Spherules Of Coccidioides Immitis Research Articles

Pulmonary Coccidioidomycosis in Immunocompetent Patient

Coccidioidomycosis is a fungal infection caused by the soil fungus, Coccidioides immitis, which is endemic to the south-western United States. However, the incidence of coccidioidomycosis has recently increased due to the increase in overseas travel to endemic areas. We report a case of pulmonary coccidioidomycosis diagnosed in an immunocompetent person. A 28-year-old female, who had lived in Phoenix, Arizona, USA for 2 years, was admitted for an evaluation of persistent cough with fever lasting for 2 weeks. The chest X-ray and Chest CT revealed multifocal patchy consolidation and ground-glass opacity in both lungs as well as multiple enlarged right hilar and paratracheal lymph nodes. A percutaneous needLe biopsy of the main mass-like consolidation confirmed mature spherules of Coccidioides immitis in lung tissue. Pulmonary coccidioidomycosis should be considered in patients presenting with persistent cough with fever and a history of travel to or immigration from an endemic area.

Disseminated coccidioidomycosis with cutaneous lesions clinically mimicking mycosis fungoides

AbstractThis report describes the second known case of disseminated coccidioidomycosis mimicking mycosis fungoides since the original description more than 100 years ago. Coccidioidomycosis should be considered in persons from an endemic area presenting with nonhealing or suspicious skin lesions. Diagnosis is established by skin biopsy and silver staining showing granulomas and classic Coccidioides immitis spherules. Disseminated coccidioidomycosis most commonly involves the skin and can have a variety of cutaneous manifestations. The first description of a Coccidioides immitis infection in 1892 was initially diagnosed as mycosis fungoides. We present the second case of coccidioidomycosis masquerading as mycosis fungoides.

The Enhancing Rim: A New Sign of a Benign Pulmonary Nodule

To describe a new radiological sign, the enhancing rim sign, that indicates that a solitary pulmonary nodule is benign rather than malignant. Sixteen adult patients underwent computed tomographic (CT) evaluation with intravenous contrast enhancement of an indeterminate solitary pulmonary nodule between May 25, 2000, and March 21, 2002, at the Mayo Clinic in Scottsdale, Ariz. In each patient, we identified a rim of enhancing soft tissue at the periphery of a solitary pulmonary nodule; this rim surrounds a homogeneous, lower-density central portion of the nodule. Surgical or biopsy specimens from 6 patients and clinical data from 10 patients were evaluated to determine whether the nodules with the enhancing rims were benign or malignant. Pulmonary nodules in 6 patients were diagnosed as benign after histological evaluations of tissue obtained by surgical resection (3 patients), bronchoscopic biopsy (1 patient), and CT-guided needle biopsy (3 patients) (1 patient had a needle biopsy and wedge resection). In another 10 patients, pulmonary nodules were diagnosed as benign during clinical follow-up (range, 2-24 months; median, 11 months): in 1 patient, the nodule disappeared; in 1 patient, the nodule's size was stable; in 3 patients, the nodule's size decreased; and in 5 patients, no further radiological studies were performed, but no symptoms or signs of pulmonary disease were present during follow-up. Coccidioides immitis spherules were identified in 2 surgical specimens and in 1 CT-guided biopsy specimen. Serologic results for C immitis were positive for 3 patients: 1 at the time of lobectomy and 2 during clinical follow-up. The enhancing rim sign, seen on chest CT with intravenous contrast enhancement of a solitary pulmonary nodule, has been useful to distinguish some benign pulmonary nodules from malignant pulmonary nodules.

Identification and cloning of an aspartyl proteinase from Coccidioides immitis.

