T-lymphocyte predominance in lesions of canine coccidioidomycosis.

Coccidioidomycosis is a systemic fungal infection endemic to the southwestern United States. Although cell-mediated immunity is considered critical in control of the infection, little is known of the cellular population in naturally occurring lesions. To characterize the lymphocytic infiltration, archived formalin-fixed, paraffin-embedded tissues (subcutis, pericardium/heart, lung, bone, and synovium) from 18 dogs with coccidioidomycosis were studied with immunohistochemistry for CD3 and CD79a. In nearly all lesions, T lymphocytes were more numerous than B lymphocytes and were distributed throughout the lesion with concentration in the periphery of granulomas, whereas B lymphocytes were mostly confined to the periphery of granulomas. The predominance of T lymphocytes in lesions of canine coccidioidomycosis was independent of the tissue evaluated, the number of intralesional organisms, and the nature or severity of the inflammatory response.

Coccidioidomycosis complicating solid organ transplantation.

Coccidioidomycosis is the most common endemic mycosis to cause disease in solid-organ transplant patients in North America. Underlying renal and liver disease, T-lymphocyte suppression from antirejection medication, and activation of immunomodulating viruses, such as cytomegalovirus, all increase the risk for coccidioidomycosis among these patients. About one half of all cases are the result of reactivation of previously acquired coccidioidal infection and occur during the first year after transplantation. Although disseminated infection is common, most cases manifest pulmonary symptoms. Culture of pulmonary secretions from bronchoscopy is frequently diagnostic. Serologic tests are particularly useful for identifying patients who are at high risk for reactivating coccidioidomycosis posttransplantation. Amphotericin B and azoles are the mainstay of therapy. Although there are no established approaches to preventing coccidioidomycosis among these patients, studies are underway examining the use of prophylactic azole antifungals with documented prior coccidioidal infection.

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.

Comparison of oral fluconazole and itraconazole for progressive, nonmeningeal coccidioidomycosis. A randomized, double-blind trial. Mycoses Study Group.

BACKGROUND: In previous open-label noncomparative clinical trials, both fluconazole and itraconazole were effective therapy for progressive forms of coccidioidomycosis. OBJECTIVE: To determine whether fluconazole or itraconazole is superior for treatment of nonmeningeal progressive coccidioidal infections. DESIGN: Randomized, double-blind, placebo-controlled trial. SETTING: 7 treatment centers in California, Arizona, and Texas. PATIENTS: 198 patients with chronic pulmonary, soft tissue, or skeletal coccidioidal infections. INTERVENTION: Oral fluconazole, 400 mg/d, or itraconazole, 200 mg twice daily. MEASUREMENTS: After 4, 8, and 12 months, a predefined scoring system was used to assess severity of infection. Findings were compared with those at baseline. RESULTS: Overall, 50% of patients (47 of 94) and 63% of patients (61 of 97) responded to 8 months of treatment with fluconazole and itraconazole, respectively (difference, 13 percentage points [95% CI, -2 to 28 percentage points]; P = 0.08). Patients with skeletal infections responded twice as frequently to itraconazole as to fluconazole. By 12 months, 57% of patients had responded to fluconazole and 72% had responded to itraconazole (difference, 15 percentage points [CI, 0.003 to 30 percentage points]; P = 0.05). Soft tissue disease was associated with increased likelihood of response, as in previous studies. Azole drug was detected in serum specimens from all but 3 patients; however, drug concentrations were not helpful in predicting outcome. Relapse rates after discontinuation of therapy did not differ significantly between groups (28% after fluconazole treatment and 18% after itraconazole treatment). Both drugs were well tolerated. CONCLUSIONS: Neither fluconazole nor itraconazole showed statistically superior efficacy in nonmeningeal coccidioidomycosis, although there is a trend toward slightly greater efficacy with itraconazole at the doses studied.

The Cryptococcus neoformans gene DHA1 encodes an antigen that elicits a delayed-type hypersensitivity reaction in immune mice.

