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provided by Universidade do Minho: RepositoriUM 25 Chrysosporium

Dongyou Liu and R.R.M. Paterson

Contents 25.1 Introduction...... 197 25.1.1 Classification and Morphology...... 197 25.1.2 Clinical Features...... 198 25.1.3 Diagnosis...... 199 25.2 Methods...... 199 25.2.1 Sample Preparation...... 199 25.2.2 Detection Procedures...... 199 25.3 Conclusion...... 200 References...... 200

25.1 Introduction thermophilum (→ Myceliophthora thermophila) [6]. Not dis- cussed here are Chrysosporium parvum (or Chrysosporium 25.1.1 Classification and Morphology parvum var. parvum) and Chrysosporium parvum var. cre­ The genus Chrysosporium (obsolete synonym: Gleno­ scens, which are considered as synonymous for Emmonsia sporella) belongs to the mitosporic Onygenales group, parva and Emmonsia crescens, respectively. To simplify the order Onygenales, class Eurotiomycetes, subphylum presentation and reduce confusion, this chapter focuses on Pezizomycotina, phylum Ascomycota, and kingdom Fungi. Chrysosporium spp. that are keratinophilic, causing subcuta- The mitosporic Onygenales group consists of nine genera: neous infections, whereas Chapter 29 deals with Emmonsia Blastomyces, Chrysosporium, Coccidioides, Emmonsia, spp. that mainly cause pulmonary adiaspiromycosis. Geomyces, Locazia, Malbranchea, Myriodontium, and Chrysosporium colonies grow moderately at 25°C, and AQ1 Paracoccidioides. In turn, the genus Chrysosporium is may appear granular, woolly, or cottony, flat, or raised, and separated into 28 recognized species: Chrysosporium folded. Colonies are white cream, yellow, or tan to pale brown articulatum, Chrysosporium carmichaelii, Chrysosporium on the front, and white to brown on the reverse. Hyphae are chiropterorum, Chrysosporium europa, Chrysosporium septate. Conidia (aleuriconidia) are hyaline, broad based, one evolceanui, Chrysosporium filiforme, Chrysosporium flu­ celled, and smooth or rough walled. Conidia are broader than viale, Chrysosporium fluviale, Chrysosporium indicum, vegetative hyphae and occur terminally on pedicels, along Chrysosporium keratinophilum, Chrysosporium lobatum, the sides of the hyphae, or in intercalary positions. Conidia Chrysosporium lucknowense, Chrysosporium mephiticum, usually have an annular frill that is the remnant of the Chrysosporium merdarium, Chrysosporium minutispo­ hyphal wall that remains after detachment from the hypha. rosum, Chrysosporium ophiodiicola, Chrysosporium Arthroconidia are abundant and larger than parent hyphae pannicola, Chrysosporium pilosum, Chrysosporium in diameter. pseudomerdarium, Chrysosporium queenslandicum, C. tropicum colonies are moderately fast growing, flat, Chrysosporium siglerae, Chrysosporium submersum, white to tan to beige, often with a powdery or granular sur- Chrysosporium sulfureum, Chrysosporium synchronum, face texture. Reverse pigment is absent or pale brownish Chrysosporium tropicum, Chrysosporium undulatum, yellow with age. Ameroconidia are hyaline, one celled, and Chrysosporium vallenarense, Chrysosporium xerophilum, produced directly on vegetative hyphae by nonspecialized and Chrysosporium zonatum, and 18 unassigned species conidiogenous cells. Conidia (6–7 μm × 3.5–4 μm) are typi- [1–3]. The teleomorphs of Chrysosporium spp. are found in cally pyriform to clavate with truncate bases and are formed the genera Aphanoascus, Nannizziopsis, and Uncinocarpus, either intercalary (arthroconidia), laterally (often on pedi- family Onygenaceae, and order Onygenales [4,5]. cels), or terminally. The obsolete species in the genus include Chrysosporium Chrysosporium zonatum (synonym: C. gourii; teleo- dermatitidis (→ Blastomyces dermatitidis), Chrysosporium morph: Uncinocarpus orissi) colonies on potato dextrose pannorum (→ Geomyces pannorus), Chrysosporium pru­ agar (PDA) at 37°C are flat and coarsely powdery, and appear AQ2 inosum (→ Sporotrichum pruinosum), and Chrysosporium yellowish white initially but darken by 14–21 days to buff