A 45 kDa protein was isolated from a soluble vaccine prepared from formaldehyde-killed spherules of Coccidioides immitis. From the N-terminal amino acid sequence, the protein yielded a 17-amino-acid peptide that was homologous to sequences of other fungal aspartyl proteinases. The coccidioidal cDNA encoding the proteinase was amplified using oligonucleotide primers designed from the 45 kDa N-terminal amino acid sequence and a fungal aspartyl proteinase consensus amino acid sequence. The PCR product was cloned and sequenced, and the remaining 5' upstream and 3' downstream cDNA was amplified, cloned, and sequenced. The cDNA encoding the coccidioidal aspartyl proteinase open reading frame was cloned and the fusion protein containing a C-terminal His-tag expressed in E. coli. The recombinant aspartyl proteinase was purified by immobilized metal affinity chromatography. This recombinant protein will be used for further studies to evaluate its antigenicity, including protective immunogenicity.

The first imported case of pulmonary coccidioidomycosis in Korea

Coccidioidomycosis is an endemic disease found in the southwestern part of North America. Travellers who visit the endemic area may carry the infection. We report a case of pulmonary coccidioidomycosis in a 74-year-old woman. She was healthy before visiting Arizona, U.S.A twice. After returning home, she began to complain of intermittent dry coughing. The symptom was mild, however, and she was treated symptomatically. Later a chest radiograph, which was taken 4 years after the onset of the symptom, showed a solitary pulmonary nodule in the right upper lobe. By percutaneous needle aspiration, a few clusters of atypical cells were noted in the necrotic background. A right upper and middle lobectomy was done. A 1.5 x 1.5 x 1.2 cm sized tan nodule was present in otherwise normal lung parenchyma. Microscopically, the nodule consisted of aggregates of multiple solid granulomas inside of which was mostly necrotic. Neutrophils and nuclear debris were scattered along the periphery of the necrotic foci. Numerous multinucleated giant cells were associated with the granulomas. In the necrotic area, mature spherules of Coccidioides immitis, which were 30-100 microm in diameter, were present. They contained numerous endospores which ranged from 5 to 15 microm and were also noted in multinucleated giant cells. The diagnosis of coccidioidomycosis was made. She is doing well after the resection.

Detecting serum antibodies to a purified recombinant proline-rich antigen of Coccidioides immitis in patients with coccidioidomycosis.

In previous work, antibodies in serum samples from patients with coccidioidomycosis were found to react with a proline-rich antigen (PRA) isolated from spherules of Coccidioides immitis, and the gene encoding this antigen was cloned. We expressed and purified recombinant PRA (rPRA) by removing the majority of amino acids contributed by the vector from the fusion protein. Purified rPRA reacted with serum IgG antibodies in 37 of 42 patients with culture-proven progressive pulmonary or extrapulmonary coccidioidal disease; specific antibodies in dilutions ranging from 1:40 to 1:102,400 were demonstrated (sensitivity, 88%). In contrast, for > 95% of patients without coccidioidomycosis reactivity of < 1:40 was demonstrated (specificity, 97%). Of 18 patients with primary self-limited coccidioidomycosis, none had detectable antibodies in serum samples collected up to 141 days after illness began. The association of antibodies to rPRA with progressive infection may have prognostic value.

Evaluation of cultures of percutaneous core needle biopsy specimens in the diagnosis of pulmonary nodules.

The value of cultures of tissue obtained by image-directed core needle biopsy of lung nodules has not been determined. Of the 250 biopsies performed during a 5-year period in an area endemic for coccidioidomycosis, 225 (90%) were diagnostic. Granulomas were identified in 75 specimens, whereas 3 specimens revealed abscess. Ziehl-Neelsen stain was positive for acid-fast bacilli (AFB) in 2 cases. Spherules of Coccidioides immitis were seen in 54 of the biopsy specimens with granulomas. Microbiologic cultures were positive for C immitis in 5 (9.6%) of 52 biopsy specimens. Both of the AFB-positive cases were negative by culture. Organisms were demonstrated in cases with abscess; however, the cultures submitted in 2 cases were negative. Cultures were uniformly negative in cases where special stains failed to reveal organisms. Cultures of core needle biopsy specimens are insensitive in the detection of specific microorganisms and need not be routinely performed.

