Identification and First Report of Inonotus (Phell
http://www.100md.com
微生物临床杂志 2005年第2期
University of Texas Health Science Center
Audie L. Murphy Division, South Texas Veterans' Health Care System, San Antonio
Baylor College of Medicine
Texas Children's Hospital, Houston, Texas
University of Nebraska Medical Center, Omaha, Nebraska
Forest Products Laboratory, USDA Forest Service, Madison, Wisconsin
Seoul National University, Seoul, Republic of Korea
ABSTRACT
Although isolates of filamentous basidiomycetes can usually be recognized in a clinical laboratory setting, identification is problematic, as they seldom exhibit diagnostic morphological features formed in nature. This paper is the first report of Inonotus (Phellinus) tropicalis inciting human disease and describes the methods used to support the identification.
CASE REPORT
A 21-year-old Hispanic man with X-linked chronic granulomatous disease (CGD) and a long history of bacterial and fungal infections presented after 2 days of painless swelling over the lower back with no history of trauma, travel outside of his lifelong home in Houston, Tex., unusual environmental exposures, or systemic problems. Previous infections had included Aspergillus osteomyelitis of the ribs and vertebral bodies (28), lymphadenitis with Staphylococcus aureus, and cutaneous and systemic infections with Serratia marcescens. Routine X rays were negative, but computed tomography and magnetic resonance imaging studies showed paraspinal and presacral inflammation and extensive sacral bony changes. He had been on chronic daily intravenous antibiotics and maintenance interferon, co-trimoxazole, and itraconazole. Between July 2001 and January 2004, a rapidly growing golden-brown filamentous mold was isolated on seven occasions. Isolation media were incubated between 25 and 30°C and consisted of Sabouraud dextrose agar, Mycosel agar, and brain heart infusion agar supplemented with 5% sheep cells, chloramphenicol, and gentamicin (Becton Dickinson, Cockeysville, Md.). Six of these isolates were forwarded to the Fungus Testing Laboratory, University of Texas Health Science Center (UTHSC) at San Antonio, for identification. Between July 2001 and March 2002, isolates were obtained from a back abscess (UTHSC 01-1419), a back lesion (UTHSC 01-1652), a bone marrow aspirate (UTHSC 02-617), and paraspinal abscess tissue (isolate not accessioned into the UTHSC stock collection). Both the bone marrow aspirate and the paraspinal abscess tissue were positive for hyphal elements histopathologically. From February 2003 to January 2004, the organism was recovered from a paraspinal abscess (UTHSC 03-453), another back wound (UTHSC 03-2972), and the paraspinal area (UTHSC 04-212). The organisms recovered were thought to represent basidiomycetes on the basis of their cultural features (rapid growth and a golden-brown color) and their growth on 10% benomyl agar (58), which paralleled that observed on potato flakes agar (PFA) (42). Somewhat unusual in our experience, however, was the ability of the isolates to grow on media containing cycloheximide. Isolate numbers UTHSC 01-1419, UTHSC 01-1652, and UTHSC 03-2972 have been deposited in the University of Alberta Microfungus Collection and Herbarium, Edmonton, Alberta, Canada, as UAMH 10436, UAMH 10437, and UAMH 10376, respectively. Complete clinical details and therapeutic regimens for this case will be presented elsewhere.
The six isolates submitted to the Fungus Testing Laboratory were initially examined on PFA and benomyl agar prepared in-house and on commercially available cycloheximide agar (Remel, Lenexa, Kans.). When grown on PFA, isolates were woolly and yellowish orange and exhibited rapid growth, filling a 60-mm-diameter plate in 10 days at 25°C (Fig. 1A). Growth also occurred on media containing 10% benomyl and 0.04% cycloheximide at 25 and 35 °C but not at 40°C. Hyphae lacked clamp connections and were either thin walled and hyaline or thick walled and yellow, and 3-week-old cultures produced golden, thick-walled, pointed setal hyphae up to 100 μm in length (Fig. 1B). When morphological and internal transcribed spacer (ITS) region sequence data (described below) indicated that the fungus was a wood-decaying basidiomycete, three isolates, i.e., UTHSC 01-1419, UTHSC 01-1652, and UTHSC 02-617, were sent to the Forest Products Laboratory, Madison, Wis., for further study. The Center for Forest Mycology Research at the Forest Products Laboratory specializes in the study of wood-decaying basidiomycetes. Strains were grown separately on 2% Bacto malt extract agar (MEA; Difco Laboratories, Detroit, Mich.), 0.5% gallic acid agar (GAA), and 0.5% tannic acid agar (TAA) (Mallinckrodt, St. Louis, Mo.) (12) according to standard protocols for cultures of decay fungi (35, 37). GAA and TAA are used in the study of wood-decaying fungi to test for the presence of polyphenol oxidases that are associated with the decay of wood (12). Colony diameters on MEA at 25°C were 43 to 46 mm at 1 week and 83 to 85 mm at 2 weeks. Mats at 2 weeks were moderately thick, raised, and densely woolly, especially around the inoculum. Mats were orange-yellow or yellowish orange with yellowish brown streaks and became thinner and arachnoid to downy toward the margin. The margin was even to slightly uneven and appressed, and the agar beneath the mat was stained a dark brown or reddish brown. No odor was noticed at 2 weeks. Advancing zone hyphae of the margin were 2 to 3.5 μm in diameter, simply septate (as opposed to having a nodose septum or developing clamp connections), and rarely branched, with thin, smooth, and hyaline walls. Aerial hyphae from the mat surface were 1.5 to 3 μm in diameter and simply septate or aseptate with thin to slightly thick and smooth walls that were hyaline at first before becoming yellowish brown. Hyphae growing in the agar were 2 to 3.5 μm in diameter, simple septate, and moderately to frequently branched, with thin, smooth, hyaline to dark-yellow walls. By 4 weeks, numerous irregular swellings were observed on the submerged hyphae (Fig. 2). No growth or reactions were observed on GAA at 1 or 2 weeks; however, a strong reaction (i.e., a dark-brown discoloration of 3 to 4 cm in diameter) was observed on TAA, with colony diameters after 1 and 2 weeks reaching 11 to 15 and 28 to 33 mm, respectively.
Extraction of DNA from the bone marrow isolate, UTHSC 02-617, was done using hyphae collected from a 7-day-old culture on Sabouraud dextrose agar (Remel) after incubation at 30°C according to procedures explained by Henry et al. (19). DNA was purified using the QIAmp blood kit (QIAGEN Inc., Valencia, Calif.) and protocols for crude cell lysates supplied by the manufacturer. Two oligonucleotide fungal primers (ITS 1 and ITS 4) described by White et al. (63) were used for amplification. Primers were synthesized by the University of Nebraska Medical Center Eppley Molecular Biology Core Laboratory. The PCR assay was performed with 5 μl of the test sample in a 50-μl total reaction volume consisting of the following: PCR buffer (20 mM Tris-HCl [pH 8.4], 50 mM KCl); 0.1 mM (each) dATP, dGTP, dCTP, and dTTP; 1.5 mM MgCl2; 0.3 μM concentrations of each primer; and 1.5 U of PlatinumTaq high-fidelity DNA polymerase (Gibco BRL Life Technologies, Gaithersburg, Md.). Thirty-five cycles of amplification were performed in a Stratagene Robocycler model 96 thermocycler after initial denaturation of DNA at 95°C for 4.5 min. Each cycle consisted of a denaturation step at 95°C for 30 s, an annealing step at 50°C for 30 s, and an extension step at 72°C for 1 min, with a final extension at 72°C for 3 min following the last cycle. DNA sequencing was performed with a Perkin-Elmer/ABI model 373 DNA sequencer by following protocols supplied by the manufacturer. The PCR products were directly sequenced using the ITS 1 and ITS 4 primers for the ITS region. The resultant nucleotide sequences were aligned with the MacVector sequence analysis software, version 6.5 (Oxford Molecular Group, Inc., Campbell, Calif.), alignment application. Amplification of the ITS 1-5.8S rRNA-ITS 2 region of the case isolate genome resulted in the production of a 680-bp PCR product. A BLAST search of the NCBI GenBank database resulted in the identification of the top eight sequences producing significant alignments as Inonotus (Phellinus) linteus strains. In the closest match, the identification showed an alignment homology of only 91% (307 of 334 bp) between the database strain and the case isolate.
