G S de Hoog

Publications of G S de Hoog

• Waterborne Exophiala species causing disease in cold-blooded animals.

  Authors: G S de Hoog, V A Vicente, M J Najafzadeh, M J Harrak, H Badali, S Seyedmousavi

  Persoonia. 12/2011; 27:46-72.

  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 orThe 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.

• Molecular identification tools for sibling species of Scedosporium and Pseudallescheria.

  Authors: M Lackner, C H Klaassen, J F Meis, A H G Gerrits van den Ende, G S de Hoog

  Medical mycology : official publication of the International Society for Human and Animal Mycology. 10/2011;

  The aim of this study was to develop molecular identification tools for currently recognized species of Pseudallescheria and Scedosporium through the use of species-specific primers and RFLP, so asThe aim of this study was to develop molecular identification tools for currently recognized species of Pseudallescheria and Scedosporium through the use of species-specific primers and RFLP, so as to enhance rapid differentiation of clinically relevant species. The variability of species was established in a set of 681 Internal Transcribed Spacer (ITS) and 349 ß-tubulin (BT2) sequences. Amplified Fragment Length Polymorphism profile clustering matched with BT2 results, whereas ITS grouping was less detailed. ITS was sufficient for the differentiation of most haplotypes of clinically relevant species (P. apiosperma, P. boydii, S. aurantiacum, S. dehoogii, and S. prolificans) and of environmental species (P. minutispora and Lophotrichus fimeti) when Restriction Fragment Length Polymorphism (RFLP) were applied. For the identification of P. apiosperma and P. boydii species-specific BT2 primers were needed. Pseudallescheria fusoidea, P. ellipsoidea and P. angusta remained difficult to distinguish from P. boydii.

• The emerging potential of melanized fungi: black yeast between beauty and the beast.

  Authors: Nina Gunde-Cimerman, Martin Grube, G S de Hoog

  Fungal biology. 10/2011; 115(10):935-6.

• Dishwashers--a man-made ecological niche accommodating human opportunistic fungal pathogens.

  Authors: P Zalar, M Novak, G S de Hoog, N Gunde-Cimerman

  Fungal biology. 10/2011; 115(10):997-1007.

  Habitats in human households may accommodate microorganisms outside the common spectrum of ubiquitous saprobes. Enrichment of fungi that may require specific environmental conditions was observed inHabitats in human households may accommodate microorganisms outside the common spectrum of ubiquitous saprobes. Enrichment of fungi that may require specific environmental conditions was observed in dishwashers, 189 of which were sampled in private homes of 101 towns or communities. One-hundred-two were sampled from various localities in Slovenia; 42 from other European countries; 13 and 3 from North and South America, respectively; 5 from Israel; 10 from South Africa; 7 from Far East Asia; and 7 from Australia. Isolation was performed on samples incubated at 37°C. Species belonging to genera Aspergillus, Candida, Magnusiomyces, Fusarium, Penicillium and Rhodotorula were found occasionally, while the black yeasts Exophiala dermatitidis and Exophiala phaeomuriformis (Chaetothyriales) were persistently and most frequently isolated. Sixty-two percent of the dishwashers were positive for fungi, and 56% of these accommodated Exophiala. Both Exophiala species are known to be able to cause systemic disease in humans and frequently colonize the lungs of patients with cystic fibrosis. We conclude that high temperature, high moisture and alkaline pH values typically occurring in dishwashers can provide an alternative habitat for species also known to be pathogenic to humans.

• Elucidation of distribution patterns and possible infection routes of the neurotropic black yeast Exophiala dermatitidis using AFLP.

  Authors: Montarop Sudhadham, A H G Gerrits van den Ende, P Sihanonth, S Sivichai, R Chaiyarat, S B J Menken, A van Belkum, G S de Hoog

  Fungal biology. 10/2011; 115(10):1051-65.

