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JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1995, p. 1979–1983 0095-1137/95/$04.0010Copyright 1995, American Society for Microbiology Diagnostic Difficulties Caused by a Nonclamped Schizophyllum commune Isolate in a Case of Fungus Ball of the Lung LYNNE SIGLER,1,2* LUIS M. DE LA MAZA,3 GRACE TAN,3 KEITH N. EGGER,4 AND RICHARD K. SHERBURNE2 University of Alberta Microfungus Collection and Herbarium, Devonian Botanic Garden,1 and Medical Microbiology & Infectious Diseases, University of Alberta,2 Edmonton, Alberta, Canada T6G 2E1; Division of Medical Microbiology, Department of Pathology, University of California, Orange, California 926683; and Department of Biology, Memorial University, St. John's, Newfoundland, Canada A1B 3X94 Received 3 March 1995/Returned for modification 17 April 1995/Accepted 3 May 1995 The presence of clamp connections on hyphae and the development of fruiting bodies in culture are primary
characters which allow identification of the basidiomycete Schizophyllum commune in cases of human infection.
The diagnostic problems presented by a nonclamped, nonfruiting isolate from a dense mass in the right upper
lobe of the lung in a female with a past history of pulmonary tuberculosis and diabetes are described. Several
features of the isolated fungus, including rapid growth rate and white, dense, cottony colonies, tolerance to the
fungicide benomyl at a concentration of 10 mg/ml, and susceptibility to cycloheximide at 400 mg/ml, suggested
that it might be a basidiomycete. Transmission electron microscopy showed the presence of a dolipore septum
with perforate pore cap characteristic of fungi in the class Holobasidiomycetes. However, species identification
remained elusive until compatibility tests with known single-basidiospore isolates confirmed the identification
of the sterile lung isolate as S. commune. Sequence analysis of the 5 internal transcribed spacer region of
ribosomal DNA further supported conspecificity.
Fungus ball of the lung, in which a mass of fungal mycelium of the lung in a patient with a history of tuberculosis and grows in a preexisting cavity, occurs in patients with underlying diabetes in whom septate, nonclamped hyphae were observed pulmonary disorders such as tuberculosis, previous infections in histopathologic sections. This report provides an example of caused by systemic fungi, recurrent bacterial pneumonia, lung the diagnostic problems presented by an isolate which is sus- abscess, or sarcoidosis. The infection is not generally diag- pected of being a basidiomycete but which fails to form the nosed by sputum culture since the fungal elements are walled characteristic macroscopic and microscopic structures which off and are not expelled, but hemoptysis is a common finding.
would allow its identification.
Most cases involve species of Aspergillus, most commonly A.
fumigatus, or Scedosporium apiospermum (Pseudallescheria MATERIALS AND METHODS
boydii) (10). Reports of infection caused by basidiomycetes arerare, and to our knowledge, no basidiomycete has been re- Case report. A 53-year-old Vietnamese female was seen in the Emergency
ported from a case of fungus ball. Other than members of the Room of the University of California, Irvine, Medical Center in September 1992 basidiomycetous yeast genera Cryptococcus and Trichosporon, complaining of chronic cough and hemoptysis for the previous 7 days. Thepatient had a history of tuberculosis diagnosed 5 years earlier, and in 1988 she probably the best-known basidiomycetous agent of infection is was found to have non-insulin-dependent diabetes. Apparently, she was treated Schizophyllum commune. Reports involving the lung include a several times over the previous 5 years with antituberculosis drugs. In December case of allergic bronchopulmonary mycosis (7) in an otherwise 1991, she was started on isoniazid, ethambutol, and rifampin.
healthy female and repeated isolation of S. commune from the A chest X ray and computed tomography scan performed in June 1992 showed a roughly spherical density in the posterior segment of the right upper lobe sputum of a patient with chronic lung disease (3). Other re- measuring approximately 2 cm in diameter. Above this level there were many ports of S. commune infection include cases of meningitis (2), smaller, sharply defined lesions scattered throughout the lung, particularly in the sinusitis (1, 8, 13), ulcerative lesions of the hard palate (12), anterior and apical segments. Leading from the larger mass toward the hilum, and possible onychomycosis (9) in both immunocompetent and there were markedly thickened bronchial walls giving the appearance of a tennisracquet, an image usually associated with tuberculosis (Fig. 1). An expectorated immunosuppressed hosts. In all of the cases reported to date, sputum specimen collected during this visit was negative for mycobacteria by the fungus has been recognized in tissue and in culture by the culture and smear.
presence of clamp connections on the hyphae and by the de- A bronchoalveolar lavage was performed in July 1992. A Gram stain showed velopment of basidiocarps (sexual fruiting bodies) in culture.