DNA sequence data for 18S rRNA, 26S rRNA, ITS, and mitochondrial rRNA have frequently been used in recent phylogenetic studies of eukaryotic cells. Among the sequenced genetic material, the ITS, which has the fastest evolutionary speed, is appropriate for the molecular identification or delimitation of fungi and is now commonly used in the systematics of species within a genus (34). A phylogenetic analysis of ITS sequences compared to sequences in the H. S. Jung Inonotus (Phellinus) database, Seoul National University (now registered sequences in GenBank), demonstrated that the clade including the case isolate UTHSC 02-617 (AY641432) was composed of two subclades representing Inonotus (Phellinus) tropicalis and I. (P.) linteus-Phellinus repandus. The former subclade was fully supported by the 100% bootstrap value, suggesting that the isolate is statistically identical to I. tropicalis (Fig. 3). The overall sequence similarity between I. tropicalis CBS 617.89 and UTHSC 02-617 was 97.8%, which was slightly lower than expected for isolates of the same species.
Mitochondrial small-subunit rRNA was amplified with the primers BMS05 and BMS173 described by Hong et al. (21). Subsequent sequence determination and phylogenetic evaluation were also done by following the method of Hong et al. (21). In the phylogenetic analysis of the mitochondrial small-subunit rRNA sequences, the two new sequences of UTHSC 01-1419 (AY598828) and UTHSC 01-1652 (AY598829) were identical, having 100% (582 of 582) similarity in sequence and forming a clade with I. tropicalis with a bootstrap value of 96% (data not shown). When UTHSC 01-1419 and UTHSC 01-1652 were compared with I. tropicalis CBS 617.89 (AY558713), the sequence similarity was 99.2% (512 of 516), suggesting that the causal fungus was actually a strain of I. tropicalis. The strain of I. tropicalis used in the analysis, CBS 617.89 from the Centraalbureau voor Schimmelcultures, was sent to the Centraalbureau voor Schimmelcultures by F. F. Lombard of the Center for Forest Mycology Research, Forest Products Laboratory, Madison, Wis., in 1989. It was collected from hardwood in Tacares, Heredia Province, Costa Rica, on 16 August 1963 by J. L. Lowe; the original collection number is L-13465. It was identified by M. J. Larsen (30).
The nuclear large-subunit rRNA of the casual fungus was also sequenced and analyzed. Extraction of the total DNA of isolates UTHSC 01-1419 and UTHSC 01-1652 and the amplification and sample preparation of the nuclear large-subunit rRNA for sequencing was done according to the procedure described by Greslebin et al. (17). The bone marrow isolate UTHSC 02-617 was not successfully amplified and thus was not evaluated. Prepared samples were sent to the University of Wisconsin Biotechnology Center for sequencing on an Applied Biosystems/Hitachi model 3730 xl DNA analyzer. Nuclear large-subunit rRNAs of UTHSC 01-1419 and UTHSC 01-1652 (AY598826 and AY598827, respectively) were identical and, when compared with I. tropicalis CBS 617.89 (AY059037), differed at only 6 out of 868 bases (99.3% sequence similarity). When both nuclear large-subunit rRNA sequences were added into the multiple sequence alignment of Wagner and Fischer (61) and analyzed with PAUP (59), they formed a clade with I. tropicalis that was fully supported by a 100% bootstrap value (data not shown), strongly indicating that the causal fungus in this investigation was I. tropicalis.
Few filamentous basidiomycetes have been authenticated as human etiologic agents. Two species documented to invade tissue include Schizophyllum commune and Coprinopsis cinerea (formerly Coprinus cinereus). Schizophyllum commune has elicited serious invasive disease in the brain (5, 41), lungs (6, 41, 53), and hard and soft palates (40). It has also been implicated in allergic bronchopulmonary disease (1, 22), chronic sinusitis (4, 25, 33, 45, 51, 52), and less serious disease of the nails (27). Coprinopsis cinerea (anamorph, Hormographiella aspergillata) (14, 18) has been the causative agent in a case of prosthetic valve endocarditis (13, 57) and in a fatal pulmonary infection (60). A Coprinus species was reported to cause a fatal infection in a leukemia patient 5 years after bone marrow transplantation (36). Another basidiomycete thought to cause pulmonary disease is Phanerochaete chrysosporium (anamorph, Sporotrichum pruinosum), although no tissue invasion has been noted (26, 56). Bjerkandera adusta, frequently isolated from pulmonary sites, may also be pathogenic in some settings (16). Fungal infections in patients with CGD are most frequently caused by Aspergillus fumigatus (7). More-recent reports have cited additional etiologic agents. These include the basidiomycete Trichosporon inkin reported by Kenney et al. in 1990 as Sarcinosporon inkin (24); the homothallic ascomycete Pseudallescheria boydii (39); Chrysosporium zonatum, an anamorph of the heterothallic ascomycete Uncinocarpus orisii (44); and other hyphomycetous species, i.e., Acremonium strictum (2), Exophiala (Wangiella) dermatitidis (23), Paecilomyces lilacinus (55), Paecilomyces variotii (8, 62), and a Paecilomyces species (54). To our knowledge, filamentous basidiomycetes have not been reported to occur in patients with CGD. This report adds the wood-destroying I. tropicalis to the list of basidiomycetes previously cited as being invasive and extends the genera of fungal organisms inciting disease in CGD patients.
Filamentous basidiomycetes are being recovered from clinical specimens with increasing frequency, and their identification in this setting remains problematic. González et al. (16), reporting in vitro antifungal susceptibility data for 44 clinical isolates of basidiomycetous fungi, indicated that, in addition to Schizophyllum commune and Coprinopsis cinerea, many arthroconidium-forming molds resembling Bjerkandera adusta were being recovered. Many more isolates, however, remained sterile and failed to produce clamp connections and/or lacked spicules suggestive of Schizophyllum commune and were reported as unidentified basidiomycetes (16). The characteristics that facilitate recognition of a basidiomycete include rapid growth; colonies that are usually white, cream, or yellow-orange pigmented; the presence of clamp connections and/or crystals; the formation of spicules along the hyphae; and mushroom- or basidiocarp-type fruiting bodies (50). Since most clinical isolates are monokaryons, neither clamps nor fruiting structures are produced, and mating studies may be required to induce these diagnostic features. The ability of most basidiomycetes to grow on agar containing 2 to 10 μg of benomyl/ml has also been employed as a screening test, although many ascomycetous fungi grow on this medium as well (58). Several basidiomycetes also fail to grow on media containing cycloheximide, a substrate commonly found in typical mycology laboratories. The isolate presented here was typical of a basidiomycete in that it exhibited rapid filamentous growth, had a yellowish to orange pigmentation, and was tolerant of benomyl. Also typical was the lack of clamp connections and basidiocarps. Somewhat unusual was the ability of the organism to grow equally well on medium containing cycloheximide. Since the organism had been isolated on numerous occasions from a profoundly compromised patient, molecular characterization was pursued.
When the sequence data of the isolates analyzed were compared to ITS and mitochondrial small-subunit rRNA sequences in GenBank, 97.8 and 99.2% matches, respectively, were made with I. tropicalis. In the sequence analysis of nuclear large-subunit rRNAs, they again showed high sequence similarity, i.e., of 99.3%, with I. tropicalis and were positively concluded to be I. tropicalis.
