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Detection of circulating antigen in experimental Candida albicans endocarditis by an enzyme linked immunosorbent assay

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Detection of

Circulating Antigen

in

Experimental

Candida

albicans

Endocarditis

by

an

Enzyme-Linked

Immunosorbent

Assay

W. MICHAEL SCHELD,l* ROBERT S. BROWN,JR.,' SALLY A. HARDING,'t

AND

MERLE A.

SANDE'

DepartmentsofMedicine'andPathology,2 University ofVirginiaSchoolofMedicine,Charlottesville,

Virginia

22908

Adouble-antibody sandwich enzyme-linked immunosorbent assay was

devel-opedfor the detection ofcirculatingCandidaalbicansantigen duringthe course

of experimental C. albicans endocarditis. The enzyme-linked immunosorbent

assay waspositive in 75% of rabbits withpolyethylene catheter-induced experi-mental aortic valve C. albicans endocarditis but wasnegative in all controls,

including catheterized animals that received intravenous Candidaorcatheterized but uninfected animals, and in rabbits with experimental fungal or bacterial

endocarditis of other etiologies. The enzyme-linked immunosorbent assay was

muchmoresensitive than bloodculturingorfever determinations inexperimental

C. albicans endocarditis. Thisassay is moresensitive than currently available serological techniques, is highly specific, and deserves further study in the diagnosis ofinvasive, disseminated C.albicansinfections,including endocarditis.

Fungal endocarditisoccursprincipallyin the following three clinical settings: after cardiac surgery,intheintravenousdrug addict,and after

prolonged intravenoustherapy, including

hyper-alimentation and treatmentfor bacterial endo-carditis(1, 8, 20,24).This disease isadevastating

complication of cardiac surgeryandappearsto

beincreasing infrequency.Thepathogenvaries with theclinicalsetting,but Candida albicans is the most commonly implicated organism in

fungalendocarditis (8, 20).Diagnosisisdifficult because blood cultures are frequently negative

(25) anddelaysare common.Thus, despitethe introduction ofnewerantifungal agentsandan

aggressive medical-surgical approach to these infections, the mortality rateremainshigh and still exceeds 80% (8, 20). Rapid and specific diagnostic tests are critically needed, and

nu-merousserological procedureshave been

evalu-ated. Antibodies directed against cell wall or

cytoplasmicantigenscanbedetectedby

agglu-tination(4, 16),precipitation(4, 5, 7, 13, 27,28), countercurrent

immunoelectrophoresis

(1 1), and radioimmunoassay (12). All of these tests are

variablysensitive andlackspecificity,withup to 40% false-positive and -negative rates (4, 27).

Theseantibodydetectiontestsrelyonanintact

host immune response andusually take weeks tobecome positive (21, 28). Detectionof circu-lating antigen offers more hope for the early

tPresentaddress:DepartmentofPathology,Richland Me-morialHospital,Columbia,SC29203.

diagnosis of systemic candidal infections. Gas-liquid chromatography (15, 17),countercurrent

immunoelectrophoresis (14), hemmagglutina-tion-inhibition (33), radioimmunoassay(19),and enzyme-linked immunosorbent assay (ELISA) (25, 30, 31) have proven highly specific in the detection ofcirculatingantigen in systemic

can-didiasis in experimental animals and patients. Due tothe relative rarity of Candida endo-carditis, evaluation of these serological proce-dures atonlyone institution isimpossible. We employed our well-characterized rabbit model (2, 21)todetermine thesensitivity and

speciflc-ity ofan ELISA method for the detection of

circulating antigeninexperimentalC. albicans endocarditis.

MATERIALS AND MEIHODS

Animalmodels. C. albicansendocarditiswas

in-duced in NewZealandwhite rabbits (2 to 3 kg) by

modification ofpreviouslypublished methods (6, 21).

