Performance of cryptococcus antigen latex agglutination kits on serum and cerebrospinal fluid specimens of AIDS patients before and after pronase treatment

0095-1137/91/020333-07$02.00/0

CopyrightC) 1991,American Society for Microbiology

Performance of Cryptococcus

Antigen

Latex

Agglutination

Kits

on

Serum

and Cerebrospinal Fluid Specimens

of AIDS Patients

before

and

after

Pronase Treatment

JOHN R.

HAMILTON,'*

ANITANOBLE,' DAVID W. DENNING,123'4 AND DAVID A. STEVENS"2'3'4

Microbiology Section, Departmentof Pathology,' and Division of InfectiousDiseases, DepartmentofMedicine,2 Santa

Clara Valley MedicalCenter, 751 SouthBascom Avenue, andCalifornia Institute for Medical Research,4 San Jose,

California 95128, and Division of InfectiousDiseases, Department ofMedicine, Stanford University School of Medicine,

Stanford, California

943053

Received 8 June 1990/Accepted 23 October 1990

Cryptococcal antigen titers in 97serumand42 cerebrospinal fluid (CSF) specimens from 37 AIDSpatients

withculture-proven cryptococcal infectionweredetermined with the Meridian kit(Meridian Diagnostics Inc.,

Cincinnati, Ohio) beforeandaftertreatmentwithpronase.The geometricmeantitersbefore and afterpronase

treatmentwere 1:45and1:588inserumand 1:97 and 1:79 in CSF, respectively. Onlyon serum(butnotCSF)

specimens afterpronasetreatmentwere(i) titers increased by 2to13 dilutionson57%of the specimens, all of

whichhadtiters of-<1:128 beforepronasetreatment, (ii) false-negative reactionson27% of specimens before

pronase treatment eliminated, all of which had titers from 1:4to 1:4,096, (iii) prozone-like reactions (titer,

<1:256)on9% ofthe specimens beforepronasetreatmenteliminated, and (iv) agglutination reactionsonall

specimensstrongerand easiertointerpret. Antifungalagentsaddedtoserum aswellasfreeze-thawcyclesdid

notchange antigen titers inserum.Aftertwoseparate tests,thesametiterswereobtainedon94%of 35serum

specimensthatweretreatedwithpronaseandon96% of 53 CSF specimens thatwerenottreated withpronase.

Atotal of 26serumspecimensand28CSF specimens from patients withnocryptococcaldiseasewerenegative

before and after pronase treatment. The IBL kit (International Biological Labs Inc., Cranbury, N.J.)was

compared with the Meridian kit on 41 serum specimens and 14 CSF specimens. Results from the twokits

agreed on 54 and 68% of serum specimens and 86 and 93% of CSF specimens before and after pronase

treatment, respectively. The IBL kit generally produced higher titers on specimens in disagreement and

produced noprozone-like reactions. Routinepronasetreatmentofserumis recommended withtheMeridian

kit in order to eliminate false-negative and unclear agglutination reactions by producing a consistent

interpretation of agglutination reactions. CSF specimens do not require pronase treatment. Titer results

producedby the kits fromthetwo different manufacturers varied considerably: the kits should notbe used

interchangeably for determining antigentiters inserumspecimens.

The detection of capsular antigen from Cryptococcus

neoformans in serum and cerebrospinal fluid (CSF) is a

sensitive and specifictest for the rapid diagnosis of

crypto-coccosis (2, 3, 5, 9, 12, 16). The sensitivity of the latex

agglutination method ranges from 10 to 157 ng of capsular

polysaccharide antigenperml,dependinguponthe reagents used (3, 12, 16). Titration of antigen in serum and CSF

specimens has been used for diagnosis, prognosis, and

monitoring of antifungal therapy(1-3, 6, 8, 11, 13). Withthe

increasing number ofC. neoformans infections caused by the AIDSepidemic, antigen detectionisanimportanttestfor the clinical microbiology laboratory. The availability of

commercial reagents has greatly contributed to the routine testing capability of laboratories; however,the

requirements

for

optimal performance

ofthevariouskits and theirrelative capabilities are notcompletely defined.