I. tropicalis is a poroid (having pores rather than gills) wood-decaying fungus that is associated with white-rot decay of various woody angiosperms. As is typical of many fungi, I. tropicalis has a complex taxonomic history. First described as Poria rickii Bres. in 1920 (3), it was given the new name Phellinus tropicalis M. Larsen et Lombard in 1988 for nomenclatural reasons (30). Phellinus ferrugineo-velutinus (Henn.) Ryv. is considered by some mycologists to be identical to Phellinus tropicalis (9, 15, 48), and its name is preferred for this taxon, based on priority. However, because the type specimen of Phellinus ferrugineo-velutinus is sterile (11), others prefer the name Phellinus tropicalis. Some mycologists also consider Phellinus flavomarginata (Murr.) Ryv. to be a synonym for Phellinus ferrugineo-velutinus (15, 48), but this is disputed by others (30, 31). I. tropicalis is common in Brazil, the type locality (30, 47), and it is reported also to occur in Mississippi (32), Florida, Georgia (15), Jamaica (46), Guadeloupe (11), Costa Rica (30), Colombia (49), East Africa (48), and Malaya, Johore, and Mawai, Malaysia (9). Phellinus is placed in the family Hymenochaetaceae and is one of the largest basidiomycetous genera of wood-destroying fungi, with over 220 named species worldwide (29). Traditionally, Phellinus is distinguished from Inonotus, a closely related genus, by its hard, woody, perennial fruiting bodies and dimitic hyphal system. Inonotus, in contrast, produces softer, annual fruiting bodies with a monomitic hyphal system. However, the morphological distinction between these two genera has become blurred over time (9, 15). Recent phylogenetic studies show that although the members of Hymenochaetaceae are a well-defined group (20), genera within this family, including Phellinus and Inonotus, are not monophyletic (43, 61). Thus, the taxonomy of Phellinus and allied genera is in a state of flux and is an area of active research (9, 10, 31, 38, 61). Wagner and Fischer's (61) phylogenetic study of Phellinus and Inonotus placed Phellinus tropicalis in the Inonotus sensu stricto clade. Thus, they transferred Phellinus tropicalis to the genus Inonotus. With the data available at this time, the best name for the casual fungus is Inonotus tropicalis; however, because most of the literature on this species refers to it as being of the genus Phellinus, we use both generic names.
This report extends the genera of basidiomycetous fungi inciting human disease and highlights the utility of molecular methods in the identification or verification of these often-unidentifiable molds when conventional taxonomic practices fail. Although the sequences from the case isolates have now been registered in GenBank, the Fungus Testing Laboratory was initially faced with locating individuals with expertise in poroid fungi who may have held genus-specific databases not in the public domain. Private and institutional databases appear to be a growing trend, and this case illustrates their usefulness for the identification of a wood-decaying fungus not previously known as a human etiologic agent.
Nucleotide sequence accession numbers. The following sequences have been deposited in the GenBank database (accession number): UTHSC-01-1419 small subunit rRNA gene (AY598828), UTHSC-01-1419 25S rRNA gene (AY598826), UTHSC-01-1652 small subunit rRNA gene (AY598829), UTHSC-01-1652 25S rRNA gene (AY598827), UTHSC-02-617 18S rRNA gene partial-ITS1complete-5.8S rRNA complete-ITS2 complete-28S rRNA partial (AY641432), and UTHSC-03-2972 18S rRNA gene partial-ITS1 complete-5.8S rRNA complete-ITS2 complete-28S rRNA partial (AY599487).
ACKNOWLEDGMENTS
We thank John Haight for obtaining the large-subunit rRNA sequences and T. Wagner for sharing the nuclear rRNA sequence database.
REFERENCES
Amitani, R., K. Nishimura, A. Niimi, H. Kobayashi, R. Nawada, T. Murayama, H. Taguchi, and F. Kuse. 1996. Bronchial mucoid impaction due to the monokaryotic mycelium of Schizophyllum commune. Clin. Infect. Dis. 22:146-148.
Boltansky, H., K. J. Kwon-Chung, A. M. Macher, and J. I. Gallin.1984. Acremonium strictum-related pulmonary infection in a patient with chronic granulomatous disease. J. Infect. Dis. 149:653.
Bresadola, G. 1920. Selecta mycologia. Ann. Mycol. 18:16-70.
Catalano, P., W. Lawson, E. Bottone, and J. Lebenger. 1990. Basidiomycetous (mushroom) infection of the maxillary sinus. Otolaryngol. Head Neck Surg. 102:183-185.
Chavez-Batista, A., J. A. Maia, and R. Singer. 1955. Basidioneuromycosis on man. An. Soc. Biol. Pernamb. 13:52-60.
Ciferri, R., A. Chavez-Batista, and S. Campos. 1956. Isolation of Schizophyllum commune from sputum. Atti Ist. Bot. Lab. Crittogam. Univ. Pavia 14:118-120.
Cohen, M. S., R. E. Isturiz, H. L. Malech, R. K. Root, C. M. Wilfert, L. Gutmasn, and R. H. Buckley. 1981. Fungal infections in chronic granulomatous disease. The importance of the phagocyte in defense against fungi. Am. J. Med. 71:59-66.
Cohen-Abbo, A., and K. M. Edwards. 1995. Multifocal osteomyelitis caused by Paecilomyces variotii in a patient with chronic granulomatous disease. Infection 23:55-57.
Corner, E. J. H. 1991. Ad Polyporaceas VII. The xanthochroic polypores. Beih. Nova Hedwigia 101:1-175.
Dai, Y.-C. 1999. Phellinus sensu lato (Aphyllophorales, Hymenochaetaceae) in East Asia. Acta Bot. Fenn. 166:1-115.
David, A., and M. Rajchenberg. 1985. Pore fungi from French Antilles and Guiana. Mycotaxon 22:285-325.
Davidson, R. W., W. A. Campbell, and D. J. Blaisdell. 1938. Differentiation of wood-decaying fungi by their reactions on gallic or tannic acid medium. J. Agric. Res. 57:683-695.
De Vries, G. A., R. F. O. Kemp, and D. C. E. Speller. 1971. Endocarditis caused by Coprinus delicatulus, p. 185. C. R. Comun. Abstr. 5th ISHAM Congr. 1971.
Gene, J., J. M. Guillamon, J. Guarro, I. Pujol, and K. Ulfig. 1996. Molecular characterization, relatedness and fungal susceptibility of the basidiomycetous Hormographiella species and Coprinus cinereus from clinical and environmental sources. Antonie Leeuwenhoek 70:49-56.
Gilbertson, R. L., and L. Ryvarden. 1987. North American polypores, vol. 2. Megasporoporia-Wrightoporia. Fungiflora, Oslo, Norway.
González, G. M., D. A. Sutton, E. Thompson, R. Tijerina, and M. G. Rinaldi. 2001. In vitro activities of approved and investigational antifungal agents against 44 clinical isolates of basidiomycetous fungi. Antimicrob. Agents Chemother. 45:633-635.
Greslebin, A., K. K. Nakasone, and M. Rajchenberg. 2004. Rhizochaete, a new genus of phanerochaetoid fungi. Mycologia 96:260-271.
Guarro, J., J. Gene, C. De Vroey, and E. Gueho. 1992. Hormographiella, a new genus of hyphomycetes from clinical sources. Mycotaxon 45:179-190.
Henry, T., P. C. Iwen, and S. H. Hinrichs. 2000. Identification of Aspergillus species using internal transcribed spacer regions 1 and 2. J. Clin. Microbiol. 38:1510-1515.
Hibbett, D. S., and R. G. Thorn. 2001. Basidiomycota: Homobasidiomycetes, p. 121-168. In D. J. McLaughlin, E. G. McLaughlin, and P. A. Lemke (ed.), The Mycota: a comprehensive treatise on fungi as experimental systems for basic and applied research, vol. VII, part B. Systematics and evolution. Springer-Verlag Berlin, Germany.
Hong, S. G., W. Jeong, and H. S. Jung. 2002. Amplification of mitochondrial small subunit ribosomal DNA of polypores and its potential for phylogenetic analysis. Mycologia 94:823-833.
Kamei, K., H. Unno, K. Nagao, T. Kuriyama, K. Nishimura, and M. Miyaji. 1994. Allergic bronchopulmonary mycosis caused by the basidiomycetous fungus Schizophyllum commune. Clin. Infect. Dis. 18:305-309.
Kenney, R. T., K. J. Kwon-Chung, A. T. Waytes, D. A. Melnick, H. I. Pass, M. J. Merino, and J. I. Gallin. 1992. Successful treatment of systemic Exophiala dermatitidis infection in a patient with chronic granulomatous disease. Clin. Infect. Dis. 14:235-242.
Kenney, R. T., K. J. Kwon-Chung, F. G. Witebsky, D. A. Melnick, H. L. Malech, and J. I. Gallin. 1990. Invasive infection with Sarcinosporon inkin in a patient with chronic granulomatous disease. Am. J. Clin. Pathol. 94:344-350.
Kern, M. E., and F. A. Uecker. 1986. Maxillary sinus infection caused by the homobasidiomycetous fungus Schizophyllum commune. J. Clin. Microbiol. 23:1001-1005.