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(grayish or brownish orange) with a light brown reverse. colonies are similar to those on PCA, with a very restricted Colony topography and color on PDA are similar at 37°C and growth at 15°C (5 mm in diameter in 14 days). At 37°C, there 25°C, but darkening of the colony obverse and growth rate is no growth. Colonies produce a strong, pungent (skunk-like) are slightly faster at 37°C (73 mm in diameter after 14 days) odor after 1 month of incubation in all the media tested. The than at 25°C (66 mm in diameter). Microscopically, the fun- fungus shows a strong keratinolytic activity. Phenotypically, gus forms solitary aleurioconidia that are borne at the ends of C. ophiodiicola is separated from the Chrysosporium ana- short, typically curved stalks or that are sessile (borne on the morph of Nannizziopsis vriesii by the absence of asperulate sides of the hyphae). Conidia (3.5–13 μm × 2.5–5 μm) are sin- fertile hyphae and globose-to-pyriform conidia sometimes gle celled, rarely two celled, smooth to slightly roughened, grouped in clusters and the presence of an odor in the colo- and clavate (club shaped) to broadly obovoid (egg shaped) nies by C. ophiodiicola [3]. and have a rounded tip and a broad, flat basal scar. Intercalary Chrysosporium species are differentiated from each other arthroconidia may be formed but are uncommon. Racquet by the texture of the colony and morphology, location, and size hyphae (hyphae showing swellings near the septa) are com- of the conidia. Chrysosporium zonatum differs from other mon [4]. The teleomorph of Chrysosporium zonatum is a members of the genus Chrysosporium by its faster growth heterothallic ascomycete Uncinocarpus orissi (synonyms, at 37°C than at 25°C and by forming darken to buff colo- Pseudoarachniotus orissi and Gymnoascus arxii) (fam- nies, and clavate, broadly truncate aleurioconidia typically ily Onygenaceae). U. orissi ascocarps are solitary, globose, borne on short, curved stalks. Chrysosporium queenslandi­ and reddish brown and are composed of pale reddish brown cum (teleomorph: Uncinocarpus queenslandicus, or Apinisia ascospores surrounded by thin-walled hyaline racket hyphae queenslandica and Brunneospora reticulata) is similar, but and conidia. Pairing of C. zonatum with U. orissi produces its colonies do not darken, and intercalary arthroconidia are ascoma-containing ascospores that are oblate (like flattened common [4]. disks) with truncate ends and appear smooth to slightly pitted C. ophiodiicola is separated from C. mephiticum by the (punctate) [4]. narrow, cylindrical-to-slightly clavate conidia of C. ophio­ Chrysosporium ophiodiicola colonies attain diameters of diicola, and the pyriform-to-subglobose conidia of C. 27–29 mm in 14 days at 25°C on potato carrot agar (PCA; mephiticum; C. ophiodiicola is differentiated from A. mephi­ 20 g potato, 20 g carrot, 15 g agar, 1 L water). Colonies are talis by the production of teleomorph during the culture of white from front and uncolored on reverse, felty, plane, A. mephitalis; and C. ophiodiicola is distinguished from and fimbriate, with a poorly defined margin. Sparse tufts Chrysosporium europae by the characteristic vinaceous, of aerial mycelium are present on the submarginal zone. buff-pigmented colonies on phytone-yeast extract agar and Vegetative hyphae (1.5–2.5 μm wide) are hyaline, branched, the absence of a strong, pungent odor of/from C. europae. septate, smooth, and thin walled. They are often disarticu- Some species such as Chrysosporium pannicola do not grow lated at maturity to form cylindrical arthroconidia (7.5– at 37°C. Chrysosporium differs from Blastomyces by being 10 μm × 2–3 μm) adjacent to each other. Fertile hyphae arise nondimorphic, from Microsporum and Trichophyton by as lateral branches. Terminal and lateral conidia are borne lacking macroconidia, from Geomyces by lacking branched, on straight or flexuous side branches of variable length (4.5– fertile hyphae on erect conidiophores, and from Sepedonium 16 μm) or are sometimes sessile. Conidia (4–9 μm × 2–3 μm) by having hyaline conidia. are unicellular, solitary, thin walled, smooth, hyaline to pale yellow, and cylindrical to slightly clavate and are released 25.1.2 Clinical Features by rhexolytic dehiscence, with broad and long basal scars. Intercalary, solitary conidia are often present, similar to the Chrysosporium spp. are soil saprophytes with broad distri- terminal and lateral ones. Racquet hyphae are scarce, and bution. They have been isolated in soil, plant material, dung, chlamydospores are not observed. On potato flake agar at and birds [6–8]. These organisms may enter hosts through 23°C, colonies are white to pale yellow, with a similarly airborne conidia and exposure to soil. Many Chrysosporium colored reverse side, are velvety to granular with age, and spp. are keratinophilic filamentous fungi involved in the produce a strong, pungent odor. Conidia are borne on stalks breakdown of shed keratinous substrates, and may cause as well as arthroconidia. On PDA (Difco Laboratories), C. skin infections and onychomycosis in humans [9–12]. ophiodiicola grows more quickly and produces denser colo- In addition, Chrysosporium species have been occasion- nies of 31–35 mm in diameter in 14 days at 25°C. Colonies ally associated with disseminated human mycosis, affecting are white to pale yellow, buff after 1 month, and powdery, the brain, lungs, sinuses, liver, and kidneys and leading to with droplets of colorless or light yellow exudates at the sinusitis, pneumonia, pleuritis, pericarditis, and osteomyeli- periphery. On phytone-yeast extract agar (BBL), colonies tis [13–15]. measure 32–39 mm in diameter in 14 days at 25°C and are Roilides et al. [4] reported the first case ofChrysosporium white and light yellow at the center, powdery, and dense, with zonatum infection in a 15-year-old boy with X-linked the presence of droplets of colorless exudate at the center and chronic granulomatous disease. The patient devel- a light brown on the reverse side. On oatmeal agar (30 g oat oped a lobar pneumonia and tibia osteomyelitis after a 2

