Rapid in vitro conversion and identification of Coccidioides immitis

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JOURNALOF CLINICAL MICROBIOLOGY,Feb.1976,p. 186-190

3,No.2 PrintedinU.S.A.

Rapid

in

Vitro

Conversion and Identification

of

Coccidioides

immitis1

S. H. SUN, M. HUPPERT,* AND K. R. VUKOVICH

MycologyResearch Laboratory, VerteransAdministrationHospital, Long Beach, California 90801;* and

Department of

Medical

Microbiology, University

of California,

Irvine,

California

90822

Received for publication15October 1975

Since manycontaminating nonpathogenicfungiresemble Coccidioides immi-tis culturally, isolatesfrompatients must be inoculated intolaboratoryanimals to confirm identification as C. immitis. This procedure is time-consuming, expensive, and notgenerallyavailable in clinicallaboratories. When cultures of C. immitiswere grown in slide cultures onmodifiedConverseliquid mediumin

purifiedagarand incubated at 40 C in a candle jar, all 57 isolates demonstrated

inhibition of mycelial growth and conversion ofarthrosporestoendosporulating

spherulesin 3to 5days. Representativeisolatesofsix speciesof nonpathogenic

fungi that resembled C. immitis culturally either did not grow or failed to

produceendosporulating spherulesunderthe sameconditions. Thisprocedureis recommended forconfirming theidentification ofa culture as C. immitis.

Coccidioidomycosis is a pulmonary disease

common in arid regions of the western

hemi-sphere. Theprevalence of infectioninthe

popu-lationfrequently exceeds 50% (5) and approxi-mately one-half of those infected develop symp-tomatic disease (11). Definitivediagnosisis es-tablishedbyisolatingthefungus, Coccidioides

immitis,orbyvisualization ofcharacteristic

en-dosporulating spherules in pathological

speci-mens. Identification of cultures involves the demonstration of arthrospores and

reproduc-tion ofthe disease inlaboratory animals. The

latterrequirementisnotacademic sincemany

nonpathogenic fungi resemble C. immitis in

cultural and morphological characteristics (6),

and asignificantnumber (15 to 20%) ofisolates ofC. immitis produce atypical cultures (8).

Re-productionofthe diseaseinlaboratory animals

is expensive, inconvenient, and

time-consum-ing (1 to 3weeks), and, in addition, many lab-oratories are not equipped to maintain

small-animal colonies.

Amethod more efficientandconvenientthan animal inoculation would be desirable. Con-verse (3, 4) described achemically defined liq-uidmedium inwhichmycelial phase growth of

C. immitiswould convertreliably to

endosporu-lating spherules. This required continuous

shake-culturing at 40C with increased CO2

tension. Roberts et al. (12), using a modified

Converse liquid medium and stationary

cul-tures, reported successful conversion to

endo-sporulating spherules with 201 isolates of C.

IVeteransAdministration projectno. 0641-01.

immitis. This would obviate the need for

ani-mal infection, but the method has not been

consistently successful in our experience.

Brosbe (2) incorporated a modified Converse liquid medium (9) into agar and reported in vitrodevelopmentofendosporulatingspherules withfour isolates of C. immitis. This was ac-complished in small petri dishes (50by 12mm)

withincubationat 40C and in a gas mixture of

20%C02-80% air.The Brosbe methodshouldbe useful in clinical laboratories. This report con-cerns confirmationof hisfindings and modifica-tionsof the proceduretomake itreadily

adapt-able foruse inclinical laboratories.

MATERIALS AND METHODS

Cultures. All57isolates of C.immitisusedinthis studywererecovered fromhumancasesof coccidioi-domycosis. Four of thesewereprimary isolates

re-isolated from inoculated mice, and the remainder had been transferredinSabouraud dextroseagarfor severalgenerations. Someof thesewereatypicalas

described earlier (8). In addition to the proven cul-turesofC.immitis,oneisolateof each ofthefollowing fungi that resembleC. immitiswasincluded:

Geotri-chum sp., Trichosporon cutaneum, Endomycopsis chodati, Myxotrichum sp., Auxarthron reticulatus, andPseudoarachniotus sp.

Media. Allcultures were grown onglucose-yeast

extract medium containing1% glucose, 0.5%yeast extract, and 1.5%agar. Cultures wereincubatedat roomtemperature untilarthrospores werepresent.

Converse liquidmedium, modified accordingtothe procedure of Levine et al. (9) wasused asthe

sus-pending liquid for arthrospores and also incorpo-ratedinto1%Ionagarno. 2(MCM-A)for conversion ofarthrospores toendosporulating spherules(2). A 186

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RAPID IDENTIFICATION OF C. IMMITIS

layerof water agar (1.5%) was used inchambersas a

sourceof moisture for incubatingslide cultures. All media weresterilized byautoclaving.