Khan, Z. U., H. S. Randhawa, T. Kowshik, S. N. Gaur, and G. A. de Vries. 1988. The pathogenic potential of Sporotrichum pruinosum isolated from the human respiratory tract. J. Med. Vet. Mycol. 26:145-151.
Kligman, A. M. 1950. A basidiomycete probably causing onychomycosis. J. Investig. Dermatol. 14:67-70.
Kline, M. W., F. C. Bocobo, M. E. Paul, H. M. Rosenblatt, and W. T. Shearer. 1994. Successful medical management of Aspergillus osteomyelitis of the spine in an 11-year-old boy with chronic granulomatous disease. Pediatrics 93:830-835.
Larsen, M. J., and L. A. Cobb-Poulle. 1990. Phellinus (Hymenochaetaceae)—a survey of the world taxa. Synop. Fungorum 3:1-206.
Larsen, M. J., and F. F. Lombard. 1988. Studies in the genus Phellinus. I. The identity of Phellinus rickii with notes on its facultative synonyms. Mycologia 80:72-76.
Loguercio-Leite, C., and J. E. Wright. 1995. The genus Phellinus (Hymenochaetaceae) on the island of Santa Catarina, Brazil. Mycotaxon 54:361-388.
Lowe, J. L. 1966. Polyporaceae of North America. The genus Poria. Technical publication no. 90. State University College of Forestry at Syracuse University, Syracuse, N.Y.
Marlier, S., J. P. Jaureguiberry, P. Aguilon, C. Carloz, J. L. Duval, and D. Jaubert. 1993. Sinusite chronique due a Schizophyllum commune au cours de SIDA. Presse Med. 22:1107.
Moncalvo, J. M., H. F. Wang, and R. S. Hseu. 1995. Phylogenetic relationships in Ganoderma inferred from the internal transcribed spacers and 25S ribosomal DNA sequences. Mycologia 87:223-238.
Nakasone, K. K. 1990. Cultural studies and identification of wood-inhabiting Corticiaceae and selected Hymenomycetes from North America. Mycol. Mem. 15:1-412.
Nenoff, P., T. Friedrich, H. Schwenke, M. Mierzwa, L.-C. Horn, and U.-F. Haustein. 1997. Rare fatal simultaneous mould infection of the lung caused by Aspergillus flavus and the basidiomycete Coprinus sp. in a leukemic patient. J. Med. Vet. Mycol. 35:65-69.
Nobles, M. K. 1948. Studies in forest pathology. VI. Identification of cultures of wood-rotting fungi. Can. J. Res. Sect. C 26:281-431.
Núez, M., and L. Ryvarden. 2000. East Asia polypores—Ganodermataceae and Hymenochaetaceae, vol. 1. Synop. Fungorum 13:1-168.
Phillips, P., J. C. Forbes, and D. P. Speert. 1991. Disseminated infection with Pseudallescheria boydii in a patient with chronic granulomatous disease: response to gamma-interferon plus antifungal chemotherapy. Pediatr. Infect. Dis. J. 10:536-539.
Restrepo, A., D. L. Greer, M. Robledo, O. Osorio, and H. Mondragon. 1971. Ulceration of the palate caused by a basidiomycete Schizophyllum commune. Sabouraudia 9:201-204.
Rihs, J. D., A. A. Padhye, and C. B. Good. 1996. Brain abscess caused by Schizophyllum commune: an emerging basidiomycete pathogen. J. Clin. Microbiol. 34:1628-1632.
Rinaldi, M. G. 1982. Use of potato flakes agar in clinical mycology. J. Clin. Microbiol. 15:1159-1160.
Rizzo, D. M., P. T. Gieser, and H. H. Burdsall, Jr. 2003. Phellinus coronadensis: a new species from southern Arizona, USA. Mycologia 95:74-79.
Roilides, E., L. Sigler, E. Bibashi, H. Katsifa, N. Flaris, and C. Panteliadis. 1999. Disseminated infection due to Chrysosporium zonatum in a patient with chronic granulomatous disease and review of non-Aspergillus fungal infections in patients with this disease. J. Clin. Microbiol. 37:18-25.
Rosenthal, J., R. Katz, D. B. DuBois, A. Morrissey, and A. Machicao. 1992. Chronic maxillary sinusitis associated with the mushroom Schizophyllum commune in a patient with AIDS. Clin. Infect. Dis. 14:46-48.
Ryvarden, L. 2000. Studies in neotropical polypores. 8. Poroid fungi from Jamaica—a preliminary check list. Mycotaxon 76:349-360.
Ryvarden, L., and A. A. R. de Meijer. 2002. Studies in neotropical polypores. 14. New species from the state of Paraná, Brazil. Synop. Fungorum 15:34-69.
Ryvarden, L., and I. Johansen. 1980. A preliminary polypore flora of East Africa. Fungiflora, Oslo, Norway.
Setliff, E. C., and L. Ryvarden. 1983. Los hongos de Colombia VII: some aphyllophoraceous wood-inhabiting species. Mycotaxon 18:509-525.
Sigler, L., and S. P. Abbott. 1997. Characterizing and conserving diversity of filamentous basidiomycetes from human sources. Microbiol. Cult. Coll. 13:21-27.
Sigler, L., J. R. Bartley, D. H. Parr, and A. J. Morris. 1999. Maxillary sinusitis caused by medusoid form of Schizophyllum commune. J. Clin. Microbiol. 37:3395-3398.
Sigler, L., S. Estrada, N. A. Montealegre, E. Jaramillo, M. Arango, C. de Bedout, and A. Restrepo. 1997. Maxillary sinusitis caused by Schizophyllum commune and experience with treatment. J. Med. Vet. Mycol. 35:365-370.
Sigler, L., L. M. de la Maza, G. Tan, K. N. Egger, and R. K. Sherburne. 1995. Diagnostic difficulties caused by a nonclamped Schizophyllum commune isolate in a case of fungus ball of the lung. J. Clin. Microbiol. 33:1979-1983.
Sillevis Smitt, J. H., J. H. Leusen, H. G. Stas, A. H. Teeuw, and R. S. Weening. 1997. Chronic bulbous disease of childhood and a Paecilomyces lung infection in chronic granulomatous disease. Arch. Dis. Child. 77:150-152.
Silliman, C. C., D. W. Lawellin, J. A. Lohr, B. M. Rodgers, and L. G. Donowitz. 1992. Paecilomyces lilacinus infection in a child with chronic granulomatous disease. J. Infect. 24:191-195.
Singh, S. M., M. Singh, and S. Mukherjee. 1992. Pathogenicity of Sporotrichum pruinosum and Cladosporium oxysporum, isolated from the bronchial secretions of a patient, for laboratory mice. Mycopathologia 117:145-152.
Speller, D. C. E., and A. G. MacIver. 1971. Endocarditis caused by a Coprinus species: a fungus of the toadstool group. J. Med. Vet. Mycol. 4:370-374.
Summerbell, R. C. 1993. The benomyl test as a fundamental diagnostic method for medical mycology. J. Clin. Microbiol. 31:572-577.
Swofford, D. L. 2002. PAUP. Phylogenetic Analysis Using Parsimony ( and other methods), version 4. Sinauer Associates, Sunderland, Mass.
Verweij, P. E., M. van Kasteren, J. van de Nes, G. S. de Hoog, B. E. de Pauw, and J. F. G. M. Meis. 1997. Fatal pulmonary infection caused by the basidiomycete Hormographiella aspergillata. J. Clin. Microbiol. 35:2675-2678.
Wagner, T., and M. Fischer. 2002. Proceedings towards a natural classification of the worldwide taxa Phellinus s. l. and Inonotus s. l., and phylogenetic relationships of allied genera. Mycologia 94:998-1016.
Williamson, P. R., K. J. Kwon-Chung, and J. I. Gallin. 1992. Successful treatment of Paecilomyces variotii infection in a patient with chronic granulomatous disease and a review of Paecilomyces species infections. Clin. Infect. Dis. 14:1023-1026.
