Melanized Fungi in Human Disease

Sanjay G Revankar; Deanna A Sutton

doi:10.1128/CMR.00019-10

. 2010 Oct;23(4):884–928. doi: 10.1128/CMR.00019-10

Melanized Fungi in Human Disease

Sanjay G Revankar ^1,^*, Deanna A Sutton ²

PMCID: PMC2952981 PMID: 20930077

Abstract

Summary: Melanized or dematiaceous fungi are associated with a wide variety of infectious syndromes. Many are soil organisms and are generally distributed worldwide, though certain species appear to have restricted geographic ranges. Though they are uncommon causes of disease, melanized fungi have been increasingly recognized as important pathogens, with most reports occurring in the past 20 years. The spectrum of diseases with which they are associated has also broadened and includes allergic disease, superficial and deep local infections, pneumonia, brain abscess, and disseminated infection. For some infections in immunocompetent individuals, such as allergic fungal sinusitis and brain abscess, they are among the most common etiologic fungi. Melanin is a likely virulence factor for these fungi. Diagnosis relies on careful microscopic and pathological examination, as well as clinical assessment of the patient, as these fungi are often considered contaminants. Therapy varies depending upon the clinical syndrome. Local infection may be cured with excision alone, while systemic disease is often refractory to therapy. Triazoles such as voriconazole, posaconazole, and itraconazole have the most consistent in vitro activity. Further studies are needed to better understand the pathogenesis and optimal treatment of these uncommon infections.

INTRODUCTION

Melanin is a ubiquitous compound found in many microbes and animals. Its functions are varied but are based on the unique molecular characteristics of its structure, which make it an extremely stable molecule, resistant to a variety of destructive physicochemical processes (83, 109, 324). In recent years its pathogenic role in fungi has become well described (123, 292, 375, 460, 546). This review will focus on fungi that are considered to be melanized as a primary feature, particularly with regard to their phenotypic appearance (macroscopic and microscopic morphologies) and appearance in tissue (histology).

The terms used to describe these fungi have evolved over the past several decades. As Sporothrix schenckii was one of the earliest melanized fungi described, “sporotrichoid” was often used to describe similar fungi, though currently it has been replaced by other, more useful terms. “Phaeoid,” “phaeo-sporotrichose,” and “dematiaceous” have also been mentioned in the literature (574). “Phaeo” comes from the Greek meaning “dark” and has been commonly used, particularly when describing infections due to these fungi as “phaeohyphomycosis,” i.e., infection caused by dark-walled fungi, as suggested by Ajello et al. (12, 630). It has been suggested that the term “dematiaceous” is not appropriate given its etymologic derivation from the Greek “deme,” meaning bundle, though it has become fairly entrenched in medical mycological literature and will likely persist in nomenclature (574). The term “melanized” has become more utilized recently, given its specific meaning. For the purposes of this review, however, the terms “dematiaceous,” “melanized,” “dark,” and “phaeoid” are used interchangeably to denote fungal elements containing melanin.

The presence of melanin alone is probably not a useful criterion for inclusion in this group of clinically important fungi, as melanin has been demonstrated in practically all “nondematiaceous” clinical fungi examined in the literature, including Histoplasma capsulatum, Paracoccidioides brasiliensis, Aspergillus spp., and even Candida albicans (293, 521, 548, 599, 757). One might contrast the fungi discussed here as heavily melanized, with brown-pigmented hyphae in tissue often discernible without the use of any staining procedure. At present, no quantitative measure of melanin is available to distinguish dematiaceous from other fungi. In addition, Sporothrix schenckii, with a yeast form in tissue, is well known and well described (177) as the agent of a unique clinical entity, sporotrichosis, and only issues regarding its mycology will be discussed here.

Melanized fungi are common in the environment (see Ecology below) and are often isolated in the microbiology lab, where they may be considered contaminants. Indeed, only 10% of dematiaceous lab isolates are likely to have clinical significance (72, 607). Clinical disease due to these fungi is uncommon, with one estimate from a large metropolitan area of one case/million persons/year (617). Despite their rarity in clinical practice, melanized fungi have become increasingly recognized as important pathogens, particularly in immunocompromised patients, though individuals with apparently “normal” immune systems have also been reported to have invasive, often fatal infections (627, 628).

The clinical syndromes caused by these fungi are differentiated based on histologic findings into eumycetoma, chromoblastomycosis, and phaeohyphomycosis. Eumycetoma is a deep tissue infection, usually of the lower extremities, characterized by the presence of mycotic granules (572). It is associated with a relatively small group of fungi. Chromoblastomycosis is caused primarily by a few species of fungi that produce characteristic sclerotic bodies in tissue and is usually seen in tropical areas (501, 572). Phaeohyphomycosis is a term generally reserved for the remainder of clinical syndromes caused by melanized fungi (623, 630). For the purposes of this review, these will be arbitrarily divided into allergic disease, superficial and deep local infections, pulmonary disease, central nervous system (CNS) infection, and disseminated disease. We do not aim to review every publication regarding melanized fungi, but rather we seek to provide a broad, yet in-depth overview of the field as it currently stands, recognizing that it will continue to evolve and expand with our increasing knowledge of and experience with these clinically important fungi.

ECOLOGY

Melanized or dematiaceous fungi as defined above are frequently considered ubiquitous saprobes inhabiting living and dead plant material and, for the most part, residing in the soil. We now know, however, that these generalized assumptions are incorrect for the group as a whole, as several etiologic agents occupy specific ecological niches or microenvironments, and the knowledge of their natural ecology contributes to our understanding of their opportunistic/pathogenic potential (175, 177, 606, 779). It has been suggested (175) that our use of the term “dematiaceous” be restricted to those ubiquitous, mostly plant-associated hyphomycetous fungi with brown hyphae (220, 221), such as Alternaria, Bipolaris, Curvularia, and Exserohilum in the order Pleosporales. The natural ecology of melanized fungi in several other orders is more restricted (187). For example, fungi in the order Calosphaeriales belong mostly to woody-plant- or wood-inhabiting genera such as Phaeoacremonium, Phialemonium, and Pleurostomophora, whereas some genera in the order Chaetothyriales, such as Exophiala, may have specific microenvironments and are characterized as “microextremophiles.” The ability of some species in this genus, such as E. xenobiotica, to grow in high concentrations of xenobiotics (606) such as xylene, toluene, or creosote-treated utility poles, as well as to cause human disease, is truly remarkable. Species in the genus Exophiala and related genera are frequently referred to as the “black yeast-like fungi” and are so named because of their ability to produce budding, yeast-like cells at some point in their life cycle as well as dark hyphae. The ecology of Pseudallescheria and Scedosporium was also recently investigated by examining the occurrence of these species in natural and human-dominated environments (393). These findings demonstrated increasing environmental recovery with increasing human habitation and a concomitant elevation in nitrogen concentrations. Another genus defined by its residence in a particular environmental niche is the halophilic genus Hortaea in the order Capnodiales. Hortaea werneckii, the agent of tinea nigra, is found in subtropical saltwater habitats and is manifested by its opportunistic adherence to the dead, salty keratin layers of the human hand (87). Thus, while several genera are considered “ubiquitous,” many prefer well-defined microenvironments which, for some genera, predispose them to causing disease where similar conditions exist in the host.

In addition, there are species that appear to be geographically restricted, such as Rhinocladiella mackenziei, which has been seen primarily in patients from the Middle East (726). While Scedosporium prolificans has been reported from many locations, most clinical cases originate from Australia and Spain, for unclear reasons (76, 336). This may be due to environmental features that preferentially support specific fungal species.

