Evaluation of the new API 20C strip for yeast identification against a conventional method

10, 0095-1137/79/09-0357/08$02.00/0

Evaluation of the New API 20C Strip for Yeast Identification

Against

a

Conventional Method

G. A. LAND,t* B. A.HARRISON,' K. L. HULME,1 B. H. COOPER,2 ANDJ. C. BYRD3

DepartmentofMicrobiology, WadleyInstitutesof MolecularMedicine, Dallas, Texas752351;Department of

Microbiology, Baylor UniversityMedicalCenter,Dallas, Texas 752462; andDepartment ofMicrobiology,St.

PaulHospital, Dallas, Texas752353

Received for publication1July1979

ThenewAPI20Cyeastidentificationsystemtogether with appropriate

micro-scopic morphology determinations achieved a97% correlation with arapid con-ventionalmethod. Whereasa groupcomposedofCandida, Torulopsis, Saccha-romyces, and Rhodotorulawasidentified withease (98%overallcorrelation), a

second group,containing Cryptococcus, Trichosporon,and Geotrichumspecies,

appeared to give the systemthe most difficulty (90% correlation). Within this

groupparticulardifficultywasencountered inidentifying varieties of Cryptococ-cusalbidus,C. terreus,C. laurentii, Trichosporonbeigelli, and Geotrichum spp. astospecies. The API20Csystemshould be incubated the full 72 h prescribed by the manufacturer. However, when used in conjunction with appropriate morphologicaltests,presumptiveidentifications ofsomeCandida andTorulopsis

species may be made at 24 to 48 h. To facilitate identifications of the more

difficultgroupofyeasts,ancillarytestsfordetermining nitrate reductase,urease,

and phenol oxidase activities should be considered as additions to the strip. Incorporating the phenol oxidasetestwould be especiallyimportant for identifi-cation ofCryptococcus neoformans,ayeastwhichshould be identifiedasquickly

andasaccuratelyaspossible. The API20Csystemwithcomputerassistance has proved to be an easy-to-inoculate, versatile, and fairly rapid method ofyeast

identification, giving results comparabletothoseobtainedby conventional meth-odologies.

Theavailabilitywithinthe pastseveralyears ofcommercialproducts whichaid in the acqui-sition andinterpretation of data for identifica-tion ofmedically

important

yeastshas rendered the task ofobtaining thisinformationmuchless demandingthanitonce was(7). Themajorityof the commercial products currently available provide carbohydrate assimilationtestsina con-venient plateorstrip form. Some products in-corporate carbohydrate assimilation as

well

as other biochemical tests, and these products eliminate the necessityforpreparingtestmedia

and

simplify

the storageof thelargevariety of

mediarequiredfor

identifying

yeastisolates(15). Althoughbased ontraditional methodology,the miniaturization of biochemical tests in these commercial kits permits the reading of results after a shorter period of incubation than was feasible with the

earlier

conventional methods (2, 13, 18, 19). Mostmanufacturersrecognize the necessity for conducting morphological exami-nations alongwithbiochemical testing and rec-ommend such procedures in the

instructions

t Presentaddress:DivisionofLaboratory Medicine, Uni-versityofCincinnati Medical Center, Cincinnati,OH 45267.

thataccompany theirproducts. Asreported in recentstudies (4, 5, 7, 15, 20), it is nowpossible with the use of these commercial systems to reliably identify most medically important yeasts within 48 to 72 h from the time biochem-icaltests are inoculated. In contrast, the more traditional methods require a maximum of 14 days forcompletion (1, 3, 9).

TheoriginalAPI20Cyeastidentification

strip

(Analytab Products,Div.of AyerstLaboratories, Plainview, N.Y.), which was one of the first commercialproductstobeintroduced fortesting medically important yeasts,

provided

media in dehydrated formfortestingboth

carbohydrate

fermentation and carbohydrate assimilation. These properties, when used in combination with morphological characteristics, permitted identification ofmany yeast species witha

re-spectable levelofreliability (5,15,20).