Cerebrospinal Fluid Antibodies Detected by ELISA against a 33-kDa Antigen from Spherules of Coccidioides immitis in Patients with Coccidioidal Meningitis

Antibodies against a 33-kDa antigen from Coccidioides immitis were detected by ELISA in patients' cerebrospinal fluid (CSF). Anti-33-kDa antibodies were detected at dilutions > 1:80 in only 1 (1.4%) of 73 patients without coccidioidal meningitis but in 74 (71.8%) of 103 with meningitis. Anti-33-kDa antibodies were detected in 53 (91.4%) of 58 patients whose anti-coccidioidal complement-fixing (CF) antibodies were detectable and in 21 (46.7%) of 45 patients whose CSF was negative by CF test (positive predictive value, 99%; negative predictive value, 71%; sensitivity, 72%; specificity, 99%). Anti-33-kDa antibodies, among which IgG1 was the dominant subclass, increased when infections worsened and decreased when patients' conditions improved. Antibody concentration appeared to be independent of most baseline findings, although only 1 of 5 patients coinfected with human immunodeficiency virus had initially detectable antibodies. Measurement of anti-33-kDa antibodies is a sensitive indicator of coccidioidal meningitis and of its clinical course.

Differences in Oxidant Release by Human Polymorphonuclear Leukocytes Produced by Stimulation with Different Phases of Coccidioides immitis

Human polymorphonuclear leukocytes (PMNL) inhibit growth of arthroconidia and young spherules but not mature spherules of Coccidioides immitis. To evaluate these differences, oxidants were measured when PMNL were exposed to C. immitis or other particulate stimuli. Concentrations of arthroconidia, young spherules, mature spherules, and Candida glabrata were adjusted to result in equivalent superoxide release. Hydrogen peroxide and hypochlorous acid were stimulated by arthroconidia or young spherules but were undetectable after exposure to mature spherules. Levels of catalase in mature spherules were too low to account for the low levels of hydrogen peroxide and hypochlorous acid. When mature spherules were added to arthroconidia or zymosan, stimulation of hydrogen peroxide and hypochlorous acid occurred. The results suggest that mature spherules stimulate superoxide release but interfere with hydrogen peroxide and hypochlorous acid production. The low levels of these antimicrobial oxidants may contribute to resistance of C. immitis to human PMNL.

Coccidioidomycosis in llamas in the United States: 19 cases (1981-1989)

Summary Coccidioidomycosis was diagnosed in 19 llamas (California, 9; Arizona, 10). All but 1 had a disseminated form, with pyogranulomas principally in the lungs, thoracic lymph nodes, liver, and kidneys. However, lesions were found in tissue specimens from most organ systems. Antemortem diagnosis was based on radiographic evaluation of the lungs, serologic testing, or presence of spherules of Coccidioides immitis in pyogranuloma aspirates, skin scrapings, or biopsy specimens. Animals residing in endemic areas of southwestern United States are at risk, and llamas appear to be highly susceptible to developing disseminated forms of coccidioidomycosis.

An immunoreactive apoglycoprotein purified from Coccidioides immitis

Deglycosylation of glycoproteins in a lysate of spherules of Coccidioides immitis has permitted purification and partial characterization of a proline-rich pronase-sensitive antigen. Moreover, soluble antigen specifically stimulated lymphocytes from persons with dermal delayed-type hypersensitivity to coccidioidal antigens. When related to reference coccidioidin by tandem two-dimensional immunoelectrophoresis, the antigen fused in the anodal region with a specific reference antigen (antigen 2). It did not show identity with coccidioidal antigens used in conventional serologic assays. Although immunoblots of the purified protein with monospecific rabbit antiserum showed a single antigen at 33 kDa, the parent spherule lysate bound the same antibody in a broad band between 70 and greater than 200 kDa, which could be explained by microheterogeneity of glycosylation. Immunoelectron microscopy using affinity-purified human antibodies localized the antigen to the cell wall and internal septa of spherules. These findings suggest that the apoglycoprotein may be important in human immune responses to coccidioidal infection.

