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Clinical and laboratory aspects of the “black yeasts”
Clinical Microbiology Newsletter October 1,1993
Vol. 15, No. 19
Clinical and Laboratory Aspects of the “Black Yeasts” Deanna A. McGough, B.S., MT,SM(ASCP), RM,SM(AAM) Departmentof Pathology Fungus Testing Laboratory University of Texas Health Science Center of San Antonio San Antonio, TX 78284
With the advent of an increasingly immunocompromised patient population has come the concomitant escalation of mycotic disease caused by a myriad of saprobic fungi, many of which are black, brown, olivaceous, or derivations thereof, i.e., dematiaceous. These dematiaceous fungi may be arbitrarily grouped together on the basis of their pigmentation, which in most cases is dihydroxynaphthalene melanin (1,2). They could also be (and frequently are) categorized by the disease processes they incite, such as the previously defined clinical entities of chromoblastomycosis, formerly chromomycosis (3), and eumycotic mycetoma (4), or the more recently described mycotic infections classified under the term phaeohyphomycosis ($6) (Table 1). Using this approach, it becomes @parent that the same organism may be responsible for more than one disease, i.e., Exophiala jeanselmei variety jeanselmei may cause chromoblastomycosis, eumycotic mycetoma, and phaeohyphomycosis. In fact, the current, preferred method of describing a mycotic condition is to cite the disease process followed by the term “caused by” or “due to,” which would indicate the etiologic agent. Placement of black moulds into any of the above groups is done, however, without regard to their method of coniCMNEEI 15(19)145-152.1993
diogenesis or conidial morphologyfactors that current taxonomy emphasizes. To further add to the confusion, several dematiaceous Hyphomycetes (those moulds whose conidia are freeborne, i.e., not enclosed in some type of conidiomata) also have the propensity to form black yeast synanamorphs (other asexual forms of the same fungus) at some stage in their development. Although the concept of “black yeasts” is complex and controversial, the focus of this article will be an attempt to define what we mean by “black yeasts,” identify which organisms may be included, describe the disease entities they elicit, provide salient histopathological, macroscopic, microscopic, and physiologic features useful for their identification, and consider therapeutic options. Definition of “Black Yeasts” In 1959, Ulson proposed the Portuguese term “leveduras pretas,” which means “black yeasts” (7). Although this term enjoyed wide acceptance as a valid mycological description, it was not until 1978 that it was adequately defined by Ajello to mean “dematiaceous filamentous fungi that, in certain stages of their development, have a unicellular form during which time multiplication is by a budding process. The colonies at this stage are pasty with some shade of black” (8). The dematiaceous or black yeasts (Hyphomycetes) may have basidiomycetous or ascomycetous affinities, deposit melanin in their cell walls, and are capable of forming myceha1 synanamorphs. These fungi are contrasted with the true yeasts (EndoEkevier
mycetales) that produce mycelia (basidiomycetous and ascomycetous species) but lack melanin in their cell walls (9). Some of the black yeasts, however, such as Phaeococcomyces nigricans, apparently exist only as Blastomycetes and fail to demonstrate hyphal synanamorphs. Although some taxonomists would include several genera under the umbrella of “black yeasts” (Table 2), McGinnis et al. (10) prefer to reserve the term for those dematiaceous fungi “that grow as solitary conidiogenous cells that collectively result in, at least initially, brown to black pasty colonies.” Thus, they proposed the family Phaeococcomycetaceae McGinnis et Schell in the class Blastomycetes, Form-division Fungi Imperfecti, to accommodate the dematiaceous yeast genera Phaeoannellomyces and Phaeococcomyces (Table 3). Adhering to this philosophy, one could apply different names to the same organism depending upon whether it displayed the yeast or
In This Issue Clinical and Laboratory Aspects of the “Black Yeasts” . . . . . . . . . . 145 An overview and update on the taxonomy, histopathology, identification, and therapy of this interesting group of organisms
Blood Culture Detection of in a Transplant Patient . . . . . . . . . . . . . . . . . . . . . . 151
Legionella
A case report
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+ 06.00
TABLE 1. Dematiaceous etidogic agents for the currently recognized clinical entities Eumycotic mycetoma (characterized by tumefaction, draining sinus tracts, and grains/granules) Curvularia spp. Exophiala jeanselmei Leptosphaeria spp. Madurella spp. Neotestudina rosatii Pseudallescheria
boya%
Chromoblastomycosis (characterized by verrucose lesions, pseudoepitheliomatous hyperplasia, and sclerotic bodies in tissue: develops over a period of years) Cla&sporium
carrionii
Exophiala jeanselmei Fonsecaea pedrosoi Fonsecaea compacta Phialophora verrucosa Rhinocladiella aquaspersa FInreohyphomycosis [characterized by “mycotic infections caused by &matiaceous fungi that, in tissue, form either yeastlike cells that are solitary or in short &aim or hyphae that are septate, often irregularly swollen to toruloid, branched or unbranched, or any combination of these forms” ( 18)la Alternaria spp. Anthopsis deltoidea Aureobasidium pullulans var. pullulans
mould phase. Not all authorities agree with this concept of providing generic names for synanamorphs, however, and point out that the application of separate names does not mean that we are dealing with distinct taxa. The production of synanamorphs among the dematiaceous Hyphomycetes illustrates another significant factor in terms of their definition. McGinnis and Schell(11) have suggested that the definition of polymorphic fungi should be based on the single most stable and distinctive anamorphy, whereas Gams (12) advocates relying upon the most differentiated anarnorphy. Several reports have indicated, however, that these “stable” or “distinctive” anamotphs may be
TABLE 2, Genera considered by some to be ‘black yeasts” but that fail to produce synaoamorpbs in the family Waeococcomscetaceae
Bipolaris spp.
Aureobasidiwn
Botryomyces caespitosis
Botryomyces de Hoog et Rubio
Chaetomium spp.
Phaeotheca Sigler, Tsuneda et Carmichael
CIadosporiwn spp. Curvuharia
Viala et Bayer
Phaeosclera Sigler, Tsuneda et Carmichael
spp.
