specific immunological test for the rapid identification of members

JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1976, p. 191-199Copyright C 1976 American Society for Microbiology'

Vol. 3, No. 2Printed in U.S.A.

Specific Immunological Test for the Rapid Identification ofMembers of the Genus Histoplasma

PAUL G. STANDARD* AND LEO KAUFMAN

Department ofParasitology and Laboratory Practice, School ofPublic Health, University ofNorth Carolina,Chapel Hill, North Carolina 27514, and Mycology Division, Center for Disease Control, Atlanta,

Georgia 30333*

Received for publication 9 October 1975

A sensitive and specific immunological method was developed for rapid identi-fication of the mycelial forms of Histoplasma capsulatum var. capsulatum, H.capsulatum var. duboisii, and H. farciminosum and for separation of thesepathogenic fungi from morphologically similar hyphomycetes and other fungalpathogens. 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. Inoculaconsisting 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 Mprecipitinogens, we detected exoantigens in 3-day-old brain heart infusion cul-ture supernatants concentrated 25 and 50 times. The ability of the procedure toidentify Histoplasma spp. was evaluated by testing 96 unknown mycelial cul-tures that grossly or microscopically resembled Histoplasma spp. Three- and six-day-old concentrated culture supernatants prepared from each unknown weretested against rabbit anti-Arthroderma tuberculatum, Chrysosporium keratino-philum, H. capsulatum var. duboisii, and Corynascus (Thielavia) sepedoniumsera and human histoplasmosis case serum. Each unknown was also identifiedby conventional laboratory procedures involving cultural and, where necessary,in vivo studies. In the comparative evaluation the immunological test wasobserved to be 100% sensitive. It permitted the accurate generic identification ofthe Histoplasma spp. within 5 days, in contrast to the average of 33 daysrequired by the routine mycological procedure.

Histoplasmosis is one of the most commonsystemic mycotic infections. Its unequivocal di-agnosis rests upon isolation of the etiologicalfungus Histoplasma capsulatum from clinicalmaterial and its subsequent identification, bothof which can be difficult at times to accomplish.Successful isolation of a hyphomycete thatbears tuberculate conidia provides data whichis only highly suggestive that the fungus is apathogen, since a number of species of sapro-phytes in the genera Arthroderma, Chrysos-porium, Corynascus (Thielavia), and Sepedon-ium grossly and microscopically resemble themycelial form of H. capsulatum. To furtherconfuse matters, these saprophytes are occa-sionally isolated from clinical specimens. Atyp-ical H. capsulatum isolates that produce onlymicroconidia may also be confused with themold form of Blastomyces dermatitidis. Differ-entiation of H. capsulatum from morphologi-cally similar saprophytes requires conversionfrom its mycelial to its yeast form. The sapro-

19:

phytes are not dimorphic. Unfortunately, invitro conversion of H. capsulatum to the yeastform cannot always be achieved and in vivoconversion must be accomplished. The latterprocess is time consuming, and expensive ani-mals must be used. It is obvious that an accu-rate and rapid immunological method for iden-tifying the mycelial form of H. capsulatumwould be a valuable asset in the diagnosticlaboratory.Kaufman and Brandt (8), using the fluores-

cent antibody test, and Li et al. (11), using thecomplement fixation and immunodiffusion (ID)tests, attempted to immunologically distin-guish H. capsulatum from the saprophytes.They were unsuccessful, however, because theywere unable to demonstrate species-specific an-tigens.Manych and Sourek (12) attempted to use a

serum-agar technique for identification of fun-gal pathogens. Their preliminary report indi-cated that such fungi might be rapidly identi-

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192 STANDARD AND KAUFMAN

fied in reactions with homologous hyperim-mune sera. However, additional studies werenecessary since some diagnostic precipitinbands did not appear until after 3 weeks ofincubation, the occurrence of cross-reactionswas widespread, reference systems for specifi-cally identifying bands were lacking, and onlyone or two representative isolates of selectedpathogens were used in the study.The aim of the present study was to deter-

mine whether the histoplasmin H and M anti-gens were specific for H. capsulatum andwhether they could be used in double diffusiontests to rapidly and accurately distinguish themold form of H. capsulatum from morphologi-cally similar saprophytes and pathogens.(A portion of this work was submitted by P.

G. S. to the University of North Carolina inpartial fulfillment of the requirements for thedegree of Doctor of Public Health in the Schoolof Public Health.)

