Abstract

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earliest melanized fungi described, “sporotrichoid” was often used to describe similar fungi, though currently it has been replaced by other, more useful terms.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 ​Table1,1, 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 featuresb
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, euseptatec, 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 distoseptad 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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^aAdapted 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).

^bOn potato flake agar at 25°C.

^cTrue septa continuous with outer wall.

^dPseudosepta 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 ​(Table2).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.

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. ​(Fig.1).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).

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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.

ANAMORPHIC HYPHOMYCETE GENERA

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. ​(Fig.2),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.”

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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. ​(Fig.3)3) (598). C. lunata is the most common clinical species, and C. lunata var. aeria (Fig. ​(Fig.4)4) may produce large, upright stroma in culture that are visible with the naked eye.

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FIG. 3.

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

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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. ​(Fig.5),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).

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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. ​(Fig.6),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. ​(Fig.7),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).

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FIG. 6.

Long, nonfragile chains of conidia as seen in Cladophialophora bantiana.

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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. ​(Fig.8)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.

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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. ​(Fig.99).

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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. ​(Fig.10)10) and P. americana (Fig. ​(Fig.11),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).

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FIG. 10.

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

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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).

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. ​(Fig.12)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.

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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. ​(Fig.1)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).

ANAMORPHIC COELOMYCETE GENERA

Pleosporales

Phoma and Phoma-like pycnidial coelomycetes.

Several genera of morphologically similar pycnidial coelomycetes are occasionally recovered in cases of human subcutaneous disease (307, 585, 704), endophthalmitis (685), and deep tissue infection (411); however, their documentation and reporting as etiologic agents is limited by a lack of adequate identification (727). They include Phoma, Pleurophoma, Pleurophomopsis, and Pyrenochaeta species, with small, hyaline, typically one-celled conidia, and Coniothyrium (411, 704), Paraconiothyrium (773), and Microsphaeropsis species (307, 585, 685), with pale brown to dark, one-celled conidia (Fig. ​(Fig.13).13). The morphologic features of species within several sections in the genus Phoma have been detailed by Boerema et al. (84). Species in these similar genera are best differentiated by ITS sequencing.

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FIG. 13.

A GMS-stained cross section of a multilocular pycnidium of a Microsphaeropsis species produced on carnation leaf agar after 5 weeks of incubation at 25°C.

TELEOMORPHIC GENERA

Microascales

Microascus.

Several pigmented Scopulariopsis species go on to produce their Microascus perithecial teleomorphs in culture. Several of these species have been documented as agents of fatal disease, particularly in transplant recipients. M. cinereus caused a brain abscess in a bone marrow transplant recipient (53), suppurative cutaneous granulomata in a patient with chronic granulomatous disease (483), and endocarditis of a prosthetic valve (129). M. cirrosus was the etiologic agent of disseminated disease in a pediatric bone marrow recipient (424), and M. trigonosporus was reported in a fatal pneumonia in another bone marrow transplant recipient (517). Microascus species are differentiated primarily by the size/shape of the perithecia, the length of the perithecial necks (Fig. ​(Fig.14),14), and the size and shape of the reddish-brown ascospores, which are orange section shaped in M. cinereus, heart shaped in M. cirrosus (Fig. ​(Fig.1515 ), and triangular in M. trigonosporus (Fig. ​(Fig.1616).

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FIG. 14.

Perithecium of Microascus trigonosporus formed on potato flake agar after 3 weeks of incubation at 25°C. Note ascospores being released from the ostiole in the neck of the perithecium.

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FIG. 15.

Heart-shaped ascospores of Microascus cirrosus produced on potato flake agar after 3 weeks of incubation at 25°C.

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FIG. 16.

Triangular ascospores of Microascus trigonosporus produced on potato flake agar after 3 weeks of incubation at 25°C.

Pseudallescheria.