White, T., T. Bruns, S. Lee, and J. Taylor. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, p. 315-322. In M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White (ed.), PCR protocols. A guide to methods and applications. Academic Press, Inc., San Diego, Calif.(D. A. Sutton, E. H. Thomp)
Audie L. Murphy Division, South Texas Veterans' Health Care System, San Antonio
Baylor College of Medicine
Texas Children's Hospital, Houston, Texas
University of Nebraska Medical Center, Omaha, Nebraska
Forest Products Laboratory, USDA Forest Service, Madison, Wisconsin
Seoul National University, Seoul, Republic of Korea
ABSTRACT
Although isolates of filamentous basidiomycetes can usually be recognized in a clinical laboratory setting, identification is problematic, as they seldom exhibit diagnostic morphological features formed in nature. This paper is the first report of Inonotus (Phellinus) tropicalis inciting human disease and describes the methods used to support the identification.
CASE REPORT
A 21-year-old Hispanic man with X-linked chronic granulomatous disease (CGD) and a long history of bacterial and fungal infections presented after 2 days of painless swelling over the lower back with no history of trauma, travel outside of his lifelong home in Houston, Tex., unusual environmental exposures, or systemic problems. Previous infections had included Aspergillus osteomyelitis of the ribs and vertebral bodies (28), lymphadenitis with Staphylococcus aureus, and cutaneous and systemic infections with Serratia marcescens. Routine X rays were negative, but computed tomography and magnetic resonance imaging studies showed paraspinal and presacral inflammation and extensive sacral bony changes. He had been on chronic daily intravenous antibiotics and maintenance interferon, co-trimoxazole, and itraconazole. Between July 2001 and January 2004, a rapidly growing golden-brown filamentous mold was isolated on seven occasions. Isolation media were incubated between 25 and 30°C and consisted of Sabouraud dextrose agar, Mycosel agar, and brain heart infusion agar supplemented with 5% sheep cells, chloramphenicol, and gentamicin (Becton Dickinson, Cockeysville, Md.). Six of these isolates were forwarded to the Fungus Testing Laboratory, University of Texas Health Science Center (UTHSC) at San Antonio, for identification. Between July 2001 and March 2002, isolates were obtained from a back abscess (UTHSC 01-1419), a back lesion (UTHSC 01-1652), a bone marrow aspirate (UTHSC 02-617), and paraspinal abscess tissue (isolate not accessioned into the UTHSC stock collection). Both the bone marrow aspirate and the paraspinal abscess tissue were positive for hyphal elements histopathologically. From February 2003 to January 2004, the organism was recovered from a paraspinal abscess (UTHSC 03-453), another back wound (UTHSC 03-2972), and the paraspinal area (UTHSC 04-212). The organisms recovered were thought to represent basidiomycetes on the basis of their cultural features (rapid growth and a golden-brown color) and their growth on 10% benomyl agar (58), which paralleled that observed on potato flakes agar (PFA) (42). Somewhat unusual in our experience, however, was the ability of the isolates to grow on media containing cycloheximide. Isolate numbers UTHSC 01-1419, UTHSC 01-1652, and UTHSC 03-2972 have been deposited in the University of Alberta Microfungus Collection and Herbarium, Edmonton, Alberta, Canada, as UAMH 10436, UAMH 10437, and UAMH 10376, respectively. Complete clinical details and therapeutic regimens for this case will be presented elsewhere.
The six isolates submitted to the Fungus Testing Laboratory were initially examined on PFA and benomyl agar prepared in-house and on commercially available cycloheximide agar (Remel, Lenexa, Kans.). When grown on PFA, isolates were woolly and yellowish orange and exhibited rapid growth, filling a 60-mm-diameter plate in 10 days at 25°C (Fig. 1A). Growth also occurred on media containing 10% benomyl and 0.04% cycloheximide at 25 and 35 °C but not at 40°C. Hyphae lacked clamp connections and were either thin walled and hyaline or thick walled and yellow, and 3-week-old cultures produced golden, thick-walled, pointed setal hyphae up to 100 μm in length (Fig. 1B). When morphological and internal transcribed spacer (ITS) region sequence data (described below) indicated that the fungus was a wood-decaying basidiomycete, three isolates, i.e., UTHSC 01-1419, UTHSC 01-1652, and UTHSC 02-617, were sent to the Forest Products Laboratory, Madison, Wis., for further study. The Center for Forest Mycology Research at the Forest Products Laboratory specializes in the study of wood-decaying basidiomycetes. Strains were grown separately on 2% Bacto malt extract agar (MEA; Difco Laboratories, Detroit, Mich.), 0.5% gallic acid agar (GAA), and 0.5% tannic acid agar (TAA) (Mallinckrodt, St. Louis, Mo.) (12) according to standard protocols for cultures of decay fungi (35, 37). GAA and TAA are used in the study of wood-decaying fungi to test for the presence of polyphenol oxidases that are associated with the decay of wood (12). Colony diameters on MEA at 25°C were 43 to 46 mm at 1 week and 83 to 85 mm at 2 weeks. Mats at 2 weeks were moderately thick, raised, and densely woolly, especially around the inoculum. Mats were orange-yellow or yellowish orange with yellowish brown streaks and became thinner and arachnoid to downy toward the margin. The margin was even to slightly uneven and appressed, and the agar beneath the mat was stained a dark brown or reddish brown. No odor was noticed at 2 weeks. Advancing zone hyphae of the margin were 2 to 3.5 μm in diameter, simply septate (as opposed to having a nodose septum or developing clamp connections), and rarely branched, with thin, smooth, and hyaline walls. Aerial hyphae from the mat surface were 1.5 to 3 μm in diameter and simply septate or aseptate with thin to slightly thick and smooth walls that were hyaline at first before becoming yellowish brown. Hyphae growing in the agar were 2 to 3.5 μm in diameter, simple septate, and moderately to frequently branched, with thin, smooth, hyaline to dark-yellow walls. By 4 weeks, numerous irregular swellings were observed on the submerged hyphae (Fig. 2). No growth or reactions were observed on GAA at 1 or 2 weeks; however, a strong reaction (i.e., a dark-brown discoloration of 3 to 4 cm in diameter) was observed on TAA, with colony diameters after 1 and 2 weeks reaching 11 to 15 and 28 to 33 mm, respectively.
Extraction of DNA from the bone marrow isolate, UTHSC 02-617, was done using hyphae collected from a 7-day-old culture on Sabouraud dextrose agar (Remel) after incubation at 30°C according to procedures explained by Henry et al. (19). DNA was purified using the QIAmp blood kit (QIAGEN Inc., Valencia, Calif.) and protocols for crude cell lysates supplied by the manufacturer. Two oligonucleotide fungal primers (ITS 1 and ITS 4) described by White et al. (63) were used for amplification. Primers were synthesized by the University of Nebraska Medical Center Eppley Molecular Biology Core Laboratory. The PCR assay was performed with 5 μl of the test sample in a 50-μl total reaction volume consisting of the following: PCR buffer (20 mM Tris-HCl [pH 8.4], 50 mM KCl); 0.1 mM (each) dATP, dGTP, dCTP, and dTTP; 1.5 mM MgCl2; 0.3 μM concentrations of each primer; and 1.5 U of PlatinumTaq high-fidelity DNA polymerase (Gibco BRL Life Technologies, Gaithersburg, Md.). Thirty-five cycles of amplification were performed in a Stratagene Robocycler model 96 thermocycler after initial denaturation of DNA at 95°C for 4.5 min. Each cycle consisted of a denaturation step at 95°C for 30 s, an annealing step at 50°C for 30 s, and an extension step at 72°C for 1 min, with a final extension at 72°C for 3 min following the last cycle. DNA sequencing was performed with a Perkin-Elmer/ABI model 373 DNA sequencer by following protocols supplied by the manufacturer. The PCR products were directly sequenced using the ITS 1 and ITS 4 primers for the ITS region. The resultant nucleotide sequences were aligned with the MacVector sequence analysis software, version 6.5 (Oxford Molecular Group, Inc., Campbell, Calif.), alignment application. Amplification of the ITS 1-5.8S rRNA-ITS 2 region of the case isolate genome resulted in the production of a 680-bp PCR product. A BLAST search of the NCBI GenBank database resulted in the identification of the top eight sequences producing significant alignments as Inonotus (Phellinus) linteus strains. In the closest match, the identification showed an alignment homology of only 91% (307 of 334 bp) between the database strain and the case isolate.
