INTRODUCTION

Top Previous Next References

Organisms of the class Zygomycetes were first noted to cause disease in humans in publications from the 1800's. Platauf (369) is credited with the first description of zygomycosis in humans in his paper entitled Mycosis Mucorina. His descriptions, in German, are detailed enough to suggest that this first case of disseminated disease in a cancer patient was caused by Absidia corymbifera. The information that emerged over the next several decades was based predominantly on tissue morphology and rarely confirmed by culture. As a result, many of the early cases, and some of the cases still reported today, relied on the morphologic tissue findings of coenocytic, angioinvasive hyphae suggesting infection with one of the Mucorales. The majority of all cases reported had no culture identification; instead, the infection was identified as a "mucormycosis," or Mucor infection, despite this lack of culture confirmation. This was further promoted because most of the pathogenic zygomycetes were originally classified as members of the genus Mucor. These organisms were later reassigned, and continue to be reassigned, into different genera and families within the order Mucorales. Even with the poor showing with culture results, it soon became obvious that Rhizopus spp., and not Mucor spp., were the predominant organisms causing disease (286, 321). Other important information was also being collected by astute clinicians and researchers regarding the association of zygomycosis with cancer (28, 209, 297), antibiotic or prednisone use (463), diabetes (181, 260), deferoxamine and desferrioxamine therapy (48), transplantation (308), and the associated forms of immunosuppressive therapies. With the development of diagnostic tools that allowed earlier diagnosis, with better surgical and antifungal interventions, and with more sophisticated laboratory methods for identifying these agents, more patients are surviving these previously fatal infections. The variety of organisms causing disease has also expanded. In addition to Rhizopus, Mucor, and Absidia, human diseases due to Rhizomucor, Apophysomyces, Saksenaea, Cunninghamella, Cokeromyces, and Syncephalastrum spp. have all been confirmed. The manifestations of disease have also evolved from primarily rhinocerebral, pulmonary, and disseminated disease to include gastrointestinal, cutaneous/subcutaneous, allergic disease, and even asymptomatic colonization.

The Entomophthorales are a very interesting order of the Zygomycetes that produce subcutaneous and mucocutaneous mycoses. The Entomophthorales derive their name from the Greek word "Entomon," meaning insect, reflecting their original identification as pathogens or parasites infecting insects. The human pathogens in this order include Basidiobolus and Conidiobolus species. Basidiobolus ranarum was described from animal and environmental sources as early as 1886 (87, 97, 128, 138, 338, 343, 460). It was not until 1956, however, that the first human case was described in a patient from Indonesia (218). Although the organism is found worldwide, there are estimated to have been only a few hundred cases of infection (320). Basidiobolus infections have historically been limited to tropical and subtropical areas, occurring as a chronic subcutaneous mycosis of the trunk and extremities in immunocompetent hosts, primarily children (175, 320, 413, 442). In recent years, not only has the geographic distribution of basidiobolomycosis expanded but also the etiology of the disease and the range of infected hosts have broadened. Infection with Basidiobolus is now reported in immunocompromised as well as immunocompetent hosts (133, 397). In addition to subcutaneous infection, basidiobolomycosis has expanded to involve other tissues including the gastrointestinal tract, lymph nodes, and muscles (41, 110, 225, 228, 320, 359). Angioinvasive disease has also been seen in one culture-confirmed case (228).

The first cases of infection caused by Conidiobolus spp. were described in the early 1960s. Conidiobolus coronatus was identified first as an agent of nasal granulomatous disease in a horse in Texas in 1961 (147), while the first human infection was reported in Jamaica in 1965 (57). As of 1991, it was estimated that at least 150 cases of chronic sinusitis conidiobolomycosis had been seen world wide (99). Of the 27 known species of Conidiobolus (486), only 4 are known to infect vertebrates (175, 214, 392).

In this review, the diversity of the zygomycetes and their disease manifestations are explored and the taxonomy and the relationship of the zygomycetes to other fungal pathogens are discussed. The epidemiology, pathogenesis, disease manifestation, diagnosis, and treatment of infections caused by the Mucorales are presented in detail, with exhaustive discussions of the individual species implicated in causing human zygomycosis; diagnostic features are summarized in table form for quick reference.

Although the Entomophthorales have some morphologic and reproductive characteristics in common with the Mucorales, this review documents that there are more differences than similarities between the Entomophthorales and the Mucorales producing disease in humans. The taxonomy of the Entomophthorales and the distinguishing features between the organisms of the Mucorales and Entomophthorales and their disease manifestations are reviewed. A detailed discussion of the epidemiologic, pathologic, and diagnostic characteristics of Basidiobolus ranarum, Conidiobolus coronatus, and Conidiobolus incongruus is also presented.

The taxonomic classification of living organisms seems to be in a state of constant flux. Early scientists first divided life into two broad groups; the kingdom Animalia, composed of motile organisms that derive nutrition by ingestion of food, and the kingdom Plantae, which was composed of nonmotile organisms that predominantly but not exclusively synthesized their own nutrition. Under this two-kingdom classification, fungi had been placed into the kingdom Plantae since, like plants, they were clearly nonmotile, with many having portions of their mycelium firmly implanted into their growth medium. However, they were considered to be "lower plants," since they lacked leaves and roots and failed to produce chlorophyll. This classification was suggested despite their clearly different methods of acquiring nutrients (photosynthesis in plants versus assimilation or absorption in the fungi) (246, 503).

As a result of the difficulties in assigning the various types of life-forms into these two limited kingdoms, a taxonomic classification dividing living organisms into five kingdoms was suggested (503). The kingdoms were designated Monera (prokaryotes, including bacteria, actinomycetes, and blue-green algae), Protista (single-celled eukaryotes including the protozoa and a variety of colonial organisms such as the slime molds), Plantae (eukaryotes that develop from embryos), Animalia (eukaryotes that develop from a blastula), and Fungi (eukaryotes that develop from spores). The assignment of various life forms into these five kingdoms helped in the placement of organisms that did not clearly meet the definition of either plant or animal. The assignment of fungi into their own kingdom allowed the separation of life on the basis of both their mode of reproduction and their method of deriving nutrition (503). As molecular techniques are increasingly used to investigate relatedness on the basis of the DNA sequences, it has become apparent that the kingdom Fungi is extremely diverse and that some organisms previously believed to be fungi are being assigned to other kingdoms while organisms originally assigned to other kingdoms are now being identified as fungi (446).

