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We report a case of Coccidioidomycosis of the cranium that presented as a cystlike structure with adjoining bone destruction in a 40-year-old patient with underlying rheumatoid arthritis that was treated with a combination of lipid amphotericin B and longterm fluconazole. We also discuss the common risk factors and presentations of this unusual extra-pulmonary form of Coccidioidomycosis.

Coccidioidomycosis is a common, environmentally acquired, pulmonary fungal infection in arid and semi-arid regions of the West, especially Arizona and California. The infection is frequently associated with striking cutaneous manifestations. Reactive, immunologically mediated eruptions include erythema nodosum, a generalized exanthem, Sweet syndrome, and reactive granulomatous dermatitis. Less commonly, the skin can harbor the actual organisms as a result of dissemination from the lungs. Dermatologists may play a key role in the recognition of coccidioidomycosis

Background: Coccidioidomycosis involving the lung is common in endemic areas. However, it is unusual for it to present as a solitary bone lesion., Cases: Two cases of Coccidioides immitis osteomyelitis clinically and radiographically mimicked a primary bone tumor. Fine needle aspiration (FNA) biopsy of these lytic bone lesions yielded diagnostic material. Aspirated material in 1 case showed a suppurative, granulomatous inflammation and scattered spherules with refractile walls, some containing endospores consistent with coccidioidomycosis. Smears in the other case showed acute inflammation and necrosis, and rare spherules were identified with the Gomori-mathenamine-silver stain. C immitis infection was confirmed by culture in both cases., Conclusion: Although rare, coccidioidomycosis can involve the bone and mimic a primary bone tumor. FNA biopsy is helpful in differentiating between inflammatory and neoplastic processes involving bone by acquiring material for cytologic studies and cultures.

Coccidioides immitis, the causal agent of coccidiodomycosis, is endemic in the arid desert regions of Mexico and the southwestern United States. Individuals may acquire the disease through the inhalation of conidia. The majority of infected patients are asymptomatic or exhibit flu-like symptoms. Two percent of infected individuals ultimately demonstrate a solitary pulmonary nodule that may be radiographically indistinguishable from neoplasms or other infectious lesions. This report describes the spectrum of cytologic findings in 73 patients who were diagnosed with pulmonary coccidiodomycosis by fine-needle aspiration (FNA) biopsy. The patients ranged in age from 30 to 92 years. Ten had a previous history of malignancy. The smears were characterized by a large amount of granular, eosinophilic debris with a paucity of acute or chronic inflammation. Granulomatous inflammation was present in only three cases. The diagnosis was confirmed by the cytologic observation of C. immitis spherules that ranged in size from approximately 20 to 200 microns. Many of these spherules had a crushed or fractured appearance, and occasional calcified forms were seen. Endospores were observed in intact spherules and were rarely observed outside these spherules. Mycelial elements occasionally were present. C. immitis were cultured in 9 of 44 cases in which fungal cultures were obtained. The cytologic differential diagnosis, which includes contaminant, other infectious diseases and malignancies, is discussed.

Cilofungin, a new antifungal drug, was found to inhibit mycelial growth of Coccidioides immitis. Light and electron microscopic observations indicated delay in development of the outer hyphal wall. Cilofungin also blocked incorporation of the chitin substrate, N-acetylglucosamine, into mycelia. However, when C. immitis was grown under conditions that induced spherule development, drug effects were dramatically decreased. Furthermore, efficacy of cilofungin in treatment of murine coccidioidomycosis could not be demonstrated. These studies indicate that glucan-synthase inhibitors have activity against C. immitis, and other compounds with different pharmacologic properties or in combination with other antifungal drugs may exploit this biologic effect.