However,

certainaspects of the systemwereless

conven-ient thanwasdesired, and the data basewhich had been usedtodevelopthe systemwas

limited,

including onlyafew isolates of certain

species

of yeasts commonly encountered in clinical labo-ratories. The main technical difficulty in

using

357

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ET

the system involved the complexities offilling

the microtubules without trapping air bubbles

in theagar,which subsequently ledto

misinter-pretation of fermentation tests (5). To

circum-venttheserelativelyminordisadvantagesandto

improve theoverallcapabilitiesofthe system,a

new generation of the API 20Cwas introduced

inearly 1978. Thenew design incorporatedthe

following modifications of the original system:

(i) the use of assimilation tests only; (ii) larger cupules for easier filling; (iii) addition of new

substrates and elimination of those that had

been shown after extensive development

evalu-ation to provide imprecise differentiations of

species based on computer-assisted

interpreta-tion oftestdata(14); and,mostimportantly, (iv)

clinical evaluation of the system withan

exten-sive data base consisting of at least 20, and in

somecases over100, isolates of each of 16yeast

taxa. With this new system, a binomial profile

numbercanbe derivedfromgrowthpatternson

19 substrates. This profile number provides a

convenient method for comparing unknown

yeast isolates with those numerical profiles in

the data base in ordertoderiveanidentification

of the unknown (14). For biotypes thatare not

foundintheprofile index,aphone-incomputer

service is available fordeterminingalikely

iden-tification of unusual isolates.

Thefollowing is areport ofanevaluation of

the revised API20C, using clinical isolates

de-rived from thediagnostic mycologylaboratories

of three hospitals along with stock isolates of

selected species. The purpose of the studywas

to ascertain the level ofreliability of the API

20C system when compared with a rapid

con-ventional method(RCM)for yeast identification (11).

MATERIALS AND METHODS

Microorganisms. All of the yeasts used in this studywereeither clinicallaboratory isolatesorstock cultures from thefollowingsources:theWadley Insti-tutes of Molecular Medicine, the Baylor University Medical Center, and the St. Paul Hospital, Dallas, Tex.,and theUniversityofOklahomaatNorman. The following American Type Culture Collection isolates

were also used: ATCC 26310, Candida albicans;

ATCC24064,Cryptococcusneoformans;ATCC16725, Rhodotorula glutinis; ATCC 10663, Trichosporon capitatum;andATCC9331, Trichosporon pullulans. Other isolatesservingasknownpositivecontrols for identification were proficiency testing samplesfrom the NewYorkCityDepartmentofHealth,Collegeof American Pathologists, Skokie, Ill., and center for DiseaseControl(Atlanta, Ga.) proficiency testing pro-grams.Allclinicalisolateswereidentifiedbyone lab-oratoryandthencoded, randomized,and distributed to the other participating laboratories for identifica-tion and comparison. To complete the double-blind

J. CLIN. MICROBIOL. study, all stock cultures and otherstrains serving as

positive controlsweretreated inthesame manner.

API20Cyeastidentificationsystem.Thetip of awooden applicator stick was used to pick up aportion ofasmallcolonyof each unknown yeast to inoculate molten (42°C) API 20C basal medium, and cupules

were filledas per the manufacturer's directions. Also

in accordance with the manufacturer's directions, yeasts wereinoculated onto cornmeal agar plates via

the Dalmau culture techniqueformorphological

de-terminations. Both morphology and assimilation tests

were incubated at30°C,and results were recordedin the manner suggested by the API package insert.

Upon the finalobservation of the API system, a profile

number was assigned to each isolate and compared with profile numbers listed in the provided quick index. Occasionally, one numerical profile was

as-signedto two or eventhree species, in which case both

morphology and ancillary biochemical tests (i.e., ni-tratereductase, fermentations, and urease production) suggested by API were used to determine the final identification. In those cases where an organism

pro-ducedaprofilethat was not listed in the API profile

register, the API supplementary computer identifica-tionsystem wasutilized. Computer-assisted

presump-tiveidentificationwasdependent upon the calculation

of likelihood of occurrence between the unknown

yeast's biochemical characteristics and isolates with

similarcharacteristics present in the computer's data

bank.

