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Vol. 30, No. 12 JOURNALOFCLINICALMICROBIOLOGY, Dec. 1992,p. 3132-3137

0095-1137/92/123132-06$02.00/0

Copyright X 1992, AmericanSocietyforMicrobiology

Detection of Serum Candida Antigens by

Enzyme-Linked

Immunosorbent Assay and

a Latex

Agglutination

Test

with

Anti-Candida albicans and Anti-Candida krusei Antibodies

SHIN-ICHIFUJITA* ANDTAKUMA HASHIMOTO

Central ClinicalLaboratory, Kanazawa UniversityHospital,

13-1,

Takara-machi,

Kanazawa

920,

Japan

Received 3 March 1992/Accepted 1 September 1992

Serum samples from 197 patients with and without candidiasis were assayed for Candida albicans mannan andCandida krusei mannan by an enzyme-linked immunosorbent assay (ELISA) and a latex agglutination test (LA) and for D-arabinitol by the enzymatic fluorometric method. Of the 43 patients positive for C. albicans mannan ('0.2 ng/ml), 34 were infected with C.albicans and 9 were infected with Candida tropicalis. C. krusei mannan (_0.3 ng/ml) was detected in 10 patients infected with Candidaparapsilosis,2patients infected with Candidaguilliermondii,and 1patient infected with C.krusei.With both anti-C. albicans antibodies and anti-C. kruseiantibodies, the sensitivities of ELISA and LA for detection of invasive candidiasis (58 patients) were 74 and38%,

respectively.

Nofalse-positive reactions were observed by the ELISA or the LA. Thesensitivityand specificity of the D-arabinitol/creatinine ratio (_1.5 ,umol/mg) to invasive candidiasis were 50 and 91%,

respectively.

The ELISA with antibodies against both C. albicans and C. krusei may be useful in diagnosing invasive candidiasis caused by

medically

important Candida strains excludingCandidaglabrata.

Invasivecandidiasis remains asignificant complication in

patients with hematologic malignancies, patients receiving total parenteral nutrition, andpatientswith solid tumors in whomabdominal surgery has been performed. The diagnosis ofinvasive candidiasis is made by the frequent isolation of Candida species from normally sterile sites; histological

evidence of Candida invasion; or positive results for cell

wall mannan (3, 9, 17), enolase (16), or D-arabinitol (3). Candida albicansmannan hasbeen detected in the sera of

patientswithC. albicansorCandida tropicalisinfections by

radioimmunoassay (17), enzyme-linked immunosorbent as-say

(ELISA)

(3, 9), and the latex agglutination test(LA) (1, 12). The specificity of the ELISA formannan was 95% or

more,butthe sensitivities variedbetweeninvestigators and

ranged between 22 and 100% (6). On the other hand, Candida

parapsilosis,

Candida

guilliermondii,

Candida

krusei,

andCandidaglabrata have also been isolatedfrom blood cultures(5). Therefore, sensitivity mightbe increased if mannan aswell as other polysaccharide antigens of

Can-didaspeciesweredetected. This report describesanELISA andanLAusedtodiagnose invasive candidiasiscausedby medically important Candida isolates.

Candida metabolites can be detected in the serum of affected patients by the enzymaticfluorometric assay (15), which is now commercially available as a D-arabinitol test kit,andby gas-liquidchromatography(3).Becausethe latter method is morecomplex, aD-arabinitol kitwasused in the present study. These resultswere compared with those of testsfor serumCandida antigens.

MATERIALSANDMETHODS

Yeast isolates. Thirty-six Candida strains isolated from clinical materials and maintained in our laboratory were

used. One reference strain ofC. albicans serotypeB (IFO 10108) was obtained from the Institute for Fermentation,

*

Corresponding

author.

Osaka, Japan. Each isolatewasidentifiedby usingCandida Check (latron Laboratories, Tokyo, Japan) with an addi-tional germ tubetest andby examining morphological char-acteristics. Microbiological observations of pseudohyphae, hyphae, and chlamydospores were made on cornmeal-Tween80 agarincubated at 35°C for 3 days.

