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Molecular Detection of Human Fungal Pathogens Olivaceous Green to Olivaceous Gray with Age; Reverse Is Dark

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Molecular Detection of Human Fungal Pathogens Olivaceous Green to Olivaceous Gray with Age; Reverse Is Dark 18 Pyrenochaeta Dongyou Liu Contents 18.1 . Introduction...................................................................................................................................................................... 145 18.1.1. Classification.and.Morphology.............................................................................................................................145 18.1.2. Clinical.Features...................................................................................................................................................146 18.1.3. Diagnosis...............................................................................................................................................................147 18.2. Methods............................................................................................................................................................................ 148 18.2.1. Sample.Preparation...............................................................................................................................................148 18.2.1.1. Procedure.for.Endophthalmitis.Samples............................................................................................... 148 18.2.1.2. Procedure.for.Keratitis.Samples............................................................................................................ 148 18.2.1.3. DNA.Extraction..................................................................................................................................... 148 18.2.2. Detection.Procedures............................................................................................................................................149 18.2.2.1. Standard.PCR.and.Sequencing.Analysis.of.ITS.Region....................................................................... 149 18.2.2.2. Real-Time.PCR.and.Sequencing.Analysis.of.ITS.Region.....................................................................149 18.3. Conclusion........................................................................................................................................................................ 149 References..................................................................................................................................................................................150 18.1 introdUCtion Nimbya,. Pithomyces,. Pyrenochaeta,. Stagonospora,. Stemphylium,. Ulocladium,. and. Unifilum.. In. turn,. the. Pyrenochaeta.is.dark-walled,.coelomycete.genus.in.the.order. genus. Pyrenochaeta. contains. eight. recognized. spe- Dothideales. that. produces. asexual. fruit. bodies. during. its. cies:. Pyrenochaeta acicola,. Pyrenochaeta gentianicola,. life.cycle..Members.of.this.genus.are.widely.distributed.in. Pyrenochaeta inflorescentiae,. Pyrenochaeta lycopersici,. the.environment,.soil,.wood,.and.plant.debris.and.are.also. Pyrenochaeta nobilis,.Pyrenochaeta romeroi,.Pyrenochaeta encountered.as.plant.pathogens..Several.Pyrenochaeta.spe- terrestris,. and. Pyrenochaeta unguis-hominis,. in. addi- cies.such.as.P. romeroi.and.P. mackinnonii.are.involved.as. tion. to. six. unassigned. species. [1].. The. teleomorphs. of. the. agents.of.chronic,.suppuratives,.and.subcutaneous.infections. Pyrenochaeta. genus. are. found. in. the. genus. Herpotrichia. in.immuno-competent.patients.after.traumatic.implantation,. (obsolete.synonyms:.Herpotrichiopsis.and.Lasiophoma).and. ultimately.leading.to.mycetoma..In.addition,.Pyrenochaeta. possibly.also.in.the.genus.Leptosphaeria,.both.of.the.family. species. are. occasionally. implicated. in. onychomycosis. and. Leptosphaeriaceae.[2,3]. keratitis. as. well. as. deep,. non-mycetomatous. infections.. Pyrenochaeta. spp.. are. saprophytic. fungal. organisms. Although. Pyrenochaeta. is. a. relatively. infrequent. cause. that. inhabit. the. soil. and. plant. debris,. particularly. in. tropi- of. human. diseases. in. comparison. with. black. yeast. genera. cal. and. subtropical. areas.. Many. Pyrenochaeta. spp.. are. Cladophialophora,.Exophiala,.and.Fonsecaea,.in.the.order. pathogenic.to.plants,.and.several.are.among.the.rare.causes. Chaetothyriales,.their.similarity.in.morphological,.biologi- of.human.infections..The.human.pathogenic.Pyrenochaeta. cal,.and.clinical.terms.make.correct.identification.of.these. species. include. Pyrenochaeta keratinophila,. Pyrenochaeta organisms.essential.in.order.to.implement.effective.control. mackinnonii,. Pyrenochaeta romeroi. (obsolete. synonym:. and.prevention.strategies.. Phlenodomus avramii),. and. Pyrenochaeta unguis-hominis. [3].. Pyrenochaeta romeroi. and. Pyrenochaeta mackinnonii. have.been.isolated.from.mycetoma,.containing.soft,.irregu- 18.1.1 ClassifiCation and morPhology lar,.and.black.grains.with.a.subhyaline.center..A.number.of. The . genus. Pyrenochaeta. is. a. dematiaceous. (dark- isolates.previously.recognized.as.Madurella grisea.have.also. walled). filamentous. fungus. belonging. to. the. mito- been.reidentified.as.Pyrenochaeta romeroi.