RCM. The RCM system of yeastidentification,as

previously described(11), consistedof four

biochemi-cal tests: a dye pour plate auxanogram, Tween-80-oxgall-caffeic acid (TOC) medium, a 10-min swab nitrate test, and a 4-h urease test with urea R broth (Difco Laboratories, Detroit, Mich.). Briefly, the tests

weredoneasfollows:amodified DPPA medium

(10-12, 16),fordetermining yeast assimilation patterns on

14different carbohydrates,wascomposed of (per liter

ofwater): 20 g of agar, 0.67 gofyeastnitrogen base, and 20 mg of bromocresol purple. Ingredients were then solubilizedbyheating, and the pH of the molten

solution was adjusted to 7.2 and sterilized. Sterile,

molten dyeagarmedium wasasepticallydispensedin

60-ml portions into sterile prescription bottles and storedat4°Cuntilrequired. Forassimilation testing,

dyepourplateauxanogrammedium in twobottles was

melted, cooledto 40to43°C,inoculatedwith 5mlof

aMacFarland no. 5 suspension of yeasts, poured into

petriplates (150 by 15 mm), and cooled. Individual

stock solutions of 14 carbohydrates were made in normal saline (pH 7) at concentrations which would

dispensein0.1 ml that amount of carbohydrate

nec-essary for itsoptimum assimilation by yeasts. Stock

solutions were filter (0.45

[Lm;

Millipore Corp.,

Bed-ford,Mass.)sterilized, and 0.1 ml of each wasplaced

individually on a sterile 0.5-inch (ca. 1.3-cm) concen-tration disk. The following carbohydrates were

ar-ranged individually (7 per petri plate) on inoculated

andsolidifieddyemediumasfollows:plate1,dextrose,

galactose, sorbose, sucrose, maltose, cellobiose, and

trehalose; plate2, lactose, melibiose,raffinose,

mele-zitose, xylose, dulcitol,and inositol. Observations were

made after24and 48hof incubation at25'C, witha positive test recorded as either a reduction of the

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VOL. 10,1979

purple dye toyellowor, iftheplate hadcompletely

reducedto yellow,appearance ofgrowtharound the

carbohydratedisk.

Microscopicobservation ofpseudohyphae, hyphae,

arthrospores, and blastospores were made on TOC

plates. TOC medium hasalsobeen reportedto

pro-motegermtubes andchlamydosporesforappropriate Candidaspecies,in additiontothe above morpholog-ical characteristics(8).Caffeic acid addedtothe

me-dium served as a substrate for the phenol oxidase

reaction,anenzymaticreactionpresumably

character-istic of C. neoformans (17).The TOC medium

con-tained10gofoxgall (Difco),20gof Davisagar,0.3g

ofcaffeic acid,and 1 ml of Tween-80broughttoaboil

in 1,000 ml of distilledwater.Thesolubilized compo-nentswerethenautoclaved,andplates containing30

ml of mediumwerepoured.Driedplateswerestreaked

with asterile swab, depositing aheavy inoculumon

one corner of the plate and thencontinuing lightly

with the characteristicDalmautechnique, finally

over-laying the lightest streak withasterilecoverslip.The

heaviest streak wasusedto rapidlydetect the char-acteristic brownpigmentof C. neoformans, whereas

the lighterstreakwasusedto monitorspecific

mor-phological changesin theyeasts astheygrew.Once

inoculated, plateswereincubatedat37°C for3h, after

whichtheywere inspectedforgerm tubeproduction

and pigmentation, whereupon they were incubated

furtherat25°C. TheTOC platesweresubsequently

observedat6, 24,and 48 h for either brown

pigmen-tation, chlamydospore formation,orother

morpholog-icalchanges.