MATERIALS AND METHODSCultures. The following isolates were used to pre-

pare precipitinogens and precipitins: H. capsulatumvar. capsulatum 6623, B-884, B-1342; H. capsulatumvar. duboisii 936, 1085; Arthroderma tuberculatum1593, 3147; Chrysosporium keratinophilum B-1512,B-1513; and Corynascus sepedonium 1106, 9787, and11814 (15). The isolates of the Histoplasma spp. weremaintained in their yeast form on Pine's agar (BBL)and in the mold form on Sabhi agar (Difco). Thenonpathogenic hyphomycetes were maintained onSabouraud dextrose agar (Difco).

Inocula. All mold form inocula were obtainedfrom a Sabouraud dextrose or Sabhi agar slant.Standardized mold form inoculum consisted of 0.5cm2 of mold form inoculated into 125 ml of Smith'sasparagine broth (6). Routine mold form inoculumconsisted of at least 2 to 4 cm2 of growth. Routineinoculum for yeast form cultures consisted of one 3-mm loopful of growth from an agar slant.

Antigen production and testing. Routine moldform inoculum from a pure full-grown unknown fun-gus culture was transferred in duplicate into 30 mlof brain heart infusion broth in 125-ml flasks. After3 and 6 days of incubation at 25 C on a gyratoryshaker rotating at 150 rpm, the cultures were killedby adding merthiolate to a concentration of 1:5,000.Incubation was continued for an additional 24 h.Then the cultures were centrifuged at 1,050 x g for20 min, the mycelial growth was removed, and eachof the supernatants was concentrated 25x and 50xwith Amicon Minicon Macrosolute concentrators B-15 (Amicon Corp., Lexington, Mass.). For the micro-ID test, the 25 x and 50 x -concentrated supernatantsfrom each broth culture were placed in adjacentlateral wells. The formation of H or H and M precip-itin bands after 24 h constituted a positive reaction.

Antisera production. Ten times-concentrated su-pernatant of 2- and 6-week-old Smith's asparaginebroth cultures of the saprophytes and lOx-concen-

trated supernatants of 6-week-old Smith's aspara-gine broth cultures of H. capsulatum var. capsula-tum and H. capsulatum var. duboisii were each usedto immunize three albino rabbits. Each rabbit wasinjected intramuscularly with 0.5 ml of supernatantantigen and 0.5 ml of Freund incomplete adjuvant(Difco) on days 1, 2, and 3 and 0.5 ml of supernatantantigen intravenously on days 7 and 14. On day 21the rabbits were bled and their sera were tested inmicro-ID tests against the homologous immunizingantigens to determine whether precipitins were elic-ited. If the precipitin response was weak or nega-tive, two additional intravenous injections consist-ing of 0.5 ml of supernatant antigen were adminis-tered 7 and 14 days after the bleeding date. Injec-tions were continued until the harvested sera dem-onstrated intense precipitin bands after reactionwith the homologous antigens. All sera were alsotested against heterologous antigens. In addition,uninoculated brain heart infusion broth and Smith'sasparagine broth concentrated 25x and 50x weretested against all the sera used in this study. Theabsence of precipitin bands with these controls re-vealed that no precipitins due to medium ingredi-ents were present in the antisera studied.ID and counterelectrophoresis tests. A modifica-

tion of Busey and Hinton's (2) micro-ID procedurewas used. Six and one-half milliliters of ID (7) agarwas poured into a plastic petri dish (15 by 100 mm)and allowed to solidify for 30 min; then 3.5 ml of agarwas poured as an overlay, and a plastic matrixcontaining 17 seven-well patterns (L.L. Pellet Co.,Dallas, Tex.) was immediately placed in the liquidagar. Using this procedure and human histoplasmo-sis case serum (CDC lot 10716) containing H and Mantibodies, we tested the concentrated supernatantsin parallel with control histoplasmin antigen (CDClot 7 or 9) for H and M precipitinogens. The antise-rum was placed in the central well and preincubatedfor 1 h at 25 C. Reference antigens were then placedin the upper and lower wells of each pattern, andunknown antigens were placed in duplicate in thelateral wells. Each plate was incubated in a moistchamber at 25 C for 24 h.The counterelectrophoresis technique of Kleger

and Kaufman (10) was used.