Various specimen treatment methods and reagents have

been reported which eliminate the false latex

agglutination

reactions caused by rheumatoid factor and other unknown

factorsinserumandCSF and allow detection and titration of

antigen (4, 7, 9, 10, 15). Aconcurrent

finding

wasthatsome

ofthese reagents increase

antigen

titers.Oneof the reagents, pronase, produces increased

antigen

titerson

specimens

not

*Correspondingauthor.

needing treatment (those with no false agglutination

reac-tions). Increased antigen titers were observed on 80.7% of

theserumspecimens andon20% oftheCSFspecimens (9).

False-negative reactions withlatexagglutination kitshave

rarely been reported. Mosthave been corrected witheither dilution ofthe specimen orpronasetreatment. A prozone-like reaction was reported on a CSF specimen (14) and a serum specimen (16) that was resolved by dilution of each

specimen: antigen titers were as high as 1:1,600. Other negative reactionshavebeenreported fromserumandCSF specimens that were converted to

positive

after the

speci-mens weretreatedwith pronase(9).Nineteen percentofthe

serumspecimenswere

falsely

negative

beforepronase

treat-ment;afterpronasetreatment titersas

high

as

1:1,024

were

obtained.

One kit

(International

Biological

Labs

[IBLI Inc.,

Cran-bury,

N.J.) has been studied more than the other three

commercially available kits have. Two of these kits have been

compared

with the IBLkit in

only

one

study (16).

The other

kit,

which is manufactured

by

Meridian

Diagnostics

Inc.,

Cincinnati,

Ohio,

wasused in

conjunction

with the IBL kit ina studyto monitor

therapy (3);

no other studies with theMeridian kit have been

reported.

The purpose of this

study

was to report our

experience

with the Meridian kit used on clinical

specimens

from

patients with AIDS and with

culture-proven

cryptococcal

333

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334 HAMILTON ET AL.

infection, the advantages and uses of pronase treatment of specimens, and comparison of the IBL and Meridian kits.

MATERIALS ANDMETHODS

Specimens. A total of42 CSF specimens and 97 serum

specimensfrom 37 AIDS patients with culture-proven

cryp-tococcal infection were tested before and after pronase treatment from late 1987 through September 1989. Serum andCSFspecimens weretestedwith the Meridian kit before

pronasetreatment andpriorto storage at -20°C or

concur-rently with andwithoutpronasetreatment or were stored at

-20°C

and then tested concurrently with and without pro-nasetreatment. Thirteen different lot numbers of the

Merid-ian kit were used. Eight lot numbers were used on the 61

specimens

thatproduced different titers after pronase

treat-ment. On56 of61 (92%) specimens, the same lot numberof

the kit (one of the eight lots) was used to test the specimen

before and after pronase treatment. The differences in titer

on these 56 specimens spanned the entire range of titer

increasesand decreases after pronasetreatment. Therefore,

the difference in titer between specimens that were treated and not treated with pronase was related to the action of

pronase and not the different lots of reagents. Specimens

tested with theIBLkitwere storedat -20°C and then tested

concurrently with and without pronase treatment. Four

different lotnumbers ofthe kit were used. Fifty-three CSF

specimens

from 20 patients were repeat tested with the

Meridian kit without pronase treatment. Of the 53 CSF

specimens,

21 were selected from the previous group of 42

CSF

specimens

from 37 patients, an additional 28 CSF

specimens

wereselected fromthose 37patients, and 4 other CSF specimens were selected from 2 additional patients.

Specimens

werestoredandtested inborosilicateglass tubes

(13 by

100mm; diSPo Tubes; American ScientificProducts,

McGawPark, Ill.). The tubes were sealed with plastic caps

(Tainertop;

Fisher Scientific, Pittsburgh, Pa.) for storage.

Agglutination titers were considered different when there

was adiscrepancy of

.2

dilutions.

CSF and serumspecimensfrompatientswith no

cryptococ-cal disease. A total of 28 CSF specimens and 26 serum

specimens

were collected from 44 patients without crypto-coccal disease. Samples were stored at -20°C and later tested with the Meridian kit concurrently with and without

pronase treatment.

