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Isolation and identification of pathogenic Aspergillus and Fusarium spp. from clinical materials provide the most accurate means for establishing a diagnosis of infections by these molds. Such efforts, however, are not always successful. Histologic diagnosis also has its limitations. In vivo the hyphae of Aspergillus and Fusarium spp. are very similar and their in situ manifestations are not pathognomonic. To improve the histologic diagnosis of infections by Aspergillus and Fusarium species, we developed polyclonal fluorescent-antibody reagents to Aspergillus fumigatus and Fusarium solani and evaluated their diagnostic utilities. Our studies revealed that A. fumigatus and F. solani share epitopes not only with one another but also with other Aspergillus and Fusarium spp. as well as with Paecilomyces lilacinus and Pseudallescheria boydii. Adsorption of the A. fumigatus conjugate with cells of Fusarium proliferatum and F. solani and F. solani antiserum with cells of Aspergillus flavus resulted in reagents that distinguished Aspergillus spp. from Fusarium spp. but that still cross-stained P. lilacinus and P. boydii. Adjunctive use of a specific P. boydii conjugate enabled the identification of Aspergillus spp., Fusarium spp., P. lilacinus, and P. boydii in formalin-fixed tissue sections from 19 humans with culture-proven cases of mycotic infection.

We studied an aberrant culture of Histoplasma capsulatum var. capsulatum isolated from synovial fluid collected from the right elbow of a patient from Kansas. Colonies on Sabouraud glucose agar and other routine mycological media were glabrous to soft, moist, heaped, deeply folded or convoluted, and orange-brown with a white, irregular margin. Microscopically, hyphae were hyaline, septate, and branched and remained totally devoid of conidiation over a period of 2 years on all mycological media. Conversion to the yeast form was achieved on Pine's medium at 37 degrees C. Colonies at early stages of growth were smooth, moist, pasty, shiny, and orange-brown but soon became wrinkled and slightly raised and produced oval, thin-walled cells measuring 2 to 3 by to 4.5 microns which multiplied by polar budding. The identity of the isolate was further confirmed by utilizing the Accuprobe DNA and the exoantigen test for H. capsulatum var. capsulatum.

Disseminated penicilliosis marneffei is an emerging opportunistic mycosis seen in severely immunocompromised human immunodeficiency virus (HIV)-infected patients and is caused by the dimorphic fungus Penicillium marneffei. Early diagnosis and treatment improve clinical outcome. Proper diagnosis is complicated by nonspecific signs and symptoms and by difficulties in histologic recognition and species identification of the pathogen. Since no established immunodiagnostic methods for penicilliosis marneffei are available, we attempted to develop separate immunodiffusion tests to detect P. marneffei antigens and antibodies in patient serum specimens and a latex agglutination test for antigenemia. Antigens consisted of 2-week-old fission arthroconidial filtrates produced in Pine's broth at 37 degrees C. Rabbit antisera were prepared against the 10 x -concentrated filtrate antigens. Studies were carried out with 17 serum specimens from HIV-seropositive adult Thai patients with penicilliosis marneffei and 15 control serum specimens from Thai persons free of HIV and P. marneffei infection. The immunodiffusion tests detected P.marneffei antigenemia in 10 (58.8%) of 17 patients, whereas the latex agglutination test detected antigenemia in 13 (76.5%) of the 17 patients. Antibody was demonstrated in only 2 of the 17 patient sera. All of the tests appeared to be highly specific, since none were positive with sera from 15 Thai control patients, six serum samples containing cryptococcal antigen, or six urine specimens positive for Histoplasma polysaccharide antigens.

The diagnosis of penicilliosis marneffei can be difficult because the clinical manifestations mimic those of tuberculosis, histoplasmosis, and other mycotic infections. Furthermore, the tissue form of Penicillium marneffei can be confused with those of Histoplasma capsulatum and Cryptococcus neoformans. To facilitate the rapid detection and identification of P. marneffei in clinical materials, we sought to develop a specific indirect fluorescent-antibody (IFA) reagent for this dimorphic pathogen. Preliminary IFA studies with yeast-like cells (fission arthroconidia) of P. marneffei indicated that these cellular elements stained with antiglobulins against culture filtrate antigens and whole yeast-like cellular antigens. Both types of antiglobulins reacted with the yeast-like cells of P. marneffei and with H. capsulatum, but not with their respective mycelial forms. The antiglobulins also failed to react with the yeast and hyphal forms of a variety of other heterologous fungi. Specific antiglobulins useful in an IFA test for identifying P. marneffei yeast-like cells in culture or in clinical materials were produced by adsorptions with yeast-form cells of H. capsulatum. The yeast-like culture filtrate antigens of P. marneffei are preferred for use in the production of the specific antiglobulins because they stained P. marneffei yeast-like elements more intensely than antiglobulins produced against intact yeast-like cells.