Sixteenanimalswerelightlyanesthetized with60mg

of sodium pentobarbital (BarberVeterinary Supply

Co., Richmond,Va.), and the right carotid arterywas

exposedandligated.A sterilepolyethylene catheter

(Radio-opaque, Intramedic,PE-90; Clay-Adams,

Par-sipanny, N.J.)waspassed across the aortic valve. After

1 hof catheter-inducedtrauma (which uniformly

re-sultsin thedepositionofnonbacteril thrombotic

en-docarditis), 103to107 colony-formingunits (CFU) of C. albicanswereinjectedthrough the catheter. The

catheterwasligated,and the woundwasclosed;

cath-etersremainedinplacefor theduration of the

exper-iments. Thetestorganism (serotype A, clone 4) was

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originally isolatedfromapatientwithprosthetic

val-vularendocarditis andwaskindly provided by Richard

A. Calderone, Georgetown University, Washington,

D.C. Yeastcellswere grownfor 24 h in Neopeptone

broth(Difco Laboratories,Detroit, Mich.),centrifuged

(3,000 rpm for 15min),and washed in0.9% NaCI twice. A10-2dilutionwasusedasthe inoculum inavolume

of 1.0 ml. This procedure uniformly results in the

development of C. albicans endocarditis.

Several control models were used, as follows. (i)

Tennoncatheterized animalsweregiven identical

in-ocula of106to 107CFU of C.albicansintravenously.

Thisprocedure failed to produce systemic infection,

andallorgans(heart,lung, liver, spleen, kidney)were

negative by culture and histologicalstudyatautopsy

(S. A. Harding, J. P. Brody, and D. E. Normansell,

Am. J.Clin. Pathol.,inpress). (ii) Five animalswere

catheterizedasabove, butnoorganismswere

admin-istered. (iii) Candida krusei endocarditis (organism

againprovided byR.A.Calderone)wasinducedbyan

identicalprocedurein threeanimalsbutrequired .108

CFUin theinoculumtoproduce consistent,uniformly

fatal disease. (iv) Experimental Staphylococcus

au-reusand Streptococcus sanguisendocarditis was

pro-ducedin 12rabbitsafter catheter-induced aorticvalve

traumaby methods described previously (22, 23). (v)

Sera from15rabbitswithexperimentalmeningitis due

to Streptococcus pneumoniae, Haemophilus

influ-enzae,orEscherichia coliafterintracisternal

inocula-tion(W. M.Scheld andM.A. Sande,J.Antimicrob.

Chemother.,inpress)werealso tested.

Experimental design. Blood was drawn for the

ELISA determination (seebelow) before inoculation

ofall animals. Thisprocedurewas repeated at24 h

after the initiation of the experiments in all groups

and every 2days thereafter. Catheterized urinewas

removedatthese times from threeadditional animals.

In addition, rectal temperatures wererecorded, and

blood(1.0 ml)wasobtainedviathecentralearartery

for cultureontrypticsoy agar (Difco) pourplatesat

these times. Animals were followed until death or

sacrificed at 30 days ifstiil alive. The hearts were

removed under sterileconditions,andtheaorticvalve

areawithvegetationswasdissectedfree, weighed,and

homogenized in saline. Serial 10-fold dilutions were

madeintrypticsoyagar(Difco),and theresultswere

expressed asCFU per gram of tissue. Small kidney

sectionswereprepared similarly.

Anti-Candidaantibodyproduction.Fiverecent

clinicalisolates(bloodorsterilebodyfluid)were

ob-tained through the microbiology laboratory at the

UniversityofVirginiaHospital.After48hofgrowth

on Sabouraud agar slants (BBL Microbiology

Sys-tems, Cockeysville, Md.), cells were harvested by

washing with phosphate-buffered saline (PBS) (pH

7.2). The cells were washed with 20 volumesofPBS

and checked for purity by subculture on chocolate

agar for48hat37°C.Theorganismswerepooledand

heatkilledbyexposureto80°Cfor1h. A1:1emulsion

ofa 20% suspension ofthe organisms with Freund

complete adjuvant was injected subcutaneously

bi-weeklyin NewZealandwhite rabbits (2.5to 3.5kg).