Fungi are eukaryotes, having a nucleus with an RNA-rich nucleolus and cytoplasmic organelles including mitochondria, vacuoles, endoplasmic reticulum, ribosomes, Golgi apparatus, and other cytoplasmic inclusions. Fungi do not have chloroplasts and do not produce chlorophyll. These organisms are delineated within the eukaryotes by their lack of flagella (nonmotile), the development of spores during asexual reproduction, and their predominantly aerobic growth requirements (251, 391, 503). The fungi produce an ergosterol-rich cell membrane and a cell wall composed of a mixture of polysaccharides including chitin, glucan, and glycoproteins. The cell wall is similar but not identical for each fungus, allowing variations in the cell wall composition to be used to differentiate one fungus form another (11, 251, 391).

The kingdom Fungi is further divided into three phyla on the basis of differences in the mode of sexual reproduction of the organisms and on the basis of morphologic features (Fig. 1). The phylum Basidiomycota is delineated by the formation of sexual basidiospores on the surface of a club-shaped basidium. These spores are formed by either sexual (meiosis) or asexual (mitosis) mechanisms. This phylum contains mushrooms, toad stools, puffballs, rusts, smuts, and other related organisms. It also includes human pathogens including the sexual stage of Cryptococcus neoformans, Filobasidiella neoformans. The Ascomycota includes the higher fungi that reproduce sexually by the production of ascospores. This phylum contains several pathogens important to humans, including the teleomorphs of the dermatophytes and Histoplasma capsulatum and Blastomyces dermatitides. A variety of other yeast and filamentous human pathogens and nonpathogens also fall into this category. The third phylum, Zygomycota, is composed of fungi that form coenocytic hyphae and reproduce sexually by the production of zygospores (251, 253, 392, 503). Further details about the Zygomycota are presented below. A catch-all category of mitosporic fungi (formerly the form phylum Deuteromyces) represents the "holding cell" for fungi whose sexual (teleomorph) phase has not yet been identified (11, 446). Since these fungi were identified only by their asexual phase ("mitosporic" indicating reproduction by mitosis only), they have been designated Fungi Imperfecti, or imperfect fungi. A large number of fungi, and most of the human fungal pathogens, fall into this category. Included in this group are the yeast-like fungi including the human pathogens Candida spp. and other related yeasts. Many filamentous fungi with septate mycelium which reproduce by formation of conidia are also thrown into this group. Included in this former "form class Coelomycetes" are both hyaline and dematiaceous fungi. Important members of this group include the agents of aspergillosis, penicillin producers, the agents of subcutaneous mycosis and chromoblastomycosis, and other fungi. It is believed that with the use of molecular techniques, these organisms will eventually be linked with their sexual phase and reassigned into their correct phyla of the kingdom Fungi (446). Neither the form phylum Deuteromyces nor its form classes are recognized taxonomic designations any longer (11, 446).

View larger version (32K): [in this window] [in a new window]   FIG. 1.   Taxonomic organization of the zygomycetes.

The zygomycetes fall into a distinctive phylum, the phylum Zygomycota. It is composed of the organisms that are characterized by the formation of wide ribbon-like aseptate hyaline hyphae (coenocytic hyphae) and sexual reproduction with the formation of zygospores. This phylum is divided into two classes, the Trichomycetes, which are obligate symbionts of arthropods and contain no human pathogens (11), and the Zygomycetes, the class containing the human pathogens. This class is subdivided into two orders, which contain the agents of human zygomycosis, the Mucorales and Entomophthorales (251) (Fig. 1).

Traditionally, the Mucorales are divided into six families of significance in causing human or animal disease: Mucoraceae, Cunninghamellaceae, Saksenaea, Thamnidiaceae, Syncephalastraceae, and Mortierellaceae (Fig. 1). Under this classification system, the vast majority of human zygomycotic disease is caused by the members of the family Mucoraceae. Members of this family include zygomycetes that produce asexual sporangiospores in a sack-like structure called sporangia. A more recently proposed reclassification of the Mucorales by von Arx (481) places the mucoraceous zygomycetes into seven families containing human pathogens. In addition to the six families mentioned in the traditional classification system, the family Absidiaceae is added based upon the presence of an apophysis, the widening of the terminal portion of the sporangiophore during sporangium formation (see Fig. 6). von Arx defined the family Mucoraceae as nonapophysate sporangium producers that may or may not produce stolons and rhizoids and includes in this family members of the genera Mucor and Rhizomucor (481). The proposed family Absidiaceae contains the zygomycetes that produce apophysate sporangia with deliquescent (dissolving) or persistent sporangial walls, produce both stolons and rhizoids, and produce zygospores with opposed suspensors. The most common pathogens in this family are in the genera Rhizopus and Absidia. Most of the recent texts on mycology adhere to the traditional taxonomic scheme in Fig. 1 (251, 446), with only a very few authors supporting the suggested reclassification scheme (11). The reader is alerted to the possibility that this reclassification may become the accepted nomenclature in time, particularly with better dissemination of its proposal.

In the family(ies) Mucoraceae/Absidiaceae, members of the genera Rhizopus, Mucor, Absidia, Rhizomucor, and Apophysomyces have all been implicated in causing human disease (251). Overall, Rhizopus species are the most commonly implicated organisms causing zygomycosis in humans (253, 279, 392, 455). In the family Cunninghamellaceae, only one species, Cunninghamella bertholletiae, has so far been proven to infect humans (495, 496). The monotypic genus Saksenaea contains Saksenaea vasiformis as its only member (400). Cokeromyces is likewise monotypic. Cokeromyces recurvatus is an unusual clinical isolate which can colonize the human colon and genitourinary tract (13, 25, 232, 289, 387). Syncephalastrum racemosum and Mortierella wolfii are two zygomycete isolates whose designations as human pathogens are tenuous. Of the few reports of these organisms causing human disease, several have been refuted as misidentifications of the organism involved and others lack complete substantiating evidence of correct identification. S. racemosum still has a single case report supporting some minimal human pathogenicity (226), while Mortierella should no longer be credited as causing human disease. We present M. wolfii in this paper to clarify the conflicting information on its pathogenicity seen in the literature.