  Distribution of populations of the opportunistic black yeast Exophiala dermatitidis was studied using AFLP. This fungus has been hypothesized to have a natural habitat in association with frugivorousDistribution of populations of the opportunistic black yeast Exophiala dermatitidis was studied using AFLP. This fungus has been hypothesized to have a natural habitat in association with frugivorous birds and bats in the tropical rain forest, and to emerge in the human-dominated environment, where it occasionally causes human pulmonary or fatal disseminated and neurotropic disease. The hypothesis of its natural niche was investigated by comparing a set of 178 strains from natural and human-dominated environments in Thailand with a worldwide selection of 107 strains from the reference collection of the CBS Fungal Biodiversity Centre, comprising 75.7% clinical isolates. Many isolates had unique AFLP patterns and were too remote for confident comparison. Eight populations containing multiple isolates could be distinguished, enabling determination of geographic distributions of these populations. Some of the populations were confined to Thailand, while others occurred worldwide. The local populations from Thailand contained strains from natural and urban environments, suggesting an environmental jump of the fungus. Strains from human brain belonged to widely dispersed populations. In some cases cerebral isolates were identical to isolates from the human intestinal tract. The possibility of cerebral infection through intestinal translocation was thus not excluded.

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

  Authors: M J Najafzadeh, V A Vicente, J Sun, J F Meis, G S de Hoog

  Fungal biology. 10/2011; 115(10):1066-76.

  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 byA 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 β-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.

• Physiological typing of Pseudallescheria and Scedosporium strains using Taxa Profile, a semi-automated, 384-well microtitre system.

  Authors: R Horré, K P Schaal, G Marklein, G S de Hoog, S-M Reiffert

  Mycoses. 10/2011; 54 Suppl 3:56-65.

  During the last few decades, Pseudallescheria and Scedosporium infections in humans are noted with increasing frequency. Multi-drug resistance commonly occurring in this species complex interferesDuring the last few decades, Pseudallescheria and Scedosporium infections in humans are noted with increasing frequency. Multi-drug resistance commonly occurring in this species complex interferes with adequate therapy. Rapid and correct identification of clinical isolates is of paramount significance for optimal treatment in the early stages of infection, while strain typing is necessary for epidemiological purposes. In view of the development of physiological diagnostic parameters, 570 physiological reactions were evaluated using the Taxa Profile Micronaut system, a semi-automatic, computer-assisted, 384-well microtitre platform. Thirty two strains of the Pseudallescheria and Scedosporium complex were analysed after molecular verification of correct species attribution. Of the compounds tested, 254 proved to be polymorphic. Cluster analysis was performed with the Micronaut profile software, which is linked to the ntsypc® program. The systemic opportunist S. prolificans was unambiguously separated from the remaining species. Within the P. boydii/P. apiosperma complex differentiation was noted at the level of individual strains, but no unambiguous parameters for species recognition were revealed.

• Reverse line blot hybridisation screening of Pseudallescheria/Scedosporium species in patients with cystic fibrosis.

  Authors: Q Lu, A H G Gerrits van den Ende, G S de Hoog, R Li, I Accoceberry, I Durand-Joly, J-P Bouchara, F Hernandez, L Delhaes

  Mycoses. 10/2011; 54 Suppl 3:5-11.

  The PCR-RLB (reverse line blot hybridisation) was applied as a molecular technique for the detection of members of Pseudallescheria and Scedosporium from sputum of patients with cystic fibrosis (CF).The PCR-RLB (reverse line blot hybridisation) was applied as a molecular technique for the detection of members of Pseudallescheria and Scedosporium from sputum of patients with cystic fibrosis (CF). Fifty-nine sputum samples were collected from 52 CF patients, which were analysed by culture and PCR-RLB. Conventional and semi-selective culture yielded five positive samples, but the PCR-RLB hybridisation assay permitted the detection of members of Pseudallescheria/Scedosporium in 32 out of 52 patients (61.5%). In total, PCR-RLB yielded 47 positives. Pseudallescheria apiosperma was detected in 20 samples, while Pseudallescheria boydii and Pseudallescheria aurantiacum were detected in 17 and eight samples, respectively. Six samples gave a positive reaction with two distinct species-specific probes and one sample with three probes. In conclusion, the PCR-RLB assay described in this study allows the detection of Scedosporium spp. in CF sputum samples and the identification of Pseudallescheria apiosperma, P. boydii, S. aurantiacum, Scedosporium prolificans and Pseudallescheria minutispora.

• Molecular identification of Penicillium marneffei using rolling circle amplification.

  Authors: Jiufeng Sun, M J Najafzadeh, Junmin Zhang, V A Vicente, Liyan Xi, G S de Hoog

  Mycoses. 09/2011; 54(6):e751-9.

  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 rollingPenicillium 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.

  Authors: M J Najafzadeh, J Sun, V A Vicente, G S de Hoog

  Mycoses. 09/2011; 54(5):e577-82.