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.

Intratracheal injection of Balb/cByJ mice with 10(4) Blastomyces dermatitidis conida produces chronic pulmonary and disseminated blastomycosis characterized by pyogranulomatous inflammation. To study the evolution of the pulmonary infection, mice were killed at varying intervals after inoculation, their lungs cultured and examined histologically. Nodular intraalveolar infiltrates of macrophages (M phi) were seen on Day 1 with occasional admixed polymorphonuclear leukocytes (PMN). Phagocytized yeast forms within M phi were evident by Day 5. By Day 28 pyogranulomas, which developed first as central microabscesses associated with a peripheral zone of M phi and giant cells containing internalized yeast, were a prominent feature of the infection. Lymphocytic and plasmacytic infiltrates, accumulating next to granulomas, formed the major peripheral component of the granuloma by Day 35. Formation of pyogranulomas was coincident with the host's failure to contain fungal growth measured by the sharp rise in colony-forming units recovered from lungs. Antibody against B. dermatitidis was first detected at Day 35 by enzyme immunoassay, but not until Day 63 by double immunodiffusion. During the 4 wk after inoculation, pulmonary lavage fluid contained > 90% M phi and < 3% PMN. On day 28, PMN rose to 17%, reaching 40% on Day 42. These data contribute to our knowledge of this model and help form the basis for investigations into the roles of fungal pathogenic and host defense mechanisms in blastomycosis.

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.

A 51-year-old female Japanese patient developed black pigmentation affecting both big toe-nails. Direct potassium hydroxide examination of the nail tissue demonstrated clusters of spherical dematiaceous cells, toruloid hyphae, and septate hyphae. Wangiella dermatitidis was repeatedly isolated from the affected toe-nail lesions. This case represents the first documented case of ungual phaeohyphomycosis, 'fungal melanonychia,' caused by the dematiaceous fungus W. dermatitidis. The patient was successfully treated with a topical solution of bifonazole.

Exophiala spinifera, one of the etiologic agents of subcutaneous phaeohyphomycosis, may be wrongly identified as Exophiala jeanselmei because of the morphologic similarity of the two species. A reagent containing a precipitin specific for E. spinifera was produced by absorbing antiserum to E. spinifera with E. jeanselmei serotype 1 antigen. Using this absorbed antiserum, we were able to correctly identify isolates of E. spinifera and differentiate them from those of Exophiala alcalophila, E. jeanselmei (serotypes 1, 2 and 3), Exophiala moniliae, Exophiala pisciphila, Exophiala salmonis, Phaeoannellomyces werneckii and Wangiella dermatitidis.

Culture filtrate antigens of Basidiobolus ranarum and Conidiobolus coronatus were analyzed by immunodiffusion (ID) with homologous rabbit antisera. B. ranarum and C. coronatus were each found to have five specific antigens. Results of tests with heterologous antisera indicated that all of the species shared at least one antigen. ID tests incorporating the specific precipitin bands as references were developed for detection of basidiobolomycosis and conidiobolomycosis. These tests were performed with sera from humans and horses with proven basidiobolomycosis and conidiobolomycosis as well as with control sera from humans and animals with and without heterologous mycotic and oomycotic infections. Only sera from cases of basidiobolomycosis and conidiobolomycosis produced lines of identity with the reference precipitates of B. ranarum and C. coronatus, respectively. The ID tests were found to be completely sensitive and specific for determining the etiology of zygomycosis caused by these two species. In addition they appeared useful for monitoring resolution of the infections.