The order Entomophthorales has two families that contain human pathogens, Ancylistaceae and Basidiobolaceae (Fig. 1) (11, 252). Similar to all zygomycetes, the Entomophthorales are characterized by the production of coenocytic hyphae and by their sexual reproduction by production of zygospores (11, 252). The Entomophthorales are distinguished from the Mucorales by their production of actively expelled asexual sporangioles and by their markedly compact and glabrous mycelial morphology. Both of these features define this order within the class Zygomycetes (11, 252). Although several species of Basidiobolus exist in nature, all cases of human disease are now known to be caused by Basidiobolus ranarum (35, 175). Conidiobolus contains several species that are pathogenic to mammals. Conidiobolus coronatus is the major human pathogen (240, 252). C. incongruus has also been implicated in several relatively invasive infections in humans (63, 136, 487). C. lamprauges is pathogenic only to horses (208, 392). A single human case of a Conidiobolus infection by another member of the species has also been described (214).

Initial designation of the diseases associated with the Zygomycetes reflected the predominance of the Mucorales in causing disease in humans. The term "mucormycosis" was commonly used to describe disease caused by these agents. This term, however, ignored the important role that the Entomophthorales play in causing disease. The use of the term "mucormycosis" additionally led physicians to identify the zygomycetes found in tissue sections as Mucor when culture confirmation was lacking. Indeed, most of the human pathogens have, at one time or another, been considered to be of the genus Mucor (M. corymbifera, M. rhizopodiformis, M. pusillus, and M. ramousus). With the ever-changing taxonomy of these fungi, organisms causing zygomycosis have since been placed into the genera Rhizopus, Absidia, and Rhizomucor, so that Mucor spp. no longer represent the majority of the human pathogens. A subsequent designation of "phycomycosis" was transiently employed to encompass the members of both the orders Mucorales and Entomophthorales. The currently accepted designation is "zygomycosis," reflecting all disease processes caused by the members of the class Zygomycetes (141), a term that unfortunately ignores the diversity of disease caused by the organisms in these two orders.

The class Zygomycetes contains hyaline fungi that produce wide ribbon-like, coenocytic hyphae in human tissues. Their asexual reproductive phase is characterized by the production of sporangiospores in sack-like structures. Sporangiospore formation occurs as a result of cleavage of the protoplasm inside the sporangium (free cell formation). This is distinguished from the process of conidiation, where hyphal elements are converted into conidial spores (412). Their sexual reproductive phase is marked by the production of zygospores. Sexual reproduction may occur within a single isolate (homothallic) or may require mating between oppositely oriented mating strains (heterothallic). Heterothallic sexual reproduction predominates among the members of the class Mucorales. Zygospore formation occurs when specialized (sexually oriented) hyphal branches called zygophores are attracted to one another. Zygophores develop near the ends of rapidly growing mycelia which secrete chemical attractants. These two elements contact one another and swell, forming the progametangia. These fuse to produce the gametangium, which undergoes plasmogamy (the mixing of cytoplasm) and karyogamy (the fusion of nuclei). The wall becomes thickened, with multiple layers, and forms the zygosporangium. It is the zygosporangium that accounts for the coloration and surface ornamentation that is characteristic for each isolate (11).

Like all fungi, the zygomycetes are eukaryotes. They lack flagella and are thus nonmotile, and they are predominantly aerobic (251, 503). While most of these organisms are considered to be saprophytic, the Mucorales may also be parasitic and predaceous, especially in regard to causing disease in humans and animals. In all cases, the fungi absorb their nutrients rather than synthesizing them. They require no light for growth (251, 503). Dimorphic conversion has been identified in some species (25, 30, 289, 363, 387, 443).

In comparison to the dimorphic fungal pathogens and agents causing chromoblastomycosis, the presence of the zygomycetes in clinical specimens need not always represent clinically significant isolates. The spores from asexual reproduction are easily airborne and may be demonstrated during sampling of both outdoor and indoor air (27, 51, 107, 265, 329). The small sporangiospore size (mean size, 6.6 µm) allows easy dissemination by the airborne route. Particles of this size have a settling rate that is very low and, as a result, may remain airborne even with very slight movements in air (329). The Mucorales may be seen in the laboratory as clinical contaminants, presumably as a result of airborne contamination of the culture medium, or they may be seen in clinical specimens as a result of oral or nasal ingestion in food or air prior to sample collection. Growth of a zygomycete in culture may therefore not represent clinically significant invasive disease. Demonstration of invasive disease by these organisms generally requires the identification of fungal elements directly in the clinical specimen or organism growth from more than one specimen obtained from a normally sterile site (497). When isolates are obtained from nonsterile sites such as sputa, culturing the same organism from multiple specimens or culturing large numbers of colonies from these specimens might suggest the diagnosis; however, these results might also reflect superficial transient colonization. A positive culture linked to a hyphal identification in cytologic specimens or tissue sections, however, is considered diagnostic (497).

The zygomycetes may be easily differentiated from other fungal agents of infection on examination of cytologic specimens or tissue sections. Zygomycetes in respiratory specimens are distinguished from the dimorphic fungal pathogens and yeasts since they do not produce a yeast phase in this site. They may be differentiated from dematiaceous fungi in clinical specimens by their lack of both darkly pigmented vegetative mycelium and septate hyphae. The major differentiation must be made between the zygomycetes, the other hyaline filamentous fungi, and members of the genus Candida. The morphology of the hyphae is important in making this distinction (Fig. 2). Zygomycetes produce wide, coenocytic, ribbon-like hyphae with wide-angle branching (Fig. 2A), while the other filamentous fungi (often times Aspergillus spp.) present as septate hyphae (Fig. 2B). Candida spp. produce pseudohyphae and blastoconidia in clinical specimens (Fig. 2C). Features that help to differentiate these fungal pathogens microscopically are summarized in Table 1.

