenetic species recognition (GCPSR)) are compared. While in geophilic dermatophytes the application of the BSC works well for species distinction and is supported by molecular data, it is not applicable for the anthropophilic and zoophilic dermatophytes where the majority of species reproduce purely asexually. Also, the application of GCPSR (an operational method to define the limits of species using molecular, multi-locus data) is problematic. GCPSR can be applied in recombining fungi even when recombination is infrequent and fungi lack phenotypic sexuality. In truly clonal fungi, however, no incongruities in multi-locus data are found, and thus separation of species may be difficult. In fungi this problem is currently taken to be non-existent, since clonality is supposed to lead to extinction. In the medically relevant, host-associated dermatophytes, however, is reason to suggest that clonal dermatophyte lineages are able to maintain ongoing populations and to follow independent evolutionary trajectories. We distinguish seasonal, short-lived and long-lived clonal species. The final goal of a species concept, in the dermatophytes as well as in other fungi, is to provide a taxonomic system that reflects the evolution of the fungal species so that the underlying biological trends elucidated in this way may be brought forward to help to guide the clinician in applying optimal therapy and prophylaxis. The application of the different species concepts may have an enormous impact on the nomenclature of dermatophytes, directly affecting the quality of communications with care providers.

Origins of microsatellite diversity in the Trichophyton rubrum-T. violaceum clade (Dermatophytes).

We analyzed the population structure of the anthropophilic dermatophyte species Trichophyton violaceum, which mainly causes tinea capitis, and T. rubrum, the most frequently isolated agent of dermatophytosis worldwide. A microsatellite marker (T1) was developed by using the enrichment technique for microsatellites. The T1 marker containing a (GT)(8-10) repeat was proven to specifically amplify both species, underlining their close kinship. Four polymorphic alleles were detected within a set of about 130 strains by using polyacrylamide gel electrophoresis with this marker. An association with geographic origin of the isolates was apparent. Given the close relatedness of both species, these data suggest an African origin of the entire T. rubrum complex, followed by the emergence of a new genotype (B) in Asia with subsequent spread of this genotype over Europe and the United States.

Postmortem isolation of Pseudotaeniolina globosa from a patient with aortic aneurysm.

We describe the isolation of the melanized meristematic fungus Pseudotaeniolina globosa from the aortic wall of a patient who died while undergoing surgery for aortic aneurysm and aortic valve regurgitation as a result of dilated cardiomyopathy. Meristematic fungi related to P. globosa have until now been considered as environmental saprobes found predominantly in ecological niches with low water activity. The isolate was identified by phenotypic analyses and by sequencing of the rDNA internal-transcribed spacer domain. The clinical significance of this isolation remains unclear but isolation of meristematic fungi from clinical specimen should be thoroughly evaluated in terms of their significance in future.

Analyses of phagocytosis, evoked oxidative burst, and killing of black yeasts by human neutrophils: a tool for estimating their pathogenicity?

The pathogenicity of several dematiaceous yeasts that have, to date, rarely been isolated in humans remains unclear. Because professional phagocytes are prominent in lesions caused by dematiaceous fungi, we address this issue by comparing phagocytosis, evoked oxidative burst and killing by human neutrophils of different black yeasts in vitro. Whereas phagocytosis of all black yeasts tested and evoked oxidative burst yielded comparable results, in contrast, the degree of killing differed significantly after 5 h. Thereby, two groups could be identified; one in which strains are killed at high rates, for example, Hortaea werneckii (81 +/- 11.6%), Exophiala castellanii (96 +/- 8.6%), Phaeoannellomyces elegans (93 +/- 9.7%), Phaeococcomyces exophialae (87 +/- 8.7%), and the other in which strains are killed to a lesser degree, for example, Exophiala dermatitidis (ATCC 34100) (61 +/- 9.5%), E. dermatitidis (CBS 207.35) (66 +/- 7.5%), E. jeanselmei (50 +/- 10.5%), E. mesophila (63 +/- 11.6%), E. bergeri (63 +/- 9.1%), and E. spinifera (57 +/- 9.6%). Non-pigmented yeasts were killed at levels comparable with those at which the white mutant strain of E. dermatitidis (ATCC 44504) was killed (95 +/- 7.5%); the yeast strains tested were Candida albicans (DSM 11943) (95 +/- 4.0% killing) and Saccharomyces cerevisiae (DSM 1333) (95 +/- 10.3%). Comparison of killing rates with the observed pathogenicity of the melanized species suggests that low killing rates might indicate or even predict a high degree of invasiveness. Although previous experiments revealed that melanization conferred killing resistance on E. dermatitidis, the differences in killing rates of other dematious fungi suggest that melanization of the cell wall is in itself insufficient to confer virulence.