RESULTSStudies were performed to determine

whether 6-week-old cultures of H. capsulatumvar. capsulatum, H. capsulatum var. duboisii,and some morphologically similar saprophytesincubated at 25 C produced H or M antigen, orboth, in Smith's asparagine broth after inocula-tion with standardized mold form inocula. Theresults of these studies are shown in Table 1. Itis evident from the data presented that H or Hand M precipitin bands were produced onlywhen lOx-concentrated Histoplasma spp. ex-oantigens or the reference histoplasmin reactedwith serum from humans with histoplasmosisor rabbit antisera to the Histoplasma spp. Noreactions were observed between the exoanti-

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RAPID IMMUNOLOGICAL TECHNIQUE FOR HISTOPLASMA

TABLE 1. Immunodiffusion reactions of 10 x-concentrated supernatant antigens ofpathogenic andnonpathogenic hyphomycetes with homologous and heterologous antisera

Precipitin bands formeda upon reactions with:

Rabbit antisera to:Control

Fungus supernatants human H. capsulatum H. capsulatum C. ker- Cory-histoplas- var capsulatum var. duboisii A. tub- atino- nascusmosis anti- ercula- i sepe-serum tUb phl don-6623 B-884 936 1085 umb dum-hume

Control HM HM HM HM HM 0 0 0histoplasmin

H. capsulatum var. capsu- HM HM H H HM 0 0 0latum 6623

H. capsulatum var. capsu- H H H H H 0 0 0latum B-884

H. capsulatum var. capsu- HM H H H H 0 0 0latum B-1342

H. capsulatum var. duboisii H H H H H 0 0 0936

H. capsulatum var. duboisii HM H H H H 0 0 01085

A. tuberculatum 1593 0 0 0 0 0 1-2 0 0A. tuberculatum 3147 0 0 0 0 0 1 0 0C. keratinophilum B-1512 0 0 0 0 0 0 2-3 0C. keratinophilum B-1513 0_1d 0 0 0 0_1d 0 2 0Corynascus sepedonium 0 0 0 0 0 0 0 2-3

1106Corynascus sepedonium 0 0 0 0 0 0 0 1-2

9787Corynascus sepedonium 0 0 0 0 0 0 0 1-3

11814

' Numerals represent number of bands formed.bAntiserum pooled from two strains.cAntiserum pooled from three strains.dBand of nonidentity inconsistently produced.

gens of the Histoplasma spp. and the rabbitantisera produced against the nonpathogens.All of the 10x-concentrated supernatant fromthe nonpathogenic hyphomycetes demon-strated one to three antigens upon reactionwith homologous antisera. Except for one iso-late of C. keratinophilum (B-1513), which in-consistently produced an antigen not identicalto H or M (Fig. 1), none of the nonpathogenichyphomycetes produced antigens reactive withHistoplasma spp. antisera. Studies with lOx-concentrated 1- , 2-, and 4-week-old culture su-pernatants produced with standardized inoculaindicated that H and M exoantigens could beproduced with 2-week-old cultures.The value of routine yeast and mold form

inocula in Smith's asparagine broth and theeffects of incubation at 25 and 37 C were alsoexamined for each of the Histoplasma spp. cul-tures. Our studies indicated that the mold forminocula of Histoplasma spp. incubated at 25 Cproduced H or H and M antigens more rapidly

(within 1 week) than did the other combina-tions of inocula and incubation temperatures.Although specific H or H and M antigens

could be consistently produced by the Histo-plasma spp., the prolonged incubation timenecessary for exoantigen formation limited itsdiagnostic value. Additional studies weretherefore conducted to determine if the anti-gens could be produced more rapidly by usingheavy inocula (routine inocula) in small vol-umes of a dehydrated, commercially availablemedium. Accordingly, 30 ml of brain heart in-fusion broth in 125-ml nalgene flasks were eachinoculated with mold form routine inocula of allstock cultures. These cultures were incubatedat 25 C on a gyratory shaker rotating at 150rpm, and merthiolate was added at 3, 6, and 10days. Supernatants were concentrated 25x and5Ox in attempts to obtain rapid results. The25 x - and 50 x -concentrated 3-, 6-, and 10-dayantigen preparations produced similar results.The modified procedure permitted the early de-

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FIG. 1. Formation of a precipitin line by C. keratinophilum B-1513 unrelated to either the H or M bands.Wells 1 and 4 contained positive histoplasmin control antigen lot 9, and well 6 contained supernatant antigenprepared from a 4-week-old culture of C. keratinophilum B-1513. Center well contained rabbit anti-H.capsulatum var. duboisii 1085 serum.

