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PubMed1999

Delayed-type hypersensitivity, in vitro T-cell responsiveness and risk of active coccidioidomycosis among HIV-infected patients living in the coccidioidal endemic area.

The prognostic importance of specific and general tests of immune function were examined among a cohort of 170 subjects infected with human immunodeficiency virus type-1 (HIV), living in an area endemic for the fungal infection coccidioidomycosis. Using the proportional hazards model and multivariate analysis, lack of expression of coccidioidal delayed-type hypersensitivity (DTH) was found to be dependent on anergy in response to the non-coccidioidal antigens mumps, Trichophyton and Candida (relative hazard 4.2, 95% CI 1.8-9.8, P=0.001). Among subjects with CD4 lymphocyte counts >/=250 microl-1 on entry into the study, the in vitro lymphocyte transformation (LT) response to the coccidioidal antigen toluene spherule lysate was 4967+/-1652 (mean counts per minute (c.p.m.)+/-SEM) in subjects with coccidioidal DTH compared with 136+/-222 in those with negative DTH (P<0.001). However, amongst those whose CD4 count was <250 microl-1, LT responses were low and there was no significant difference based on coccidioidal DTH (P=0.965). Using the proportional hazards model and multivariate analysis, only a CD4 count <250 microl-1 was prognostically associated with the development of either active coccidioidomycosis or AIDS. These data indicate that immunodeficiency, particularly a CD4 lymphocyte count <250 microl-1, is the most important factor in the lack of expression of specific immunity to coccidioidomycosis and in the development of active coccidioidomycosis among HIV-infected individuals living in the coccidioidal endemic area.

Arizona↗
PubMed2006

Phenotypic characterization and ecological features of Coccidioides spp. from Northeast Brazil.

This study extends phenotypic and ecological knowledge of Coccidioides spp., by describing its recovery from soils of Ceará State (Northeast Brazil) and analyzing the in vitro features of the growth of its vegetative phase. Following a human coccidioidomycosis case, Coccidioides spp. strains were isolated from 3 of 14 soil samples collected in an armadillo's burrow. Mycological analysis showed colonies with glabrous, velvety or cottony texture and an increasing quantity of arthroconidia. The overall growth rates of the strains were slower in 8% NaCl medium, maximum growth rate was obtained at 30 degrees C, and their pH tolerance ranged from 4.0 to 11.0. Several carbohydrates and polyalcohol sources could be efficiently metabolized by Coccidioides spp. strains in the mycelial form. Total absence of growth was observed in media supplemented with either L-aspartic acid or L-histidine. Whereas intense growth was found when strains were incubated with any other aminoacid sources studied. Coccidioides spp. strains did not grow in the presence of Tween 60 and Tween 80, but exhibited intense growth in Tween 20. Nicotinic acid and the toxic compounds caffeic acid and phenol could not be metabolized by any strain. All of the strains were positive for urease production and displayed intense growth in media containing cycloheximide concentrations ranging from 0.01 and 0.05%, but did not grow at 0.1 and 0.2%. The present findings confirm the importance of armadillos burrows in the ecology of Coccidioides spp. in Northeast Brazil and indicate that the fungus is a very physiologically versatile organism.

Animals↗
PubMed2001

Dendritic cells pulsed with Coccidioides immitis lysate induce antigen-specific naive T cell activation.

Coccidioidomycosis, an infection endemic to the southwestern United States, is caused by the fungus Coccidioides immitis. Coccidioidal infection is overcome by the development of cell-mediated immunity. This study evaluated the role of dendritic cells (DCs) in the initiation of coccidioidal immunity in nonimmune individuals. It was demonstrated that DCs pulsed with the coccidioidal antigen preparation, toluene spherule lysate (TSL), induce DC maturation, autologous lymphocyte proliferation, and antigen-specific lymphocyte responses from nonimmune donors. Furthermore, TSL-primed lymphocytes secreted interferon-gamma after restimulation with TSL or antigen 2/proline-rich antigen, a subcomponent of TSL, but they did not do so when restimulated with ovalbumin or unpulsed DCs. The results demonstrate that DCs generated from individuals not exposed to C. immitis can specifically prime lymphocytes for coccidioidal antigens and that the response generated by the lymphocytes is characteristic of a cellular immune response.

Antigens, Fungal↗
PubMed1996

Use of a recombinant Coccidioides immitis complement fixation antigen-chitinase in conventional serological assays.