The presence of nitrate reductase in yeasts was

determinedbyswabs saturated witha fivefold-concen-tratedliquidmedium(pH5.8to6.0)containing2gof

KNO3,11.7gofNaH2PO4,1.14gofNa2HPO4,and 1.2

ml ofa17%solution ofZephiran chloridein 200mlof water(11).Dried swabswereinoculatedby sweeping

themacrossseveral colonies ofaplate and then

swirl-ingthemagainst the bottom ofanempty testtube (13

by150mm)toensurecontactbetweenorganisms and

substrate. The tube and swabwere incubated for10

minat45°C,andthe swabwasthenplacedinasecond

tubecontaining twodropseach of0.5%

ca-naphthyla-API EVALUATION 359

mineand 0.8% sulfanilicacid, each in5N acetic acid.

Apositivetestwasindicated by the swab tip turning

bright cherry red.

For theurease test,onevial ofureaR brothwas

reconstituted with distilledwateronthe day itwasto

be used. A0.2-mlamountof the resultant liquidwas

dispensed into eachwell ofa96-well microtitertest

plate(Microtiter II; Falcon Plastics, Oxnard, Calif.).A

heavy inoculum(3to4small colonies)of the unknown

yeastwastransferred viaawooden applicator tipto

the microtiter wells containing ureaR broth. Wells

weresealed with clearsealingtapeand incubated for

4hat37°C. Observationsweremade hourly, withany

change of thestraw-colored medium topink

consid-eredapositivetest(11).

RESULTS

Theoverallresults obtained with thenewAPI

20C correlated very closely with the RCM.

There were 1,063positive identifications outof 1,093 total cultures (97%), using the API bio-chemical testsin conjunction with morphology and anitrate reductase test (Table 1).

Ninety-two percent of these yeasts were compatible

with profiles appearing in the system's profile index; the remaining profiles (5%) weresimilar

to profiles stored in the computer's data base. Using the biochemical tests alone, 75% of the

isolates could be correctly identified with the

commercial system. Theaveragetime to positiv-ity for Candida, Torulopsis, Saccharomyces, and Rhodotorula species (group 1 yeasts) was

31 h, as opposed to 72 h for a more difficult group(group 2), composedofCryptococcus,

Tri-chosporon,andGeotrichumspecies.C.albicans

and Torulopsis glabrata could routinely be

identified within 24hby usingthe APIstrip in

conjunction with appropriate morphological changes. Approximately 5% of the yeasts

sur-veyed requiredcomputerassistancefor

identifi-cation, and 3%werenotidentifiablebyAPI20C.

TABLE 1. Correlationof API 20C with RCM in the identification of 1,093 yeasts from six medically

importantgenera

Correlation' Avg timetopositive Identification method No.of isolates

B B/M/NO3 API(h) RCM(h)

Quickindex

Group 1b 707 547(77) 677(96) 31 18

Group2C 386 275(71) 337(87) 72 30

Total 1,093 822(75) 1,014(92)

Computeraided

Group1b 21 (2.0)

Group2C 28(2.6)

Overallcorrelation 1,093 822(75) 1,063 (97)

a The numerical and percent (in parentheses) correlations of API 20CwithRCMby biochemicaltestsonly

(B)orby biochemistry, morphology, and nitrate(B/M/NO3).

b IsolatesrepresentingCandida, Torulopsis, Saccharomyces, and Rhodotorula species.

cIsolatesrepresenting Cryptococcus, Trichosporon, and Geotrichum species.

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360 LAND ET AL.