Soluble culture extracts of Bipolaris australiensis, B. hawaiiensis, B. spicifera, Exserohilum longirostratum, E. mcginnisii, E. rostratum, and Helminthosporium solani were used to prepare reference antisera in New Zealand White rabbits. The absorbed reference antisera were tested by a microimmunodiffusion method against concentrated culture filtrates prepared from 115 environmental and clinical isolates of Alternaria spp., Bipolaris spp., Curvularia spp., Dactylaria sp., Drechslera spp., Embellisia spp., Exserohilum spp., Fusarium sp., Helminthosporium sp., Microsporum sp., Scolecobasidium sp., and Scopulariopsis sp. Cross-reactivity did not occur between isolates of the genera tested except for some Bipolaris and Curvularia spp. Antigens shared by species of Bipolaris and Curvularia correlated with their morphologic similarity and phylogenetic closeness. Cross-reactivity was observed among isolates of B. australiensis, B. hawaiiensis, and B. spicifera and among isolates of E. longirostratum, E. mcginnisii, and E. rostratum. The exoantigen test is valuable for differentiating these fungi at the generic level.

Dactylaria gallopava and Scolecobasidium constrictum were reduced to varietal status under the new combination of Dactylaria constricta (Abbott) Dixon et Salkin var. gallopava (Cooke) Salkin & Dixon, and D. constricta (Abbott) Dixon et Salkin var. constricta, primarily on the basis of the morphologic similarity of their two-celled, dematiaceous, blastic conidia. To appraise this taxonomic change, we studied the antigenic relationship of D. gallopava to S. constrictum using the exoantigen procedure. Exoantigens were prepared from 20 isolates of D. gallopava, seven isolates of S. constrictum and two isolates of S colecobasidium tschawytschae and were tested against reference rabbit anti-D. gallopava and anti-S. constrictum antisera in the presence of their homologous antigens using the micro-immunodiffusion technique. All D. gallopava isolates produced two to three distinct, identical exoantigens. The seven isolates of S. constrictum also produced two to three distinct exoantigens. None of the seven isolates of S. constrictum was reactive against the D. gallopava reference system. Three of the 20 D. gallopava culture filtrate antigens produced one or two precipitin bands of nonidentity with the S. constrictum reference reagents. Both isolates of S. tschawytschae were nonreactive with the D. gallopava and S. constrictum reference reagents. In addition, D. gallopava differed from S. constrictum in the production of a reddish-brown diffusible pigment, growth up to 45 degrees C, and sensitivity to cycloheximide. Based on these physiologic differences and little or no antigenic relatedness between D. gallopava and S. constrictum, we conclude that these two species should be retained as separate entities rather than be considered as varieties of a single species.

We analyzed the exoantigens of 54 isolates belonging to the pathogenic Cladosporium species. Cladophialophora ajelloi was found to be antigenically identical to Cladosporium carrionii. All human and cat isolates of C. bantianum and C. trichoides were found to share the same antigens. Although cross-reactions were observed among the four species of Cladosporium: C. carrionii, C. bantianum, c. herbarum, and C. cladosporioides they were identified and differentiated specifically with four monospecific factor sera obtained through absorption procedures. The exoantigen serologic procedure also permitted us to identify cultures of these four Cladosporium spp. within 8 days.

A practical, sensitive, and specific immunodiffusion test was developed for diagnosing and monitoring pythiosis in horses. Culture filtrates, a soluble cell mass, and trypsinized Pythium sp. antigens were evaluated against prepared rabbit anti-Pythium sp. serum and pythiosis horse case sera. The culture filtrate antigens demonstrated the greatest capacity for detecting precipitins and the greatest stability during storage. In contrast, the trypsinized antigens had the weakest capability for detecting multiple precipitins and the poorest stability. The 13 sera from horses with proven active pythiosis were positive in immunodiffusion tests with the culture filtrate antigens. Each serum contained from three to six precipitins. Treated horses lost precipitins, and some became antibody negative. No false-positive reactions were noted in tests with sera from normal horses and humans or with sera from a variety of heterologous horse and human infections.

We evaluated commercial serodiagnostic test reagents from Greer Laboratories (GL), Lenoir, NC; Immuno-mycologics, Inc. (IMI), Norman, OK; and Scott Laboratories (SL), Fiskville, RI; for their ability to detect Aspergillus spp. exoantigens and group them in their proper series. We detected 87 culture extracts from coded cultures of Aspergillus groups and heterologous fungi against anti-A. fumigatus, A. flavus, A. nidulans, A. niger, and A. terreus sera in the presence of their corresponding antigens. Parallel control studies were performed using serological reagents from both laboratories. The IMI reagents accurately grouped all the isolates. The A. nidulans and A. terreus reactions, however, were significantly weaker than those noted with the control reagents. GL and SL do not supply A. nidulans and A. terreus reagents. Their available reagents correctly grouped the A. fumigatus, A. flavus, and A. niger isolates. Our results indicate that the commercial serodiagnostic reagents for aspergillosis can be effectively used to accurately immunoidentify the medically important Aspergillus spp.