Sarcinomyces Liidner
Dactylaria constricta var. gallqxzva sensu Dixson et al. (Ochroconis gallopavum)
Hormonema Lag&erg
Exophiala spp.
Lecythophora Nannfeldt
et Mehn
Exserohilwn spp. Hormonema &matioi&s Lecythophora spp. Nattrassia mangiferae (Hendersonula toruloidea) Phaeoannellomyces
spp.
Phialemonium spp. Phialophora spp. Phoma spp. Pleurophoma spp. Scedosporiuw prolijicans (Sceakxporiwn injlatwn) Scytalidium dimidiatwn
LThislist is not inclusive of all agents.
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Phaeoannellomyces McGinnis et Schell 1985 (for those isolates that consist of percurrently prolliferating conidiogenous dematiaceous yeast cells [anneliides and annelloconidial]) Phaeococcomyces de Hoog 1977 (for those isolates whose dematiaceous yeast cells form blastoconidia)
Wangielhz akrmatitidis Xylohypha spp.
146
TABLE 3. Genera of C‘blackyeasts” as defined by McGinnis and Scbell io tbe family Pb aeococcomyrce*eae
8 1993 Elsevier Scimce PublishingCo., Inc.
Clinical Microbiology Ncwsktter 15:19,1993
TABLE 4. Wack yeasts” and their associated mould synanamorphsaTb Mould synanamorphs
Black yeast Phaeoannellomyces eLegans Schelll985 (PE)
McGiis
et
Exophiala jeanselmei (Langeron) McGinnis et Padhye var. jeanselmei de Hoog 1977 (WJ) Exophiala jeanselmei (Langeron) McGinnis et Padhye var. lecanii-corni (Benedek et Specht) de Hoog 1977 (EILC)
Phaeoannellomyces
elegans
Phaeoannellomyces
elegans
Exophiala jeanselmei (Langeron) McGinnis et Padhye var. hetermotpha (Nannfeldt) de Hoog 1977 (BIH)
Phaeoannellomyces
elegans
Exophiala jeanselmei (Langeron) McGinnis et Padhye var. castellanii (Iwatsu, Nishimura et Miyaji) Iwatsu et Udagawa 1990 (EIC)
Phaeoannellomyces
sp.
Exophiala moniliae de Hoog 1977 (EM)
Phaeoannellomyces
sp.
Exophiala spinifera (Neilsen et Conant) McGinnis 1977 (ES)
Phaeoannellomyces
sp.
Exophialapisciphila 1974 (EP)
Phaeoannellomyces
sp. (?)
Exophiala angulospora Iwatsu, Udagawa et Takase 1991 (13Qc
Phaeoannellomyces wemeckii (Horta) McGinnis et Schelll985 (PW) Phaeoannellomyces 1977 (PEK)
exophialae de Hoog
Exophiala werneckii
McGinnis et Ajello
(Horta)von Arx 1970
VW) Wangiella dermatitidis (K.ano) McGinnis (WD)
aAdapted from reference 16. bInitials after name refer to organisms in Table 6. ‘No published reports but possibly pathogenic to humans based upon the coenzyme Q system reported by Yamada (20.21).
highly dependent upon such factors as incubation temperature,media, degree of aeration, inoculum size, and light (13-H), and that there is not general agreement as to what constitutes the most stable and distinct anatomic forms.
“Black Yeast” Taxa Table 4 lists organisms that most authorities would consider in the “black yeast” category. Currently, the yeast anarnorphsare found in the genera Phaeoannellomyces and Phaeococcomyces, whereas their corresponding mould synanamorphsate species of Exophiala and Wangiella dermatitidis. Some would also contend that species of Phialophora exhibit polymorphism, although this view is not shared by all. It seems only reasonable that additional genera may be required to adequately accommodate this complex, heterogeneous group of fungi as we become more knowledgeable of their makeup at the molecular level (17). Clinical Microbiology Nmvsletkr 1519.1993
Clinical Spectrum of “Black Yeast” Infections Infections caused by “black yeasts” may be separatedinto three main categories based upon the disease processes they elicit: (i) chromoblastomycosis, characterized by verrucose lesions, pseudoepitheliomatous hyperplasia, and sclerotic bodies (muriform cells) in tissue; (ii) eumycotic mycetoma, characterized by tumefaction, draining sinus tracts, and grains or granules composed of mycelial aggregates; or (iii) phaeohyphomycosis, which may be superficial, cutaneous, subcutaneous, or systemic (and disseminated). Phaeohyphomycosis encompasses a distinct, heterogenous group of mycotic infections in which the etiologic agents occur in tissue as typically dematiaceous yeastlike cells, pseudohyphae, or short hyphal elements that may be distorted (toruloid), swollen (monilifonn), or any combination of the above characteristics. Several histological stains are useful for Q 1993 Elsevier Science Publishing Co., Inc.
the detection of these fungal elements, with the Grocott-Gomori methenamine silver stain possibly providing the best contrast for staining the fungi black. To document the dematiaceous nature of these elements, however, one must be able to discern the pigment through periodic acid Schiff, hematoxylin and eosin-stained, or unstained tissue sections. The use of the Fontana-Masson stain for human melanin has considerable utility in this setting provided appropriate fungal controls are used and there is adequate staining time for fungal melanin (19). With our patient population becoming ever more compromised, phacohyphomycosis may well be the most frequently seen entity, particularly in developed countries. Superficial disease is usually confined to the stratum comeum with little or no tissue response. In cutaneous or cornea1 phaeohyphomycosis keratinized tissue is invaded, nonliving layers are involved, and extensive tissue destruction is frequently present. Subcutaneous disease frequently results from the traumatic implantation of foreign material and lesions often remain localized with the formation of abscesses. Systemic disease may be the result of dissemination to various organs, occurs most frequently in the host with a severely abro-
TABLE 5. Clinical classification of disease processes caused by “black yeasts” and their etiologic agents Chromoblastomycosis Exophiala jeanselmei Eumycotic mycetoma Exophiala jeanselmei Phaeohyphomycosis Superficial, Tmea nigra Phaeoannellomyces Cutaneous
werneckii
and cornea1
Exophiala jeanselmei Subcutaneous Exophiala jeamelmei Exophiala moniliae Exophiala spinifera Wangiella dermatitidis systemic Exophiala jeanselmei Wangiella afermatitidis
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TABLE 6. IdenWmtion
chart
-Co1 diamb
Figure
EW
25oC
35T
+
+
4OT
NlT
+
(mm) >lO
+
+
+
>lO
3.3-5.9
1.5-2.4
Conidia elongate, formed primarily from intercalary conidiogenous loci.