In the clinical portion of this study, 98% of group 1 yeasts were identified by the combina-tion ofAPI,morphology, and nitrate reductase, whereas86% of the organisms could be identified onthe basisofassimilationresults alone(Table 2). One isolate ofCandida solani and four of eight isolates of Saccharomyces cerevisiae, whose profiles were not inthe data base, were particularly difficult to identify with the API system.Group2yeasts identified with the three combinedtestsalsohad a 98% correlation rate withRCM(Table 3). Morphology wasextremely

TABLE 2. Clinicalcomparisonofthe API 20C yeast

identification systemversusRCM: group1

(Candida,Torulopsis,Saccharomyces,and

Rhodotorula)

Organism No. of iso-

Correlation'

lates B

B/M/NO3

C. albicans 64 89 100

C.tropicalis 80 100 100

C.parapsilosis 28 96 100

C. krusei 15 0 100

C. stellatoidea 3 100 100

C.guilliermondii 3 0 100

C.pseudotropicalis 3 100 100

C.lipolytica 3 0 100

C. solani 1 0 ob

T.glabrata 73 100 100

T.candida 2 0 100

S. cerevisiae 8 50 50b

R.glutinis 3 100 100

R. rubra 1 100 100

Avg 86 98

aPercent correlation of API 20C with RCM by

biochemical tests only (B) orbybiochemistry,

mor-phology, and nitrate(B/M/NO3).

bSomebiotypeswerenotin thecomputer.

TABLE 3. Clinicalcomparison of theAPI 20Cyeast

identificationsystemversusRCM:group2

(Cryptococcus,Trichosporon,andGeotrichum)

No.of iso- Correlation'

Organism lates

lts B B/M/NO3

C.neoformans 26 85 100

C.albidusvar.al- 3 100 100 bidus

C.albidusvar. 3 100 100

diffluens

C.laurentii 2 50 50

T.beigelii 3 67 100

T.capitatum 3 0 100

G.candidum 1 100 100

Avg 74 98

aPercent correlation of API 20C with RCM by

biochemical testsonly (B) or by biochemistry,

mor-phology,andnitrate (B/M/NO3).

important in the identification of these

orga-nisms, since only 74% could be determined by

biochemical tests alone. One clinical isolate of

Cryptococcus laurentii could not be identified

by both the combined tests and computer

as-sistance.

Results obtained in the clinical study

sug-gested that the data obtained for theAPIstrip

were often insufficient for separating Candida krusei,Candidalipolytica, and T.capitatumas well asin identifying Cryptococcus, Trichospo-ron, and Geotrichumspecies.Thestockculture

portion of the study was weighted in favor of

theseorganisms in ordertoprovidea severetest

of the capabilities of the API system. Among those yeasts in group 1 of the stock culture study, Candida stellatoidea proved to be the

most difficult species for the API system to

identify consistently (Table 4). Approximately 58% of the isolates utilized the API trehalose; however,accordingtothe API percentagechart, only 1% should have reacted positively. The

RCMtrehalose, onthe otherhand,was

assimi-latedby all isolates of C. stellatoidea. Moreover,

some13%of thesewerenegativeonAPImaltose,

whereas according to the percent chart and RCM all isolates should have grown on the

substrate. Thesediscrepanciesgavethe API

sys-tema51% overallefficiency rating for the

iden-tification of C. stellatoidea. Several organisms werefound which had no correlating profile in

the API database including: 4 of 15 isolates of

S.cerevisiae,2 of 11isolates of R.glutinis, 1of

2 isolates of R. rubra, 3 of 80 isolates of C.

TABLE 4. Comparisonof identification of stock

cultures by the API 20C system with RCM: group 1

(Candida,Torulopsis,Saccharomyces, and Rhodotorula)

No. of

Correlation'

Organism ioae_{isolates B}

_B/M/NO3

C. albicans 80 88.8 96.2

C.parapsilosis 44 95.4 95.5

C.tropicalis 80 100 100

C. krusei 60 0 100

C.stellatoidea 27 51.8 51.8

C.guilliermondii 10 60 100

C.pseudotropicalis 3 100 100

C.lipolytica 8 0 100

T.glabrata 73 100 100

T.candida 2 0 100

S. cerevisiae 15 46.6 74.0

R.glutinis 11 27.2 73.0

R.rubra 7 14.2 86.0

Avg 71 94

aPercent

correlation of API 20C with RCM by

biochemicaltests only (B) orby biochemistry,

mor-phology,and nitrate(B/M/NO3).