Rabbits with a whole cellagglutinin titer of-1:640 (17)at8to 12weekswerebledweeklyforanadditional

8weeks,and the sera were pooled. Purified

immuno-globulinG was prepared by chromatography on

dieth-ylaminoethylcellulose (Whatman DE52), adjusted to

1mg/mlwith PBS,and stored at -70°Cuntil used as

coating antibody or horseradish

peroxidase-conju-gated antibody intheELISAprocedure.

ELISA procedure. A double-antibody sandwich

ELISA for the detection of circulating antigen was

usedinthese studies. Anti-Candida antibody (200

i1

containing 2 Mg of immunoglobulin G in PBS) was

usedtocoatpolyvinylchloride microtiter wells (Cooke

Engineering Co. [Dynatech Corp.], Alexandria, Va.)

for3 h at37°C. After three washes with PBS-0.05%

Tween 20, a

200-j1

heat-inactivated(56°Cfor 30 min) serumsamplewasaddedto each well and incubated

for3 h at37°C. PBS-Tween washes were repeated.

Anti-Candidaantibody was conjugated to horseradish

peroxidase type VI by the method of Nakane and

Kawaoi (18). Fluorodinitrobenzene-blocked

peroxi-dasewasoxidized with sodiumperiodate to form

al-dehydegroups.Peroxidase-aldehyde was bound to free

amino groups ofimmunoglobulin G unidirectionally

withhigh affinities. This peroxidase-labeled

immuno-globulin retainedboth its enzymatic and

immunologi-calproperties. Working dilutionsof unlabeled coating

antibody and peroxidase-labeled immunoglobulin G

weredetermined bycheckerboard titrations in

micro-titerwells with known antigen-positive and -negative

sera.Peroxidase-labeled immunoglobulin G was then

addedtoeach well(~0.5 MgofimmunoglobulinG per

ml in 200

pl)

and incubated for3 h at37°C or held

overnightat4°C. After washingwithPBS-Tween,200

ul offreshly preparedsubstrate(5-aminosalicylicacid

with 0.05% H202 [9:1,vol/vol]) was added and

incu-bated for1h at25°C. The reactionwasstoppedwith

50

pd

of3NNaOH. Positivereactionswereeasilyread

visually by the appearance of a dark brown color or

spectrophotometrically by absorbance twice known

negative controls at 450 nm. Knownpositivecontrols

(rabbitserumspiked with10Mugof purified C. albicans

mannanperml, kindlyprovided by J. E. Bennet, NIH,

Bethesda, Md.) were included on each plate with

knownnegative controls (PBSandnormal rabbit

se-rum). Purified antigens, including extracts of

Asper-gillusfumigatus, Aspergillus flavus, and Aspergillus

niger(Hollister-Stier Laboratories, Spokane,Wash.),

pneumococcalvaccine(MerckSharp& Dohme, West

Point, Pa.), and group A meningococcal vaccine (J.

Colabro, University ofRochester, Rochester, N.Y.),

failedtoyield positive reactions with thisprocedure.

RESULTS

Clinical and microbiological

character-istics of experimental C. albicans endocar-ditis. All 16 animals followed a clinical course similar to that described previously (21). The median duration of survivalwas 22days (range, 8 to230). Fouranimalssurvived tosacrifice at 30 days. All hearts contained grossly visible,

friablevegetations weighing upto 0.86g. Quan-titative aortic valvevegetationculturesrevealed

a mean+standard deviation Candida

concen-tration oflogo7.4+0.9CFU/g.All animals with

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endocarditis had renal involvementgrossly visi-bleas multiple cortical abscesses. Quantitative kidneycultureswereallpositive witha mean ± standarddeviationfungal concentration oflogo 4.4 ± 1.6 CFU/g. Despite such evident

pathol-ogy, fever andpositive blood cultureswere

un-usual. Only 22 out of 146 (17%) temperature recordingswereabove39.6°C,theupperlimitof

normal in the New Zealand white rabbit. In

addition, only 1 out of96 (1%) 1-mlblood

cul-turesobtainedwaspositivefor C. albicans.

EUISA determination of Candida

anti-gen. ThisELISAprocedurewascapableof de-tecting1ngofpurifiedmannanpermlunder the conditions described. If the heat inactivation step was omitted, the level of sensitivity de-creased to 100 ng ofpurified mannanper ml. This heat-labile inhibitor in normal rabbitserum

hasnotbeencharacterized further.