+
+
t-
>6 9-16
2.6-4.2 x 1.6-2.5
Budding cells give rise to secondary conidia. Loose chains of globose cells of variable length.
+
+
+
<6
Conidia (~.rm) 1X1-5.0 x 0.9-2.9
x
x
2.5-5.0
1.5-3.0
Comments Annellides taper, med. length_ Conidia from apex accumulate in balls. Assimilates melezitose.
Young col. wet, mucoid. Old col. wrinkled, brain-like. Conidiierous
pegs
EM
V
+
>lO
1 S-2.5
x
1 O-1.5
Conidia short. Swollen conidiogenous cell. Tyrosinc + 3 wk.
Es
+
+
>lO
l&3.9
x
1 O-2.9
Long spinelike conidiophores ceils at base.
with dark
EP
-
+
>lO
2.5-4-O
x
6.0-8.0
Large conidia. Conidiophores those as in ESd,’
resemble
EA
-
+
2.54.0
x
2.0-3.0
Angular conidia. Conidial apparatus differentiated, swollen!
3.0-6.8
Conidia 2celled. Growth in 15% NaCl. Agent of tinea nigra.
<6
PW +
WD
PE
+
+
PM
+
+
+
1.5-3.0x
6-16
2.5-6.1 x 2.9-3.9
Conidiogenous cell phialide without collarette. Young col. wet, mucoid may remain so.g
.lO
2.1 x 4.3
Young, hyaline, thin-walled; mature, black, thick-walled, usually single annellidic conidiogenous locus.
6-16
3-6
Mucaid, slimy colonies. Globose. cells, l-3 loci.
%ce Table 4 for organismnames. b Sabouranddextroseagar, 14 days, 27°C. ’ All species may be somewhatyeastlike when young. Growthon potato dextroseagaror potatoflakes agarappearsto promotemore yeastlike colonies &an does Sabonrauddextroseagar at 25°C. d Fish pathogencausing human disease.
‘Fish pathogensand other species of Exophdalacking yeast synanamorphsomittedfrom this table. Consult reference 16. f Possible human pathogen. g Not agreed upon by all taxonomists.
gated immune system, and carries a grave prognosis.
How to Identify Them The detection of compatible dematiaceous fungal elements in tissue is tantamount to authenticating mycotic disease caused by “‘blackyeasts.” Having documented this prerequisite, growth of the organism in culture and its subsequent identification confirms the etiologic agent. Table 6 is a compilation of macroscopic, microscopic, and physiologic features that defme these organisms. We should describe here, however, the differences between tbe conidiogenous cells in Exophiula and 148
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Phaeoanneliomyes,which are annellides, and those seen in Wangiella, which are phialides without collarettes. Annelloconidia are produced from a flask-shaped cell that extends by percurrent proliferation (growth of inner layers through outer layers) thus becoming longer and narrowerwith the production of each new conidium. Rings or annellations of outer cell-wall ma&&i are laid down in the process. I%ialocomdia &I!3 n=FJd=dftQm that do not incmase in length or taper. Both of the above representblastic production. This distinction is useful with light microscopy because the actual anQ 1993Elsevicr Scimcc F’ublishing Co., Inc.
nellations are difficult to discern withouttheuseofa scanning electron microscope. Blastocomdia, as seen in Phaeococcomyces,ofiginate“as a blownout portion of the mother cell. The developing ce& isrecognizable before being rIe&&ed by ase@un~.~The daughter ceil mayseparate completely or remain attacbedto form eha&’ (22). Itisalsoimporta&toreitera@that this is an extremely polytrmqbic group of H:M tbey&s@ayat~ygiveRtime~ highly &pendent upon a variety of cultural factors. Our expfzriencewit&EXOphialajeanselmei vaFie(ylecanii-corni Cliiical Mcmbiology Newsletter 15:19,1993
Figure I. Colonial morphology of Exophiala jeanselmei variety castellanii grown for 4 wk on potato flakes agar at 25°C. Note the combinadon of yeastlike, black, pasty areas (usually seen when colonies are young) coupled with filamentous areas (develops as cultures matures).
and Phaeoannellomyces
Figure 2. Exophiala jeanselmei variety jeanselmei showing medium-length annellides as well as annelloconidia being produced from intercalary conidiogenous loci (those on the hyphae). Nomarski optics x 1250.
elegans has
shown that the most “stable characteristics” for each of these anamorphs are not very stable at all, and may be manipulated at will by altering the environmental conditions.Bearing this in mind, one could apply different names to the same organism as it exhibits its various phenotypic morphologies. One additional tool that exists for separating some species of “black yeasts” is the exoantigen. Its utility in the routine laboratory is limited, however, as reagents are not commercially available. It also remains to be seen if DNA probes or other molecular technologies will be useful in separating the various taxa.
Figure 3. Exophiala jeanselmei variety lecanii-comi showing conidia being producedprimarily from intercalaty conidiogenous loci. Cells of black yeast synanamorph aLso present. Nomarski optics x 1250.
Therapeutic Considerations Defining therapeutic regimens is beyond the scope of this +cle; however, our in vitro experience at the Fungus Testing Laboratory coupled with anecdotal reports would suggest that the recently approved triazole, itraconzaole (Sporanox), may be an alternative to more toxic agents for the “black yeasts.” Treatment with this antifungal may need to be long term, however, and in the case of systemic disease, probably in high dose. Darkly pigmented imperfect fungi that exhibit “black yeast” synanamorphs comprise a complex group of organisms that often are difficult to Clinical Microbiology Newsletter 15:19,1993
Figure 4. Exophiala jeanselmei variety castellanii. Lateral annellated conidiferous pegsproducing annelloconidia. Nomarski optics x 1250.
