An Analysis of Skin Test Responses to Spherulin-Based Coccidioidin (Spherusol®) Among a Group of Subjects with Various Forms of Active Coccidioidomycosis.

BACKGROUND: A reformulated skin test for coccidioidomycosis, Spherusol®, was recently approved for use in the USA. We hypothesized that it could be useful in predicting severity of illness and outcome in various types of coccidioidomycosis. METHODS: Subjects with non-meningeal coccidioidomycosis attending a clinic in the coccidioidal endemic region were skin tested with Spherusol® and clinical data were collected at the time of testing and at follow-up. RESULTS: Twenty-seven subjects were studied, eight of whom had extrathoracic dissemination. A total of 15 subjects had positive tests, including 11 of 19 subjects with non-disseminated pulmonary disease and four with extrathoracic disseminated coccidioidomycosis. Among those with non-disseminated pulmonary disease, age ≥ 65 years, female sex, and antifungal therapy were significantly associated with a negative test on univariate but not multivariate analysis. For 23 subjects, there was a trend for those not on antifungal therapy at the time of follow-up to have a positive test but no association with coccidioidal complement-fixation titer or overall outcome. CONCLUSIONS: Not all subjects with non-disseminated pulmonary coccidioidomycosis were found to be skin test positive and half of those with extrathoracic disseminated disease manifested dermal hypersensitivity. In this small study, the results of the skin test were not clinically predictive of disease severity or outcome.

A reformulated spherule-derived coccidioidin (Spherusol) to detect delayed-type hypersensitivity in coccidioidomycosis.

The ability of spherule-derived coccidioidin containing 0.4 % phenol and 0.0001 % thimerosal in buffered saline to induce delayed-type hypersensitivity (DTH) was evaluated in four separate studies. The skin test antigen was titrated in 20 adult volunteers with a recent history of pulmonary coccidioidomycosis using intradermal doses of 0.4, 0.8, and 1.6 µg of antigen, based on total dry weight. Based on these data, a dose of 1.27 µg was shown to elicit a mean ± SEM induration response of 23.5 ± 2.3 mm at 48 h, similar to the 23.6-mm response after 48 h of the U. S. Reference coccidioidin last tested approximately 13 years ago. The 1.27 µg dose in 0.1 mL of the spherule-derived antigen (Spherusol) was then examined in three separate groups of adult volunteers to determine the sensitivity and specificity of the product. Fifty-nine of 60 individuals living in a non-endemic area for coccidioidomycosis were skin test negative to Spherusol. Twelve subjects with a recent history of pulmonary histoplasmosis were skin test negative to Spherusol. Finally, 51 of 52 individuals with a recent diagnosis of acute pulmonary coccidioidomycosis were skin test positive to Spherusol. Within this group, prior therapy with fluconazole did not appear to reduce the reactivity to Spherusol. No serious adverse events were observed in the four studies. From these data, Spherusol was found to be safe and has an overall observed sensitivity and specificity of ≥ 98 % in detecting DTH in coccidioidomycosis.

Clinical comparison of two Mexican coccidioidins.

Coccidioidin, an extract from the saprophytic mycelial form of Coccidioides spp., has been a very useful antigen preparation both for skin and serological tests for coccidioidomycosis. Unfortunately, coccidioidin is not currently available for skin testing in the United States. Coccidioidin has been produced commercially in Mexico by a vaccine and reagents laboratory of the Mexican Federal Government. It also has been produced at the Microbiology Laboratory of the Faculty of Medicine, Universidad Nacional Autónoma de México exclusively as an antigen for research projects. The objective of the study was to compare both coccidioidins in their reactivity and safety when applied in humans. One hundred and eighty-four volunteers were tested; median age was 33 (range 14-82). When the cutoff point is set in 5 mm, 88 subjects (47.8%) had a positive test for the commercial coccidioidin and 76 (41.3%; CI(95%) 0.50, 1.15; P = 0.20) were positive with the research antigen. Seventy-five subjects were positive for both antigens and 96 were negative for both. Fifty-nine subjects (31.3%) reported an adverse reaction after the application of the antigen; they were mostly very mild local reactions. Mexican research coccidioidin is a safe and reliable antigen that can be used for the detection of coccidioidomycosis infection in mammals.