View larger version (70K): [in this window] [in a new window]   FIG. 2.   Microscopic morphology of Rhizopus spp., Aspergillus spp., and Candida spp. in tissue. (A) Rhizopus spp. in tissue section stained with GMS. The mucoraceous zygomycetes produce wide ribbon-like aseptate hyphae in tissues. There is a great deal of variation of hyphal width. Branching occurs at wide angles nearing 90° (arrowheads). A frothy or bubbly tissue appearance may be seen in areas of tissue where the hyphae are cross-sectioned (upper left hand corner of the frame). (B) Aspergillus spp. in tissue section stained with GMS. Aspergillus spp. produce thin hyphae with relatively consistent diameters. Hyphae are septate, with no constriction of the fungus seen at the point of septation (arrowheads). Blastoconidia are not produced, although areas where hyphae are cross-sectioned may be confused with yeast cells (asterisk). Hyphae branch at acute angles of about 45° (arrow). (C) Candida spp. in tissue section stained with GMS. Fungal elements in tissue appear as pseudohyphae with blastoconidia. Fungal elements constrict or "bud" at sites of septation (arrowheads). Branching occurs at acute angles (arrow). Pseudohyphae are thin, and their diameter is very similar to that seen for the true hyphae of the Aspergillus spp. All three panels are the same magnification. Bar, 10 µm.

For the most part, the Mucorales are considered to be opportunistic pathogens. They require a breakdown in the immune defenses, particularly disease processes that lead to neutropenia or neutrophil dysfunction. Although neutrophil dysfunction induced by ketoacidosis underlies the majority of cases of human zygomycosis, neutropenia induced by bone marrow suppression during chemotherapy or immunosuppression induced following transplantation is causing a growing proportion of cases (60, 160, 279, 308, 455). Disease is occasionally seen in competent hosts, however, usually associated with antibiotic use or a breakdown in the mucocutaneous barrier. The Mucorales are relatively uncommon causes of invasive human infections, falling far behind Aspergillus spp., Candida spp., and other opportunistic yeasts. In several large studies of the occurrence of fungal infections in high-risk populations, infections with the agents of zygomycosis represented only 5 to 12% of all fungal infections (24, 182, 482). In an occasional study, zygomycosis represents up to 25 to 44% of all invasive fungal disease (318, 358). The zygomycetes should be in the differential diagnosis for causing disease in the immunocompromised host. Similar to Aspergillus spp. and Candida spp., the zygomycetes will cause a spectrum of disease, often leading to angioinvasion and systemic dissemination.

EPIDEMIOLOGY OF THE ZYGOMYCETES

Top Previous Next References

Modes of Transmission

The major mode of disease transmission for the zygomycetes is presumed to be via inhalation of spores from environmental sources. Experimentally, when rabbits were infected by nasal instillation of a spore solution, they developed upper and lower respiratory disease with subsequent spread to the central nervous system (32, 382, 485). Inhalation of spores in dust likewise provides the exposure seen in the allergic interstitial pneumonitis or alveolitis syndrome seen in employees of both the malt and lumber industries (12, 34, 333, 509) and in outbreaks of rhinocerebral or pulmonary zygomycosis linked to excavation, construction, or contaminated air conditioning filters (113, 151, 271, 313).

Percutaneous routes of exposure are also very important in causing infection by the zygomycetes. Traumatic implantation of spores in dirt has been seen in a number of patients (6, 9, 66, 77, 93, 171, 178, 207, 210, 276, 280, 309, 342, 350, 453). Needle-stick exposures have been implicated in zygomycotic infections occurring at the site of medicine injection (77, 158, 215, 361, 474), catheter insertion sites (160, 234, 237, 262, 332), injection sites for illicit drug use (7, 163, 189, 205, 365, 401, 426, 465, 476, 514), and tattooing (357). Insect bites or stings have also been implicated in disease transmission in cases of cutaneous and subcutaneous zygomycosis (33, 152, 159, 199, 203, 233, 372, 494). The development of wound zygomycosis has been seen with a variety of adhesive products used in the hospital setting (52, 53, 168, 234, 292, 353, 423, 479).

The ingestion of fermented milk with dried bread products (259, 321) or fermented porridges and alcoholic drinks derived from corn may play a role in promoting gastric zygomycosis (445). Spore-contaminated herbal or homeopathic remedies have likewise been linked to gastrointestinal disease (344). A series of cases were presumably transmitted orally by spore contaminated tongue depressors used for oropharyngeal examinations in a hematology/oncology clinic (261). Consumption of moldy hay or grains is the most likely way in which infection is acquired in animals (8, 402).

Although healthy humans have a strong natural immunity to infection with the zygomycetes, risk factors for developing zygomycosis were recognized several decades ago. Very early on, diabetes mellitus was seen as the major risk factor for developing rhinocerebral zygomycosis (181). In the original publication by Gregory et al., two out of the three patients reported had presented with diabetic ketoacidosis and the third patient was thought to have an undiagnosed case of diabetes. This publication served as the first description of fulminant rhinocerebral zygomycosis (181).

The mechanism behind this association of diabetic ketoacidosis and the development of zygomycosis has been extensively studied in diabetic animal models using Rhizopus arrhizus as the representative zygomycete. The strongest association occurs during diabetic ketoacidosis. In the diabetic-rabbit model, subcutaneous inoculation of R. arrhizus into acutely diabetic animals leads to disseminated disease and death whereas metabolically normal rabbits contained the infection at the sites of inoculation and spontaneous healing was seen (424). Intranasal infection of animals with R. arrhizus spores likewise led to disseminated zygomycosis in experimentally induced diabetic animals but not in normal controls (32, 381, 483, 484). The increased risk for developing zygomycosis seems to involve two main processes: failure to suppress the germination of spores and subsequent failure to kill proliferating hyphal elements. In normal hosts, macrophages prevent the initiation of infection by phagocytosis and oxidative killing of the spores. In hosts with diabetes, the monocytes/macrophages are dysfunctional and fail to suppress this spore germination process (264, 483, 484). In the diabetic-mouse model, serum also permits spore germination, while normal sera are relatively inhibitory (483). Once infection is established, neutrophils play a pivotal role in fighting fungal infections in the normal host. Despite the large size of the hyphal elements and their subsequent inability to be ingested by the inflammatory cells, neutrophils are still able to mediate fungal killing. Neutrophils are chemotactically attracted to the hyphae on which they attach and spread. Using their oxidative cytotoxic system, neutrophils damage and kill the fungal elements without accompanying phagocytosis (119, 264). In diabetes, each of the four phases of neutrophil activation is impaired (26). Chemotaxis, the phagocytic functions (adherence and spreading), and finally the oxidative burst are all inhibited in the ketoacidotic state, essentially inducing functional neutropenia (26, 64, 310, 424).