Culture conditions.Candida isolatesweregrownin Yeast

Nitrogen Base broth (Difco Laboratories, Detroit, Mich.)

with0.3% glucosefor 24 hat35°C. Thepelletswerewashed

withsteriledistilledwater,and about105washed cellswere

inoculated into 2 ml of Yeast Nitrogen Base broth with glucose andinoculatedon ashaker(150 rpm) at35°Cfor 22

h. Afterboilingina waterbath for5min,the cultureswere centrifugedat3,000 xgfor 10min.Then,the supernatants werediluted 1:2indistilledwaterforD-arabinitol

measure-ment, and serial 10-fold dilutions (1:10, 1:100, and 1:1,000) weremade for antigen detection.

Antiserum preparation.Six Candida isolates (C albicans serotypeA,C. tropicalis, C. parapsilosis, C. guilliennondii,

C.

knrsei,

and C.

glabrata)

from blood cultureswere used.

First, each of the isolateswasgrown on Candida GS agar

plates(Eiken ChemicalCo. Ltd., Tokyo,Japan),harvested

by centrifugation, and washed three times in sterile saline.

Next, Japanese White rabbits were inoculated subcutane-ouslywith a1:1 emulsion(1 ml)ofa10% salinesuspension

of heat-killed (80°C for 30

min)

cells and Freund

complete

adjuvant. Two weeks later, the rabbits received another inoculation in the same manner. Twoweeks after the last

inoculation, intravenous injectionsof about 108 heat-killed cells were given weekly, and the rabbits were tested for

antibodyresponsesbythehomologousagglutinationtestand thehemagglutinationtest

(Hoffmann-La Roche, Basel,

Swit-zerland)4 daysafter each intravenous inoculation. Whena

homologous agglutination titer of 1:128 or more was

ob-tained, blood was collected, and the serum was stored at

-80°Cuntiluse.

Patients and controls. During a 5-year period from July

1987toJuly 1992,540serumsampleswereobtained from 197 3132

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hospitalized patients, and all sera were frozen at -80°C.

Seraweretakenat theacutestageofinfection, andatleast

twoserum samples perpatientwere tested. When positive resultsfor antigens or theD-arabinitol:creatinine ratio were

obtained, sera were taken twice a week thereafter. Sera drawn during the administration of D-mannitol were ex-cluded. Patients were divided intofivegroups on thebasis of clinical andmicrobiological information. Group 1 (invasive

candidiasis)consistedof16patients (72serumsamples)with

deep-seated candidiasis confirmed by histological evidence atautopsy or surgery and 42patients (178 serum samples)

who had two or more positive blood cultures drawn over a

period of days. Group 2consisted of 20 patients (52 serum

samples) with possibleinvasive candidiasis, defined by sus-tained feverdespite antibacterial therapy. Antifungalagents

such as amphotericin B, miconazole, or fluconazole were administered parenterally to all patients in this group for treatment of suspected candidiasis. Cultures of sputum(12 patients), urine(2patients), orcathetertip(4 patients)were

positive forC. albicans. C. parapsilosis was isolated from

theperitonealfluidofonepatientand from the cathetertipof onepatient. Group 3(transient candidemia) consisted of 10

patients who had only one positive blood culture for Can-dida species (6 patientswithC. albicans,2patientswithC. parapsilosis, and 1 patient each with C glabrata and C. tropicalis) and whose temperature had fallen to normal

within 2 days of the candidemia. Group 4 consisted of 54

patients with bacterial sepsis.Thesepatients madefavorable progress after parenteral administration of antibiotics. Group 5 consisted of 55 cancer patients without clinical

evidence of infection. Forty-two normalhealthy adults were also includedinthestudy.

ELISA for antigens. TheELISA technique for detecting Candida antigens is similartothatdescribedpreviously(4). Briefly, polystyrene microtitration plates (Immulon II;

Dynatech) were coatedwith0.1 ml of the diluted

immuno-globulin G (IgG)

(anti-C.

albicans IgG, 5 ,ug/ml; anti-C krusei IgG,10

,ug/ml)

in0.1Mbicarbonatebuffer(pH 9.6)at 25°C for 2.5 h and then at4°C overnight. Theplates were washed with

phosphate-buffered

saline (PBS) containing 0.05% Triton X-100 (PBST) by using a microplate washer.