CLASSIFICATION, TAXONOMY, AND NOMENCLATURE

Classification of fungi is simply their assignment into defined categories. A classification system is composed of hierarchical groups which may be further subdivided to indicate degrees of relationships. The basic unit of classification is the species, although there is currently no universally acceptable definition for this unit. Taxonomy is the arrangement of these fungi into a classification. With multilocus sequencing providing classification insights unavailable to the phenotypic systematists (those who study the relationships and classification of organisms and the processes by which they have evolved), new phylogenetic classification schemes have emerged. Taylor et al. have provided an excellent treatment of the phylogenetic concepts underlying the definition of species in fungi (737). The abbreviated classification scheme to the ordinal level for ascomycetous melanized fungi covered in this review is based upon the most recent work of Hibbett et al. (340) and the Myconet “Outline of Ascomycotya” (463).

Kingdom: Fungi

Phylum: Ascomycota
- Subphylum: Pezizomycotina
  - Class: Dothideomycetes
    Orders: Capnodiales, Dothideales, Pleosporales, Botryosphaeriales
  - Class: Eurotiomycetes
    Order: Chaetothyriales
  - Class: Sordariomycetes
    Orders: Microascales, Sordariales, Calosphaeriales, Ophiostomatales

As seen above and in Table 1, clinically significant melanized fungi span several ascomycetous orders in the kingdom Fungi.

TABLE 1.

Salient features of selected clinically significant dematiaceous fungi^a

Genus type	Order	Genus or species	Salient phenotypic and/or diagnostic features^b
Anamorphic (asexual) hyphomycete (conidia	Capnodiales	Hortaea werneckii	Colonies olivaceous to black, mucoid to yeast-like, restricted; broad hyphae, wide annellated zones produce pale brown 1- to multicelled annelloconidia
borne free)		Cladosporium spp.	Colonies olivaceous to black, velvety; conidiophores simple or branched, with or without nodes or swellings; ramoconidia (“shield cells”) give rise to branching chains of fragile, dark, mostly 1- or 2-celled conidia with prominent attachment scares (hila)
	Dothideales	Aureobasidium pullulans	Colonies of A. pullulans var. pullulans mucoid, cream to pink initially and later brown to black, while those of A. pullulans var. melanigenum black at the outset; hyaline blastoconidia borne synchronously from hyaline hyphae; dark, thick-walled chlamydospores; DNA sequencing necessary for reliable differentiation of A. pullulans and H. dematioides
		Hormonema dematioides	Colonies similar to those seen in A. pullulans; hyaline blastoconidia produced asynchronously by percurrent proliferation from hyaline and dark hyphae
	Pleosporales	Alternaria spp.	Colonies woolly, pale to olivaceous to black, with rapid growth; large, dark, euseptate^c, muriform conidia in chains; A. infectoria conidia may be sparse and have long apical beaks serving as secondary conidiogenous cells
		Bipolaris spp.	Colonies woolly, gray to black, with rapid growth, bipolar germination, geniculate conidiophores, flattened hilum; B. spicifera has 3 distosepta^d and 4 cells, while B. hawaiiensis has predominately 5 distosepta and 6 cells
		Curvularia spp.	Colonies woolly, gray to black, with rapid growth; geniculate conidiophores; conidia euseptate and curved (pronounced to subtle) due to swollen middle cell which is darker in C. lunata; C. lunata var. aeria produces large stroma visible with the naked eye
		Exserohilum spp.	Colonies woolly, gray to black, with rapid growth; geniculate conidiophores, truncate protruding hilum; E. rostratum has 7-9 distosepta; 8-10 cells; prominent dark basal and distal septa; E. longirostratum has longer conidia with central curvature; E. mcginnisii has subtle warty projections on conidia
	Chaetothyriales	Exophiala spp.	Colonies mucoid initially, later more filamentous; conidiogenous cells predominately annellidic; phialides sometimes present; annellated black yeast synanamorph often present; many species very similar microscopically; nitrate positive; DNA sequencing of ITS region facilitates species identification; maximum temp varies; most frequently seen clinical species include E. xenobiotica, E. oligosperma, E. lecanii-corni, and E. phaeomuriformis
		Exophiala dermatitidis	Colonies black, mucoid; nitrate negative; growth at 40°C; black yeast E. exophialae synanamorph present; most common clinical Exophiala species; accurately identified by phenotypic features; obsolete name Wangiella dermatitidis
		Cladophialophora spp.	Colonies black, velvety; growth rates and temperatures vary for individual species; microscopically similar to Cladosporium spp. but lack conidiophores, “shield cells,” and prominent hila; conidia are nonfragile (remain intact in chains); neurotropic species include C. bantiana and C. modesta; other human pathogenic species include C. arxii, C. boppii, C. carrionii, C. devriesii, C. emmonsii, C. mycetomatis, C. samoënsis, andC. saturnica
		Fonsecaea spp.	Colonies olivaceous to black, velvety; conidia formed from swollen denticles giving rise to secondary and tertiary conidia in chains of up to four conidia; conidia also formed on sympodial conidiophores like in Rhinocladiella and occasionally from discrete phialides like in Phialophora; F. pedrosoi an agent of chromoblastomycosis, F. monophora an agent of both chromoblastomycosis and cerebral phaeohyphomycosis
		Ochroconis gallopava	Colonies are brownish, velvety, with a maroon diffusing pigment; 2-celled, clavate conidia borne from denticles; growth at 45°C; no growth on media containing cycloheximide; neurotropic; obsolete names Dactylaria gallopava, D. constricta var. gallopava
		Phialophora spp.	Colonies olivaceous to black, velvety; three species are clinically significant; P. verrucosa has dark, funnel-shaped collarettes; P. americana has deep, vase-shaped collarettes; the slow-growing P. europaea has very short collarettes
		Rhinocladiella spp.	Colonies olivaceous to black, velvety; long, erect, brown, unbranched sympodial conidiophores; 1-celled pale ellipsoidal conidia borne on crowded denticles; an Exophiala yeast synanamorph may be present; R. mackenziei is a neurotropic species in the genus with relatively few conidia per fertile part of the geniculate conidiophore; conidia 1-celled, pale brown, ellipsoidal with a prominent truncate hilum; poor growth at 25°C, good growth at 40°C; obsolete name Ramichloridium mackenziei; other pathogenic species include R. aquaspersa and R. similis
		Veronaea botryosa	Colonies gray to blackish-brown, woolly; long, brown conidiophores; pale brown, 2-celled conidia with a rounded apex and truncate base borne from closely spaced intercalary conidiogenous cells
	Microascales	Scedosporium spp.	Colonies pale to yellowish-gray to darker gray, some with orange reverse, woolly; conidiogenous cells annellidic; some species produce a Pseudallescheria teleomorph and a Graphium synanamorph; similar human pathogenic species in the Pseudallescheria boydii species complex as defined by recent molecular studies include S. apiospermum, S. boydii, S. aurantiacum, and S. dehoogii; S. prolificans (obsolete name S. inflatum) possessing inflated annellidic conidiogenous cells, is unrelated to members of the P. boydii species complex
		Scopulariopsis spp.	Colonies gray to olivaceous-brown, woolly; conidiogenous cells annellidic; several very similar dark species are anamorphs of various Microascus spp.
	Sordariales	Madurella mycetomatis	Colonies very slow growing and often heaped; dark brown to black; diffusible brown pigment; unlike M. grisea, M. mycetomatis grows at 40°C and fails to assimilate sucrose; precise identification facilitated by ITS sequencing
		Myceliophthora thermophila	Colonies light brown, powdery; ill-defined margin; conidia borne from ampulliform swellings are hyaline and smooth initially becoming rough and brown at maturity; growth at 48°C
		Acrophialophora fusispora	Colonies centrally dark front and reverse; unbranched, erect, brown, echinulate conidiophores are anchored by a foot cell; chains of conidia with fine or coarse spirals produced from apex of brown conidiophores and inflated phialides on hyaline hyphae; growth at 40°C
	Calosphaeriales	Phialemonium spp.	Colonies buff to gray to yellow; conidiogenous cells phialides and adelophialides (reduced phialides lacking a basal septum); P. obovatum has obovate conidia and a green diffusing pigment; sporodochia-producing isolates of P. curvatum have been reported
		Phaeoacremonium spp.	Colonies range from buff to pale yellow to pale or dark pink to various shades of brown; hyphae brown; conidiophores often have small warts (exudates); three distinct types of phialides may be present (types I, II, and III); polyphialides may also be present; 1-celled conidia aggregate at apices of phialides and are commonly reniform (kidney shaped) to allantoid (sausage shaped); human pathogenic species that grow at 40°C include P. parasiticum (obsolete name Phialophora parasitica), P. rubrigenum, P. alvesii, P. amstelodamense, P. krajedenii, P. tardicrescens, and P. venezuelense
		Pleurostomophora spp.	Colonies of P. richardsiae (obsolete name Phialophora richardsiae) dark brown, velvety; phialides with prominent flaring collarettes bear globose, brown conidia while phialides with indistinct collarettes bear pale allantoid to cylindrical conidia; colonies of P. repens (obsolete name Phialophora repens) pale brown, phialides lack flaring collarettes, and conidia are pale, allantoid to cylindrical
	Coniochaetales	Lecythophora spp.	Colonies moist, salmon to orange; conidiogenous cells primarily adelophialides; conidia aggregate at apices of conidiogenous cells; L. mutabilis distinguished from L. hoffmannii by dark chlamydoconidia
	Ophiostomatales	Sporothrix spp.	Colonies initially cream-colored, moist, with a finely wrinkled surface, becoming brownish-grayish with the production of dark sessile conidia; hyaline, budding cigar-shaped yeast cells present in host and at 35°C; S. schenckii is a species complex as determined by calmodulin sequencing; human pathogenic species include S. schenckii (sessile conidia triangular to oval); S. globosa (sessile conidia globose, no growth at 37°C); S. brasiliensis (sessile conidia subglobose, geographically restricted to Brazil); S.luriei (dark sessile conidia absent)
Anamorphic (asexual) coelomycete (conidia borne within enclosed or semienclosed structures; organisms treated here have pycnidial conidiomata; frequently acquired by traumatic implantation)	Pleosporales	Phoma, Pleurophoma, Pleurophomopsis spp.	Colonies pale to light brownish-gray to darker gray, woolly; pycnidia brown to black; conidia small (4-6 μm), oblong, sometimes slightly curved, hyaline, often guttulate (containing small droplets); species are very similar and best differentiated by ITS sequencing
		Coniothyrium, Paraconiothyrium, Microsphaeropsis spp.	Colonies pale gray to grayish-brown to brownish-black, some producing dark diffusible pigments into the agar, woolly; pycnidia brown to black; conidia mostly oblong of various sizes, pale brown to dark; species are very similar and best differentiated by ITS sequencing
		Pyrenochaeta spp.	Colonies olivaceous to gray-black, restricted, velvety; pycnidia brown to black with setae surrounding the ostiole; conidia 1-celled, hyaline
	Botryosphaeriales	Lasiodiplodia theobromae	Colonies grayish-black, woolly; pycnidia ostiolate, sometimes with setae; conidiogenous cells annellidic; large conidia 20-30 by 10-15μm, initially aseptate and hyaline; 1 septate, dark, longitudinally striate at maturity; obsolete name Botryodiplodia theobromae
		Neoscytalidium dimidiatum	Colonies woolly, black, with rapid growth, filling plate within a few days; an otherwise similar hyaline variant is also referred to as N. dimidiatum; 1- and 2-celled, dark or hyaline arthroconidia not separated by disjunctor cells; thin hyaline and wide (10-12 μm) dark or hyaline hyphae; multilocular pycnidial coelomycetous synanamorph requires several weeks to mature on plant-based media and produces versicolored conidia (middle cell darker); no longer referred to as Nattrassia mangiferae as this organism is an unrelated fruit pathogen now known as Neofusicoccum mangiferae
		Macrophomina phaseolina	Colonies gray, woolly, with a dark diffusing pigment and small black dots representing immature/mature sclerotia; pycnidia and conidia usually not formed in culture; identification by sequencing
	Sordariales	Phomopsis spp.	Colonies pale to light brown or gray, woolly; pycnidia brown to black, may be multilocular; conidia of two types, alpha conidia ellipsoidal while beta conidia long, filamentous, curved
Teleomorphic (sexual) (produce ascomata, asci, and ascospores in culture)	Sordariales	Chaetomium spp.	Colonies olivaceous to grayish-brown, woolly; ascomata perithecial (opening at top) and covered with setae (hairs); large, reddish-brown elliptical ascospores; C. globosum, setae coiled, ascospores subglobose, growth at 35°C, no growth at 42°; C. atrobrunneum, neurotropic, setae mostly straight, ascospores narrowly fusoidal, growth at 42°C; C. perlucidum, neurotropic, very similar to C. atrobrunneum in colony morphology setae, and ascospore size; growth at 42°C
		Achaetomium strumarium	Colonies pale to light brown with reddish-brown diffusing pigment after 2 weeks, woolly; ascomata perithecial with long, slightly curved setae; ascospores hyaline to dark, 13-17.5 by 8.5-11 μm, fusoidal; neurotropic species with growth at 40°C
	Pleosporales	Leptosphaeria spp.	Colonies dark, velvety to slightly woolly, slow-growing; ascomata cleistothecial (no opening); ascospores hyaline, mostly with 4-6 septa; L. senegalensis and L. thompkinsii distinguished by ascospore features
	Microascales	Microascus spp.	Colonies initially white to gray to brownish; ascomata perithecial, developing as black dots in concentric rings on the agar, and may exude a cirrhus of red ascospores at maturity; species treated here have very similar, dark Scopulariopsis anamorphs; M. cinereus, short perithecial necks and orange-segment-shaped ascospores; M. cirrosus, longer perithecial necks with heart-shaped ascospores; M. trigonosporus, longer perithecial necks with triangular ascospores
		Pseudallescheria spp.	Colonies pale to yellowish gray to gray to brownish, woolly; ascomata cleistothecial; Scedosporium and Graphium anamorphs present; human pathogenic species as defined by recent molecular studies are Pseudallescheria boydii (anamorph Scedosporium boydii), Pseudallescheria apiosperma (anamorph Scedosporium apiospermum, heterothallic, does not form a teleomorph in culture, d-ribose negative), P. ellipsoidea