0 1993 Elsevier Sciwce Publishing Co., Inc.
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classify, name, and identify. Attempts to standardize media and environmental conditions would appear to facilitate establishing more tmiform identification and nomenclature schemes for these polymorphic fungi by microbiologists from diverse mycological backgrounds. References
FigrcIIZ 5. Exophiala moniliae. Note moniliform (swollen) conidiogenous xl2 50.
cells. Nomarksi cw its
1.
Dixon. D. M., P. J. Szaniszlo, and A. Polak. 1991. Dihydroxynaphthalene (DHN) melanin and its relationship with virulence in the early stages of phawhyphomycosis, p. 297-318. In G. T. Cole and H. C. Hoch (ed.), The fungal spore and disease initiation in plants and animals. plenum Ress, New York.
2.
Polak, A. 1990. Melanin as a virulence factor in pathogenic fungi. Mycoses 33:215-224.
3.
Terra F. et al. 1922. Novo typo de dermatite verrucosa mycose por Acrotheca corn associacao de leishmaniosa. BraxilMedica. 2~253-259.
4.
McGinnis, M. R. and R. C. Fader. 1988. Mycetoma: a contemporary concept, Infect. Dis. Clin. N.A. 23939-954.
5.
Ajello L. et al. 1974. A case of phawhyphomywsis caused by a new species of Phialophora. Mycologia. 66~490-498.
6.
McGinnis, M. R. 1983. Chromoblastomycosis and phaeohyphomycosis: new concepts, diagnosis, and mycology. J. Am. Acad. Dermatol. 81-16.
7.
Ulson, CM. 1959. Contribuicao para o estudo das chamadas “leveduras pretas.” Tese, Department0 de Publicatoes, FacuIdade de Medicina, Universidade de Sao Paulo, Sao Paulo, Brasil.
8.
Ajello, L. 1978. The black yeasts as disease agents: historical perspective, p. 916. In The black and white yeasts. Scientific Publication NO. 356. Pan American Health Organization, Washington, D.C.
9.
De Hcog, G. S, and M. R. McGiiis. 1987. Aswmycetous black yeasts. Studies Mycol. 30~187-199.
Figswe 6. Exophiala spinifera. Note long, spine-like conidiophores that are darkened at the base. Non wrski optics x 625.
10. McGinnis, M. R., W. A. Schell, and J. Carson. 1985. Phaeoannellomyces and the Phaeococwmycetaceae, new dematiaceous blastomycete taxa. J. Med. Vet. Mycol. 23:179-188.
Figwe 7. Exophiala pisciphila. Long conidiophores resembling those of Exophiala spin5 era but not the large size of annelloconidia. Nomarski optics x 625.
150
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8 1993 Elm&r Science PublishingCo., Inc.
11. McGinnis, M. R. and W. A. Schell. 1980. The genus Fonrecaea a& its relationship to the genera C~porium, Phialophora, Ramichloridiwn and Rhinocladiella. p. 215-234. Scientifz Publication No. 3%. Pan American He&h Organization, Washington, D.C.
Clinical Microbiology Newsletter 15:19,1993
12. Gams, W. 1982. Generic names for synanamorphs? Mycotaxonomy 15:459464. 13. Hardcastle, R.V. and P.J. Szaniszio. 1974. Characterization of dimorphism in Cludosporium werneckii. J. Bacteriol, 119:294-302. 14. Houston, M. R. et al. 1969. Dimorphism in Cladosporium werneckii. Sabouraudia. 7:195-198. 15. McGough, D. A., A. W. Fothergill, and M. G. Rinaldi. Abstr. Annu. Meet. Am. Sot. Microbial. 1991, F7, p. 409. 16. Dixson, D. M. and A. Polak-Wyss. 1991. The medically important dematiaceous fungi and their identification. Mycoses. 34:1-18. 17. Kawasaki, M. et al. 1990. Mitochondrial DNA analysis of Exophiulu jeunselmei and Exophialu dermatitidis. Mycopathologia. 110: 107-l 12.
Figure 8. Wangiella dermatitidis. Note prominent yeast cells, few hypae, and conidia being produced from cells that do not taper.
18. Jorgensen, J. H. and M. G. Rinaldi. 1986. A Clinician’s dictionary of bacteria and fungi. Eli Lilly and Company, Indianapolis, IN. 19. Wood, C. and B. Russel-Bell. 1983. Characterization of pigmented fungi by melanin staining. Am. J. Dermatopathol. 5:77-81. 20. Iwatsu, T., S. Udagawa, and T. Takase. 199 1. A new species of Exophiala recovered from drinking water. Mycotaxonomy. XLI:321-328. 21. Yamada, Y. et al. 1989. Coenzyme Q systems in ascomycetous black yeasts. Antonie van Leeuwenhoek. 56:349356. 22. Dixon, D. and R. A. Fromtling. 1991. Morphology, taxonomy, and classification of fungi, p. 579-587. In Balows et al. (ed.), Manual of clinical microbiology, 5th Pd., American Society for Microbiology, Washington, D.C.
Figure 9. Yeast cell of Phaeoannellomyces elegans showing hvo distinct unnellations and bud separating from the mother cell. Scanning electron microscopy x 22,100.