41 erythrocytes but no organisms. Bacterial cultures were positive for a mixedflora. Cultures and direct smears were negative for mycobacteria. Viral cultures However, Kamei et al. (7) suggested that diagnosis of infection were positive for cytomegalovirus and adenovirus. Fungal cultures were negative.
caused by S. commune may be difficult because of (i) the failure The cytopathological analysis was negative for malignancy, and acid-fast bacillus to observe clamp connections on hyphae which otherwise ap- stains were negative.
pear similar to those of Aspergillus, (ii) the absence of fruiting In October 1992 a right upper lobe lobectomy was performed. The patient had a mild pneumothorax after surgery and was discharged 10 days following surgery.
body formation in the dark, and (iii) the possibility of identi- Histopathology. The pathological report of the frozen intraoperative specimen
fication only when the fungus is a dikaryon capable of produc- was described as a necrotizing granuloma, with a fungal form consistent with ing basidiocarps. We present a case of necrotizing granuloma Aspergillus sp. present. Hematoxylin and eosin (H&E)-stained tissue samplesfrom the lobectomy specimen showed histopathological lesions typical of a my-cobacterial infection including the presence of caseous calcified granulomas withLanghans cells. Stains for acid-fast bacilli were positive. H&E and the Gomori- * Corresponding author. Mailing address: University of Alberta Mi- methenamine silver (GMS) stains showed that the center of the lesion was crofungus Collection and Herbarium, Devonian Botanic Garden, Ed- occupied by a large mass of hyphae which were present in the cavity but which monton, AB, Canada T6G 2E1. Phone: (403) 987-4811. Fax: (403) did not invade the surrounding tissues. The hyphae were septate, varying in width 987-4141. Electronic mail address: [email protected].
from 1.5 to 5 mm but mostly measuring between 1.5 and 3 mm in diameter,

SIGLER ET AL.
J. CLIN. MICROBIOL.
FIG. 1. Computed tomography scan of the chest showing a dense mass in the right upper lobe and thickened bronchial walls giving the appearance of a tennisracket.
FIG. 3. Colony on unamended SDA (A) and SDA amended with 10 mg of benomyl per ml (B).
sometimes showing irregularly shaped swellings up to 7 mm wide or having short,rounded protrusions, rarely branched at an acute angle or dichotomous. In was also compared with known isolates of S. commune. A dikaryotic isolate, GMS-stained material, some hyphae showed variation in intensity of staining, UAMH 7796 (CDC B-5575), was obtained from a brain abscess; monokaryotic with an individual hypha showing short segments of darkly stained areas alter- isolates were obtained from the Forest Products Laboratory, U.S. Department of nating with lighter-stained areas (Fig. 2).
Agriculture, Madison, Wis., as single-basidiospore isolates UAMH 7692 (7599 Mycology. Cultures of the surgical specimen yielded several colonies of a
ss-1), UAMH 7693 (7599 ss-7), UAMH 7694 (FP-74612 ss-2), and UAMH 7695 white, rapidly growing fungus on inhibitory mold agar containing ciprofloxacin (FP-74612 ss-3).
(Dimed Corporation, St. Paul, Minn.) and on Sabouraud dextrose agar (SDA) Additional studies. For transmission electron microscopy (TEM), the sample
containing no antibiotics (BBL/Beckton Dickinson Microbiology Systems, Cock- was prepared by the methodology of Currah and Sherburne (4) and was exam- eysville, Md.).
ined with a Philips model 410 transmission electron microscope. For scanning Further studies of the lung isolate were conducted at the University of Alberta.