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Adapted from Table 14-1 from reference 724 with permission. This list is not all inclusive. Pictures of all organisms are available at doctorfungus.org or on the CD-ROM of the Atlas of Clinical Fungi, pilot version of 3rd ed. (174).

On potato flake agar at 25°C.

True septa continuous with outer wall.

Pseudosepta where only inner walls are involved.

Nomenclature refers to assigning formal scientific names. This process is regulated by the International Code of Botanical Nomenclature (ICBN) (http://www.bgbm.org/iapt/nomenclature/code/default.htm) to facilitate a stable naming system and to avoid and reject names which are in error or are ambiguous (789). Lack of adherence to these requisites often invalidates a taxon name and results in multiple names for the same organism. Other reasons for name changes include the placement of fungi into new genera as determined by phylogenetic studies, which frequently occurs within this heterogeneous group of fungi. When this occurs, the species epithet is retained, but it may require modification in keeping with the rules of Latin grammar. An example of recent changes for melanized fungi include the movement of Phialophora richardsiae to Pleurostomophora richardsiae and of Phialophora parasitica to Phaeoacremonium parasiticum. Discovery of a previously unrecognized teleomorph (sexual or meiotic state) may also precipitate a name change. A recent example is found in the discovery of the teleomorph for Scedosporium apiospermum, which was incorrectly thought to be Pseudallescheria boydii. We now know through the work of Gilgado et al. that the teleomorph for S. apiospermum is the heterothallic ascomycete Pseudallescheria apiosperma, as evidenced by the production of cleistothecia (round sexual structures containing asci and ascospores) and ascospores (the sexual reproductive propagules) between compatible mating strains of S. apiospermum (283). As the teleomorph name takes precedence over the anamorph (asexual, mitotic) name, the correct binomial would be the sexual state. Whether this name would be adopted by clinicians in everyday usage remains problematic.