The coccidioidal complement fixation (CF) antigen has been cloned previously, and the fusion protein has been expressed in Escherichia coli. The recombinant CF (rCF) antigen was affinity purified by adsorption-desorption to chitin, and its reactivity was studied by using sera containing coccidioidal antibodies. The affinity-purified rCF antigen formed a line of identity with an immunodiffusion (ID) CF reference antigen (coccidioidin) derived from mycelial-phase Coccidioides immitis and was reactive with human, canine, and equine sera containing coccidioidal antibody. The affinity-purified rCF antigen yielded no detectable reaction with Blastomyces of Histoplasma antiserum by ID. The affinity-purified rCF antigen fixed complement with positive human sera and, even when used at lower concentrations, yielded titers comparable to those obtained with the coccidioidin. The reactivity of the affinity-purified rCF antigen was further evaluated by enzyme immunoassay, in which it manifested good sensitivity (96.9%) and specificity (100%) when evaluated with 43 human patients' sera. Thus, the affinity-purified rCF antigen has yielded reactions comparable to those of crude coccidioidal antigens in conventional CF, IDCF, and enzyme immunoassay.

Animals↗
PubMed1986

Development of dermal hypersensitivity to coccidioidal antigens associated with repeated skin testing.

Four antigens prepared from Coccidioides immitis were found to evoke no dermal reactions in 1,245 volunteers on first testing, but 2 months later, upon retesting, 18.6% of the subjects reacted to 1 or more of the antigens. In no subject did other findings indicate an intercurrent coccidioidal infection. Conversion of a skin test to reactive significantly (p less than 0.018) increased with subject age. No association was found with either years of residence within areas endemic for C. immitis or occupational exposure, associations that would be expected if conversion was due to latent hypersensitivity from a prior coccidioidal infection. Possible explanations for our findings include either boosting of immunity to a noncoccidioidal stimulus or primary sensitization from the skin testing itself. Until this phenomena is understood more fully, physicians should be reluctant to accept interval conversion of coccidioidal skin tests as a means of diagnosing recent coccidioidal infection.

Adolescent↗
PubMed2002

Reversal of coccidioidal anergy in vitro by dendritic cells from patients with disseminated coccidioidomycosis.

Coccidioides immitis is a pathogenic, dimorphic fungus found in the southwestern United States and is the causative agent of coccidioidomycosis. Extrathoracic dissemination of coccidioidomycosis is associated with a lack of cellular immunity. Dendritic cells (DCs) have been shown to initiate and modulate cellular immune responses. To determine whether DCs could modulate or initiate the immune response in this disease, monocyte-derived DCs were generated from coccidioidal Ag nonresponsive patients with disseminated coccidioidomycosis and healthy nonimmune individuals. DCs generated from both groups demonstrated phenotypes characteristic of DCs and stimulated strong allogeneic MLR. DCs from patients and healthy nonimmune individuals pulsed with the coccidioidal Ag preparation T27K induced lymphocyte proliferation. Mature DCs were much more efficient than immature DCs in these stimulations. Furthermore, restimulation of T27K-primed PBMC with Ag-pulsed DCs generated a C. immitis-specific cellular immune response in PBMC from patients with disseminated coccidioidomycosis as well as healthy nonimmune individuals. These results show that 1) DCs have the capacity to stimulate specific cellular immune responses from patients with disseminated coccidioidomycosis who are nonresponsive to coccidioidal Ag and healthy nonimmune individuals in vitro; 2) DCs can be used to screen coccidioidal Ags as candidates for human vaccine development; and 3) DC therapy may be useful in the treatment of disseminated coccidioidomycosis.

Antigens, Fungal↗
PubMed2004

Endocarditis caused by Coccidioides species.

Endocarditis caused by Coccidioides species has been reported rarely. Herein, we report 2 such cases and summarize 4 published reports. Coccidioidal endocarditis was found in patients who had prolonged or disseminated infection. Vegetations were identified on mitral or aortic valves or valvular rings, and embolic phenomena were observed. Diagnosis was hindered by uniformly negative results from blood cultures. The patients had a wide range of serologic titers for Coccidioides species (1:1-1:2048). The infection was fatal in 4 of the 6 patients whose cases we reviewed. We conclude that coccidioidal endocarditis is an uncommon manifestation of Coccidioides infection that connotes a poor prognosis.

Adult↗
PubMed1977

Unusual manifestation of Coccidioides immitis infection.

Cavitary pulmonary disease secondary to coccidioidomycosis occurs in endemic areas of the southwestern United States. Significant hemoptysis requiring pulmonary resection may develop in patients with cavitary lesions. In rare instances hemoptysis may be due to the development of a fungus ball within the cavity, and such mycetomas may contain spherule and mycelial forms of Coccidioides immitis. A patient with a documented coccidioidal cavity who had hemoptysis is described in this report. Chest radiograph revealed a fungus ball within a left lower lobe cavity. The patient underwent left lower lobectomy and recovered without complications. Examination of the resected left lower lobe disclosed a cavity containing a fungus ball comprised of mycelial and spherule forms of Coccidioides immitis. A review of the literature found three reports of biphasic growth of this condition within a coccidioidomycosis cavity and two additional cases of radiographically diagnosed coccidioidal mycetoma. Discussion of the potentially contagious nature, the medical and surgical management, and the prognosis of coccidioidal mycetoma form the basis of this report.

