Agar block smear preparation: a novel method of slide preparation for preservation

Transcription

1 JCM Accepts, published online ahead of print on 21 July 2010 J. Clin. Microbiol. doi: /jcm Copyright 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. 1 2 Agar block smear preparation: a novel method of slide preparation for preservation of native fungal structures for microscopic examination and long-term storage Patrick C. Y. Woo, 1,2,3,4 Antonio H. Y. Ngan, 4 Hon-Kit Chui, 4 Susanna K. P. Lau, 1,2,3,4 Kwok-Yung Yuen 1,2,3,4 * State Key Laboratory of Emerging Infectious Diseases, 1 Research Centre of Infection and Immunology, 2 Carol Yu Centre of Infection, 3 and Department of Microbiology, 4 The University of Hong Kong, Hong Kong Running title: Agar block smear preparation PCY Woo and AHY Ngan contributed the same to the manuscript. *Corresponding author. Mailing address: State Key Laboratory of Emerging Infectious Diseases, Department of Microbiology, The University of Hong Kong, University Pathology Building, Queen Mary Hospital, Hong Kong. Phone: (852) Fax: (852) hkumicro@hkucc.hku.hk. 1

2 ABSTRACT We describe a novel method of fungal slide preparation, named agar block smear preparation. Five hundred and ten agar block smears of 25 fungal strains obtained from culture collections, 90 QC fungal strains and 82 clinical fungal strains from our clinical microbiology laboratory, which included a total of 137 species of yeasts, molds and thermal dimorphic fungi, were prepared and examined. In contrast to adhesive tape preparation, agar block smears preserved the native fungal structures, such as intact conidiophores of Aspergillus species and arrangement of conidia in Scopulariopsis breviculis. Furthermore, agar block smears allowed examination of fungal structures embedded in the agar, such as the ascomatum with ascomal hairs in Chaetomium funicola; pycnidium of Phoma glomerata; the intercalary ovoidal chlamydospores arranged in chains of Fusarium dimerum and the lateral, spherical chlamydospores arranged in pairs of Fusarium solani. After one year of storage, morphological integrity was maintained in 459 (90%) of the 510 agar block smears. After three years of storage, morphological integrity was maintained in 72 (71%) of the 102 smears prepared in Agar block smear preparation preserves the native fungal structures and allows long-term storage and examination of fungal structures embedded in the agar, hence overcoming the major drawbacks of adhesive tape preparation. The major roles of agar block smear should be for diagnosis of difficult cases, accurate diagnosis of fungal species for clinical management of patients and epidemiological studies and long-term storage for transportation of slides and education purposes. 2

3 INTRODUCTION Accurate identification of fungi is the cornerstone of selecting and prescribing appropriate antifungal drugs to patients with fungal infections. In contrast to bacteria for which antibiotic susceptibility testing is routinely performed when a clinically significant bacterium is isolated, antifungal susceptibility testing is not commonly performed due to the relatively poor correlation between in vitro susceptibility results and clinical response. On the other hand, the choice of antifungal drugs in patients with fungal infections relies heavily on the identification of the corresponding isolates. Although molecular methods, such as internal transcribed spacer region and 18S rrna sequencing, have been increasingly used for fungal identification, these technologies are still expensive and require the corresponding expertise (5-7). Therefore, most clinical microbiology laboratories still rely on phenotypic methods for identification of fungi. Identification of molds in clinical microbiology laboratories is most commonly performed by culture on agar plates followed by microscopic examination of lactophenol cotton blue stained adhesive tape preparations of the fungal colonies for direct visualization of characteristic microscopic morphological features. However, adhesive tape preparations are associated with three major drawbacks. First, fungal structures may be squashed and damaged during the preparation procedures, hence affecting the accurate identification of the fungus. Second, as a result of drying of the smear, adhesive tape preparations can only be used for microscopic examination within a few hours. Third, fungal structures embedded in the agar cannot be observed. To overcome these and other drawbacks, methods such as the microslide method, are sometimes employed when 3