Also confusing for clinicians and laboratorians alike is the naming convention that permits the use of more than one name for the same fungus. This is allowed when a particular form of the fungus is the one more commonly seen in the laboratory. Fungi recovered in culture commonly display only an anamorphic state. They may be either heterothallic isolates with no known teleomorph or homothallic strains failing to produce their sexual state in vitro. A few clinically significant homothallic melanized fungi do form both anamorphs and teleomorphs in culture. In this situation, as mentioned above, the teleomorph name takes precedence over the anamorph name, e.g., Pseudallescheria boydii rather than Scedosporium boydii and Microascus cinereus rather than Scopulariopsis cinerea. Additionally, in some genera, such as Pseudallescheria, two separate anamorphs which are distinctively different microscopically may be produced, and these are referred to as synanamorphs (another asexual form of the same fungus). Some homothallic strains, however, lack anamorphs and are known only by the name of the sexual state. Examples would include members of the genus Chaetomium. The advent of sequencing characterization has provided the tools necessary to reevaluate the evolutionary relationships of these black molds, and today multilocus molecular phylogenetic studies are clearly redefining previously described entities, uncovering new species and varieties, and correlating these with their natural habitats.

IDENTIFICATION OF ETIOLOGIC AGENTS

Over 150 species and 70 genera of dematiaceous fungi have been implicated in human and animal disease (Table 2). As the number of patients immunocompromised as a result of diseases and medical therapy increases, additional species are being reported as causes of human disease, expanding an already long list of potential pathogens. Identification of melanized etiologic agents known to cause human or animal disease has traditionally been based upon phenotypic features of the isolate observed in culture (175, 177, 220, 221). This practice continues to be the mainstay of fungal identification in most routine settings. More recently, molecular techniques employed for classification purposes and those provided by research facilities have provided additional tools for the characterization of these molds. Extensive sequencing for some genera has illustrated the concept of “species complexes,” or the inclusion of several separate species into what was formerly referred to as a single species. This has been clearly demonstrated in the genera Exophiala (825), Scedosporium (281-283), and Phaeoacremonium (525). The “splitting” of these species into separate taxa has of necessity changed our reporting practices. As an example, laboratories previously comfortable with discriminating only between Exophiala (Wangiella) dermatitidis and E. jeanselmei are now aware of several other clinically significant species that are not easily separated by phenotypic features alone (177, 184, 825) and that E. jeanselmei is in fact one of the less frequent agents of disease. Therefore, species other than E. dermatitidis are best reported as an Exophiala sp., not E. dermatitidis, unless sequencing has provided a species identification. These “new and improved” reporting techniques, however, must be communicated to clinicians in a manner consistent with their understanding of current organism terminology and the associated mycoses.

TABLE 2.

Melanized fungi in human disease^a

Genus	Species
Achaetomium^d	A. strumarium
Acrophialophora^b	A. fusispora
Alternaria^b	A. alternata, A. chlamydospora, A. dianthicola, A. infectoria, A. longipes, A. tenuissima
Anthopsis^b	A. deltoidea
Arnium^d	A. leporinum
Arthrinium^b	A. phaeospermum
Ascotricha^d	A. chartarum
Aureobasidium^b	A. pullulans
Bipolaris^b	B. australiensis, B. hawaiiensis, B. papendorfii, B. spicifera
Botryomyces^b	B. caespitosus
Chaetomium^d	C. atrobrunneum, C. funicola, C. globosum, C. murorum, C. perlucidum
Cladophialophora^b	C. arxii, C. bantiana, C. boppii, C. carrionii, C. devriesii, C. emmonsii, C. modesta, C. mycetomatis, C. saturnica, C. samoënsis
Cladorrhinum^b	C. bulbillosum
Cladosporium^b	C. cladosporioides, C. herbarum, C. oxysporum, C. sphaerospermum
Colletotrichum^c	C. coccodes, C. crassipes, C. dematium, C. gloeosporioides, C. graminicola
Coniothyrium^b	C. fuckelii
Corynespora^b	C. cassiicola
Curvularia^b	C. brachyspora, C. clavata, C. geniculata, C. inequalis, C. lunata, C. pallescens, C. senegalensis, C. verruculosa
Cyphellophora^b	C. laciniata, C. pluriseptata
Dichotomophthora^b	D. portulacae
Dichotomophthoropsis^b	D. nymphaearum
Dissitimurus^b	D. exedrus
Drechslera^b	D. biseptata
Exophiala^b	E. asiatica, E. attenuata, E. bergeri, E. castellanii, E. dermatitidis, E. jeanselmei, E. lecanii-corni, E. moniliae, E. oligosperma, E. phaeomuriformis, E. pisciphila, E. salmonis, E. spinifera, E. xenobiotica
Exserohilum^b	E. longirostratum, E. mcginnisii, E. rostratum
Fonsecaea^b	F. monophora, F. pedrosoi
Hormonema^b	H. dematioides
Hortaeae^b	H. werneckii
Lasiodiplodia^c	L. theobromae
Lecythophora^b	L. hoffmannii, L. mutabilis
Leptosphaeria^d	L. senegalensis, L. thompkinsii
Macrophomina^c	M. phaseolina
Madurella^b	M. grisea, M. mycetomatis
Microascus^d	M. cinereus, M. cirrosus, M. trigonosporus
Monilliella^b	M. suaveolens
Microsphaeropsis^c	M. arundinis, M. olivacea
Myceliophthora^b	M. thermophila
Mycocentrospora^b	M. acerina
Mycoleptodiscus^b	M. indicus
Neoscytalidium^c	N. dimidiatum
Neotestudina^d	N. rosatii
Nigrospora^b	N. sphaerica
Ochrocladosporium^b	O. elatum
Ochroconis^b	O. gallopava, O. humicola, O. tshawytschae
Oidiodendron^b	O. cerealis
Phaeoacremonium^b	P. alvesii, P. amstelodamense, P. griseorubrum, P. krajdenii, P. parasiticum, P. rubrigenum, P. sphinctrophorum, P. tardicrescens, P. venezuelense
Phaeosclera^b	P. dematioides
Phaeotrichoconis^b	P. crotalariae
Phialemonium^b	P. curvatum, P. obovatum
Phialophora^b	P. americana, P. bubakii, P. europaea, P. reptans, P. verrucosa
Phoma^b	P. cruris-hominis, P. dennisii var. oculo-hominis, P. eupyrena, P. glomerata, P. herbarum, P. minutella, P. minutispora, P. sorghina
Piedraia^d	P. hortae
Pleurophoma^c	P. cava
Pleurophomopsis^c	P. lignicola
Pleurostomophora^b	P. repens, P. richardsiae
Pseudochaetosphaeronema^b	P. larense
Pseudomicrodochium^b	P. suttonii
Pyrenochaeta^b	P. mackinnonii, P. romeroi, P. unguis-hominis
Rhinocladiella^b	R. aquaspersa, R. basitona, R. mackenziei, R. similis
Sarcinomyces^b	S. phaeomuriformis
Scedosporium^a	S. prolificans
Scopulariopsis^b	S. asperula, S. brumptii, S. fusca
Sphaeropsis^c	S. subglobosa
Stenella^b	S. araguata
Taeniolella^b	T. stillbospora
Tetraploa^b	T. aristata
Thermomyces^b	T. lanuginosus
Ulocladium^b	U. chartarum
Veronaea^b	V. botryosa

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Some doubtful cases have been omitted; the list may not be all inclusive. Some genera that are outside the taxonomic orders discussed in the text but that contain melanized structures are included. Adapted from reference 493 with permission of the publisher.

Anamorphic hyphomycete.

Anamorphic coelomycete.

Teleomorphic ascomycete.

