Evaluation of five commercially available immunodiffusion kits for detection of Coccidioides immitis and Histoplasma capsulatum antibodies.

Five commercial test kits for the serodiagnosis of coccidioidomycosis and histoplasmosis based upon immunodiffusion were evaluated. The correlation of results with the test kits in the Clinical Laboratory varied from 71 to 100% for coccidioidomycosis. The correlation for coccidioidomycosis immunodiffusion testing varied from 57 to 83% when results from the test kits and the Mycology Research Laboratory were compared. Only 81% correlation was noted between the two laboratories when the same reference system was used. Results with the test kits for Histoplasma serodiagnosis and results from the Mycology Research Laboratory showed a correlation of 52 to 75%. There were no false-positive results with any system. All of the commercial kits were 100% specific for the diagnosis of both coccidioidomycosis and histoplasmosis, but the sensitivity of the immunodiffusion tests varied with the system used.     

Cross-Reactivity Between Antigens of Coccidioides immitis, Histoplasma capsulatum, and Blastomyces dermatitidis in Lymphocyte Transformation Assays

The kinetics of phagocytosis and killing of four fungal forms with varying virulence by two types of phagocytic cells was examined. Human monocytes ingested Saccharomyces cerevisiae, Candida tropicalis, and the blastospores of Candida albicans more rapidly than did human neutrophils. There was no difference in the rate of phagocytosis of C. albicans pseudohyphae by these two cell types. Intracellular killing of each of the four fungal forms was consistently and significantly more rapid by monocytes than by neutrophils. Neutrophils were unable to destroy ingested C. albicans pseudohyphae. These experiments suggest that the monocyte plays an important role in host defenses against fungal diseases and that the relative virulence of the pathogenic yeasts in human disease may be related to the ability of these organisms to survival after being ingested by circulating phagocytes.     

Deactivation of the dimorphic fungi Histoplasma capsulatum, Blastomyces dermatitidis and Coccidioides immitis using hydrogen peroxide vapor.

Hydrogen peroxide vapour (HPV) has been proposed as an alternative to formaldehyde fumigation for the decontamination of biosafety level (BSL) III laboratories. The aim of this study was to evaluate the efficacy of HPV against the dimorphic fungi Histoplasma capsulatum, Blastomyces dermatitidis and Coccidioides immitis. Working inside a class II biological safety cabinet (BSC) within a BSL III laboratory, inocula containing approximately 5-log(10) cfu/ml from the mould form of each organism suspended in RPMI medium were deposited on stainless steel discs and allowed to air dry. The organisms were exposed to HPV inside a BSC using a BIOQUELL ClarusS HPV generator. In three replicate experiments, individual discs were transferred into liquid media at timed intervals during a 105 minute HPV exposure period. Control- and HPV-exposed discs were incubated in RPMI media at 30 degrees C for 6 weeks to determine if any viable organisms remained. Positive cultures were confirmed using specific nucleic acid hybridization probes. Results indicate that H. capsulatum, B. dermatitidis and C. immitis were killed within 30 minutes of HPV exposure.     

Recombinant Coccidioides immitis complement-fixing antigen: detection of an epitope shared by C. immitis, Histoplasma capsulatum, and Blastomyces dermatitidis.

We undertook an investigation to assess the utility of a recombinant Coccidioides immitis complement-fixing (CF) antigen for detecting CF antibody in sera from patients with coccidioidomycosis. Enzyme-linked immunosorbent assays established that recombinant CF antigen and, for comparison, a commercially available coccidioidin were reactive with 19 of 19 sera from patients with active coccidioidomycosis. The recombinant antigen was significantly more sensitive than coccidioidin. The median titer obtained when patients' sera were assayed against recombinant CF antigen was 1:51,200 compared to 1:25,600 with coccidioidin (P < 0.027). The recombinant antigen was also more effective in distinguishing the antibody levels in sera from patients with pulmonary coccidioidomycosis than in sera from those with disseminated disease. Whereas patients with pulmonary disease showed a median antibody titer of 1:25,600, those with multifocal disease showed a median titer of 1:102,400 (P < 0.028). The recombinant CF antigen was found, however, to express an epitope(s) that reacted with sera from 6 of 12 patients with histoplasmosis and 2 of 12 patients with blastomycosis.     

Comparative evaluation of chemiluminescent DNA probe assays and exoantigen tests for rapid identification of Blastomyces dermatitidis and Coccidioides immitis.