4 greater morphological detail is necessary. However, microslide cultures are also not suitable for long-term storage. Throughout the years, modifications of microslide cultures have been suggested, but these methods were still far from ideal (1, 2). Although an improvement to adhesive tape preparations, the double-layer tape prep, which extended the useful life of the tape preparation to several weeks, was suggested recently, this is still not suitable for long-term storage, sending the slide out for consultation and examination of fungal structures embedded in agar (3). In this article, we describe a novel method of slide preparation, named agar block smear preparation, which preserves the native structures of molds and yeasts, and allows long-term storage and examination of fungal structures embedded in the agar, hence overcoming all three drawbacks of adhesive tape preparation. Downloaded from on October 6, 2018 by guest 4

5 MATERIALS AND METHODS Strains. Twenty-five fungal strains obtained from culture collections, 90 QC fungal strains and 82 fungal strains isolated from patients in the clinical microbiology laboratory of Queen Mary Hospital in Hong Kong were included in the present study (Table 1). A total of 137 species of yeasts, molds and thermal dimorphic fungi were included. Culture medium. The transparent, extremely low nutrition culture medium for detection of Acanthamoeba species in ophthalmologic specimens (Acanthamoeba agar), which contained 12% (w/v) NaCl, 4% (w/v) MgSO 4 7H 2 O, 14.2% (w/v) Na 2 HPO 4, 13.6% (w/v) KH 2 PO 4 and 4% (w/v) CaCl 2 2H 2 O, was used for agar block smear preparation unless otherwise stated (4). Agar block smear preparation method. Fungus was inoculated onto Acanthamoeba agar in a class II biological safety cabinet and incubated at optimal conditions for the specific fungus. Spore formation and other characteristic structures were checked every two to seven days, depending on the growth rate of the fungus, by agar block examination of agar plates under light microscope (40 or 100 magnification) (Fig. 1a). When the fungus was mature, photos could be taken directly using the microscope camera and the plates were used for subsequent agar block cutting. For known or suspected biosafety level 3 fungi such as Coccidioides immitis, the agar plate was fumigated using 37% formaldehyde for 48 h before agar block cutting. In a class II biological safety cabinet, a 15 mm 15 mm agar block was cut using sterile dissecting knife and placed on a glass slide (Fig. 1b). After adding a drop of 5

6 lactophenol cotton blue stain or other stains such as calcofluor white stain, a cover slip (18 mm 18 mm) was put onto the agar block (Fig. 1c). The agar block was examined under light microscope (400 or 1000 magnification) (Fig. 1d). For long-term storage, the block was air-dried at room temperature in a class II biological safety cabinet until the thickness of the agar block reached 0.5 mm, which usually took about 48 h (Fig. 1e). The four sides of the agar block under the cover slip were filled with Permount Mounting Medium (Fisher Scientific, NJ, USA) (Fig. 1f). After drying of the mounting medium, the agar block smear was examined under light microscope (400 or 1000 magnification). Downloaded from on October 6, 2018 by guest 6

7 RESULTS Direct examination of agar plate under microscope before cutting the agar block. This allows observation of very delicate fungal structures of diagnostic significance. It should be noted that for very fragile and delicate conidial structures, their arrangements on conidiophores may still be destroyed even by agar block smear preparation. For example, all the conidia of Fusarium chlamydosporum were dislodged from the polyphialides during agar block smear preparation (Fig. 2a), whereas their flower-like arrangement on the polyphialides was well preserved when the whole agar plate was observed under light microscope before cutting the agar block (Fig. 2b). Similarly, the conidia chains of Fusarium verticllioides that were attached to the phialides (Fig. 2c) and the unique conidia chains resulting from basipetal growth in Trichothecium roseum (Fig. 2d) were well preserved when the whole agar plate was observed under light microscope, but they were destroyed by agar block smear preparation. Additionally, direct examination of the agar plate under microscope was particularly useful for observation of the conidial head of Aspergillus species and sporangiophores arrangement in members of Mucorales, both being key features for their speciation. Observed with a lateral light source, the conidial heads of Aspergillus niger was radiate (Fig. 2e) while those of Aspergillus fumigatus were columnar (Fig. 2f). As for the Mucorales, the sporangiophores of Lichtheimia corymbifera were branched in umbel (Fig. 2g) while the unbranched sporangiophores of Rhizopus microsporus group were found directly above the rhizoids (Fig. 2h). 7