Apositive ELISAwasdefined whena serum

dilution of 21:2 on two sequential samples

yielded a readily visible brown-color reaction noted by two independent observers and was

confirmedspectrophotometrically.Bythese cri-teria, 12 out of 16 animals (75%) with

experi-mental C. albicans endocarditisproduceda

pos-itive ELISA. Urine samples were positive for antigen in all threeanimalsstudied.Allcontrols

were negative; these included the 10 animals that received 106 to 107 CFU of C. albicans intravenouslywithoutprior catheterization,the

5uninfected catheterizedrabbits, animals with

experimental C. krusei(3

animals),

S.aureus (6 animals),orS.sanguis(6

animals)

endocarditis,

and animals with experimental meningitis due

toS.pneumoniae (6animals), H. influenza (4 animals),orE. coli(4animals).

Rabbits with endocarditis and a positive

ELISA revealed titers of1:4to1:8at24hafter

injection(Fig. 1).Titers thenprogressivelyrose

and reacheda median level of 1:512 by5 to 7

days(range,1:256to1:1,024).Titerswere main-tained in thisrangeuntil the animaldiedor was sacrificed. Urine titers were generally lower (range, 1:16 to 1:256). A total of2 out of 10 animalsthatreceived 106to107 CFU of C.

albi-cansintravenouslyin theabscence of catheter-inducedaortic valvetraumaproducedatiter of

51:2 soon after injection; the other 8 animals

produced consistently negative results. These

results were considered negative because the titers were all sl:2 ononly one determination

andquantitativeheart andkidneycultureswere

negativeatautopsy.

DISCUSSION

Thisstudy demonstrates that an ELISA

de-termination ofcirculating antigenin

experimen-1:2048 1:1024 1:512

MEDIAN 1:256 ELISA 4:128 TITER 1:64

OF s1:32

CANDIDA 1:16

ANTIGEN 1:8

1:4

1:2

UNDIL

-<~~~~~~0

PRE 1 2 3 4 5 6 7 21

DAYS POST-INJECTION

FIG. 1. Median maximal serum ELISA titer of

Candida antigen versus day after injection of1i&

CFUofC. albicans. Symbols:*,patternfollowed by

12outof16rabbits with endocarditis (ENDO);O,

patternfollowed by2outof 10 uncatheterized rabbits

after intravenous(IV injection. UNDIL, Undiluted;

PRE,preinjection. Bars represent range of ELISA determinations.

tal C.albicans endocarditis is feasible, relatively rapid and sensitive, simple, and highly specific. Themethodwascapable of detecting cl ng of

purified mannan per ml and was much more

sensitive than bloodculturinginthis

experimen-talmodel.

The experimental modelofendocarditis

em-ployedinthese studies is similarto the disease inhumansandpursues anindolentcourse.Only 17% of thetemperature recordingswerepositive in the animalsused in this study; thisfigureis

lower than those reported forpostcardiac sur-gery C. albicans endocarditis in patients, but <50% of the patients werefebrile, and another 25% hadonly transitory feverinone series (24).

Other investigations have found fever in

vir-tually all patients (1, 20) with well-established disease.

Methods forthe detection ofcirculating anti-body in systemiccandidiasis have included such standard techniquesas agglutination,

immuno-diffusion, and countercurrent immunoelectro-phoresis (4, 5, 7, 11-13, 16, 27, 28), butail are unsatisfactoryduetothefollowingproblems: (i) lack ofantigen standardization, often employing cell wallorcytoplasmiccomponentsorboth; (ii) variablesensitivity in the detectionofinvasive

disease

(s40

to 93%, depending on the

tech-nique); (iii) poor specificity, with inability to

differentiate colonization from systemic

infec-tion (up to 40% faLse-positives in some series); (iv) inabilitytodetect disease early(require 12

daysto turnpositiveinexperimentalcandidiasis

[21,27] and asimilarinterval in patients), thus

precludingrapid institution of specific therapy; (v) dependence on an intact immune response

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forformation, oftenseverely attenuated in the

immunosuppressedpatient, where systemic

can-didiasisisprevalent; and (vi) persistent positiv-ity for months (or longer). Thus, these tests cannotbe usedto assesstheresponse to antifun-galtherapy.