Induction of tumor necrosis factor alpha by spherules of Coccidioides immitis.

The cytokine tumor necrosis factor alpha (TNF-alpha) functions as an immunomodulatory protein and as a mediator of cachexia. We report that viable or Formalin-killed spherules of Coccidioides immitis induced the secretion of TNF-alpha by peritoneal-exudate cells from BALB/c mice. The identification of the cytokine as TNF-alpha was based on its lytic activity against the TNF-alpha-sensitive LS murine fibrosarcoma cell line but not the TNF-alpha-resistant LR cell line, its neutralization by rabbit anti-TNF-alpha, and its secretion by peritoneal cells having characteristics of macrophages. The induction of TNF-alpha was to spherules and not to contaminating lipopolysaccharide (endotoxin), as evidenced by the finding that polymyxin B, a reagent that blocks the TNF-alpha-inducing component of lipopolysaccharide, did not negate the production of TNF-alpha in response to spherules, whereas pretreatment of spherules with hyperimmune goat antiserum to spherulin neutralized the induction of TNF-alpha by these cells. The demonstration that C. immitis activates macrophages to secrete TNF-alpha in vitro is a new finding and warrants studies to determine whether this cytokine is produced during active coccidioidomycosis.

Immunoreactivity of a surface wall fraction produced by spherules of Coccidioides immitis

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.

Extraction of serologic and delayed hypersensitivity antigens from spherules of Coccidioides immitis.

We have used an aqueous toluene extraction procedure to obtain antigens from mature spherules of Coccidioides immitis. This extract contained many antigens as determined by immunoblotting and two-dimensional immunoelectrophoretic studies. These included antigens with specificity for tube precipitin-type antibodies having molecular weights greater than or equal to 100 KDa. The extract also displayed lymphocyte-transforming activity when tested on human peripheral blood mononuclear leukocytes from donors who react to coccidioidal skin tests but elicited no such stimulation of cells from persons whose coccidioidal skin tests were nonreactive. At high concentrations of the extract, lymphocyte transformation did not occur, a finding that could not be explained by nonspecific toxicity. When gel filtration was employed to separate antigens by size, tube precipitin-like activity and specific coccidioidal delayed-type hypersensitivity displayed overlap, although only the latter activity was apparent in lower molecular weight pools.

Isolation and morphology of an immunoreactive outer wall fraction produced by spherules of Coccidioides 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.

Invasive aspergillosis diagnosed by immunohistochemistry with monoclonal and polyclonal reagents

Therapies differ for invasive infection due to the various filamentous fungi. However, histopathologic identification of Aspergillus, Pseudallescheria, Fusarium, Trichosporon, dematiacious Hyphomycetes, Candida, and Zygomycetes may be confused, and microbiologic isolation is often delayed. To improve diagnosis, we examined the utility of polyclonal and monoclonal antibodies to identify Aspergillus organisms by peroxidase immunohistochemical techniques. Tissues obtained from 68 infected patients were examined. Fungal antigen was detected in all 26 cases of aspergillosis by the monoclonal immunohistochemical test. Hyphae of Aspergillus organisms exhibited strong intracellular and cell wall staining, and extracellular localization was also observed. In contrast, negative tests were observed in tissues from five cases of infection with Pseudallescheria boydii, three with Trichosporon, three with Zygomycetes, six with Candida species, and one with Curvularia, from as well as 20 cases of nonfungal pneumonia. However, cross-reactivity to Coccidioides immitis spherules was noted in three cases. Staining of Candida was observed with the polyclonal antibody but not the monoclonal reagent. This is the first immunohistochemical demonstration of aspergillus antigen with a monoclonal reagent. The study indicates that the monoclonal immunohistochemical technique can distinguish Aspergillus species from other filamentous fungi and may facilitate the clinical diagnosis of invasive aspergillosis.