8 Microscopic examination of slides prepared by agar block smear preparation method. A total of 510 agar block smears of 137 fungal species were prepared (Table 1), with 102 smears prepared in 2006, 153 smears in 2007 and 255 smears in Slides that were prepared using the agar block smear preparation method were compared to those using the adhesive tape method. Preservation of native structural features. Intact conidiophores with foot cells of Aspergillus versicolor were well preserved in agar block smears. As a result, the length of conidiophores, which is one of the key features for Aspergillus speciation (Fig. 3a), could be measured easily. On the other hand, conidiophores, especially the longer ones, were usually broken due to the tearing force during preparation of adhesive tape smear (Fig. 3b). For Scopulariopsis breviculis, its conidiogenous cells (annelides) were well preserved in agar block smears (Fig. 3c). They were either single or in brush-like clusters with short conidiophores. From the annelides, truncated conidia in chains were produced basipetally. On the other hand, the annelides were overlapped with each other and their native states were destroyed adhesive tape smears (Fig. 3d). In the agar block smear of Phialophola verrucosa, its phialides, like a dozen of vases of flowers, were distributed alongside the vegetative hyphae (Fig. 3e). The phialides are flask-shaped with funnel-like collarettes. Round to oval conidia accumulated in clusters at their apices. However, in the adhesive tape smear of P. verrucosa, artificial overlapping of philalides were observed (Fig. 3f). 8

9 For Cladophialophora bantiana, the elliptical conidia arranged in chains and their origins were well preserved in agar block smears (Fig. 3g). On the other hand, many acropetal conidia chains, with the youngest conidia at tip, were detached and conidiophores were not observed in adhesive tape smears (Fig. 3h). Observation of fungal structures embedded under agar surface. For Chaetomium funicola, the ascomatum with typical ascomal hairs that was partially embedded in agar was well shown by agar block smear (Fig. 4a). The arrangements of the straight, stiff ascomal hairs that were dichotomously branched repeatedly were better preserved in agar block smears (Fig. 4a) than in tease mounts. They differed from the non-branched, undulate ascomal hairs of a more common Chaetomium species, C. globosum. Under high power magnification, its limoniform ascospore and the distinctly verrucose ascomal hairs were observed (Fig. 4b). For Phoma glomerata, a coelomycete which produces conidia by conidiogenous cells lining the inner cavity of its asexual fruiting bodies called pycnidia, a spherical pycnidium embedded partially under the agar surface was observed with two ostioles in agar block smear (Fig. 4c). It is darkly pigmented around the ostioles from which conidia were released. A brown chlamydospore with longitudinal and transverse septa (muriform), an additional key structure for identification of P. glomerata, was also well demonstrated (Fig. 4d). For Pyrenochaeto unguis-hominis, also a coelomycete but differs from P. glomerata by its setose pycnidium, was also well shown in agar block smear (Fig. 4e). Unlike other Pyrenochaeto species with setae tapering towards the tip, the setae observed in P. unguis-hominis were obtuse at their apices (Fig 4f). 9

10 The arrangement and position of chlamydospores are one of the critical characteristics for Fusarium speciation. For F. dimerum, the intercalary chlamydospores arranged in a chain were ovoidal in shape (Fig. 4g), but for F. solani, the lateral chlamydospores were usually arranged in pairs and spherical in shape (Fig. 4h). These chlamydospores were often found on the submerged hyphae. Microscopic examination of agar block smears after long term storage. After one year of storage, 459 (90%) out of 510 agar block smears were kept in good state. After three years of storage, 72 (71%) out of the 102 smears prepared in 2006 were still in good state. Fig. 5a and b showed an agar block smear of C. immitis stored for more than three years. Under low power magnification, chains of arthroconidia with alternate disjuncter cells were well displayed (Fig. 5a). Raquet hyphae were also observed. Under high power magnification, conidiogenesis of alternate arthroconidia chains was clearly demonstrated (Fig. 5b). They were formed by fragmentation of hyphae through disjunctor cells dissolution. Barrel-shaped arthroconidia were usually attached with annular frill on both ends (Fig. 5b). Fig. 5c showed an agar block smear of Bipolaris hawaiiensis stored for more than one year. In contrast to other Bipolaris species, its macroconidia showed more than three distosepta. They were pseudosepta and differentiated from the true one by the cytoplasm contraction of distoseptate conidia and becoming angular with lactophenol cotton blue staining. Fig. 5d showed an agar block smear of Exophiala dermatitidis stored for more 10