Ail of theseproblemscould be avoidedbythe

useofanassayforcirculating antigen. Milleret al.(17)werethefirsttoreportantigendetection

insystemic candidiasis.Theydemonstrated ab-normalpeaks inserumbygas-liquid

chromatog-raphyin6outof6patients with candidemia and

2 out of4 patients with documented invasive disease; thepeakswereabsent ifsuperficial col-onization alone was present. Similarly, an

ex-tremelyspecifichemagglutination-inhibition as-saydetected circulating Candida mannanin 4 outof14patientswithsystemiccandidiasis(33). Recently, a radioimmunoassay capable of de-tecting 100 ng ofCandida antigenperml was

positive in 70% of mice withsystemiccandidiasis (19), andcountercurrentimmunoelectrophoresis

detection ofcirculatingcell wallpolysaccharide

Candida antigen waspositive in 13 patients, 8 of whomwereproventohaveinvasive candidi-asis(14).Althoughconsiderableoverlapdid oc-cur,gas-liquid chromatography detected21 ,ug

ofD-arabinitol perml in 15 outof20 patients

with systemic candidiasis versus 3 out of 28

patients withonlysuperficial colonization (15).

Thesemethods deserve furtherevaluation in the

diagnosisofsystemicCandida infection. Warrenet al. (30) employedanELISA tech-niquesimilartothemethod usedin these

exper-iments to detect circulating antigen in

experi-mental systemiccandidiasisin mice and intwo

rabbits. These earlierexperiments didnot eval-uatenonlethalinfection,antigentiter,or

patho-logiccorrelations, however. Althoughthe

anti-gen was not identified, later resultssuggested

that the ELISA detected antigens other than

mannan (31). In addition, the procedure

re-vealedunacceptablefalse-positiveand-negative rates when evaluatedagainst unknown human

sera (32). The methods ofantibody production,

antibody andsera addition inthe ELISA, and enzymeconjugation(alkalinephosphatase)were

different from thoseinthe proceduresoutlined in theseexperiments. The ELISA reported here

is much moresensitive (clversuss250 to500 ng ofpurified mannanperml),requires20times lesscoatingantibody, and isequallyasspecific

astheprocedureusedbyWarren andcolleagues. Recently, an ELISA-inhibition technique

re-vealedcirculating Candidaantigenin allseven

patients withsystemicdisease(25);however, the

method is complicated and lesssensitive (225

ng of mannanperml),andresults are not

avail-ablefor 24 h.

TheELISA, originally described in 1972 (3),

is as sensitive and specific as the radioimmu-noassayinmany situations, butrequires nonra-dioactive materials and inexpensive reagents, andhas shownexciting promise in the detection

ofvarious infectious diseases (29). The ELISA

methodreported here detected sl ng of purified

mannanper ml and was positiveforcirculating antigeninover70% ofanimalswithexperimental C. albicans endocarditis. However, 4 out of16

animals withendocarditisproduced negative

re-sults; quantitative aorticvalve and kidney

cul-tures were allpositive and revealed no differ-enceswhencompared withthe 12 rabbits

posi-tivebytheELISA. Thismay represent a detec-tion ofdifferent antigens because awhole-cell immunization schemewas employed,and

anti-bodies directed against different parts of the

Candida yeast cell orpseudohypha have been described (10, 26).Alternatively, blocking anti-body ornonspecific reactions with serum

pro-teins mayrender theELISA falselynegative(9). These possibilitiesrequire further analysis. The

test employedinthisstudywas negative in all controls, demonstrating high specificity. The procedure is easy to perform, inexpensive,

re-quires relatively simple reagents,and can

pro-vide rapid results (7 h). The ELISA deserves

further study in thedetectionofcirculating an-tigen in systemic candidiasis, including

endocar-ditis.A similar method revealed 100% sensitivity

and specificity in the detection of circulating antigen in experimental systemic candidiasis in

imimunosuppressedrabbitspretreated with

cor-tisone(Hardingetal.,inpress). The ELISA may

be of great value in the diagnosis of Candida

endocarditis and in following the course after

the institution of therapy because circulating antigen, unlike antibody, should decline and

eventually disappear with successful interven-tion.