Meridian kit. The Meridian Diagnostics cryptococcal

an-tigen latex agglutination kit, CALAS, contains

anticrypto-coccal globulin reagent (latex particles coated with rabbit

anticryptococcal

globulin), normal globulin reagent (latex

particles

coated with rabbit normal globulin), antiglobulin control (goat anti-rabbit serum), negative control (normal

human serum), cryptococcal antigen control (purified

polysaccharide

antigen from cultures of C.neoformans),and

glycine-buffered

diluent withalbuminat pH 8.4.

The kit was usedaccordingto theinstructionsprovided by the manufacturer. Briefly, CSFspecimens were placed in a

boiling water bath for 3 min, and serum specimens were

heatedat56°C for 30min. Twenty-five-microliter samplesof CSF or serum were mixed with an

equal

volumeof

anticryp-tococcal reagent or normal globulin reagent. Positive and

negative controls weretested daily. The slide was placed on

arotating platformat 120 rpm for 5

min,

andthereactionwas read within 15 to 30 s without moving the slide. The

agglutination reaction was graded negative or 1+ to 4+.

Pictures of each reaction were provided in the kit, which

helped

to produce a consistent interpretation ofthe

reac-tions. A negative reactionwas interpretedas eithera homo-geneous suspension with no visible clumps or a fine granu-lation against a milky background (1+ reaction). The agglutinations of 2+ to 4+ were graded by the

increasing

size ofthe clumps and the degree ofbackground

clearing;

these were interpreted as positive. Specimens were diluted in the glycine buffer by using a twofold dilution series to

determine the antigen titer.

IBL kit. CSF and serum specimens were tested

according

to the procedures supplied with the Crypto-LA Kit

(IBL

Laboratories) anddefined

previously

(15). Resultswere read

immediately after mechanical rotation with minimal hand

rotation. Reactions were interpreted asdefined in the kit. A negative reaction was interpreted as a fine granular

back-ground or milky suspension with the absence of

agglutina-tion. A positive reaction was interpreted as distinct large

clumpsagainstaclearorslightlymilky background, orsmall but definite clumps against a milky background. Four

dif-ferent lot numbers were used.

Pronase preparation and use. The pronase reagent

(Pro-nase protease; 53702; Calbiochem, San Diego, Calif.) was

prepared as described previously (9), but was stored at

-20°C

and not lyophilized. The glycine-buffered saline (pH

8.2)supplied with the Meridian kit was used to suspend the pronase. The reagent was divided into 0.5-ml portions in

glass tubes, sealed with plastic caps, andfrozen at

-20°C

for

later use. Prior to pronase treatment, all specimens were

prepared according to the instructions supplied with each latex agglutination kit. The pronase reagent was thawed at

room temperature,added to an equal volume(0.2ml)ofCSF or serum specimen in glass tubes, incubated for 15

min

at

56°C,

and then heated in a boiling water bath for 5

min

to

inactivate the pronase enzyme. Pronase-treated specimens

were not saved beyond the initial day of testing. If repeat testing was performed on alater day, aportionof theoriginal

specimen was treated with pronase and tested. Since the specimen is diluted with an equal volume of the pronase reagent, this dilution factor was incorporated into all calcu-lations of antigen titer. The reagent was held at room temperature for the day of use and then discarded. Studies conducted over a 45-day period showed that the reagent was stable after reconstitution when held at -20 or 2 to

8°C.

RESULTS

No cryptococcal disease. The results from 28 CSF speci-mens and 26 serum specispeci-mens from 44 patients with no cryptococcal disease were negative when tested before and after pronase treatment by using the Meridian kit.

Serum tested with Meridian kit. Results from 97 serum

specimens from 35 patients with culture-proven cryptococ-cal disease tested with the Meridian kit are shown in Fig. 1. Before pronase treatment of specimens, most agglutination reactions were 1 to 3+ in reactivity, producing titers that ranged from negative to 1:65,536 with a median and geomet-ric mean of 1:16 and 1:45, respectively. After pronase treatment agglutination reactions were consistently 3+ to 4+ and produced approximately the same range of titers, but the median and geometric mean increased to 1:512 and

1:588,

respectively.

