48.1 Introduction
48.1.1 Classification and Morphology
48.1.2 Clinical Features
48.1.3 Diagnosis
48.2 Methods
48.2.1 Sample Preparation
48.2.2 Detection Procedures
48.3 Conclusion
Reference

Liu, Dongyou

Paterson, R. R. M.

Universidade do Minho: RepositoriUM

38948.1  IntroductIon48.1.1  ClassifiCation and MorphologyThe genus Chaetomium consists of dematiaceous (dark-walled) fungi classified in the family Chaetomiaceae, order Sordariales, class Sordariomycetes, subphylum Pezizomycotina, phylum Ascomycota, and kingdom Fungi. Of the 35 recognized and 69 unassigned species within the genus, the following are most common: Chaetomium atrobrunneum (obsolete synonym: C. fusisporale and C. rectopilium), Chaetomium funicola, Chaetomium globosum (obsolete synonyms: C. caprophilum and C. cinnamomeum), Chaetomium perlucidum, Chaetomium piluliferum (a teleomorph of Botryotrichum piluliferum), and Chaetomium strumarium (obsolete synonyms: Achaetomium cristalliferum, Achaetomium strumarium, C. spinulosum, and C. sulphureum) [1,2].Chaetomium species are saprobic ascomycetes with worldwide distribution and are commonly found in dung, straw, paper, bird feathers, seeds, plant debris, soil, and air. Some Chaetomium spp. are thermophilic and neurotropic in nature and are among the fungi causing phaeohyphomyco-sis in humans, with clinical presentations ranging from brain abscess, peritonitis, cutaneous lesions, onychomycosis, to fatal deep mycoses.Chaetomium species grow rapidly at between 25°C and 35°C, with some species responsible for invasive human disease growing well at 35°C–37°C, and others showing a predilection for the central nervous system growing at up to 42°C–45°C. Chaetomium colonies are cottony and white in color initially and become gray to olive with age. The colony reverse is tan to red or brown to black. Hyphae are septate. Perithecia are large, dark brown to black, fragile, and glo-bose to flask shaped and have filamentous, hair-like, brown to black appendages (setae) on their surface. Perithecia have ostioles (small-rounded openings) and contain asci and ascospores inside. Asci are clavate to cylindrical in shape and rapidly dissolve to release their ascospores (4 to 8 in number). Ascospores are one celled, olive brown, and lemon shaped [2].Features fundamental to the identification of Chaetomium species include the presence of hairs or setae covering the ascomata, the size and shape of ascospores, and the presence and position of germ pores. Among the similar Chaetomium species (i.e., C. gangligerum, C. raii, C. jodhpurense, and C. fusisporum), C. gangligerum produces broader ascospores (12–15 μm × 7.5–9.5 μm) and usually forms chlamydospores; C. raii has smaller ascospores (10–13 μm × 5.5–7.5 μm), and C. jodhpurense and C. fusisporum have larger ascospores (14–19 μm × 6–8 μm and 14–17 × 7–8 μm, respectively).C. strumarium is distinguished by its formation of ascocarps covered with pale, thin-walled, flexuous hairs, a feature contributing to its original placement in the genus Achaetomium. Presence of pinkish exudate droplets and/or crystals associated with hyphae or ascocarps, good growth at 42°C, and production of small conidia further separate C. strumarium from other species in the Chaetomium genus [3].Furthermore, C. perlucidum produces globose (spherical) to subglobose or ovoid ascomata (108–220 μm × 90–200 μm), with undulate hairs and a wide ostiole (30–50 μm in diame-ter) and eight-spored asci (20–40 μm × 7–18 μm). The asco-spores (12.0–15.0 μm × 6–7.5 μm) are elliptical and olive brown and contain a subapical germ pore. These features help differentiate C. perlucidum from other invasive species of Chaetomium [4].C. globosum is distinguished by its lack of growth at elevated temperatures (<35°C) and its generation of lemon-shaped ascospores.48 ChaetomiumDongyou Liu and R.R.M. Patersoncontents48.1 Introduction ..................................................................................................................................................................... 38948.1.1 Classification and Morphology ............................................................................................................................ 