electron microscopy (SEM), the methodology followed the same procedure Colonial features were examined by subculture onto phytone-yeast extract agar described above for TEM except that the sample was rinsed in distilled water (PYE) (BBL), Mycosel agar (BBL), 2% malt extract agar (MEA; Difco Bacto following the 1% (wt/vol) osmium tetroxide fixation step and was further incu- malt extract, 25 g; agar, 18 g), potato dextrose agar (PDA; Difco), and Pablum bated with 1% tannic acid in distilled water. The specimen was then rinsed in cereal agar (CER) (11) and incubation at 258C. Growth rates at temperatures of water and was again incubated in the osmium tetroxide fixative. After a further 37 and 438C were determined with PDA. Incubation was in alternating condi- wash, the sample was dehydrated, dried to the critical point, mounted, and tions of light and dark except at 438C, at which incubation was entirely in the examined in a Hitachi model S 4000 field emission scanning electron microscope dark. Susceptibility to cycloheximide at a concentration of 400 mg/ml was deter- at an accelerating potential of 2.5 kV.
mined by growing the fungus on Mycosel agar, and tolerance to benomyl was To test compatibility between UAMH 7287 and the four monokaryotic strains determined by assessing growth rates on unamended SDA compared with those and among the monokaryotic isolates themselves, the isolates were paired in all on SDA amended with benomyl at concentrations of 2 and 10 mg/ml as described possible combinations. A plug of mycelium of 3 mm in diameter was removed by Summerbell (15). Additional media were used in attempts to induce sporu- from the margin of a colony grown on PDA to one-half of a new PDA plate; a lation in the sterile isolate, and cultures were incubated for 3 to 4 months under plug from a second isolate was placed on the other half. Microscopic mounts various light conditions. Microscopic observations were made in slide cultures on from the contact zone between advancing mycelia were examined for the pres- CER. The isolate from this patient was deposited in the University of Alberta ence of clamp connections (dikaryotization).
Microfungus Collection and Herbarium as UAMH 7287. The unknown isolate Total genomic DNA was extracted from plugs of mycelium removed from colonies grown on PDA and was freeze-dried for 24 h. The internal transcribedspacer 1 (ITS1) region was amplified with primers ITS1 (17) and ITS10mun (5).
The procedures for DNA extraction, amplification, and sequence analysis fol-lowed the methodology of Egger and Sigler (5).
Nucleotide sequence accession numbers. Representative sequences are depos-
ited in GenBank under the following accession numbers U21483 (UAMH 7287and UAMH 7693), U21484 (UAMH 7694), and U21485 (UAMH 7695).
Description of the isolate from this case. Macroscopically,
the isolate grew rapidly on all media, reaching diameters of 50to 60 mm in 7 days on PDA or PYE and 40 mm on 2% MEA.
Growth at 378C was similar, with a colony diameter of 60 mm,but growth was slightly faster at 438C (diameter, 70 mm) onPDA. Daily growth rates on PDA at temperatures of 25, 37,and 438C were 8.6, 8.6, and 10.1 mm, respectively. Colonies onPDA or PYE were dense, cottony, white, and slightly raisedwith a central umbo. The fungus was susceptible to cyclohex-imide but was tolerant to benomyl at 10 mg/ml (Fig. 3). Itproduced a strong, unpleasant odor that was easily detectablewhen the plates were incubated in plastic bags or containers.
Microscopically, features of the hyphae in agar culture con-formed to those of the hyphae observed in histopathology, FIG. 2. GMS-stained section showing hyphae of varying widths. Note that some segments also show variations in staining intensity (arrow). Bar, 20 mm.
demonstrating (i) variation in width, ranging from 1.5 to 5 mm,

DIAGNOSTIC DIFFICULTIES CAUSED BY S. COMMUNE ISOLATE FIG. 6. Scanning electron micrograph showing dome-shaped pore cap with FIG. 4. Slide culture preparation showing segments of hyphae with small, perforations. Bar, 1 mm.
rounded swellings (arrow) and rounded protrusions resembling clamps. Bar, 10 SEM of intact hyphae verified the presence of the distinctiverounded swellings on some hyphae (Fig. 7).