flakes, 1 g MgSO4·7H2O, 1.5 g KH2PO4, 15 g agar, 1 L water), month prophylactic therapy with γ-interferon. The patient

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presented with pain in his right shoulder and distal part of 25.2 Methods his right tibia, ­infrequent cough, and fever. A computed tomography (CT) scan of the chest showed a well-demar- 25.2.1 Sample Preparation cated large mass in the right lower lobe, enlarged left hilar Chrysosporium and other fungal isolates are revived from lymph nodes, and lingular pneumonitis. A tibia x ray was either freeze-dried or frozen (vapor phase of liquid nitrogen) diagnostic for osteomyelitis. A biopsy of the tibia lesion stock and grown at 25°C on petri plates containing pablum revealed granulomatous tissue and a few short and thick cereal agar for 14–21 days. Blocks (1 × 1 cm) of mycelium and hyphae. Chrysosporium zonatum was grown from sputum agar from cultures of Chrysosporium species are excised from and biopsy specimens, which was identified by its thermo- the culture plates and transferred to sterile snap-cap polypro- tolerance, darkening colonies ­(yellowish white to buff) and pylene tubes (12 mm × 75 mm; Fisher Scientific). The myce- club-shaped terminal aleurioconidia borne at the ends of lial blocks are freeze-dried by using an Edwards Moduylo short, curved stalks. Therapy with amphotericin B, itra- freeze-dryer. Freeze-dried blocks of agar and Chrysosporium conazole, and then liposomal amphotericin B subdued the mycelium (100 mg) are placed in 1.5 mL tubes and ground to osteomyelitis, pneumonia, pericarditis, and pleuritis. a fine powder by using a 200-μL capacity pipettor tip. The Guerrero Palma et al. [15] described a case of inva- fungal material is rehydrated with 500 μL of DNA extraction sive sinusal mycosis due to Chrysosporium tropicum in a buffer (50 mM Tris, 10 mM EDTA, 1% sarcosyl, pH 8.0) with patient with acquired immunodeficiency. Levy et al. [16] gentle agitation for 10 min. An equal volume of 1:1 chloro- also reported a case of aggressive fungal rhinosinusitis form–phenol is added to each tube, and mixed by shaking for caused by Chrysosporium sp. in a patient with acute lym- 20 min. Then the aqueous and organic phases are separated in phocytic leukemia. Histopathological and microbiological a microcentrifuge for 5 min at 14,000 × g. The aqueous phase studies permitted the identification of the culprit organ- is pipetted into a clean tube, and 0.1 volume of 3 M sodium ism. In addition, Warwick et al. [17] presented an inva- acetate (pH 6.0) and 1.3 volumes of ethanol are added. The sive Chrysosporium infection in an 18-year-old patient tube is sealed, and the contents are mixed by inverting the after allogeneic sibling bone marrow transplant for T lin- tube several times. Precipitated nucleic acids are pelleted by eage acute lymphoblastic leukemia. The infection started centrifugation at 14,000 × g for 