38948.1.2 Clinical Features .................................................................................................................................................. 39048.1.3 Diagnosis ............................................................................................................................................................. 39048.2 Methods ........................................................................................................................................................................... 39048.2.1 Sample Preparation .............................................................................................................................................. 39048.2.2 Detection Procedures ........................................................................................................................................... 39048.3 Conclusion ....................................................................................................................................................................... 391References ................................................................................................................................................................................. 391K10626_C048.indd   389 1/20/2011   9:22:29 AM390	 Molecular	Detection	of	Human	Fungal	Pathogens48.1.2  CliniCal featuresDespite being saprophytic ascomycetes with only occasional involvement in human disease processes, Chaetomium spe-cies are capable of inducing a broad spectrum of mycoses including onychomycosis, sinusitis, empyema, pneumonia, and fatal disseminated cerebral disease, especially in immu-nocompromised patients and intravenous drug users [5–9].Chaetomium atrobrunneum is a notably invasive, neuro-tropic species, and its ability to grow at elevated temperatures may contribute to its neurotropism [10–13]. Thomas et al. [9] described a case of fatal brain abscess due to C. atrobrun-neum in a bone marrow transplant patient. The rapid pro-gression of cerebral infection indicates that the brain tissue provides a favorable environment for growth and prolifera-tion of the fungus.Chaetomium strumarium is another invasive, neurotropic species. Abbott et al. [3] reported three C. strumarium-related cases of fatal cerebral mycosis in males with prior histories of intravenous drug use from the United States and Australia. C. strumarium was detected by histopathology and isolated from the brain tissue.Chaetomium perlucidum is recently confirmed as a neu-rotropic species. Barron et al. [4] documented the first two cases of invasive human mycoses caused by this phaeoid ascomycete. The first case concerned a 45-year-old female patient with acute myelogenous leukemia, who had an unre-lated, 4/5 HLA-matched umbilical cord blood transplant. The patient became disoriented and febrile, and computed tomography of the chest revealed a 3 × 2 cm mass in the right lower lobe. After suffering a massive right-sided intrapa-renchymal hemorrhage, the patient died. Autopsy revealed disseminated invasive fungal infection in the lungs, brain, and myocardium; and cultures from the surgically obtained lung tissue yielded C. perlucidum. The second case involved a 78-year-old female with a history of asthma and chronic bronchiectasis. The patient underwent a lobectomy due to worsening symptoms, and cultures from the lung tissue grew C. perlucidum. The patient showed no further manifestations of disease after the lobectomy.Chaetomium globosum is an occasional agent of onycho-mycosis [14,15]. In addition, Teixeira et al. [16] reported that C. globosum was responsible for a systemic infection with enlargement of the axillary and cervical lymph nodes in a chronic myeloid leukemia patient, who underwent an allo-geneic sibling-matched bone marrow transplant. Culture of the aspirates from both lymph nodes resulted in the growth of C. globosum. The infection was successfully treated with amphotericin B.48.1.3  diagnosisConsiderable similarities exist between mycosis caused by Chaetomium and Aspergillus from radiographical and his-topathological