[4]..Pyrenochaeta sporic. Pleosporaceae. group,. family. Pleosporaceae,. unguis-hominis.has.been.isolated.from.the.infected.nails.of. order. Pleosporales,. subclass. Pleosporomycetidae,. class. some. cases.. Pyrenochaeta keratinophila. is. a. new. species. Dothideomycetes,. subphylum. Pezizomycotina,. phylum. that.was.recently.isolated.from.corneal.scrapings.of.a.case.of. Ascomycota,.kingdom.Fungi..The.mitosporic.Pleosporaceae. keratitis.in.Spain.[3]. group. covers. 13. genera:. Alternaria,. Dendryphiella,. Pyrenochaeta. colonies. grow. moderately. rapidly. and. Dendryphion,. Drechslera,. Embellisia,. Exserohilum,. appear.flat,.woolly.to.cottony,.white.initially.and.becoming. 145 © 2011 by Taylor & Francis Group, LLC 146 Molecular Detection of Human Fungal Pathogens olivaceous .green.to.olivaceous.gray.with.age;.reverse.is.dark.. cells. (12–18.×.2–3.5.μm). are. phialidic,. mostly. cylindri- Hyphae.are.septate,.hyaline.to.subhyaline..Pycnidia.(singular. cal.. Conidia. (2–4.×.1–2.μm). are. whitish. in. mass,. ellipsoi- pycnidium,. which. is. a. round. or. flask-shaped. fruiting. body. dal,.wide,.straight.or.slightly.curved,.hyaline,.and.smooth. containing. conidia). are. globose. to. flask. shaped,. ostiolate,. walled.[2]. brown.to.black,.with.setae.(rigid.hair.located.on.the.pycnidia). Although. P. keratinophila. and. P. unguis-hominis. have. arising. from. their. upper. portion.. Phialides. arise. from. the. similar.conidiophores.and.conidia,.they.can.be.distinguished. inner.lining.of.the.pycnidia..Conidia.(2–4.×.1–2.μm).are.one. by.the.color.of.the.colonies.and.the.location.of.the.setae.on. celled,. oval. to. cylindrical,. hyaline,. and. straight. or. slightly. the.pycnidia..P. keratinophila.colonies.on.OA.are.gray.oli- curved.[5,6]. vaceous. to. greenish. olivaceous,. while. P. unguis-hominis. At.the.species.level,.Pyrenochaeta keratinophila.colonies. colonies.are.brown.vinaceous.to.fawn..The.pycnidial.setae.of. reach.a.diameter.of.24–30.mm.on.oatmeal.agar.(OA).at.20°C. P. keratinophila.are.scarce.and.placed.mainly.near.the.ostiole,. for.10.days,.with.even.margin,.and.are.colorless,.flat;.immersed. while.those.of.P. unguis-hominis.are.usually.more.abundant. mycelium. is. pale. gray. olivaceous. to. greenish. olivaceous,. and.more.dispersed.over.the.entire.surface.of.the.pycnidium.. aerial. mycelium. is. concolorous,. diffuse,. woolly,. floccose;. P. keratinophila.also.produces.conidia.from.the.mycelium,.a. reverse.is.olivaceous.gray.to.olivaceous.black..Colonies.reach. feature.unknown.in.P. unguis-hominis.or.any.other.species. a.diameter.of.18–23.mm.on.malt.extract.agar.at.20°C.for.10. of.the.genus.Pyrenochaeta. P. mackinnonii.is.distinguished. days,.with.even.margin.and.buff.color;.immersed.mycelium. from.P. keratinophila.by.having.more.restricted,.raised,.or. is.olivaceous.black.or.brown.vinaceous;.aerial.mycelium.is. wrinkled.colonies..P. romeroi.is.distinguished.by.its.grayish- dense,.felty.to.woolly,.pale.to.olivaceous.gray;.reverse.is.dark. sepia.to.fuscous-black.colonies.and.the.presence.of.discrete. hazel..Superficial.pycnidia.are.abundant..On.OA,.pycnidia. conidiogenous.cells.[3]. (100–400.μm. in. diameter). are. olivaceous. brown. to. almost. black,.globose.or.flask.shaped,.single.or.confluent,.with.one. 18.1.2 CliniCal features to.three.ostioles.(10–25.μm.in.diameter),.displaying.hyaline,. thick-walled. periphyses;. pycnidial. walls. of. textura. angula- The . term. “dematiaceous. fungi”. refers. to. a. heterogeneous. ris. comprise. cells. (4–9.mm. in. diameter). with. dark-brown. group.of.fungal.organisms.with.black.filaments.(dematiaceae). intercellular.material..The.outer.surface.of.the.pycnidial.wall. that.may.cause.opportunistic.superficial.or.deep.infections. displays. scarce,. brown,. slightly. roughened,. septate. setae. in.humans,.producing.a.diverse.range.of.clinical.syndromes. (20–35.×.2.5–4.μm),. with. a. blunt. apex,. mostly. positioned. (collectively. known. as. phaeohyphomycosis,. chromoblasto- near. the. ostiole.. Conidiogenous. cells. (12–23.×.2–3.5.μm). mycosis,.and.mycetoma)..To.date,.about.130.species.belong- arise.from.the.entire.inner.surface.of.the.pycnidial.wall.and. ing. to. 70. dematiaceous. fungal. genera. have. been. shown. to. are.rarely.discrete,.ampuliform.to.doliform,.mostly.cylindri- induce.these.clinical.diseases. cal,.and.integrated.in.conidiophores,.which.are.branched.at. Mycetoma. is. a. highly. debilitating. disease. that. is. char- the.base,.acropleurogenous.(i.e.,.having.terminal.and.lateral.
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