Schizophyllum radiatum, an Emerging Fungus from Human Respiratory Tract

Abstract

Schizophyllum is an important genus of basidiomycetes that, apart from being of genetic and biotechnological interest, is also reported to be a plant and animal pathogen. Schizophyllum commune is the best-known species and the only one reported from clinical specimens thus far, being recovered mainly from the respiratory tract. The aim of this study was to determine the species diversity of 23 clinical isolates of Schizophyllum from the United States using multilocus phylogenetic analysis and their in vitro susceptibilities to six drugs. The markers used for sequencing were the internal transcribed spacer (ITS), a portion of the nuclear large subunit (LSU) of ribosomal DNA, the RNA polymerase II second-largest subunit (RPB2), and the translation elongation factor 1α (EF-1α) gene. The analyses revealed that 22 of the clinical isolates were in the Schizophyllum radiatum clade with high support values and 1 isolate was in the S. commune clade. This is the first report of this species in clinical samples. The two species mentioned above showed very similar morphological features in culture (i.e., white, cottony, unsporulated colonies composed of hyphae with clamp connections), making morphological discrimination between the two impossible. An epitype is designed for S. radiatum, and its sequences have been deposited in GenBank. The antifungal that showed the greatest in vitro activity against the strains tested was shown to be amphotericin B. In general, the strains of S. radiatum showed higher MICs than S. commune.

INTRODUCTION

Schizophyllum (Schizophyllaceae, Agaricales) comprises one of the most commonly found mushrooms on the planet (1). The most common species of the genus is Schizophyllum commune, a ubiquitous fungus well-known as a wood decay organism able to cause white rot (1, 2) but also by its biotechnological applications (3). It has been used as an ethanol producer (4), and some of its metabolites have anticancer (5) and antimicrobial (6, 7) properties. It also has been studied for many years as a model for understanding mating interactions (8, 9). In recent years, S. commune has become clinically relevant as an etiological agent of respiratory infections in humans (10–15). However, it has been reported to be an etiological agent of other types of infections and conditions, such as a brain abscess (16, 17), meningitis (18), an eye infection (19), palate ulceration (20), and onychomycosis (21). Infections have also been reported in other mammals (22–24) and in both immunocompromised and immunocompetent individuals (25, 26).

The most effective treatment options for infections by S. commune are yet to be determined, although various treatment modalities have been used (14, 25–29). Most susceptibility patterns for these fungi are restricted to individual cases; however, studies by Gonzalez et al. (30) and Chowdhary et al. (31) with 5 and 26 strains, respectively, indicated that amphotericin B (AMB), itraconazole (ITC), and voriconazole (VRC) had good in vitro activity against the isolates tested, while elevated MICs were observed for flucytosine and fluconazole.

The identification of S. commune and other filamentous basidiomycetes from clinical specimens is often problematic. Key morphological features necessary for the recognition of these fungi are the presence of clamp connections on hyphae and the development of fruiting bodies (32). However, many clinical isolates of S. commune do not form such structures and in culture only show colonies with white cottony surfaces, a high growth rate, and droplets of exudate (33), which are clearly not distinctive for either species or genus recognition. Schizophyllum includes nearly 20 species (http://www.mycobank.org). However, ex-type strains for many of those species do not exist; only a limited number of strains for a few species identified by specialists (i.e., S. commune, S. fasciatum, S. radiatum, and S. umbrinum) have been deposited into different public culture collections. In addition, some species, such as S. commune and S. radiatum, have long been considered conspecific due to their morphological similarity (2). Recently, molecular methods have more successfully discriminated between various basidiomycetous molds (13, 33, 34). Singh et al. (34) proposed sequencing the internal transcribed spacer (ITS) and D1/D2 regions for the identification of basidiomycetes. However, when the sequences of these two regions are submitted to the GenBank database for comparison, there is a low level of discrimination mainly due to the lack of reliable sequences, therefore making a conclusive identification impossible (35). Although it is well-known that databases may contain poor-quality sequences, the discordant results may also be related to the deposition of misidentified strains. Therefore, the occurrence of S. commune may have been overestimated and other species may in fact have been involved in both human and animal infections. Hence, the real distribution of the species of this genus in clinical samples is unknown. Therefore, the aim of this study was to assess the spectrum of Schizophyllum spp. in a set of clinical isolates from the United States by comparing representative reference strains of different Schizophyllum species and using a multilocus phylogenetic analysis to clarify their taxonomy. Additionally, the in vitro antifungal susceptibilities of the fungi investigated to six antifungal agents were evaluated to determine if the susceptibility profiles of the U.S. strains differ from those of strains isolated from other parts of the world.