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VOL. 10, 1979

albicans, and 3 of 44 isolates of Candida

par-apsilosis. Thesebiochemicallyvariable isolates ofC. albicansandC.parapsilosisalsoexhibited aberrantmorphologies, making their identifica-tions by eithersystemdifficult.

In addition, itwas difficult to identify as to

species stock culturesbelongingtogroup2with the APIyeast system. Seventy-one percent of this group were identified on the basis of

bio-chemicaltestsalone,and 86%wereidentifiedby using the threecombined tests(Table 5).

Cryp-tococcus terreus and Geotrichum candidum werethemost difficult isolates toidentify.

Iso-lates ofC.terreusvaried inabilitytoassimilate inositol, with only 57% becoming positive after 96hofincubation. 2-Ketogluconate,asubstrate

utilized by thesameyeaststhat metabolize ino-sitol, also demonstratedthesamevariability in

assimilationasdid inositol, further compounding

the problems in

identifying

theseyeasts. Geotri-chum species did not regularly assimilate API glycerolandxylose, making their identification also difficult. The problems experienced with the API system in

identifying

other group 2

organisms alsorelatedtofalse negative assimi-lations ofinositol orother key substrates. For example,3outof164isolatesof C.neoformans

didnotassimilateinositol after 96 h of

incuba-tion,

and 10 of 81 Cryptococcus albidus var.

albidus

and 5 of 26 C. laurentii

(Table 6)

also

failedtoassimilate thiscarbohydrate. Twelve of

TABLE 5. Comparison of the identification of stock cultures by the API 20C system with RCM: group 2

(Cryptococcus, Trichosporon, and Geotrichum)

No. of Correlationa

Organism

isolates B R/M/NO3

C.neoformans 138 93.1 94.3

C. albidus var. al- 78 67.4 79

bidus

C.albidus var. dif- 34 78.5 78.5

fluens

C.laurentii 24 37.5 87.5

C.terreus 7 28.5 28.5

C.uniguttulatus 3 100 100

T.beigelii 41 51.5 88

T. capitatum 13 0 100

T.penicillatum 2 100 100

G.candidum 5 20 20

Avg 71 86

aPercent

correlation of API 20C with RCM by

biochemical tests only (B) orby biochemistry,

mor-phology, and nitrate(B/M/NO3).

TABLE 6. Comparison of selected positive assimilations in the API 20C system with RCM

Assimilation(%positive) Organism No.ofisolates Substratea

Predicted value API RCM

C. albicans 144 Mlz 2 13 0

Gly 9 0 b

Ara 3 11

-C.parapsilosis 72 Miz 99 100 100

Gly 74 99

-Ara 99 94

-C.tropicalis 160 Mlz 100 100 100

Gly 11 35

-Ara 3 7

-Cel 12 8 86

C.stellatoidea 30 Tre 1 58 100

C.neoformansc 164 Ins 97 90 100

Ara 8 14

-Xlt 0 12

-C.laurentiic 26 Ins 84 80 100

Ara 99 95

-Xlt 84 87

-C. albidusvar.albidusc 81 Ins 83 88 100

Ara 88 80

-Xlt 11 14

aMlz, Melezitose; Gly, glycerol; Ara, arabinose; Cel,

cellobiose;

Tre,trehalose; Ins,inositol; Xlt,xylitol.

b_,

SubstratenotpresentintheRCM protocol.

'A numberof the cryptococci had to be incubated for an additional 24h (i.e., 96-htotal) inorder for the

inositol reaction to become positive.

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TABLE 7. Yeasts identifiableby RCM which generated profile numbers not compatible with the API data base

No. of Comments

Organism

iolate APIprofileno.