When mice are vaccinated with a culture filtrate from Cryptococcus neoformans (CneF), they mount a protective cell-mediated immune response as detected by dermal delayed-type hypersensitivity (DTH) to CneF. We have identified a gene (DHA1) whose product accounts at least in part for the DTH reactivity. Using an acapsular mutant (Cap-67) of C. neoformans strain B3501, we prepared a culture filtrate (CneF-Cap67) similar to that used for preparing the commonly used skin test antigen made with C. neoformans 184A (CneF-184A). CneF-Cap67 elicited DTH in mice immunized with CneF-184A. Deglycosylation of CneF-Cap67 did not diminish its DTH activity. Furthermore, size separation by either chromatography or differential centrifugation identified the major DTH activity of CneF-Cap67 to be present in fractions that contained proteins of approximately 19 to 20 kDa. Using N-terminal and internal amino acid sequences derived from the 20-kDa band, oligonucleotide primers were designed, two of which produced a 776-bp amplimer by reverse transcription-PCR (RT-PCR) using RNA from Cap-67 to prepare cDNA for the template. The amplimer was used as a probe to isolate clones containing the full-length DHA1 gene from a phage genomic library prepared from strain B3501. The full-length cDNA was obtained by 5' rapid amplification of cDNA ends and RT-PCR. Analysis of DHA1 revealed a similarity between the deduced open reading frame and that of a developmentally regulated gene from Lentinus edodes (shiitake mushroom) associated with fruiting-body formation. Also, the gene product contained several amino acid sequences identical to those determined biochemically from the purified 20-kDa peptide encoded by DHA1. Recombinant DHA1 protein expressed in Escherichia coli was shown to elicit DTH reactions similar to those elicited by CneF-Cap67 in mice immunized against C. neoformans. Thus, DHA1 is the first gene to be cloned from C. neoformans whose product has been shown to possess immunologic activity.

Genetic transformation of Coccidioides immitis facilitated by Agrobacterium tumefaciens.

Agrobacterium tumefaciens was used to facilitate genetic transformation of Coccidioides immitis. A gene cassette containing the gene encoding hygromycin phosphotransferase (hph) was cloned into a T-DNA vector plasmid and introduced into A. tumefaciens, and the resultant strain was used for cocultivation with germinated arthroconidia. This procedure produced numerous colonies 60- to >500-fold more resistant to hygromycin than untransformed mycelia. Both polymerase chain reaction and Southern blot analysis of the transformants indicated that all contained hph, usually as a single genomic copy. A transformation frequency of 1 per 10(5) arthroconidia was obtained by varying the germination time prior to cocultivation and altering the bacterium: fungus ratio. This approach requires no special equipment that might complicate biocontainment. Furthermore, transformation does not require digestion of fungal cell walls, further simplifying this procedure. A. tumefaciens-facilitated transformation should make possible the development of tagged mutagenesis and targeted gene disruption technology for C. immitis and perhaps other fungi of medical importance.

Practice guideline for the treatment of coccidioidomycosis. Infectious Diseases Society of America.

Management of patients diagnosed with coccidioidomycosis involves defining the extent of infection and assessing host factors that predispose to disease severity. Patients with relatively localized acute pulmonary infections and no risk factors for complications often require only periodic reassessment to demonstrate resolution of their self-limited process. On the other hand, patients with extensive spread of infection or at high risk of complications because of immunosuppression or other preexisting factors require a variety of treatment strategies that may include antifungal therapy, surgical debridement, or both. Amphotericin B is often selected for treatment of patients with respiratory failure due to Coccidioides immitis or rapidly progressive coccidioidal infections. With other more chronic manifestations of coccidioidomycosis, treatment with fluconazole, itraconazole, or ketoconazole is common. Duration of therapy often ranges from many months to years, and, for some patients, chronic suppressive therapy is needed to prevent relapses.

Resistance to Coccidioides immitis in mice after immunization with recombinant protein or a DNA vaccine of a proline-rich antigen.

Two inbred strains of mice (BALB/c and C57BL/6) were vaccinated with either recombinant expression protein of a Coccidioides immitis spherule-derived proline-rich antigen (rPRA) in monophosphoryl lipid A-oil emulsion adjuvant or a DNA vaccine based on the same antigen. Four weeks after vaccination, mice were infected intraperitoneally with arthroconidia. By 2 weeks, groups of mice receiving saline or plasmids with no PRA insert exhibited significant weight loss, and quantitative CFUs in the lungs ranged from 5.9 to 6.4 log10. In contrast, groups of mice immunized with either rPRA or DNA vaccine had significantly smaller pulmonary fungal burdens, ranging from 3.0 to 4.5 log10 fewer CFUs. In vitro immunologic markers of lymphocyte proliferation and gamma interferon (IFN-gamma) release after splenocytes were stimulated with rPRA correlated with protection. Also, plasma concentrations of rPRA-specific total immunoglobulin G (IgG), IgG1, and IgG2a showed increases in vaccinated mice. These studies expand earlier work by demonstrating protection in mice which differ in H-2 background, by using an adjuvant that is potentially applicable to human use, and by achieving comparable protections with a DNA-based vaccine. Our in vitro results substantiate a Th1 response as evidenced by IFN-gamma release and increased IgG2a. However, IgG1 was also stimulated, suggesting some Th2 response as well. PRA is a promising vaccine candidate for prevention of coccidioidomycosis and warrants further investigation.