Adult↗
PubMed1991

Interaction of human peripheral blood mononuclear cells with Coccidioides immitis arthroconidia.

We explored the in vitro interaction of human peripheral blood mononuclear cells with the arthroconidial stage of the fungus Coccidioides immitis. Fresh peripheral blood monocytes in an adherent monolayer were capable of ingesting C. immitis. Further, peripheral blood monocytes from either skin-test-positive or skin-test-negative donors significantly decreased the in vitro growth of C. immitis when coccidioidal arthroconidia were incubated with monocytes. Peripheral blood mononuclear cells also reduced fungal incorporation of the chitin precursor N-acetyl glucosamine. Cell fractions consisting predominantly of monocytes were significantly more active in this regard than fractions containing predominantly lymphocytes. Moreover, this activity was independent of the coccidioidal skin-test status of the donor. We conclude that human fresh peripheral blood mononuclear cells are able to phagocytize C. immitis arthroconidia and have the ability to inhibit its growth in vitro. That these abilities are independent of the immune status of the donor supports the possibility that the peripheral blood monocyte may contribute to the early defense against initial coccidioidal infection.

Acetylglucosamine↗
PubMed1988

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.

Animals↗
PubMed1975

Delayed-type hypersensitivity responses to a cell wall fraction of the mycelial phase of Coccidioides immitis.

A skin test-active fraction was isolated from the mycelial-phase cell walls of Coccidioides immitis. This alkali-soluble, water-soluble antigen (C-ASWS) elicited positive reactions in 22 of 24 (92%) of the Coccidioides-sensitized guinea pigs whereas only 14 (54%) of the same guinea pigs reacted to commercial coccidioidin (BioCox). None of the 21 Histoplasma-sensitized guinea pigs cross-reacted with the C-ASWS antigen. Footpad tests in mice actively infected with Coccidioides further established the efficacy of the C-ASWS antigen in eliciting a delayed-type hypersensitivity response. One-microgram doses of C-ASWS produced reactions comparable to 100-mug doses of nondialyzable coccidioidin (Smith's lot 64 D4). The C-ASWS fractions isolated from three different C. immitis strains showed similar reactivity in terms of the number of positive reactions produced in Coccidioides-sensitized guinea pigs. However, the induration responses (diameter in millimeters) elicited by the C-ASWS fraction of one strain were significantly less than those elicited by the C-ASWS fractions of the other two C. immitis strains.

Administration, Intranasal↗
PubMed1991

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.

Antigens, Fungal↗
PubMed1992

Killing of Coccidioides immitis by human peripheral blood mononuclear cells.

The ability of human peripheral blood mononuclear cells (MNL) obtained from healthy donors to kill the fungus Coccidioides immitis was examined in vitro with an assay that uses a single fungal particle per well. MNL killed 25.0% +/- 3.5% of a coccidioidal arthroconidial target, compared with the 4.7% +/- 2.9% killed by polymorphonuclear leukocytes obtained from the same donors (P = 0.012). Arthroconidial killing by MNL was not dependent on donor delayed dermal hypersensitivity to spherulin. Killing of another fungal target, Candida glabrata, was not significantly different between MNL and polymorphonuclear leukocytes (P = 0.783). Depletion of monocytes from MNL with Sephadex G-10 resulted in a significant reduction in arthroconidial killing (21.4% +/- 13.6% versus 2.4% +/- 3.4%; P = 0.025), while enrichment of monocytes by Percoll density gradient centrifugation or plastic adherence resulted in significantly increased arthroconidial killing compared with that by MNL (P = 0.005 and 0.001, respectively). Killing of 96-h spherules by MNL was 7.3% +/- 3.1%, significantly less than the 21.4% +/- 2.8% killing of arthroconidia in the same experiments (P = 0.016). Incubation of MNL with human recombinant gamma interferon or tumor necrosis factor alpha did not result in increased MNL killing of coccidioidal arthroconidia under various conditions. These results suggest that MNL have an inherent ability to kill coccidioidal arthroconidia in vitro which is not dependent on prior host exposure to C. immitis. This activity appears to reside in peripheral blood monocytes.

Coccidioides↗
PubMed2006

Urease produced by Coccidioides posadasii contributes to the virulence of this respiratory pathogen.