Slide culture procedure. A small block of agar (approximately 10by 10 by3 mm) was cutfrom a

poured plateof MCM-A and transferredto asterile microscope slide in a sterile petri dish. The agar blockwas inoculated onthe upper surface with a

loop of arthrospore suspensionprepared by addinga

drop of Converse liquid medium, as modified by Levine et al. (9), tothe top of a cultureon glucose-yeast extractmedium, allowing the sporesto float up, and picking them up with the loop. A sterile glasscoverslipwasplacedontopofthe inoculated block of agarand pressed gentlyto secure it tothe agar and to flatten the agar slightly. In separate experiments, the slides were placed either in an

anerobic incubator (National Appliance Co., model 3640-5) orinacandle jar. When the anaerobic incu-batorwasused, slide cultureswereplacedin amoist chamber (a petri dish, 150by 15mm)containing a

layer of water agar and two applicator sticks to

supportthe slides(maximum of four slides per moist chamber).Disheswereplacedinthe anaerobic

incu-batorand incubated ateither37 or 40C withagas mixtureof 20%C02-80%air. Asmallcylindrical jar (18 cm high by 12 cm in diameter) wasused as a

candle jar.Adoublelayer of filter paperwasplaced in the bottom and saturated with sterile water to

supply moisture. Slide cultures were placedin an

upended rectangular glass slide holder (from a

staining jar)sothat slides remained horizontal. This wasplacedinthecylindrical jar andasmallcandle standingin a smallglass petri dish wasplacedon

topof the slide holder. Athinroll ofmodeling clay wasattached to therimof thecylindrical jar. After the candlewaslighted, aglass petri dishcover was

placed on top of the cylindrical jar and sufficient pressure was applied to seal the unit. The entire

unit wasplacedinanincubator eitherat37 or 40C. Slide cultureswereobservedat zerotimeand every

24hfordevelopment ofendosporulating spherules. Photomicrography was accomplished witha Nikon Microflex model AFM microscope at x600 magnifi-cationwith KodakRoyalPan film(10.2by12.7cm).

RESULTS

The sequence ofmorphological changes

un-der different experimental conditions were

identical for all of the cultures of C. immitis

tested.Inaddition, thesequenceof

morphologi-calevents wasessentially thesamefor all of the

nonpathogenic fungi (onlyminorand

nonsigni-ficant differences). Therefore, only typical

ex-amples will be described.

C. immitis. Arthrospore suspensionsat zero timecontained single spores, chains of several spores, and very few hyphal strands(Fig. la).

When incubated at room temperature,

germi-nation andhyphae wereapparent at 24 h (Fig. lb) with branching prominent after 48 h. Most isolates showed typical arthrospore formation in 4to 5 days (Fig. lc). When incubated at 37 C

ineither the anaerobic incubator or the candle jar, endosporulating spherules werepresent in 4 to 5days, but the slide culturewasovergrown

with hyphae and many newly formed

arthro-spores had developed. Morphological changes were identical when slide cultures were incu-bated at 40 C in both the anaerobic incubator and the candlejar. Arthrospores had become round cells at 24 h (Fig. ld), and these had enlarged and theircontentsappearedgranular at 48 h (Fig. le). Endosporulating spherules

were obvious in 4 to 5 days (Fig. lf). Hyphal

formation was markedlyinhibited at 40 C and nonewly formed arthrospores were apparent.

Nonpathogenic fungi. Suspensions at zero

time contained arthrospores, singly and in

chains, as well as short strands of hyphae(Fig. 2a). Continuous incubation at room tempera-ture resulted in germination, formation of hy-phae, and development of new arthrospores in4 to 5days (Fig. 2b). A few of these cultures could

be easily confusedwith C. immitisby

inexperi-encedpersonnel. Some species did not grow at 37 or 40C, and the remainder developed

simi-larlyinboth the anaerobic incubator and

can-dle jar. Round cells were formed at 24 h (Fig.

2c). These either degenerated in 4 to 5 days

(Fig. 2d) or germinated into hyphae with chla-mydospores (Fig. 2e) and newly formed arthro-spores (Fig. 2f). None ever formed endosporu-lating spherules.

DISCUSSION

Three primary questions were considered in

this study. Would all isolates of C. immitis

develop endosporulating spherules on the

MCM-A medium? Would fungus species that

resembled C.immitisfailtodo this? Whatwere

the optimal conditions for consistent

morpho-genesis to endosporulating spherules and

con-venient use in clinical laboratories?