On 41 of 97 (43%) specimens, results were within 1 dilution of each other before and after pronase treatment. Titers ranged 1:16 to 1:65,536, with most (39 of 41; 95%) being at a titer of .1:256.

On 55 of 97 (57%) specimens, titers were higher after

pronase treatment compared with titers before pronase

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PRONASE AND CRYPTOCOCCAL ANTIGEN TESTING 335

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FIG. 1. Comparison ofcryptococcal antigen titers on 97 serum specimens (U)and 42CSF specimens (0) tested with the Meridian kit before and after pronase treatment.

treatmentby 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 dilutions on4,6,7, 13, 5, 1, 5, 2, 5, 3, 3, and 1specimen, respectively. Titers on all 55 specimens before pronase treatment were s1:128. After pronase treatment, titers on these 55 speci-mens ranged from 1:4 to 1:4,096. Titers on 26 of 55

speci-mens (from 10 patients) before pronase treatment were

interpreted asnegative. On many of these specimens,a 1+

agglutination (negative interpretation) was observed that

produced an endpoint titer equal to that observed after pronase treatment. After pronase treatment, titers on these

specimens, whichwere negative before pronase treatment,

ranged from 1:4 to 1:4,096.

On 1of97(0.8%) specimens,pronasetreatment produced a fourfold lower titer (1:2,048) compared with the titer

(1:8,192) before pronase treatment.

Randomly selected serum specimens (35 specimensfrom 16patients) with titersthatrangedfrom 1:8 to 1:32,768 were repeat tested todetermine thereproducibilityoftiter results

afterstorage at-20°Cand then pronasetreatment. Titers on 33 of35 specimens werewithin 1 dilution ofthepreviously obtained titer. On2of 35 specimens, eachfrom adifferent

patient,the repeat titerdiffered from thepreviousone by 2

dilutions;one wasgreater(initial titer, 1:4,096; repeattiter, 1:16,384); the other was less (initial titer, 1:32,768; repeat

titer, 1:8,192).

Effect of multiple freeze-thaw cycles on antigen titer. In

ordertodeterminewhethermultiple freeze-thaw cycleson a

specimen

could cause changes in

titer,

two

patient

speci-mens (titers, 1:1,024 and 1:32,768) were thawed, and a

portion was removed for testing and then refrozen. The

cyclewasrepeated weeklyfor 4 weeks. Eachweekly sample

waspronase treatedand then tested with the Meridian kit. Nochangesin titerwereobservedatanyofthe

testing

times. All titerswerewithin 1 dilutionoftheinitial titer.

CSF tested with Meridian kit. Results on 42 CSF

speci-mens from 19 patients with culture-proven

cryptococcal

meningitis tested with the Meridian kit before and after

pronase treatment are shown in Fig. 1. The range of titers

before and after pronase treatment was approximately the same,undilutedto 1:8,192. The median and geometric mean

titersbefore andafterpronase treatment were 1:128 and 1:97 and 1:128 and 1:79, respectively. On 37 of 42 (88%) speci-mens,titersafter pronase treatment were within 1 dilution of

thetiters obtained before pronasetreatment. Pronase treat-ment did not enhance reactivity or increase antigen titers.

On5of42 specimens, eachfrom adifferent patient, results were 2 to 3dilutions lower after pronase treatment.

Fifty-three randomly selected CSF specimens from 20

patientswere repeat tested without pronase treatment after storage at -20°C. Titers ranged from undiluted to 1:4,096.

Titers from 51 of 53 (96%) specimens after storage were

within 1 dilutionofthepreviously obtained titer. Two of 53

specimens, each from a different patient, differed by 2

dilutions;onewas lower(initial titer, 1:32; repeattiter, 1:8);

the other washigher(initial titer,1:256; repeat titer, 1:1,024). Prozone-like reactions. Only 9 of97 (13%) serum

speci-mens (from seven patients) andnone of42CSF specimens produced a prozone-like reaction (no agglutination in low

dilutions ofthespecimen) beforepronase treatment with the

Meridian kit. All specimens were from patients who had

meningitis. Theresultsareshown inTable 1. Sixofthenine

resultsoccurred on all

specimens

oron multiple

specimens

submitted from four patients. The other three ofnine

pro-zone-like results occurred on

only

one of the

multiple

specimens

from three

patients.