Urease activity during in vitro growth in the saprobic and parasitic phases of Coccidioides spp. is partly responsible for production of intracellular ammonia released into the culture media and contributes to alkalinity of the external microenvironment. Although the amino acid sequence of the urease of Coccidioides posadasii lacks a predicted signal peptide, the protein is transported from the cytosol into vesicles and the central vacuole of parasitic cells (spherules). Enzymatically active urease is released from the contents of mature spherules during the parasitic cycle endosporulation stage. The endospores, together with the urease and additional material which escape from the ruptured parasitic cells, elicit an intense host inflammatory response. Ammonia production by the spherules of C. posadasii is markedly increased by the availability of exogenous urea found in relatively high concentrations at sites of coccidioidal infection in the lungs of mice. Direct measurement of the pH at these infection sites revealed an alkaline microenvironment. Disruption of the urease gene of C. posadasii resulted in a marked reduction in the amount of ammonia secreted in vitro by the fungal cells. BALB/c mice challenged intranasally with the mutant strain showed increased survival, a well-organized granulomatous response to infection, and better clearance of the pathogen than animals challenged with either the parental or the reconstituted (revertant) strain. We conclude that ammonia and enzymatically active urease released from spherules during the parasitic cycle of C. posadasii contribute to host tissue damage, which exacerbates the severity of coccidioidal infection and enhances the virulence of this human respiratory pathogen.

Animals↗
PubMed2006

Spherules derived from Coccidioides posadasii promote human dendritic cell maturation and activation.

Previous studies have shown that dendritic cells (DC) pulsed with T27K, an antigenic preparation derived from spherules (of Coccidioides posadasii), activate peripheral blood mononuclear cells (PBMC) from nonimmune subjects as well as from patients with disseminated coccidioidomycosis. In this study, we have assessed the interaction between human DC and C. posadasii spherules in order to better understand the initial response between Coccidioides and the human host. Whole autoclaved spherules induced lymphocyte transformation in PBMC obtained from immune but not from nonimmune donors. Immature DC (iDC) bound fluorescein isothiocyanate-labeled spherules in a time- and temperature-dependent manner. This binding was blocked by the addition of mannan, suggesting mannose receptor involvement in the DC-Coccidioides interaction. Binding was subsequently associated with ingestion and intracellular processing of spherules. Coculturing of spherules with iDC was associated with the development of mature DC that were morphologically, phenotypically, and functionally similar to those induced by tumor necrosis factor alpha and prostaglandin E2. Finally, spherules incubated with iDC induced activation of PBMC from nonimmune donors. These data indicate that human DC are capable of binding, internalizing, and presenting antigens from Coccidioides spherules and suggest that DC may play a critical early role in the formation of a cellular immune response in human coccidioidomycosis.

Cell Adhesion↗
PubMed2006

Identification of Histoplasma capsulatum, Blastomyces dermatitidis, and Coccidioides species by repetitive-sequence-based PCR.

The performance of repetitive-sequence-based PCR (rep-PCR) using the DiversiLab system for identification of Coccidioides species, Blastomyces dermatitidis, and Histoplasma capsulatum was assessed by comparing data obtained to colony morphology and microscopic characteristics and to nucleic acid probe results. DNA from cultures of 23 Coccidioides, 24 B. dermatitidis, 24 H. capsulatum, 3 Arthrographis, and 2 Malbranchea isolates was extracted using a microbial DNA isolation kit as recommended by Bacterial Barcodes, Inc. Rep-PCR and probe results agreed for 97.2% of the dimorphic fungi when > or =85% similarity was used as the criterion for identification. Two H. capsulatum isolates were not identified, but no isolates were misidentified. From 43 of those cultures (15 Coccidioides, 14 B. dermatitidis, 14 H. capsulatum, 3 Arthrographis, and 2 Malbranchea), DNA also was extracted using an IDI lysis kit, a simpler method. Rep-PCR and probe results agreed for 97.7% of the dimorphic fungi when a criterion of > or =90% similarity was used for identification. One H. capsulatum isolate could not be identified; no isolates were misidentified. Using > or =85% similarity for identification resulted in one misidentification. These data suggest that the DiversiLab system can be used to identify Coccidioides and B. dermatitidis and, possibly, H. capsulatum isolates.

Blastomyces↗
PubMed2004

Amplification of coccidioidal DNA in clinical specimens by PCR.

Coccidioides DNA was amplified from serum by a PCR using coccidioid-specific primers. A 239-bp product was visualized when 10 fg of exogenous coccidioidal DNA was subjected to amplification. This product was demonstrated in some human and mouse sera prior to the detection of coccidioidal antibodies.

Animals↗
PubMed1975

Coccidioides immitis. A rare cause of pulmonary mycetoma.

Coccidioides immitis occurring as a fungus ball within a chronic pulmonary cavity has previously been reported twice and must be rare. We report a mycetoma composed of both spherule and mycelial forms of Coccidioides immitis, proved by culture and histologic sections. Fungus ball due to Coccidioides immitis is a logical progression from mycelia seen in the cavity wall in the active pulmonary coccidioidal lung cavity.

Coccidioides↗