All 57isolates ofC. immitis convertedreadily from arthrospores to endosporulating spherules

but none of the nonpathogenic species did so.

Some of thelatterdidproducelarge round cells

comparable insize tospherulesand,therefore,

it isabsolutely necessary that an unknown

iso-lant be observed to produce and release

endo-spores (Fig. lf) before it is identified as C. immitis. This was detectable in some isolates

by3daysand in almost all by 4 days.Roberts et al. (12) reported 95% conversion by 3 days with their liquid culture method, so our method is

approximately 1 day slower. Significantly,

however, we found that the most recent iso-lantsdeveloped endosporulating spherules the mostrapidly.

Theculture medium and environmental con-187 VOL.3,1976

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188 SUN, HUPPERT, AND VUKOVICH

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FIG. 1. Morphologicalstages inthe developmentofC. immitis: (a) thearthrospore inoculum;(b) arthro-sporesgerminatingafter24hatroomtemperature; (c)typicalnewarthrosporeformation after 4 to 5days at roomtemperature;(d) round cellsformed from arthrosporesafter24 h of incubation at 40 C with 20%CO2;

(e) enlarged round cells with cleavage planes after 48 to 72 h of incubation at 40 C with 20% CO2; (/9

endosporulatingspherulesafter4to 5daysat 40 C with 20%CO2.

ditions used in this study resulted in consistent temperature, spherulation occurred readily and conversion of arthrospores to endosporulating mycelial formation was inhibited (see also

ref-spherules. Althoughthis occurred at both incu- erences 1, 4). Mycelia developed regularly

dur-bation temperatures, there were distinct ad- ingincubation at 37 C and, in addition, arthro-vantages favoring incubation at 40 C. At this spores were formed. These represent a definite

J. CLIN. MICROBIOL.

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RAPID IDENTIFICATION OF C. IMMITIS

hazard for infection of laboratory personnel whenslide cultures are removed from the incu-bation chamber for microscope observation.

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FIG. 2. Representative morphological stages in thedevelopment ofnonpathogenicfungi: (a) the

arthro-sporeinoculum; (b) newcropof arthrospores after5daysatroomtemperature; (c) round cellsformed from arthrospores after24hat40C with 20%C0,; (d) degeneratinground cellsafter5days at40C with 20%

C02;(e)formationofhyphaeandchlamydospores after5daysat40C with20%CO,;(/9 germinatinground

cells,hyphae,andnewcrop ofarthrospores after5daysat40C with20%CO,. ThefungiillustratedareA. umbrinum (a),A. reticulatus(b, /9,Pseudoarachniotussp. (c, d),andMyxotrichumsp. (e).

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190 SUN, HUPPERT, AND VUKOVICH

culturing demonstrated that all cultures were killed by this treatment. Thisprocedure is convenient, however, because only a single in-cubation time can be used or replicate slide

cultures must be prepared for each isolate.

Therefore, incubation at 40 C is recommended.

The increased CO2 tension hasbeen shown by

several investigators to favor development of

spherules (1, 10). In this regard, the simple

candlejar arrangement proved as effective as

thecontrolled gas mixture.

In our opinion, the slide culture method is more convenient than the liquid culture proce-dure described by Roberts et al. (12). Liquid

culturesrequired the use of 50-mlErlenmeyer

flasks, preliminary observation through the

bottom of the flask onthestage ofan inverted microscope todetermine that large round cells

hadformed, andpreparationof slidesto

estab-lish that endosporulating spheruleswere

pres-ent. Incontrast, a number of slide cultures can

betested simultaneouslyin a single candle jar

and theslidescanberemoved

daily

for

micros-copy andreturnedtothe candle jar for

contin-ued incubation if spherulation has not

oc-curred. Both methods require an incubator set

at 40 C, butonlyasmall one isneeded forthe slide culturemethod.

Although

webelievethat slide cultures incubated at40C do not repre-sent apotential for laboratory infection, some

personnelmay be uncomfortable with

handling

such preparations. If so, the original Brosbe

method can beused; i.e.,MCM-A insmallpetri

dishes and incubation ina candlejar at40C.

These dishes can be observed

daily

with

low-power objectives without

opening

the lidsand,

when large round cellsare seen, slides canbe

made to determine whether endosporulating

spherules are present. This would be an

ap-proach similar tothat of Robertsetal.:itoffers

theadvantage of smaller culturecontainers but

lacksthe convenience of slide cultures.

