Mannose in body fluids as an indicator of invasive candidiasis

0095-1137/81/110557-06$02.00/0

Mannose in Body

Fluids

as an

Indicator

of Invasive

Candidiasis

THOMAS P. MONSONt* AND KATHERINE P. WILKINSON

Veterans Administration Medical Center andDepartmentofMedicine, University ofArkansas for Medical

Sciences,

Little

Rock,

Arkansas 72206 Received2March1981/Accepted12June 1981

Usinggas-liquid chromatography, wemeasured freemannose inthe serumof

six nondiabeticpatients withautopsy-proveninvasivecandidiasis. In all patients

serummannoseconcentrationswerehigher thanmannoselevels found inserum

from normal adults andchildrenorfrom patients with catheter-associated

can-didemia, mucosalcandidiasis, and othermycoses.Spinal fluid fromtwopatients

with Candida meningitis showed increased freemannose ascomparedtoseven

non-mflammatoryspinal fluid samples. However, freemannose in theserum of

poorlycontrolleddiabetics(blood glucose of -300 mg/dl) did overlap

concentra-tions inpatients withinvasivecandidiasis. In vitro culture of Candidaalbicans

demonstratedincreasing concentrations ofmannoseassociated with growth of the

organism. We conclude that physical and chemical assayfor mannose in body

fluidsmaybeausefultechniquetoassist inthediagnosis in invasive candidiasis.

Severe diseaseproduced

by

Candida species

has become an

increasing problem during

the last decade,

although

its association with

com-promised hostshasbeenrecognized for wellover

a century

(2).

High-dosage antibiotic, steroid,

and immunosuppressive

therapy

has

signif-icantly contributedtothis

development

(12)and

will

likely

continue to doso.

Complicating

rec-ognition of this clinical

problem

istheproclivity of

Candida

tocolonize or cause mild infection ofthe mucous membranes and skin.

Thus,

ag-gressiveproceduresareoftenrequiredtoobtain

adefinite

diagnosis

of invasive candidiasis.

Serological techniques involving

Candida

an-tibody

detection have been evaluated as

diag-nostic

aids,

but theresults andthe

interpreta-tions have been

conflicting (4, 5).

Serological

procedures for detection of

circulating

microbial

polysaccharides

have been showntobe

clinically

usefulinseveral

settings (7,

17). Studies

utilizing

gas-liquid

chromatography

havesuggested that

patients

with invasive candidiasismaybe distin-guished

by

having

elevated serum

concentra-tions ofD-arabinitol

(10, 18)

and that increased

serum mannose concentrations are present in

patientswithcandidemia (14).Inanattemptto

confirm the association of elevated serum

man-noseconcentration with invasivecandidiasis,we

have used gas-liquid chromatography to

mea-suretheconcentration offreeorunboundbody

fluid mannose in controlpopulationsand in

pa-t Address reprinpa-t requespa-ts pa-to: Thomas P. Monson, 300E.

RooseveltRd.,LittleRock,AR72206.

tientswithdifferenttypes of Candida infection.

We havealso demonstrated the in vitro produc-tion of free mannose

by

culture of Candida

albicans.

(Thesefindingswerepresentedin part at the

Southern Sectionmeeting of the American

Fed-erationfor ClinicalResearch,New

Orleans,

La.,

26January1978.)

MATERIALS AND METHODS

Sera from thefollowing subjectswerestudied:121

adult male andfemale blood donors and23 normal childrenaged1 to19yearsservedasnormalcontrols;

2subjectshad intravenouscatheter-associated

candi-demia; 42 had mucosalvaginalcandidiasis;6had sys-temiccandidiasis; 2subjects hadbloodcultures and

lungaspirate positiveforCandidatropicalis,

respec-tively; 7 had untreated non-Candida mycotic

infec-tions;4patientshad disseminated breast carcinoma;

11 patients were poorly controlled diabetics; and 33 weremiscellaneous intensive care unit patients.

Cere-brospinalfluid(CSF)from sixchildren andoneadult,

obtained for the purpose ofexcluding meningitis but showing normal parameters, was also evaluated.

Thediagnosisof invasive candidiasiswasbasedon

histological evidence of infection with yeastlike orga-nisms resemblingCandida in autopsy or biopsy tissue, otherthan skinor mucosalsurfaces (six cases). Cath-eter-associated candidemiawasdiagnosedonthebasis

offeverwithpositive bloodcultures, whichpromptly

remitted inbothpatientswithremoval of the catheter.

