Measurement of Cellular Immunity in Human Coccidioidomycosis

Measurement of cellular immune response in human coccidioidomycosis, a disease of the Western Hemisphere caused by the soil-dwelling fungus Coccidioides, began more than 75 years ago. Numerous studies have shown that measurement of coccidioidal cellular immunity is a useful epidemiologic and clinical tool. The first widely used coccidioidal skin-test reagent, coccidioidin, was derived from a filtrate of cultured mycelia of Coccidioides by Smith and colleagues in the 1940's. It remained the standard until the 1970's, when spherulin, obtained from the spherule form of the fungus, was found to be more sensitive. Both reagents are specific indicators of coccidioidal cellular immunity. Since then, other coccidioidal antigens have been identified and in vitro tests of cellular immunity have been shown to yield comparable results to skin testing. In vitro assays have also begun to open a window on the specific immunologic events of coccidioidal cellular immunity. Persistent expression of coccidioidal cellular immune response appears to augur an improved clinical outcome. Despite this, a study of a coccidioidal vaccine failed to demonstrate efficacy. Current and future studies are focused on modulating the coccidioidal immune response in vitro and in vivo and on developing an improved vaccine.     

Coccidioidomycosis.

Coccidioidomycosis is a systemic fungal infection endemic to the southwestern United States and other parts of the western hemisphere. Although producing a wide range of disorders in healthy persons, immunosuppression predisposes to especially severe disease. Thus, a knowledge of the pathogenesis of coccidioidal infections and its relation to the normal immune responses is useful to understand the diversity of problems that Coccidioides immitis may cause. Diagnosis usually requires laboratory studies such as fungal culture or specific serologic testing. Fortunately, many patients do not need to be treated for the infection to resolve. Therapy for the more severe forms of coccidioidal infection was once limited to amphotericin B but now includes azole antifungal agents. These expanded alternatives now require physicians to weigh many factors in determining the best management for specific patients.     

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.     

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.     

COCCIDIOIDIN SKIN TESTING DURING PREGNANCY AND IN INFANTS AND CHILDREN

Results of a study of 220 newborn infants in an endemic area indicate coccidioidal infection is not transmitted congenitally.Skin testing of 595 infants and children disclosed that coccidioidal infection may be acquired at a very early age—as early as one month. The children tested were divided into various age groups, and the incidence of infection increased gradually from the lowest age group to the highest.     

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.     

An Archived Lot of Coccidioidin Induces Specific Coccidioidal Delayed-type Hypersensitivity and Correlates with in vitro Assays of Coccidioidal Cellular Immune Response

No test for assessing cellular immune response in coccidioidomycosis is currently available in the United States. In the present study, we tested 49 healthy subjects living in the coccidioidal endemic region with a 1:55.8 dilution of a single lot of coccidioidin archived since the 1970s. In this group, 23 evaluable subjects demonstrated ≥5 mm of induration at 24, 48 or 72 h, with a mean±SEM maximum induration of 18.4±4.0 mm. The induration results among 14 subjects reactive at 24 h were compared to those from 179 individuals in an historical cohort studied in the 1980s using a reference lot of coccidioidin. Results were within 5% and not significantly different (P=0.924). The maximum induration response of all evaluable subjects correlated significantly with the results of in vitro tests of coccidioidal cellular immunity using supernatant interferon-gamma concentration and CD69 expression on T cells (Spearman rank correlation coefficients 0.69 and 0.68, respectively; P<0.01 for both). These data suggest that archived coccidioidin retains its potency and specificity and that in vitro test of coccidioidal immunity may have utility in the measurement of coccidioidal cellular immunity.     

In vitro modulation of cytokine production by lymphocytes in human coccidioidomycosis