Phenotypic Identification

The level to which black molds can or should be identified in the routine laboratory may depend on several factors, such as the genus of the organism recovered, whether or not an epidemiologic investigation is warranted, and/or the level of identification required for appropriate patient management. The phenotypic identification of black molds is based primarily upon their macroscopic morphology (color, growth rate, and growth characteristics on standardized media), their microscopic morphology (hyphae, conidiogenous cells [specialized cells that produce the conidia], conidia [asexual reproductive propagules], etc.), and a limited number of physiologic features (primarily cycloheximide tolerance, nitrate assimilation, urea hydrolysis, and growth at various salt concentrations). Only genus-level identification may be possible or required for genera with several similar, closely related species, such as described above for Exophiala. In some other genera, certain species are clearly associated with a particular type of mycosis, and a combination of morphologic features, temperature, and physiology can provide a species-level identification. This is the case for the agent of cerebral phaeohyphomycosis, Cladophialophora bantiana.

Macroscopic morphology.

The medium (see “Isolation Procedures and Culture” in Diagnosis below) is an important consideration in the identification of melanized fungi. The use of a medium that promotes growth most consistently matching the original description of the organism is preferred, and this typically is a plant-based medium. The most commonly used is potato dextrose agar (PDA) or variations thereof. It provides colony colors that are close to those originally described, and it is usually adequate for conidiation. Other plant-based media include malt extract agar, V-8 juice agar, cereal agar, carnation leaf agar, cornmeal dextrose agar, and others. A more complete list of media and reagents may be found in the Manual of Clinical Microbiology, 9th ed. (441), and in the Atlas of Clinical Fungi, 2nd ed. (175). Phaeoid molds vary considerably in their colony colors. Although this characteristic is highly dependent upon environmental conditions, it is one that can be useful in the initial separation of genera/species. While most species are various shades of pale gray to dark gray to black, others may be brown or very pale or may turn darker only with the production of certain structures. Others may be some shade of purple or distinguished by diffusible pigments. Etiologic agents which are typically brown on PDA include Ochroconis gallopava, Pleurostomophora richardsiae, Pleurostomophora repens, some Phaeoacremonium species, Wallemia sebi, Myceliophthora thermophila, and Veronaea botryosa. The “pale list” includes fungi which seldom turn dark, such as Phialemonium species. Lecythophora mutabilis remains lightly colored until the production of dark chlamydospores. Ochroconis gallopava exudes a wine-red pigment into the agar (more pronounced on Sabouraud dextrose agar [SDA]), and several Phaeoacremonium species exhibit purple to lavender colonies.

Microscopic morphology and pleomorphism.

Variable microscopic morphology in the same fungus, also referred to as pleomorphism or pleoanamorphism, is another feature useful in the phenotypic identification of black molds. Some fungi may display more than one form, such as yeast-like growth initially and more filamentous growth subsequently. This is common in the black yeasts such as Exophiala and related genera. Pleoanamorphism may also be exhibited by different types of anamorphic structures (synanamorphs), such as the Graphium state in Pseudallescheria or variably shaped conidia in Pleurostomophora richardsiae (Fig. 1). Identification of homothallic ascomycetes is typically based on the type of ascomata produced (primarily cleistothecia [round, closed structures containing asci and ascospores] or perithecia [round to pear-shaped structures with an opening or ostiole containing asci and ascospores], as in Pseudallescheria or Chaetomium/Achaetomium/Microascus, respectively) and differences in ascospore morphology. Ascospores may be of various sizes, shapes, colors, and ornamentations. The bulk of clinical black molds, however, are heterothallic ascomycetes. These mitosporic fungi are identified mostly by their methods of conidiogenesis and the morphology of their conidia. The majority of mitosporic isolates are hyphomycetes with their conidia borne free in the aerial mycelium. Also seen are coelomycetes, whose conidia are borne within asexual structures known as conidiomata. The methods of conidiogenesis (blastic [blown-out = blastoconidia, as seen in many genera] or thallic [formed from preexisting hyphae = arthroconidia, as in Neoscytalidium]), the types of conidiogenous cells (primarily annellidic [Scedosporium, Scopulariopsis, and Hortaea] or phialidic [many genera]), and the morphology of the conidia are taken in aggregate to form the basis for a morphologic identification. Annelloconidia are formed from percurrent, indeterminate conidiogenous cells that produce rings or annellations and become longer and narrower with the production of conidia, while phialoconidia are formed from conidiogenous cells with collarettes that may be quite distinct or subtle, and the conidiogenous cell remains the same size and shape with conidial production. It should be pointed out, however, that these morphologic features used to identify anamorphic species lacking teleomorphs are strictly phenotypic and do not define their phylogenetic placement within the order (157).

FIG. 1. — Conidiogenous cells of *Pleurostomophora richardsiae*, demonstrating prominent flaring collarettes as well as the two types of conidia (oval and globose) produced by this species. (Unless otherwise noted, in this and subsequent figures light microscopy photomicrographs of conidiogenous cells and/or conidia were taken from slide culture preparations grown on potato flakes agar for 7 days at 25°C.)

Physiologic features.

Physiologic characteristics may also assist in separation of various genera/species. However, only those that are available in routine laboratories are widely employed. The ability or inability of isolates to grow on media containing cycloheximide (referred to as cycloheximide tolerance), nitrate assimilation, urease activity, and salt tolerance, particularly for halophilic strains, are all useful adjuncts to the morphologic examination. Larger reference labs and research facilities also may use a battery of carbon assimilation profiles. Temperature tolerance is also useful in segregating potential pathogens. Those that fail to grow at 35°C are more likely to be recovered from superficial sites, while those capable of growth at this temperature have the potential for more invasive human disease. Several clinically significant dematiaceous molds are thermotolerant to thermophilic, with maximum growth temperatures to 45°C and beyond. A partial listing of these potentially neurotropic species includes E. dermatitidis, O. gallopava, C. bantiana, C. modesta, C. emmonsii, R. mackenziei, Acrophialophora fusispora, Fonsecaea monophora, and some aggressive Achaetomium and Chaetomium species.

Molecular Characterization

Molecular characterization of fungi is a mature discipline in the molecular systematics arena, with multilocus datasets, extensive taxon sampling, and rigorous analytical methods being the norm (340). Its use in the clinical laboratory, however, is mostly restricted to epidemiologic studies and to identification of unusual/uncommon or difficult-to-identify isolates. Molecular identification of most species relies on sequencing of ribosomal genes and comparison with published databases, notably those in GenBank; however, over 10% of these deposits may be erroneous (176). Private databases are also sometimes utilized for particular genera; however, these are difficult to access and may also contain incorrect deposits. Also, various methods and genes or gene regions such as the internal transcribed spacer regions ITS1 and ITS2, the D1/D2 domains, β-tubulin, actin, calmodulin, manganese superoxide dismutase, ATPase subunit 6, chitin synthase, mitochondrial small-subunit (SSU) rRNA, translation elongation factor 1α, and others are utilized, so that interlaboratory standardization of sequencing is lacking. Several International Society for Human and Animal Mycology (ISHAM) working groups are addressing standardization of fungal sequencing (58) as is the Clinical and Laboratory Standards Institute (CLSI) (146). Genera for which substantial sequencing data are available and for which species distinction appears to be satisfactory include those known as black yeasts, i.e., Exophiala and related genera (ITS) (825), Sporothrix species (calmodulin) (480), Phaeoacremonium species (β-tubulin and actin) (525), and Pseudallescheria/Scedosporium species (ribosomal DNA [rDNA] gene cluster, β-tubulin, calmodulin, and translation elongation factor 1α) (330). Molecular characterization should always be evaluated in light of phenotypic features, and sequence data for uncommon and/or potentially new species should be compared with those for ex-type strains.