Case Report
Blood Culture Detection of Legionella in a Transplant Patient Donna M. Carter, M.T., A.S.C.P. Department of Pathology Loyola University Medical Center Foster G. McGuw Hospital Muywood, IL 60153
Clinical Microbiology Newsletter 15:19,1993
Case Report A 56-yr-old immunosuppressed male underwent a cadaveric renal transplant for chronic renal failure at Loyola University Medical Center. His course was complicated on the sixth postoperative day when he developed an oral temperature of 39.7”c associated with rigors and a productive cough. The
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Science Publishing Co., Inc.
chest X-ray confirmed the diagnosis of pneumonia. The sputum Gram stain showed many white blood cells, but no organisms. The initial antimicrobial regimen used was erythromycin and aztreonam. Because of the patient’s increasing lethargy and seizures, rifampin was added to his treatment regimen on the ninth post-operative day. Routine
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Vol. 15, No. 19
Clinical and Laboratory Aspects of the “Black Yeasts” Deanna A. McGough, B.S., MT,SM(ASCP), RM,SM(AAM) Departmentof Pathology Fungus Testing Laboratory University of Texas Health Science Center of San Antonio San Antonio, TX 78284
With the advent of an increasingly immunocompromised patient population has come the concomitant escalation of mycotic disease caused by a myriad of saprobic fungi, many of which are black, brown, olivaceous, or derivations thereof, i.e., dematiaceous. These dematiaceous fungi may be arbitrarily grouped together on the basis of their pigmentation, which in most cases is dihydroxynaphthalene melanin (1,2). They could also be (and frequently are) categorized by the disease processes they incite, such as the previously defined clinical entities of chromoblastomycosis, formerly chromomycosis (3), and eumycotic mycetoma (4), or the more recently described mycotic infections classified under the term phaeohyphomycosis ($6) (Table 1). Using this approach, it becomes @parent that the same organism may be responsible for more than one disease, i.e., Exophiala jeanselmei variety jeanselmei may cause chromoblastomycosis, eumycotic mycetoma, and phaeohyphomycosis. In fact, the current, preferred method of describing a mycotic condition is to cite the disease process followed by the term “caused by” or “due to,” which would indicate the etiologic agent. Placement of black moulds into any of the above groups is done, however, without regard to their method of coniCMNEEI 15(19)145-152.1993
diogenesis or conidial morphologyfactors that current taxonomy emphasizes. To further add to the confusion, several dematiaceous Hyphomycetes (those moulds whose conidia are freeborne, i.e., not enclosed in some type of conidiomata) also have the propensity to form black yeast synanamorphs (other asexual forms of the same fungus) at some stage in their development. Although the concept of “black yeasts” is complex and controversial, the focus of this article will be an attempt to define what we mean by “black yeasts,” identify which organisms may be included, describe the disease entities they elicit, provide salient histopathological, macroscopic, microscopic, and physiologic features useful for their identification, and consider therapeutic options. Definition of “Black Yeasts” In 1959, Ulson proposed the Portuguese term “leveduras pretas,” which means “black yeasts” (7). Although this term enjoyed wide acceptance as a valid mycological description, it was not until 1978 that it was adequately defined by Ajello to mean “dematiaceous filamentous fungi that, in certain stages of their development, have a unicellular form during which time multiplication is by a budding process. The colonies at this stage are pasty with some shade of black” (8). The dematiaceous or black yeasts (Hyphomycetes) may have basidiomycetous or ascomycetous affinities, deposit melanin in their cell walls, and are capable of forming myceha1 synanamorphs. These fungi are contrasted with the true yeasts (EndoEkevier
mycetales) that produce mycelia (basidiomycetous and ascomycetous species) but lack melanin in their cell walls (9). Some of the black yeasts, however, such as Phaeococcomyces nigricans, apparently exist only as Blastomycetes and fail to demonstrate hyphal synanamorphs. Although some taxonomists would include several genera under the umbrella of “black yeasts” (Table 2), McGinnis et al. (10) prefer to reserve the term for those dematiaceous fungi “that grow as solitary conidiogenous cells that collectively result in, at least initially, brown to black pasty colonies.” Thus, they proposed the family Phaeococcomycetaceae McGinnis et Schell in the class Blastomycetes, Form-division Fungi Imperfecti, to accommodate the dematiaceous yeast genera Phaeoannellomyces and Phaeococcomyces (Table 3). Adhering to this philosophy, one could apply different names to the same organism depending upon whether it displayed the yeast or
In This Issue Clinical and Laboratory Aspects of the “Black Yeasts” . . . . . . . . . . 145 An overview and update on the taxonomy, histopathology, identification, and therapy of this interesting group of organisms
Blood Culture Detection of in a Transplant Patient . . . . . . . . . . . . . . . . . . . . . . 151
Legionella
A case report
01%4399/93/$0.00
+ 06.00
TABLE 1. Dematiaceous etidogic agents for the currently recognized clinical entities Eumycotic mycetoma (characterized by tumefaction, draining sinus tracts, and grains/granules) Curvularia spp. Exophiala jeanselmei Leptosphaeria spp. Madurella spp. Neotestudina rosatii Pseudallescheria
boya%
Chromoblastomycosis (characterized by verrucose lesions, pseudoepitheliomatous hyperplasia, and sclerotic bodies in tissue: develops over a period of years) Cla&sporium
carrionii
Exophiala jeanselmei Fonsecaea pedrosoi Fonsecaea compacta Phialophora verrucosa Rhinocladiella aquaspersa FInreohyphomycosis [characterized by “mycotic infections caused by &matiaceous fungi that, in tissue, form either yeastlike cells that are solitary or in short &aim or hyphae that are septate, often irregularly swollen to toruloid, branched or unbranched, or any combination of these forms” ( 18)la Alternaria spp. Anthopsis deltoidea Aureobasidium pullulans var. pullulans
mould phase. Not all authorities agree with this concept of providing generic names for synanamorphs, however, and point out that the application of separate names does not mean that we are dealing with distinct taxa. The production of synanamorphs among the dematiaceous Hyphomycetes illustrates another significant factor in terms of their definition. McGinnis and Schell(11) have suggested that the definition of polymorphic fungi should be based on the single most stable and distinctive anamorphy, whereas Gams (12) advocates relying upon the most differentiated anarnorphy. Several reports have indicated, however, that these “stable” or “distinctive” anamotphs may be
TABLE 2, Genera considered by some to be ‘black yeasts” but that fail to produce synaoamorpbs in the family Waeococcomscetaceae
Bipolaris spp.
Aureobasidiwn
Botryomyces caespitosis
Botryomyces de Hoog et Rubio
Chaetomium spp.
Phaeotheca Sigler, Tsuneda et Carmichael
CIadosporiwn spp. Curvuharia
Viala et Bayer
Phaeosclera Sigler, Tsuneda et Carmichael
spp.