Chemiluminescent DNA probe (Accuprobe) assays developed by Gen-Probe, Inc. (San Diego, Calif.), for the rapid identification of Blastomyces dermatitidis and Coccidioides immitis were evaluated and compared with the exoantigen test by using 74 mycelial cultures of B. dermatitidis and 72 mycelial cultures of C. immitis. Seventeen isolates of the dimorphic pathogen Paracoccidioides brasiliensis were included because of their gross morphologic and antigenic relatedness to B. dermatitidis. The heterologous fungi, namely, species of Chrysosporium, which are often confused with B. dermatitidis, and species of Malbranchea, which morphologically resemble C. immitis, were tested. All 74 of the B. dermatitidis mycelial isolates were correctly identified by the Accuprobe assay for B. dermatitidis within 2 h. However, the B. dermatitidis probe cross-hybridized with rRNA extracts of 10 of the 17 P. brasiliensis isolates, misidentifying them as B. dermatitidis. All 72 of the C. immitis isolates were identified correctly with the C. immitis probe. None of the other heterologous fungi belonging to Chrysosporium spp., Malbranchea spp., Onychocola canadensis, and Geotrichum sp. were cross-reactive with the B. dermatitidis and C. immitis probes. The exoantigen tests specifically identified 74 B. dermatitidis, 72 C. immitis, and 17 P. brasiliensis isolates within 48 to 72 h and differentiated the related heterologous fungi from the three dimorphic fungal pathogens.     

Identification of Histoplasma capsulatum, Blastomyces dermatitidis, and Coccidioides species by repetitive-sequence-based PCR

The performance of repetitive-sequence-based PCR (rep-PCR) using the DiversiLab system for identification of Coccidioides species, Blastomyces dermatitidis, and Histoplasma capsulatum was assessed by comparing data obtained to colony morphology and microscopic characteristics and to nucleic acid probe results. DNA from cultures of 23 Coccidioides, 24 B. dermatitidis, 24 H. capsulatum, 3 Arthrographis, and 2 Malbranchea isolates was extracted using a microbial DNA isolation kit as recommended by Bacterial Barcodes, Inc. Rep-PCR and probe results agreed for 97.2% of the dimorphic fungi when > or =85% similarity was used as the criterion for identification. Two H. capsulatum isolates were not identified, but no isolates were misidentified. From 43 of those cultures (15 Coccidioides, 14 B. dermatitidis, 14 H. capsulatum, 3 Arthrographis, and 2 Malbranchea), DNA also was extracted using an IDI lysis kit, a simpler method. Rep-PCR and probe results agreed for 97.7% of the dimorphic fungi when a criterion of > or =90% similarity was used for identification. One H. capsulatum isolate could not be identified; no isolates were misidentified. Using > or =85% similarity for identification resulted in one misidentification. These data suggest that the DiversiLab system can be used to identify Coccidioides and B. dermatitidis and, possibly, H. capsulatum isolates.     

Evaluation of commercial reagents to identify the exoantigens of Blastomyces dermatitidis, Coccidioides immitis, and Histoplasma species cultures

Commercially available individual serologic reagents (Nolan/Scott Biological Laboratories, Inc.) and an "exoantigen identification system" (a kit supplied by Immuno-Mycologics, Inc.) were evaluated at two laboratories for their possible application in the microimmunodiffusion test to identify Blastomyces dermatitidis, Coccidioides immitis, and Histoplasma species. Both laboratories accurately identified all the cultures, and comparison of the results obtained with the commercial products and those established with the conventional methods performed by a third control laboratory demonstrated full agreement. The use of these two commercial products is recommended for the immunologic identification of cultures suspected of being B. dermatitidis, C. immitis, and Histoplasma species. It was observed, however, that the exoantigen reactions for four B. dermatitidis and two C. immitis isolates were much sharper and more readable with the individual reagents than the reactions observed with the kit.     

Evaluation of Gen-Probe's Histoplasma capsulatum and Cryptococcus neoformans AccuProbes.

Gen-Probe's DNA probes were evaluated for use in the identification of clinical isolates of Histoplasma capsulatum var. capsulatum and Cryptococcus neoformans. Ninety-five mould-phase fungi were probed, including 41 isolates of H. capsulatum var. capsulatum. Similarly, 98 yeasts, including 42 C. neoformans isolates, were examined by using the C. neoformans DNA probe. In the study, both probes demonstrated 100% specificity and 100% sensitivity. Their use in the clinical laboratory may significantly reduce the time required for definitive identification of fungi.     