As discussed above for the anamorphic genus Scedosporium, Cortez et al. extensively reviewed Pseudallescheria/Scedosporium in 2008 (153), and so only subsequent taxonomic changes will be discussed here. The human pathogenic species as defined by recent molecular studies are as follows: Pseudallescheria boydii (anamorph Scedosporium boydii), Pseudallescheria apiosperma (anamorph Scedosporium apiospermum, heterothallic, not forming its teleomorph in culture, and d-ribose negative), and Pseudallescheria ellipsoidea (281-283). Other species of clinical interest in the P. boydii species complex include S. aurantiacum (190, 281) and S. dehoogii (282).

PATHOGENESIS

Surveys of outdoor air for fungal spores routinely show dematiaceous fungi (687). This suggests that all individuals are exposed, though few develop disease. Exposure is primarily from inhalation or minor trauma, which is frequently not even noticed by the patient. Relatively little is known regarding the pathogenic mechanisms by which melanized fungi cause disease, particularly in immunocompetent individuals.

DIAGNOSIS

The timely and accurate diagnosis of fungal infections by melanized fungi consists of a multifaceted approach. With the exponential increase in immunocompromised individuals, particularly those in tertiary care cancer centers (72), this becomes imperative to prevent potentially fatal outcomes. Standard conventional diagnostic procedures include direct microscopy, histopathological stains to document tissue invasion, radiographic and computerized tomography (CT) findings, and isolation procedures to recover the fungus and identify the etiologic agent. The clinical presentation and diagnostic findings segregate these infections into the major categories of eumycetoma, chromoblastomycosis, and phaeohyphomycosis. Phaeohyphomycosis maybe further delineated depending upon whether infections are superficial or deep, by their anatomic location, and by the host's response. The microscopic features seen in phaeohyphomycosis, however, are similar regardless of the anatomic site. The confusion surrounding the placement of members of the Sporothrix schenckii and Pseudallescheria boydii species complexes within the dark molds is related to their tissue presentation as yeast cells and hyaline hyphae, respectively. As the term phaeohyphomycosis is commonly used to describe fungi with dark hyphae in tissue, these organisms would be excluded; however, both produce melanized conidia in culture. As a thorough review of infections caused by Pseudallescheria/Scedosporium species has recently been published (153), this paper will concentrate on taxonomic changes and new species documented as etiologic agents subsequent to that review.

Initial Specimen Processing

The appropriate specimen collection, transport, and processing procedures are important considerations in the demonstration of melanized fungi in tissue and their recovery in culture. The most useful diagnostic specimens are those collected at the source of infection; however, specimens peripheral to the site of infection, such as blood cultures in hematogenously disseminated disease, may also be diagnostic in the absence of focal manifestations or when foci are not easily accessible. Appropriate specimens for the recovery of fungi are detailed elsewhere in several reference works (723). Specimens commonly obtained for recovery of melanized fungi include tissue biopsy specimens, aspirates, and body fluids. Surgically obtained specimens should always be cultured as well as processed for histopathology, and the inoculum should be finely sliced or minced rather than ground (as in the case for recovery of H. capsulatum). Gross examination may occasionally reveal evidence of melanized fungi as well (Fig. ​(Fig.17).17). Small volumes of sterile body fluids may also be concentrated by syringe filtration (0.2 μm). Several blood culture systems are available, and the maximum amount of blood recommended should always be used. Swab cultures from superficial sites are usually not representative of the disease process, frequently contain indigenous contaminating mycobiota, and should generally be avoided. Grains or granules should also be washed several times in antimicrobial-containing saline to avoid bacterial overgrowth (504). Also compromising etiologic agent recovery is a delay in specimen transport. Optimally, most specimens should be processed within 2 h of collection, and cerebrospinal fluid should never be refrigerated (723).

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FIG. 17.

Bipolaris spicifera colonies in stomach mucosa of patient with disseminated disease (autopsy). (Reproduced from reference 624 [original Fig. ​Fig.1515-​-6A]6A] with kind permission of Springer Science and Business Media.)