Sarcinomyces Liidner
Dactylaria constricta var. gallqxzva sensu Dixson et al. (Ochroconis gallopavum)
Hormonema Lag&erg
Exophiala spp.
Lecythophora Nannfeldt
et Mehn
Exserohilwn spp. Hormonema &matioi&s Lecythophora spp. Nattrassia mangiferae (Hendersonula toruloidea) Phaeoannellomyces
spp.
Phialemonium spp. Phialophora spp. Phoma spp. Pleurophoma spp. Scedosporiuw prolijicans (Sceakxporiwn injlatwn) Scytalidium dimidiatwn
LThislist is not inclusive of all agents.
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Phaeoannellomyces McGinnis et Schell 1985 (for those isolates that consist of percurrently prolliferating conidiogenous dematiaceous yeast cells [anneliides and annelloconidial]) Phaeococcomyces de Hoog 1977 (for those isolates whose dematiaceous yeast cells form blastoconidia)
Wangielhz akrmatitidis Xylohypha spp.
146
TABLE 3. Genera of C‘blackyeasts” as defined by McGinnis and Scbell io tbe family Pb aeococcomyrce*eae
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Clinical Microbiology Ncwsktter 15:19,1993
TABLE 4. Wack yeasts” and their associated mould synanamorphsaTb Mould synanamorphs
Black yeast Phaeoannellomyces eLegans Schelll985 (PE)
McGiis
et
Exophiala jeanselmei (Langeron) McGinnis et Padhye var. jeanselmei de Hoog 1977 (WJ) Exophiala jeanselmei (Langeron) McGinnis et Padhye var. lecanii-corni (Benedek et Specht) de Hoog 1977 (EILC)
Phaeoannellomyces
elegans
Phaeoannellomyces
elegans
Exophiala jeanselmei (Langeron) McGinnis et Padhye var. hetermotpha (Nannfeldt) de Hoog 1977 (BIH)
Phaeoannellomyces
elegans
Exophiala jeanselmei (Langeron) McGinnis et Padhye var. castellanii (Iwatsu, Nishimura et Miyaji) Iwatsu et Udagawa 1990 (EIC)
Phaeoannellomyces
sp.
Exophiala moniliae de Hoog 1977 (EM)
Phaeoannellomyces
sp.
Exophiala spinifera (Neilsen et Conant) McGinnis 1977 (ES)
Phaeoannellomyces
sp.
Exophialapisciphila 1974 (EP)
Phaeoannellomyces
sp. (?)
Exophiala angulospora Iwatsu, Udagawa et Takase 1991 (13Qc
Phaeoannellomyces wemeckii (Horta) McGinnis et Schelll985 (PW) Phaeoannellomyces 1977 (PEK)
exophialae de Hoog
Exophiala werneckii
McGinnis et Ajello
(Horta)von Arx 1970
VW) Wangiella dermatitidis (K.ano) McGinnis (WD)
aAdapted from reference 16. bInitials after name refer to organisms in Table 6. ‘No published reports but possibly pathogenic to humans based upon the coenzyme Q system reported by Yamada (20.21).
highly dependent upon such factors as incubation temperature,media, degree of aeration, inoculum size, and light (13-H), and that there is not general agreement as to what constitutes the most stable and distinct anatomic forms.
“Black Yeast” Taxa Table 4 lists organisms that most authorities would consider in the “black yeast” category. Currently, the yeast anarnorphsare found in the genera Phaeoannellomyces and Phaeococcomyces, whereas their corresponding mould synanamorphsate species of Exophiala and Wangiella dermatitidis. Some would also contend that species of Phialophora exhibit polymorphism, although this view is not shared by all. It seems only reasonable that additional genera may be required to adequately accommodate this complex, heterogeneous group of fungi as we become more knowledgeable of their makeup at the molecular level (17). Clinical Microbiology Nmvsletkr 1519.1993
Clinical Spectrum of “Black Yeast” Infections Infections caused by “black yeasts” may be separatedinto three main categories based upon the disease processes they elicit: (i) chromoblastomycosis, characterized by verrucose lesions, pseudoepitheliomatous hyperplasia, and sclerotic bodies (muriform cells) in tissue; (ii) eumycotic mycetoma, characterized by tumefaction, draining sinus tracts, and grains or granules composed of mycelial aggregates; or (iii) phaeohyphomycosis, which may be superficial, cutaneous, subcutaneous, or systemic (and disseminated). Phaeohyphomycosis encompasses a distinct, heterogenous group of mycotic infections in which the etiologic agents occur in tissue as typically dematiaceous yeastlike cells, pseudohyphae, or short hyphal elements that may be distorted (toruloid), swollen (monilifonn), or any combination of the above characteristics. Several histological stains are useful for Q 1993 Elsevier Science Publishing Co., Inc.
the detection of these fungal elements, with the Grocott-Gomori methenamine silver stain possibly providing the best contrast for staining the fungi black. To document the dematiaceous nature of these elements, however, one must be able to discern the pigment through periodic acid Schiff, hematoxylin and eosin-stained, or unstained tissue sections. The use of the Fontana-Masson stain for human melanin has considerable utility in this setting provided appropriate fungal controls are used and there is adequate staining time for fungal melanin (19). With our patient population becoming ever more compromised, phacohyphomycosis may well be the most frequently seen entity, particularly in developed countries. Superficial disease is usually confined to the stratum comeum with little or no tissue response. In cutaneous or cornea1 phaeohyphomycosis keratinized tissue is invaded, nonliving layers are involved, and extensive tissue destruction is frequently present. Subcutaneous disease frequently results from the traumatic implantation of foreign material and lesions often remain localized with the formation of abscesses. Systemic disease may be the result of dissemination to various organs, occurs most frequently in the host with a severely abro-
TABLE 5. Clinical classification of disease processes caused by “black yeasts” and their etiologic agents Chromoblastomycosis Exophiala jeanselmei Eumycotic mycetoma Exophiala jeanselmei Phaeohyphomycosis Superficial, Tmea nigra Phaeoannellomyces Cutaneous
werneckii
and cornea1
Exophiala jeanselmei Subcutaneous Exophiala jeamelmei Exophiala moniliae Exophiala spinifera Wangiella dermatitidis systemic Exophiala jeanselmei Wangiella afermatitidis
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TABLE 6. IdenWmtion
chart
-Co1 diamb
Figure
EW
25oC
35T
+
+
4OT
NlT
+
(mm) >lO
+
+
+
>lO
3.3-5.9
1.5-2.4
Conidia elongate, formed primarily from intercalary conidiogenous loci.