The antigen titers on the specimens with

prozone-like

reactions ranged from 1:1,024 to

1:32,768

before pronase treatment.The titerof the

prozone-like

reaction

ranged

from

undiluted to 1:256. There was no correlation between the

antigen titer and the presence or titer of the prozone. On seven ofnine

specimens,

the

agglutination

reactions on the dilutions in theprozonewerenot

clearly

negative;

a border-line(1+)

reactivity

wasobserved. On the othertwo of nine

specimens, each from different

patients,

a clear

negative

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336 HAMILTON ET AL.

TABLE 1. Prozone-like reactionsonnineserum specimenstested with the Meridian and IBL kits before and afterpronase treatment

Meridiankit IBL kit

Patient Specimens' Titerbefore Titerafter Titerbefore Titerafter

Prozoneb Antigen Prozone Antigen Prozone Antigen Prozone Antigen

1 1/1 128 2,048 No 2,048 No 1,024 No 4,096

2 1/1 1 8,192 No 2,048 No 4,096 No 4,096

3 2/2 128 8,192 No 8,192 No 8,192 No 4,096

64 8,192 No 4,096 No 4,096 No 4,096

4 2/3 32 8,192 No 16,384 No 8,192 No 16,384

32 1,024 No 1,024 NTC NT NT NT

5 1/2 256 2,048 No 4,096 No 8,192 No 4,096

6 1/5 64 4,096 No 8,192 NT NT NT NT

7 1/5 8 32,768 No 16,384 No 32,768 No 32,768

"Number

of specimens withprozonereaction/totalnumberofpatientspecimenstested. bTheendpointdilution oftheprozone-likereactioninwhich noagglutinationwasobserved.

NT,Specimenswere not tested.

agglutination reaction was observed only on the undiluted

specimen; the other dilutions in the prozone showed 1+

reactivity. Pronase treatment eliminated the prozone-like reaction on all dilutions, produced a 3+ to 4+ reactive

agglutination to the endpoint dilution, but did not increase

antigen titer.

Effect of antifungal agents on antigen testing. Antifungal agentsandserafromapatient treatedwithitraconazolewere

mixedwithanantigen-positive specimenand tested withthe

Meridian kit in order to determine whether these agents caused adirect reduction in antigen titer. Equal portions of an antigen-positive (1:1,024) serum specimen were mixed

with (i) serum from a patient with Coccidioides immitis

infectiontreated withitraconazole, (ii)normal human serum

supplemented withitraconazole (10

,ug/ml),

and(iii) normal

human serum supplemented withamphotericin B (2 ,ug/ml) andflucytosine (100 jig/ml). Specimens were tested before and after treatment with pronase. Titers before and after

pronase treatment in all test conditions equaled the

begin-ning titerafter the dilution factorwas calculated.

Serum specimens tested with the IBL and Meridian kits.

Forty-one serum specimens from 29 patients were tested with both kits. The serum specimens that represented four groups oftestresults obtained with the Meridian kit before andafterpronase treatment wereselected. These specimens were tested with the IBL kit before and after pronase treatment.Overall titers produced with the two kits were the

sameordiffered by only 1dilution on 22(54%) and 28 (68%) of serum specimens before and after pronase treatment,

respectively. On serum specimens in which the two kits produced discrepant titer results, the IBL kit produced

higher titersthan the Meridian kit did by 2 to >5 dilutions on 18 of19 specimens before pronase treatment and by 2 to 4

dilutionson 11 of 13 specimens after pronase treatment.

