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Aphanoascus Fulvescens (Cooke) Apinis The ultimate benchtool for diagnostics. Introduction Introduction of ATLAS Introduction CLINICAL FUNGI Introduction The ultimate benchtool for diagnostics Introduction Introduction Introduction Sample pages Introduction G.S. de Hoog, J. Guarro, J. Gené, S. Ahmed, Introduction A.M.S. Al-Hatmi, M.J. Figueras and R.G. Vitale 1 ATLAS of CLINICAL FUNGI The ultimate benchtool for diagnostics Overview of approximate effective application of comparative techniques in mycology Use Strain Variety Species Genus Family Order Class Keyref Cell wall Tax Kreger & Veenhuis (191) Pore Tax Moore (198) Karyology Tax Takeo & de Hoog (1991) Co- Tax Yamada et al. (198) Carbohydrate pattern Tax eijman & Golubev (198) Classical physiology Tax Yarrow (1998) API 32C Diag Guého et al. (1994b) API-Zym Diag Fromentin et al. (1981) mole% G+C Tax Guého et al. (1992b) SSU seq Tax Gargas et al. (1995) SSU-RFLP Tax Machouart et al. (2006) LSU Diag Kurtzman & Robnett (1998) ITS seq/RFLP Diag Lieckfeldt & Seifert (2000) IGS Epid Diaz & Fell (2000) Tubulin Tax Keeling et al. (2000) Actin Tax Donnelly et al. (1999) Chitin synthase Tax Karuppayil et al. (1996) Elongation factor Diag Helgason et al. (2003) NASBA Tax Compton (1991) nDNA homology Epid Voigt et al. (199) RCA Epid Barr et al. (199) LAMP Tax Guého et al. (199) MLPA Diag Sun et al. (2010) Isoenzymes (MLEE) Epid Pujol et al. (199) Maldi-tof Diag Schrödl et al. (2012) Fish Diag Rigby et al. (2002) RLB Diag Bergmans et al. (2008) PCR-ELISA Diag Beifuss et al. (2011) Secondary metabolites Tax/Diag Frisvad & Samson (2004) SSR Epid Karaoglu et al. (2005) rep-PCR Epid MacDonald et al. (2000) rt-PCR Diag Bergmans et al. (2010) RAPD / UP-PCR Tax Doherty et al. (2003) M-13 Epid/Tax eising et al. (1995) T3B Epid Gäser et al. (2000) Microsatellites Epid/Tax Cai et al. (2013) mtDNA RFLP Epid Ishizaki et al. (1996) AFLP Epid Savelkoul et al. (1999) Karyotyping Epid Franzot et al. (1998) MLST Epid Meyer et al. (2009) AFLP = Amplifi ed Fragment Length Polymorphism; Co-Q = Coenzyme Q; IGS = InterGeneric Spacer; ITS = Internal Transcribed Spacer; LAMP = Loop-medicated Isothermed Amplifi cation; LSU = Large SubUnitMaldi-tof= Matrix-assisted Laser-Desorption/Ionisation time-of-fl ight; MLEE = MultiLocus Enzyme Electrophoresis; MLPA = Multiplex Ligation - Dependent Probe Amplifi cation; MLST = Multi-locus Sequence Typing; NASBA = Nucleic Acid Sequence-Based Amplifi cation; RAPD = Random Amplifi ed Polymorphic DNA; RCA = Rolling Circle Amplifi cation; RFLP = Restric- tion Fragment Length Polymorphism; SSU = Small SubUnit rDNA; UP-PCR = Universally Primed PCR.Diag = Diagnostics; Epid = Epidemiology; Tax = Taxonomy. Recipes of recommended media (all recipes based on 1 litre medium) BCPCG: Bromocresol purple casein glucose agar skimmed milk 80 g bromocresol purple 1% in ethanol 2 mL glucose 40 g agar 30 g pH 6.8 BHI: Brain-heart infusion agar commercial BHI agar 52 g final pH 7.4 CEA: Casamino acids erythritol agar casamino acids (Bacto) 3 g MgSO4 0.1 g KH2PO4 1.8 g meso-erythritol 10 g albumin 10 mL agar 15 g final pH 6.8 CDBT: Creatine dextrose bromothymol blue thymine agar Solution A: 2 ATLAS of CLINICAL FUNGI Clinical pathology fused with secondary skin infections caused by systemic inflammation occurs. According to the appearance of the fungi treated below. causative agent as seen in native preparations, the follow- ing subdivision is made: Dermatophyte infections of the dermis Hyalohyphomycosis. The fungal elements are colourless. Phaeohyphomycosis. The fungal elements are melanised Dermatophytes are normally confined to cutis, hair and (special staining often necessary). nails, but occasionally the dermis is involved. Chromoblastomycosis Introductions Majocchi’s granuloma. Perifollicular granulomatous inflammation. The dermatophyte may be present in the The disease occurs mostly on the extremities and is char- cutis, but also deeper skin layers are involved. acterized by localized, slowly expanding lesions. Superfi- cial, warty to cauliflower-like tumours and deformations Kerion (pseudomycetoma). Subcutaneous, elevated, develop, due to hyperkeratosis and acanthosis. The le- spongy lesion by dermatophytes with local necrosis and sions are greyish, crusted and dry in appearance. The fun- with presence of hyphae or grain-like structures. gus occurs as dark, muriform cells in the tissue. Causative agents: Fonsecaea monophora, F. nubica, F. pedrosoi, Clado- Non-ulcerative infections by diverse fungi phialophora carrionii, Phialophora verrucosa, Rhinocladiella aquaspersa. The following types can be distinguished, of These concern local, non-ulcerative infections caused by which several may occur in the same patient (adapted a variety of fungi. The fungus is present in the form of from Queiroz Telles