Guidelines regarding the handling of potentially infectious fungi in the laboratory setting are available. It is suggested that cultures of certain well-known pathogenic fungi, such as Coccidioides immitis/posadasii and H. capsulatum, be worked with in a biosafety level (BSL) 3 facility, which requires a separate negative-pressure room, though clinical samples may be handled under BSL 2 conditions (130). Recently, certain agents of phaeohyphomycosis, in particular C. bantiana, have been included in the list of fungi that should be kept under BSL 2 containment (130), and in Europe this mold is considered a hazard category 3 agent (one that can cause severe human disease) (231). This seems reasonable given its propensity, albeit rare, for causing life-threatening infection in healthy individuals.

Histopathology and Special Stains

Several histopathological stains are useful for the demonstration of melanized fungi (670). The most frequently used hematoxylin-and-eosin (H&E) stain demonstrates pigmentation in hyphae that are strongly melanized (Fig. ​(Fig.18).18). In fungi that are only lightly pigmented, hyphae may be misidentified as hyaline rather than dark. The melanin Fontana-Masson stain (Fig. ​(Fig.19)19) is useful in these situations to visualize the phaeoid nature of hyphae in tissue, though other molds may occasionally stain strongly as well (414). An additional stain useful for dark hyphae is the periodic acid-Schiff (PAS) stain (Fig. ​(Fig.20).20). It is frequently preferred over H&E due to the more vivid colors of hyphae, which stain a bright pink-purple against a green background; however, it may overshadow the melanin when present. For practically all fungal histopathology, a Gomori methenamine silver (GMS) stain is ordered. Its utility is in the dramatic visualization of hyphae as dark elements against a green background; however, it fails to discriminate between pigmented and nonpigmented fungal elements (Fig. ​(Fig.21).21). Note that in Fig. ​Fig.1919 and ​and2121 many of the fungal elements are either short stubby hyphae, pseudohyphae, or moniform (bead-like) hyphae. This is not an uncommon tissue presentation with several dematiaceous genera and is quite different from that seen in aspergillosis, fusariosis, or zygomycosis. Chromoblastomycosis (Fig. ​(Fig.22)22) presents in tissue as brown, compact muriform hyphal elements with horizontal and vertical cross walls variously referred to as sclerotic bodies, Medlar bodies, or “copper pennies” (610).

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FIG. 18.

H&E stain of melanized, moniliform hyphal elements of Cladophialophora bantiana from a brain abscess.

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FIG. 19.

Bipolaris spicifera in lung tissue (Fontana-Masson stain; magnification ×100). (Reproduced from reference 624 [original Fig. ​Fig.1515-13B] with kind permission of Springer Science and Business Media.)

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FIG. 20.

PAS stain of Ochrocladosporium elatum, formerly Cladosporium elatum, from sinus tissue.

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FIG. 21.

GMS stain of Rhinocladiella mackenziei from a brain abscess. Note the many moniform hyphal elements often seen with melanized fungi.

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FIG. 22.

GMS stain of sclerotic bodies produced by Fonsecaea pedrosoi.

IN VITRO ANTIFUNGAL SUSCEPTIBILITY

In vitro antifungal susceptibility testing has advanced considerably in the past several years, especially when one considers that a standardized method for testing yeasts was not available until 1997 (534), the first standardized method for filamentous fungi was not available until 2002, and both were updated in 2008 (147, 148). Due to the relatively recent development of antifungal susceptibility testing, the available in vitro data for dematiaceous fungi are relatively sparse, and often rely on small numbers of isolates per species. An important issue is that much of the older literature is often inconsistent with regard to methodology, making reliable observations difficult. In addition, as defined interpretive breakpoints are not available for any of the molds, guidelines for interpreting in vitro data frequently rely on close approximations to breakpoints for Candida species, as well as achievable concentrations of the drug using standard dosing regimens. A MIC of ≤1 μg/ml is often used as an indicator of potential susceptibility for most drugs used to treat black molds, excluding flucytosine (5-FC) (<50 μg/ml), recognizing that there are significant differences in pharmacological properties between the various agents as well as differences in drug concentrations tested. Lower MICs typically suggest better activity. The in vitro activities of several antifungal agents against a variety of dematiaceous fungi are presented in Table ​Table3.3. The data are from a compilation of the current literature (49, 50, 161-163, 224-228, 250, 266, 294, 315, 382-384, 391, 532, 553, 625, 781, 782); however, clinical correlates are not available. Antifungal susceptibility testing of etiologic agents, when warranted, may assist in appropriate patient management.