Isolates

Candidahumicoli 6 6777773 Same profileas C.laurentii,but differentmorphology

Candidasolani 1 6102231

Candidautilis 1 6404273

Saccharomyceschevalieri 3 2040022 Same assimilation profile as S. cerevisiae with API,

butseparable by RCM

Saccharomyceschampagni 1 2040032 Same asS.chevalieri

Kluveromyces fragilis 4 6660422 Sameprofileas Candida pseudotropicalis with API, butascospore positive and separable by RCM

Kluveromycesbulgaricus 3 6060422 Sameas Kluveromyces fragilis

1 6662422 1 6460422

Kluveromyces lactis 1 6046673 Ascospore positive

Pichiaohmeri 4 6156372 Ascospore positive

Rhodotorula 2 2670063

Rhodotorulaglutinis 1 6672062 Nitrate reductase positive

Aureobasidiumsp. 3 6777773 May at first be white and have same profile as C.

1 6773373 laurentii and T. beigelii, but becomes dematiaceous

with characteristicmorphology upon aging

Ustilago sp. 1 2747573

81 isolates ofC. albidusvar.albidus, whichwere

lactose positive by RCM, werenegativeon the

corresponding API substrate andwere forthat reasoninseparable from their sibling species C.

albidusvar.diffluens.

Of thoseorganismswhichwerenotidentified

by theAPI20Csystem,50%were

ascosporogen-ousyeasts(Table 7). Three isolatesidentifiedas

S. cerevisiae by API20C weretermed S.

chev-alieriiby the RCM. These organismshad simi-lar assimilation profiles but differed in their

fermentation patterns. Kluveromyces fragilis

(four strains) and K. bulgaricus (three strains)

hadthesameassimilationprofilesasand similar

morphology to Candida pseudotropicalis, but they differed in that they formed ascospores.

Candida humicolagenerated profilessimilarto

those of both C. laurentii and Trichosporon

beigellii, soidentification reliedheavilyupon a

critical evaluation ofmorphology, regardless of

the system used. Another yeastlike organism

which was confused with C. laurentii was

Au-reobasidiumspecies.These isolatesappearedin

early cultureas awhiteyeastwith biochemical

properties identicaltoC. laurentiiorT.beigelii,

but after extended culture they formed both

hyalineanddematiaceoushyphae.

DISCUSSION

The newAPI 20C systemhad a high degree

ofcorrelation with conventional methodology,

providing that adjunctive tests of morphology

andnitratereductasewereused. The lattertest

wasespecially important inidentifying Crypto-coccus and Rhodotorula species. Pinello et al.

haveplacedspecial emphasisuponshowingthat morphological examinations in conjunction with the APIbiochemicaltests arenecessaryfor

com-plete yeast identification (14). Thisnecessityfor morphological examination in yeast identifica-tionwasagainunderscored inourstudy bythe

fact that only 75% of these yeasts could be identified on the basis ofbiochemical activity alone. To emphasize this point, the data have

beenpresentedboth with and without morpho-logical characteristics being taken into consid-eration. The overall correlation of API with

conventional methodologyof97%wasin

agree-mentwith whatBueschingetal. found (96%) in evaluatingthe systemagainst505organisms (6). Parallelingourexperience,Buesching etal. also found that fresh isolatesappearedtogrowmore

rapidly and to give fewer ambiguous reactions than did stock cultures.

Thereare twoclusters ofmedically important yeastswhich areofparticular importance, and, for this reason, theymust be rapidlyand

accu-rately identified.The first clusterconsists of C. albicans, C.parapsilosis, and Candida tropi-calis, since it is possible for them to exhibit similarmorphologies and assimilation patterns

on traditional media and substrates. The API

system is designedto separatemembers of this groupby their respective utilizations of melezi-tose,glycerol, and arabinose.Melezitose, accord-ingto the APIdatabase, is assimilated by

vir-tually all isolatesofC. tropicalisandC. parap-silosis but by 2% of C. albicans isolates. We found that the degree of melezitose

positivity

among C. albicans was much higher than the

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VOL. 10,1979

predicted percentage and would have ledtoan identification ofC. tropicalisorC.parapsilosis, especially for those isolates which didnotform chlamydospores(Table6).Glycerol,asubstrate used for delineating C. parapsilosis from C. albicans and C.tropicalis,yielded similar incon-sistencies. Glycerolwasassimilatedby virtually all C. parapsilosis isolates, butnotby C. albi-cans, whereasoverone-third of the C.tropicalis isolates were positive, instead of the expected 11%. Itappeared thatarabinoseassimilationwas not asvariable a characteristic as those above and servedas anexcellentmeans ofseparating C.parapsilosis from C. albicans and C. tropi-calis.