After 0.1 ml of heat-treated (9) or protease-heat treated serum samples was added to the wells, the plates were

incubatedat25°Cfor2.5h. Eachwell was washedfivetimes withPBST, after which 0.1 ml of diluted biotin-linked IgG(in PBSTcontaining 0.5%bovine serumalbuminand 1% poly-ethylene glycol 6000)was added, and theplateswere

incu-batedat25°Cfor 1.5 h. Afterfive morewashes,0.1 ml ofa 1:2,000 dilution ofstreptavidin-horseradish peroxidase

con-jugate(BethesdaResearchLaboratories)wasaddedand the

plateswere incubatedat25°C for 30min. Todetermine the

optimal dilutions of the biotin-linked IgG and

streptavidin-enzymeconjugate, checkerboard titrations were done. The unbound conjugate was removed by washing thoroughly,

and a substrate (0.1 M citrate buffer [pH 4.5]) containing 0.1%

o-phenylenediamine

and 0.012% hydrogen

peroxide

wasadded at 0.1 mlper well. Theenzyme-substratereaction was allowed toproceed at25°C for 15 min, and the actual

A450 was measured on a spectrophotometer. Pooled sera

containingantigensatconcentrationsrangingfrom 0.125 to 4

ng/ml

were included on each plate. The concentrations of

antigensin the serum samples were extrapolated from the standard curve.

Candidapolysaccharide antigensofC. albicans serotype A,

C.

glabrata,

C.

guilliermondii,

C.

krusei,

Cparapsilosis,

andC.tropicaliswereprepared bythe method of Meisteret

al. (10). Carbohydrate was measured by the anthrone

reac-tion by using D-mannose as a standard (7). For thin-layer chromatography, samples of thecarbohydrate preparations

(5 mg/ml in 2 N HCl)were hydrolyzed for 2 h in a boiling water bath. Thin-layer chromatography was carried out on

cellulose F 254 (Merck, Darmstadt, Federal Republic of

Germany) with pyridine-ethyl acetate-water (26:66:8; by volume) asthesolvent(10). Protease and heat treatmentof

serum samples was performed as follows: 0.3 ml ofeach samplewasmixedwith40,ulofprotease(20 mg/mlinPBST

[typeVIII;Sigma]), incubated for 30min at45°C, andplaced

in a boilingwater bath for 5 min. The mixture was then centrifuged, and thesupernatantwasused for antigen detec-tion.

LAfor antigens. Polystyrenebeads (0.8

pum

in diameter;

Difco) were coated with antibodies against C. albicans (LA-CAreagent) orC. krusei (LA-CK reagent). Inbrief, 1

volumeof latex beads was added to the same volumeofIgG

solution diluted with glycine-buffered saline (0.85% NaCl

and0.75% glycine [pH8.4]),and themixturewasincubated

at roomtemperaturefor2h. Aftercentrifugationat3,000 x

g for 10 min, the pelleted latex beads were washed three

times with glycine-buffered saline and resuspended in 2 volumes of glycine-buffered saline containing 0.1% bovine

serumalbumin and0.05% sodium azide. The sensitivitiesof

the LA reagents were assessed for each run by testing pooled serum containing mannan antigens at final

concen-trations of 0.5, 1,2,or4ng/ml. To25 ,ul ofprotease-treated

serum, 25

RI

of latex reagent was added and mixed on aslide

for 10min at 160 rpm in a moist chamber.

D-Arabinitolmeasurement.D-Arabinitol was measuredby

using acommercialtestkit fromNacalaiTesqueCo., Ltd., Kyoto,Japan.Inbrief, 0.5ml ofsamplewasmixedwith1ml of 0.05 mol ofcitrate buffer(pH4.0)per liter. The mixture was placed in a boiling water bath for 5 min and was

centrifuged at 1,500 x gfor 10min. Then, 2 ml ofreaction

mixture containing resazurin, NAD, and diaphorase was

addedto0.3 ml of the

supernatant.

Afteradding 0.05 ml of D-arabinitol dehydrogenase solution to the mixture, the increase influorescencewasmeasured witha spectrofluoro-photometer (15).

RESULTS

Homologousagglutination titers of 1:256 to1:2,048

(indi-recthemagglutinationtiters of 320 to20,480)wereobtained.

The ELISA withantiserumagainst

C.

albicansor C.

tropi-calis detected both

C.

albicans antigens and C.

tropicalis

antigenswith almost the same sensitivity

(Table

1). In the

detection of Candidaantigens

excluding C.

albicans and

C.

tropicalis, ELISAwith anti-C krusei antibodies was some-what more sensitive than that with antibodies against

C.

parapsilosis,

C. guilliermondii,

or C.

glabrata.