DNA sequence data for 18S rRNA, 26S rRNA, ITS, and mitochondrial rRNA have frequently been used in recent phylogenetic studies of eukaryotic cells. Among the sequenced genetic material, the ITS, which has the fastest evolutionary speed, is appropriate for the molecular identification or delimitation of fungi and is now commonly used in the systematics of species within a genus (34). A phylogenetic analysis of ITS sequences compared to sequences in the H. S. Jung Inonotus (Phellinus) database, Seoul National University (now registered sequences in GenBank), demonstrated that the clade including the case isolate UTHSC 02-617 (AY641432) was composed of two subclades representing Inonotus (Phellinus) tropicalis and I. (P.) linteus-Phellinus repandus. The former subclade was fully supported by the 100% bootstrap value, suggesting that the isolate is statistically identical to I. tropicalis (Fig. 3). The overall sequence similarity between I. tropicalis CBS 617.89 and UTHSC 02-617 was 97.8%, which was slightly lower than expected for isolates of the same species.
Mitochondrial small-subunit rRNA was amplified with the primers BMS05 and BMS173 described by Hong et al. (21). Subsequent sequence determination and phylogenetic evaluation were also done by following the method of Hong et al. (21). In the phylogenetic analysis of the mitochondrial small-subunit rRNA sequences, the two new sequences of UTHSC 01-1419 (AY598828) and UTHSC 01-1652 (AY598829) were identical, having 100% (582 of 582) similarity in sequence and forming a clade with I. tropicalis with a bootstrap value of 96% (data not shown). When UTHSC 01-1419 and UTHSC 01-1652 were compared with I. tropicalis CBS 617.89 (AY558713), the sequence similarity was 99.2% (512 of 516), suggesting that the causal fungus was actually a strain of I. tropicalis. The strain of I. tropicalis used in the analysis, CBS 617.89 from the Centraalbureau voor Schimmelcultures, was sent to the Centraalbureau voor Schimmelcultures by F. F. Lombard of the Center for Forest Mycology Research, Forest Products Laboratory, Madison, Wis., in 1989. It was collected from hardwood in Tacares, Heredia Province, Costa Rica, on 16 August 1963 by J. L. Lowe; the original collection number is L-13465. It was identified by M. J. Larsen (30).
The nuclear large-subunit rRNA of the casual fungus was also sequenced and analyzed. Extraction of the total DNA of isolates UTHSC 01-1419 and UTHSC 01-1652 and the amplification and sample preparation of the nuclear large-subunit rRNA for sequencing was done according to the procedure described by Greslebin et al. (17). The bone marrow isolate UTHSC 02-617 was not successfully amplified and thus was not evaluated. Prepared samples were sent to the University of Wisconsin Biotechnology Center for sequencing on an Applied Biosystems/Hitachi model 3730 xl DNA analyzer. Nuclear large-subunit rRNAs of UTHSC 01-1419 and UTHSC 01-1652 (AY598826 and AY598827, respectively) were identical and, when compared with I. tropicalis CBS 617.89 (AY059037), differed at only 6 out of 868 bases (99.3% sequence similarity). When both nuclear large-subunit rRNA sequences were added into the multiple sequence alignment of Wagner and Fischer (61) and analyzed with PAUP (59), they formed a clade with I. tropicalis that was fully supported by a 100% bootstrap value (data not shown), strongly indicating that the causal fungus in this investigation was I. tropicalis.
Few filamentous basidiomycetes have been authenticated as human etiologic agents. Two species documented to invade tissue include Schizophyllum commune and Coprinopsis cinerea (formerly Coprinus cinereus). Schizophyllum commune has elicited serious invasive disease in the brain (5, 41), lungs (6, 41, 53), and hard and soft palates (40). It has also been implicated in allergic bronchopulmonary disease (1, 22), chronic sinusitis (4, 25, 33, 45, 51, 52), and less serious disease of the nails (27). Coprinopsis cinerea (anamorph, Hormographiella aspergillata) (14, 18) has been the causative agent in a case of prosthetic valve endocarditis (13, 57) and in a fatal pulmonary infection (60). A Coprinus species was reported to cause a fatal infection in a leukemia patient 5 years after bone marrow transplantation (36). Another basidiomycete thought to cause pulmonary disease is Phanerochaete chrysosporium (anamorph, Sporotrichum pruinosum), although no tissue invasion has been noted (26, 56). Bjerkandera adusta, frequently isolated from pulmonary sites, may also be pathogenic in some settings (16). Fungal infections in patients with CGD are most frequently caused by Aspergillus fumigatus (7). More-recent reports have cited additional etiologic agents. These include the basidiomycete Trichosporon inkin reported by Kenney et al. in 1990 as Sarcinosporon inkin (24); the homothallic ascomycete Pseudallescheria boydii (39); Chrysosporium zonatum, an anamorph of the heterothallic ascomycete Uncinocarpus orisii (44); and other hyphomycetous species, i.e., Acremonium strictum (2), Exophiala (Wangiella) dermatitidis (23), Paecilomyces lilacinus (55), Paecilomyces variotii (8, 62), and a Paecilomyces species (54). To our knowledge, filamentous basidiomycetes have not been reported to occur in patients with CGD. This report adds the wood-destroying I. tropicalis to the list of basidiomycetes previously cited as being invasive and extends the genera of fungal organisms inciting disease in CGD patients.
Filamentous basidiomycetes are being recovered from clinical specimens with increasing frequency, and their identification in this setting remains problematic. González et al. (16), reporting in vitro antifungal susceptibility data for 44 clinical isolates of basidiomycetous fungi, indicated that, in addition to Schizophyllum commune and Coprinopsis cinerea, many arthroconidium-forming molds resembling Bjerkandera adusta were being recovered. Many more isolates, however, remained sterile and failed to produce clamp connections and/or lacked spicules suggestive of Schizophyllum commune and were reported as unidentified basidiomycetes (16). The characteristics that facilitate recognition of a basidiomycete include rapid growth; colonies that are usually white, cream, or yellow-orange pigmented; the presence of clamp connections and/or crystals; the formation of spicules along the hyphae; and mushroom- or basidiocarp-type fruiting bodies (50). Since most clinical isolates are monokaryons, neither clamps nor fruiting structures are produced, and mating studies may be required to induce these diagnostic features. The ability of most basidiomycetes to grow on agar containing 2 to 10 μg of benomyl/ml has also been employed as a screening test, although many ascomycetous fungi grow on this medium as well (58). Several basidiomycetes also fail to grow on media containing cycloheximide, a substrate commonly found in typical mycology laboratories. The isolate presented here was typical of a basidiomycete in that it exhibited rapid filamentous growth, had a yellowish to orange pigmentation, and was tolerant of benomyl. Also typical was the lack of clamp connections and basidiocarps. Somewhat unusual was the ability of the organism to grow equally well on medium containing cycloheximide. Since the organism had been isolated on numerous occasions from a profoundly compromised patient, molecular characterization was pursued.
When the sequence data of the isolates analyzed were compared to ITS and mitochondrial small-subunit rRNA sequences in GenBank, 97.8 and 99.2% matches, respectively, were made with I. tropicalis. In the sequence analysis of nuclear large-subunit rRNAs, they again showed high sequence similarity, i.e., of 99.3%, with I. tropicalis and were positively concluded to be I. tropicalis.