Immune system compromise also was linked to the development of zygomycosis in the mid-1900's. Many of the original observations were made in patients with solid tumors, leukemias, and lymphomas (64, 182, 209, 297). Researchers linked the acquisition of fungal disease to the onset of neutropenia due to either the progression of the underlying disease or the chemotherapy used in the treatment of the cancerous state (60, 166). The presence of neutropenia with an absolute neutrophil count of less than 1000/µl for 1 week or more poses the major risk for these patients (60). This association was nicely demonstrated for pulmonary zygomycosis by Tedder et al. in a retrospective review of published cases (455). Prior to the 1950s, there were relatively few cases of pulmonary zygomycosis. By the 1980s, chemotherapy represented the underlying condition associated with 13% of the cases reported (455). The widespread use of steroids had also resulted in an increased incidence of zygomycosis in this population of patients chronically or acutely treated with this class of drugs (166, 438, 463). The mechanism by which the corticosteroids enhance susceptibility to developing zygomycosis is probably twofold. First, steroids suppress the normal inflammatory cell response that would otherwise occur, and second, they may induce a diabetic state (291). Other immunocompromised states such as organ or bone marrow transplantation also contribute to the underlying risk factors for developing disease with these opportunistic infections (54, 160, 190, 196, 308). The use of broad-spectrum antibiotics has also been associated with an increased risk of developing zygomycosis. Presumably, elimination of the normal flora by antibiotic use allows the fungi to establish an infection in the absence of bacterial competition (166, 291, 463). For patients in whom myelosuppressive therapies have been administered and neutropenia and fever have persisted for longer than 7 to 10 days despite antibiotic therapy, a diagnosis of a fungal infection, including zygomycosis, should be suspected (60).

In the late 1980s, physicians began to notice the occurrence of zygomycosis in patients on dialysis who were receiving deferoxamine/desferrioxamine for iron or aluminum overload (48, 102, 139, 317, 374, 510). The more liberally the iron chelator was used, the more likely zygomycosis was to develop (48). A large body of evidence supports the theory that the zygomycetes are able to utilize iron bound to iron chelators to enhance their growth (49, 50, 470, 473). The effect is most prominent in patients with chronic renal failure on dialysis, a situation which leads to the presence of ferrioxamine in the serum for an extended period (50). Even in the absence of renal failure, deferoxamine/desferrioxamine therapy carries a risk for developing zygomycosis. In the guinea pig model, administration of deferoxamine and iron in experimentally infected animals lead to decreased survival and decreased response to amphotericin B (470). The presence of acidosis together with deferoxamine therapy may also be a fatal combination (15). By inhibiting the binding and sequestration of iron by transferrin, acidosis also serves to keep the concentrations of iron in the plasma high, allowing its use as a growth factor by the zygomycetes (23). It has also been demonstrated that iron overload states, such as hemochromatosis, even in the absence of chelator usage, may pose a slightly increased risk for the development of zygomycosis in man (153, 212, 260, 274).

Percutaneous risks for developing cutaneous and subcutaneous disease with these fungi include several methods of breakdown of the cutaneous barrier. Burn wounds have emerged as a significant risk. Aside from the obvious risk incurred by the breach in cutaneous integrity, burn wound victims are at additional risk for developing zygomycosis due to the administration of Sulfamylon cream and broad-spectrum antibiotics to prevent the occurrence of Pseudomonas wound infections. An overall 10-fold increase in fungal infections in burn wounds was seen since the introduction of topical antibacterial preparations in 1964 (318). Also implicated in the increased risk for developing zygomycosis in the burn patient is the development of a condition called "burn stress pseudodiabetes," which is marked by the occurrence of persistent hyperglycemia and glucosuria (22, 375).

The association between intravenous drug use and zygomycosis has also been well described (7, 189, 205, 365, 476, 514). Whether this represents the introduction of spores into the patient from the cutaneous puncture or the injection of spores contained in the illicit drugs remains a point of conjecture. The fact that many of these infections do occur at sites remote from the needle stick (heart valves and the brain are often involved) suggests that the latter theory is probably correct. The role of deficient T-cell immunity in this process is likewise unknown, but notably, several of these cases involve individuals with human immunodeficiency virus (HIV) infection (205, 476).

The risk factors associated with gastrointestinal zygomycosis are varied. Protein calorie malnutrition (261, 306, 321, 432, 445, 491), diarrhea, typhoid fever, and gastric or intestinal ulcers (5, 67, 103, 111, 222, 259, 281, 307, 321, 432), and amebic colitis (321) have all been associated with the development of gastrointestinal disease by the zygomycetes. Gastrointestinal zygomycosis only occasionally occurs in the setting of diabetes (103, 111, 222).

Rhinocerebral disease represents one-third to one-half of all cases of zygomycosis (367, 438). The process originates in the sinuses following inspiration of fungal spores. It is estimated that 70% of the cases of rhinocerebral zygomycosis occur in the setting of diabetic ketoacidosis (291). Disease starts with symptoms consistent with sinusitis. Sinus pain, drainage, and soft tissue swelling are initially seen. The disease may become rapidly progressive, extending into neighboring tissues. Involved tissues become red, then violaceous, and finally black as vessels are thrombosed and the tissues undergo necrosis. Extension into the periorbital region of the face and ultimately into the orbit are often found, even at presentation (181, 260). Periorbital edema, proptosis, and tearing are early signs of tissue involvement. Ocular or optic nerve involvement is first suggested by pain, blurring, or loss of vision in the infected eye. Cranial nerve palsies may also be seen. Extension from the sinuses into the mouth often occurs, producing painful, black, necrotic ulcerations into the hard palate. A bloody nasal discharge is generally the first sign of that the disease has invaded through the terbinates and into the brain. Patients may demonstrate an altered mental state due either to ketoacidosis or to central nervous system invasion (243). Once the eye is infected, fungal disease can readily progress up the optic nerve, again gaining access to the central nervous system. Angioinvasion is often seen and may result in systemically disseminated disease (181, 243, 260, 291, 367). Decidedly uncommon forms of rhinofacial disease published in the literature include isolated sinusitis (172) and calcified fungal ball of the sinus (176). Early cases with rhinocerebral zygomycosis were almost uniformly fatal (181, 260, 356). There is still a high mortality rate with rhinocerebral disease, but curative interventions have been made with early diagnosis and aggressive surgical and antifungal treatment (61, 356). The nature of the underlying disease is the most important determinant of survival. In a study of 179 patients with rhinocerebral zygomycosis, 75% of patients with no underlying immune compromise survived therapy while 60% of those with diabetes and only 20% of patients with other systemic disease were cured of their disease (45).