From the resultsgiveninTable 1,bothantibodies

against

C. albicans

and antibodiesagainst

C.

kruseiwerechosen for the detec-tion of Candida antigens. The

patterns

obtained by

thin-layer chromatographywere identical for each antigen and showed one strong spot whichcorrespondedwithmannose.

Broth culturesof C albicans serotypeB,C. parapsilosis, C.

guilliermondii,

C.

krusei,

and C.

glabrata

revealed C.

albicans antigen levels of 0.01 ,ug/ml or less (Table 2). C.

krusei

antigen was not detected from the broth culture of

C.

glabrata.

D-Arabinitol concentrations

ranging

from 84to

480

p.mol/liter

weredetected in broth cultures ofC. albicans serotypes A and B, C

tropicalis,

C.

parapsilosis,

and C.

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3134 FUJITA AND HASHIMOTO

TABLE 1. Absorbance of brothcultures of Candida isolates A450byELISAwith antibodies against: Broth culture (1:100)

Calbicans C.glabrata C guilliennondii C krusei C. parapsilosis C. tropicalis

C. albicans serotypeA 0.985 0.147 0.179 0.337 0.161 1.099

C. glabrata 0.044 0.289 0.052 0.099 0.106 0.033

C guilliermondii 0.055 0.162 0.344 0.515 0.059 0.055

C. krusei 0.064 0.097 0.191 >1.500 0.179 0.071

C.parapsilosis 0.065 0.162 0.143 0.286 0.298 0.081

C. tropicalis 1.488 0.575 0.291 0.297 0.099 1.433

YNWB 0.032 0.054 0.084 0.057 0.055 0.038

aYNB,YeastNitrogenBasebrothwith 0.3% glucose.

guilliennondii, whereas cultures of C krusei and C. glabrata showed D-arabinitolconcentrations of 10 ,umol/literorless.

Figure 1 shows the standardcurvesof C. albicans and C

krusei mannans according to serum treatment. The upper

limit of negativity was determined by adding 3 standard

deviations of about0.06 eachtothe absorbance of the pooled

serumsamples from normal healthy adults. With the prote-ase and heat treatment, this value was 0.2 ng/ml for C.

albicans mannanand was 0.3 ng/ml for C krusei mannan.

With heattreatmentof thesamples, the sensitivity limitwas

0.3ng/ml for C. albicansmannan,whereasitwas0.5 ng/ml

for C. kruseimannan.From theseresults,proteaseand heat treatmentwaschosenforsubsequent studies. The

sensitiv-ities of the LA-CA and LA-CKtests were 1 and 4 ng/ml, respectively.

The results oftestsfor antigens and D-arabinitol in various

groups are given in Table 3. C. albicans mannan was

detected in 31 patients (116 serum samples) with invasive

candidiasis and in 12 patients (29 serum samples) with

possible invasive candidiasis. C. albicansmannanlevels in

serumranged from 0.2to4.8 ng/ml. C. krusei mannan was

positive in 12 patients with invasive candidiasis (34 serum

samples) and 1 patient with possible invasive candidiasis (3

serumsamples). The concentrations of C kruseimannan in serum ranged from 0.3 to 2.3 ng/ml. Of 56 antigenemic patients, persistentorintermittentantigenemiawasshown in

48 patients with invasive candidiasis and 8 patients with possible invasive candidiasis. In accordance with the reduc-tion in fever, the antigenemia disappeared in six patients with invasive candidiasis and two patients with possible invasive candidiasis.

The LA-CA and LA-CKtests showedpositive results in 22patients (15 patients with C. albicans and 7 patients with C tropicalis) and 5 patients (4 patientswithCparapsilosis

and 1patient with C krusei), respectively.

The definition of a positive test result was a sample

concentration with a D-arabinitol/creatinine ratio greater

than themean + 3 standard deviations in patients without infections. This valuewas 1.5 p.mol/mg.Of 46patients with elevatedD-arabinitol/creatinine ratios, 29 patients had inva-sive candidiasis. Elevated D-arabinitol/creatinine ratios

re-turned tonormal in five patients with invasive candidiasis, three patients with possible invasive candidiasis, three

pa-tients with transient candidemia, and three patients with bacteremia. Although no false-positive reactionswere

ob-served in either theELISAorthe LA, 10 control patients in

groups 4 and 5 had elevated D-arabinitollcreatinine ratios. Among 58 patients with invasive candidiasis, 43 showed positive results for ELISA with antibodies against C.

albi-cansorC krusei, whereas only 22 had positive LA results.