Diagnosis of coccidioidomycosis largely depends on serologic tests. In this investigation, the enzyme-linked immunosorbent assay (ELISA) was used to detect patient immunoglobulin M (IgM) precipitin antibody binding to a 120-kilodalton (kDa) fraction previously isolated from an alkali-soluble, water-soluble extract of the arthroconidial wall and mycelial culture filtrate plus toluene lysate of Coccidioides immitis. Results of the serologic response to this tube precipitin antigen (TP-Ag) in the ELISA correlated well with results of immunodiffusion assays of 30 serum samples from patients. Immunoelectron microscopic examinations of arthroconidia and spherules were performed with patient IgM precipitin antibodies isolated from sera eluted over a solid-phase immunosorbent column containing the purified 120-kDa TP-Ag. The antibody probe located the 120-kDa TP-Ag on the walls of in vitro-grown arthroconidia and spherules. Pronase digestion and heating (100 degrees C, 5 min) had no apparent effect on the activity of the 120-kDa TP-Ag, while periodate oxidation resulted in total loss of its immunodiffusion-TP activity. Analysis of the carbohydrate composition of the TP-Ag revealed xylose, 3-O-methylmannose (3-O-MM), mannose, galactose, and glucose. Competitive inhibition ELISAs were used to demonstrate that 3-O-MM is largely responsible for the reactivity of IgM precipitin antibodies with the 120-kDa TP-Ag. Synthetic 3-O-MM may be a useful probe for detection of anti-Coccidioides precipitin antibodies in the ELISA.

Pathogenesis and electron microscopic changes of spherulogenesis of Coccidioides immitis were determined. The organism showed a strong affinity for lung tissue, with granuloma developing in 100% (n = 40) of animals after subcutaneous and intraperitoneal inoculation. Intranasal instillation of the arthrospores resulted in moderate granuloma, with a markedly low frequency rate of infection in 11 animals (55%). Intradermal inoculation and skin scarification did not induce systemic infection. Unlike other routes used to induce infection, only 30% of the mice showed cutaneous changes, whereas in the group infected by skin scarification, 25% of the animals revealed pathologic changes. Indurated tiny nodular lesions developed in the skin at the site of the inoculation. The lesions did not ulcerate, and complete resolution was observed in 4 to 6 weeks. Electron microscopy of the spherulogenesis showed that the tissue form of the fungus replicated in the host tissues by segmentation of the innermost stratum of the wall of the spherule. The layer appeared to function as a germinating center, and as many as 8 spores of irregular size and shape were produced within a kidney-shaped segment. Many segments were in each spherule. The spores were separated, pushed more centrally, and became rounded to become the endospores. When the endospores were freed, each developed into a spherule.

We previously reported isolation of a serine proteinase from the soluble conidial wall fraction of Coccidioides immitis. The purified proteinase was identified as a polypeptide band of 36,000 Mr by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In this study, we raised monospecific antiserum in rabbits against the purified proteinase for use in immunoelectron microscopy. We showed that immunolabel was localized in the cell wall of both the saprobic and parasitic phases but was most concentrated in the wall of the segmentation apparatus of spherules just prior to endospore differentiation. The total wall fractions of the mycelial phase, as well as those of presegmented and endosporulating spherules, were isolated from in vitro grown cells and then treated with a proteinase inhibitor (phenylmethylsulfonyl fluoride [PMSF]) which irreversibly binds to the residual proteolytic enzyme in the wall isolates. Each fraction was dialyzed, lyophilized, and separately incubated with the active, purified 36,000-Mr proteinase. The reaction mixtures were examined spectrophotometrically (A280) for decomposition of the substrates. Only the PMSF-treated wall isolated from endosporulating spherules was significantly digested. Active, 36,000-Mr proteinase was isolated from intact and viable, endosporulating spherules by brief extraction of the cells with 1% octyl-beta-D-thioglucoside, a nonionic detergent. The serine proteinase may be partly responsible for autolysis of the segmentation apparatus of mature spherules, a morphogenetic process which is pivotal for release of endospores and subsequent proliferation of the pathogen.

The fungus, Coccidioides immitis, differs from other dimorphic pathogens in that its parasitic stage is a complex morphogenic cycle, raising the question that changes and composition during morphogenesis might influence host responses. As a prelude to examining the interaction of fungal morphogenesis and host responses, the life cycle of this fungus has been examined in greater detail than previously accomplished. During saprobic development, alternating enterothallic arthroconidia are formed as infectious propagules. The outer wall is broken and loosely adherent. Under in vitro conditions supporting the parasitic cycle, multinucleate arthroconidia transform into uninucleate round cells. Rapid, synchronous, nuclear replication is initiated, accompanied by increase in cell mass and deposition of new cell wall substance. As karyokinesis ceases, morphologic differentiation begins with invagination of the inner layers of the spherule wall and then is progressive, eventually segmenting the protoplasm into uninucleate endospores grouped in clusters within a hyaline membrane. Endospores, escaping through a break in the spherule wall, are held in aggregates by fibrils which are stretched and broken as endospores separate. It would seem that rapid production of hundreds of progeny from an original single cell, protected during development by an enclosing spherule wall and then released in clusters, should favor establishment of the fungus in a host, and dynamic changes in the cell wall during morphogenesis should influence the host response.