LITERATURE CrIED

1. Andriole,V.T., IL M. Kravetz, W. C. Roberts, and J. P.Utz.1962.Candidaendocarditis.Clinical and patho-logic studies. AmJ.Med.32:251-285.

2.Calderone,R.A.,M.F.Rotondo, and M. A. Sande. 1978. Candida albicans endocarditis: ultrastructural studies ofvegetation formation. Infect. Immun. 20:279-289.

3. Engvall, E., and P. Perlmann. 1972. Enzyme-linked immunosorbent assay, ELISA. J. Immunol. 109:129-135.

4. Everett,E.D., F. M.Laforce, and T. C. Eickhoff. 1975.

Serologicstudies in suspected visceral candidiasis. Arch. Intern.Med.135:1075-1078.

5. Filice, G.,B.Yu, and G. Armstrong. 1977. Immunodif-fusion andagglutinationtestsforCandida in patients withneoplastic disease: inconsistentcorrelation of re-sultswith invasive infections. J.Infect.Dis. 135:349-357.

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6. Freedman, LR.,and M. L Johnson. 1972.

Experimen-talendocarditis.IV.Tricuspidandaortic valve infection withCandida albican8 in rabbits. Yale J.Biol. Med. 45:163-175.

7. Gaines, J.D., and J. S.Remington. 1973.Diagnosis of deep infection with candida.Astudy of candida

precip-itins. Arch. Intern. Med. 132:699-702.

8.Harford, C. G. 1974. Postoperative fungal endocarditis. Fungemia,embolism,andtherapy. Arch. Intern. Med. 134:116-120.

9. Hasenclever, H. F. 1978. Theinvitro interactionsof Candida albicans withnonspecificserumproteins.

My-copathologia65:169-176.

10. Ho,Y.M., M. H.Ng,andC.T.Huang.1979.Antibodies

togerminatingandyeastcells of Candida albicans in

humanand rabbit sera. J.Clin.Pathol.32:399405. 11.Hopfer, R. L, and D. Grôschel. 1979. Detection by

counterinmunoelectrophoresisof anti-candida

precipi-tins in sera from cancerpatients.Am. J.Clin. Pathol.

72:215-218.

12.Huang, S. Y., C. W. Berry, J. T. Newman,W. H. Cooper, and N. Y.Zachariah. 1979.A radioimmu-noassay method for the rapid detection of Candida

antibodiesin experimentalsystemic candidiasia. My-copathologia67:55-58.

13.Ianninm,P.B., E. D.Everett, G.Pappas,and F. M. LaForce.1976.Candidaprecipitinsas adiagnosticaid inCandidaendocarditis.J.Am. Med. Assoc. 236:2518-2520.

14.Kerkering, T. M., A. Espinel-Ingroff, and S. Sha-domy. 1979. Detection of Candida antigenemia by counterimmunoelectrophoresis inpatients with

inva-sivecandidiais.J. Infect.Dis.140:659-664.

15.Kiehn,T.E.,E. M.Bernard,J. W. M.Gold,and D.

Armstrong.1979.Candidiasis: detectionbygas-liquid

chromatography ofD-arabinitol,afungalmetabolite,in

humanserum.Science 206:577-580.

16. Merz, W.G.,G. LEvans,S.Shadomy,S.Anderson,

LKaufman,P. J.Kozlni,D. W.Mackenzie,W. P. Protzman,and J. S.Remington. 1977.Laboratory

evaluation ofserologicaltestsfor systenuccandidisis.

Acooperative study.J.Clin. Microbiol.5:596-603. 17.Miller,G.G.,M. W.Witwer,A. LBraude,andC.E.

Davis.1974.Rapid identification ofCandidaalbican8 septicemia in manby gas-liquid chromatography. J. Clin. Invest.54:1235-1240.