MATERIALS AND METHODS

Fungal isolates.

A total of 33 Schizophyllum strains were included in this study; 23 of these were clinical isolates recovered from across the United States and received in the Fungus Testing Laboratory at the University of Texas Health Sciences Center at San Antonio (UTHSCSA) for identification, antifungal susceptibility testing, or both. The U.S. isolates were of human (n = 21) and animal (n = 2) origin and were mainly recovered from the respiratory tract (n = 19, 82.6%), i.e., sinuses (43.5%), bronchoalveolar lavage fluid (26.1%), sputum (8.7%), and lung (4.3%). Additionally, one isolate was from a lymph node, another isolate was from a spinal mass, and two other isolates were of unknown origin. The remaining 10 isolates were obtained from different international culture collections as reference strains for comparison (Table 1).

TABLE 1.

Origins and GenBank accession numbers of the sequences of Schizophyllum strains included in this study^a

Species (no. isolates)	Strain	Origin	GenBank accession no.
			ITS	LSU	EF-1α	RPB2
S. commune (8)	CBS 132304	Human, India	LT217530	LT217561	LT217595	LT217629
	CBS 476.64	Unknown, USA	LT217531	LT217562	LT217596	LT217630
	FMR 14713	Sputum, India	LT217532	LT217563	LT217597	LT217631
	MUCL 20578	Fagus sylvatica, Belgium		LT217564	LT217598	LT217632
	MUCL 29305	Human, Brazil	LT217533	LT217565	LT217599	LT217633
	MUCL 30748	Saccharum officinarum, Africa	LT217534	LT217566	LT217600	LT217634
	MUCL 31016	Hay, Belgium	LT217535	LT217567	LT217601	LT217635
	UTHSCSA DI14-5	Sinus-nasal cavity, USA	LT217536	LT217568	LT217602	LT217636
S. radiatum (23)	CBS 301.32	Unknown, Panama	LT217537	LT217569	LT217603	LT217637
	UTHSCSA DI14-1	Unknown, USA	LT217539	LT217571	LT217605	LT217639
	UTHSCSA DI14-2	Lymph node,b USA	LT217540	LT217572	LT217606	LT217640
	UTHSCSA DI14-3	Lung,b USA	LT217541	LT217573	LT217607	LT217641
	UTHSCSA DI14-4	BAL, USA		LT217574	LT217608	LT217642
	UTHSCSA DI14-6	Sputum, USA	LT217542	LT217575	LT217609	LT217643
	UTHSCSA DI14-7	Maxillary sinus, USA		LT217576	LT217610	LT217644
	UTHSCSA DI14-8	BAL, USA	LT217543	LT217577	LT217611	LT217645
	UTHSCSA DI14-9	Sinus-nasal cavity, USA	LT217544	LT217578	LT217612	LT217646
	UTHSCSA DI14-10	BAL, USA	LT217545	LT217579	LT217613	LT217647
	UTHSCSA DI14-11	Sinus-nasal cavity, USA	LT217546	LT217580	LT217614	LT217648
	UTHSCSA DI14-12	Sphenoid sinus, USA	LT217547	LT217581	LT217615	LT217649
	UTHSCSA DI14-13	Spinal mass, USA	LT217548	LT217582	LT217616	LT217650
	UTHSCSA DI14-14	Sphenoid sinus, USA	LT217549	LT217583	LT217617	LT217651
	UTHSCSA DI14-15	BAL, USA	LT217550	LT217584	LT217618	LT217652
	UTHSCSA DI14-16	BAL, USA	LT217551	LT217585	LT217619	LT217653
	UTHSCSA DI14-17	Sputum, USA	LT217552	LT217586	LT217620	LT217654
	UTHSCSA DI14-18	Abscess, USA	LT217553	LT217587	LT217621	LT217655
	UTHSCSA DI14-19	BAL, USA	LT217554	LT217588	LT217622	LT217656
	UTHSCSA DI14-20	Maxillary sinus	LT217555	LT217589	LT217623	LT217657
	UTHSCSA DI14-22	Sinus-nasal cavity, USA	LT217556	LT217590	LT217624	LT217658
	UTHSCSA DI14-23	Ethmoid sinus, USA	LT217557	LT217591	LT217625	LT217659
	UTHSCSA DI14-26	Maxillary sinus, USA	LT217558	LT217592	LT217626	LT217660
S. fasciatum	CBS 267.60	Unknown, USA	LT217559	LT217593	LT217627	LT217661
S. umbrinum	MUCL 43017	Unknown, USA	LT217560	LT217594	LT217628	LT217662