The role of the propagule in the initiation of fungal disease is not fully appreciated. Knowledge of the nature of early interactions between infectious propagules and host tissue during onset of respiratory mycoses is particularly scanty. This stems partly from lack of sufficient data on the structure, chemical composition and immunological activity of the microbial surface. In this study, a technique is described for isolation and subsequent ultrastructural examination of the surface wall components of infectious propagules (conidia) produced by the respiratory fungal pathogens, Coccidioides immitis and Aspergillus fumigatus. The SEM has proved very useful in monitoring the cell shearing procedure employed for isolation of the outer conidial wall fractions.

During studies of both saprobic and parasitic cycles of Coccidioides immitis, we found that the hyphae contained septa with simple pores, Woronin bodies, pinocytotic vesicles and/or lomasomes. The alternating thallic arthroconidia were released by fracturing of the adjacent sterile cells. The endospores were formed by progressive cleavage of the spherules. The taxonomic classification of C. immitis still remains obscure.

In vitro-cultivated spherules of Coccidioides immitis were disrupted in a Braun homogenizer. The resultant wall fragments were separated from cell sap and endospores by centrifugation. Extraction of the walls with phosphate-buffered saline yielded a subcellular fraction that was immunogenic in mice. This fraction, which contained polysaccharide and protein, gave substantial protection against intranasal challenge with a usually lethal dose (1,000) of arthrospores, provided that it was administered with adjuvant.

The membranous spherule outer wall (SOW) isolated from liquid cultures of Coccidioides immitis has been shown to elicit reactivity with human anti-Coccidioides antibody by immunofluorescence and the immunodiffusion-tube precipitin assay. The serologically reactive components were extracted from SOW with the nonionic detergent N-octyl-beta-D-glucopyranoside (OG). The OG-soluble fraction of SOW was shown to be reactive with immunoglobulin G in 25 serum samples from coccidioidomycosis patients by an enzyme-linked immunosorbent assay. The isolated SOW and OG-soluble fraction of SOW were also demonstrated to be capable of eliciting lymphocyte blastogenesis. The antigenic and protein compositions of the OG-soluble fraction were examined by two-dimensional immunoelectrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), respectively. Two antigens which were extracted from SOW were identified as antigens 2 and CS on the basis of the coccidioidin-anticoccidioidin reference system. The latter was isolated earlier and shown to correspond to a molecular mass (Mr) of 19 X 10(3) by SDS-PAGE under reducing conditions. This same electrophoresis band was shown to be reactive with sera from coccidioidomycosis patients by immunoblot analysis. One other SDS-PAGE component of the OG-soluble fraction of SOW with an Mr of 66 X 10(3) was shown to be reactive with sera from patients by immunoblot analysis. The SOW of C. immitis represents an important reservoir of immunoreactive wall components which has not previously been reported.

BALB/c mice were inoculated by intranasal challenge with viable arthroconidia of C. immitis and in-vivo morphogenesis of the fungal pathogen was investigated by electron-microscopic examination of pulmonary lavage and cryofractured lung specimens. Samples were prepared at intervals over an 11-day period. Stages of spherule and endospore development were easily identified by scanning and thin-section electron microscopy. Details of morphogenesis of the pathogen in vivo closely resemble developmental aspects reported from in-vitro studies.

The authors report the first case in which the spherules and arthroconidia of Coccidioides immitis were identified from an indwelling cerebrospinal fluid reservoir. The organism was recovered in culture. Present as isolated meningitis, the patient's infection disseminated over a 12-year period despite intrathecal and intravenous administration of amphotericin B. The unusual findings in this patient are compared with findings in 95 cases of infections containing arthroconidia reported since 1940. Arthroconidia is most likely to be found in the tissues of white male patients who are in the third or fourth decade of life and have pulmonary lesions.