Coccidioidomycosis: a regional disease of national importance. Rethinking approaches for control.

Coccidioidomycosis is an increasingly important health problem because of the migration of large numbers of persons to portions of the southwestern United States in which the disease is endemic and because of the increasing numbers of immunosuppressed patients. Most infections due to Coccidioides immitis, although causing significant illness, are self-limited and resolve over a period of weeks to months without specific treatment. It is not known whether antifungal treatment of early infections hastens resolution of the primary illness or prevents complications. Even so, diagnosis of early infections is of value for allaying patient anxiety, lessening the need for further diagnostic studies, decreasing empirical use of antibacterial agents, and facilitating early identification of patients with complications that are more serious. Patients who develop chronic coccidioidal pneumonia or extrapulmonary infection often have complicated courses that require the involvement of various medical, surgical, and radiologic subspecialties for management. Improvement of the ability to control the problem of coccidioidomycosis will require research into the molecular and cellular biology of C. immitis, vaccine development to prevent coccidioidal infection, a better understanding of the soil ecology that supports the fungus in its endemic regions, and discovery of new antifungal drugs. In addition, government agencies, colleges, the military, and employers could improve public health by initiating education programs about the most common manifestations of the disease among persons at risk for infection.

Proline-rich vaccine candidate antigen of Coccidioides immitis: conservation among isolates and differential expression with spherule maturation.

A proline rich antigen (PRA), which protects mice against Coccidioides immitis, has been analyzed for differential expression and variation among isolates. Northern blots of RNA from different stages of growth were probed with previously cloned cDNA and showed that mRNA for PRA increased as spherules transformed and matured from mycelia. This pattern corresponds to the relative potency of whole cell vaccines from similar preparations. The PRA gene was then cloned from a genomic library of the Silveira strain of C. immitis and its sequence analyzed. Eight other coccidioidal isolates, selected for diversity in geographic origin and resulting clinical disease, demonstrated heterogeneity in Southern blots and in sequences of polymerase chain reaction products. Silveira differed from other California isolates at 33 of 555 bases, whereas it differed from non-California isolates by <=2 bases. Only one of these substitutions affected protein sequence. Thus, tests or vaccines based on this gene are likely to cover most isolates.

Detecting serum antibodies to a purified recombinant proline-rich antigen of Coccidioides immitis in patients with coccidioidomycosis.

In previous work, antibodies in serum samples from patients with coccidioidomycosis were found to react with a proline-rich antigen (PRA) isolated from spherules of Coccidioides immitis, and the gene encoding this antigen was cloned. We expressed and purified recombinant PRA (rPRA) by removing the majority of amino acids contributed by the vector from the fusion protein. Purified rPRA reacted with serum IgG antibodies in 37 of 42 patients with culture-proven progressive pulmonary or extrapulmonary coccidioidal disease; specific antibodies in dilutions ranging from 1:40 to 1:102,400 were demonstrated (sensitivity, 88%). In contrast, for > 95% of patients without coccidioidomycosis reactivity of < 1:40 was demonstrated (specificity, 97%). Of 18 patients with primary self-limited coccidioidomycosis, none had detectable antibodies in serum samples collected up to 141 days after illness began. The association of antibodies to rPRA with progressive infection may have prognostic value.

Evaluation of the proline-rich antigen of Coccidioides immitis as a vaccine candidate in mice.

We have expressed the proline-rich antigen (PRA) from Coccidioides immitis in Escherichia coli and evaluated its potential as a vaccine candidate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the recombinant protein (rPRA) revealed two bands, which exhibited virtually identical primary amino acid sequences. T cells from rPRA-immunized BALB/c mice showed a significant in vitro proliferative response to rPRA. A small but statistically significant proliferative response was also induced by rPRA in T cells from mice immunized with whole-cell coccidioidal vaccines. BALB/c mice immunized with rPRA and challenged intraperitoneally with virulent C. immitis had a greatly reduced fungal burden in their lungs and spleens compared to unvaccinated mice. The number of organisms in the lungs was reduced 500-fold, and similar reductions were observed in the spleens of immunized mice. These studies support the continued development of rPRA as a candidate vaccine for prevention of coccidioidomycosis.