1 min. Ethanol is decanted, as a facial swelling and extended into the central nervous and the pellet is dried by inverting the tube over absorbent system. Despite antifungal treatment, the disease spread paper for 5 min. Nucleic acids are dissolved in 100 μL of TE rapidly, and an autopsy revealed fungal invasion of brain, buffer (10 mM Tris, 1 mM EDTA, pH 8.0), and 250 μL of a lungs, liver, and kidneys. saturated NaI solution and 10 μL of glass milk (Gene Clean kit, Bio 101) are added to the tube. The tube is inverted peri- odically, and DNA is adsorbed to the glass milk for 20 min. 25.1.3 Diagnosis The glass milk is pelleted and rinsed, and the genomic DNA Conventional techniques for the identification of is eluted into 50 μL of 1/10-strength TE. DNA is stored at Chrysosporium spp. and other fungi rely on microscopic −20°C until used [18]. observation of mycotic elements in clinical specimens fol- Alternatively, a loopful of Chrysosporium mycelium is lowed by in vitro culture. Chrysosporium colonial morphol- transferred from SDA to a 1.5 mL tube containing 2.5 mg ogy and growth rate are assessed on PDA at 25°C and 37°C, zymolase (Sigma–Aldrich) in 250 μL zymolyase lysis buf- and tolerance to cycloheximide is tested by measuring the fer [10 mM Tris/HCl (pH 8.0), 1 mM EDTA] and incubate growth rate at 25°C on Mycosel agar (Becton Dickinson). for 45 min at 37°C. Subsequently, 200 μL is transferred to Microscopic features of Chrysosporium isolates are exam- another tube for DNA extraction using Qiagen kit, resulting ined in slide culture preparations with pablum cereal agar in 100 μL of DNA extract [20]. (pablum precooked mixed cereal 10%, agar 1.5%). The abil- ity of Chrysosporium isolates to digest hairs in the hair per- foration assay is diagnostic. 25.2.2 Detection Procedures Molecular methods for the determination of Pounder et al. [19] described a real-time PCR with SYBR Chrysosporium identity include sequencing analysis of green DNA-binding dye and amplicon melting temperature small subunit (SSU) and large subunit (LSU) of ribosomal analysis for fungal detection using pan-fungal primers ITS1 AQ3 RNA genes as well as internal transcribed spacer (ITS) forward (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 regions [3,18,19]. PCR amplification of ITS2 using primers reverse (5′-TCCTCCGCTTATTGATATGC-3′). The identity ITS86 (5′-GTGAATCATCGAATCTTTGAAC-3′) and ITS4 of the fungi is verified by subsequent sequencing analysis. (5′-TCCTCCGCTTATTGATATGC-3′), restriction digestion with BstUI (CG/CG), and restriction fragment length poly- Procedure morphism (RFLP) assessment by capillary electrophoresis permit discrimination of Chrysosporium from dermatophyte 1. PCR mixture is composed of 1× Lightcycler FastStart fungi such as Arthroderma, Epidermophyton, Microsporum, DNA Master Hybridization Probes mixture (Roche and Trichophyton [20]. Applied Science) (containing deoxynucleoside