11 than one year. Torulose hyphae with rare spherical phialides were demonstrated. Being a dematiaceous fungus, dark pigment of melanin was obvious on its cell walls. Fig. 5e, f, g and h showed agar block smears stained with calcofluor white and stored for more than six months. The fluorescence did not fade and the different colors of fluorescence were due to different barrier wavelengths used in the fluorescent microscope (430 nm for blue and 520 nm for green fluorescence respectively). Fig. 5e showed an agar block smear of Graphium eumorphum, the asexual synanamorph of Pseudallescheria boydii. Conidiophores were aggregated into a compound stalk called synnemata. A sterile basal part and a fertile head producing cylindrical terminal conidia were showed in green fluorescence. Fig. 5f showed an agar block smear of Exophiala moniliae in blue fluorescence. Annellated taping, which protruded and became rather long with age, was clearly observed as deeply pigmented tip due to melanin deposit. Fig. 5g showed an agar block smear of Phialemonium curvatum in blue fluorescence. Short adelophialides without basal septum, a characteristic of P. curvatum, were clearly observed, which was different from Acremonium strictum which showed long phialides with basal septum (Fig. 5h). 11

12 DISCUSSION The marked increase in the density of intact native structures of fungi observed in agar block smears markedly increased the accuracy of laboratory diagnosis of pathogenic fungi. The integrity and native arrangement of conidiophores and sporulating structures are of paramount importance for microscopic speciation of pathogenic fungi. The length of conidiophores is important for speciation of some fungi, such as the Aspergillus species, whereas the arrangement of conidia is crucial to identification of others, such as S. breviculis. During the process of adhesive tape smear preparations that primarily demonstrates fungal structures on the surface of agar plates, many native structures of fungi are squashed and torn, leading to breaking of most conidiophores and loss of the native arrangement of the sporulating structures. As for fungal structures partially or completely embedded inside the agar, such as the fruiting bodies of some ascomycetes and coelomycetes, although they could be dug out by tease mounts, the native orientation of the fruiting structures is often destroyed. On the other hand, for agar block smear preparations, the whole thickness of agar was gradually compressed to 0.5 mm. This allows the preservation and examination of the native structures of most fungi, both formed on the surface and embedded in the agar. Furthermore, it also markedly increases the chance of recognizing rare structures, such as the spherical phialides of Exophiala dermatitidis. As for the medium, when the concept of agar block smear was first conceived in 2006, a number of transparent culture media, including Sabouraud dextrose agar, carnation leaf medium and Acanthamoeba medium were tried for growing the fungi for agar block smear preparations. Our preliminary study showed that Acanthamoeba 12

13 medium was best in promoting conidiogenesis of most molds because of its extremely low nutrition. For slow growing dermatophytes, longer incubation time was required for spore formation. For example, at least three weeks of incubation was necessary for the observation of the macroconidia of Microsporum canis and Microsporum gypseum. Good preservation of the native fungal structures for agar block smears with respect to time allows the sending of slides out for consultation and education purposes. In July 2006, the concept of agar block smear was conceived and the first agar block smear was made from a strain of Fusarium solani. Up to now, more than 500 smears have been prepared from 137 fungal species of yeasts, hyaline molds with septate hyphae, dematiaceous molds with septate hyphae, members of Mucorales with rarely septate hyphae and thermal dimorphic fungi. In our experience, more than 90% of the smears were still in good conditions after the first one year. After one year of storage, the most serious problem that could affect the quality of the smears was air leakage through the mounting medium, with the resultant air bubbles and drying effect jeopardizing the spore structures. Other minor problems include fading of stain color and degeneration of mycelium. The major roles of agar block smear should be for diagnosis of difficult cases, accurate diagnosis of fungal species for epidemiological and clinical studies and longterm storage for transportation of slides and education purposes. Despite the advantages of agar block smear, it cannot replace adhesive tape smear. Adhesive tape smear is easy and quick to perform and inexpensive. With an agar plate culture, a preliminary identification of the molds to the genus level can often be achieved within 15 min using 13