^aBAL, bronchoalveolar lavage fluid specimen; CBS, CBS Fungal Biodiversity Centre (The Netherlands); FMR, Facultat de Medicina de Reus (Spain); MUCL, Université Catholique de Louvain (Belgium); UTHSCSA, University of Texas Health Science Center (San Antonio, TX, USA); ITS, internal transcribed spacer regions of the rDNA and 5.8S regions; LSU, partial large subunit of the rDNA; EF-1α, partial translation elongation factor gene; RPB2, partial RNA polymerase II second largest subunit.

^bAnimal origin.

DNA extraction, amplification, and sequencing.

Total genomic DNA was extracted from potato dextrose agar (PDA; Pronadisa, Madrid, Spain) cultures after 7 days of incubation at 25°C using a FastDNA kit and a FastPrep instrument (MP Biomedicals, Irvine, CA, USA), according to the manufacturer's specifications. Four DNA targets were amplified using the following primer pairs: ITS4 and ITS5 for internal transcribed spacer 1 (ITS1), the 5.8S gene, and the ITS2 regions (36); LR0R and LR5 for a portion of the large subunit (LSU) gene of ribosomal DNA (rDNA) (37); EF-983F and EF-2218R for the translation elongation factor 1α (EF-1α) gene (38); and 5F and 7CR for the RNA polymerase II second-largest subunit (RPB2) gene (39). PCR products were sequenced in both directions, using the same primers, at Macrogen Europe (Macrogen Inc., Amsterdam, The Netherlands). The sequences were assembled and edited using the Sequencher (v.4.1.4) program (Gene Codes Corporation, Ann Arbor, MI, USA).

Molecular identification and phylogenetic analyses.

The phylogenetic analyses were carried out first individually for each gene, and after the topologies proved to be congruent, a concatenated study was performed. All sequences used for the analyses were obtained from the strains included in this study. For multiple-sequence alignment, the ClustalW tool was used together with the MUSCLE tool in MEGA (v.6) software (40) with manual adjustments. The maximum likelihood (ML) phylogenetic method, as well as the estimation of the best nucleotide substitution method, was also run with MEGA (v.6) software. Support of the internal branches was assessed by the bootstrap (bs) method with 1,000 replications, where values of ≥70 were considered significant. The Bayesian inference (BI) method was performed using MrBayes (v.3.1.2) software (41). The evolutionary models that best fit each gene were assessed by MrModelTest (v.2) software (42). Markov chain Monte Carlo (MCMC) sampling was performed with two simultaneous runs for 3 million generations, with samples taken every 100 generations. The 50% majority rule consensus trees and posterior probability (pp) values were calculated after removing the first 25% of the resulting trees for burn-in. A pp value of ≥0.95 was considered in the tree.

Antifungal susceptibility testing.

The in vitro susceptibility profiles of the isolates were determined by the CLSI M38-A2 method with a few modifications (43). The modifications included working inocula, obtained from colonies on PDA with 7 to 10 days of incubation at 30°C, of 2.5 × 10⁴ to 5.0 × 10⁴ hyphal fragments/ml, and the microplates were incubated at 35°C for 72 h. The six antifungal agents tested were amphotericin B (AMB; Sigma-Aldrich Quimica S.A., Madrid, Spain), itraconazole (ITC; Jansen Pharmaceuticals, Beerse, Belgium), posaconazole (PSC; Schering-Plough Research Institute, NJ, USA), voriconazole (VRC; Pfizer S.A., Madrid, Spain), caspofungin (CFG; Merck & Co., Inc., Rahway, USA), and terbinafine (TBF; Sigma-Aldrich Química S.A., Madrid, Spain). The MIC was defined as the lowest drug concentration that produced 100% inhibition of visible fungal growth for AMB and the azoles (ITC, PSC, and VRC) or 80% inhibition for TBF. The minimum effective concentration (MEC) was determined for CFG and was defined microscopically to be the lowest concentration of drug that led to the growth of small, rounded, compact hyphal forms rather than the long, unbranched hyphal clusters that were seen in the growth control. Two quality control strains (Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258) were used in each test, and their MIC ranges were within the CLSI reference ranges. All tests were carried out in duplicate on different days for determination of reproducibility. Results were statistically analyzed using Prism software for Windows (v.6.0; GraphPad Software, San Diego, CA).