Experience With Needle Biopsy for Coccidioidal Lung Nodules

Three hundred forty-eight patients with solitary pulmonary nodules underwent needle biopsy of the lung in an endemic area for coccidioidomycosis. The purposes of the study were to establish the value of smears and cultures from needle biopsy specimens in the diagnosis of solitary coccidioidal granuloma and to determine how the final diagnosis was established for patients without a clear-cut diagnosis after initial biopsy. Spherules of Coccidioides immitis were initially identified in 49 of the 96 patients with coccidioidomas. Only three of 35 cultures from biopsy-proved coccidioidal lesions were positive (8.6% yield); nevertheless, cultures were cost-effective in nondiagnostic cases. Thoracotomy should be delayed until the results of cultures are known if findings of skin tests, serologic studies, and roentgenography make a benign diagnosis plausible.

Pure spherules of Coccidioides immitis in continuous culture.

Investigation of host-parasite relationships involving the parasitic form of Coccidioides immitis has been difficult because, previously, spherules and endospores have not been grown continuously in tissue culture medium without detectable formation of hyphae. Arthroconidia were harvested from mycelial cultures and inoculated into tissue culture flasks which contained RPMI 1640 medium supplemented with 10% calf serum and N-Tamol (Rohm & Haas Co., Philadelphia, Pa.). Flasks were purged with 5% CO2, sealed, and placed on a reciprocating shaker at 35 degrees C. Hyphae which arose during incubation were removed by filtration. Arthroconidia readily converted to the spherule-endospore form within 12 days. Six days after complete conversion, spherules and endospores were transferred to RPMI 1640 without N-Tamol. The spherule-endospore cycle was maintained in tissue culture medium for 84 days without the formation of detectable hyphae.

Amphotericin B in the treatment of coccidioidomycosis.

In the 25 years since its introduction, amphotericin B has demonstrated clear value in the management of coccidioidomycosis. However, its effectiveness is less certain than in diseases due to other fungal aetiological agents, even when the loci of infection and in vitro drug susceptibilities are identical. The refractoriness of coccidioidomycosis may relate to the unique ability of each Coccidioides immitis spherule to release hundreds of endospores, thus maximally challenging host defence mechanisms. Amphotericin B is most likely to be effective where there is evidence of intact cell-mediated immunity against C. immitis (i.e. positive coccidioidin or spherulin skin test; low titre of complement fixing antibody), and structural damage to tissues. When bones and joints are involved, as is frequently the case, adjunctive surgical management is generally required. Patients with structural lung disease (i.e. cysts and/or cavities) show variable, often minimal, response to treatment. Amphotericin B has transformed coccidioidal meningitis from a routinely fatal disease to one where prolonged survival is possible. However, the drug must be given by the intrathecal route, and for periods of years, before the possibility of cure can be considered. Relapses of bone, joint and meningeal coccidioidomycosis are common and should be anticipated, especially in patients with impaired immunity.

Rapid Diagnosis of Pulmonary Coccidioidomycosis

In 26 patients with culture-proved pulmonary coccidioidomycosis, cytologic preparations of respiratory secretions were significantly more sensitive (38.5%) than potassium hydroxide (KOH) preparations (15.4%) in identifying spherules of Coccidioides immitis. Those patients able to produce sputum or having multiple-lobe pneumonitis on chest roentgenogram appeared to be more likely to have spherules identified in the sputum by either technique. In the absence of sputum production or multiple-lobe pneumonitis, neither cytologic nor KOH preparations were likely to disclose spherules. Because of its greater sensitivity, a cytologic preparation should be examined in addition to a KOH preparation when pulmonary coccidioidomycosis is a clinical consideration.