standpoints. A melanin-specific stain (i.e., the Masson-Fontana stain) is helpful reputedly for distinguishing the melanin-containing Chaetomium from most Aspergillus species. However, this is contradicted by reports of melanin from Aspergillus (e.g., Aspergillus fumigatus) [17], and so the information obtained may have limited value in differentiat-ing the causal fungi.In vitro culture techniques offer a slow but valuable way to isolate and propagate Chaetomium organisms for subsequent macroscopic and microscopic characterization. Inoculation of sterilized plant material with ascospore suspension may enhance induction of mature perithecia, leading to the pro-duction of well-developed ascomata on the surface of the substrate.Molecular methods such as (i) PCR and (ii) sequenc-ing analysis of rRNA and internal transcribed spacer (ITS) regions provide an approach for the rapid and accurate iden-tification of Chaetomium species from other fungi.48.2  Methods48.2.1  saMple preparationClinical specimens are examined by microscopy with a mel-anin-specific stain (i.e., the Masson-Fontana stain). However, as mentioned above, a melanin stain may not be particularly useful. Portions of the samples are inoculated on Sabouraud glucose agar with or without antibiotics. The resulting iso-lates are identified on the basis of macroscopical and micro-scopical features.For DNA extraction, a small amount of fungal pellet is suspended in 600 μL extraction buffer (200 mM Tris–HCl, pH 7.5, 25 mM EDTA, 0.5% w/v sodium dodecyl sulfate [SDS], and 250 mM NaCl) and vortexed for 15 s. The mix-ture is then incubated at 100°C for 15 min, chilled on ice for 60 min, and centrifuged at 13,000 rpm for 15 min. The supernatant is transferred to a new tube, and the solution is extracted with phenol–chloroform–isoamyl alcohol (25:24:1 v/v). DNA is precipitated with cold isopropanol (−20°C), air dried, and resuspended in 100 μL distilled water.48.2.2  deteCtion proCeduresArzanlou et al. [18] utilized the universal primers ITS1 and ITS4 to amplify the ITS region of the nuclear ribosomal RNA operon, including the 3′ end of the 18S rRNA gene, the first ITS region (ITS1), the 5.8S rRNA gene, the second ITS region (ITS2), and the 5′ end of 28S rRNA gene. Subsequent sequencing analysis allows identification of fungal organ-isms including Chaetomium species.Procedure 1. PCR mixture (25 μL) is composed of 0.5 U Taq poly-merase (Bioline), 1× PCR buffer, 0.5 mM MgCl2, 0.2 mM of each dNTP, 5 pmol of each primer, and approximately 10–15 ng of fungal genomic DNA. 2. Amplification is performed on a GeneAmp PCR System 9700 (Applied Biosystems) with primary denaturation at 96°C for 5 min; 36 cycles of 96°C for K10626_C048.indd   390 1/20/2011   9:22:30 AMChaetomium	 39130 s, 52°C for 30 s, and 72°C for 60 s; a final exten-sion at 72°C for 7 min. 3. The amplicons are sequenced using BigDye Terminator v. 3.1 (Applied Biosystems,) or DYEnamicET Terminator (Amersham Biosciences) Cycle Sequencing Kits and analyzed on an ABI Prism 3700 (Applied Biosystems). 4. Newly generated sequences are subjected to a Blast search of the NCBI databases, sequences with high similarity are downloaded from GenBank, and comparisons are made on the basis of the alignment of the obtained sequences. 5. Phylogenetic analysis is performed with PAUP (Phylogenetic Analysis Using Parsimony) v. 4.0b10 using the neighbor-joining algorithm with the uncorrected (“p”), the Kimura 2-parameter, and the HKY85 substitution models. Alignment gaps longer than 10 bases are coded as single events for the phy-logenetic analyses; the remaining gaps are treated as missing data. Any ties are broken randomly when encountered. 6. Phylogenetic relationships are also inferred with the parsimony algorithm using the heuristic search option with simple taxon additions and tree bisec-tion and reconstruction (TBR) as the branch-swap-ping algorithm; alignment gaps are treated as a fifth character state and all characters are unordered and of equal weight. Branches of zero length are col-lapsed and all multiple, equally parsimonious trees are saved. 