We evaluated the diagnostic value of antibody to Blastomyces dermatitidis A precipitin arcs for identifying B. dermatitidis cultures by the exoantigen technique with 88 isolates from the United States and Canada, 12 from Africa, and 1 each from India and Israel. In addition, we studied 190 mycelial-form isolates of other fungi which could be confused antigenically or morphologically with B. dermatitidis. Antigen extracts from all of the North American isolates, the Indian and Israeli isolates, and one of the African isolates reacted with the B. dermatitidis A antibody. The anti-A serum did not react with antigens from the other 11 African isolates or with antigens from any of the 190 heterologous fungi. Studies with unadsorbed and adsorbed antisera to selected North American and African isolates of B. dermatitidis showed that the North American isolates not only produced the distinct A antigen, but also formed another antigen that was common to all 11 of the antigenically less complex African isolates. The results of this study indicate that at least two serotypes of B. dermatitidis exist. All of the North American, Indian, and Israeli isolates and one of the African isolates contained the A antigen and were designated serotype 1. Most of the African isolates (11 of 12) constituted a second serotype that was deficient in A but showed an antigen designated K.

Ninety-two slant extracts prepared from 2-week-old cultures of seven Aspergillus groups, nonsporulating "albino-type" A.fumigatus, Blastomyces dermatitidis, Histoplasma capsulatum, 3 Penicillium spp., 2 Pseudallescheria spp., 3 Paecilomyces spp., and Acremonium sp., were analyzed concurrently against antisera to A. fumigatus, A. flavus, A. nidulans, A. niger, and A. terreus. The extract of each of the aforementioned five pathogenic Aspergillus spp. produced 2-11 specific antigen-antibody complexes after reacting with its homologous antiserum. Antisera prepared against extracts to these pathogenic aspergilli demonstrated variable intra-generic crossreactivity, which were eliminated by adsorption with heterologous Aspergillus spp. antigens. Fewer nonspecific reactions were noted with the reference antigen-antibody precipitates of A. nidulans and A. Terreus than with reference precipitates of the other Aspergillus spp. Exoantigen extracts of "albino-type" isolates of A. fumigatus produced two to five specific precipitins against anti-A. fumigatus serum only. Based on the specific reactions produced by the eight albino-type isolates and 1 Aspergillus sp. isolate, all these fungi were identified correctly and placed in the A. fumigatus group. Analyses of conventionally identified A. fumigatus, A. flavus, A. nidulans, A. niger, and A. terreus including an isolate of A. parasiticus, enabled us correctly to classify them (51/51, or 100%) into their respective groups. Specific Aspergillus spp. antigen-antibody precipitates were characterized and used as references in exoantigen tests. None of the fungi in the genera other than Aspergillus produced antigens identical to the selected reference antigens.

Within a decade of its introduction, the exoantigen technique has won general acceptance for accurate and rapid identification of fungal pathogens. This acceptance is emphasized by the fact that positive exoantigen results obtained with the dimorphic pathogenic fungi B. dermatitidis, C. immitis, H. capsulatum varieties capsulatum, duboisii, and farciminosum, and P. brasiliensis are no longer considered presumptive evidence but are considered definitive data for species identification. Technical problems associated with poor sensitivity and false-positives in some of the early tests have been resolved. The test expands the diagnostic capabilities of the laboratory. We encourage the establishment of libraries of antisera for species identification and, where appropriate, for serotyping. Since the test is simple and reagents for many of the pathogens are commercially available, the test can be performed in most laboratories. As highly defined antigens are produced, more standardized and specific tests will be developed. Hybridoma technology may provide the means for producing specific antibodies without the need for highly purified antigens, which have been difficult to produce. The identification of numerous fungi could be facilitated by application of exoantigen techniques. Specific antisera should be developed to achieve this goal and to obtain antigenic data useful for elucidating taxonomic relationships. Some fungi cannot be classified on the basis of morphologic and biochemical qualities alone. Supplementary data obtained with exoantigen analyses could undoubtedly aid in resolving such problems.

