A.D. van Diepeningen

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.

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.

One fungus, which genes? Development and assessment of universal primers for potential secondary fungal DNA barcodes

The aim of this study was to assess potential candidate gene regions and corresponding universal primer pairs as secondary DNA barcodes for the fungal kingdom, additional to ITS rDNA as primary barcode. Amplification efficiencies of 14 (partially) universal primer pairs targeting eight genetic markers were tested across > 1 500 species (1 931 strains or specimens) and the outcomes of almost twenty thousand (19 577) polymerase chain reactions were evaluated. We tested several well-known primer pairs that amplify: i) sections of the nuclear ribosomal RNA gene large subunit (D1–D2 domains of 26/28S); ii) the complete internal transcribed spacer region (ITS1/2); iii) partial β -tubulin II (TUB2); iv) γ-actin (ACT); v) translation elongation factor 1-α (TEF1α); and vi) the second largest subunit of RNA-polymerase II (partial RPB2, section 5–6). Their PCR efficiencies were compared with novel candidate primers corresponding to: i) the fungal-specific translation elongation factor 3 (TEF3); ii) a small ribosomal protein necessary for t-RNA docking; iii) the 60S L10 (L1) RP; iv) DNA topoisomerase I (TOPI); v) phosphoglycerate kinase (PGK); vi) hypothetical protein LNS2; and vii) alternative sections of TEF1α. Results showed that several gene sections are accessible to universal primers (or primers universal for phyla) yielding a single PCR-product. Barcode gap and multi-dimensional scaling analyses revealed that some of the tested candidate markers have universal properties providing adequate infra- and inter-specific variation that make them attractive barcodes for species identification. Among these gene sections, a novel high fidelity primer pair for TEF1α, already widely used as a phylogenetic marker in mycology, has potential as a supplementary DNA barcode with superior resolution to ITS. Both TOPI and PGK show promise for the Ascomycota, while TOPI and LNS2 are attractive for the Pucciniomycotina, for which universal primers for ribosomal subunits often fail.

Stenotrophomonas maltophilia: a new potential biocontrol agent of Ralstonia solanacearum, causal agent of potato brown rot

Stenotrophomonas maltophilia was isolated from the rhizosphere of eggplant in the Nile Delta of Egypt, and its antagonistic potential against Ralstonia solanacearum race 3 biovar 2, the causal agent of potato brown rot, was in vitro evaluated on KB agar medium and in vivo on potato plants. In vitro, four isolates of S. maltophilia (PD3531, PD3532, PD3533, and PD3534) appeared antagonistic. The isolate (PD3533) was screened as the most promising antagonist for the in vivo tests. In the greenhouse, the antagonist was applied directly to soil or by bacterization of potato eyepieces. Stenotrophomonas maltophilia significantly suppressed potato brown rot in Egyptian clay soil but not in Dutch clay soil. Survival of a rifampicin and chloramphenicol-resistant S. maltophilia strain PD4560 was investigated in two pairs of clay soils, conventionally and organically managed, from Egypt and the Netherlands. The survival of S. maltophilia was significantly less in Dutch than in Egyptian soils, while the converse occurred for R. solanacearum. These results are in agreement with those obtained in the in vivo biocontrol tests. In conclusion, S. maltophilia may be useful for control of brown rot in the area where it was originally isolated, the Nile Delta in Egypt.

Revision of agents of black-grain eumycetoma in the order Pleosporales

Eumycetoma is a chronic fungal infection characterised by large subcutaneous masses and the presence of sinuses discharging coloured grains. The causative agents of black-grain eumycetoma mostly belong to the orders Sordariales and Pleosporales. The aim of the present study was to clarify the phylogeny and taxonomy of pleosporalean agents, viz. Madurella grisea, Medicopsis romeroi (syn.: Pyrenochaeta romeroi), Nigrograna mackinnonii (syn. Pyrenochaeta mackinnonii), Leptosphaeria senegalensis, L. tompkinsii, and Pseudochaetosphaeronema larense. A phylogenetic analysis based on five loci was performed: the Internal Transcribed Spacer (ITS), large (LSU) and small (SSU) subunit ribosomal RNA, the second largest RNA polymerase subunit (RPB2), and translation elongation factor 1-alpha (TEF1) gene. In addition, the morphological and physiological characteristics were determined. Three species were well-resolved at the family and genus level. Madurella grisea, L. senegalensis, and L. tompkinsii were found to belong to the family Trematospheriaceae and are reclassified as Trematosphaeria grisea comb. nov., Falciformispora senegalensis comb. nov., and F. tompkinsii comb. nov. Medicopsis romeroi and Pseudochaetosphaeronema larense were phylogenetically distant and both names are accepted. The genus Nigrograna is reduced to synonymy of Biatriospora and therefore N. mackinnonii is reclassified as B. mackinnonii comb. Nov. Mycetoma agents in Pleosporales were phylogenetically quite diverse despite their morphological similarity in the formation of pycnidia, except for the ascosporulating genus Falciformispora (formerly in Leptosphaeria). Most of the species diagnosed from human mycetoma were found to be related to waterborne or marine fungi, suggesting an association of the virulence factors with oligotrophism or halotolerance.