tection of H or H and M antigens in 3-day-oldBHI broth supernatants of Histoplasma spp.(Table 2). No apparent antigenic crosses wereobserved between the 25x-concentrated super-natants from the Histoplasma spp. and the an-tisera to the nonpathogens. The concentratedsupernatants from the 3-day-old nonpathogensproduced precipitin bands only when they weretested against the homologous antisera.A study was carried out to determine the

sensitivity and specificity of the newly devel-oped immunological technique and to comparethe times necessary to identify Histoplasmaspp. immunologically and by routine culturalmethods. The immunological and conventionalidentification procedures were performed inparallel on 96 unidentified fungi that grossly ormicroscopically resembled Histoplasma spp.These unknowns were received from manyareas of the world. They had been isolated fromhuman, animal, and environmental sources,and their identity was unknown to us. Forty-eight of the 96 unknowns produced H or H andM antigens when tested against a histoplasmo-

sis case serum. Of these 48 immunologicallypositive isolates, 41 were identified as H. capsu-latum var. capsulatum and seven as H. capsu-latum var. duboisii on the basis of their mor-phology either in vitro or in vivo or both. Five ofthe 41 cultures morphologically identified as H.capsulatum var. capsulatum were received asH. farciminosum from reliable sources. All buttwo of the Histoplasma isolates produced H orH and M antigens within 3 days, and the lattercultures produced H antigen in 6 days. None ofthe supernatants from these 48 fungi reactedwith antisera to A. tuberculatum, C. keratino-philum, or Corynascus sepedonium. Figure 2shows the typical ID reactions when superna-tant antigens of the unknown Histoplasma spp.were reacted with Histoplasma antiserum.Supernatants from five of the unknowns pro-

duced a band that was unrelated to the H andM reference bands. Immunologically thesewere not identified as Histoplasma spp. Mor-phologically they were identified as Cocci-dioides immitis. Forty-three of the unknownsdid not produce H or M antigens (Fig. 2), and

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TABLE 2. Immunodiffusion reactions of25 x-concentrated 3-day-old supernatant antigens ofpathogenic andnonpathogenic hyphomycetes with antisera to H. capsulatum var. capsulatum and nonpathogenic

hyphomycetesPrecipitin bands formed' upon reaction with antisera to:

Supermatant antigens of: No. of cultures H.capu A Corynas-latum var. A. tubercu- C. keratin- Crnscapsula- latumb ophiliumb douse

tumr

H. capsulatum var. capsulatumH. capsulatum var. duboisiiA. tuberculatumC. keratinophilumCorynascus sepedonium

3 H or HM2 H or HM2 023

00

o o 0o 0 0

2-3 0 00 1 0O 0 2-3

a Numerals represent number of bands formed.b Antiserum pooled from two strains.r Antiserum pooled from three strains.

FIG. 2. Immunological identification of unknown Histoplasma spp. cultures and differentiation of Histo-plasma spp. from morphologically similar nonpathogens. Wells 1 and 4 contained positive control histoplas-min lot 9, well 2 contained supernatant antigen produced from S. chrysospermum ATCC 13378, well 3contained supernatant antigen produced from C. keratinophilum ATCC 14803, and wells 5 and 6 containedhistoplasmin produced from two unknown Histoplasma spp. cultures. Center well contained human histo-plasmosis case serum containing H and M antibodies.

they were not immunologically identified asHistoplasma spp. Morphological studies onthese 43 unknowns confirmed the immunologi-cal test results and showed them to be either

saprophytes or pathogens other than the Histo-plasma spp. It is of interest to note that, of the48 Histoplasma spp., 25 (52%) failed to produceconidia (macro- and microconidia).

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Of the 43 fungi whose supernatants did notreact with the antiserum to H or M antigens,only five produced bands upon reaction withantisera to the saprophytes: one with A. tuber-culatum antiserum, two with the C. keratino-philum antiserum, and two with the Corynas-cus sepedonium antiserum. In contrast, themorphological studies indicated that 10 of the43 fungi were members of the aforementionedgenera.