(ii) the presence of small rounded swellings occurring on some Comparison with S. commune. Differences between the
hyphae (crenate hyphae) (Fig. 4) which in the stained sections dikaryotic (fruiting) isolate and monokaryotic isolates were appeared as variations in staining intensity (Fig. 2), and (iii) evident in growth rates and colonial and microscopic morphol- short, rounded protrusions somewhat resembling aborted ogies. The dikaryotic isolate (UAMH 7796) grew slightly more clamp connections (Fig. 4). In slide culture preparations, nar- slowly at all temperatures, showing daily growth rates of 7.1 row hyphae often formed loose knots. The isolate failed to mm at 258C, 5.1 mm at 378C, and 8.3 mm at 438C. Colonies produce either sexual or asexual structures on any medium and were densely woolly and had highly irregular (lobate) margins.
initially could not be identified. However, the general cultural By 7 days, several fruiting bodies formed on PDA at 258C, but features and tolerance to benomyl suggested the sterile isolate not at 378C, after 2 weeks under conditions of alternating light would be more likely to be basidiomycetous than ascomyce- and dark. Average daily growth rates for the monokaryotic isolates at each temperature were 6.2, 10, and 11.4 mm. How- An oblique section of the hypha examined by TEM demon- ever, differences in growth rates were observed between the strated the presence of a dolipore septum with a dome-shaped single basidiospore isolates obtained from the same fruiting pore cap showing perforations or openings (Fig. 5). Fracture of body; for example, UAMH 7692 showed daily growth rates of a hyphal cell adjacent to the septum and examination by SEMallowed a unique three-dimensional view of the pore cap show-ing the perforations in the cap (Fig. 6). Lower-magnification FIG. 5. Transmission electron micrograph of oblique section of hypha show- FIG. 7. Scanning electron micrograph of colony showing hyphae with small, ing dolipore septum with perforate pore cap. Bar, 10 mm.
rounded swellings (arrow). Bar, 10 mm.
SIGLER ET AL.
J. CLIN. MICROBIOL.
FIG. 8. Consensus sequence for the ITS1 region of the nuclear-encoded rRNA genes amplified with primers ITS1 and ITS10mun. The 18S rRNA and 5.8S rRNA sequences flanking the ITS1 region are indicated in lowercase letters. Variable positions are indicated by using the standard ambiguity code of the International Unionof Pure and Applied Chemistry: R 5 A or G, Y 5 C or T, M 5 A or C, and W 5 A or T.
6.4, 8.1, and 8.6 mm at each temperature, respectively, com- found to be a useful characteristic in assessing the affinities of pared with respective growth rates of 11.4, 14.3, and 14.3 mm an unknown isolate to either the ascomycetes or the basidio- for strain UAMH 7693. Colonies were densely cottony to mycetes, especially when other characteristics are absent. Most woolly and, in contrast to the dikaryotic isolate, margins were ascomycetous fungi, for example, A. fumigatus, have simple, entire (i.e. smooth and regular). A strong, unpleasant odor was single-pored septa. In contrast, among the basidiomycetes, sev- produced by all isolates and was detectable, in some instances, eral different types of septal structure are recognized and are through the closed door of the incubator. Clamp connections important characters in the taxonomy. The dolipore septum were present on hyphae of the dikaryotic isolate; in addition, with a multiperforate pore cap confirmed in our isolate by some hyphae bore short, fine pegs or spinulose projections TEM and SEM (Fig. 5 and 6) is typical of members of the class arising at right angles. This highly distinctive morphologic fea- Holobasidiomycetes (producing meiospores on nonseptate ba- ture of S. commune hyphae was absent from all monokaryotic sidia), which includes the orders Aphyllophorales and Agari- isolates which also lacked clamp connections. Hyphae of the latter showed considerable variation in width, ranging from 1.5to 5 mm, and were thin or thick walled, and in slide culture Among the Aphyllophorales, the best-known agent of infec- preparations narrow hyphae often formed loose knots. No tion is S. commune. Although it is sometimes difficult to eval- conidia or chlamydospores were observed.
uate the significance of isolation of S. commune from clinical In compatibility tests, the isolate from the case patient specimens (6, 7, 9), there have been a number of well-docu- (UAMH 7287) formed clamp connections when it was paired mented reports especially involving the nasal musosa, hard with each of the monokaryotic isolates, thus demonstrating a palate, and lung (1, 7, 8, 13). To date, all confirmed cases of S. process of dikaryotization. Compatibility among the mono- commune infection have been based on isolates which form karyotic isolates occurred only between single basidiospore characteristic fruiting bodies in culture. Additional features isolates obtained from different fruiting bodies (i.e., UAMH which allow the identification of an isolate as S. commune 7692 formed clamp connections with UAMH 7694 and UAMH include clamp connections and narrow hyphal pegs or spicules 7695 but not UAMH 7693). No pair of isolates formed ba- (1, 2) present on some hyphae. Greer (6) reported that the sidiocarps, and no changes in colonial characteristics occurred pegs could also be observed on hyphae growing in tissue. Nei- in the dikaryotized mycelia.