The sensitivities of the ELISA, the LA, and the enzymatic D-arabinitol test for invasive candidiasis were 74, 38, and

50%, respectively. The specificities of the ELISA, the LA, and the D-arabinitolassay were100, 100, and 91%,

respec-tively.

DISCUSSION

Previous studies have shown that Candidamannan

anti-gen detection in the diagnosis of invasive candidiasis is inconsistent probably because of the low detection limits (generally above 1 ng/ml) of variousdetection systems(1, 4, 8). InourELISA, the detection limitwas0.2ng/ml, whereas

itwas0.8ng/ml inaprevious study (4). This improvementin

sensitivitymaybe duetoantisera withhigh titers against C albicans mannan and the use ofprotease treatment rather than the boiling method for the dissociation of antigen-antibody complexes. Antimannan serum was readily

ob-tainedby intravenous (9, 13)orsubcutaneous(17) injections ofheat-treated C. albicans. However, serological tests for Candida antibody such as agar gel diffusion, whole-cell agglutination, and LA have not been reliable, and the

necessity for standardizedreagentsforantibody titration has beenstressed (11). Weused acommerciallyavailable

indi-TABLE 2. Antigen and metabolite concentrationsofmedicallyimportantCandidaspeciesinbrothcultures

Meanantigenconcn(pg/ml[range]) detected by D-Arabinitol

Organisms No. of strains ELISA with antibodies against: concn(pmoV

tested ltr[ag]

C.

albicans C

kusei

liter [range])

C. albicans serotypeA 13 16(2.0-56) 0.70(0.68-0.75) 200(84-350)

C. albicans serotypeB 1 <0.01 0.39 260

C.glabrata 5 <0.01 <0.01 <0.05

C.guilliennondii 5 <0.01 0.38(0.31-0.49) 270(230-310)

C kusei 1 <0.01 28 9.2

C.parapsilosis 7 <0.01 1.5(0.5-2.5) 280(170-360)

C. tropicalis 5 29(7.0-42) 0.19(0.11-0.38) 250(160-360)

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B

Protease

Tr

T

A

1 / ; Boiling

A0~~~~~~~

JL ~ I

0.125 0.25 05 2 4

C.albicans mannan, ng/ml C.krusei mannan, ng/nl

FIG. 1. Standard curvesof theenzymeimmunoassay analyzed according tothe mode oftreatment ofserum samples (0, boiling; 0,

protease), preparedinduplicatewithknownconcentrations of C. albicansmannan(A)and C. k,useimannan(B)inpooledserafrom normal healthyadults. Means standarddeviationsaregiven,and dotted lines indicatedetection limits.

rect hemagglutination test kit and obtained antiserum with antibody titers of 1:20,480, whereaswe obtained a titer of

1:2,560 inourpreviousstudy (4). Heattreatmentof diluted

serum containing EDTA has been reported togive assays greater sensitivity than the proteaseand heat treatment or

heat extraction methods(9). However, the detection limit of the EDTA and boiling method was about twofold greater

than that of theproteasemethod inourstudy. This reduction

insensitivitymayhave resulted from thetrapping of antigen within the precipitates, the incomplete dissociation of

se-rum-antigen complexes, orthe dilution (1:1) ofserum

sam-ples.

Because of thesimplicity of the LA, this method has been applied for the detection of C. albicansmannan(1, 12). The

LA formannan,which hadadetection limit of 1.25ng/mlin bufferedsaline, had been reportedtobepositive in 17 (81%) of21patientswith disseminatedcandidiasis(1). On the other hand, Phillipsetal.(12) reported thatnoneof theirpatients withcandidiasis showed positive results by LA formannan.

In our study, 22 (51%) of 43 C. albicans mannan-positive

patients showed positive results for LA-CA which could detect1 ngofmannanpermlinpooled serum.TheELISA was about three- to eightfold more sensitive than the LA, andserafrom48% ofantigen-positivepatientswerenegative

fortheantigen bythe LA.Therefore, the diagnosticvalue of

theLA is limited.