DISCUSSIONID studies with H. capsulatum var. capsula-

tum and H. capsulatum var. duboisii mycelial-form exoantigens showed them to share H or Hand M precipitinogens (Tables 1 and 2). Thesefindings are in agreement with those of Ehr-hard and Pine (5), who reported that H. capsu-

latum var. duboisii produced H and M anti-gens. Antigenic analysis of the supernatants ofH. capsulatum var. capsulatum and H. capsu-

latum var. duboisii suggest that the exoanti-gens are identical. Attempts to demonstratespecies-specific precipitinogens were unsuccess-ful. A new and interesting finding was that H.farciminosum exoantigens reacting with hu-man histoplasmosis case sera also produced Hor H and M bands (Table 3).The exoantigens ofthe nonpathogenic hypho-

mycetes A. tuberculatum, C. keratinophilumand Corynascus sepedonium did not produce Hor M bands upon reaction with rabbit or humanHistoplasma spp. antiserum (Tables 1-3), nor

J. CLIN. MICROBIOL.

did the exoantigens of the nonpathogens elicitproduction of H or M antibodies after injectioninto rabbits (Tables 1 and 2). These results are

not consistent with the findings of Li et al. (11),who reported that rabbit anti-C. keratino-philum ATCC 14803 and anti-Sepedoniumchrysospermum ATCC 13378 produced M bandsafter reacting with histoplasmin produced fromone isolate of H. capsulatum but did not pro-

duce precipitins when reacted with histoplas-min produced from eight other H. capsulatumisolates. Because of such reactions, these twosaprophytic fungi were included in this investi-gation (Table 3; Fig. 2). The present study didnot substantiate the implications of Li et al.(11) that C. keratinophilum and S. chrysosper-mum shared antigens with H. capsulatum. Theexoantigens produced by the isolates used by Liet al. (11) did not produce M precipitin bands or

any other bands when tested against the H.capsulatum reference antiserum used in thisstudy (Fig. 2).The precipitin bands observed by Li et al. (11)

could be similar to the band noted earlier withC. keratinophilum B-1513 reacting with H. cap-sulatum antiserum (Fig. 1). The line of noni-dentity noted with the supernatants of C. kera-tinophilum B-1513 and that noted with some ofthe C. immitis isolates (Table 3) represent theonly apparent exoantigen cross-reactions occur-

ring with the H. capsulatum antisera. The dif-ference in the results obtained by Li et al. (11)and by us might be due to the fact that homoge-

TABLE 3. Evaluation with 96 unknown hyphomycetes of the accuracy of the immunological procedure foridentifying Histoplasma spp. as compared to conventional methods

No of un-known

Type of precipitin re- fungus su- Identification by im- Identification by con- No. iden- Aactions pernatants munological test ventional test tified mee-

producing mentreaction

H or HM bands 48a Histoplasma spp. H. capsulatum var. cap- 41c 100sulatumb

H. capsulatum var. du- 7boisii

Band noted, but not H 5 Not Histoplasma spp. C. immitis 5 100or M

No bands 43 Not Histoplasma spp. B. dermatitidis 9 100Chrysosporium-Sepe- 10donium complexd

C. immitis 6Mycelia sterilia 2P. brasiliensis 9Saprophytes 7

a All but two supernatants showed H or M bands in 3 days.b An average of 33 days was required for identification.c Five of the 41 cultures identified as H. capsulatum were received as H. farciminosum.d Includes ATCC cultures C. keratinophilum 14803 and S. chrysospermum 13378.

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nate antigens were used in the former studyand supernatant antigen was used in the latter.The differentiation of fungi producing such an-tigens from Histoplasma spp. poses no problemwhere the immunological test is used, sincenone produce H or M antigens.

In studies with histoplasmin skin test anti-gens, Palmer et al. (13), Asgari and Conant (1),and Edwards et al. (4) implied that, in certainhuman and animal cases, false positive delayedhypersensitivity reactions occurred and mightbe attributed to encounters with saprophyticfungi sharing common antigens with H. capsu-latum, rather than to encounters with the lat-ter pathogen. The results presented in our in-vestigation revealed extrageneric reactivity be-tween H. capsulatum and some isolates of C.keratinophilum and C. immitis. None of thesereactions was due to H and M antigens. Toobviate false positive skin test reactions withhistoplasmin, it is recommended that purifiedhistoplasmin containing only H and M antigensand no common antigens be used for skin test-ing (9).White fluffy fungal isolates that do not sporu-

