Immunohistologic identification of Aspergillus spp. and other hyaline fungi by using polyclonal fluorescent antibodies.

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.

An aberrant variant of Histoplasma capsulatum var. capsulatum.

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.

Diagnostic antigenemia tests for penicilliosis marneffei.

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.

Development of specific fluorescent-antibody test for tissue form of Penicillium marneffei.

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.

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.

Development of pulmonary infection in mice inoculated with Blastomyces dermatitidis conidia.

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.

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.

Fungal melanonychia: ungual phaeohyphomycosis caused by Wangiella dermatitidis.

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.

Exoantigen test for the rapid identification of Exophiala spinifera.

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.

Immunodiffusion test for serodiagnosing subcutaneous zygomycosis.

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.

Differentiation of medically important isolates of Bipolaris and Exserohilum with exoantigens.

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.

Antigenic relationship of Dactylaria gallopava to Scolecobasidium constrictum.

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.

Antigenic relationship of Penicillium marneffei to P. primulinum.

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.

Exoantigen tests for the rapid and specific identification of Apophysomyces elegans and Saksenaea vasiformis.

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.

An immunological procedure for the rapid identification of Phaeoannellomyces werneckii.

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.

Evaluation of commercial serologic test reagents for immunoidentification of medically important aspergilli.

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.

Specific and rapid identification of medically important fungi by exoantigen detection.

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.

Immunodiffusion test for diagnosing and monitoring pythiosis in horses.

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.

Reliability of exoantigens for differentiating Blastomyces dermatitidis and Histoplasma capsulatum from Chrysosporium and geomyces species.

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.