ANIMAL MODELS OF INFECTION

There are relatively few animal studies with dematiaceous fungi. One of the earliest studies was a murine model of infection with E. dermatitidis and F. pedrosoi (603). Amphotericin B and 5-FC were active alone or in combination, though ketoconazole was not. In another study, 5-FC had the broadest activity against C. bantiana, O. gallopava, and E. dermatitidis in mice, followed by amphotericin B and fluconazole (despite resistance in vitro) (202). Terbinafine was ineffective in vivo, despite good in vitro activity (202).

More recent studies have focused on therapy with posaconazole in refractory mycoses due to R. mackenziei, C. bantiana, and E. dermatiditis. Al-Abdely et al. found posaconazole to be more effective than amphotericin B or itraconazole in murine models of central nervous system infection with R. mackenziei and C. bantiana (14, 15). Posaconazole was also found to be effective in a model of disseminated E. dermatiditis infection (301). In another murine model of C. bantiana infection, posaconazole and flucytosine improved survival alone, though the combination of posaconazole, flucytosine, and micafungin yielded the greatest benefit (481). In a recent murine model of F. monophora, posaconazole was associated with significantly better survival than amphotericin B or itraconazole (113). Posaconazole was associated with improved survival compared with amphotericin B or caspofungin in a murine model of Exophiala infection (633).

S. prolificans was studied in a murine model, and the combination of micafungin with either voriconazole or amphotericin B was associated with improved survival, though the triple combination of all three agents was not more effective (637). Posaconazole with granulocyte-macrophage colony-stimulating factor (GM-CSF) against S. prolificans did not improve survival in one study (698), though liposomal amphotericin B with G-CSF did improve survival in a murine model (560).

CLINICAL SYNDROMES AND THEIR MANAGEMENT

A wide variety of clinical syndromes have been associated with melanized fungi, reflecting their diverse nature (Table ​(Table4).4). The number of published articles relating to these fungi has risen steadily in recent years. They may be considered opportunists or true pathogens, and many of the various clinical presentations can occur in both healthy and immunocompromised individuals. In reviewing the literature, it is seen that case reports often lack crucial details of medical history, diagnostic studies, therapy, and especially clinical follow-up. This limits their usefulness in determining the efficacy of the therapy. However, since randomized trials are not practical given the rarity of these infections, we are left to manage with the available data.

TABLE 4.

Clinical syndromes, associated dematiaceous fungi, and suggested therapy^a

Clinical syndrome	Commonly associated fungal genera or species	Therapyb
Eumycetoma	Madurella, Pyrenochaetae, Leptosphaeria	Azole ± Terb
Chromoblastomycosis	Fonsecaea(F. pedrosoi), Phialophora, Rhinocladiella	Azole ± Terb
Phaeohyphomycosis
Allergic fungal sinusitis	Bipolaris, Curvularia	Surgery + steroids ± Itra
Allergic bronchopulmonary mycosis	Bipolaris, Curvularia	Steroids ± Itra
Onychomycosis	Alternaria, Scopulariopsis	Itra or Terb ± topical agents
Tinea nigra	Hortaea werneckii, Stenella araguata	Topical agents
Subcutaneous nodules	Alternaria, Exophiala, Phialophora	Surgery ± azole
Keratitis	Curvularia, Bipolaris, Exserohilum	Topical natamycin ± topical azole
Bone and joint infection	Scedosporium prolificans, Alternaria	Vori ± Terb
Peritonitis	Curvularia, Exophiala, Alternaria	Catheter removal ± AmB or azole
Pneumonia	Ochroconis, Exophiala, Chaetomium	Vori (L-AmB if severe)
Brain abscess	Cladophialophora bantiana, Rhinocladiella mackenziei, Ochroconis	Azole + L-AmB or echinocandin ± 5-FC (see text)
Disseminated disease	Scedosporium prolificans, Bipolaris, Exophiala	Vori + Terb ± echinocandin, Vori ± echinocandin or L-AmB (see text)