A group ofeight serum specimens (from eight patients) that were negative before pronase treatment but positive after pronase treatment (tested with the Meridian kit) were

selected; titers ranged from 1:8 to 1:1,024. The IBL kit produced titers that ranged from negative to 1:4,096 before pronase treatment and from negative to 1:16,384 after pro-nasetreatment. The differences in antigen titer between the

twokits are shown in Fig. 2A. The titer results from the two

kits were in agreement on 2 (25%) of the specimens before pronase treatment and on 5 (63%) of the specimens after pronase treatment. Before pronase treatment, two of eight

specimenswerealso negative when tested with the IBL kit;

onthe other six specimens, the IBL kit had positive titers at

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FIG. 2. Cryptococcalantigentiter differences (lower [-], higher [+]) betweenthe IBL andMeridian kits tested before(U)and after (O) pronase treatment on specimens selected on the basis oftest resultsobtainedwith the Meridian kit. (A) Eight serumspecimens that were negative before pronase treatment and positive after pronasetreatment; (B) 9 serum specimens that had titers that were

.2dilutions higherafter pronase treatment; (C) 17 serum specimens that hadthe sametitersbeforeand after pronase treatment; (D) 14 randomly selectedCSF specimens.

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2, 4, and >5 dilutions on 1, 2, and 3 specimens, respectively. After pronase treatment, seven of eight specimens were positive with the IBL kit; however only five of seven specimens had equal titers with both kits. On two of seven specimens, the IBL kit produced higher titers than the Meridian kit did by 2 and 4 dilutions. On the one of eight specimens that was negative before and after pronase treat-ment and tested with the IBL kit, the Meridian kit produced a titer that was 5 dilutions higher after pronase treatment than that before pronase treatment.

A group of nine serum specimens (from nine patients) that produced increased titers by -2 dilutions after pronase treatment (tested with the Meridian kit) were selected. Titers ranged from 1:1 to 1:64 before pronase treatment and from 1:32 to 1:2,048 after pronase treatment. The IBL kit pro-duced titers that ranged from 1:4 to 1:2,048 before pronase treatment and from 1:64 to 1:4,096 after pronase treatment. Five of the nine specimens tested with the IBL kit had increased titers as a result of pronase treatment. The differ-ences in antigen titer between the two kits are shown in Fig. 2B. The titer results from the two kits were in agreement on two (22%) of the specimens before pronase treatment and on three (33%) of the specimens after pronase treatment. Before pronase treatment seven of nine specimens had higher titers with the IBL kit than they did with the Meridian kit by 3, 4, and 5 dilutions on three, one, and three specimens, respec-tively. After pronase treatment the IBL kit produced higher titers compared with those produced by the Meridian kit on six of nine specimens; five of the six titer increases were by 2 dilutions; the other was a 3-dilution increase.

A group of 17 serum specimens (from 12 patients) that produced the same titer before and after pronase treatment (tested with the Meridian kit) were selected; titers ranged from 1:16 to 1:16,384. The IBL kit produced equal titers before and after pronase treatment on 15 of 17 specimens; titers ranged from 1:64 to 1:65,536 before and after pronase treatment. The differences in antigen titer between both kits are shown in Fig. 2C. The titer results from the two kits were in agreement on 12 (71%) of the specimens before pronase treatment and on 13 (77%) of the specimens after pronase treatment. Before pronase treatment, titers werehigher with the IBL kit than they were with the Meridian kit on 4of 17 specimens by 2 dilutions, but were lower than those obtained with the Meridian kit on 1 of 17 specimens by 3 dilutions. After pronase treatment titers were higher with the IBL kit than they were with the Meridian kit on 3 of 17 specimens by 2 to 3 dilutions but were lower on 1 specimen by 3 dilutions. A group of seven serum specimens (from sixpatients) that produced a prozone-like reaction before pronase treatment (tested with the Meridian kit) were selected. Titers ranged from 1:2,048 to 1:32,768 with the Meridian kit and did not increase after pronase treatment. Pronase treatment elimi-nated the prozone-like reaction on alldilutions. The IBL kit produced equal titers before and after pronase treatment on six of seven specimens; titers ranged from 1:1,024 to 1:32,768 before pronase treatment and from 1:4,096 to 1:32,768 after pronase treatment, as shown inTable 1. The titer results from the two kits were inagreement onsix (86%) of the specimens before pronase treatment and on seven (100%) of the specimens after pronase treatment. Antigen titers between the two kits agreed on sixofseven specimens before pronase treatment and on seven ofseven specimens after pronase treatment. In contrast to theMeridian kit, no prozone-like reactions were observed with the IBL kit.