ANAMORPHIC HYPHOMYCETE GENERA

Capnodiales

Hortaea

Hortaea werneckii is the etiologic agent of tinea nigra, an asymptomatic, superficial mycosis causing hyperchromic plaques without keratinolysis in the dead keratin layers of the skin (186, 685) and mostly restricted to the palms of the hands (tinea nigra palmaris) and soles of the feet (tinea nigra plantaris) (87). It is a halophilic organism whose natural habitat is in tropical and subtropical hypersaline environments (823), and it is thought be acquired through superficially abraded skin (186). Colonies are restricted, black, moist, and yeast-like initially, later becoming filamentous. Wide hyphae are densely septate, thick walled, and brown. Intercalary or lateral annellidic conidiogenous cells produce brown, two-celled ellipsoidal conidia with a darkened central septum. ITS sequencing facilitates molecular identification (823) and clearly distinguishes H. werneckii from other closely related halophilic and acidophilic (H. acidophila) nonpathogenic species (347).

Cladosporium.

The genus Cladosporium has recently undergone molecular and morphologic scrutiny (157), with many organisms being reassigned to other genera. One example is the transfer of Cladosporium elatum to Ochrocladosporium elatum. The genus is extremely ubiquitous, and although it is an agent of allergic disease in indoor settings, few species are documented to cause disease. The species complexes Cladosporium cladosporioides and C. oxysporum are the ones most commonly cited in cases of cutaneous and subcutaneous disease (313, 586, 641, 776) and occasionally deeper infections (396, 429); however, they are commonly contaminants, making the nature of reports doubtful. The inability of Cladosporium species to grow on media containing cycloheximide, their prominent “shield cells,” and conidia that are fragile (easily detached) and possess dark hila (attachment scars) are all features distinguishing Cladosporium from the unequivocally pathogenic Cladophialophora species.

Dothideales

Aureobasidium.

Recent molecular characterization of Aureobasidium pullulans and closely related organisms by multilocus sequence analysis (ITS, partial 28S rDNA, β-tubulin, translation elongation factor 1α, and elongase), expanding the work of de Hoog and Yulova and of Yulova et al. (173, 820), has shown that the genus Aureobasidium contains a single species and several varieties containing differing amounts of melanin and having various salt (820, 823) and temperature (824) tolerances. The mode of conidiogenesis is primarily synchronous rather than percurrent, as in Hormonema; however, features of conidiogenesis are difficult to ascertain with certainty. Sequencing is usually required for a definitive identification. Two varieties are human pathogens, A. pullulans var. pullulans, and A. pullulans var. melanigenum. In the former, colonies remain pink for approximately 1 week, tolerate 15% salt, and have a maximum temperature of 30°C, while in the latter, colonies are black at the outset, tolerate 10% salt, and have a maximum temperature of 35°C. Aureobasidium is an opportunistic pathogen of humans and animals recovered in cases of catheter-related septicemia (117, 360), disseminated infections (344, 663), chromoblastomycosis (616), and peritonitis (144, 367).

Hormonema.

As noted above, Hormonema species are phenotypically similar to Aureobasidium pullulans; however, conidiogenesis is primarily percurrent rather than synchronous. There are rare reports of cutaneous phaeohyphomycosis (149) and fungal peritonitis (690) due to this organism, both of which were reported prior to molecular characterization.

Pleosporales

Alternaria.

Alternaria is a large genus of plant pathogen species that are only occasionally implicated in opportunistic human disease. Cutaneous and subcutaneous phaeohyphomycosis in immunosuppressed individuals is the most common presentation (275, 577, 587, 798). Organ transplantation (280) and Cushing's syndrome appear to be major risk factors for cutaneous/subcutaneous disease, while bone marrow recipients are at risk for sinusitis (577). Ocular disease in individuals exposed to soil and garbage (577) is the next most common presentation, while onychomycosis is rarely reported. There are also occasional reports of allergic fungal sinusitis (67). While several species, such as A. chlamydospora (65, 703), A. longipes (275), and A. tenuissima (124, 642, 644), have been reported, most clinical isolates have been shown to be either A. alternata (176, 497, 710) or A. infectoria (99, 551, 648). ITS region sequences have demonstrated that A. longipes and A. tenuissima cannot be distinguished from A. alternata. Conidial production by Alternaria infectoria is sparse, and colonies may be pale.

Bipolaris.

The most common mycosis attributed to Bipolaris spp. is allergic fungal sinusitis (125, 246, 417, 444, 508, 580). Other disease associations include subcutaneous lesions, keratitis, and peritoneal dialysis-associated peritonitis (508). Extension to the central nervous system via the nasal sinuses highlights the neurotropic potential of the genus, though this is very rare (260, 817). Clinically significant species inciting human disease include B. spicifera, B. hawaiiensis (Fig. 2), and B. australiensis. They are differentiated morphologically by conidial size and the number of distoseptations (pseudosepta where only inner walls are involved) (20). Conidia demonstrate bipolar germination, hence the genus name “Bipolaris.”

FIG. 2. — Conidia of *Bipolaris hawaiiensis*, demonstrating mostly five distosepta and six cells being borne from a geniculate conidiophore/conidiogeous cell.

Curvularia.

Curvularia species are common in dead plant material and may cause a variety of human mycoses, including fungal keratitis, invasive sinusitis (215), onychomycosis, black grain eumycotic mycetoma (378), endocarditis (104), subcutaneous disease (813), and peritonitis (98, 241, 631) as well as systemic infections (175, 177). Additional reports involved fatal cerebral phaeohyphomycosis in an immunocompetent individual (121), endophthalmitis (579), and contaminated saline-filled breast implants (392). Clinical isolates include C. geniculata, C. lunata, C. pallescens, C. senegalensis, C. brachyspora, C. clavata, C. verruculosa, and C. inaequalis (Fig. 3) (598). C. lunata is the most common clinical species, and C. lunata var. aeria (Fig. 4) may produce large, upright stroma in culture that are visible with the naked eye.

FIG. 3. — Conidia of *Curvularia inaequalis* with mostly five septa and six cells borne from a geniculate conidiogenous cells.

FIG. 4. — Conidia of *Curvularia lunata* var. *aeria* borne from a geniculate conidiogenous cell. Note that the middle cell is slightly enlarged, and septa are eusepta (true septa continuous with the outer wall).

Exserohilum.

Three Exserohilum species recovered from humans are E. rostratum, E. longirostratum, and E. mcginnisii, although molecular studies suggest that they may be the same species (175, 177). The genus is characterized by its long, multidistoseptate conidia and a protruding hilum. E. rostratum exhibits darkened basal and distal septa, and E. longirostratum has conidia that are noticeably longer and centrally curved (Fig. 5), while E. mcginnisii has conidia with warty projections on their outer walls. Not all authorities agree that E. rostratum and E. longirostratum are separate species. Species are opportunistic and are etiologic agents of sinusitis (566), which may extend to the central nervous system (46), and keratitis (488), as well as cutaneous and subcutaneous mycoses (359, 508, 580). A fatal disseminated case was reported in a patient with aplastic anemia (37).

FIG. 5. — Multidistoseptate conidia of *Exserohilum longirostratum*, demonstrating a prominent basal septum (true septum) and a protruding hilum.

Chaetothyriales

Exophiala.