+
+
t-
>6 9-16
2.6-4.2 x 1.6-2.5
Budding cells give rise to secondary conidia. Loose chains of globose cells of variable length.
+
+
+
<6
Conidia (~.rm) 1X1-5.0 x 0.9-2.9
x
x
2.5-5.0
1.5-3.0
Comments Annellides taper, med. length_ Conidia from apex accumulate in balls. Assimilates melezitose.
Young col. wet, mucoid. Old col. wrinkled, brain-like. Conidiierous
pegs
EM
V
+
>lO
1 S-2.5
x
1 O-1.5
Conidia short. Swollen conidiogenous cell. Tyrosinc + 3 wk.
Es
+
+
>lO
l&3.9
x
1 O-2.9
Long spinelike conidiophores ceils at base.
with dark
EP
-
+
>lO
2.5-4-O
x
6.0-8.0
Large conidia. Conidiophores those as in ESd,’
resemble
EA
-
+
2.54.0
x
2.0-3.0
Angular conidia. Conidial apparatus differentiated, swollen!
3.0-6.8
Conidia 2celled. Growth in 15% NaCl. Agent of tinea nigra.
<6
PW +
WD
PE
+
+
PM
+
+
+
1.5-3.0x
6-16
2.5-6.1 x 2.9-3.9
Conidiogenous cell phialide without collarette. Young col. wet, mucoid may remain so.g
.lO
2.1 x 4.3
Young, hyaline, thin-walled; mature, black, thick-walled, usually single annellidic conidiogenous locus.
6-16
3-6
Mucaid, slimy colonies. Globose. cells, l-3 loci.
%ce Table 4 for organismnames. b Sabouranddextroseagar, 14 days, 27°C. ’ All species may be somewhatyeastlike when young. Growthon potato dextroseagaror potatoflakes agarappearsto promotemore yeastlike colonies &an does Sabonrauddextroseagar at 25°C. d Fish pathogencausing human disease.
‘Fish pathogensand other species of Exophdalacking yeast synanamorphsomittedfrom this table. Consult reference 16. f Possible human pathogen. g Not agreed upon by all taxonomists.
gated immune system, and carries a grave prognosis.
How to Identify Them The detection of compatible dematiaceous fungal elements in tissue is tantamount to authenticating mycotic disease caused by “‘blackyeasts.” Having documented this prerequisite, growth of the organism in culture and its subsequent identification confirms the etiologic agent. Table 6 is a compilation of macroscopic, microscopic, and physiologic features that defme these organisms. We should describe here, however, the differences between tbe conidiogenous cells in Exophiula and 148
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Phaeoanneliomyes,which are annellides, and those seen in Wangiella, which are phialides without collarettes. Annelloconidia are produced from a flask-shaped cell that extends by percurrent proliferation (growth of inner layers through outer layers) thus becoming longer and narrowerwith the production of each new conidium. Rings or annellations of outer cell-wall ma&&i are laid down in the process. I%ialocomdia &I!3 n=FJd=dftQm that do not incmase in length or taper. Both of the above representblastic production. This distinction is useful with light microscopy because the actual anQ 1993Elsevicr Scimcc F’ublishing Co., Inc.
nellations are difficult to discern withouttheuseofa scanning electron microscope. Blastocomdia, as seen in Phaeococcomyces,ofiginate“as a blownout portion of the mother cell. The developing ce& isrecognizable before being rIe&&ed by ase@un~.~The daughter ceil mayseparate completely or remain attacbedto form eha&’ (22). Itisalsoimporta&toreitera@that this is an extremely polytrmqbic group of H:M tbey&s@ayat~ygiveRtime~ highly &pendent upon a variety of cultural factors. Our expfzriencewit&EXOphialajeanselmei vaFie(ylecanii-corni Cliiical Mcmbiology Newsletter 15:19,1993
Figure I. Colonial morphology of Exophiala jeanselmei variety castellanii grown for 4 wk on potato flakes agar at 25°C. Note the combinadon of yeastlike, black, pasty areas (usually seen when colonies are young) coupled with filamentous areas (develops as cultures matures).
and Phaeoannellomyces
Figure 2. Exophiala jeanselmei variety jeanselmei showing medium-length annellides as well as annelloconidia being produced from intercalary conidiogenous loci (those on the hyphae). Nomarski optics x 1250.
elegans has
shown that the most “stable characteristics” for each of these anamorphs are not very stable at all, and may be manipulated at will by altering the environmental conditions.Bearing this in mind, one could apply different names to the same organism as it exhibits its various phenotypic morphologies. One additional tool that exists for separating some species of “black yeasts” is the exoantigen. Its utility in the routine laboratory is limited, however, as reagents are not commercially available. It also remains to be seen if DNA probes or other molecular technologies will be useful in separating the various taxa.
Figure 3. Exophiala jeanselmei variety lecanii-comi showing conidia being producedprimarily from intercalaty conidiogenous loci. Cells of black yeast synanamorph aLso present. Nomarski optics x 1250.
Therapeutic Considerations Defining therapeutic regimens is beyond the scope of this +cle; however, our in vitro experience at the Fungus Testing Laboratory coupled with anecdotal reports would suggest that the recently approved triazole, itraconzaole (Sporanox), may be an alternative to more toxic agents for the “black yeasts.” Treatment with this antifungal may need to be long term, however, and in the case of systemic disease, probably in high dose. Darkly pigmented imperfect fungi that exhibit “black yeast” synanamorphs comprise a complex group of organisms that often are difficult to Clinical Microbiology Newsletter 15:19,1993
Figure 4. Exophiala jeanselmei variety castellanii. Lateral annellated conidiferous pegsproducing annelloconidia. Nomarski optics x 1250.