14 adhesive tape smear. This preliminary identification of clinically significant molds is of paramount importance to the choice of antifungal agents and has high impact on patient management. On the other hand, preparation of agar block smear requires subculturing of the fungus and the time for preparation of the smear. Therefore, agar block smear cannot replace adhesive tape smear in clinical microbiology laboratories on a day-to-day basis because of the turn-around-time and economic considerations. On the other hand, for accurate determination at the species level so as to elucidate, for example, the differential antifungal susceptibility of different species to antifungal agents, agar block smear would be more preferred than adhesive tape smear. Downloaded from on October 6, 2018 by guest 14

15 ACKNOWLEDGEMENTS This work is partly supported by a Research Grants Council Grant; University Development Fund, The University of Hong Kong; and the HKSAR Research Fund for the Control of Infectious Diseases of the Health, Welfare and Food Bureau. Downloaded from on October 6, 2018 by guest 15

16 REFERENCES 1. Connell, S. L., and D. E. Padgett An improved technique for making permanent slide cultures of fungi. Mycopathologia 101: Harris, J. L Modified method for fungal slide culture. J. Clin. Microbiol. 24: Hughes, A. D., G. D. Lorusso, and D. L. Greer The 'double-layer tape prep': an improvement to a standard technique. J. Med. Microbiol. 53: Isenberg, H. D Clinical Microbiology Procedures Handbook, second edition. ASM Press, Washington, D.C. 5. Lau, S. K., P. C. Woo, S. K. Chiu, K. W. Leung, R. W. Yung, and K. Y. Yuen Early diagnosis of Exophiala CAPD peritonitis by 18S ribosomal RNA gene sequencing and its clinical significance. Diagn. Microbiol. Infect. Dis. 46: Woo, P. C., S. K. Lau, A. H. Ngan, H. Tse, E. T. Tung, and K. Y. Yuen Lasiodiplodia theobromae pneumonia in a liver transplant recipient. J. Clin. Microbiol. 46: Woo, P. C., S. Y. Leung, K. K. To, J. F. Chan, A. H. Ngan, V. C. Cheng, S. K. Lau, and K. Y. Yuen Internal transcribed spacer region sequence heterogeneity in Rhizopus microsporus: implications on molecular diagnosis in clinical microbiology laboratories. J. Clin. Microbiol. 48:

17 LEGENDS TO FIGURES FIG. 1. Preparation of agar block smears. Panel a, spore formation and other characteristic structures were checked by agar block examination of agar plates under light microscope. Panel b, a 15 mm 15 mm agar block was cut using sterile dissecting knife. Panel c, a cover slip (18 mm 18 mm) was put onto the agar block after lactophenol cotton blue staining. Panel d, the agar block with cover slip was examined under light microscope. Panel e, the block was air-dried until the thickness of the agar block reached 0.5 mm. Panel f, the agar block under the cover slip was filled with mounting medium. FIG. 2. Direct examination of agar plate under microscope before agar block cutting. Panel a and b, Fusarium chlamydosporum, showing the dislodged conidia from the polyphialides during agar block smear preparation (Panel a) and the well preserved flower-like arrangement on the polyphialides when the whole agar plate was observed under light microscope before cutting the agar block (Panel b). Panel c, Fusarium verticllioides, showing the conidia chains attaching to the phialides. Panel d, Trichothecium roseum, showing the unique conidia chains resulting from basipetal growth. Panel e, Aspergillus niger, showing the radiating conidial heads. Panel f, Aspergillus fumigatus, showing the columnar conidial heads. Panel g, Lichtheimia corymbifera, showing the sporangiophores that are branched in umbel. Panel h, Rhizopus microsporus, showing the unbranched sporangiophores directly above the rhizoids. FIG. 3. Preservation of native structural features of fungi by agar block smears. Panel a, Aspergillus versicolor, prepared by agar block smear method, showing intact 17