Species in the genus Exophiala are frequently referred to as “black yeasts” due to the ability of several species to form a budding yeast-like synanamorph as well as hyphal forms. Colonies are olivaceous-black with a black reverse and are initially moist or yeast-like, later becoming velvety at maturity. Asexual replication is by annellidic conidiogenous cells, and conidia are formed in clusters both from intercalary conidiogenous loci and at the tips of annellides. Some species may occasionally form conidia in chains (182) (catenate) on nutritionally deficient media or display phialides as well as annellides (183). Species are very similar microscopically, and unequivocal differentiation is facilitated by physiologic features such a temperature tolerance and nitrate assimilation and by molecular characterization. Some waterborne psychrophilic species such as E. pisciphila are pathogens of fish (436, 438), while others such as E. mesophila are found in dental unit water lines (604) and municipal drinking water (300). The most clinically important species are thermotolerant (719). In a recent study of U.S. clinical isolates, reidentification of strains by ITS sequencing showed the most common species to be E. dermatitidis (29%), E. xenobiotica (20%), and E. oligosperma (19%) (94, 825). While many clinical isolates are reported as E. jeanselmei, which has been regarded as a major agent of subcutaneous phaeohyphomycosis, this species made up only 8% of the isolates, and molecular studies clearly showed E. jeanselmei to be a heterogeneous complex of species (184, 780). Exophiala jeanselmei has been redefined clinically as an agent of traumatic cutaneous infection eventually leading to eumycetoma (27, 52, 683). Exophiala dermatitidis is distinguished phenotypically by its mostly mucoid colonies, ability to grow at 40°C, lack of nitrate assimilation (569), and yeast cells surrounded by capsules (819), which it shares with another aggressive species, E. spinifera (180, 214, 536, 699). The range of mycoses incited by E. dermatitidis include neurotropic infections in young, immunocompetent individuals (restricted to Asia) (138, 345, 492, 494), systemic lymphadenitis (13), cutaneous and subcutaneous infections in mostly immunocompromised individuals (346, 492), colonization of airways in cystic fibrosis patients (597), and mycoses related to continuous ambulatory peritoneal dialysis (CAPD) (783). It is also an opportunist in lungs of cystic fibrosis patients (320, 355) and may be recovered from the stool in patients with diarrhea (178). It has been recovered from Turkish steam baths (489) and associated with free-living amoebae in hospital water (128). E. phaeomuriformis, which is similar in morphology to E. dermatitidis, can grow at a maximum temperature of 38°C (490). Exophiala spinifera and the similar E. attenuata (780) have long, spine-like conidiophores. E. spinifera is an agent of serious disseminated mycoses in adolescents (180) and of cases of subcutaneous phaeohyphomycosis (327, 699). E. xenobiotica, which is capable of growing in the presence of high concentrations of xenobiotics such as xylene, toluene, or creosote-treated utility poles, was the agent of subcutaneous phaeohyphomycosis in a non-Hodgkin lymphoma patient (36). E. asiatica is a newly described species causing a fatal, disseminated cerebral phaeohyphomycosis in China (452).

Cladophialophora.

Cladophialophora species, although morphologically similar to Cladosporium species, are differentiated by belonging to a different order, the Chaetothyriales rather than the Capnodiales; by lacking conidiophores, “shield cells,” or prominent hila (attachment points); by their ability to grow on media containing cycloheximide; and by having dry, nonfragile chains of conidia. The genus has recently been reevaluated by multilocus sequencing and currently contains seven species associated with humans (51). C. bantiana, (Fig. 6), previously characterized at the molecular level (279), is a neurotropic species with growth at 40°C and is the causative agent of numerous cases of cerebral phaeohyphomycosis (204, 272, 353, 395, 628, 733), many of which occur in immunocompetent individuals and most of which are fatal. The species has also been reported as an agent of eumycetoma (89), along with Madurella mycetomatis (51). Less common species occasionally incriminated in deep and superficial mycoses include C. modesta, C. arxii, C. devriesii, C. emmonsii (Fig. 7), C. boppii, and C. saturnica (47, 51, 295, 505, 516, 568, 748). C. carrionii and the recently described C. samoënsis are agents of chromoblastomycosis (51, 229, 446, 610, 826). C. yegresii is considered a closely related environmental sister species to C. carrionii (181, 782).

FIG. 6. — Long, nonfragile chains of conidia as seen in *Cladophialophora bantiana*.

FIG. 7. — Long, nonfragile chains of conidia produced by a less common species of *Cladophialophora*, *C. emmonsii.* Note that conidiophores and prominent hila (attachment scars) are absent.

Fonsecaea.

The genus Fonsecaea is comprised of two species (174, 533). F. pedrosoi is known almost exclusively as an agent of chromoblastomycosis (45, 515, 610, 695), while the newly described F. monophora (Fig. 8) is known as an agent of chromoblastomycosis (808, 809, 810) and subcutaneous disease and, more recently, cerebral phaeohyphomycosis (721, 733). Prior reported cases of central nervous system and/or other deep tissue infections (520, 545, 701) should most likely be attributed to F. monophora. A murine model of disseminated infection with F. monophora was recently reported (113). Both species form conidia from swollen denticles which give rise to secondary and tertiary conidia in short chains of up to four conidia. Conidia may also be formed from sympodial conidiophores, as in Rhinocladiella, and in balls from discrete phialides with collarettes, as in Phialophora. Molecular characterization is required for unequivocal differentiation.

FIG. 8. — Conidial formation in *Fonsecaea monophora.* Conidia are formed from swollen denticles which give rise to secondary and tertiary conidia in chains of up to four conidia. The same type of conidiogenesis occurs in *F. pedrosoi*.

Ochroconis.

O. gallopava was initially observed to cause central nervous system disease in poultry (354). It has subsequently been shown to be an etiologic agent of neurotropic infections in immunocompromised humans (692) as well as pulmonary infections in immunocompetent hosts (348, 554). O. gallopava has colonies that are brownish rather than gray or olivaceous, produces a maroon diffusing pigment more pronounced on SDA than on PDA, grows at 40°C, fails to grow on media containing cycloheximide, and displays clavate, two-celled, hyaline conidia borne on long denticles (Fig. 9).

FIG. 9. — Two-celled, clavate (club-shaped) conidia of *Ochroconis gallopava* borne on long, thin denticles.

Phialophora.

Some human pathogens with phialidic conidiogenesis previously assigned to Phialophora (263) have been moved to other genera, namely, Phaeoacremonium (525) and Pleurostomophora (777), leaving only those species that are filamentous throughout their life cycle. Both P. verrucosa and P. americana produce their conidia from phialides with conspicuous darkened collarettes; these are funnel shaped and vase shaped in P. verrucosa (Fig. 10) and P. americana (Fig. 11), respectively. Sequencing has demonstrated a close relatedness, suggesting that the species may be synonymous (185, 811). P. verrucosa is primarily an agent of chromoblastomycosis (257, 770), although other reported infections include endocarditis, keratitis, and osteomyelitis (209, 760). A recently described species implicated in superficial infections, P. europaea, has very short collarettes (179).

FIG. 10. — Dark, funnel-shaped collarettes at the tips of the conidiogenous cells (phialides) in *Phialophora verrucosa.* Also note the oval-shaped conidia.

FIG. 11. — Deep, dark, vase-shaped collarettes in *Phialophora americana*.

Rhinocladiella.

Four species of Rhinocladiella are known agents of human disease. R. mackenziei (formerly Ramichloridium mackenziei and also thought to be synonymous with Ramichloridium obovoideum, which is now unrelated in the genus Pleurothecium as P. obovoideum) (43) is a frequently fatal neurotropic organism previously thought to be restricted to individuals residing in or immigrating from Middle Eastern countries (114, 394, 726). It has now been reported as the etiologic agent of a brain abscess in a man from India, an area where it is not endemic, who reported no travel outside the country (48). R. aquaspersa is an occasional agent of chromoblastomycosis (39, 589, 693). R. basitona was recovered from subcutaneous lesions in a man from Japan (43). R. similis (184) appears to be the agent reported under the name R. atrovirens in cases of mycetoma (535) and cerebral phaeohyphomycosis in an AIDS patient (193).

Veronaea.

Initial reports of infection due to Veronaea botryosa were clustered in China; however, a more global distribution is now recognized, with cases seen in Libya, Philippines, an island in the Indian Ocean, and the United States. Two cases are noteworthy as agents of subcutaneous disease in heart (725) and liver (251) transplant recipients. The genus has recently been reexamined at the molecular level by Arzanlou et al. (43).

Microascales

Scedosporium.