Theaddition ofa germ tube test to API20C wouldprovideagood backuptestfor cornmeal-Tween-80 agarmorphologyandwould also help to split C. albicansawayfrom C.parapsilosis and C. tropicalis (7, 17). This additional mor-phologicaltestin tandem withmelezitose, glyc-erol, and arabinose assimilations wouldhelpto remove some of the ambiguity in relying upon assimilation entirelyas a meansofidentification. Cellobiose assimilation has beenshown to be an efficientmeansofseparating C. tropicalis from C. albicansandC.parapsilosis (4-6, 11), andit could alsoaugmentthe otherkeyAPIsubstrates iftheproblem of itsvariable assimilation could beovercome. The assimilation ofcellobiose as wellas other carbohydrates byyeastshas been shown to be afunctionof aspecificconcentration range for each substrate and of the nitrogen contentof themedium (12).

The

failure

toutilizethe optimum

concentra-tionforeach substrate as wellas therelatively high nitrogencontentof the mediummight also explain the failure of several other yeasts to grow onsubstratesthatshouldhave been assim-ilated. Trehalose, commonlymetabolized by C. stellatoideainother systems and used by

all

of theseisolatesonRCM, had a 50%falsenegative rate onAPI. Furthermore, inositol, a key sugar in theconventional identification of Cryptococ-cusspecies (3, 11, 13, 17), also was not utilized by 100% of cryptococci grown on APImedium. Particularlynoteworthy was the fact that only 26% ofC. terreus isolates utilized this carbohy-drate.Among thecryptococci, inositol

assimila-tionswere soweak that they had to be held an extra day to be considered positive, and 10 to 15% didn't grow at all. 2-Ketogluconate, a backuptestfor inositol assimilation, had a sim-ilar 10 to 15% false negative rate instead of the near 100% positive rate expected from the per-centage chart. These false negative

assiimilations

led to somedifficulty in identifying the various cryptococci, anexperience also noted by

Buesch-API 20C STRIP EVALUATION

ingetal.(6).

Thesecondclusterof yeasts ofmedical inter-est arecomposed of C.neoformans, C.laurentii, and C. albidus.These species appear to be fairly separableby the API system. Xylitol appears to be anadequate substrate for delineating C. lau-rentiifromC. neoformans and C. albidus, with 100% of C. laurentii utilizing the substrate whereas onlyabout 14% of the other cryptococci werepositive.Arabinose was assimilated by 12% of the C.neoformansisolates in this study rather than the predicted0%. This wouldstillbe a fair characteristic foridentification, since 95% of the C. laurentiiand80% ofthe C. albidus metabo-lizedthe substrate. However, due to the medical importance of determining the presence of C. neoformans in a clinical specimen, we feel an adjunctive test for phenol oxidase activity should be provided with the kit. This test is presumably specific for the identification of C. neoformans and couldeasily be adaptedtothe API20C (8,11, 17).

Baseduponour experience with the new API 20Cyeastidentificationsystem, weconclude the following: this identification system, together with the recommendedmorphologicaltests, cor-relates well with conventional methodology. However,someof the biochemicaltestschosen by the computer as a means ofseparating cer-tain taxa and the computer'suseof these data differconsiderably from conventional testsand dichotomies butmay, withtime,proveequalto

conventional methodsorperhaps even tobe a more accurate approachtothe identification of

medically

importantyeasts.

ACNOWLEDGMENT

Thisworkwassupportedinpartbythe Sammons Foun-dation,Dallas, Tex.

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