late are frequently obtained by diagnostic my-cology laboratories. These nonsporulatingfungi could be atypical H. capsulatum isolatesand might be erroneously reported as contami-nants. Fifty-two percent (25 of 48) of the un-known Histoplasma isolates used in this studywere nonsporulating. Some of the pathogensstudied were not primary isolates. Convention-ally, the only way these nonsporulating Histo-plasma spp. cultures could be correctly identi-fied was by persistent in vitro or in vivo effortsto convert them to their yeast form. In thoseinstances where conversion attempts provedunsuccessful, the diagnosis would be equivocal.The immunological test eliminates the need forconversion and reduces the time necessary foridentifying Histoplasma spp. to 5 days, as com-pared with the average of 33 days required byroutine morphological procedures.The reagents developed in this study proved

to be of little value for the immunological iden-tification of the saprophytic Arthroderma spp.,Chryrosporium spp., and Corynascus spp. Ap-plication of the antisera prepared against thestock cultures of the nonpathogenic hyphomy-cetes to concentrated supernatant antigens pre-pared from numerous isolates of Arthrodermaspp., Chrysosporium spp., and Corynascus spp.received during the study failed in most cases toproduce precipitin bands. Serologically, thisgroup of fungi appeared to be very heteroge-neous. Consequently, the accurate identifica-tion of these fungi must still be based uponmorphological criteria (3).

None of the supernatant antigens of the my-celial form of the pathogens B. dermatitidis, C.immitis, and Paracoccidioides brasiliensisshared H or H and M antigens with the Histo-plasma spp. (Table 3). This observation is inter-esting in light of the recent studies of Restrepoand Moncada (14). They noted that some para-coccidioidomycosis case sera, when reactedwith paracoccidioidin, formed three bands des-ignated 1, 2, and 3. Band 3, which was closest tothe serum well, was considered to be identicalto the histoplasmosis M band. However, of 35patients' sera producing band 3, only eightcross-reacted with histoplasmin. In the presentstudy, no cross-reactions were noted when con-centrated supernatant antigens from 3- and 6-day cultures grown from 21 isolates ofP. brasi-liensis were tested against H. capsulatum anti-sera. The consistent absence of M antigens inthe P. brasiliensis supernatant antigens pres-ently studied and the fact that M precipitinswere not consistently demonstrated in the para-coccidioidomycosis case sera studied by Res-trepo and Moncada (14) suggest that the Mbands noted in the latter study were not pro-duced by infection with P. brasiliensis but bycoincidental or earlier infection with H. capsu-latum.By using a dense mycelial mass for inoculum

and small volumes of brain heart infusion brothas substrate, we developed a procedure thatpermitted the rapid production ofH or H and Mantigens by Histoplasma spp. The immunologi-cal procedure for identification of Histoplasmaspp. based upon detection of H or H and Mantigens was 100% sensitive and specific whencompared with the results obtained by the con-ventional laboratory procedures (Table 3).Ninety-six unknown cultures that consisted of48 Histoplasma spp. and 48 morphologicallysimilar hyphomycetes and other fungal patho-gens were used in evaluating this immunologi-cal procedure. Of the latter 48 fungi, none pro-duced H or M antigens and none was reportedas Histoplasma spp. However, five superna-tants from what proved to be C. immitis cul-tures did react with Histoplasma spp. antisera,producing precipitin bands that were nonident-ical to the H or M reference bands. This did notpose any diagnostic problem. The five C. immi-tis supernatants also produced lines of noni-dentity with all the saprophyte ID referencesused in this study. These results indicate theneed for establishing lines of identity in mak-ing an immunological diagnosis.The immunological method does not permit

the delineation of Histoplasma spp. Althoughrepresentative isolates of the two species andtheir varieties occasionally produced bands

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other than H or M, they were not consistentlyobserved. This study indicates that the culturesupernatants of the mycelial forms ofH. capsu-latum var. capsulatum, H. capsulatum var.duboisii, and H. farciminosum consistentlycontained the common antigens H or H and M.The strong antigenic and morphological simi-larities displayed by these fungi. appear to jus-tify their being classified together as species ofHistoplasma or varieties of H. capsulatum.The fact that two Histoplasma spp. cannot be

differentiated immunologically does not pose adiagnostic problem in the Americas, since theonly Histoplasma spp. found here is H. capsu-latum var. capsulatum. In Africa, immunologi-cal identification of a fungus as a Histoplasmaspp. would only signify that the agent was oneof the two varieties of H. capsulatum or thespecies H. farciminosum. In such a case, in vivoand morphological studies would be needed forhuman isolates to distinguish H. capsulatumvar. capsulatum and H. capsulatum var. du-boisii from each other. If the fungus was iso-lated from a soliped, morphological and clinicalmanifestations would be necessary to distin-guish H. capsulatum var. capsulatum and H.farciminosum.