Accession numbers.

The sequences newly generated in this study were deposited in GenBank under the accession numbers shown in Table 1.

RESULTS

The phylogenetic analyses of the individual markers proved that the rDNA regions tested (ITS and LSU) were much conserved and did not discriminate well between the closely related species S. commune and S. radiatum. The LSU, EF-1α, and RPB2 markers showed consistency and were used to perform a concatenated study. The ITS region was not included in the combined alignment because, apart from not being informative for differentiating between the above-mentioned species, some strains were difficult to amplify and sequence. For example, the ITS sequence could not be obtained for MUCL 20578, UTHSCSA DI14-4, and UTHSCSA DI14-7 (Table 1).

The concatenated sequence alignment consisted of 2,622 bp (LSU, 838 bp; EF-1α, 971 bp; RPB2, 813 bp), from which 539 bp were variable sites (LSU, 30 bp; EF-1α, 248 bp; RPB2, 261 bp) and 282 bp were parsimony informative (LSU, 2 bp; EF-1α, 116 bp; RPB2, 164 bp). The topologies of the trees obtained with ML and BI analyses were basically the same, with only minor differences in the support values of internal nodes being observed. Figure 1 shows the phylogenetic tree constructed using the LSU, EF-1α, and RPB2 markers. Two main clades were observed in the phylogenetic tree. One clade included the strains of S. commune (i.e., 7 reference strains and 1 clinical isolate), and the other clade included the only reference strain of S. radiatum (CBS 301.32) used in the study together with the 22 remaining clinical isolates. Both clades showed high support values (values of bs/pp of 99/1 and 100/0.87, respectively). The reference strains of S. umbrinum and S. fasciatum acted as outgroups since the genetic distance to the clades of S. radiatum and S. commune were elevated (about 11.5% for S. umbrinum and about 9% for S. fasciatum for the LSU, EF-1α, and RPB2 markers). The average genetic distance between the clades of S. radiatum and S. commune was 4.0%, and the differences within the clades were 1.8% and 2.7% for S. radiatum and S. commune, respectively.

FIG 1.

Maximum likelihood tree obtained from the combined LSU, EF-1α, and RPB2 sequences of the isolates. Branch lengths are proportional to the phylogenetic distance. Bootstrap support values/Bayesian posterior probability scores over 70/0.95 are indicated on the nodes. The fully supported branches (bootstrap support value/Bayesian posterior probability score, 100/1) and reference strains are shown in bold. CBS, CBS Fungal Biodiversity Centre (The Netherlands); FMR, Facultat de Medicina de Reus (Spain); MUCL, Université Catholique de Louvain (Belgium); UTHSCSA, University of Texas Health Science Center (San Antonio, TX, USA).

In general, all the drugs tested showed activity against the Schizophyllum isolates studied. However, the antifungal with the greatest potency was AMB, followed by CFG and TBF (geometric mean [GM] MICs, 0.29 μg/ml, 0.58 μg/ml, and 0.79 μg/ml, respectively), while the least effective agents were ITC and PSC (GM MICs, 1.67 μg/ml and 2.93 μg/ml, respectively). Significant variation in in vitro activity was noted among the strains, especially for TBF, with MIC values ranging from 0.03 to 16.0 μg/ml. The drug displaying the most consistent results was CFG, with MEC values ranging from 0.25 to 1.0 μg/ml. Schizophyllum radiatum showed clearly higher GM MICs for all the antifungals tested than S. commune, especially for ITC and PSC. The results of the in vitro susceptibility test are summarized in Table 2.

TABLE 2.

Results of in vitro antifungal susceptibility test for 31 isolates of Schizophyllum spp.