The modulation of the cytokine response to coccidioidal antigen by lymphocytes from donors with coccidioidomycosis was examined. In initial experiments, samples from 13 healthy immune donors and seven donors with active coccidioidomycosis anergic to the coccidioidal antigen T27K were assessed for CD3 lymphocyte expression of intracellular IFN-gamma using whole blood analysis. Addition of 10 ng/ml of recombinant IL-12 significantly increased response to T27K among immune and anergic subjects (p<0.05), but the percent of cells expressing IFN-gamma was still significantly greater for immune subjects. Among immune donors, the percentage of CD3 lymphocytes expressing IFN-gamma was significantly reduced with the addition of 10 ng/ml of recombinant IL-4, IL-10, TGF-beta, or their combination (for all, p<0.05). Among anergic donors, addition of 10 ng/ml of anti-IL-10 significantly increased IFN-gamma production (p<0.05), but addition of anti-IL-4 or anti-TGF-beta did not. Among immune donors, the percent of both CD3 lymphocytes and NK cells expressing IFN-gamma after 24h of T27K was increased above control (p<0.05), while the percent of NK cells producing TNF-alpha in response to T27K was not greater than control. Depletion of NK cells from peripheral blood mononuclear cells resulted in significant increases in TNF-alpha and IL-10 (for both, p<0.05) but resulted in no significant decrease in IFN-gamma or IL-2. These data demonstrate a differential response to stimulation with the coccidioidal antigen T27K among donors with coccidioidomycosis that can be manipulated by cell type and cytokine environment.     

Association of ABO blood group and outcome of coccidioidal infection

Dissemination of fungal infection due to Coccidioides immitis has been previously shown to be related to hereditary factors. Two associations reported to date are race (e.g., Filipino and black ancestry) and HLA histocompatibility type (HLA-19). In the present study of 105 patients a significant association of blood group B and dissemination is demonstrated. C. immitis is known to possess antigens with blood group A activity. Previous epidemiologic studies have also shown HLA-A9 and blood group B are both more common in persons of black and Filipino ancestry. Further studies are needed to define whether these are independent variables, and may define subgroups at particularly high risk following coccidioidal infection.     

Meningeal Coccidioidomycosis Diagnosed by Real-Time Polymerase Chain Reaction Analysis of Cerebrospinal Fluid

We describe two cases of coccidioidal meningitis (CM) diagnosed using real-time polymerase chain reaction (PCR) analysis of cerebrospinal fluid. These cases highlight the promise of PCR as a diagnostic method to assist in the rapid diagnosis of CM.     

Antigens of Coccidioides posadasii as an Important Tool for the Immunodiagnosis of Coccidioidomycosis

Serologic diagnosis has been presented as a safe alternative for coccidioidomycosis. However, commercial kits based on coccidioidal antibodies available in the USA are considered too expensive for laboratories outside that country. In this study, we describe the preparation of antigens for detection of human coccidioidal antibodies by the immunodiffusion test (ID) and enzyme immunoassay (EIA). Antigens were tested against serum samples from patients with coccidioidomycosis, histoplasmosis and paracoccidioidomycosis, as well as healthy individuals. The highest reactivity in the ID tests was seen in the F0-90 antigen. In the EIAs, the best results were obtained with the F60-90 antigen. None of the serum samples from healthy individuals were recognized by any of the antigen extracts tested by ID or EIA. In conclusion, the F0-90 and F60-90 antigens have the potential to be commercially employed in presumptive diagnosis of coccidioidomycosis by ID or EIA, respectively. The tests could improve serological diagnosis of coccidioidomycosis in South America.     

Immunochemical properties of the extracellular hydrolases (protease and alkaline phosphatase) of Coccidioides immitis]

Extracellular hydrolases (protease and alkaline phosphatase) of the coccidioidal fungus possessed antigenic properties and caused production of the corresponding antibodies. Phosphatase-antiphosphatase-substrate system apparently has future prospects for the elaboration of immunobiochemical methods for the diagnosis of coccidioidomycosis.     

COCCIDIOIDAL MENINGITIS—The Use of Amphotericin B in Treatment

Amphotericin B is the first agent to alter favorably the course of coccidioidal meningitis. The morbidity and toxicity of the drug are at present its chief limiting factors.Although no cures were obtained in a series of 11 cases, significant remissions usually followed a course of therapy. Comparison with similar groups showed a significant prolongation of life in adequately treated cases.     

COCCIDIOIDOMYCOSIS AS A COMPLICATION OF PREGNANCY

Records of 33 cases of coccidioidomycosis occurring during pregnancy were reviewed. In this group the incidence of dissemination of the disease was considerably greater than the reported incidence of dissemination in all cases of coccidioidomycosis.The incidence of dissemination was higher in the patients who contracted coccidioidomycosis late in pregnancy than it was in those in whom onset of the disease occurred earlier in gestation; but dissemination occurred in all Negro patients in the group, regardless of the time of onset during pregnancy.The chief complicating effect when onset was in the first trimester of pregnancy was a tendency to abortion. In cases in which onset was in the third trimester, the incidence of premature labor was extremely high.There was no evidence of congenital infection in any of the babies, but in one case invasion of the placenta by coccidioidal spherules was observed.     