Rapid conversion of Histoplasma capsulatum, Blastomyces dermatitidis and sporothrix schenckii in tissue culture.

A simple method for positive identification of Histoplasma capsulatum, Blastomyces dermatitidis, and Sporothrix schenckii is given. Primary tissue cultures of guinea pig peritoneal macrophage were inoculated with the mycelial phase of each organism and after 24 h the cells were stained and observed microscopically. The characteristic yeast phase could then be observed allowing for positive identification.     

Improved version of the exoantigen test for identification of Coccidioides immitis and Histoplasma capsulatum cultures.

A rapid and simple method for extracting specific cell-free antigens of Coccidioides immitis and Histoplasma capsulatum from agar slant cultures was developed. The extracts were analyzed in immunodiffusion tests for the presence of C. immitis or H. capsulatum specific antigens. These extracts were compared with culture filtrates of brain heart infusion broth subcultures in tests with 32 isolates of C. immitis and H. capsulatum and 13 other fungi which might be morphologically confused with them. The studies showed that the slant extracts were as useful as the culture filtrates and allowed more rapid identification of C. immitis and H. capsulatum. In every case, when an identification was made by conventional morphological methods, the immunological test yielded correlating results. The immunological tests were positive for two isolates that were converted to their yeast forms only after 4 months of study by conventional tests. The new procedure permits the identification of C. immitis and H. capsulatum 2 days after receipt of a pure mycelial-form culture. The test is recommended for the presumptive identification of C. immitis and H. capsulatum cultures.     

Immunoidentification of Histoplasma capsulatum and Blastomyces dermatitidis with commercial exoantigen reagents. Effect of culture age

We have studied the effect of the length of incubation on the reliability of commercial exoantigen test reagents (Nolan/Scott Biological Laboratories Inc, and Immuno-Mycologics Inc). Antigen extracts, tested in each commercial system, were prepared from duplicate sets of cultures of Histoplasma capsulatum (12 isolates) and of Blastomyces dermatitidis (11 isolates) after one to six weeks of growth. For isolates of H capsulatum, antigens necessary for immunoidentification were detected in most cultures in two weeks and in all cultures after four weeks of incubation for both Nolan and Immuno-Mycologics reagents, and continued to be detected for at least six weeks. Positive results could usually be obtained from exoantigen tests for H capsulatum before dimorphism could be demonstrated by conversion of the mold to yeast. Diagnostically significant antigen was not uniformly present in all cultures of B dermatitidis during the test period, but ten of 11 isolates were positive at some time during the six weeks using the Nolan reagents; antigen of only one of 11 isolates was detected by the Immuno-Mycologic reagents. Results of exoantigen tests for B dermatitidis were usually not available until after the identity had been confirmed by conversion of the mold to yeast phase on cottonseed agar.     

Interaction of Blastomyces dermatitidis, Sporothrix schenckii, and Histoplasma capsulatum with Acanthamoeba castellanii

Several dimorphic fungi are important human pathogens, but the origin and maintenance of virulence in these organisms is enigmatic, since an interaction with a mammalian host is not a requisite for fungal survival. Recently, Cryptococcus neoformans was shown to interact with macrophages, slime molds, and amoebae in a similar manner, suggesting that fungal pathogenic strategies may arise from environmental interactions with phagocytic microorganisms. In this study, we examined the interactions of three dimorphic fungi with the soil amoeba Acanthameobae castellanii. Yeast forms of Blastomyces dermatitidis, Sporothrix schenckii, and Histoplasma capsulatum were each ingested by amoebae and macrophages, and phagocytosis of yeast cells resulted in amoeba death and fungal growth. H. capsulatum conidia were also cytotoxic to amoebae. For each fungal species, exposure of yeast cells to amoebae resulted in an increase in hyphal cells. Exposure of an avirulent laboratory strain of H. capsulatum to A. castellanii selected for, or induced, a phenotype of H. capsulatum that caused a persistent murine lung infection. These results are consistent with the view that soil amoebae may contribute to the selection and maintenance of certain traits in pathogenic dimorphic fungi that confer on these microbes the capacity for virulence in mammals.     

Evaluation of a chemiluminescent probe assay for identification of Histoplasma capsulatum isolates.