Comparison of the in vitro activities of the echinocandin LY303366, the pneumocandin MK-0991, and fluconazole against Candida species and Cryptococcus neoformans.

Two new glucan synthesis inhibitors, the echinocandin LY303366 and the pneumocandin MK-0991 (formerly L-743,872), were studied for their antifungal activities in vitro in relation to each other and in relation to the activity of the triazole fluconazole. Systematic analysis of broth macrodilution testing by varying the starting inoculum size, medium composition, medium pH, temperature of incubation, length of incubation, or selection of endpoints failed to identify significant differences in antifungal activity for either LY303366 or MK-0991 in comparison to the activity under standard test conditions specified for other antifungal agents in National Committee for Clinical Laboratory Standards (NCCLS) document M27A. Under standardized conditions, both drugs exhibited prominent activity against Candida species including Candida glabrata and Candida krusei but showed little activity against Cryptococcus neoformans. This spectrum of activity differed from that of fluconazole, which exhibited marginal activity against C. glabrata and C. krusei but prominent activity against other Candida species and C. neoformans. For individual strains, broth microdilution MICs of LY303366 and MK-0991 were similar to but frequently higher than broth macrodilution results. In contrast, fluconazole broth microdilution MICs were often lower than broth microdilution results. We conclude that the test conditions specified in NCCLS document M27A are applicable to these two new glucan synthesis inhibitors and that systematic differences between broth microdilution procedures and the broth macrodilution reference standard will need to be addressed before the two test methods can be used interchangeably.

Invasive Nattrassia mangiferae infections: case report, literature review, and therapeutic and taxonomic appraisal.

We report on a case of subcutaneous infection of the arm caused by the coelomycetous fungus Nattrassia mangiferae (formerly Hendersonula toruloidea) in a steroid-dependent diabetic man with chronic obstructive lung disease. The man was a resident of Arizona, where the fungus is known to be endemic on Eucalyptus camaldulensis and on citrus trees. Diagnosis of fungal infection was made by observation of narrow hyphal filaments by histopathology of biopsy specimens and isolation of a fast-growing black mold which demonstrated hyphae and arthroconidia of varying widths typical of the Scytalidium synanamorph (S. dimidiatum). The formation of pycnidia, which at maturity expressed conidia with a central median dark band, allowed for the confirmation of the isolate as N. mangiferae. Remission of the lesions occurred following intravenous therapy with amphotericin B, followed by topical clotrimazole treatment. We use this patient's case report as an opportunity to review the literature on cases of deep infection caused by Scytalidium species, to evaluate the antifungal susceptibilities of a spectrum of Scytalidium isolates, and to review the taxonomy of Scytalidium species isolated from human infections.

Development of interpretive breakpoints for antifungal susceptibility testing: conceptual framework and analysis of in vitro-in vivo correlation data for fluconazole, itraconazole, and candida infections. Subcommittee on Antifungal Susceptibility Testing of the National Committee for Clinical Laboratory Standards.

The availability of reproducible antifungal susceptibility testing methods now permits analysis of data correlating susceptibility in vitro with outcome in vivo in order to define interpretive breakpoints. In this paper, we have examined the conceptual framework underlying interpretation of antimicrobial susceptibility testing results and then used these ideas to drive analysis of data packages developed by the respective manufacturers that correlate fluconazole and itraconazole MICs with outcome of candidal infections. Tentative fluconazole interpretive breakpoints for MICs determined by the National Committee for Clinical Laboratory Standards' M27-T broth macrodilution methodology are proposed: isolates for which MICs are < or = 8 microg/mL are susceptible to fluconazole, whereas those for which MICs are > or = 64 microg/mL appear resistant. Isolates for which the MIC of fluconazole is 16-32 microg/mL are considered susceptible dependent upon dose (S-DD), on the basis of data indicating clinical response when > 100 mg of fluconazole per day is given. These breakpoints do not, however, apply to Candida krusei, as it is considered inherently resistant to fluconazole. Tentative interpretive MIC breakpoints for itraconazole apply only to mucosal candidal infections and are as follows: susceptible, < or = 0.125 microg/mL; S-DD, 0.25-0.5 microg/mL; and resistant, > or = 1.0 microg/mL. These tentative breakpoints are now open for public commentary.

Analysis of autoantibodies to T-cell receptors among HIV-infected individuals: epitope analysis and time course.