In one case, the catheterwascultured and grew C. albicans. The diagnosis of vaginal candidiasis was basedontypical clinicalpresentationtogether witha

KOH preparation of vaginal exudate demonstrating organisms consistent with Candida. Patients with

blastomycosis (two), histoplasmosis (two),

dissemi-557

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AND

nated aspergillosis(one),andcryptococcalmeningitis (two) were diagnosed after isolationof the organism fromappropriate specimens.

AnisolateofC.albicans originally obtained froma

patient with systemic candidiasis wasmaintained in

yeast phase onSabouraud agar.For in vitrostudies, the organism was inoculated into 1 liter of Eagle minimal essential medium which had been supple-mentedwith nonessential amino acids(GIBCO, Grand Island, N.Y.). Subcultures and Gram stainsweredone

toconfirmpurity oftheculture.Samples of10mlwere

removedat42, 48, 72, 96, and 120 h. With a Coleman

model9nephelo colorimeter (Coleman Instruments,

Maywood,Ill.) optical densityof the broth culture was

determinedinduplicate. After determination of opti-cal density, the organisms were separated from the

supernatantbycentrifugationat7,000xgfor30min

at 4°C; the supernatant was then passed through a 0.45-,um pore size membrane filter

(Millipore Corp.,

Bedford, Mass.). Theresulting filtratewas storedat

-30°C before analysis bygas-liquid chromatography. Unbound mannose was determined by gas-liquid chromatography of the aldononitrile derivativeas de-scribedpreviously (15). A Hewlett-Packard gas-liquid chromatograph equipped with dual flame ionization detectorswasused(Hewlett-Packard, Avondale, Pa.) Coiled 1.8-m glass columns (2 mmID) were packed

with 2% neopentyl-glycol-succinate on 80/100-mesh Chromosorb W(HP).Withhydroxylamine

hydrochlo-ride and acetic anhydride with pyridine usedas the

solvent,thederivativewasprepared,dried, and then reconstituted intrichloromethane with 3

jul

injected

ontothe column.Assayswereperformedinduplicate. Theareaunder themannose curvewasintegrated and convertedtomicrograms ofmannosepermilliliterof serum byusingastandard curve. Day-to-day

repro-ducibilities of between 2 and 12% formannose were attainable forquantititesof100to900ng.The sensi-tivitywassuch that 10mg/liter couldbedetected in

a0.1-mlsample ofserum.

RESULTS

The invitro productionofmannose inbroth

cultureof C. albicansis shown inFig. 1.There was nomeasurable mannose atthe time of cul-tureinoculation,butmannoseconcentrationsin

the broth supernatant increasedconcomitantly

withgrowth of the organismasrepresented by theoptical densityof the broth. The appearance ofmannosewasnoted withoutsignificant

trans-formationtothemycelial phase.

The mannose concentration for the different

control groups is summarized in Fig. 2. Serum

from normal blood donors (mean, 1.3 ug/ml),

normalchildren(mean,

0.0Og/ml),

and

patients

with vaginalcandidiasis (mean, 0.3 ug/ml) had

low concentrations. Although we studied only

two patients with catheter-associated

candide-mia(mean,36.1

isg/ml),

theirvalueswerereadily

distinguishablefrompatientswithinvasive

can-didiasis.Clinicalcharacteristics, includingrenal

function, of the patients withinvasive candidi-70

60

501

401

301

201

I0

S

oSàO1

Ov

W

1.0 k

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FIG. 1. In vitrogeneration of mannose assayed by gas-liquid chromatography (O) concurrent with growthof C. albicans cultureasmeasured byoptical density(El).

asis, the range of mannose concentrations where

morethanoneserumwasavailable, andvalues

for the threeCSF specimensevaluatedare

sum-marized inTable 1.

At least three serial serum specimens were available for each of two patients to relate the role of free mannose to progress of Candida

infection. Patient 1 showed a progressive in-crease in mannose concentration despite

nega-tive bloodcultures untilshortly beforehe died,

whenblood cultures becamepositive and anti-fungal therapywas initiated. Patient 5 showed increasingmannoseconcentrations until

ampho-tericin Btherapywasbegun. Shortly after

ther-apy wasinitiated,mannoseconcentrations were

elevatedbeforebody fluidculturesbecame

pos-itive.