Species (no. isolates)	Parameter	MIC or MEC (μg/ml) fora:
		AMB	CFG	ITC	PSC	TBF	VRC
S. commune (8)	GM	0.09	0.41	0.37	0.20	0.61	0.20
	MIC range	0.03–0.25	0.25–1.0	0.25–1.0	0.12–0.5	0.12–>16	0.12–0.5
	Mode	0.25	0.25	0.25	0.25	0.25	0.25
S. radiatum (23)	GM	0.43	0.66	2.82	7.46	0.86	1.72
	MIC range	0.06–2.0	0.25–1.0	0.12–>16.0	0.25–>16.0	0.03–>16.0	0.06–>16.0
	Mode	0.5	1.0	>16.0	>16.0	0.5	>16.0
	MIC90	2.0	1.0	>16.0	>16.0	>16.0	>16.0
Total (31)	GM	0.29	0.58	1.67	2.93	0.79	0.99
	MIC range	0.03–2.0	0.25–1.0	0.12–>16.0	0.12–>16.0	0.03–>16.0	0.06–>16.0
	Mode	0.5	1.0	0.25	>16.0	0.25	0.12
	MIC90	2.0	1.0	>16.0	>16.0	>16.0	>16.0

^aAMB, amphotericin B; CFG, caspofungin; ITC, itraconazole; PSC, posaconazole; TBF, terbinafine; VRC, voriconazole; MEC, minimum effective concentration, which is provided instead of the MIC for CFG; GM, geometric mean.

Taxonomy.

Schizophyllum was described by Fries in 1815 (44) with S. commune as the type species. The same author transferred the morphologically similar species Agaricus radiatum to Schizophyllum (45), which was accepted by Linder in 1933 (46). In contrast, Cooke in 1961 considered both to be varieties of the same species and treated S. radiatum as a synonym of S. commune (2). However, in the absence of modern taxonomic studies on this genus, due mainly to the lack of ex-type strains, the Index Fungorum (http://www.indexfungorum.org) and MycoBank (http://www.mycobank.org) databases list the species as different taxa.

According to our phylogenetic analysis, the clade formed by the confirmed strain of S. radiatum and most of the clinical isolates from the United States constitutes a well-supported monophyletic lineage with enough genetic differences to be considered a species distinct from S. commune, which forms a sister clade. For the purpose of taxonomic stability of the name S. radiatum and based on the current recommendations (47, 48), we selected strain CBS 301.32 as the epitype (CBS H-22699, MBT372269) for this species. This strain was collected in the same region as the type strain (Panama and Jamaica, respectively) and was identified by Linder (46) on the basis of the original description provided by Fries (45). Among numerous specimens, Linder examined the authentic material of S. radiatum, which is deposited in the Farlow Herbarium at Harvard University (46). Sequence data from the ex-epitype may be useful for future phylogenetic studies of Schizophyllum species, especially for the identification of nonsporulating isolates of S. radiatum involved in human infections, and to determine whether this species is also predominant among the Schizophyllum species in other parts of the world.

Our results also show that S. commune represents a species complex, which was already mentioned by Linder (46), since the genetic variation within the clade ranged from 0.9% (between MUCL 20578 and UTHSCSA DI14-5) to 3.5% (between MUCL 31016 and FMR 14713), with a mean variation of 2.7%. Nonetheless, further studies are needed to solve the taxonomic differentiation within the S. commune clade.

DISCUSSION

Schizophyllum is a very common and cosmopolitan genus of basidiomycetes that has been associated with human infections for over 60 years (11, 21). The most recent reviews of infections by S. commune were by Buzina et al. (33), who reported 16 published cases, and Chowdhary et al. (13), who reported 71 cases. More recently, 15 additional cases have been reported (14, 15, 17, 19, 49–59). Here, we studied 23 isolates of Schizophyllum from different clinical specimens, and although histopathological evidence for the corresponding infections is not available, this number of isolates from a single country reinforces the growing importance of Schizophyllum in the clinical setting. The different clinical manifestations highlight the versatility of this pathogen (13); however, the pulmonary and respiratory sites appear to be the most common sites of recovery. In Colombia, S. commune was the second most common agent of fungal rhinosinusitis, following Aspergillus (60). In our study, 43.5% of the strains were isolated from sinuses, and a total of 82.6% of strains were from patients with respiratory conditions. Interestingly, in a study on the distribution and seasonal diversity of pathogenic fungi in outdoor air in the northeastern United States, which used quantitative real-time PCR plus pyrosequencing, S. commune was the most abundant species, particularly in spring (61). However, the identification of S. commune exclusively in all those studies should be taken with caution due to the lack of phylogenic studies that have clarified the taxonomy of closely related species in Schizophyllum, such as S. commune and S. radiatum.