Individuals seropositive for human immunodeficiency virus type 1 (HIV) express elevated levels of autoantibodies (AAbs) directed against recombinant T-cell receptors (TCRs) and synthetic peptide epitopes duplicating beta chain markers. We performed longitudinal studies of anti-TCR AAbs in HIV-1-infected individuals, making comparisons with uninfected sera and sera from other individuals infected with a nonviral agent. We determined levels of autoantibodies by titration using enzyme-linked immunosorbent assay (ELISA) and developed a means for characterizing "autoantibody CDR recognition spectrotypes" for individual sera. Antibody levels against certain defined synthetic epitopes were substantially elevated in HIV-infected subjects relative to reactivities by control groups. Individual sera showed relatively high AAb levels to a subset of CDR1 peptide epitopes. Two patients who subsequently developed AIDS showed particular reactivity to Vbeta2.1, 8.1, 10.1, and 22.1 epitopes. Our results show that production AAbs to TCR Vbeta epitopes is a general consequence of HIV infection. The response is individual but shows some restriction and shifts in AAb subpopulations often occur with time.

In vitro studies of activities of the antifungal triazoles SCH56592 and itraconazole against Candida albicans, Cryptococcus neoformans, and other pathogenic yeasts.

We investigated the effects of various assay conditions on the activities of two antifungal drugs, SCH56592 and itraconazole, against seven species of fungi by the broth macrodilution testing procedure proposed by the National Committee for Clinical Laboratory Standards (NCCLS). For both drugs, which are insoluble in water, the concentration and type of solubilizing agent produced differences in drug activity. Starting inoculum size differences from 10(2) to 10(5) yeast cells per ml resulted in approximately a fourfold effect on the MIC of both drugs, but other significant differences were not observed with variations in synthetic medium composition, pH, buffering reagent, or incubation temperature. Under standardized conditions of reference method M27-T with 1% polyethylene glycol as the solubilizing agent, median MICs of SCH56592 and itraconazole of 60 and 125 mg/ml, respectively, were demonstrated for 110 strains (12 to 23 strains for each of seven species). Broth microdilution results were typically severalfold higher than broth macrodilution results. We conclude that the NCCLS standard reference method can be applied without modification to the testing of SCH56592 and itraconazole, but particular attention to solubilizing the agents is critical to obtaining consistent results.

Microdilution antifungal susceptibility testing of Candida albicans and Cryptococcus neoformans with and without agitation: an eight-center collaborative study.

The growth patterns observed in the trailing wells when fluconazole is being tested may give rise to readings that suggest resistance or increased MICs for known susceptible strains. We conducted a multicenter study to evaluate the intralaboratory and interlaboratory reproducibilities of a method that uses agitation to disperse these types of growth. Ten strains of Candida albicans and five strains of Cryptococcus neoformans were tested against fluconazole, flucytosine, and amphotericin B by using a microdilution adaptation of the proposed reference method of the National Committee for Clinical Laboratory Standards for yeasts (M27-T). The endpoint criterion used before agitation was consistent with the M27-T recommendation, while a criterion of 50% or more reduction of growth compared with the control was used after agitation. The results of this study showed that use of agitation and the modified endpoint criterion both improved intralaboratory and inter-laboratory agreement and increased the frequency of interpretable MICs. The MICs obtained by this method were comparable to those obtained by the broth macrodilution M27-T method. Like M27-T, this method was not able to definitely distinguish amphotericin B-susceptible from -resistant strains, although the MICs for the resistant strains were consistently higher than those for the susceptible ones. The findings imply that agitation should be seriously considered when antifungal agents, particularly fluconazole, are tested in a microdilution format.

Cytokine production by peripheral blood mononuclear cells in human coccidioidomycosis.

The production and mRNA expression of the cytokines interferon-gamma (IFN-gamma) and interleukin (IL)-4, -10, and -12 by peripheral blood mononuclear cells (PBMC) after incubation with the coccidioidal antigen toluene spherule lysate (TSL) from various subjects were measured. The IFN-gamma concentration in PBMC supernatants incubated for 72 h from 8 subjects with disseminated coccidioidomycosis was significantly less than that from 7 healthy, coccidioidal-immune subjects (P = .015). No differences were seen among the subject groups in the concentrations of IL-4, -10, or -12. By use of competitive polymerase chain reaction, PBMC from subjects with disseminated coccidioidomycosis also expressed less mRNA for IFN-gamma and IL-12 than did cells from healthy, immune subjects. These data suggest that patients with disseminated coccidioidomycosis have a diminished T helper lymphocyte type 1 response.