Although the six patients with autopsy or

biopsy evidence, orboth, ofcandidiasis all had

C.albicansonisolation, it is importanttorealize

that other Candidaspecies may produce

inva-sive disease.Wehavestudiedtwopatients with

C. tropicalisinfectioninwhomtissuediagnosis

was not available. A 45-year-old female with

acuteleukemia had blood cultures positive for

C. tropicalis

during therapy-induced

leuko-penia. Therewas noevidence for

catheter-asso-ciated infection. Serum mannose concentration

atthe timeblood cultures weredrawnwas82.8

fig/ml;

thisvaluedecreased to 29.5

jug/ml

after

9days ofamphotericinBtherapy and thereturn

ofperipheral neutrophils. The second patient, a

34-year-old male, presented with progressive

pneumonitis. Lung aspirate was cultured and

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PATIENT

GROUPS

EVALUATED

FIG. 2. Serum mannoseconcentrations inpatients with invasive candidiasis and in control groups. 1, Invasivecandidiasis;2, normalchildren; 3,blooddonors;4, mucosalcandidiasis;5, cathetercandidiasis;6, intensivecareunitpatients; 7, breastcancerpatients;8, other fungal infections; 9, ketoacidosis patients.

revealed C.tropicalis togetherwithFrancisella

tularensis. Serummannose atthetime of

aspi-ration was 32.5

itg/ml.

Thepatientdiedshortly

thereafter.

Concentrations ofmannosefoundin the seven

"normal" CSFspecimensranged from0 to 10.5

,ug/ml witha meanvalue of3.1,ug/ml

(data

not

shown). Twoofoursix

patients

had

histological

andculturalevidenceforC.albicans

meningitis.

In oneof these (patientno.6)the serum

concen-tration offree mannose was considerably less

(49.4

itg/ml)

than in our other five patients,

while CSFmannose wasmodestly elevated.This

may reflect the autopsy findings thatonly the

meningeswereinvolved with the Candida

infec-tion. Patientno.4, our otherpatientwith central

nervous system candidiasis but with higher

se-rum and CSF mannose values than no. 6, had

grossly more extensive central nervous system

involvement with

multiple

Candida brain

ab-scesses inadditiontomeningitis.

Serum from

patients

with non-Candida

my-coses showed mannose values that were less

thanthose seen with systemic candidiasis (mean,

16.8,ug/ml).

Evidence has been presented showing

in-creased concentrations oftotal blood mannose

(including glycoproteins)inpatients with breast

carcinoma (16). We measured free serum

man-nosein four patients with metastatic breast

car-cinoma and found concentrations to be only

slightly above those of normal blood donors

(mean,8.2,ug/ml).

Sera were obtained from 32 consecutive

pa-tientsadmittedtotheVeteransAdministration

MedicalCenterintensivecareunit. Thisgroup

was comprised primarily ofpatients having

is-chemic heart disease, renal insufficiency, and

respiratory failure with or without pulmonary

infection. Thesepatientswere receivinga

vari-ety ofpharmacologicalagentsincluding

anti-ar-rhythmic drugs and antimicrobial agents.

Ail

fourpatientswith renalinsufficiency had

man-nose concentrationsless than20,g/mland did

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not

overlap

with

patients

having

systemic

can-didiasis. With the exception of one

patient

in

diabeticketoacidosis, the intensivecarepatients

had a mean maximum mannose concentration

of13.4,g/mlof serum.

Because thepatientinketoacidosiswasfound

to have a serum mannose concentration of 46

,ug/ml, wecarried out a preliminary evaluation

of serum from 30 diabetic patients. Only in

poorly controlled diabetic patients (glucose of 2300 mg/dl) were mannose concentrations found whichoverlapped values found in candi-diasispatients.Hemoglobin

Ac

valueswerenot

availablefor thesepatients. None ofourpatients

withinvasive candidiasiswas known tobe dia-betic.

DISCUSSION

Thepresence ofcirculating Candida antigen

wasfirstreported by AxelsenandKirkpatrickin apatient with chronic mucocutaneous

candidi-asis (1).Usinganti-Candida antibodiescoupled

toagarose, Fischeretal. (6) reported the

detec-tion ofa Candida

polysaccharide

from thesera

of6of23children withchronic mucocutaneous

candidiasis. Miller and co-workers (14) noteda

unique gas chromatograph

"fingerprint"

in

pa-tients with blood cultures positive for C.

albi-cans. They suggested that the unique peaks

werederivatives ofmannose,consistentwiththe

high percentage of mannose-containing

com-pounds known to be present inthe cell wall of

C.albicans (3). Kiehnand

colleagues

(10) have

usedgas

chromatography

todescribe

D-arabini-tolin the seraofpatientswith invasive

candidi-asis. They found 15of20patientstohave

con-centrations greaterthan 1

,ug/ml,

although

pa-tients with renal

insufficiency

and colonization withCandida could have elevated values.Roboz

and co-workersreportedsimilarresults,

detect-ingconcentrations ofarabinitolof21.2

,ug/ml

in

9 of11 serafrom patients diagnosed as having

invasivecandidiasis (18).