18 conidiophores with well preserved foot cells (arrow). Panel b, A. versicolor, prepared by adhesive tape method, showing broken conidiophores. Panel c, Scopulariopsis breviculis, prepared by agar block smear method, showing its annelides arranged either singly or in brush-like clusters and truncated conidia produced in chain basipetally. Panel d, S. breviculis, prepared by adhesive tape method, showing its annelides artificially overlapping with each other (arrow). Panel e, Phialophola verrucosa, prepared by agar block smear method, showing its flask-shaped phialides well distributed along the vegetative hyphae with funnel-like collarettes (arrow). Panel f, P. verrucosa, prepared by adhesive tape method, showing artificial overlapping of philalides and air bubbles (arrow). Panel g, Cladophialophora bantiana, prepared by agar block smear method, showing elliptical conidia arranged in chains from indistinct conidiophores. Panel h, C. bantiana, prepared by adhesive tape method, showing many detached acropetal conidia chains with the youngest conidia at tip (arrow), and the presence of tape ripples making it unable to take photos at the same focal plane. FIG. 4. Observation of fungal structures embedded under agar surface in agar block smears. Panel a, Chaetomium funicola, showing the ascomatum with typical straight, stiff, dichotomously branched ascomal hairs partially embedded in agar. Panel b, Chaetomium globosum, showing the distinctly verrucose ascomal hairs (arrow) and its limoniform ascospore (arrow). Panel c and d, Phoma glomerata, showing a spherical pycnidium embedded partially under the agar surface with two ostioles (arrows) and a brown chlamydospore with longitudinal and transverse septa. Panel e and f, Pyrenochaeto unguis-hominis, showing the characteristic setose pycnidium and setae 18

19 with obtuse apices (arrow). Panel g, Fusarium dimerum, showing the intercalary ovoidal chlamydospores arranged in chains. Panel h, Fusarium solani, showing the lateral spherical chlamydospores arranged in pairs. FIG. 5. Microscopic examination of agar block smears after long-term storage. Panel a and b, Coccidioides immitis, showing chains of arthroconidia with alternate disjuncter cells, raquet hyphae (arrow) and alternate arthroconidia chains with barrelshaped arthroconidia usually with annular frill (arrow) on both ends. Panel c, Bipolaris hawaiiensis, showing macroconidia with more than three distosepta (arrow). Panel d, Exophiala dermatitidis, showing the torulose hyphae with rare spherical phialides (arrow) and dark pigment of melanin on its cell walls. Panel e, Graphium eumorphum stained with calcofluor white, showing conidiophores aggregated into a compound stalk called synnemata and cylindrical terminal conidia. Panel f, Exophiala moniliae stained with calcofluor white, showing annellated taping (arrow) which protruded and became rather long with age. Panel g, Phialemonium curvatum stained with calcofluor white, showing the short adelophialides without basal septum (arrow). Panel h, Acremonium strictum stained with calcofluor white, showing the long phialides with basal septum (arrow). 19