18.Nakane,P. K., and A. Kawaoi. 1974. Peroxidase-la-belled antibody: a new method ofconjugation.J. His-tochem.Cytochem.22:1084-1094.

19. Poor, A.H.,and J. E.Cutler. 1979.Partiallypurified

antibodiesusedinasolid-phaaeradioimmunoassayfor

detectingcandidalantigenemia.J.Clin. Microbiol.9: 362-368.

20.Rubenstein, E.,E. R.Noriega,M.S.Simberkoff,R.

Holzman, and J. J. Rahal, Jr. 1977. Fungal endocar-ditis:analysis of 24 cases and review of the literature. Medicine (Baltimore)54:331-334.

21. Sande,M.A., C.R.Bowman,and R. A.Calderone. 1977. Experimental Candida albicans endocarditis:

characterization of the disease and response to therapy. Infect. Immun.17:140-147.

22. Sande, M. A., and M.L. Johnson.1975.Antimicrobial therapy ofexperimental endocarditiscaused by

Staph-ylococcusaureus.J.Infect. Dis. 13:367-375.

23. Scheld,W.M.,J.A. Valone, and M. A. Sande. 1978.

Bacterialadherence in thepathogenesis ofendocarditis.

Interactionof bacterial dextran, platelets and fibrAn. J. Clin. Invest.61:1394-1404.

24. Seeling, M. S., C. P. Speth, P. J.Kozinn,E. F.Toni,

and C. L Taschdjian. 1973. Candida endocarditis

after cardiac surgery. Clues to earlier detection. J. Thorac.Cardiovasc. Surg.65:583-601.

25. Segal,E., R. A. Berg, P. A.Pizzo,and J. E. Bennett. 1979.Detection ofCandida antigen in sera of patients

with candidiasisby anenzyme-linkedimmunosorbent assay-inhibition technique. J. Clin. Microbiol.

10:116-118.

26. Syverson,R.E., H. R.Buckley,and C.C. Campbell.

1975. Cytoplasmic antigens unique to the mycelial or yeast phase of Candidaalbicans. Infect. Immun. 12: 1184-1188.

27. Syverson, R.E., H. R. Buckley, and J. R. Gibian. 1978. Incressmg thepredictive value positive of the precipitin test for thediagnosisofdeep-seated candidi-ais.Am. J.Clin. Pathol. 70:826-831.

28.Taschdjian,C.L, P. J. Kozinn, M. B. Cuesta, and E.

F. Toni. 1972. Serodiagnosis ofcandidalinfections. Am. J. Clin. Pathol.57:195-205.

29.Voiler,A.,D. E.Bidwell,and A. Bartlett. 1976. En-zyme immunoassays in diagnostic medicine. Theory andpractice.Bull. W.H.O.53:55-65.

30.Warren, R.C.,A.Bartlett, D. E.Bidweil,M. D.

Rich-ardson,A.Voiler,and LO. White.1977.Diagnosis ofinvasivecandidiasisby enzymeimmunoassayof se-rumantigen.Br. Med. J.1:1183-1185.

31. Warren, R.C.,M. D.Richardson, and L O.White. 1978.Enzyme-linkedimmunosorbent assay of antigens from Candida albicans circulating in infected mice and

rabbits:the role of mannan. Mycopathologia 66:179-182.

32. Warren, R. C., L O.White, S. Mohan, andM. D.

Richardson. 1979. The occurrenceand treatment of

falsepositive reactionsinenzyme-linked immunosor-bent assays(ELISA)for the presence of fungal antigens inclinicalsamples.J.Immunol. Methods 28:177-186. 33. Weiner,M.H.,and W. J. Yount. 1976. Mannan

anti-genemiainthediagnosis of invasive candida infections. J.Clin. Invest.58:1045-1053.

VOL.12,1980

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References

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The RSCH shear strains predicted by the models used to develop APA rut depth criteria were compared with the Asphalt Institute (AI) criteria for evaluating rut resistance using

The branch instruction transfer control to the d ata memory address cal­. culated by adding a displacement to the