Finally, oneshould beaware that theuseof

refinedagaryieldsbetter resultsthan the usual

bacteriologicalagar. Brosbe

reported

that

Iona-gar no. 2 (Colab Laboratories, Inc.), purified

agar (Difco), and agarose (Bausch and Lomb,

Inc.) supported conversion of arthrospores to

endosporulating spherules equally well. Our

experience has been similar

and,

in

addition,

we have evaluated thesethree agars with re-spect to formation of

endosporulating

spher-ules. They are rated in descending order of

efficiency

as follows: Ionagar no. 2, agarose,

purified agar.

The conditions that are necessary to induce morphogenesis of arthrospores to endosporulat-ingspherules are relatively simple. These con-sist ofthe culture medium (MCM-A) and incu-bation at 40 C in an atmosphere withincreased CO2 tension. Thetemperature in the incubator around the cultures should be checked. If it exceeds 41 C, little or no growth will result.

The agar should be inoculated with a spore

suspensionsufficiently dense to ensure ease of locating cells undergoing morphogenesis; the denser the suspension, the better the conver-sion. In ourexperience, more than 90% of the arthrospores will convert to endosporulating

spherules. Furthermore, since completing

these studies, we have taught this method to

two persons from other laboratories and they

have achieved successfulconversions.

LITERATURE CITED

1. Breslau, A. M., and M. Kubota. 1964. Continuous in vitrocultivation of spherules of Coccidioides immitis. J.Bacteriol. 87:468-472.

2. Brosbe, E.A.1967. Useof refined agar for the invitro propagation of the spherule phase of Coccidioides immitis. J.Bacteriol. 93:497-498.

3. Converse, J. L. 1955. Growth ofspherules of Cocci-dioides immitis in a chemically defined liquid me-dium. Proc. Soc. Exp.Biol. Med. 90:709-711. 4. Converse, J. L. 1956. Effect ofphysico-chemical

envi-ronment on spherulation ofCoccidioides immitis in a chemically defined medium.J.Bacteriol.72:784-792. 5. Edwards,P. Q., and C. E. Palmer. 1957. Prevalence of sensitivity tococcidioidin with special referenceto specificandnonspecific reactions of coccidioidinand histoplasmin. Dis. Chest 31:35-60.

6. Emmons, C. W. 1967. Fungi which resemble Cocci-dioidesimmitis, p. 333-337.InL.Ajello (ed.), Coccidi-oidomycosis. University of Arizona Press, Tucson. 7. Friedman, L. D., D. Pappagianis, R. J. Berman, and C.

E.Smith.1953.Studies onCoccidioidesimmitis, mor-phology and sporulation capacity of forty-seven strains.J.Lab. Clin. Med.42:438-444.

8. Huppert,M., S. H. Sun, and J. W. Bailey. 1967. Natu-ralvariabilityinCoccidioidesimmitis, p. 323-328.In L.Ajello(ed.), Coccidioidomycosis. University of Ari-zonaPress, Tucson.

9. Levine, H. B., J. M. Cobb, and C. E. Smith. 1960. Immunitytococcidioidomycosis inducedinmiceby purified spherule, arthrospore, and mycelial vac-cines.Trans.N.Y.Acad. Si.22:436-449.

10. Lones, G. W., and C. L. Peacock. 1960. Role of carbon dioxideinthedimorphism of Coccidioidesimmitis.J. Bacteriol.79:308-309.

11. Pappagianis, D. 1967. Epidemiological aspects of respi-ratorymycotic infections. Bacteriol. Rev. 31:25-34. 12. Roberts, J. A., J. M.Counts,and H. G. Crecelius. 1970.

Productioninvitroof Coccidioidesimmitisspherules andendosporesas adiagnostic aid. Am. Rev.Resp. Dis. 102:811-813.

J. CLIN. MICROBIOL.

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ERRATA

Effect of

Types of Media on the

Production of Acid from

Glucose by

So-Called Glucose-Negative

Strains of Neisseria

gonorrhoeae

ELLEN S. BARON AND ARTHUR K. SAZ*

Georgetown University, Department of Microbiology, Schools of Medicine andDentistry,

Washington,

D.C.,20007

Volume3, no. 3,p.333,reference11:Changeto"Cultivation properties ofNeisseriagonorrhoeae

grown inchemically defined media. Can. J. Microbiol. 18:1087-1090."

Rapid

in

Vitro

Conversion

and

Identification of

Coccidioides immitis

S. H. SUN, M. HUPPERT,* AND K. R. VUKOVICH

Mycology ResearchLaboratory, Veterans AdministrationHospital, Long Beach, California 90801*; and

Department

of Medical

Microbiology,

University

ofCalifornia, Irvine, California90822

Volume 3, no. 2, p. 186,paragraphbeginning with"Cultures," line 11: Change "reticulatus" to

'umbrinum."

Volume 3, no. 2, p. 189, legend forFig. 2: Change"reticulatus"to"umbrinum."