Pulmonary disease is also a common manifestation with this group of organisms. Leukemia, lymphoma, and diabetes mellitus underlie the majority of cases with primary pulmonary involvement (455). A wide variety of pulmonary disease manifestations exist. Isolated solitary nodular lesion (167), lobar involvement (40, 374, 379, 455), cavitary lesions (192, 235, 279, 301, 354, 440), and disseminated lesions (48, 286, 455) have all been described. Cases of fatal hemoptysis associated with erosion of the fungus into the pulmonary artery have also been described (192, 315, 351). Upper respiratory disease may present as tracheal involvement (17, 40) and chronic endobronchial zygomycosis (202). In many cases of pulmonary zygomycosis, the diagnosis is missed, at least initially, and the patient is treated for a bacterial pneumonia. Chest X-rays may present a lobar picture, only to reveal a granulomatous process later on. Wedge infarcts of the lung may be seen, particularly following thrombosis of the pulmonary vessels with fungal angioinvasion (279). Extensive necrosis and subsequent bleeding into the involved tissues are often seen. For isolated pulmonary disease, the death rate is lower than for zygomycosis overall (65 and 80%, respectively) (455). This is due to the availability of good surgical and medical treatments. Localized disease is more likely to be curable by surgery, providing a large survival advantage for this group of patients (117, 455). This requires early identification of disease before dissemination occurs. In a large study of patients with pulmonary zygomycosis, however, only 44% were diagnosed premortem with a 20% overall survival (455). In a study where premortem diagnosis was improved to 93%, survival rates improved but were still only 73% (356). In patients who receive no treatment, death usually results from the manifestations of disseminated disease before pulmonary failure occurs (455). The notable exception to this are those rare cases of massive hemoptysis (192, 315, 351).

Cutaneous disease may occur from primary inoculation or as a result of disseminated disease. The clinical presentation will be quite different for these two disease processes. Growth of the fungus in a preexisting lesion produces an acute inflammatory response with pus, abscess formation, tissue swelling, and necrosis. The lesions may appear red and indurated but often progress to form black eschars. Necrotic tissue may slough off and produce large ulcers. Infections may be polymicrobic and are generally rapidly aggressive even in the face of appropriate debridement and medical treatment. Occasionally, cutaneous lesions will produce an aerial mycelium that may be visible to the naked eye. Primary cutaneous disease may be very invasive locally, involving not only the cutaneous and subcutaneous tissues but also the fat, muscle and fascial layers beneath. Necrotizing fasciitis may occur secondary to cutaneous or subcutaneous zygomycosis. When present, necrotizing fasciitis due to the Mucorales has a very high mortality rate (80% in one study) (362). Direct extension into adjacent bone and other tissues has also been described. With vessel invasion, frankly disseminated disease may arise. Due to the superficial site of infection, this disease manifestation is fairly likely to be diagnosed and appropriately treated. Curative surgical intervention may, however, be quite disfiguring or may require amputation of the involved limb (89, 362). Individuals with operable sites of involvement, especially limbs, are more likely to survive than those with trunk or head involvement (89). In burn patients, both superficial colonization of the eschar and invasive disease may be seen. Superficial colonization may serve as the precursor to truly invasive disease and is thus an important process to identify and treat in these patients (318). While cutaneous or wound zygomycosis may be seen with most of the zygomycetes, Apophysomyces elegans, Saksenaea vasiformis, the Mucor spp., Basidiobolus ranarum, and Conidiobolus spp. tend to have the greatest predilection for these sites. Cutaneous disease arising from disseminated disease, in comparison to the wound zygomycosis, usually presents as nodular subcutaneous lesions which may ulcerate.

Disseminated zygomycosis may originate from any of the primary sites of infection. Lung involvement is the single most common site of infection associated with disseminated disease, however. In the infected host, the zygomycetes rapidly invade vessels and may metastasize wildly by the hematogenous route. There is a very high death rate associated with this disease manifestation, with 96% mortality noted in one study (455) and 100% in another (438).

Gastrointestinal disease caused by the zygomycetes is relatively uncommon. Two syndromes have been described with gastric involvement. Gastric colonization occurs with superficial involvement in ulcerative lesions. These colonized ulcers often have a velvety surface texture. This syndrome is marked by a lack of vessel invasion and a good survival rate (259). Invasive zygomycosis has been identified more commonly and presents with fungal invasion into the mucosa, submucosa, and vessels. Necrotic gastric (5, 103, 139, 222, 259, 314, 491) or intestinal (67, 263, 306, 307) ulcers are produced, which may rupture, causing peritonitis. This disease manifestation is often fatal, with only 2% of patients surviving the infection (281, 511).

The zygomycetes may cause infection in virtually any body site. Brain involvement in the absence of sinus involvement has also been demonstrated, primarily in the intravenous drug abusers (7, 189, 514). Isolated renal involvement has been described in several cases, again associated with intravenous drug use (476). Cardiac infections may occur as a manifestation of disease disseminated from the lungs (358, 478) or secondary to intravenous drug use (7, 189) or may occur as an isolated cardiac mycosis (478). Of 60 cases of fungal cardiac infection, 12% represented disease by the zygomycetes (24). Several cases of postcardiac surgery zygomycosis have also been noted (24, 478). Isolated pleural involvement following a surgical intervention has also been seen (180).

Other disease manifestations are decidedly uncommon and involve predominantly noninvasive infection. Oncomycosis has been seen with infections due to Mucor circinelloides (443), while external otitis has been seen with infections due to Mucor spp. (78, 209, 355) and Absidia corymbifera (200).