  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,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.

• Black yeast-like fungi in skin and nail: it probably matters.

  Authors: D M Saunte, B Tarazooie, M C Arendrup, G S de Hoog

  Mycoses. 07/2011; 55(2):161-7.

  Black yeast-like fungi are rarely reported from superficial infections. We noticed a consistent prevalence of these organisms as single isolations from mycological routine specimens. To investigateBlack yeast-like fungi are rarely reported from superficial infections. We noticed a consistent prevalence of these organisms as single isolations from mycological routine specimens. To investigate the prevalence of black yeast-like fungi in skin, hair and nail specimens and to discuss the probability of these species to be involved in disease. Slow-growing black yeast-like fungi in routine specimens were prospectively collected and identified. A questionnaire regarding patient information was sent to physicians regarding black yeast-like fungus positive patients. A total of 20 746 dermatological specimens were examined by culture. Black yeast-like fungi accounted for 2.2% (n = 108) of the positive cultures. Only 31.0% of the samples, culture positive for black yeast-like fungi were direct microscopy positive when compared with overall 68.8% of the culture positive specimens. The most prevalent species were Phialophora europaea (n = 29), Coniosporium epidermidis (n = 12), Ochroconis cf. humicola (n = 6) and Cladophialophora boppii (n = 4). These are not common saprobes and thus less likely to be coincidental colonizers. In 10/30 cases, discolouration of nail/skin had been noticed. A limited number of black yeast-like fungi were repeatedly isolated from routine specimens suggesting that they may play a role in superficial infections or as colonizers.

• Melanization of a meristematic mutant of Fonsecaea monophora increases tolerance to stress factors while no effects on antifungal susceptibility.

  Authors: Jiufeng Sun, Junmin Zhang, M J Najafzadeh, Hamid Badali, Xiqing Li, Liyan Xi, G S de Hoog

  Mycopathologia. 06/2011; 172(5):373-80.

  Melanin is a complex polymer, which is widely distributed in nature, and is known as an important virulence factor in opportunistic and pathogenic fungi. In this study, three melanin mutants ofMelanin is a complex polymer, which is widely distributed in nature, and is known as an important virulence factor in opportunistic and pathogenic fungi. In this study, three melanin mutants of Fonsecaea monophora from a case of chromoblastomycosis were generated from a parent strain that lacked hyphal morphology but was meristematic instead. Two albino mutants, one of which (CBS 125187) produced secreted melanin and another (CBS 125149) lacked melanin, grew faster than a mutant with cell-wall-associated and secreted melanin (CBS 125188) and than the meristematic parent strain (CBS 122845) (P < 0.05). The albino strains were also more sensitive to low pH, high UV radiation, and oxidative stress (P < 0.05). However, susceptibility testing against eight antifungal agents showed no statistical difference (P > 0.05). The discovery of three melanin mutants of a single meristematic mutant provided an alternative way to study the role of cell-wall-associated and secreted melanins in the pathogenesis of black fungi.

• Microdilution in vitro antifungal susceptibility of Exophiala dermatitidis, a systemic opportunist.

  Authors: H Badali, G S de Hoog, M Sudhadham, J F Meis

  Medical mycology : official publication of the International Society for Human and Animal Mycology. 05/2011; 49(8):819-24.

  The in vitro activities of eight antifungal agents were determined against clinical (n = 63 genotype A, n = 3 genotype B) and environmental (n = 2 genotype A, n = 13 genotype B) strains of ExophialaThe in vitro activities of eight antifungal agents were determined against clinical (n = 63 genotype A, n = 3 genotype B) and environmental (n = 2 genotype A, n = 13 genotype B) strains of Exophiala dermatitidis. The resulting MIC(90)s for all strains (N = 81) were, in increasing order, as follows: posaconazole, 0.125 μg/ml; itraconazole, 0.25 μg/ml; voriconazole, 0.5 μg/ml; amphotericin B, 0.5 μg/ml; isavuconazole, 1 μ/ml; caspofungin, 8 μg/ml; anidulafungin, 8 μg/ml and fluconazole, 16 μg/ml. There were no significant differences in the patterns of susceptibility between genotypes A and B, environmental and clinical strains, isolates recovered from cutaneous and deep locations and strains from different geographical areas (P > 0.05). The difference in the MIC(90)s between each of these groups was not more than one dilution. The present study demonstrated that, based on in vitro activity, posaconazole and itraconazole have the highest activity against this fungus. In addition, voriconazole and the experimental broad-spectrum antifungal triazole, isavuconazole, both of which are available as intravenous preparations, have adequate activity against E. dermatitidis. However, in vivo efficacy remains to be determined.