A recent study suggested that Chrysosporium species have the same diagnostic antigens as Histoplasma capsulatum and Blastomyces dermatitidis and, thus, compromise the antigenic identification of these pathogens. In light of these findings, studies were undertaken to determine the reliability of the exoantigen tests for identifying B. dermatitidis and H. capsulatum organisms from cultures. Sixty-three slant or shake culture extracts, or both, were derived from C. asperatum, C. keratinophilum, C. parvum, C. pruinosum, C. parvum var. crescens, Geomyces (Chrysosporium) pannorus, B. dermatitidis, and H. capsulatum. These were analyzed by use of a commercial exoantigen kit and exoantigen test reagents obtained from a commercial source. The results of these analyses were compared with those obtained with Centers for Disease Control reagents. Many of the extracts derived from nonpathogenic fungi produced nonspecific precipitin bands when reacted with the kit and reference antisera, particularly the B. dermatitidis antisera. None, however, produced antigens identical to the specific B. dermatitidis A and H. capsulatum H and M antigens. Our findings indicate that the properly controlled immunoidentification procedure is 100% specific for B. dermatitidis and H. capsulatum, and that cross-reacting antigens derived from morphologically similar saparophytic fungi do not pose identification problems.

Coccidioides immitis produces two unrelated heat-stable antigens which are diagnostically useful in immunodiffusion tests. One, the tube precipitin antigen, is valuable for specifically detecting antibody and diagnosing early primary cases of coccidioidomycosis. The other heat-stable antigen, designated HS, is the most useful coccidioidin antigen for specifically immunoidentifying C. immitis cultures. Both of the antigens were compared and evaluated for their usefulness in exoantigen and serologic immunodiffusion tests. Our studies indicated that the detection of tube precipitin antigen is of limited value for immunoidentifying C. immitis isolates because the antigen is common to certain gymnoascaceous saprophytes, such as Arachniotus, Auxarthron, and Malbranchea species, that form alternate arthroconidia. Antibodies to HS antigens are infrequently found in human sera from patients with coccidioidomycosis and are thus of little serodiagnostic value.

Penicillium marneffei (ATCC 24100) was first isolated from a naturally acquired human infection in the U.S.A. by DiSalvo et al. in 1973. In 1979, this isolate was studied by Pitt who reidentified it as Penicillium primulinum. This prompted us to examine the antigenic relationship between P. marneffei and P. primulinum by comparing their exoantigens. The antigenic relationships of 11 isolates, including the type strains of P. marneffei and P. primulinum, were examined. Each exoantigen extract was tested simultaneously against rabbit anti-P. marneffei and P. primulinum antisera in the presence of the appropriate reference antigens. All eight isolates of P. marneffei, including ATCC 24100, produced two to four specific precipitin lines against their homologous antiserum. However, four of the eight extracts cross-reacted with unabsorbed antiserum to P. primulinum. The P. primulinum extracts contained specific exoantigens which only reacted with the anti-P. primulinum antiserum. Non-specific cross-reactions were eliminated by absorption procedures. On the basis of the specific exoantigens produced by both species, P. marneffei and P. primulinum were found to be antigenically distinct. Isolate ATCC 24100 was shown to be closely related to the other P. marneffei isolates and distinct from P. primulinum. Our results confirm the original identification of ATCC 24100 as P. marneffei and not P. primulinum as Pitt had concluded.

Double immunodiffusion (DID) is a useful, inexpensive antibody screening test that is more specific than complement fixation for blastomycosis. Seven of 10 patients with proved blastomycosis had positive results on DID assays for antibody to the A antigen of Blastomyces dermatitidis. Sputum cytologic examination and skin or other tissue biopsies may be diagnostic if the organism is demonstrated. Culture remains the sina qua non of diagnosis, and a negative DID test result for A precipitin does not rule out this infection. Follow-up serologic studies of patients in remission and in relapse should be undertaken to determine the evolution of the immune response to the A antigen.

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.

An immunological procedure for the rapid and specific identification of Coccidioides immitis isolates has been developed. The specificity of the procedure is based on the fact that C. immitis produces antigens that are not produced by morphologically similar fungi. The procedure involved the transfer of heavy mold-form inocula to flasks that contained small volumes of brain heart infusion broth. Shake cultures were grown at 25 degrees C on a gyratory shaker at 150 rpm. The concentrated supernatant antigens so obtained from broth cultures were tested in parallel with coccidioidin by a microimmunodiffusion technique against C. immitis antiserum. The ability of the immunological procedure to identify C. immitis cultures was evaluated by testing the supernatant antigens of 166 unknown isolates, many of which, by gross or microscopic examination or both, resembled C. immitis or belonged to the family Gymnoascaceae. Each culture was also identified by conventional laboratory procedures. Comparative evaluation showed that the immunological test for C. immitis was 100% sensitive and specific. The diagnostic precipitin bands that were produced by the interaction of the C. immitis supernatants and antisera were shown to consist of (i) a heat-stable antigen comparable to that active in the tube precipitin test and (ii) two heat-labile antigens, one of which was associated with complement fixation reactivity. With pure cultures, this immunological procedure permitted the identification of C. immitis isolates within 5 days.