0 1993 Elsevier Sciwce Publishing Co., Inc.
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classify, name, and identify. Attempts to standardize media and environmental conditions would appear to facilitate establishing more tmiform identification and nomenclature schemes for these polymorphic fungi by microbiologists from diverse mycological backgrounds. References
FigrcIIZ 5. Exophiala moniliae. Note moniliform (swollen) conidiogenous xl2 50.
cells. Nomarksi cw its
1.
Dixon. D. M., P. J. Szaniszlo, and A. Polak. 1991. Dihydroxynaphthalene (DHN) melanin and its relationship with virulence in the early stages of phawhyphomycosis, p. 297-318. In G. T. Cole and H. C. Hoch (ed.), The fungal spore and disease initiation in plants and animals. plenum Ress, New York.
2.
Polak, A. 1990. Melanin as a virulence factor in pathogenic fungi. Mycoses 33:215-224.
3.
Terra F. et al. 1922. Novo typo de dermatite verrucosa mycose por Acrotheca corn associacao de leishmaniosa. BraxilMedica. 2~253-259.
4.
McGinnis, M. R. and R. C. Fader. 1988. Mycetoma: a contemporary concept, Infect. Dis. Clin. N.A. 23939-954.
5.
Ajello L. et al. 1974. A case of phawhyphomywsis caused by a new species of Phialophora. Mycologia. 66~490-498.
6.
McGinnis, M. R. 1983. Chromoblastomycosis and phaeohyphomycosis: new concepts, diagnosis, and mycology. J. Am. Acad. Dermatol. 81-16.
7.
Ulson, CM. 1959. Contribuicao para o estudo das chamadas “leveduras pretas.” Tese, Department0 de Publicatoes, FacuIdade de Medicina, Universidade de Sao Paulo, Sao Paulo, Brasil.
8.
Ajello, L. 1978. The black yeasts as disease agents: historical perspective, p. 916. In The black and white yeasts. Scientific Publication NO. 356. Pan American Health Organization, Washington, D.C.
9.
De Hcog, G. S, and M. R. McGiiis. 1987. Aswmycetous black yeasts. Studies Mycol. 30~187-199.
Figswe 6. Exophiala spinifera. Note long, spine-like conidiophores that are darkened at the base. Non wrski optics x 625.
10. McGinnis, M. R., W. A. Schell, and J. Carson. 1985. Phaeoannellomyces and the Phaeococwmycetaceae, new dematiaceous blastomycete taxa. J. Med. Vet. Mycol. 23:179-188.
Figwe 7. Exophiala pisciphila. Long conidiophores resembling those of Exophiala spin5 era but not the large size of annelloconidia. Nomarski optics x 625.
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8 1993 Elm&r Science PublishingCo., Inc.
11. McGinnis, M. R. and W. A. Schell. 1980. The genus Fonrecaea a& its relationship to the genera C~porium, Phialophora, Ramichloridiwn and Rhinocladiella. p. 215-234. Scientifz Publication No. 3%. Pan American He&h Organization, Washington, D.C.
Clinical Microbiology Newsletter 15:19,1993
12. Gams, W. 1982. Generic names for synanamorphs? Mycotaxonomy 15:459464. 13. Hardcastle, R.V. and P.J. Szaniszio. 1974. Characterization of dimorphism in Cludosporium werneckii. J. Bacteriol, 119:294-302. 14. Houston, M. R. et al. 1969. Dimorphism in Cladosporium werneckii. Sabouraudia. 7:195-198. 15. McGough, D. A., A. W. Fothergill, and M. G. Rinaldi. Abstr. Annu. Meet. Am. Sot. Microbial. 1991, F7, p. 409. 16. Dixson, D. M. and A. Polak-Wyss. 1991. The medically important dematiaceous fungi and their identification. Mycoses. 34:1-18. 17. Kawasaki, M. et al. 1990. Mitochondrial DNA analysis of Exophiulu jeunselmei and Exophialu dermatitidis. Mycopathologia. 110: 107-l 12.
Figure 8. Wangiella dermatitidis. Note prominent yeast cells, few hypae, and conidia being produced from cells that do not taper.
18. Jorgensen, J. H. and M. G. Rinaldi. 1986. A Clinician’s dictionary of bacteria and fungi. Eli Lilly and Company, Indianapolis, IN. 19. Wood, C. and B. Russel-Bell. 1983. Characterization of pigmented fungi by melanin staining. Am. J. Dermatopathol. 5:77-81. 20. Iwatsu, T., S. Udagawa, and T. Takase. 199 1. A new species of Exophiala recovered from drinking water. Mycotaxonomy. XLI:321-328. 21. Yamada, Y. et al. 1989. Coenzyme Q systems in ascomycetous black yeasts. Antonie van Leeuwenhoek. 56:349356. 22. Dixon, D. and R. A. Fromtling. 1991. Morphology, taxonomy, and classification of fungi, p. 579-587. In Balows et al. (ed.), Manual of clinical microbiology, 5th Pd., American Society for Microbiology, Washington, D.C.
Figure 9. Yeast cell of Phaeoannellomyces elegans showing hvo distinct unnellations and bud separating from the mother cell. Scanning electron microscopy x 22,100.
Case Report
Blood Culture Detection of Legionella in a Transplant Patient Donna M. Carter, M.T., A.S.C.P. Department of Pathology Loyola University Medical Center Foster G. McGuw Hospital Muywood, IL 60153
Clinical Microbiology Newsletter 15:19,1993
Case Report A 56-yr-old immunosuppressed male underwent a cadaveric renal transplant for chronic renal failure at Loyola University Medical Center. His course was complicated on the sixth postoperative day when he developed an oral temperature of 39.7”c associated with rigors and a productive cough. The
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Science Publishing Co., Inc.
chest X-ray confirmed the diagnosis of pneumonia. The sputum Gram stain showed many white blood cells, but no organisms. The initial antimicrobial regimen used was erythromycin and aztreonam. Because of the patient’s increasing lethargy and seizures, rifampin was added to his treatment regimen on the ninth post-operative day. Routine
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