• Exophiala oligosperma involved in a refractory chronic rhinosinusitis.

  Authors: H Badali, M T Hedayati, M Bahoosh, A Kasir, M Ghasemi, J Motahari, J F Meis, G S De Hoog

  European review for medical and pharmacological sciences. 03/2011; 15(3):319-23.

  Fungal rhinosinusitis refers to a wide variety of conditions caused by fungal infections of the paranasal sinuses. Allergic fungal rhinosinusitis and sinus fungus balls are mostly noted in healthyFungal rhinosinusitis refers to a wide variety of conditions caused by fungal infections of the paranasal sinuses. Allergic fungal rhinosinusitis and sinus fungus balls are mostly noted in healthy individuals. Aspergillus species are supposed to be the most common etiologic agents of the disorder, but melanized fungi also occur, and these potentially are able to lead to fatal dissemination into brain parenchyma. We report on a case of fungus ball in a 20-year-old female with refractory chronic rhinosinusitis (RCRS) and bronchial asthma due to the black yeast Exophiala oligosperma which was confirmed by mycological and molecular (sequences of ITS rDNA) investigations. Exophiala oligosperma has previously not been reported to cause fungus balls or invasive fungal rhinosinusitis. Patient underwent functional endoscopic sinusitis surgery and the hypertrophic mucosa was removed completely. Without antifungal therapy, successful cure was achieved after spray therapy with corticosteroids for 1 month, without any relapse after a 6 month-follow up.

• Acremonium phylogenetic overview and revision of Gliomastix, Sarocladium, and Trichothecium.

  Authors: R C Summerbell, C Gueidan, H-J Schroers, G S de Hoog, M Starink, Y Arocha Rosete, J Guarro, J A Scott

  Studies in mycology. 01/2011; 68:139-62.

  Over 200 new sequences are generated for members of the genus Acremonium and related taxa including ribosomal small subunit sequences (SSU) for phylogenetic analysis and large subunit (LSU) sequencesOver 200 new sequences are generated for members of the genus Acremonium and related taxa including ribosomal small subunit sequences (SSU) for phylogenetic analysis and large subunit (LSU) sequences for phylogeny and DNA-based identification. Phylogenetic analysis reveals that within the Hypocreales, there are two major clusters containing multiple Acremonium species. One clade contains Acremonium sclerotigenum, the genus Emericellopsis, and the genus Geosmithia as prominent elements. The second clade contains the genera Gliomastixsensu stricto and Bionectria. In addition, there are numerous smaller clades plus two multi-species clades, one containing Acremonium strictum and the type species of the genus Sarocladium, and, as seen in the combined SSU/LSU analysis, one associated subclade containing Acremonium breve and related species plus Acremonium curvulum and related species. This sequence information allows the revision of three genera. Gliomastix is revived for five species, G. murorum, G. polychroma, G. tumulicola, G. roseogrisea, and G. masseei. Sarocladium is extended to include all members of the phylogenetically distinct A. strictum clade including the medically important A. kiliense and the protective maize endophyte A. zeae. Also included in Sarocladium are members of the phylogenetically delimited Acremonium bacillisporum clade, closely linked to the A. strictum clade. The genus Trichothecium is revised following the principles of unitary nomenclature based on the oldest valid anamorph or teleomorph name, and new combinations are made in Trichothecium for the tightly interrelated Acremonium crotocinigenum, Spicellum roseum, and teleomorph Leucosphaerinaindica. Outside the Hypocreales, numerous Acremonium-like species fall into the Plectosphaerellaceae, and A. atrogriseum falls into the Cephalothecaceae.

• Rapid identification and characterization of Penicillium marneffei using multiplex ligation-dependent probe amplification (MLPA) in paraffin-embedded tissue samples.

  Authors: Jun-Min Zhang, Jiu-Feng Sun, Pei-Ying Feng, Xi-Qing Li, Chang-Ming Lu, Sha Lu, Wen-Ying Cai, Li-Yan Xi, G S de Hoog

  Journal of microbiological methods. 01/2011; 85(1):33-9.