Phaeoannellomyces werneckii is identified on the basis of its annellidic conidiogenous cells and the production of two-celled conidia. Without the proper recognition of its annellidic mode of conidiogenesis, isolates of P. werneckii can be confused with other species of Phaeococcomyces, Exophiala, and Scolecobasidium constrictum. To overcome this problem, we developed exoantigen reagents capable of specifically identifying P. werneckii. Rabbit P. werneckii antiserum was rendered specific by adsorptions with antigens of Exophiala jeanselmei serotype 1, Exophiala moniliae, and Wangiella dermatitidis. Eighty-two dematiaceous yeast-like fungi, including 18 P. werneckii cultures, were tested in the immunodiffusion test against P. werneckii antisera. Exoantigen tests permitted the rapid and specific identification of the 18 P. werneckii isolates. Negative results were obtained with the 64 heterologous antigen extracts.

Phialophora verrucosa and P. americana, two dematiaceous hyphomycetes, are known to cause chromoblastomycosis. Even though most medical mycologists consider P. americana as synonymous with P. verrucosa, others maintain them as two distinct species on the basis that the phialides of P. americana have deeper collarettes than those of P. verrucosa. Thirty-two isolates, identified either as P. americana or P. verrucosa, were studied for their morphologic, physiologic, and antigenic characteristics to evaluate their taxonomic status. Collarette morphology was found to be a variable character in 12 of the 32 isolates. Those 12 produced phialides with both shallow and deep collarettes. All of the isolates hydrolysed urea within 7 days at 25 degrees C, and failed to liquefy gelatin after 3 weeks at 25 degrees C. None of the isolates decomposed casein, xanthine, or hypoxanthine at the end of 3 weeks at 25 degrees C. All decomposed tyrosine. They grew at 25 degrees C and 37 degrees C, but failed to grow at 40 degrees C. The antigenic relationship between the two species was studied by the exoantigen procedure. The 32 isolates showed close antigenic relatedness. Adsorptions of antisera with homologous and heterologous antigens rendered the antisera free of precipitin bands when studied by the microimmunodiffusion test. The depth of phialide collarettes produced by the two species, being found to be variable character, and the identical nature of the two species with respect to their physiologic and antigenic characteristics, led us to conclude that P. americana should be considered as a synonym of P. verrucosa.

Seventy-nine mycelial-form stock cultures of Blastomyces dermatitidis, Coccidioides immitis, Histoplasma capsulatum, and morphologically similar fungi were extracted and tested by using commercial macroimmunodiffusion exoantigen test kits and the Centers for Disease Control (CDC) reference system for identifying fungal isolates. Results showed 100% correlation between the two systems. Specific exoantigens of C. immitis and H. capsulatum extracted from agar slant cultures (slant extraction method) readily were identified. In eight of 26 cultures of B. dermatitidis, broth culture filtrates (broth-shake-flask method) were required to demonstrate the specific bands of identity. No false-negative reactions or cross-reactivity among the pathogens and other fungi were observed. The commercial test kits provided a rapid and specific method for identifying or confirming suspected fungal pathogens.

A sensitive and specific immunological method was developed for rapid identification of the mycelial forms of Histoplasma capsulatum var. capsulatum, H. capsulatum var. duboisii, and H. farciminosum and for separation of these pathogenic fungi from morphologically similar hyphomycetes and other fungal pathogens. This method is based on the fact that all of the Histoplasma spp. produce H and M histoplasmin antigens, whereas the other fungi do not. Inocula consisting of heavy mycelial growth from a pure, full-grown culture were transferred into flasks containing small volumes of brain heart infusion broth. These cultures were placed on a shaker and grown at 25 C. Using the micro-immunodiffusion technique and antisera containing antibodies to H and M precipitinogens, we detected exoantigens in 3-day-old brain heart infusion culture supernatants concentrated 25 and 50 times. The ability of the procedure to identify Histoplasma spp. was evaluated by testing 96 unknown mycelial cultures that grossly or microscopically resembled Histoplasma spp. Three- and six-day-old concentrated culture supernatants prepared from each unknown were tested against rabbit anti-Arthroderma tuberculatum, Chrysosporium keratinophilum, H. capsulatum var. duboisii, and Corynascus (Thielavia) sepedonium sera and human histoplasmosis case serum. Each unknown was also identified by conventional laboratory procedures involving cultural and, where necessary, in vivo studies. In the comparative evaluation the immunological test was observed to be 100% sensitive. It permitted the accurate generic identification of the Histoplasma spp. within 5 days, in contrast to the average of 33 days required by the routine mycological procedure.