Dimorphism in the fungus Coccidioides immitis Rixford et Gilchrist under the action of saprophytic bacilli]

It is shown that there are specific substances produced and secreted into the environment by saprotrophic bacilli. These inhibit the growth of the coccidioidal fungus in its mycelial form and some cells are converted into the yeast form, which leads to the destruction of the fungi (in natural environment) or, if the conditions allow, to their growth in the yeast form. This phenomenon, existence of a large amount of bacilli antagonistic to Coccidioides immitis, may be one of reasons why the latter has not been isolated so far from the soil in the territory of the USSR.     

Liposomal Amphotericin B as Monotherapy in Relapsed Coccidioidal Meningitis

Coccidioidal meningitis remains a difficult clinical problem, and despite life-long therapy with triazole antifungals, relapses of disease and medication intolerance occur necessitating salvage treatment. We report two patients with recurrent coccidioidal meningitis who improved following a 2-week course of liposomal amphotericin B monotherapy and discuss potential advantages of this treatment option.     

Symptoms and routine laboratory abnormalities associated with coccidioidomycosis.

To assess the relationships of various symptoms and other early findings to the diagnosis of primary coccidioidomycosis, we devised a 40-question survey that was completed by 556 college students seeking medical care for illness possibly due to Coccidioides immitis. The results of routine laboratory studies on these patients were also compiled. Of 269 who had coccidioidal antibody determinations and other diagnostic tests, coccidioidomycosis was diagnosed in 36 (13%). By logistic regression procedures, an elevated erythrocyte sedimentation rate, male gender, "red lumps on shins," recent arrival to an endemic area, acuteness of symptoms, and decreased total peripheral blood lymphocyte counts were independent factors positively associated with infection (P less than .05). Relative risk analysis indicated that 60% of patients with four or more of these factors were found to have coccidioidomycosis. Other significantly but not independently associated factors were an increased total leukocyte count, chest pain with breathing, fever, an absence of hoarseness, and an abnormal chest roentgenogram.     

Immunologic and clinical improvement of progressive coccidioidomycosis following administration of transfer factor

Three patients with progressive coccidioidomycosis were given preparations of transfer factor (TF). Adverse reactions to TF were minimal. Following TF administration two of these patients had prolonged clinical remissions in their coccidioidal disease. Cellular immune responses were sequentially evaluated by coccidioidininduced delayed-type skin tests, lymphocyte blast transformation and macrophage inhibition factor production (MIF). These three patients each exhibited different cellular immune patterns before and after TF administration. Two patients converted their coccidioidin skin tests, and one converted lymphocyte transformation response to coccidioidin. Also, TF apparently favorably affected the MIF response in all three patients.     

Coccidioidomycosis

Coccidioidomycosis is an endemic fungal infection whose incidence and impact have been increasing in the southwestern United States. Advances in understanding its epidemiology and mycology are continuing. New diagnostic tests have been developed and experience with newer therapeutic agents continues to accumulate, but numerous clinical questions remain. Timely recognition and diagnosis of coccidioidomycosis are still problematic. With the rise in incidence, changing demographics, an aging population, and the increase in immunocompromised patients, coccidioidomycosis continues to be a fungal infection worthy of further study. This review summarizes the pathogenesis of coccidioidomycosis; the epidemiologic, diagnostic, and therapeutic approaches for coccidioidal infections; and the management challenges of select at-risk populations.     

Intravenous and Intrathecal Miconazole Therapy for Systemic Mycoses

Ten patients with systemic mycoses, including five with fungal meningitis, were treated with intravenously or intrathecally administered miconazole, or both. Minimal inhibitory concentrations of miconazole for clinical isolates of Coccidioides immitis, Cryptococcus neoformans and Candida albicans were less than 0.6 µg per ml. Except for pruritis of variable degrees, the drug was well tolerated both intravenously and intrathecally by all patients. No measurable impairment of renal, hepatic or bone marrow function was observed in patients after 4½ months of intravenous therapy. No hematological or biochemical abnormalities and no evidence of recurrent coccidioidal osteomyelitis were observed in 16 months of follow-up in our first patient treated with this drug. Miconazole is apparently an effective antifungal drug of low toxicity and is a potentially useful agent for treatment of human systemic mycoses.