ther of these distinctive hyphal features was present in the Alignment of ITS1 sequences indicated that UAMH 7287, isolate from our case patient or in any of the single basidios- UAMH 7693, UAMH 7694, and UAMH 7695 are conspecific.
pore isolates; moreover, these monokaryotic isolates demon- Only five of 221 positions were variable (Fig. 8), for a total strated dissimilar colonial features. Since monokaryons are the variation of 2.26%. No insertions or deletions were observed.
most common basidiomycetous isolates encountered in the The sequences of UAMH 7796 and UAMH 7692 are not diagnostic laboratory (14, 15), the possibility that our fungus reported here. After two attempts, we were unable to obtain an was a monokaryotic S. commune isolate was considered but unambiguous sequence for the dikaryotic isolate, and the orig- was initially ruled out by these apparent differences. This false- inal isolate of UAMH 7692 sent to K. N. Egger was found to negative result was corrected only when single-basidiospore be a basidiomycetous contaminant. Since a subculture of the isolates were obtained for comparison and compatibility original isolate demonstrated compatibility in mating tests as reported above, the sequencing was not repeated for the pu- The DNA sequence data confirmed the results of the mating rified isolate UAMH 7692.
tests. A level of base substitution of 2.26% is well within therange of intraspecific variation commonly observed in fungi (5). The ITS1 region is also highly susceptible to insertion or Macroscopic features of rapid growth and white woolly col- deletion events. Although insertions or deletions are occasion- onies, tolerance to the fungicide benomyl, and susceptibility to ally found within species, they are particularly common be- cycloheximide were suggestive initially that the isolate was a tween species. The low level of base substitution and the ab- basidiomycete. However, since it remained sterile under all sence of insertions or deletions indicates that the isolates are conditions, a definite identification was not possible. A diag- nosis of aspergillosis could easily have been made in this case Our finding of a monokaryotic isolate of S. commune caus- since the hyphae in the histopathology section demonstrated ing infection supports the contention of Kamei et al. (7) that only subtle differences and white, sterile isolates of A. fumiga- many cases of infection caused by basidiomycetes may be mis- tus are isolated occasionally from patients with chronic lung diagnosed. Any white, rapidly growing, sterile isolate showing disorders. However, the hyphae of the fungus in the necrotic good growth at 378C with tolerance to benomyl, susceptibility lesion were consistent with those of the isolate in culture, to cycloheximide, and a pronounced odor should be suspected which was proven to be a basidiomycete by ultrastructural of being S. commune. Since compatibility (dikaryotization) be- evidence of the dolipore septum.
tween monokaryotic mycelia derived from different fruiting If clamp connections are present, the identification of an bodies reaches almost 100% (16), the compatibility test, as isolate as a basidiomycete is simple. However, clamp connec- described in this report, could be used effectively to allow rapid tions are absent in monokaryotic isolates, and many basidio- confirmation of the identity of a suspected monokaryotic S. mycetes lack them completely. Septal ultrastructure has been DIAGNOSTIC DIFFICULTIES CAUSED BY S. COMMUNE ISOLATE 1994. Allergic bronchopulmonary mycosis caused by the basidiomycetous
fungus Schizophyllum commune. Clin. Infect. Dis. 18:305–309.
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ucts Laboratory, U.S. Department of Agriculture, for providing single- Invest. Dermatol. 14:67–70.
basidiospore isolates of S. commune. L. Sigler and K. N. Egger grate- 10. Kwon-Chung, K. J., and J. E. Bennett. 1992. Medical mycology. Lea &
fully acknowledge financial support from the Natural Sciences and 11. Padhye, A. A., A. S. Sekhon, and J. W. Carmichael. 1973. Ascocarp produc-
Engineering Research Council, Canada.
tion by Nannizzia and Arthroderma on keratinous and non-keratinous media.
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Chronic maxillary sinusitis associated with the mushroom Schizophyllum man. Anais Soc. Biol. Pernambuco 13:52–60.
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lum commune from sputum. Atti Inst. Bot. Lab. Crittogam. Univ. Pavia other Hyphomycetes with arthroconidia. Mycotaxon 4:349–488.
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