Apophysomyces elegans and Saksenaea vasiformis frequently fail to sporulate on routine media used in clinical laboratories, thus delaying or preventing their specific identification. We have developed exoantigen test reagents capable of identifying non-sporulating isolates of A. elegans and S. vasiformis. A reagent containing three specific A. elegans precipitins was produced when A. elegans antiserum was absorbed with S. vasiformis. Antigenic analyses showed that at least two serotypes of S. vasiformis exist. Only six of 10 S. vasiformis isolates studied produced bands of identity with S. vasiformis (B-2190) (serotype 1) antiserum absorbed with S. vasiformis (B-2189) (serotype 2) antigens. The remaining four isolates, however, produced bands of identity with S. vasiformis B-2189 antiserum absorbed with B-2190 antigens. Antigenic differences among the isolates of the two groups could be correlated to some extent with the morphologic differences observed, especially with respect to the size of the sporangia and sporangiospores.

Currently, exoantigen test procedures for identifying mycelial form cultures of pathogenic molds require that the fungi being extracted be treated with thimerosal to render them safe for handling. Recent studies have demonstrated that thimerosal may not be fungicidal. In view of these reports, we investigated the effects of thimerosal and formaldehyde on a variety of exoantigen preparations. Mature mycelial form fungal cultures, including cultures of Blastomyces dermatitidis, Coccidioides immitis, and Histoplasma capsulatum and morphologically similar fungi, were grown on Sabouraud dextrose agar slants and treated with 0.02, 0.04, and 0.08% thimerosal for 24 and 48 h and with 0.2 and 0.5% formaldehyde for 24 and 48 h. We found that 0.5% formaldehyde killed all of the fungi studied, whereas 0.2% formaldehyde permitted the growth of only one fungus; 0.02, 0.04, and 0.08% thimerosal were fungistatic. Furthermore, 0.2 and 0.5% formaldehyde and 0.08% thimerosal affected certain antigens adversely. For those investigators who prefer to use 0.02% thimerosal and to work with sterile extracts, we recommend that the procedure be modified, and we advocate sterilization of extracts by passage through membrane filters.

Microbial penetration of sterile packs was studied by using double-wrap (two layers each) muslin, single-wrap (two layers) muslin inner covering with single-wrap (one layer) two-way crepe paper outer covering, and single-wrap (two layers) muslin inner covering with single-layer BAR-BAC wrappers to wrap 20 gauze sponges (2 by 2 in.). These packs were stored on open shelves of a central sterile supply department of a hospital and processed for sterility at weekly intervals. Microorganisms penetrated the double-wrap muslin as early as 28 days, the single-wrap muslin and single-wrap two-way crepe paper combination in 77 days, and the single-wrap muslin and single-layer BAR-BAC combination in 63 days.

Species of Hansenula and Candida are resistant to high concentrations of anionic alkylbenzene sulfonates and degrade subinhibitory concentrations of these detergents.

Microbial penetration of sterile packs was studied using single-wrap (two layers) muslin, double-wrap (four layers) muslin, and two-way crepe paper (single layer) to wrap 20 gauze sponges (2 by 2 inch). These packs were stored in the central sterile supply departments of two hospitals and processed for sterility at predetermined intervals. Microorganisms penetrated single-wrap muslin as early as 3 days and double-wrap muslin and single-wrap two-way crepe paper in 21 to 28 days stored in open shelves. The time required for microbial penetration was at least twice as long when closed cabinets were used. Single-wrap muslin packs stored in sealed, impervious plastic bags remained sterile for at least 9 months. All sterile materials in pervious wrappers should be handled as little as possible and then only with extreme care and caution. Closed cabinets offer more protection than open shelves, and single wrappers are not recommended.