The process involved in the in vivo conversion of the arthroconidia of Coccidioides immitis into endosporulating spherules was studied with the aid of a scanning electron microscope. By the fifth and sixth day after inoculation of laboratory mice, complete conversion had occurred in their kidneys, lungs, and spleens. The progressive stages of cleavage that occurred in the enlarged arthroconidia were initiated by invagination at several points of the cytoplasmic membrane that covers the inner surface of the developing spherule's cytoplasm was divided into progressive smaller segments. These segments were aggregated in packets and enclosed in a membranous sac. At maturity the membrane dissolved, and the endospores were freed within the spherules. Finally, the spherule wall ruptured, and the endospores were released.

Coccidioides immitis is a unique fungal pathogen, characterized by a saprobic mycelial phase that gives rise to infectious arthroconidia which in host tissue, convert into a morphologically distinct spherule-endospore phase. This multiphasic cycle presents a formidable challenge to the host. The cell wall of C. immitis is a major reservoir of immunoreactive antigens and thus figures prominently in host-parasite interaction in this disease. Extracts of cell walls have yielded antigens reactive with the TP and CF antibodies, antigens that elicit in vivo and in vitro T-cell responses, and antigens that augment or suppress host response. By using techniques of 2D-IEP and Western blotting, investigators have provided evidence that the TP antigen has a predominant polysaccharide composition, with a molecular weight range of 130-330 kD. This antigen may have tolerogenic activity, as suggested by the induction of IgM antibody early during the disease, which then wanes and is followed by a modest or nondemonstrable IgG antibody response. In contrast, the CF antigen is highly immunogenic, inducing IgG levels within 1 to 3 months of clinical illness, which then persist throughout the course of disease. Compositional analyses of the CF antigen indicate that this heat-labile precipitinogen is a glycolipoprotein complex with a molecular weight range of 43-48 kD. The skin test active component(s) of C. immitis has not been purified, but preliminary evidence suggests that it may be a polymeric antigen designated as Ag2. This polymeric antigen is susceptible to enzymatic treatment with mannosidase and protease, indicating a mannoprotein composition, although other constituents may be present as well. Despite differences in the chemical composition of Ag2 and the TP antigen, these two components coelute on molecular sieve, ion-exchange, and lectin affinity columns and both antigens are solubilized from cell walls with alkali. These similar chromatographic profiles suggest that Ag2 and the TP antigen are intimately associated and perhaps present as a complex. Comparative analyses of morphological forms of C. immitis have provided evidence that extracts from spherules are more potent than those from the mycelial phase in eliciting immune responses. These differences may, however, be attributed to quantitative rather than qualitative differences in the antigenic composition of these cell types. In summary, many advances have been made in the isolation and characterization of antigens from this pathogenic fungus. However, additional work is required to fully delineate their production and expression during the morphogenetic phases of C. immitis and to assess their role in the pathophysiology of coccidioidomycosis.

The authors studied the ultrastructure of the Coccidiodes immitis spherules in the sections of the abdominal cavity foci and pneumonic foci of Cricetus auratus W. and in albino mice infected intraperitoneally with a lethal dose of the mycelium fungus phase. It was found that the spherules of coccidiod fungus represented multinuclear cells with a well-organized protoplast included into a complicated supermembrane structure--the cell wall. Endospores (which copied the spherule structure in miniature) formed as a result of division of the cytoplasm by entire sections into polygonal segments with the subsequent separation of the latter. Due to the rupture of the cell wall of mature sporulating spherules the endospores are released into the surrounding tissue and are attacked there and phagocytized by polynuclears and macrophages. Phygocytosis of the fungi is mostly incomplete.

To assess the influences of leukocytes on the dimorphism of Coccidioides immitis, mycelial elements (arthroconidia) or spherules were incubated with polymorphonuclear leukocytes (PMNLs) or mononuclear leukocytes. In the presence of human leukocytes, arthroconidia became separated spherules as determined by electron microscopy but grew as mycelia when viable leukocytes were absent. Plasma was not required for this effect. In a new assay, PMNLs, lymphocytes, or monocytes from human blood as well as rabbit alveolar macrophages inhibited spherules from forming germ tubes. In flat-bottomed glassware, maximal inhibition occurred with PMNL:spherule ratios above 15:1, whereas in conical-bottomed glassware a ratio of 3:1 was maximally suppressive with PMNLs as well as mononuclear leukocytes. Cytochalasin B and colchicine blocked the effect of PMNLs but not that of other blood leukocytes on spherules. These studies indicate that leukocytes influence coccidioidal dimorphism toward the spherule phase. This effect may be important in host defenses against C. immitis.