C. albicans and C. tropicalis are important pathogenic

yeaststrainsinimmunocompromised patients.Inourseries,

55 (71%) of78 patientswith invasive or possible invasive

candidiasis were affected by these two Candida species. FourCandida species, C. glabrata, C. krusei, C.

parapsilo-sis, and C. guilliermondii, have reportedly been isolated from 52 of 200 fungemic patients (5). Horn et al. (5) have

reported that most fungemic patients with C. albicans, C tropicalis,orC. krusei infections had disseminatedorlocal

diseaseatautopsy,whereasnoneof thosewith C. parapsi-losisorC. glabrata infections did (5). Inourpresentstudy,

nopatients with histologicallyprovenC.glabrata infections were found. Of12patientswith C. parapsilosis infections,

however, 1 had histologically proven endocarditis with in-termittentantigenemia, and 8 patients showed persistentor

intermittent antigenemia, suggesting invasion of the tissues by Candida strains.

Broth cultures of C. glabrata showed negative results in testsforantigens and D-arabinitol, suggesting the nonvalue of these tests in diagnosing C. glabrata infections. The ELISA with antibodies against C. knrsei detected C.

albi-cansandC. tropicalis antigens in broth cultures, but it failed

to detect C. krusei antigens in patients infected with C. albicans or C. tropicalis. One of the reasons for these

negative results may be antigen concentrations that were

below the detection limit.

Although D-arabinitol is a major metabolic product of

Candida species, a higher D-arabinitol/creatinine ratio in

serafromhigh-risk patientswithout candidiasis than that in

serafrom normalsubjectswasobserved(3).Inourstudy,13 of 109 patients without invasive candidiasis had elevated

D-arabinitol/creatinine ratios. Heavy colonization by Can-A

A450

1.0

O

.94

Protease

0.8-

0.7-

0.6-

0.5-

0.4-

0.3-

0.2- 0.1-Boiling

0.2

-0.1

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3136 FUJITA AND HASHIMOTO

TABLE 3. Results ofantigen and metaboliteassays inpatients with and without candidiasis and normalhealthyadults No.of casespositivefora:

Group andCandidaisolate No. of casesa Antigensdetectedby Antigens detected byLA Elevated ELISA with antibodies withantibodies against: D-arabinitolcreatendne

against: D-ara tio

ratio C.albicans C.krusei C.albicans Ckrusei

Invasive candidiasis

C. albicans 26(18) 22(19) 0 11(8) 0 12(9)

C. glabrata 6(1) 0 0 0 0 1

C. guilliernondii 4(2) 0 2(1) 0 0 2(1)

C. krusei 1(1) 0 1(1) 0 1(1) 0

C.parapsilosis 10(6) 0 9(3) 0 3(2) 6(4)

C. tropicalis 11(10) 9(7) 0 7(7) 0 8(8)

Possibleinvasive candidiasis

C. albicans 18(6) 12(6) 0 4(4) 0 4

C.parapsilosis 2 0 1 0 1 0

Transient candidemia 10 0 0 0 0 3

Bacteremia 54 0 0 0 0 5

Patients without infections 55 0 0 0 0 5

Normalhealthy adults 42 0 0 0 0 0

a Valuesinparenthesesindicate the number ofpatientswhodiedwith sustained fever.

dida species at mucosal sites may have resulted in the

production of sufficient D-arabinitol to cause a significant rise in concentrations inserum, because high-risk patients

were readily colonized with Candida species (14). More-over, the enzymatic fluorometric assay for D-arabinitol demonstrated positive results in cultures of C. krusei from which no D-arabinitol production was detected (2). These

positive resultsmight belinked to the assayformat;

D-ara-binitol dehydrogenase alsooxidizedserumD-mannitol, and

D-mannitol generated NADH as efficiently as D-arabinitol

did(15).

In conclusion, the ELISA with antibodies against C.

albicans in combination with the ELISA with antibodies

against C. krusei is a useful method for the diagnosis of

invasive candidiasis caused bymedically important Candida

strains excludingC.glabrata. Because of the time-consum-ing nature of the ELISA method, however, a simple and

rapid methodother than the ELISA is in use in our labora-tory.

ACKNOWLEDGMENT

This workwas supported in part by a grant from the Clinical Pathology Research FoundationofJapan.

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