  Penicillium marneffei infection is a deadly disease and early diagnosis leads to prompt and appropriate antifungal therapy. To develop a sensitive method to diagnose P. marneffei infection, aPenicillium marneffei infection is a deadly disease and early diagnosis leads to prompt and appropriate antifungal therapy. To develop a sensitive method to diagnose P. marneffei infection, a multiplex ligation-dependent probe amplification (MLPA) assay was adapted. This method can rapidly and specifically detect P. marneffei DNA in cultured cells and paraffin-embedded tissue samples. Three pairs of probes were designed for amplifying the internally (intergenic) transcribed spacer (ITS) region of P. marneffei rRNA using a systematic phylogenetic analysis. These three probe sets produced three amplicons of 198, 166, and 152 bp, respectively, specific for P. marneffei. In contrast, there was only one 198 bp amplicon produced for Talaromyces stipitatus, and one 152 bp amplicon for P. funiculosum, T. intermedius and T. derxii. The probes did not amplify any other reference strains. An array of 40 P. marneffei strains isolated from human patients, bamboo rat, and the local environment was tested by using MLPA, and all were positively identified. Most importantly, P. marneffei in paraffin-embedded tissue specimens from infected human patients was positively amplified by MLPA. The sensitivity and specificity of the MLPA assay could be a useful tool for prompt diagnosis, pathogen characterization, and epidemiological studies of fungal infections.

• Molecular identification of Clonorchis sinensis and discrimination with other opisthorchid liver fluke species using multiple Ligation-depended Probe Amplification (MLPA).

  Authors: Jiufeng Sun, Jin Xu, Pei Liang, Qiang Mao, Yan Huang, Xiaoli Lv, Chuanhuan Deng, Chi Liang, G S de Hoog, Xinbing Yu

  Parasites & vectors. 01/2011; 4:98.

  Infections with the opisthorchid liver flukes Clonorchis sinensis, Opisthorchis viverrini, and O. felineus cause severe health problems globally, particularly in Southeast Asia. Early identificationInfections with the opisthorchid liver flukes Clonorchis sinensis, Opisthorchis viverrini, and O. felineus cause severe health problems globally, particularly in Southeast Asia. Early identification of the infection is essential to provide timely and appropriate chemotherapy to patients. In this study we evaluate a PCR-based molecular identification method, Multiplex Ligation-dependent Probe Amplification (MLPA), which allows rapid and specific detection of single nucleotide acid differences between Clonorchis sinensis, Opisthorchis viverrini and O. felineus. Three probe pairs were derived from the Internally Transcribed Spacer 1 (ITS1) of three opisthorchid liver flukes using a systematic phylogenetic analysis. Specific loci were detected in all three species, yielding three amplicons with 198,172 and 152 bp, respectively, while no cross reactions were observed. A panel of 66 C. sinensis isolates was screened using MLPA. All species were positively identified, and no inhibition was observed. The detection limit was 10(3) copies of the ITS gene for the three liver flukes, or about 60 pg genomic DNA for Clonorchis sinensis. Amplification products can be detected by electrophoresis on agarose gel or in a capillary sequencer. In addition, genomic DNA of Clonorchis sinensis in fecal samples of infected rats was positively amplified by MLPA. The flexibility and specificity make MLPA a potential tool for specific identification of infections by opisthorchid liver flukes in endemic areas.

• Identification of Pseudallescheria and Scedosporium species by three molecular methods.

  Authors: Qiaoyun Lu, A H G Gerrits van den Ende, J M J E Bakkers, Jiufeng Sun, M Lackner, M J Najafzadeh, W J G Melchers, Ruoyu Li, G S de Hoog

  Journal of clinical microbiology. 12/2010; 49(3):960-7.

