0095-1137/80/11-0679/05$02.00/0
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
22908Adouble-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 arevariablysensitive 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 incubatedfor3 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 heldovernightat4°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. Positivereactionswereeasilyreadvisually 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(6animals)
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 thetech-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.
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VOL.12,1980
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