et al., 2009): septate hyphae or hyphal elements. In the case of hy- Nodular. Moderately elevated, fairly soft, dull to pink phomycotic cysts the affected area is surrounded by a violaceous growth. Surface smooth, verrucous or scaly. fibrous, collagenous secretion of the host, and hence no With time lesions may gradually become tumorous. Main types of histopathological response to subcutaneous and deep infection Subcutaneous Special features Purulence Granuloma Fibrosis Necrosis - + - -,w Hyphae with melanin - + - -,w Hyphae without melanin + + + + Grains - + + - Muriform cells + + + + Asteroid bodies, rabbit ears - + - - ide hyphal elements, oedema Pulmonary + + - + Hyphae without melanin + + + - Yeast cells with wide base + + + - Spherules + + + + Pilot wheel/Mickey Mouse - + + - Intracellular yeast - + + - Adiaspores - + - - Intracellular arthroconidia Rhinoorbital + - - + ide hyphal elements, oedema - + + - Splendore-Hoeppli - + + - Hyphae Systemic yeast infection + + - + Yeast cells, pseudohyphae - + - - Capsular yeast cells Overview of histopathological features in subcutaneous, deep and disseminated infections in immunologically competent individuals: host responses and fungal tissue forms. 3 ATLAS of CLINICAL FUNGI The ultimate benchtool for diagnostics Phylum Basidiomycota The life cycle of a prototypical basidiomycete is depicted. Thethallus consists of a septate, dikaryotic mycelium, often provided with clamp connections; these are bridges between adjacent cells to provide each of them with a nucleus derived from one of the parent strains. The septa are perforated by a single, central pore. The wall of the pore canal is often characteristically swollen; such a structure is called a dolipore. Spores each produce a short-lived, haploid myce- lium. Cells of suitable mating type show plasmogamy, but karyogamy is postponed. Consequently a heterokaryon with clamp connections is formed. This condition is maintained during the major part of the life cycle, including fruitbody production. Karyogamy, immediately followed by meiosis, takes place in the basidium, which produces meiospores (basidiospores) exogenously, the spores often being forcibly discharged. Prototypical life cycle Basidiomycota 4 ATLAS of CLINICAL FUNGI Basidio filamentous Basidio filamentous Bjerkandera adusta, CBS 230.93. A. Fruitbody in natural habitat on rotten wood, left carpophores, right backside with pores; B. colony (MEA, 1 wk, 24°C, blue light), obverse; C. colony (OA, 4 wk, 24°C, blue light), obverse, with fruiting structures formed near colony edge; D-F. details of fruiting structures; G. section of fruiting structure; H-K. basidia with basidiospores; L. arthroconidia; M. clamp connection. Scale bars = 10 µm. 5 ATLAS of CLINICAL FUNGI The ultimate benchtool for diagnostics A. longipes CBS 540.94 A. gossypina CBS 104.32 A. gaisen CBS 632.93 A. arborescens CBS 102605 A. alstroemeriae CBS 118809 A. burnsii CBS 107.38 A. tomato CBS 114.35 Section A. jacinthicola CBS 133751 Alternaria A. doliconidium KUMCC 17-0263 100 A. alternata CBS 916.96 A. betae-kenyensis CBS 118810 A. eichhorniae CBS 489.92 A. iridiaustralis CBS 118486 A. hordeicola CBS 121458 A. montsantina FMR 17060 A. graminicola CBS 119400 A. conjuncta CBS 196.86 A. caespitosa CBS 177.80 A. intercepta CBS 119406 A. cesenica MFLUCC 13-0450 A. novae-zelandiae CBS 119405 A. broccoli-italicae CBS 118485 A. dactylidicola MFLUCC 15-0466 A. fimeti FMR 17110 A. ventricosa CBS 121546 A. quercicola CBS 141466 A. triticina CBS 763.84 A. triticimaculans CBS 578.94 A. merytae CBS 119403 Section A. pseudoventricosa FMR 16900 Infectoriae A. lawrencei FMR 17004 A. alternarina CBS 119396 A. curvata FMR 16901 A. daucicaulis CBS 119398 A. ethzedia CBS 197.86 A. hordeiaustralica CBS 119402 A. infectoria CBS 210.86 A. slovaca CBS 567.66 x3 A. aconidiophora FMR 17111 99 A. metachromatica CBS 553.94 A. arbusti CBS 596.93 A. incomplexa CBS 121330 84 A. oregonensis CBS 542.94 A. pobletensis FMR 16448 97 A. cetera CBS 121340 A. obclavata CBS 124120 Section 99 A. malorum CBS 135.31 Chalastospora A. breviramosa CBS 121331 A. abundans CBS 534.83 94 A. armoraciae CBS 118702 80 A. chlamydospora CBS 491.72 A. phragmospora CBS 274.70 A. limaciformis CBS 481.81 Section A. molesta CBS 548.81 Phragmosporae A. mouchaccae CBS 119671 A. papavericola CBS 116606 A. penicillata CBS 116608 0.01 Maximum Likelihood (ML) tree of four sections of the genus Alternaria based on confidently aligned rDNAITS sequences using ClustalW. The sub- stitution model is Kimura 2-parameter and gamma distributed (K2+G). The tree was bootstrapped 1000 times and values above 80% are indicated near of the nodes and with thick branches. The tree is rooted withAlternaria papavericola and A. penicillata. Note that the general barcoding marker ITS does not allow species
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