Sexual and vegetative compatibility genes in the aspergilli

Gene flow within populations can occur by sexual and/or parasexual means. Analyses of experimental and in silico work are presented relevant to possible gene flow within the aspergilli. First, the discovery of mating-type (MAT) genes within certain species of Aspergillus is described. The implications for self-fertility, sexuality in supposedly asexual species and possible uses as phylogenetic markers are discussed. Second, the results of data mining for heterokaryon incompatibility (het) and programmed cell death (PCD) related genes in the genomes of two heterokaryon incompatible isolates of the asexual species Aspergillus niger are reported. Het-genes regulate the formation of anastomoses and heterokaryons, may protect resources and prevent the spread of infectious genetic elements. Depending on the het locus involved, hetero-allelism is not tolerated and fusion of genetically different individuals leads to growth inhibition or cell death. The high natural level of heterokaryon incompatibility in A. niger blocks parasexual analysis of the het-genes involved, but in silico experiments in the sequenced genomes allow us to identify putative het-genes. Homologous sequences to known het- and PCD-genes were compared between different sexual and asexual species including different Aspergillus species, Sordariales and the yeast Saccharomyces cerevisiae. Both het- and PCD-genes were well conserved in A. niger. However some point mutations and other small differences between the het-genes in the two A. niger isolates examined may hint to functions in heterokaryon incompatibility reactions.

Geographic differences in trichothecene chemotypes of Fusarium graminearum in the Northwest and North of Iran

The diversity and prevalence of Fusarium species and their chemotypes on wheat in the North-West and North of Iran was determined. Wheat in these areas is severely affected by Fusarium head blight, with Fusarium graminearum as prevalent species causing 96% of the infections in the North-West and 50% in the Northern provinces. Fungal isolates were identified based on morphological characters and sequences of the internal transcribed spacer region, and parts of translation elongation factor 1-α and RNA polymerase subunit II sequences. Phylogenetic and phylogeographic analyses show little haplotype variation between the F. graminearum strains collected from the different locations, but the isolates differ significantly in their trichothecene chemotypes as determined with a multilocus genotyping assay. F. graminearum strains producing 15-acetyldeoxynivalenol were abundant in Ardabil (North-West of Iran), while in Golestan province (North of Iran) at the other side of the Caspian Sea especially nivalenol producing strains and a variety of other Fusarium species were observed. Strains producing 3-acetyldeoxynivalenol were rarely found in both areas. This is the first detailed study on Fusarium infections in Iranian wheat, showing large differences in prevalent etiological agents and in mycotoxin chemotypes geographically.

DNA barcoding of clinically relevant Cunninghamella species

Mucormycosis caused, in part, by representatives of the genus Cunninghamella is a severe infection with high mortality in patients with impaired immunity. Several species have been described in the literature as etiologic agents. A DNA barcoding study using ITS rDNA and tef-1α provided concordance of molecular data with conventional characters. The currently accepted Cunninghamella species were well supported in phylogenetic trees of both markers except for C. septata with ITS that clustered in the C. echinulata clade. Sequence variability was distinctly higher for the ITS than for tef-1α. Intraspecific ITS variability of some of the species exceeded that between some closely related species, but the marker remained applicable for species identification. The most variable species for both markers was C. echinulata. Cunninghamella bertholletiae is the main pathogenic species; infections by C. blakesleeana, C. echinulata, and C. elegans are highly exceptional.

Tintelnotia, a new genus in Phaeosphaeriaceae harbouring agents of cornea and nail infections in humans

Phaeosphaeriaceae is a family in the order Pleosporales containing numerous plant pathogens, endophytes, lichenised fungi, and environmental saprobes. A novel genus, Tintelnotia is introduced containing two species, one of which caused an eye infection and several nail infections in humans. All species of Tintelnotia produce conidia in soft pycnidia with a wide ostiole. The generic type species is T. opuntiae causing necrotic spots on cactus plants. The isolates of the human opportunist T. destructans showed variable susceptibility pattern to a panel of common antifungal agents. The MICs of amphotericin B, voriconazole, posaconazole and itraconazole were 1 μg/mL, complemented by an in vitro MEC of 16 μg/mL against caspofungin; the MIC of terbinafine was 0.125 μg/mL. The latter compound contributed to the successful therapy in the ocular mycosis refractory to standard antifungal therapy, the benefit of terbinafine should be highlighted as a therapeutic option especially in difficult‐to‐treat fungal keratitis.

Proximal subungual onychomycosis caused by Fusarium falciforme successfully cured with posaconazole

Proximal subungual onychomycosis (PSO) is a fungal infection on the inner layer of the nail plate which starts from the eponychium and nail matrix.(1) Onychomycosis can be caused by a variety of dermatophytes, yeasts and non-dermatophyte molds (NDMs) including species of Aspergillus, Acremonium, Scopulariopsis, Chaetomium, and Fusarium.(2) Fusarium and Acremonium species require special attention, because of their intrinsically low susceptibility to most common antifungal agents and their ability to disseminate in severely compromised hosts, using the nail as a potential source of infection.(3,4) This article is protected by copyright. All rights reserved.