Although in nature these fungi mainly adopt the form of a gilled mushroom (62), when they are isolated from clinical specimens and growing in culture, Schizophyllum strains often remain sterile (63). This precludes their morphological identification. In routine laboratories, the induction of sporulation usually requires about 3 weeks and is associated with high failure rates (34). Therefore, molecular methods, such as DNA sequence analysis, are required for accurate identification of this kind of fungus that fails to sporulate (13). The most commonly sequenced markers in Schizophyllum are the ITS and D1/D2 regions of the LSU (13, 33, 34). However, Schizophyllum has a highly conserved ITS region, which is often difficult to amplify and to sequence. Singh et al. (34) reported this difficulty, being unable to amplify 3 of 27 strains that they identified as S. commune. Although the LSU region is more informative, in general, its resolution is only at the genus level. In addition, Romanelli et al. (35) demonstrated, with filamentous basidiomycetes that included Schizophyllum, that a comparison with sequences in the GenBank database can cause disagreement between results when both the ITS and LSU regions of the same strain are searched; of the 15 Schizophyllum strains (9 S. commune and 6 S. radiatum strains) identified by ITS sequencing, only 1 strain of S. radiatum agreed with the identification by use of the LSU region. Moreover, two strains identified as belonging to Phlebia by use of the ITS were identified to be Schizophyllum spp. by use of the LSU region. The inconsistency of results made it impossible to assign a conclusive identification to over 70% of the isolates included in that study (35). It would seem clear, then, that other, more appropriate markers need to be adopted for reliable species identification. In the present study, the use of three markers combined (LSU, EF-1α, and RPB2) provided enough phylogenetic information for this purpose. We were able to demonstrate that more than one species may be involved in Schizophyllum infections, and, contrary to general belief, S. radiatum was shown to be, almost exclusively, the species identified from clinical isolates of a U.S. reference center. In addition, this study provides a set of 33 strains (4 species) of Schizophyllum spp. for future comparison.

The optimum management of infections by S. commune has not yet been determined (14, 31, 34). However, treatment with antifungal drugs has given encouraging results. AMB is reported to be the most potent in vitro against S. commune, and other antifungal drugs have also shown good activity (14, 16, 31, 64). It is worth mentioning, however, that at least one patient with allergic bronchopulmonary infection treated with ITC for 10 months showed no clinical improvement (65, 66). In fact, a correlation between in vitro data and clinical efficacy has not been well established (54). In agreement with the findings of other studies (30, 31), AMB showed the highest in vitro potency against most Schizophyllum isolates tested. The strains of S. commune tested here had low MICs for PSC and VRC (0.20 μg/ml for both), similar to previous reports (30, 31). For S. radiatum, our results revealed high degrees of variability in the activities of the agents tested among strains, especially for the azoles and for TBF (Table 2). In the case of PSC, MIC values of S. radiatum were significantly higher than those of S. commune (GM MICs, 7.46 μg/ml and 0.20 μg/ml, respectively; P < 0.0001) when they were compared using the Mann-Whitney U test. For ITC and VRC, the differences were not statistically significant, even though the MICs of S. radiatum were apparently higher than those of S. commune (GM MICs, 2.82 μg/ml and 0.37 μg/ml, respectively, for ITC, and 1.72 μg/ml and 0.20 μg/ml, respectively, for VRC). For the echinocandin tested, CSP, in general, low MECs were observed (range, 0.25 to 1.0 μg/ml), contrary to the findings of Singh et al. (34), who reported high values (range, 2 to 8 μg/ml) of the same drug for S. commune. Considering that echinocandins have been reported to be ineffective against clinically relevant basidiomycetes (67, 68), our discrepant results might be due to variations in the methods used (variations in incubation times, methods of preparation of the inoculum with hyphal fragments, etc.). Since there is no standardized method for antifungal susceptibility testing of filamentous basidiomycetes and nonsporulating fungi (68), studies often modify the CLSI protocol (31, 34) in an attempt to obtain reproducible results, as in our case. Therefore, before standardization of these protocols, interlaboratory results cannot be properly compared.

In conclusion, the importance of Schizophyllum in the clinical setting has been demonstrated by the number of reported cases and strains isolated. The present study with a collection of isolates from the United States has shown that S. radiatum, rather than S. commune, is the most frequently recovered species and provides informative targets for molecular discrimination between the two species.