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triphosphates), FastStart Taq DNA polymerase, Microscopic observation of mycotic elements in clinical

and 1 mM MgCl2 (additional MgCl2 is added to a specimens and in vitro isolation of related fungal strains have final concentration of 4.6 mM), 0.4 μM each of ITS1 been the main approaches for identification and ­diagnosis of forward and ITS4 reverse primers, 1× SYBR green Chrysosporium infections. More recently, molecular tech- (Molecular Probes), and 3 μL template DNA. niques such as PCR, sequencing, and RFLP analysis have 2. Thermal cycling parameters include 95°C for been developed and applied for improved determination 10 min; 50 cycles of 95°C for 5 s, 60°C for 20 s, and of Chrysosporium and other fungal organisms. Through 76°C for 30 s; and a final extension at 72°C for 2 min. examination of nucleotide sequences in the SSU and LSU 3. The quality of the amplicon is determined using the of ribosomal RNA genes as well as ITS regions, the iden- derivative of the melt analysis curve (55°C–99°C, tity of Chrysosporium spp. can be rapidly and unequivocally 45-s hold at 55°C, 5 s/°C) using the RotorGene 3000 ascertained. (Corbett Robotics, Inc.). 4. The amplified product is purified for bidirectional References sequencing using ExoSAP-IT (USB Corp.). Five microliters of Big Dye Terminator Ready Reaction 1. http://www.uniprot.org//taxonomy// AQ4 Mix v. 1.1 (Applied Biosystems) is added to 4 μL of 2. Al-Musallam A, Tan CS, Chrysosporium zonatum, a new each primer (0.8 pmol/μL) and 3 μL of purified PCR keratinophilic fungus. Persoonia. 1989;14:69–71. 3. Rajeev S et al., Isolation and characterization of a new fungal product. Cycle sequencing is performed with a 9700 species, Chrysosporium ophiodiicola, from a mycotic granu- thermal cycler (ABI), using 25 cycles of 96°C for loma of a black rat snake (Elaphe obsoleta obsoleta). J Clin 10 s, 50°C for 5 s, and 60°C for 4 min. Sequencing Microbiol. 2009;47(4):1264–1268. reaction products are passed through a Sephadex 4. Roilides E. et al., Disseminated infection due to Chrysosporium G-50 fine column to remove unincorporated dye ter- zonatum in a patient with chronic granulomatous disease and minators. Purified sequencing reaction products are review of non-Aspergillus fungal infections with this disease. run on an ABI Prism 3100 Genetic Analyzer with a J Clin Microbiol. 1999;37:18–25. 50 cm capillary array. 5. Abarca ML et al., Cutaneous hyalohyphomycosis caused by a Chrysosporium species related to Nannizziopsis vrie- 5. Sequences are analyzed with the SmartGene sii in two green iguanas (Iguana iguana). Med Mycol. Integrated Database Network software version 2008;46:349–354. 3.2.3 vr. SmartGene is a web-based software and 6. Chabasse D, De Gentile L, Bouchara JP, Pathogenicity of some database system with reference sequences derived Chrysosporium species isolated in France. Mycopathologia. from the National Center for Biological Information 1989;106(3):171–177. (NCBI) GenBank repository. 7. Qiu WY et al., Fungal spectrum identified by a new slide cul- ture and in vitro drug susceptibility using Etest in fungal kera- titis. Curr Eye Res. 2005;30(12):1113–1120. Note: Sequence-based identifications are defined by percent 8. Singh I, Mishra A, Kushwaha R, Dermatophytes, related identity: species, ≥99%; genus, 93%–99%; and inconclusive, ­keratinophilic and opportunistic fungi in indoor dust of houses ≤93%. and hospitals. Indian J Med Microbiol. 