  The major clinically relevant species in Scedosporium (teleomorph Pseudallescheria) are Pseudallescheria boydii, Scedosporium aurantiacum, Scedosporium apiospermum, and Scedosporium prolificans,The major clinically relevant species in Scedosporium (teleomorph Pseudallescheria) are Pseudallescheria boydii, Scedosporium aurantiacum, Scedosporium apiospermum, and Scedosporium prolificans, while Pseudallescheria minutispora, Petriellopsis desertorum, and Scedosporium dehoogii are exceptional agents of disease. Three molecular methods targeting the partial β-tubulin gene were developed and evaluated to identify six closely related species of the S. apiospermum complex using quantitative real-time PCR (qPCR), PCR-based reverse line blot (PCR-RLB), and loop-mediated isothermal amplification (LAMP). qPCR was not specific enough for the identification of all species but had the highest sensitivity. The PCR-RLB assay was efficient for the identification of five species. LAMP distinguished all six species unambiguously. The analytical sensitivities of qPCR, PCR-RLB, and LAMP combined with MagNAPure, CTAB (cetyltrimethylammonium bromide), and FTA filter (Whatman) extraction were 50, 5 × 10(3), and 5 × 10(2) cells/μl, respectively. When LAMP was combined with a simplified DNA extraction method using an FTA filter, identification to the species level was achieved within 2 h, including DNA extraction. The FTA-LAMP assay is therefore recommended as a cost-effective, simple, and rapid method for the identification of Scedosporium species.

• Subcutaneous phaeohyphomycotic cyst caused by Pyrenochaeta romeroi.

  Authors: H Badali, J Chander, N Gulati, A Attri, R Chopra, M J Najafzadeh, S Chhabra, J F G M Meis, G S de Hoog

  Medical mycology : official publication of the International Society for Human and Animal Mycology. 08/2010; 48(5):763-8.

  Pyrenochaeta romeroi is a rare agent of chronic, suppurative subcutaneous infections which ultimately lead to mycetoma. It has only rarely been reported from deep, non-mycetomatous infections. WePyrenochaeta romeroi is a rare agent of chronic, suppurative subcutaneous infections which ultimately lead to mycetoma. It has only rarely been reported from deep, non-mycetomatous infections. We describe a case of a subcutaneous phaeohyphomycotic cyst in a 45-year-old Indian female who suffered from verrucous plaque and a swelling (30 mm in diameter) on the right forearm that gradually increased in size over a period of 3 months. Direct microscopic examination with 10% KOH and histopathological investigation of exudates revealed septate hyphae without granules, the hallmark of mycetoma. The lesion appeared to be a subcutaneous phaeohyphomycotic cyst caused by P. romeroi. The suspected agent was recovered in culture, identified on the basis of morphologic features and its identification confirmed by sequencing of the internal transcribed spacer regions of rDNA. Treatment consisted of surgical excising of the cyst without any antifungal therapy. There was no relapse during a one-year follow-up and the patient was successfully cured. In vitro antifungal susceptibility tests demonstrated that itraconazole (0.5 microg/ml), isavuconazole (0.125 microg/ml) and posaconazole (0.5 microg/ml) had potent activity against this isolate of P. romeroi. High MICs were found with amphotericin B (4 microg/ml), fluconazole (>64 microg/ ml), voriconazole (4 microg/ml) and caspofungin (8 microg/ml). However, their clinical effectiveness in the treatment of P. romeroi infections remains to be evaluated.

• Fonsecaea nubica sp. nov, a new agent of human chromoblastomycosis revealed using molecular data.

  Authors: M J Najafzadeh, J Sun, V Vicente, L Xi, A H G Gerrits van den Ende, G S de Hoog

  Medical mycology : official publication of the International Society for Human and Animal Mycology. 03/2010; 48(6):800-6.

  A new species of Fonsecaea, Fonsecaea nubica, morphologically similar to F. pedrosoi and F. monophora, is described using multilocus molecular data including AFLP profiles, sequences of the ribosomalA new species of Fonsecaea, Fonsecaea nubica, morphologically similar to F. pedrosoi and F. monophora, is described using multilocus molecular data including AFLP profiles, sequences of the ribosomal internal transcribed spacers (ITS), and partial sequences of the cell division cycle (cdc42), beta-tubulin (tub1) and actin (act1) genes. A phylogenetic approach was used to evaluate species delimitation. Topologies of the trees were concordant. Fonsecaea strains could be classified into three major entities, i.e., one representing Fonsecaea pedrosoi isolates, another consisting of strains of F. monophora, and a third, unnamed group comprising isolates mostly recovered from cases of chromoblastomycosis in South America and China. F. nubica is part of this latter group. Based on strains analyzed thus far, we have found that the pathologies of these three Fonsecaea species are somewhat different in that F. pedrosoi and F. nubica are preponderantly associated with chromoblastomycosis, while F. monophora may also act as a systemic opportunist in cases involving brain infections. The latter species is also the most frequently recovered of the three from environmental samples.