7. Other measures calculated include tree length, con-sistency index, retention index, and rescaled con-sistency index (TL, CI, RI, and RC, respectively). The robustness of the obtained trees is evaluated by 1000 bootstrap replications. Bayesian analysis is performed. The best nucleotide substitution model is determined using MrModeltest v. 2.2. MrBayes v. 3.1.2 is used to perform phylogenetic analyses, using a general time-reversible (GTR) substitution model with inverse gamma rates, dirichlet base frequen-cies, and the temp value set to 0.5.Note. Part of the large-subunit 28S rRNA (LSU) gene may be also amplified with primers LR0R and LR5 followed by sequencing analysis.48.3  conclusIonThe genus Chaetomium consists of a large group of dema-tiaceous (dark-walled) fungi that are present in a variety of environments, including dung, straw, paper, bird feathers, seeds, plant debris, soil, and air. Several Chaetomium spp. are opportunistic human pathogens, causing phaeohypho-mycosis, with clinical manifestations of brain abscess, peri-tonitis, cutaneous lesions, onychomycosis, and fatal deep mycoses. Since conventional methods for identification of Chaetomium spp. require lengthy incubation and demand specialized skill, molecular procedures have been increas-ingly applied for culture-independent detection and differen-tiation of Chaetomium spp. The use of melanin stains alone may be inconclusive.references 1. http://www.uniprot.org/taxonomy/ 2. von Arx, J. A., J. Guarro, and M. J. Figueras. 1986. The ascomycete genus Chaetomium. Beih. Nova Hedwigia 84:1–162. 3. Abbott, S. P. et al. 1995. Fatal cerebral mycoses caused by the ascomycete Chaetomium strumarium. J. Clin. Microbiol. 33:2692–2698. 4. Barron, M. A. et al. 2003. Invasive mycotic infections caused by Chaetomium perlucidum, a new agent of cerebral phaeohy-phomycosis. J. Clin. Microbiol. 41(11):5302–5307. 5. Hoppin, E. C., E. L. McCoy, and M. G. Rinaldi. 1983. Opportunistic mycotic infection caused by Chaetomium in a patient with acute leukemia. Cancer 52:555–556. 6. Anandi, V. et al. 1989. Cerebral phaeohyphomycosis caused by Chaetomium globosum in a renal transplant recipient. J. Clin. Microbiol. 27:2226–2229. 7. Yeghen, T. et al. 1996. Chaetomium pneumonia in a patient with acute myeloid leukaemia. J. Clin. Pathol. 49:184–186. 8. Aru, A., L. Munk-Nielsen, and B. H. Federspiel. 1997. The soil fungus Chaetomium in the human paranasal sinuses. Eur. Arch. Otorhinolaryngol. 254:350–352. 9. Thomas, C. et al. 1999. Fatal Chaetomium cerebritis in a bone marrow transplant patient. Hum. Pathol. 30:874–879. 10. Guarro, J., L. Soler, and M. G. Rinaldi. 1995. Pathogenicity and antifungal susceptibility of Chaetomium species. Eur. J. Clin. Microbiol. Infect. Dis. 14:613–618. 11. Friedman, A. H. 1998. Cerebral fungal infections in the  immunocompromised host: A literature review and a new pathogen—Chaetomium atrobrunneum: Case report—Comment. Neurosurgery 43:1469. 12. Guppy, K. H. et al. 1998. Cerebral fungal infections in the immunocompromised host: A literature review and a new pathogen—Chaetomium atrobrunneum: Case report. Neurosurgery 43:1463–1469. 13. Rock, J. P. 1998. Cerebral fungal infections in the immu-nocompromised host: A literature review and a new patho-gen—Chaetomium atrobrunneum: Case report—Comment. Neurosurgery 43:1469. 14. Stiller, M. J. et al. 1992. Onychomycosis of the toenails caused by Chaetomium globosum. J. Am. Acad. Dermatol. 26:775–776. 15. Lesire, V. et al. 1999. Possible role of Chaetomium globosum in infection after autologous bone marrow transplantation. Intensive Care Med. 25:124–125. 16. Teixeira, A. B. et al. 2003. Phaeohyphomycosis caused by Chaetomium globosum in an allogeneic bone marrow trans-plant recipient. Mycopathologia 156(4):309–312. 17. Youngchim, S. et al. 2004. Production of melanin by Aspergillus fumigatus. J. Med. Microbiol. 53:175–181. 18. Arzanlou, M. et al. 2007. Phylogenetic and morphotaxonomic revision of Ramichloridium and allied genera. Stud Mycol. 58:57–93.AQ1K10626_C048.indd   391 1/20/2011   9:22:30 AMAUTHOR QUERY[AQ1] Please provide accessed date for reference [1].K10626_C048.indd   392 1/20/2011   9:22:30 AM

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