It is obvious from the data presented that thenew immunological procedure will provide thelaboratory with an additional tool for rapidlyidentifying atypical H. capsulatum isolatesthat fail to sporulate or convert to the yeast-form. If this test is used, fewer false negativeswill be reported. Preliminary studies haveshown that the time required for the immuno-logical identification (5 days) can be shortenedby almost 24 h by using counterelectrophoresisin lieu of ID techniques (10).For performance of the immunological test, it

is recommended that two flasks of brain heartinfusion broth be inoculated for each isolate.Then, if the 3-day culture supernatant is nega-tive for H or H and M antigens, merthiolatetreatment of the second culture supernatantwould be carried out in 6 days, and then thesupernatant could be concentrated and tested.Supernatants should be concentrated 25x and50 x. For the most part, in the present study the25 x and 50 x concentrates yielded equally po-tent precipitinogens. In rare cases weak Mbands were demonstrated in 50x supernatantantigens but not in the 25x concentrates. Hbands, however, were seen in both 25 x and 50 xconcentrates of Histoplasma spp. In somecases, 50 x concentration was excessive andtended to drive the precipitin reaction into thecenter well of the ID pattern, whereas the 25xconcentration yielded well-separated precipi-tins.

In performing the immunological test, it isrecommended that supernatants of only twosuspected Histoplasma spp. isolates be placedin each ID pattern (Fig. 1 and 2). Since someHistoplasma spp. produce potent antigens in 3days, the bands produced may extend and runin front of adjacent wells containing negativesupernatants. Such a phenomenon could bemisinterpreted and be a source of false posi-tives. To avoid such false results, we recom-mend that the 25x- and 50x-concentrated su-pernatants from each unknown isolate beplaced in the adjacent wells on either side of thereference wells. For example, the supernatantsof one isolate should be run in wells 2 and 3 andthe supernatants of the other isolate in wells 5and 6 (see numbered patterns in Fig. 1 and 2).

ACKNOWLEDGMENTSWe gratefully acknowledge the assistance of the staffs of

the Mycology Training Branch and Mycology Division,CDC, as well as the numerous investigators who contrib-uted cultures.

LITERATURE CITED

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2. Busey, J. F., and P. F. Hinton. 1965. Precipitins inhistoplasmosis. Am. Rev. Resp. Dis. 92:637-639.

3. Carmichael, J. W. 1962. Chrysosporium and some otheraleuriosporic hyphomycetes. Can. J. Bot. 40:1137-1173.

4. Edwards, L. B., F. A. Acquaviva, and V. T. Livesay.1973. Further observations on histoplasmin sensitiv-ity in the United States. Am. J. Epidemiol. 98:315-325.

5. Ehrhard, H., and L. Pine. 1972. Factors influencing theproduction of H and M antigens by Histoplasma cap-sulatum: development and evaluation of a shake cul-ture procedure. Appl. Microbiol. 23:236-249.

6. Harrell, W. K., H. Ashworth, and L. E. Britt. 1973.Procedural manual for production of bacterial, fungaland parasitic reagents. U.S. Department of Health,Education, and Welfare, Washington, D.C.

7. Kaufman, L. 1972. Micro agar-gel immunodiffusiontests, p. 8-14. In Manual of standardized serodiagnos-tic procedures for systemic mycoses. Pan AmericanHealth Organization Scientific Publication, Wash-ington, D.C.

8. Kaufman, L., and B. Brandt. 1964. Fluorescent anti-body studies of the mycelial form ofHistoplasma cap-sulatum and morphologically similar fungi. J. Bacte-riol. 87:120-126.

9. Kaufman, L., D. McLaughlin, and R. T. Terry. 1969.Immunological studies with a M-deficient histoplas-min skin-test antigen. Appl. Microbiol. 18:307-309.

10. Kleger, B., and L. Kaufman. 1973. Detection and iden-tification of diagnostic Histoplasma capsulatum pre-cipitates by counterelectrophoresis. Appl. Microbiol.26:231-238.

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specific immunological test for the rapid identification of members

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