Fifty-three of 54 isolates of fungi were correctly identified with an acridinium ester-labelled probe for Histoplasma capsulatum (Accuprobe; Gen-Probe, San Diego, Calif.). One isolate of Aspergillus niger was incorrectly identified as H. capsulatum. Age of the culture, medium for isolation, and morphologic state did not affect the results.     

Cell wall studies of Histoplasma capsulatum and Blastomyces dermatitidis using autologous and heterologous enzymes.

Enzymes capable of hydrolyzing cell walls of Blastomyces dermatitidis and chemotypes I and II of Histoplasma capsulatum were prepared in the laboratory or obtained from commercial sources. They included chitinases, beta-1,3-glucanases, beta-1,6-glucanase, and Pronase. Monosaccharides and disaccharides of glucose released from the cell walls by the enzymes were determined qualitatively by paper and gas-liquid chromatography, and monosaccharides were quantitated by the latter technique as well. An enzyme system isolated from Streptomyces sp. containing both chitinase and glucanase released maximum amounts of glucose and N-acetylglucosamine from the cell walls of H. capsulatum chemotype I. A chitinase preparation, free of glucanase, from Serratia marcescens released only chitobiose and N-acetylglucosamine from chemotype I cell walls, but the total quantity of N-acetylglucosamine released was about 60% less than that released by the Streptomyces system. A beta-1,3-glucanase from Bacillus circulans hydrolyzed the cell walls of H. capsulatum chemotype I, but a beta-1,6-glucanase failed to release glucose from the same walls. Autolytic enzymes, viz., beta-1,3-glucanases and several glycosidases were detected as constitutive enzymes in both yeast and mycelial phases of B. dermatitidis and H. capsulatum chemotypes I and II. No difference in the amount of activity was found between cell sap and culture filtrate preparations. The beta-glucanases prepared from the Histoplasma and Blastomyces strains were active on the cell walls of the yeast phases of H. capsulatum chemotypes I and II, releasing laminaribiose and glucose, but were essentially inactive on the cell walls of B. dermatitidis. Chitinase, beta-1,6-glucanase, alpha-glucanase, and alpha-glucosidase activities were absent from these fungal enzyme preparations.     

Evaluation of the exoantigen test for identification of Histoplasma species and Coccidioides immitis cultures

Recently it was demonstrated that certain fungal pathogens could be rapidly and accurately identified on the basis of their exoantigens. Mycelial-form cultures of the Histoplasma species consistently appear to produce either H or M antigens, or both, whereas cultures of Coccidioides immitis produce tube precipitinogen, heat-labile precipitinogen, or heat-labile F antigens. Three laboratories each evaluated 48 cultures by the exoantigen procedure for the identification of Histoplasma spp. and C. immitis. One hundred forty-four cultures were tested, and 143 (99.3%) were correctly identified as Histoplama spp. or C. immitis, or neither. The simple and rapid exoantigen test appears to be a sensitive and specific method for identification of these fungi.     

Detection of Blastomyces dermatitidis and Histoplasma capsulatum from culture isolates and clinical specimens by use of real-time PCR

Blastomyces dermatitidis and Histoplasma capsulatum are dimorphic fungi that often cause self-limited respiratory infections. However, they may also cause severe disseminated disease, depending on the level of the exposure to the organism and the host immune status. In addition, patients with infections caused by these fungi may have very similar clinical presentations. Although microbiologic culture is a standard method for detecting these pathogens, their recovery may require days to weeks, and the manipulation of cultures presents a significant safety hazard to laboratory personnel. Therefore, the goal of this study was to design a rapid, real-time PCR assay to detect and differentiate B. dermatitidis and H. capsulatum from culture isolates and directly from clinical specimens. Primers and fluorescence resonance energy transfer hybridization probes were designed to target the histidine kinase and glyceraldehyde-3-phosphate dehydrogenase genes of B. dermatitidis and H. capsulatum, respectively. The analytical sensitivity of the assay was determined to be 100 copies/μl for both fungi. From culture isolates, the assay demonstrated 100% specificity and 100% sensitivity for B. dermatitidis and 100% specificity and 94% sensitivity for H. capsulatum. Detection directly from 797 clinical specimens demonstrated specificities and sensitivities of 99% and 86% for B. dermatitidis and 100% and 73% for H. capsulatum compared with the results for culture. This real-time PCR assay provides a rapid method for the detection of B. dermatitidis and H. capsulatum from culture isolates and directly from clinical specimens.     