Treatment of zygomycosis requires several simultaneous approaches: surgical intervention, antifungal therapy, and medical management or correction of the underlying condition that is predisposing the patient to the disease. A retrospective study of 255 cases of pulmonary disease compared patient survival for individuals treated with or without surgical interventions. Surgical resection for patients with isolated pulmonary disease greatly improved survival compared to that of patients who received antifungal therapy alone (455). Response to surgery is best in cases of localized disease without dissemination (117, 455). Although there are several case reports of a single treatment modality producing cure, these are the exception and not the rule. There are notable cases of zygomycosis being cured by surgery only (77, 373, 455, 462). Similarly, antifungal therapy alone has been used successfully when surgical intervention is not possible or not preferable due to the site of the infection (159, 272, 293, 324, 357, 372, 477, 498). The vast majority of cases where successful treatment has been administered link aggressive surgical intervention with antifungal therapy and rigorous medical management of the patient.

Amphotericin B is the first-line drug of choice for most cases of zygomycosis caused by the Mucorales. Amphotericin mediates its antifungal action by modifying fungal cell walls. This drug binds to ergosterol and causes increased cell wall permeability. With permeabilization, ions leak from the cell and the membrane depolarizes. Lethal effects of amphotericin B occur at concentrations of drug higher than that causing increased permeability (55, 360). Secondary or indirect mechanisms responsible for its lethal antifungal action include the stimulation of the oxidative pathway in the immune response. Monocyte/macrophage stimulation by amphotericin B increases production of hydrogen peroxide and free radicals, which may then have killing action on the fungal elements, again mediated by cell wall changes (55). Amphotericin B is not effective in treatment of all cases, particularly if the patient presents late in the disease course and has inoperable or disseminated disease. The therapeutic activity of amphotericin B is limited by its potentially severe side effects. Impaired renal function often leads to cessation of therapy. The liposomal preparation of amphotericin B may help to alleviate this problem and allow for higher doses of medication to be administered (269). Although synergism of amphotericin B and rifampin in treating zygomycosis has been suggested by some authors (84, 324, 398), this has not been conclusively demonstrated in clinical trials.

Antifungal therapy has produced variable results, dependent upon the organism and the drug selected. It has become evident that the azoles should not be used in treating zygomycosis due to the lack of both in vitro and in vivo susceptibility (149, 348, 434). Early successful attempts at antifungal therapy for infection with the Mucorales included the use of oral saturated potassium iodide and local applications of tincture of iodide for cutaneous sites (98, 221, 398). This mirrors the treatment regimen successfully used to treat infections with Basidiobolus and Conidiobolus spp. (454). Similarly, topical treatment of Mucor-associated otomycosis has been optimal with mercurochrome applications (preferable to clotrimazole and miconazole) after adequate cleaning of the auditory canal (78). Oils applied topically into the auditory canal have also been reported to be sporostatic (216). All the zygomycetes tested demonstrated resistance to saperconazole (347), 5-fluorocytosine, and naftitfine (348, 434). Assessing the impact that isolated pharmacological therapy has had on patient outcome is impossible since multiple simultaneous interventions are used for most patients. With Rhizopus spp. as the representative for the zygomycete infections, in vitro studies have demonstrated a lack of effect by pneumocadin L-743,872 (114), 5-fluorocytosine, and the echinocadins (364) in inhibiting fungal growth.

Hyperbaric oxygen treatments have been touted as an appropriate addition to standard surgical, medical, and antifungal therapy, particularly for rhinocerebral disease. Unfortunately, there has not been any extensive experience with this treatment modality. In a retrospective analysis of the cases seen in a single medical center, the addition of hyperbaric oxygen treatments to the standard treatments improved survival in the patients who received them. While four of the seven patients not receiving hyperbaric oxygen therapy died, only two of the six patients who received this treatment died (157). The mechanism behind the success of this therapy rests upon the presumed improved neutrophilic killing achieved by the higher oxygen delivery achieved in these patients (100). Additionally, the direct growth-suppressive effects of 10 atm of oxygen may play a role in successful treatment. Hyperbaric oxygen either delays or totally inhibits the growth of fungal spores and mycelium in vitro (393). The addition of hyperbaric oxygen treatment to systemic drug therapy could aid in direct fungal killing or at least diminish the fungal growth rate, allowing the natural host immune defenses to recover. Other scattered case reports also support the use of hyperbaric oxygen (178, 322, 342) and suggest that further trials with this treatment modality may be warranted.

Treatment of the underlying disease process placing the patient at risk for opportunistic infections with the zygomycetes can not be underestimated. Correction of diabetic ketoacidosis helps to restore neutrophil function that is temporarily impaired by the acidotic environment (64, 310). Occasionally, extraordinary measures such as continuous infusion of insulin have been utilized (504). Anecdotal reports of improved recovery from infection by correcting neutropenia either with granulocyte transfusions (47, 398) or by enhancing endogenous neutrophil production using growth factors (174, 262, 266) have been published, but the data are insufficient to suggest that this should be the standard of care. Discontinuation of iron chelation therapy or immunosuppressive therapy, particularly steroids, is often warranted in patients receiving these therapies when a diagnosis of zygomycosis is made.

GENERAL DIAGNOSTIC FEATURES OF THE ZYGOMYCETES

Top Previous Next References

Microscopic Examination of Clinical Specimens

Demonstration of fungal elements from cytologic preparations (i.e., sputa, inflammatory fluid aspirates from abscesses or sinusitis infection, and genitourinary and gynecologic specimens) may be quite difficult due to the difficulty in extracting fungal elements from invaded tissues. Fungal elements may be rare in cytologic specimens and when present are often fragmented. Additionally, hyphae may be very focal and may appear in only part of the specimen. The key features associated with the Zygomycetes on direct examination of cytologic specimens is the presence of wide, ribbon-like aseptate, hyaline hyphal elements, often in the setting of extensive necrotic debris. The width of the hyphal element varies substantially. Branching of the hyphae is seen, with wide-angle (generally around 90°) bifurcations noted. Yeast formation (blastoconidial formation) has been observed with Cokeromyces recurvatus, which presents as budding yeast that may be confused with Paracoccidioides braziliensis (102, 289, 387). In addition, some Mucor species are dimorphic, producing budding yeast at 37°C (443).

The stains most commonly used in identifying yeast directly from patient cytologic specimens include calcofluor white stain, Gomori methenamine silver stain (GMS), periodic acid shiff (PAS), Gram's stain, and Papanicolaou stain (PAP stain). Although the zygomycetes may be demonstrated on either PAP or Gram's stain, these are not generally the stains of choice. Demonstration of the fungal elements with fungal specific stains such as calcofluor white or GMS is recommended. Phase-contrast microscopy, although not widely used, has also been successful in identifying fungal elements in cytologic clinical specimens (394).

