The Return of Delayed-Type Hypersensitivity Skin Testing for Coccidioidomycosis.

A skin test that detects dermal hypersensitivity in persons with past infection with Coccidioides species is again available for clinical use. Nearly all of the clinical studies with similar materials were published prior to the 1990s, and as a result, many practicing physicians will be unfamiliar with how skin testing for coccidioidomycosis might be useful in patient management or as a research tool. We review clinical and epidemiological studies with past skin test antigens, the composition of past and current skin test preparations with particular attention to differences in the preservatives, and how the current preparation could be used today.

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.

Disseminated coccidioidomycosis with peritonitis in a patient with acquired immunodeficiency syndrome. Prolonged survival associated with positive skin test reactivity to coccidioidin.

Coccidioidomycosis involving the lungs and the meninges occurred as the sole opportunistic infection in a patient with the acquired immunodeficiency syndrome (AIDS). Skin test reactivity to coccidioidin was present, but antibody response to coccidioidal antigens was markedly distinguished. Treatment with amphotericin B, administered intravenously for 3 1/2 months and intrathecally for 13 months, resulted in a disease-free interval of one year. Subsequently, coccidioidal peritonitis developed, which responded to treatment with amphotericin B. However, 29 months after the initial diagnosis, the patient died of complications of hepatic encephalopathy resulting from alcoholic cirrhosis. To our knowledge, this patient represents the first reported case of coccidioidal peritonitis in AIDS and involves the most prolonged survival of a patient with coccidioidomycosis and AIDS. The presence of positive skin test reactivity to coccidioidin may have been a predictor of prolonged survival in this patient.

Clinical usefulness of lymphocyte transformation in patients with coccidioidomycosis.

The development of an appropriate host defense in coccidioidomycosis is predicated on the presence of a positive delayed skin test reaction to coccidioidin. In severe and/or disseminated disease, coccidioidin reactions are routinely negative. By employing serial in vitro spherulin-induced lymphocyte blast transformation (LT) studies in a group of eight severely-ill coccidioidomycosis patients, prognostic clinical data were provided which could not have been obtained from their skin test status alone. Four of the eight demonstrated positive LT responses early in the course of their disease, quickly converted their skin tests to positive, and were cured of their disease. Two patients had negative LT responses until their skin test converted after several months of therapy. The final two have continued to demonstrate negative LT values despite several years of therapy and have experienced exacerbations of their disease when treatment was discontinued. The use of LT data in such patients can be very helpful in guiding therapeutic decisions in this difficult clinical problem.

The maternal immune response in coccidioidomycosis. Is pregnancy a risk factor for serious infection?

Seven subjects with prior coccidioidal disease and three with active Coccidioides immitis infection during their first trimester were studied during pregnancy and postpartum to determine their general and antigen-specific cell-mediated immune status. All ten were white and carried their pregnancies to term without incident. Decreases in total lymphocytes and T-helper and T-suppressor subsets were noted during the third trimester, presumably secondary to an increase in plasma volume. Lymphocyte responses to the mitogens phytohemagglutinin, concanavalin A, and pokeweed were mildly decreased late in pregnancy, with significant intrasubject and intersubject variation. Responses to tetanus antigen were consistently and significantly lower as pregnancy progressed, rising above first trimester levels by 12 weeks postpartum. A similar pattern of response was noted with spherulin antigen for the seven subjects with previously demonstrated coccidioidal immunity. The three subjects with active coccidioidomycosis either failed to mount a significant spherulin immune response or demonstrated an early response that fell as pregnancy progressed. This antigen-specific immune suppression continued for up to 16 months postpartum despite the fact that there was no clinical evidence of coccidioidal activity beyond the first trimester. Thus, while all three completed pregnancy without complication, the data suggest that significantly increased maternal risk may be present when active coccidioidomycosis and pregnancy occur together. This risk may be greatest among darker-skinned individuals who become infected during the latter half of pregnancy.

Antigenemia in primary coccidioidomycosis.

Clinical findings in patients with primary infection due to Coccidioides immitis have suggested that fungal antigenemia may occur. To study this possibility, we devised an inhibition enzyme-linked immunosorbent assay (ELISA) to measure antigenic activity compared to the activity of antigens derived from spherules. Twenty-one of 27 sera from 19 infected patients showed antigenic activity above that found in sera from uninfected persons. Activity was highest early in the course of illness. In other studies, anti-spherulin antibody activity was measured by ELISA, which demonstrated an association of antigen activity with IgM, but not IgG or IgA. One possible explanation for this phenomenon is that IgM antibody and antigen are present as circulating immune complexes.

Paracoccidioidin sensitization in Guyana--a preliminary skin test survey in hospitalized patients and laboratory workers.

Thirty-one percent of a group of 49 hospitalized patients or laboratory workers in Guyana showed positive intradermal paracoccidioidin tests in the presence of negative histoplasmin reactions. In 2 patients (4%), the intradermal reactions to paracoccidioidin were greater than 10 mm in diameter. The prevalence of positive reactors in a selected population suggests that paracoccidioidomycosis may be endemic in Guyana although no clinical case has been reported from the country. A further survey in a larger, unselected population would lead to a clearer understanding of the problem.

Delayed cutaneous hypersensitivity in the dog: reaction to tuberculin purified protein derivative and coccidioidin.