Specificity of immunoglobulin E in coccidioidomycosis and correlation with disease involvement.

Serum immunoglobulin E (IgE) antibodies were quantitated in 26 patients with active pulmonary coccidioidomycosis, 59 patients with active disseminated disease, 12 patients in clinical remission, and 91 healthy subjects. Significant differences were obtained in IgE serum levels of patients with active disease versus healthy subjects (P less than 0.0001). Patients with pulmonary coccidioidomycosis did not differ in their IgE levels when compared with patients with disseminated disease. However, serum IgE levels were significantly increased in patients with disease involving two or more organ systems when compared with patients with pulmonary disease or extrapulmonary disease involving a single organ system (P less than 0.02). Total serum IgE correlated with anti-Coccidioides IgE (P less than 0.001), but with only six exceptions, patients with anti-Coccidioides IgE also exhibited IgE antibodies to 1 or more of 12 common allergens. The correlation between hyperproduction of IgE and disease severity coupled with the depressed cell-mediated immune status of patients with this disease suggests a defect(s) in the T-lymphocyte population which functions to regulate IgE synthesis.

In vivo and in vitro cell-mediated responses in coccidioidomycosis. I. Immumologic responses of persons with primary, asymptomatic infections.

Chest roentgenograms of 58 children who were skin test positive to coccidioidin and resided in an area endemic for coccidioidomycosis revealed that 34 per cent had roentgenographic evidence of an inflammatory process, 14 per cent showed calcific densities, and 52 per cent showed no evidence of infection. The in vitro lymphocyte responses of children who had evidence of an inflammatory process (Group I) were compared with those of children who had calcific densities (Group II); those of children who were coccidioidin skin test negative and had normal chest roentgenograms (Group III); and those of patients who had active coccidioidomycosis (Group IV). The mean lymphocyte transformation responses (expressed as cpm times 10-(4)) of Groups I, II, III, and IV to a coccidioides antigen were 16.8, 19.5, 4.2, and 7.0, respectively. The mean migration inhibitory factor responses of these groups were 22.4, 20.0, 1.2, and 4.0 per cent, respectively. Thus, the over-all responses of children in Groups I and II were comparable to each other, whereas the responses of patients in Group IV were depressed to the extent that they were indistinguishable from those of coccidioidin skin test-negative donors in Group III. Follow-up chest roentgenograms taken 3 months after the immunologic assays were performed revealed that the one subject in Group I who had been nonresponsive in the lymphocyte assays had now stabilized his infection, as evidenced by calcifications. In contrast, the 2 subjects in Group I who had yet to stabilize their infection had exhibited strong in vitro lymphocyte responses. These findings suggest that primary, asymptomatic coccidioidomycosis is not associated with an immunologically nonresponsive state. However, patients with active, progressive coccidioidomycosis do have a depressed immunologic response to coccidioides antigens.

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.

Cutaneous sensitivity and in vitro responsiveness of lymphocytes in patients with disseminated coccidioidomycosis.

Sixty-eight of 74 patients with disseminated coccidioidomycosis demonstrated cutaneous reactivity to intradermal injectionof coccidioidin. Lymphocytes of all patients with positive skin test results underwent blast transformation in vitro in response to coccidioidin. No correlation was found between the size of skin test induration and the magnitude of in vitro lymphocyte response. Six patients with disseminated disease failed to demonstrate cutaneous reactivity to coccidioidin; however, lymphocytes of three of these patients responded with blast transformation in vitro. The lymphocyte reactivity of two of these three patients was abrogated by addition of autologous plasma to the culture medium. Autologous plasma did not block the patient's in vitro response to phytohemagglutinin or allogeneic lymphocytes, and blocking did not appear to be attributable to direct neutralization of coccidioidin.