2009;27(3):242–246. For strains producing discrepant identification 9. Gan GG et al., Non-sporulating Chrysosporium: An oppor- between the methods based on phenotypic characteris- tunistic fungal infection in a neutropenic patient. Med J tics and ITS sequence analysis, the D1–D2 region of the Malaysia. 2002;57(1):118–122. large-subunit RNA gene is amplified with primers NL1 10. Maghazy S, Incidence of dermatophytes and cyclohexamide resistant fungi on healthy children hairs and nails in nurseries. (5 -GCATATCAATAAGCGGAGGAAAAG-3 ) and NL4 ′ ′ Mycopathologia. 2002;154(4):171–175. (5′-GGTCCGTGTTTCAAGACGG-3′) and sequenced for 11. Stebbins WG, Cutaneous adiaspiromycosis: A distinct derma- species clarification [3]. tologic entity associated with Chrysosporium species. J Am Acad Dermatol. 2004;51(5 Suppl):S185–S189. Erratum in: J Am Acad Dermatol. 2004;51(6):1040. 25.3 Conclusion 12. Manzano-Gayosso P et al., Onychomycosis incidence in type 2 diabetes mellitus patients. Mycopathologia. The genus Chrysosporium contains a large number of fila- 2008;166(1):41–45. mentous saprophytic fungi classified in the mitosporic 13. Stillwell WT, Rubin BD, Axelrod JL. Chrysosporium, a new Onygenales group, order Onygenales. The teleomorphs of the causative agent in osteomyelitis. A case report. Clin Orthop Chrysosporium genus are found in the genera Aphanoascus, Relat Res. 1984;(184):190–192. Nannizziopsis, and Uncinocarpus, family Onygenaceae, 14. Tomsiková A. Causative agents of nosocomial mycoses. Folia and order Onygenales. Many members of the genus Microbiol (Praha). 2002;47(2):105–112. Chrysosporium are thermotolerant and keratinophilic, and 15. Guerrero Palma MA et al., Invasive sinusal mycosis due to Chrysosporium tropicum. Acta Otorrinolaringol Esp. occasionally implicated in human diseases, producing clini- 2007;58(4):164–166. cal symptoms ranging from skin infections, onychomycosis, 16. Levy FE et al., Invasive Chrysosporium infection of the sinusitis, pneumonia, pleuritis, pericarditis, osteomyelitis, to nose and paranasal sinuses in an immunocompromised host. other disseminate infections. Otolaryngol Head Neck Surg. 1991;104(3):384–388.

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17. Warwick A et al., Presumptive invasive Chrysosporium infec- 19. Pounder JI et al., Discovering potential pathogens among tion in a bone marrow transplant recipient. Bone Marrow fungi identified as nonsporulating molds. J Clin Microbiol. Transplant. 1991;8(4):319–322. 2007;45(2):568–571. 18. Peterson SW, Sigler L. Molecular genetic variation in 20. De Baere T et al., Evaluation of internal transcribed spacer Emmonsia crescens and Emmonsia parva, etiologic agents 2-RFLP analysis for the identification of dermatophytes. J of adiaspiromycosis, and their phylogenetic relationship Med Microbiol. 2010;59:48–54. to Blastomyces dermatitidis (Ajellomyces dermatitidis) and other systemic fungal pathogens. J Clin Microbiol. 1998;36(10):2918–2925.

AUTHOR QUERIES [AQ1] Please check if “is separated into” can be changed to “consist of” in the sentence beginning “In turn, the genus…” [AQ2] Please check the insertion of spelled out form of PDA, that is, potato dextrose agar. [AQ3] Please check that intergenic spacer has been replaced by internal transcribed spacer. [AQ4] Please provide accessed date for Ref. [1].

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