Trichoderma from Brazilian garlic and onion crop soils and description of two new species: Trichoderma azevedoi and Trichoderma peberdyi.

Fifty four Trichoderma strains were isolated from soil samples collected from garlic and onion crops in eight different sites in Brazil and were identified using phylogenetic analysis based on combined ITS region, tef1-α, cal, act and rpb2 sequences. The genetic variability of the recovered Trichoderma species was analysed by AFLP and their phenotypic variability determined using MALDI-TOF. The strain clusters from both typing techniques coincided with the taxonomic determinations made from phylogenetic analysis. The phylogenetic analysis showed the occurrence of Trichoderma asperellum, Trichoderma asperelloides, Trichoderma afroharzianum, Trichoderma hamatum, Trichoderma lentiforme, Trichoderma koningiopsis, Trichoderma longibrachiatum and Trichoderma erinaceum, in the soil samples. We also identified and describe two new Trichoderma species, both in the harzianum clade of section Pachybasium, which we have named Trichoderma azevedoi sp. nov. and Trichoderma peberdyi sp. nov. The examined strains of both T. azevedoi (three strains) and T. peberdyi (12 strains) display significant genotypic and phenotypic variability, but form monophyletic clades with strong bootstrap and posterior probability support and are morphologically distinct from their respective most closely related species.

Sporotrichosis caused by Sporothrix brasiliensis in Argentina: Case report, molecular identification and in vitro susceptibility pattern to antifungal drugs.

Sporotrichosis is considered a neglected disease of humans and animals in many regions of the world and is the most frequent implantation mycosis in Latin America. OBJECTIVES: To illustrate the zoonotic importance of the disease, describing a case involving a veterinarian and an infant that acquired the disease from a domestic cat and to describe, genotype and characterize these new isolates. METHODS: Direct examination of tissue samples from the two patients and feline lesions revealed the presence of Sporothrix yeast-like organisms. Fungal cultures and molecular identification of the strains were performed. Since antifungal susceptibility data of animal-borne isolates are scarce, the in vitro susceptibility testing by a microdilution reference method was determined against azoles, amphotericin B and terbinafine. RESULTS: Fungal culture and sequence analysis of the ITS region of rDNA and calmodulin and ß-tubulin genes confirmed the diagnosis and the causative agent as Sporothrix brasiliensis. In all cases, terbinafine was the most active drug, followed by posaconazole, itraconazole and voriconazole; the least active drugs were amphotericine B and fluconazole. Lack of clinical response in the veterinarian and in the infant to itraconazole and potassium iodide, respectively was observed. CONCLUSIONS: This study contributed to the molecular epidemiology of Sporothrix species in Argentina and the characterization of the in vitro susceptibility pattern of S. brasiliensis isolates recovered from a cat and two humans involved in this case of zoonotic sporotrichosis. Bearing in mind the "One Health" concept, the experience described in the present study highlights the need for future strategies for sporotrichosis treatment, control and prevention.

Consequences of Misnomer or Mistakes in Identification of Fungal Species.

The author, as a reviewer of many international journals, describes his long-standing experiences with incorrect identification of mushroom and fungal species and the resultant incorrect naming of those species that served as experimental models. From his own praxis, he selected several characteristic examples that sometimes ended in a curious situation. Some recommendations to authors of publications and persons responsible for the proper naming of mushrooms under study are summarized.

Emerging Trichosporon asahii in elderly patients: epidemiological and molecular analysis by the DiversiLab system.

Trichosporon asahii has been recognized as an emerging opportunistic agent for invasive infections, mainly in immunocompromised patients. Urinary tract infections by this pathogen may also occur, especially in patients with urinary obstruction or those undergoing vesical catheterization and antibiotic treatment. Many outbreaks of Trichosporon spp. have been detected after urinary catheter manipulations. We report the molecular-epidemiological characterization of T. asahii in our institution using the DiversiLab system for the molecular strain typing and compare three different methods for susceptibility testing. Our results present T. asahii as an emergent pathogen in elderly patients with urinary drainage devices that can be adequately treated with triazoles, with voriconazole being the most active. Broth dilution and Vitek 2 had good concordance, while Etest showed more discrepancies. In addition, the DiversiLab system for clonal strain typing may be a useful tool for fast and accurate management of nosocomial outbreaks.

Development of a molecular approach to describe the composition of Trichoderma communities.

Trichoderma and its teleomorphic stage Hypocrea play a key role for ecosystem functioning in terrestrial habitats. However, little is known about the ecology of the fungus. In this study we developed a novel Trichoderma-specific primer pair for diversity analysis. Based on a broad range master alignment, specific Trichoderma primers (ITSTrF/ITSTrR) were designed that comprise an approximate 650bp fragment of the internal transcribed spacer region from all taxonomic clades of the genus Trichoderma. This amplicon is suitable for identification with TrichoKey and TrichoBLAST. Moreover, this primer system was successfully applied to study the Trichoderma communities in the rhizosphere of different potato genotypes grown at two field sites in Germany. Cloning and sequencing confirmed the specificity of the primer and revealed a site-dependent Trichoderma composition. Based on the new primer system a semi-nested approach was used to generate amplicons suitable for denaturing gradient gel electrophoresis (DGGE) analysis and applied to analyse Trichoderma communities in the rhizosphere of potatoes. High field heterogeneity of Trichoderma communities was revealed by both DGGE. Furthermore, qPCR showed significantly different Trichoderma copy numbers between the sites.