Other workers (13, 23) have used

hemagglu-tination inhibition todemonstrate the presence

ofcirculating mannansin 30to60% ofpatients

with invasive candidiasis. The enzyme-linked

immunosorbent assay and

radioimmunoassay

havebeenusedtodemonstrate Candida

antigen

in human serum. Warren et al. reported that

blastospore antigens could be detected in the sera oftwo patients with invasive candidiasis

(21), and Stevens et al.reported12of19patients

tohavemeasurable levels ofasoluble

cytoplas-mic protein antigen (20). Segal and colleagues

used an enzyme-linked immunosorbent

assay-inhibition technique to assay for mannan

anti-gen in seven patientswith evidence of

candidi-asis at autopsy (19). Weiner andCoats-Stephens

have developed a radioimmunoassay for Can-dida mannan and detected antigenemia in 6 of

14patients with invasive candidiasis (22).

Kerkering and co-workers have demonstrated

a Candida polysaccharide antigen in eight

pa-tients with invasive disease by using counter-immunoelectrophoresis (9), whereas Lehmann and Reiss were unable to detect Candida

pep-tidoglucomannan in six patients with systemic

candidiasis by using immunodiffusion,

counter-immunoelectrophoresis, andenzyme-linked im-munosorbent assay (11).

Wepresent data thatquantitation of free

man-noseidentifiedallsix patients with invasive

can-didiasis. Fiveof the six tissue-proven cases had

markedly elevated serum concentrations, and

twopatients hadincreased CSF concentrations.

Although a larger variety and number of CSF

specimens must be evaluated, our present data suggest that increased free CSF mannose

con-centrations may accompany Candida

involve-ment ofthe central nervous system.

Ofthe twopatientswithless definite evidence

for invasive candidiasis, one had elevated

con-centrations of mannose which decreased with

amphotericin B therapy and the return of pe-ripheral neutrophils. This is consistent with the

known antigenic similarity of C. tropicalis and

C. albicans (8).

The mannose assay also qualitatively distin-guished patients with mucosal infection and

catheter colonization from those with invasive

disease. Itshould be noted, however, thatonly

twopatients withcathetercandidiasiswere

eval-uated. Patients with renalinsufficiency did not

have mannose values overlapping those of

pa-tients with candidiasis. Likewise, there was no

correlationbetween mannoseconcentration and

renal insufficiency in the patients described in

Table 1. Thus far, poorly controlled diabetics are the only patientswho have been found to

have elevated mannose concentrations similar to those ofpatients with systemic candidiasis.

No evidence of infection with Candida or other

fungicould befoundinthese diabetic patients. As a physicochemical assay, our method

avoidssome

problems

inherent with

immunoas-says for

antigen.

Wheat and co-workers (24)

haverecently reported their inability to

consis-tentlydemonstrateantigenemia in patients with

staphylococcal endocarditisbyusing a radioim-munoassay, presumably a result of preexisting

antibodiesformingimmunecomplexes.

Preexist-ing antibodies to Candida sp. might have a

similar effect onsensitivity of immunoassays for

Candidaantigen, althoughacid-heatextraction

ofserum before assay may improve sensitivity

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http://jcm.asm.org/

(22). A physicochemical assay such as we have

described measures a relatively simple

com-pound thoughttoderive from the Candida cell

wall, whereas

immunoassays

measure more

complexantigens. Thus, specificity isoften less

than immunoassays, and precautions must be

taken to remove unwanted sources of antigen

(in this case, mannose) during sample

prepara-tion. Even then, some degree of overlap with

otherpatientpopulations maynotbeavoidable,

aswith ourpoorlycontrolled diabetics.

Our data suggestthatfurther effortis justified

indeveloping physicochemicalassaysfor

micro-bialproducts. Suchproceduresmightrepresent

a

supplementary

or alternative method for

ex-istingtechniquesofantigendetection.

ACKNOWLEDGMENT

This work was supported in partby Veterans

Administra-tion Research Funds, projectno.5782-001.

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