I. tropicalis is a poroid (having pores rather than gills) wood-decaying fungus that is associated with white-rot decay of various woody angiosperms. As is typical of many fungi, I. tropicalis has a complex taxonomic history. First described as Poria rickii Bres. in 1920 (3), it was given the new name Phellinus tropicalis M. Larsen et Lombard in 1988 for nomenclatural reasons (30). Phellinus ferrugineo-velutinus (Henn.) Ryv. is considered by some mycologists to be identical to Phellinus tropicalis (9, 15, 48), and its name is preferred for this taxon, based on priority. However, because the type specimen of Phellinus ferrugineo-velutinus is sterile (11), others prefer the name Phellinus tropicalis. Some mycologists also consider Phellinus flavomarginata (Murr.) Ryv. to be a synonym for Phellinus ferrugineo-velutinus (15, 48), but this is disputed by others (30, 31). I. tropicalis is common in Brazil, the type locality (30, 47), and it is reported also to occur in Mississippi (32), Florida, Georgia (15), Jamaica (46), Guadeloupe (11), Costa Rica (30), Colombia (49), East Africa (48), and Malaya, Johore, and Mawai, Malaysia (9). Phellinus is placed in the family Hymenochaetaceae and is one of the largest basidiomycetous genera of wood-destroying fungi, with over 220 named species worldwide (29). Traditionally, Phellinus is distinguished from Inonotus, a closely related genus, by its hard, woody, perennial fruiting bodies and dimitic hyphal system. Inonotus, in contrast, produces softer, annual fruiting bodies with a monomitic hyphal system. However, the morphological distinction between these two genera has become blurred over time (9, 15). Recent phylogenetic studies show that although the members of Hymenochaetaceae are a well-defined group (20), genera within this family, including Phellinus and Inonotus, are not monophyletic (43, 61). Thus, the taxonomy of Phellinus and allied genera is in a state of flux and is an area of active research (9, 10, 31, 38, 61). Wagner and Fischer's (61) phylogenetic study of Phellinus and Inonotus placed Phellinus tropicalis in the Inonotus sensu stricto clade. Thus, they transferred Phellinus tropicalis to the genus Inonotus. With the data available at this time, the best name for the casual fungus is Inonotus tropicalis; however, because most of the literature on this species refers to it as being of the genus Phellinus, we use both generic names.
This report extends the genera of basidiomycetous fungi inciting human disease and highlights the utility of molecular methods in the identification or verification of these often-unidentifiable molds when conventional taxonomic practices fail. Although the sequences from the case isolates have now been registered in GenBank, the Fungus Testing Laboratory was initially faced with locating individuals with expertise in poroid fungi who may have held genus-specific databases not in the public domain. Private and institutional databases appear to be a growing trend, and this case illustrates their usefulness for the identification of a wood-decaying fungus not previously known as a human etiologic agent.
Nucleotide sequence accession numbers. The following sequences have been deposited in the GenBank database (accession number): UTHSC-01-1419 small subunit rRNA gene (AY598828), UTHSC-01-1419 25S rRNA gene (AY598826), UTHSC-01-1652 small subunit rRNA gene (AY598829), UTHSC-01-1652 25S rRNA gene (AY598827), UTHSC-02-617 18S rRNA gene partial-ITS1complete-5.8S rRNA complete-ITS2 complete-28S rRNA partial (AY641432), and UTHSC-03-2972 18S rRNA gene partial-ITS1 complete-5.8S rRNA complete-ITS2 complete-28S rRNA partial (AY599487).
ACKNOWLEDGMENTS
We thank John Haight for obtaining the large-subunit rRNA sequences and T. Wagner for sharing the nuclear rRNA sequence database.
REFERENCES
Amitani, R., K. Nishimura, A. Niimi, H. Kobayashi, R. Nawada, T. Murayama, H. Taguchi, and F. Kuse. 1996. Bronchial mucoid impaction due to the monokaryotic mycelium of Schizophyllum commune. Clin. Infect. Dis. 22:146-148.
Boltansky, H., K. J. Kwon-Chung, A. M. Macher, and J. I. Gallin.1984. Acremonium strictum-related pulmonary infection in a patient with chronic granulomatous disease. J. Infect. Dis. 149:653.
Bresadola, G. 1920. Selecta mycologia. Ann. Mycol. 18:16-70.
Catalano, P., W. Lawson, E. Bottone, and J. Lebenger. 1990. Basidiomycetous (mushroom) infection of the maxillary sinus. Otolaryngol. Head Neck Surg. 102:183-185.
Chavez-Batista, A., J. A. Maia, and R. Singer. 1955. Basidioneuromycosis on man. An. Soc. Biol. Pernamb. 13:52-60.
Ciferri, R., A. Chavez-Batista, and S. Campos. 1956. Isolation of Schizophyllum commune from sputum. Atti Ist. Bot. Lab. Crittogam. Univ. Pavia 14:118-120.
Cohen, M. S., R. E. Isturiz, H. L. Malech, R. K. Root, C. M. Wilfert, L. Gutmasn, and R. H. Buckley. 1981. Fungal infections in chronic granulomatous disease. The importance of the phagocyte in defense against fungi. Am. J. Med. 71:59-66.
Cohen-Abbo, A., and K. M. Edwards. 1995. Multifocal osteomyelitis caused by Paecilomyces variotii in a patient with chronic granulomatous disease. Infection 23:55-57.
Corner, E. J. H. 1991. Ad Polyporaceas VII. The xanthochroic polypores. Beih. Nova Hedwigia 101:1-175.
Dai, Y.-C. 1999. Phellinus sensu lato (Aphyllophorales, Hymenochaetaceae) in East Asia. Acta Bot. Fenn. 166:1-115.
David, A., and M. Rajchenberg. 1985. Pore fungi from French Antilles and Guiana. Mycotaxon 22:285-325.
Davidson, R. W., W. A. Campbell, and D. J. Blaisdell. 1938. Differentiation of wood-decaying fungi by their reactions on gallic or tannic acid medium. J. Agric. Res. 57:683-695.
De Vries, G. A., R. F. O. Kemp, and D. C. E. Speller. 1971. Endocarditis caused by Coprinus delicatulus, p. 185. C. R. Comun. Abstr. 5th ISHAM Congr. 1971.
Gene, J., J. M. Guillamon, J. Guarro, I. Pujol, and K. Ulfig. 1996. Molecular characterization, relatedness and fungal susceptibility of the basidiomycetous Hormographiella species and Coprinus cinereus from clinical and environmental sources. Antonie Leeuwenhoek 70:49-56.
Gilbertson, R. L., and L. Ryvarden. 1987. North American polypores, vol. 2. Megasporoporia-Wrightoporia. Fungiflora, Oslo, Norway.
González, G. M., D. A. Sutton, E. Thompson, R. Tijerina, and M. G. Rinaldi. 2001. In vitro activities of approved and investigational antifungal agents against 44 clinical isolates of basidiomycetous fungi. Antimicrob. Agents Chemother. 45:633-635.
Greslebin, A., K. K. Nakasone, and M. Rajchenberg. 2004. Rhizochaete, a new genus of phanerochaetoid fungi. Mycologia 96:260-271.
Guarro, J., J. Gene, C. De Vroey, and E. Gueho. 1992. Hormographiella, a new genus of hyphomycetes from clinical sources. Mycotaxon 45:179-190.
Henry, T., P. C. Iwen, and S. H. Hinrichs. 2000. Identification of Aspergillus species using internal transcribed spacer regions 1 and 2. J. Clin. Microbiol. 38:1510-1515.
Hibbett, D. S., and R. G. Thorn. 2001. Basidiomycota: Homobasidiomycetes, p. 121-168. In D. J. McLaughlin, E. G. McLaughlin, and P. A. Lemke (ed.), The Mycota: a comprehensive treatise on fungi as experimental systems for basic and applied research, vol. VII, part B. Systematics and evolution. Springer-Verlag Berlin, Germany.
Hong, S. G., W. Jeong, and H. S. Jung. 2002. Amplification of mitochondrial small subunit ribosomal DNA of polypores and its potential for phylogenetic analysis. Mycologia 94:823-833.
Kamei, K., H. Unno, K. Nagao, T. Kuriyama, K. Nishimura, and M. Miyaji. 1994. Allergic bronchopulmonary mycosis caused by the basidiomycetous fungus Schizophyllum commune. Clin. Infect. Dis. 18:305-309.
Kenney, R. T., K. J. Kwon-Chung, A. T. Waytes, D. A. Melnick, H. I. Pass, M. J. Merino, and J. I. Gallin. 1992. Successful treatment of systemic Exophiala dermatitidis infection in a patient with chronic granulomatous disease. Clin. Infect. Dis. 14:235-242.
Kenney, R. T., K. J. Kwon-Chung, F. G. Witebsky, D. A. Melnick, H. L. Malech, and J. I. Gallin. 1990. Invasive infection with Sarcinosporon inkin in a patient with chronic granulomatous disease. Am. J. Clin. Pathol. 94:344-350.
Kern, M. E., and F. A. Uecker. 1986. Maxillary sinus infection caused by the homobasidiomycetous fungus Schizophyllum commune. J. Clin. Microbiol. 23:1001-1005.