Giant forms of Blastomyces dermatitidis in the pulmonary lesions of blastomycosis. Potential confusion with Coccidioides immitis

Typical yeast-phase cells of Blastomyces dermatitidis have a characteristic appearance in tissue sections. Fungal morphologic variation occurs infrequently in the lesions of blastomycosis, yet it can complicate the differential diagnosis, particularly if fresh tissue is not available for microbiologic culture. The authors report a case of pulmonary blastomycosis, confirmed by culture and direct immunofluorescence, in which some of the yeast-like cells were abnormally large. These giant yeast-like cells exceeded the size range accepted for the tissue forms of B. dermatitidis; therefore, coccidioidomycosis was considered initially in the differential diagnosis. Otherwise characteristic morphologic features of these cells, in particular multinucleation and the production of broad-based blastoconidia, helped resolve the differential diagnosis. The diagnosis can be confirmed by direct immunofluorescence or microbiologic culture.     

Comparative evaluation of a chemiluminescent DNA probe and an exoantigen test for rapid identification of Histoplasma capsulatum.

A chemiluminescent DNA probe (Accuprobe) assay developed by Gen Probe, Inc., for the rapid identification of Histoplasma capsulatum was evaluated and compared with the exoantigen test by using 162 coded cultures including Histoplasma capsulatum var. capsulatum, Histoplasma capsulatum var. duboisii, Histoplasma capsulatum var. farciminosum, Blastomyces dermatitidis, Coccidioides immitis, Paracoccidioides brasiliensis, and morphologically related saprobic fungi. Each test uses a chemiluminescent, acridinium ester-labeled, single-stranded DNA probe that is complementary to the rRNA of the target organism. Lysates of the test cultures were prepared by sonication with glass beads and heat treated. After the rRNA was released from the target organism, the labeled DNA probe combined with the target H. capsulatum rRNA to form a stable DNA-RNA hybrid. A hybridization protection assay was used, and the chemiluminescence of hybrids was measured initially with a Leader 1 luminometer as relative light units and later during the investigation with a probe assay luminometer as probe light units. Of the 162 coded mycelial cultures tested by the Accuprobe assay, 105 were identified as H. capsulatum. The test could be performed with an inoculum of a few square millimeters (1 to 2 mm2) of growth. In the primary evaluation, the Accuprobe identified 103 of the 105 cultures as H. capsulatum within 2 h. The remaining two cultures, contaminated with bacteria, had to be purified before the Accuprobe assay identified them correctly as H. capsulatum. Since each coded culture was concurrently tested for H. capsulatum, B. dermatitidis, and C. immitis exoantigens, the identification of all three dimorphic pathogens was provided simultaneously. Of the 162 coded cultures tested, 105 were identified by the exoantigen test as H. capsulatum, 12 were identified as B. dermatitidis, 13 were identified as C. immitis, and 32 were negative for H. capsulatum, B. dermatitidis, and C. immitis. The bacterial contamination in two isolates did not interfere with the exoantigen testing. The exoantigen test required 7- to 10-day-old colonies and required 48 to 72 h of incubation before definitive identification was obtained.     

DNA probe for the identification of Histoplasma capsulatum

A 1.85-kilobase HindIII nuclear DNA probe from Histoplasma capsulatum G217B detected polymorphic restriction fragments within whole-cell DNA from different clinical isolates of H. capsulatum, consistent with the previous system of classification. The probe failed to hybridize to DNA from Blastomyces dermatitidis, Candida spp., Saccharomyces cerevisiae, Sepedonium chrysospermum, and Chrysosporium keratinophilum under low-stringency conditions and therefore may have value as a diagnostic reagent to identify H. capsulatum.     

Real-time PCR assay for identification of Blastomyces dermatitidis in culture and in tissue

The TaqMan real-time PCR assay was developed from the Blastomyces dermatitidis BAD1 gene promoter. The assay identified all haplotypes of B. dermatitidis and five of six positive paraffin-embedded tissues. The assay sensitivity threshold was 1 pg genomic DNA of the mold form and 2 CFU of the yeast form of B. dermatitidis. No cross-reactivity was observed against other fungal DNA. The assay allowed rapid (5-h) identification of B. dermatitidis from culture and from clinical specimens.