Antigenic analysis of coccidioidin and spherulin determined by two-dimensional immunoelectrophoresis.

Immunological tests are valuable aids for diagnosis of mycotic infections and, in some cases, as objective guides for clinical management and prognosis. The usefulness of these procedures is limited to the extents that crude antigen preparations are employed, that these are difficult to standardize uniformly, and that they contain antigens common to several species of pathogenic fungi. Analysis by two-dimensional immunoelectrophoresis methods of the two crude preparations used for coccidioidomycosis demonstrated that coccidioidin contained at least 26 antigens, with 10 of these found also in spherulin. In addition, spherulin contained two antigens not demonstrated in coccidioidin. No single test detected all antigens present, and multiple procedures were required to display the complete array of antigens. A reference system was established for coccidioidin and precipitated immunoglobulins from a burro hyperimmunized with coccidioidin. Evaluation of the reference system demonstrated that it was highly reproducible with respect to the reagents used, to repeated tests by the same person, and to comparative tests by two individuals using the same reagents. Applications of this reference system for standardization of reagents, for detecting common antigens, for monitoring successive steps during fractionation of crude preparations, and for fingerprinting strains for ecological and epidemiological studies are presented.

Comparison of coccidioidin and spherulin in complement fixation tests for coccidioidomycosis.

Coccidioidin, an extract from the saprophytic mycelial form of Coccidioides immitis, has been a very useful antigen preparation in serological tests for coccidioidomycosis. Its sensitivity has been very good for detecting most types of clinical disease, but tests with coccidioidin have been negative for 40% or more of patients with chronic pulmonary disease, the clinical entity which must be differentiated from other cavitary, nodular, or fibrotic pulmonary disease, e.g., tuberculosis and cancer. The specificity of coccidioidin has also been good although it results in positive tests for an average of 16% among patients with noncoccidioidal mycoses. Recently spherulin, an extract from the parasitic endosporulating spherule form of C. immitis, was reported to be more sensitive than coccidioidin in concurrent complement fixation tests with sera from selected cases. We have compared coccidioidin and spherulin in concurrent complement fixation tests with 614 sera submitted routinely for coccidioidal serology and with 159 selected sera from patients with noncoccidioidal mycoses. Among the former, spherulin was positive with 25% and coccidioidin with 23%, and correlation of titer scores was highly significant. Statistical analysis revealed no significant differences with respect to frequency of positive specimens, titer scores, or diagnosis for current coccidioidomycosis. The results with sera from noncoccidioidal mycoses revealed marked differences. Coccidioidin was positive with 20%, and spherulin was positive with 48%. The titer scores with spherulin were consistently and significantly higher, and there was no correlation for results with the two antigens. Thus, coccidioidin and spherulin were equally sensitive, but spherulin was considerably less specific.

Lung weight parallels disease severity in experimental coccidioidomycosis.

Evidence provided by histopathological study of lesions is a valuable adjunct for evaluating chemotherapeutic efficacy in experimental animal models, In addition, this should be correlated with a measure of disease severity in the same animal. The latter could be obtained by homogenization of infected organs and quantitative enumeration of viable cells of the etiological agent, but this would preclude histopathological studies in the same animal. Progression of disease in pulmonary infection is associated with replacement of air space by fluid, cells, and cellular debris. Therefore, an increase in lung weight should reflect severity of disease. Results with the murine model of coccidioidomycosis demonstrate that increasing lung weight parallels the increasing census of fungus cells in the lungs of both treated and nontreated infected mice. This was supported with evidence obtained from microscopic studies of lesions indicating that specific chemotherapy limited spread of the infection and inhibited multiplication of the fungus in the lung. Therefore, lung weight can be used as a measure of disease severity in the murine model of coccidioidomycosis.

Effect of amphotericin B and rifampin against Coccidioides immitis in vitro and in vivo.

Amphotericin B, the principal drug used for treating systemic mycoses, possesses undesirable toxic properties. The ability of this antibiotic to potentiate antifungal activity of other compounds suggests that lower doses of amphotericin B could be used in combination with a second drug without loss of therapeutic efficacy. In vitro tests demonstrated that amphotericin B potentiated rifampin against the mycelial growth phase of Coccidioides immitis but not against the spherule-endospore phase. Therapy for murine coccidioidomycosis with a combined amphotericin B-rifampin regimen was not better than treatment with amphotericin B alone; in fact, combined drugs may have been even less effective. This would have clinical significance for therapy of concurrent infections.

Rapid in vitro conversion and identification of Coccidioides immitis.

Since many contaminating nonpathogenic fungi resemble Coccidioides immitis culturally, isolates from patients must be inoculated into laboratory animals to confirem identification as C. immitis. This procedure is time-consuming, expensive, and not generally available in clinical laboratories. When cultures of C. immitis were grown in slide cultures on modified Converse liquid medium in purified agar and incubated at 40 C in a candle jar, all 57 isolates demonstrated inhibition of mycelial growth and conversion of arthrospores to endosporulating spherules in 3 to 5 days. Representative isolates of six species of nonpathogenic fungi that resembled C. immitis culturally either did not grow or failed to produce endosporulating spherules under the same conditions. This procedure is recommended for confirming the identication of a culture as C. immitis.

Combined amphotericin B-tetracycline therapy for experimental coccidioidomycosis.

Although amphotericin B is the principal antibiotic for treating systemic mycoses, its clinical use is restricted, primarily because of the toxicity associated with the required prolonged therapy. Other investigators have reported results from in vitro experiments demonstrating that amphotericin B can potentiate antifungal activity of other antibiotics which are ineffective when used alone. In the present study, amphotericin B was used in combination with tetracycline for treating experimental coccidioidomycosis in mice. The results show that the combination of antibiotics is effective with a dosage of amphotericin B reduced 2.5 to 4 times of that required for effective chemotherapy with amphotericin B alone.

Early experience effects upon cortical dendrites: a proposed model for development.

We studied the effects of environmental stimulation on the development of rat cortical pyramidal cell synaptic loci (dendritic spines) and the number of such cells staining by the rapid Golgi technique. Stimulation three to five times a day from the day of birth increased the number of spines per micrometer in 8-day-old animals and increased the number of neurons stanining at 8 to 16 days of age. This effect of afferent input upon development of the dendritic spine may represent the neuroanatomical basis for the influence of early experience on subsequent behavior. The number of neurons staining by the rapid Golgi technique appears to be related to those that are functionally involved at the time of tissue preparation.