The diagnosis of zygomycosis is easily made on tissue section. Involved tissue demonstrates focal areas of infection and may appear nodular or may produce extensive areas of necrosis with accompanying hemorrhage into the tissue. Abscess formation with central tissue necrosis, acute inflammatory exudate, and peripheral tissue invasion by hyphal elements is quite common. An acute inflammatory exudate often accompanies these infections in nonneutropenic patients (Fig. 3A). Invasion of the blood vessels (angioinvasion) by hyphal elements is generally seen in infections with the Mucorales (Fig. 3) but usually not with the Entomophthorales. The hallmark of a zygomycosis includes the demonstration of wide, ribbon-like, hyaline, predominantly aseptate hyphae with wide-angle (45 to 90°) branching (Fig. 2A and 3C). The hyphae often are not preserved well and may become crinkled or gnarled in the tissue sections. This is often referred to as a "crinkled cellophane" appearance of the hyphal elements. To the inexperienced observer, these artifactual folds in the hyphae may be confused with septations. Cross sections of hyphal elements often give tissues a vacuolated appearance. These cross sections vary in diameter and may be confused with yeast cells (Fig. 2A). In hematoxylin and eosin (H&E)-stained tissue section, the Entomophthorales demonstrate hyphal encasement by eosinophilic material. This Splenore-Hoeppli material may be the first indication that a patient has an infection with either Basidiobolus or Conidiobolus instead of one of the Mucorales, which rarely demonstrate this phenomenon in tissues.

View larger version (211K): [in this window] [in a new window]   FIG. 3.   Angioinvasion by mucoraceous zygomycetes. (A) H&E-stained section of a vessel filled with hyphae and inflammatory cells. Tissues infected with the mucoraceous zygomycetes often demonstrate extensive angioinvasion by fungal elements. Neutrophils are the predominant inflammatory cells responding to an infection with these agents. The inflammatory cells stain basophilic (dark), while the hyphae demonstrate an inconspicuous hyaline staining that can often be overlooked. Pale-staining hyphae (arrowheads) often look like hole or bubbles in the tissue. The hyphal elements are nearly obscured on this H&E section due to the inflammatory process seen. For these reasons, it is recommended that a fungus-specific stain such as GMS or PAS be performed to better characterize fungal elements in tissues having thrombosed vessels, extensive tissue necrosis, or hemorrhage. (B) H&E-stained section of a vessel with typical zygomycete hyphal elements in a patient with neutropenia. In contrast to panel A, the hyphal elements are much more prominent in this H&E preparation despite their hyaline staining, due largely to the absence of the inflammatory cells. (C) GMS-stained section of a vessel containing coenocytic fungal hyphae typical of the zygomycetes. Wide ribbon-like hyphae with broad-angle branching is seen (arrowhead). GMS provides excellent contrast to help distinguish hyphae, which stain black or dark gray, from the tissue elements or inflammatory cells, which stain with the pale counterstain. (D) GMS-stained section of a vessel with fungal hyphae penetrating the vessel wall. Infections originate in mucocutaneous sites, from which they may spread hematogenously. Fungal elements invade through the vessel walls from their tissue sites. Mycotic emboli disseminate and may thrombose small vessels in which they are lodged. Fungal elements may invade normal or devitalized tissues at these remote sites by directly penetrating the vessel wall (arrow). All four panels are the same magnification. Bar, 50 µm.

In comparison to the other hyaline molds that cause disease in man, the zygomycetes stain more poorly or inconsistently with the stains typically used. In H&E-stained tissue sections, zygomycetes generally stain pink with empty or clear lumina. The infected tissue may appear frothy due to these clear spaces. Hyphae may be difficult to demonstrate convincingly using this stain. The diagnosis is more likely to be suggested by the bubbly and frothy appearance generated by the nonstaining hyphal interior than by the hyaline staining of the hyphae themselves (Fig. 3). Variability in staining with H&E may be seen. Although the zygomycete elements usually stain pink (eosinophilic) with H&E, purple (hematoxylyniophilic) staining may also be noted. The H&E stain should always be confirmed with a more fungus-specific tissue stain such as GMS or PAS. Although either of these stains will readily demonstrate zygomycete elements, the relative staining may be less intense than would be seen with other hyaline fungi. There is also some variability in staining intensity within a single clinical specimen. Sporulation in tissues, if it truly does occur, is exceedingly rare.

Growth characteristics. The Mucorales grow well on both nonselective and fungal selective media. When fungal infections are suspected, it is recommended that fungal selective media be used to suppress the growth of bacterial elements potentially present in the sample (e.g., Inhibitory mold agar). The growth of the Mucorales tends to be rapid, with mycelial elements expanding to cover the entire plate in only a few (1 to 7) days. Organisms of the order Mucorales are characterized by an erect aerial mycelium that is described as fibrous or "cotton candy-like." The mycelium tends to be quite high, with some isolates reaching the lid of the petri dish at mature growth. It is this vigorous growth characteristic that is responsible for the group being designated "lid lifters" (Fig. 4A and C). These organisms are hyaline, with the reverse side of the plate demonstrating light coloration (tan to yellow for most species) (Fig. 4D). A great deal of inter- and intraspecies variation may be seen in height, rate of growth, and degree of pigmentation.

View larger version (139K): [in this window] [in a new window]   FIG. 4.   Gross morphology of Rhizopus, Mucor, and Absidia isolates in culture. (A) Rhizopus gross morphology on SABHI medium. Rhizopus spp. typically produce a very high, fibrous colony that rapidly fills the entire petri dish. This isolate has expanded to the lid (known as a "lid lifter"). It has produced abundant pigmented sporangia, which are seen as the dark areas peppering the otherwise pale mycelium. This morphology is characteristic of the Rhizopus spp. (B) Low-growing Mucor variant gross morphology on SABHI medium. Mucor spp. will show variation from culture to culture. This particular isolate has produced a low-growing, fibrous colony morphology that readily demonstrates the "woolly" or floccose growth characteristic of the Mucoraceae. Pigmentation is also variab