Khan, Z. U., H. S. Randhawa, T. Kowshik, S. N. Gaur, and G. A. de Vries. 1988. The pathogenic potential of Sporotrichum pruinosum isolated from the human respiratory tract. J. Med. Vet. Mycol. 26:145-151.
Kligman, A. M. 1950. A basidiomycete probably causing onychomycosis. J. Investig. Dermatol. 14:67-70.
Kline, M. W., F. C. Bocobo, M. E. Paul, H. M. Rosenblatt, and W. T. Shearer. 1994. Successful medical management of Aspergillus osteomyelitis of the spine in an 11-year-old boy with chronic granulomatous disease. Pediatrics 93:830-835.
Larsen, M. J., and L. A. Cobb-Poulle. 1990. Phellinus (Hymenochaetaceae)—a survey of the world taxa. Synop. Fungorum 3:1-206.
Larsen, M. J., and F. F. Lombard. 1988. Studies in the genus Phellinus. I. The identity of Phellinus rickii with notes on its facultative synonyms. Mycologia 80:72-76.
Loguercio-Leite, C., and J. E. Wright. 1995. The genus Phellinus (Hymenochaetaceae) on the island of Santa Catarina, Brazil. Mycotaxon 54:361-388.
Lowe, J. L. 1966. Polyporaceae of North America. The genus Poria. Technical publication no. 90. State University College of Forestry at Syracuse University, Syracuse, N.Y.
Marlier, S., J. P. Jaureguiberry, P. Aguilon, C. Carloz, J. L. Duval, and D. Jaubert. 1993. Sinusite chronique due a Schizophyllum commune au cours de SIDA. Presse Med. 22:1107.
Moncalvo, J. M., H. F. Wang, and R. S. Hseu. 1995. Phylogenetic relationships in Ganoderma inferred from the internal transcribed spacers and 25S ribosomal DNA sequences. Mycologia 87:223-238.
Nakasone, K. K. 1990. Cultural studies and identification of wood-inhabiting Corticiaceae and selected Hymenomycetes from North America. Mycol. Mem. 15:1-412.
Nenoff, P., T. Friedrich, H. Schwenke, M. Mierzwa, L.-C. Horn, and U.-F. Haustein. 1997. Rare fatal simultaneous mould infection of the lung caused by Aspergillus flavus and the basidiomycete Coprinus sp. in a leukemic patient. J. Med. Vet. Mycol. 35:65-69.
Nobles, M. K. 1948. Studies in forest pathology. VI. Identification of cultures of wood-rotting fungi. Can. J. Res. Sect. C 26:281-431.
Núez, M., and L. Ryvarden. 2000. East Asia polypores—Ganodermataceae and Hymenochaetaceae, vol. 1. Synop. Fungorum 13:1-168.
Phillips, P., J. C. Forbes, and D. P. Speert. 1991. Disseminated infection with Pseudallescheria boydii in a patient with chronic granulomatous disease: response to gamma-interferon plus antifungal chemotherapy. Pediatr. Infect. Dis. J. 10:536-539.
Restrepo, A., D. L. Greer, M. Robledo, O. Osorio, and H. Mondragon. 1971. Ulceration of the palate caused by a basidiomycete Schizophyllum commune. Sabouraudia 9:201-204.
Rihs, J. D., A. A. Padhye, and C. B. Good. 1996. Brain abscess caused by Schizophyllum commune: an emerging basidiomycete pathogen. J. Clin. Microbiol. 34:1628-1632.
Rinaldi, M. G. 1982. Use of potato flakes agar in clinical mycology. J. Clin. Microbiol. 15:1159-1160.
Rizzo, D. M., P. T. Gieser, and H. H. Burdsall, Jr. 2003. Phellinus coronadensis: a new species from southern Arizona, USA. Mycologia 95:74-79.
Roilides, E., L. Sigler, E. Bibashi, H. Katsifa, N. Flaris, and C. Panteliadis. 1999. Disseminated infection due to Chrysosporium zonatum in a patient with chronic granulomatous disease and review of non-Aspergillus fungal infections in patients with this disease. J. Clin. Microbiol. 37:18-25.
Rosenthal, J., R. Katz, D. B. DuBois, A. Morrissey, and A. Machicao. 1992. Chronic maxillary sinusitis associated with the mushroom Schizophyllum commune in a patient with AIDS. Clin. Infect. Dis. 14:46-48.
Ryvarden, L. 2000. Studies in neotropical polypores. 8. Poroid fungi from Jamaica—a preliminary check list. Mycotaxon 76:349-360.
Ryvarden, L., and A. A. R. de Meijer. 2002. Studies in neotropical polypores. 14. New species from the state of Paraná, Brazil. Synop. Fungorum 15:34-69.
Ryvarden, L., and I. Johansen. 1980. A preliminary polypore flora of East Africa. Fungiflora, Oslo, Norway.
Setliff, E. C., and L. Ryvarden. 1983. Los hongos de Colombia VII: some aphyllophoraceous wood-inhabiting species. Mycotaxon 18:509-525.
Sigler, L., and S. P. Abbott. 1997. Characterizing and conserving diversity of filamentous basidiomycetes from human sources. Microbiol. Cult. Coll. 13:21-27.
Sigler, L., J. R. Bartley, D. H. Parr, and A. J. Morris. 1999. Maxillary sinusitis caused by medusoid form of Schizophyllum commune. J. Clin. Microbiol. 37:3395-3398.
Sigler, L., S. Estrada, N. A. Montealegre, E. Jaramillo, M. Arango, C. de Bedout, and A. Restrepo. 1997. Maxillary sinusitis caused by Schizophyllum commune and experience with treatment. J. Med. Vet. Mycol. 35:365-370.
Sigler, L., L. M. de la Maza, G. Tan, K. N. Egger, and R. K. Sherburne. 1995. Diagnostic difficulties caused by a nonclamped Schizophyllum commune isolate in a case of fungus ball of the lung. J. Clin. Microbiol. 33:1979-1983.
Sillevis Smitt, J. H., J. H. Leusen, H. G. Stas, A. H. Teeuw, and R. S. Weening. 1997. Chronic bulbous disease of childhood and a Paecilomyces lung infection in chronic granulomatous disease. Arch. Dis. Child. 77:150-152.
Silliman, C. C., D. W. Lawellin, J. A. Lohr, B. M. Rodgers, and L. G. Donowitz. 1992. Paecilomyces lilacinus infection in a child with chronic granulomatous disease. J. Infect. 24:191-195.
Singh, S. M., M. Singh, and S. Mukherjee. 1992. Pathogenicity of Sporotrichum pruinosum and Cladosporium oxysporum, isolated from the bronchial secretions of a patient, for laboratory mice. Mycopathologia 117:145-152.
Speller, D. C. E., and A. G. MacIver. 1971. Endocarditis caused by a Coprinus species: a fungus of the toadstool group. J. Med. Vet. Mycol. 4:370-374.
Summerbell, R. C. 1993. The benomyl test as a fundamental diagnostic method for medical mycology. J. Clin. Microbiol. 31:572-577.
Swofford, D. L. 2002. PAUP. Phylogenetic Analysis Using Parsimony ( and other methods), version 4. Sinauer Associates, Sunderland, Mass.
Verweij, P. E., M. van Kasteren, J. van de Nes, G. S. de Hoog, B. E. de Pauw, and J. F. G. M. Meis. 1997. Fatal pulmonary infection caused by the basidiomycete Hormographiella aspergillata. J. Clin. Microbiol. 35:2675-2678.
Wagner, T., and M. Fischer. 2002. Proceedings towards a natural classification of the worldwide taxa Phellinus s. l. and Inonotus s. l., and phylogenetic relationships of allied genera. Mycologia 94:998-1016.
Williamson, P. R., K. J. Kwon-Chung, and J. I. Gallin. 1992. Successful treatment of Paecilomyces variotii infection in a patient with chronic granulomatous disease and a review of Paecilomyces species infections. Clin. Infect. Dis. 14:1023-1026.
White, T., T. Bruns, S. Lee, and J. Taylor. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, p. 315-322. In M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White (ed.), PCR protocols. A guide to methods and applications. Academic Press, Inc., San Diego, Calif.(D. A. Sutton, E. H. Thomp)