A previously undescribed, immunoreactive, membranous spherule outer wall (SOW) fraction produced by Coccidioides immitis (strains 634 and 735) grown in culture was isolated. Both this fraction and intact spherules were reactive with sera from coccidioidomycosis patients, as demonstrated by immunofluorescence microscopy. The serological activity of SOW was also demonstrated by its reactivity with human anti-C. immitis tube precipitin in a standardized immunodiffusion assay. Extraction of SOW with the nonionic detergent N-octyl-beta-D-glucopyranoside (OG) permitted the isolation of an OG-soluble fraction which was reactive in the immunodiffusion assay. Rabbit antisera raised against the OG-soluble fraction were used in immunofluorescence and immunoelectron-microscopic studies of the parasitic cycle to confirm that the immunoreactive components of the solubilized fraction of SOW were associated with the inner and outer layers of the spherule wall as well as with distinct cytoplasmic organelles observed in thin sections of spherules. The immunoreactivity of SOW with sera from patients suggested that infected individuals are exposed to this surface wall material isolated from in vitro-grown spherules.

Intranasal infection of BALB/c mice with Coccidioides immitis resulted in the induction of delayed-type hypersensitivity to coccidioidin within 9 days of infection and in the subsequent development of anergy. The acquired anergy was coincident with detection of circulating Coccidioides cell wall antigen and could be passively transferred to normal, syngeneic mice by intravenous injection with sera from infected donors. Suppression of T lymphocyte reactivity was also induced by injecting BALB/c mice intravenously with coccidioidin or a soluble extract of C. immitis cell walls. Suppression by intravenously administered antigen occurred via the activation of a splenic suppressor cell, as evidenced by suppressed T cell responses in recipients of spleen cells from antigen-injected donors. These collective results provide evidence for an immunosuppressive role of C. immitis cell wall antigens at levels comparable to those produced during the course of active coccidioidomycosis.

Scanning and transmission electron microscopy was performed on the various phases of Coccidioides immitis, exposed for different periods of time to the imidazole antifungals miconazole and ketoconazole. The development of spherules into endospores, which takes place in cultures under normal growth conditions, was suppressed in the drug treated cultures. Typical ultrastructural changes were localized at the cell periphery and in the vacuolar system. The drugs did induce changes in mature, resting endospore cultures and in cultures incubated statically at room temperature. Aerobically growing endospores were not susceptible to either drug. The transformation of arthroconidia into mycelium was fully prevented after treatment. Mycelial cells were most susceptible to the antifungals for necrosis was induced in a substantial part of the hyphae after exposure for 24 h.

Comparative lipid content, cell wall yield, neutral monosaccharide, glucosamine, and protein (amino acid) contents of arthrospores, mycelia, and spherules of Coccidioides immitis Cash were studied. Cellular lipid contents were found in the decreasing order: spherules, arthrospores, mycelia. Lipid content of mycelia did not reach the level of arthrospores or spherules even when mycelia were grown on relatively rich media. Cell wall yields of spherules were lower than for mycelia when grown on comparable media. Cell walls of arthrospores, mycelia, spherules, and spherule culture filtrate all contained 3-O-methylmannose, mannose, and glucose, but in varying amounts. Cell wall yield and cell wall glucose content increased in mycelia grown in increasingly rich media, whereas mannose content either decreased or remained constant.

Human polymorphonuclear leukocytes (PMNLs), when added to arthroconidial suspensions of Coccidioides immitis, markedly inhibit fungal incorporation of the cell-wall precursor N-acetylglucosamine. This effect does not require serum but is facilitated by a heat-labile serum component(s), probably by promoting PMNL attachment to the arthroconidia. Inhibition is entirely reversible within 24 hr. In parallel with this finding, PMNLs exhibited virtually no killing of arthroconidia and (as determined by electron microscopy) did not produce damage to the fungal ultrastructure. PMNLs from a patient with chronic granulomatous disease suppressed fungal incorporation of N-acetyl-glucosamine by only 0%-22% under various conditions, as compared with 54%-85% suppression by PMNLs from normal donors. Our studies demonstrate that PMNLs can influence the metabolism of C immitis and raise the possibility that their presence in coccidioidal histopathology may be an important immediate host defense in limiting progression early after infection.