The genus Scedosporium and its associated teleomorph Pseudallescheria were extensively reviewed by Cortez et al. in 2008 (153); therefore, the information provided here will augment that previously published and/or highlight new taxonomy, distribution, and disease. Scedosporium prolificans, which is closely related but not a member of this complex, appears to occupy a more restricted geographic range, with infections occurring mainly in Australia, Spain, and the United States (747). Clinical discussion (see Clinical Syndromes and Their Management below) will be limited to S. prolificans, as related species have been extensively reviewed elsewhere (153) and may not reveal phaeoid hyphae in tissue, in contrast to the case for S. prolificans. Infection with this organism is of major concern in all settings due to its refractoriness to antifungal therapy and associated high mortality (18, 29, 76, 96, 119, 153, 312, 475, 538, 664, 802, 806). A recent review of 162 cases reported in the literature summarizes major risk factors as malignancy (46%), cystic fibrosis (12%), and solid organ transplantation (9%) and chief clinical presentations as disseminated infection (44%) and pulmonary mycoses (29%), followed by bone and joint infections (10%) (638). All disseminated infections were in individuals with underlying disease, primarily hematological malignancies; 70% of these had positive blood cultures, and mortality in this group was 88%. Molecular characterization by ITS, D1/D2, translation elongation factor 1α, and the chitin synthase genes for 20 cases of S. prolificans infection occurring in Germany between 1993 and 2007 suggests the possibility of two or three distinct genotypes (747). This finding may further our understanding of the epidemiology of this organism. Multiple genotypes were previously suggested by inter-simple-sequence-repeat (ISSR) fingerprinting (708). Increased numbers of infections with S. prolificans have also been reported from France (304) and Australia (190). Inflated annellides, a key microscopic feature in the identification of this organism, may be subtle in some isolates and easily overlooked. However, the colony color of S. prolificans is always darker than for other Scedosporium species.

Scopulariopsis.

The genus Scopulariopsis is unusual in containing both hyaline and dark species. Most pigmented species associated with disease are anamorphs of various Microascus species detailed in Teleomorphic Genera below. Scopulariopsis shares an annellidic method of conidiogenesis with Scedosporium species but can be differentiated from this genus by conidial formation in chains rather than in clumps.

Sordariales

Madurella.

An agent of dark grain mycetoma primarily in West Africa, M. mycetomatis has recently been proven to be a member of the Sordariales (172), unlike M. grisea, which resides in the Pleosporales. Isolates are very slow growing, produce a brown diffusible pigment, grow at 40°C, and frequently remain sterile in culture; however, lateral phialides and globose conidia are occasionally produced. Precise identification is facilitated by DNA sequencing. Molecular characterization of 38 different M. mycetomatis isolates from Sudan has shown them to have identical DNA patterns, suggesting that host susceptibility rather than differential virulence is the determining factor in clinical presentations (10).

Myceliophthora.

Myceliophthora thermophila is a thermophilic fungus common in high-temperature areas such as compost and exhibits growth at 50°C. Colonies are pale brown, and conidia are borne from ampulliform swellings. Reports suggest that its recovery from tissue, even with a heavy fungal burden, may be difficult (196). The organism is also uncommonly seen in the laboratory and may provide identification challenges. It has been fatal in a disseminated case (95) and in a patient with aortic involvement with medial necrosis (234). A severe case of osteomyelitis was also reported following extensive injury to a knee and distal femur following a barnyard pitchfork injury (196).

Acrophialophora.

Acrophialophora fusispora is an uncommonly seen agent occasionally microscopically misidentified as Scedosporium prolificans. The two species have similarly inflated conidiogenous cells, although they are phialidic versus annellidic and conidia are produced in chains rather than clusters in A. fusispora and S. prolificans, respectively. The organism grows at 40°C, colonies display a striking darkening centrally (both front and reverse), and it produces finely echinulate conidia demonstrating various degrees of spiral banding. It has been reported as an agent of cerebral phaeohyphomycosis in a leukemic child (24), as an agent of keratitis (691), and as an agent of keratouveitis in association with a retained intraocular lens (41).

Calosphaeriales

Phialemonium.

The genus Phialemonium was initially described to accommodate organisms closely resembling Acremonium spp. but containing pigmentation, although colonies often remain pale (264). Colonies are typically moist to slightly filamentous, and conidiogenous cells are a mixture of medium-length phialides and adelophialides (short phialides lacking a basal septum). The genus currently contains two species of clinical interest, P. obovatum and P. curvatum. P. obovatum produces a green, diffusible pigment; has obovate conidia (like an upside-down egg); and has been reported as an agent of fatal endocarditis in a neonate (273). P. curvatum isolates range from cream to yellowish to pale brownish and have allantoid (curved) conidia. Infections attributed to P. curvatum include cutaneous and subcutaneous disease, disseminated infection, endophthalmitis, peritonitis, arthritis, and fungemia (167, 264, 308, 793). Also reported are cases of hemodialysis-associated endovascular infections (608) and endocarditis (561), with some cases linked to intracavernous penile injections in men frequenting impotence clinics (717). Several recent cases have demonstrated sporodochial formation in P. curvatum, a feature not previously seen in this species (167, 608, 793). Rivero et al. have recently reviewed published Phialemonium cases (634).

Phaeoacremonium.

The genus Phaeoacremonium initially accommodated isolates with features similar to those seen in both Acremonium and Phialophora (159). It differs from the former by having pigmented hyphae and conidiophores and from the latter by having indistinct collarettes and warty conidiogenous cells. A recent morphologic and molecular characterization of the genus using β-tubulin sequences (525) has more clearly defined the genus and provided differential features for clinically significant species. Human pathogens include P. parasiticum (obsolete Phialophora parasitica) (Fig. 12) (335), P. alvesii (567), P. amstelodamense, P. griseorubrum, P. krajdenii (525), P. rubrigenum (491), P. tardicrescens, and P. venezuelense (309, 525). Infections caused by P. parasiticum include subcutaneous abscesses (245), thorn-induced arthritis (651), and disseminated infection (54). Colony colors may range from yellowish brown to orange-brown to brown to lavender.

FIG. 12. — Melanized hyphae, demonstrating warts (bottom), long robust phialides, and allantoid (curved) conidia of *Phaeoacremonium parasiticum*.

Pleurostomophora.

Clinically significant species in the mostly wood-inhabiting genus Pleurostomophora include P. richardsiae (obsolete Phialophora richardsiae) and P. repens (obsolete Phialophora repens), and individuals acquiring these mycoses are commonly immunocompromised (369, 601, 815). Species are anamorphs of the genus Pleurostoma. P. richardsiae is characterized microscopically by distinctive flaring collarettes (Fig. 1) and both globose and oval conidia. The colonies of both species tend to be brown rather than gray or olivaceous. Human infections include subcutaneous cases (311) and bone disease (761).

Coniochaetales

Lecythophora.

Two Lecythophora species, L. mutabilis and L. hoffmannii, are of clinical significance. Both produce orange, moist colonies initially, with central darkening in L. mutabilis as pigmented chlamydospores are produced. Organisms are agents of endophthalmitis (677), sinusitis (485), and prosthetic valve endocarditis (207). Recent large-subunit rDNA sequencing confirms the association of Lecythophora species with teleomorphs in the genus Coniochaeta (792) in the order Coniochaetales (361).

Ophiostomatales

Sporothrix.

Sporotrichosis occurs worldwide, with the primary agent of disease being Sporothrix schenckii. The disease is commonly acquired by implantation of the fungus from various types of woody/plant material. Lymphocutaneous lesions are the norm; however, pulmonary disease and disseminated infections may occur in patients with underlying diseases (177). As a dimorphic fungus, it exhibits cigar-shaped yeasts in tissue and at 35°C and filamentous growth in culture. Only the sessile conidia borne along the sides of the hyphae are melanized. In a recent study characterizing the genus by calmodulin sequencing (480) and critically reviewing morphologic/physiologic features, these sessile conidia were shown to vary according to species within the S. schenckii species complex (479). They are elongate to triangular in S. schenckii and globose to subglobose in S. brasiliensis and S. globosa.