A quantitative skin test for delayed-type hypersensitivity was developed in the dog. The test procedure involved testing animals in the pinna of the ear and quantitating the reaction by measuring the change in ear thickness. Skin test reactions to tuberculin-purified protein derivative and coccidioidin were found to be specific and to correlate with the immunization histories of the 27 dogs tested. Kinetic studies on the tuberculin reaction indicated that ear thickness increased slowly following antigen injection, reaching a peak at about 48 hours. Cellular infiltrates at reaction sites were primarily responsible for the increase in ear thickness. They consisted predominantly of mononuclear cells, although a marked number of neutrophils were also present. Multiple skin tests with tuberculin-purified protein derivative and coccidioidin on 8 nonimmunized (normal) dogs indicated that a skin test was capable of actively sensitizing a portion of the animals tested.

[Histoplasmin and paracoccidioidin reactions in Serra de Pereiro. (Ceara State--Brazil)]. / Reações à histoplasmina e paracoccidioidina na Serra de Pereiro (Estado do Ceará--Brasil).

Intradermal tests using histoplasmin and paracoccidioidin antigens were performed in 138 persons from Pereiro, Ceará, Brazil. The results were positive in 61.5% and 32.5% with histoplasmin and paracoccidioidin antigens respectively. These results suggest infection by H. capsulatum and P. brasiliensis in the people living in the studied area. New studies are necessary to detect histoplasmosis and paracoccidioidomycosis as clinical diseases in that region.

[Intradermal tests and epidemiologic survey of South American blastomycosis]. / Testes intradérmicos e inquerito epidemiológico blastimicose sul americana.

This paper is part of the author's free-teaching theses presented in 1974, in which he has studied 436 persons not carriers of the B.S.A. and other diseases, resident in Campos, Conceição de Macabu and Quissamã (urban and rural zones). Tests were applied in these patients by using standardized antigen of polysaccharide of Paracoccidioides brasiliensis of histoplasmine. The author concluded that there has been existence of the B.S.A.-infection which means what is called "INFEKT". He emphasizes that the epidemiologic researches have important role in the estimation of the incidence of the B.S.A.

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.

Transfer factor: failure to transfer reactivity in normal human subjects.

Transfer factor was prepared from highly selected normal donors. One lot was made from donors strongly reactive to coccidioidin and negative to Dharmendra antigen in in vivo and in vitro testing. The other lot was made from donors without reactivity to coccidioidin and strongly reactive to Dharmendra. Aliquots of each lot were injected into eight normal recipients. Eight additional normal recipients were given placebo injections. Before and after injection, skin test reactivity and in vitro testing were evaluated by an individual who did not know which preparation the patient received. Changes in immunologic reactivity in subjects receiving transfer factor could not be distinguished from those in subjects receiving placebo. I conclude that transfer factor does not cause enhancement of immunologic reactivity in normal subjects. A well designed, critical study is needed to determine whether or not it does, in fact, cause enhancement of immunologic reactivity in patients with impaired cellular immune reactivity.

Antigenic identity of biologically active antigens in coccidioidin and spherulin.

We recently reported the isolation of three clinically relevant antigens from coccidioidin; viz., the antigen that is reactive in the immunodiffusion (ID) assay for detecting tube precipitin (TP) antibody (designated IDTP); the antigen that is reactive in the ID assay for detecting complement-fixing antibody (designated IDCF); and the heat-stable (HS) antigen which, when demonstrated in soluble extracts of fungal cultures by using the IDHS assay, establishes the mycologic identification of Coccidioides immitis. This investigation was undertaken to determine whether the IDTP, IDCF, and IDHS antigens isolated from coccidioidin are of antigenic identity with components in spherulin. By employing the purified coccidioidin antigens in line-immunoelectrophoresis with spherulin and by assaying coccidioidin and spherulin by tandem two-dimensional immunoelectrophoresis against antisera produced to the purified antigens, we report that these three coccidioidin-derived antigens are antigenically identical to precipitinogens in spherulin.

An evaluation of reactivity to Coccidioides immitis skin tests in subjects with diabetes mellitus.

Persons with diabetes mellitus have an increased incidence of progression and dissemination of infection with Coccidioides immitis. This increased incidence of infection suggests that diabetic subjects may have a defect in cell-mediated immunity (CMI) to coccidioidomycosis. Skin test response is also determined by CMI. A negative coccidioidin skin test is often seen with serious infections and is used as evidence that therapy should be initiated. To evaluate CMI in diabetic subjects we investigated the reactivity to coccidioidin and spherulin of 54 diabetic and 56 nondiabetic subjects. There was no difference between the two groups in the prevalence or size of positive reactions to coccidioidin. These results support the continued use of coccidioidin to evaluate the need for therapy in diabetic subjects who have coccidioidomycosis. Spherulin antigen, however, produced fewer positive reactions in the diabetic group. This decreased spherulin reactivity may be associated with the increased incidence of severe coccidioidal infection in diabetic subjects.

Cutaneous responses to antigens and irritants in patients of leprosy.

Forty seven patients of various types of leprosy were skin tested with PPD, Dharmendra lepromin, DNCB, Coccidiodin, histamine and croton oil. Twenty five age matched normal controls were also included in the study. All types of leprosy patients reacted in smaller numbers and with decreased response to all antigens and irritants as compared to normal controls. Depression of response being minimum in tuberculoid and maximum in lepromatous group. None of the patients or controls reacted to coccidiodin. Details are given.