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^aAdapted from reference 625 with permission of Expert Reviews Ltd.

^bAbbreviations: Vori, voriconazole; Itra, itraconazole; Terb, terbinafine; L-AmB, lipid amphotericin B; 5-FC, flucytosine; azole, voriconazole, posaconazole, or itraconazole; +, with; ±, with or without.

CONCLUSIONS

Melanized fungi remain uncommon causes of infection in humans but have become increasingly recognized in a wide variety of clinical syndromes. Many species across a broad range of genera are associated with disease, which leads to daunting challenges in diagnostic testing. However, relatively few are responsible for the majority of clinical cases. Alternaria is a frequent cause of subcutaneous lesions, Bipolaris and Curvularia are often associated with allergic disease, and C. bantiana and S. prolificans are the most common causes of brain abscess and disseminated disease, respectively. Taxonomy is constantly evolving as molecular methods shed new light on relationships between species. Melanin appears to be an important virulence factor for these fungi, though much additional work is needed to better understand the pathogenic mechanisms underlying these infections, particularly in immunocompetent patients. Life-threatening infections are rare but may be seen even in individuals with no apparent risk factors, especially in cases of brain abscess. As these are typically soil organisms and common laboratory contaminants, sometimes they are disregarded as nonpathogenic. However, the clinical setting in which they are isolated should always be considered when evaluating their potential as etiologic agents and before making decisions regarding therapy. Diagnosis depends on a high degree of clinical suspicion and careful mycological and pathological examination of clinical specimens. Molecular diagnostic techniques are progressing but are not standardized or reliable for the diverse species encountered.

Therapy for many infectious syndromes has evolved with the advent of several new antifungal agents in recent years. The oral triazoles voriconazole, posaconazole, and itraconazole demonstrate the most consistent in vitro activity against this group of fungi and are widely used, though voriconazole is usually the drug of choice in most clinical settings. High doses of amphotericin B lipid formulations may have a role in the treatment of refractory cases or for severe infections in unstable patients, though it is usually not effective as a single agent. Once the patient is stable, “consolidation” therapy with a broad-spectrum oral azole is often employed until a complete response is achieved. Terbinafine has broad activity against melanized fungi, and interest in its use beyond dermatophyte infections is increasing. It appears to provide synergistic activity with azole antifungals, and this may be a useful strategy against refractory subcutaneous infections such as chromoblastomycosis and mycetoma that often do not respond to conventional monotherapy. In addition, the use of terbinafine with voriconazole for disseminated S. prolificans infection has been successful with what is otherwise an almost universally fatal infection. It should be pointed out that in these disseminated cases, recovery of immune function, especially phagocytic cells, is critically important as well. Flucytosine has limited activity against dematiaceous fungi, though it may have a role in therapy of chromoblastomycosis and of brain abscess due to C. bantiana, in particular. Echinocandins do not appear to be useful as single agents but may be considered in combination therapy of difficult cases. Combination therapy is a potentially useful therapeutic strategy for refractory infections, particularly brain abscess and disseminated disease. However, it is not clear which antifungal drug combinations are most effective. Therapy is evolving for many of the clinical syndromes described, and randomized clinical trials to address this issue are impractical given the sporadic nature of cases. Detailed case reporting of both successful and unsuccessful clinical experiences will be important in attempting to define optimal therapy for infections caused by dematiaceous fungi.

REFERENCES