Audit of laboratory mycology services for the management of patients with fungal infections in the northwest of England.

BACKGROUND: Fungal infection is increasingly recognised as an important cause of morbidity and mortality, especially in immunocompromised patients. Little information exists on laboratory services available and the methods used by general microbiology laboratories to diagnose these important infections. AIM: To investigate the services microbiology laboratories in northwest England provide towards the diagnosis and management of superficial and deep fungal infections. METHODS: A questionnaire was sent to laboratories to get a holistic view of the support given to clinicians looking after patients with fungal infections. The aim was not to investigate details of each laboratory's standard operating procedures. The completed questionnaires, which formed the basis of this report, were returned by all 21 laboratories which were recruited. This study was conducted between March 2004 and September 2004. RESULTS: Services were provided to District General Hospitals and to six tertiary centres, including eight teaching hospitals by 16 laboratories. Their bed capacity was 250-1300 beds. Total specimens (including bacterial and viral) processed annually were 42 000-500,000 whereas fungal ones were 560-5400. CONCLUSION: In most microbiology laboratories of northwest England, clinicians were aware of the potential of fungal pathogens to cause infections especially in immunocompromised patients. Additional measures such as prolonged incubation of samples were introduced to improve fungal yield from patients at high risk. It is necessary to train and educate laboratory and medical staff about the role of serology and molecular methods in diagnosis and management of patients with fungal infection.

[Utilization of tomato juice agar (V8 agar) in the presumptive identification of Candida dubliniensis]. / Utilização do ágar suco de tomate (ágar V8) na identificação presuntiva de Candida dubliniensis.

We evaluated the capacity of tomato juice agar (V8 agar) to differentiate Candida dubliniensis from Candida albicans based on chlamydospore production. Candida albicans (n= 93) and Candida dubliniensis (n= 26) were studied; 100% of Candida dubliniensis showed chlamydospores and in 92.5% of Candida albicans isolates these elements were absent. These results suggest this medium as an alternative tool for presumptive differentiation between these species.

Utilizacão do ágar suco de tomate (ágar V8) na identificacão presuntiva de Candida dubliniensis / Utilization of tomato juice agar (V8 agar) in the presumptive identification of Candida dubliniensis

Avaliou-se a capacidade do ágar suco de tomate (ágar V8) em diferenciar Candida dubliniensis de Candida albicans com base na producão de clamidoconídios. Noventa e três isolados de Candida albicans e vinte e seis de Candida dubliniensis foram incluídos; 100 por cento de Candida dubliniensis formaram clamidoconídios e 92,5 por cento de Candida albicans não evidenciaram estas estruturas. Estes resultados permitem sugerir este meio como recurso alternativo na identificacão presuntiva de Candida dubliniensis.

Utilization of AFLP markers for PCR-based identification of Aspergillus carbonarius and indication of its presence in green coffee samples.

AIMS: The objective of this work was to test whether ochratoxin A (OTA) production of Aspergillus niger and A. carbonarius is linked to a certain genotype and to identify marker sequences with diagnostic value aiding identification of A. carbonarius, a fungus of major concern regarding OTA production in food and food raw materials. METHODS AND RESULTS: Aspergillus niger and A. carbonarius were isolated mainly from Brazilian coffee sources. The ability of isolates to produce OTA was tested by thin layer chromatography (TLC). Strains were genetically characterized by AFLP fingerprinting and compared with each other and with reference strains. Cluster analysis of fingerprints showed clear separation of A. niger from A. carbonarius strains. To obtain marker sequences, AFLP fragments were isolated from silver stained polyacrylamide gels, cloned and sequenced. Sequences obtained were used to develop species- specific PCR primers for the identification of A. carbonarius in pure culture and in artificially and naturally infected samples of green coffee. CONCLUSIONS: No clear correlation between genetic similarity of the strains studied and their potential to produce OTA was found. The PCR assays designed are a useful and specific tool for identification and highly sensitive detection of A. carbonarius. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed PCR assays allow specific and sensitive detection and identification of A. carbonarius, a fungus considered to be one of the major causative agents for OTA in coffee and grape-derived products. Assays may provide powerful tools to improve quality control and consumer safety in the food processing industry.

The use of Niger seed agar to screen for Candida dubliniensis in the clinical microbiology laboratory.

Candida dubliniensis is a recently described pathogenic yeast that is closely related to C. albicans. The germ tube test is used routinely in diagnostic laboratories for the identification of C. albicans, and C. dubliniensis may also produce germ tubes under the same conditions. We evaluated a previously described method for differentiating between the two species using Niger seed agar (Staib agar). The aim was to find a useful, user-friendly and cost-effective method for use in diagnostic work. C. albicans produces only yeast cells on this medium after 24 h at 37 degrees C, while C. dubliniensis produces extensive hyphal and pseudohyphal growth that is easily observed. Of 495 yeasts isolated in, or sent for identification to, a diagnostic mycology laboratory 9 isolates (1.8%) were found to be C. dubliniensis. The method was found to be valuable for screening yeasts before proceeding to further identification if positive for hyphal/pseudohyphal growth on Niger seed agar. This method is therefore suitable for the screening of selected yeast isolates in order to identity C. dubliniensis and will further our understanding of the clinical importance of this species.