Safety evaluation of a lactase enzyme preparation derived from Kluyveromyces lactis.

Neutralact(R), the DSM brand name of a lactase enzyme preparation, obtained from a homologous rDNA strain of Kluyveromyces lactis, was subjected to a series of toxicological tests to document the safety for use as a processing aid in the dairy industry. The enzyme preparation was examined for subacute oral toxicity and mutagenic potential. As a result of these tests, no evidence of oral toxicity, mutagenicity or clastogenicity was found. Administration of the lactase enzyme preparation at doses of 500, 3000 and 10,000 mg/kg body weight/day for 28 days did not induce noticeable signs of toxicity. The no-observed-adverse-effect level (NOAEL) of the enzyme preparation in the acute toxicity study was 10,000 mg/kg body weight/day (equivalent to 114,000 NL units/kg body weight/day). It can be concluded that no safety concerns were identified in the studies conducted with this lactase enzyme preparation derived from Kluyveromyces lactis under controlled fermentation conditions.

Case report. Atypical cutaneous pseudallescheriosis refractory to antifungal agents.

We report on a 65-year-old male heart transplant recipient who was otherwise in good condition. The patient was immunocompromised secondarily due to cyclosporin, prednisolone and azathioprine when widespread pustular skin lesions with erythematous margins subsequently developed on his left forearm. There was no history of trauma or septic temperature. Bacterial cultures were sterile and the results of native and cultural investigation studies were negative. A biopsy specimen of the lesion demonstrated hyalohyphomycosis with numerous septate hyphae within granulomas throughout the dermis. Subcutaneous tissues were not involved. Culture plates inoculated with pus and skin from the punch biopsy showed growth of a mould yielding Pseudallescheria boydii. Sensitivity testing was performed with miconazole, ketoconazole and itraconazole showing the best in vitro activity against P. boydii. In spite of treatment with itraconazole, the erythema and pustules continued to spread and therapy was changed to intravenous miconazole. Due to ongoing progression after 3 months of antifungal therapy surgical debridement was required. After 2 years of follow up, he had no recurrence.

Development of a species-specific PCR-restriction fragment length polymorphism analysis procedure for identification of Madurella mycetomatis.

Madurella mycetomatis is the commonest cause of eumycetoma in Sudan and other countries in tropical Africa. Currently, the early diagnosis of mycetoma is difficult. In attempting to improve the identification of M. mycetomatis and, consequently, the diagnosis of mycetoma, we have developed specific oligonucleotide primers based on the sequence of the internal transcribed spacer (ITS) regions spacing the genes encoding the fungal ribosomal RNAs. The ITS regions were amplified with universal primers and sequenced, and then two sets of species-specific primers were designed which specifically amplify parts of the ITS and the 5.8S ribosomal DNA gene. The new primers were tested for specificity with DNA isolated from human mycetoma lesions and DNA extracted from cultures of M. mycetomatis reference strains and related fungi as well as human DNA. To study the genetic variability of the ITS regions of M. mycetomatis, ITS amplicons were obtained from 25 different clinical isolates and subjected to restriction fragment length polymorphism (RFLP) analysis with CfoI, HaeIII, MspI, Sau3AI, RsaI, and SpeI restriction enzymes. RFLP analysis of the ITS region did not reveal even a single difference, indicating the homogeneity of the isolates analyzed during the current study.

Database on the structure of large subunit ribosomal RNA.

The Antwerp database on large subunit ribosomal RNA now contains 607 complete or nearly complete aligned sequences. The alignment incorporates secondary structure information for each sequence. Other information about the sequences, such as literature references, accession numbers and taxonomic information is also available. Information from the database can be downloaded or searched on the rRNA WWW Server at URL http://rrna.uia.ac.be/

Database on the structure of small subunit ribosomal RNA.

Over 11 500 complete or nearly complete sequences are now available from the Antwerp database on small subunit ribosomal RNA. All these sequences are aligned with one another on the basis of the adopted secondary structure model, which is corroborated by the observation of compensating substitutions in the alignment. Literature references, accession numbers and taxonomic information are also compiled. The database can be consulted via the World Wide Web at URL http://rrna.uia.ac.be/ssu/