20 343 TABLE 1. Fungal species used in the present study Fungal species Source NEQAS UK QC CAP USA QC Hyaline molds (nondermatophytes) Aspergillus candidus Our clinical microbiology laboratory Strains from culture collections Aspergillus clavatus Aspergillus flavipe Aspergillus flavus (ATCC ) Aspergillus fumigatus Aspergillus gaucus Aspergillus niger Aspergillus restrictus Aspergillus sydowii Aspergillus terreus Aspergillus ustus Aspergillus versicolor Emericella nidulans Eurotium chevalieri (CBS ) Eurotium cristatum (CBS ) Eurotium rubrum Neosartorya fischeri Neosartorya pseudofischeri Acremonium strictum Arthrinium phaeospermum Beauveria bassiana Botrytis cinerea Cephalotheca foveolata Chrysosporium keratinophilum Fusarium chlamydosporum Fusarium dimerum Fusarium nygamai (FRC-M7492) Fusarium oxysporum (NRRL-28973) Fusarium proliferatum (FRC-M6992) Fusarium solani (CBS ) Fusarium verticillioides (ATCC MYA 3629) Gliocladium species Lecythophora hoffmannii Malbranchea species Monilia species Nectria haematococca Paecilomyces lilacinus Paecilomyces variotii (ATCC MYA 3630) Penicillium species Scopulariopsis brevicaulis Thermomyces species Trichoderma species Trichothecium roseum 20

21 Hyaline molds (dermatophytes) Epidermatophyton floccosum Microsporum canis (CBS ) Microsporum ferrugineum Microsporum gypseum Microsporum nanum Microsporum persicolor Trichophyton erinacei Trichophyton fischeri Trichophyton mentagrophyte Trichophyton rubrum (CBS ) Trichophyton schoenleinii Trichophyton soudanense Trichophyton tonsurans Trichophyton verrucosum Trichophyton violaceum Dematiaceous molds Alternaria species Aureobasidium pulluans Bipolaris hawaiiensis Chaetomium funicola Chaetomium globosum Cladophialophora bantiana Cladophialophora boppii Cladosporium carrionii Cladosporium cladosporioides Cladosporium herbarum Curvularia species Cyphellophora pluriseptata Epicoccum species Exophiala dermatitidis Exophiala jeanselmei Exophiala moniliae Exserohilum species Fonsecaea pedrosoi Hormonema dematioides Hortaea werneckii Lasiodiplodia theobromae Nigrospora species Ochroconis constricta Ochroconis gallopavum Phaeoacremonium parasiticum Phialemonium curvatum Phialophora fastigiata Phialophora richradsiae Phialophora verrucosa Phoma glomerata Pithomyces species 21

22 Pseudollescheria boydii (CBS ) Pyrenochaeto unguis-hominis Rhinocladiella aquaspersa Scedosporidium prolificans Scytalidium dimidiatum Scytalidium hyalinum Ulocladium species Mucorales Absidia coerulea (MUCL10045) Cunninghamella bertholletiae Cunninghamella species Lichtheimia corymbifera (MUCL10046) Lichtheimia blakesleeana (CBS ) Lichtheimia hyalospora (CBS ) Mucor species Rhizomucor pusillus Rhizopus azygosporus Rhizopus microsporus var. microsporus Rhizopus microsporus var. (CBS ) oligosporus Rhizopus microsporus var. (CBS631.82) chinensis Rhizopus microsporus var. (CBS343.29) rhizopodiformis Rhizopus oryzae (CBS ) Rhizopus stolonifer Syncephalastrum racemosum Yeasts Blastoschizomyces capitatum Candida albicans (ATCC 90028) Candida dubliniensis Candida glabata Candida guilliermondii Candida kefyr Candida krusei (ATCC 6258) Candida lusitaniae Candida parapsilopsis (ATCC 22019) Candida tropicalis Cryptococcus albidus Cryptococcus neoformans (CBS 132) Cryptococcus uniguttulatus Geotrichum candidum Malassezia pachydermatis Malassezia furfur (CBS 1878) Prototheca wickerhamii Rhodotorula rubra Saccharomyces cerevisiae Trichosporon species 22

23 Ustilago species Dimorphic fungi Blastomyces dermatitidis Coccidioides immitis Penicillium marneffei Sporothrix schenckii 344 Downloaded from on October 6, 2018 by guest 23

24 FIG. 1. Downloaded from on October 6, 2018 by guest

25 FIG. 2. Downloaded from on October 6, 2018 by guest

26 FIG. 3. Downloaded from on October 6, 2018 by guest

27 FIG. 4. Downloaded from on October 6, 2018 by guest

28 FIG. 5. Downloaded from on October 6, 2018 by guest