On all specimens that had equal titers before and after pronase treatment (tested with theMeridian kit)(the

preced-ingtwogroups), resultsfrom the two kits agreed on 18 of 24

(75%) specimens before pronase treatment and 20 of 24

(83%) specimens after pronase treatment.

CSF specimens tested with the IBL and Meridian kits. Fourteen CSF specimens from 13 patients were tested with

both kits beforeandafterpronase treatment.Titersobtained with the Meridian and IBL kits ranged from 1:8 to 1:4,096 and 1:16 to 1:16,384 before pronase treatment and from 1:8 to 1:8,192 and 1:16 to 1:16,384 after pronase treatment,

respectively. The differences in titers obtained with the two kits are shown in Fig. 2D. Antigen titers obtained with the two kits were the same on 12 (86%) specimens before pronase treatmentand on 13 (93%) specimens after pronase treatment. Before pronase treatment the Meridian kit pro-ducedhigher titersthan the IBL kit did on 2 of 14specimens

by 2 dilutions. After pronase treatment, the IBL kit

pro-duced higher titers than the Meridian kit did on 1 of 14 specimens by 3 dilutions. On this specimen, both kits

produced equaltiters (1:8,192) before pronase treatment, but

afterpronase treatmentthe Meridian and IBL kitsproduced titers of1:2,048 and 1:16,384, respectively.

DISCUSSION

This is the first extensive study ofcryptococcal antigen testing in the AIDS era. It may have uncovered adiagnostic

problem that is unique to these patients and their disease process, as demonstrated with the Meridian and the IBL kits. Pronase treatment of serum specimens enhanced the

detection and titration ofcryptococcal antigen by uncover-ing and resolvuncover-ing false-negative results, eliminating poten-tially false-negative results from a prozone-like reaction, and eliminating the variable endpoint determinationsfor titration

of antigen. No identifiablefactors werefoundtoexplain the effects of pronase treatment. The Meridian and IBL kits were shown to produce different results even after pronase treatmentof specimens.

Pronase treatment of specimens, as originally described, was used to eliminate false-positive reactions caused by rheumatoid and other factors. Only one study (9) has shown the effects of pronase treatment of all specimens onantigen detection and titration. The IBL kit was used to test 57 serum specimens and 70 CSF specimens before and after pronase treatment. More of the serum specimens (81%) in that study had increased titers (.2 dilutions) after pronase treatment compared with those (57%) in the present study tested with the Meridian kit. It is interesting inboth studies that the range of titers before pronase treatment were the same. On CSF specimens tested after pronase treatment in the previous study (9), titers were increased on 20% of the specimens, none haddecreasedtiters, butone specimenthat was negative before pronase treatment was positive. These findings are in contrast to theresultsof the presentstudy, in which titers decreased after pronasetreatment on 12% of the specimenstested with the Meridian kit. The greater effect of pronase treatment shown in the previous study may reflect variables with individual patients and, perhaps, not differ-ences in the two kits. It isinteresting that in the present and previous studies, pronase treatment of CSF changed titers regardless of the degree of titer before pronase treatment. There is no explanation for the opposite effect of pronase treatment on CSFspecimens tested with the Meridian kit in the present study. Results from the previous study (9) recommend theuse ofpronase treatment of CSFspecimens.

Results from this study show that on CSFspecimens tested

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338 HAMILTON ET AL.

with the Meridian kit, routine pronase treatment is not

needed.

In theprevious study (IBL kit)(9), in which all specimens

were treated with pronase, specimens that were negative before pronase treatment but positive after pronase

treat-ment were found on 19% of serum specimens and 1.4% of CSF specimens. Very similar results were obtained in the present study on serum specimens tested with the Meridian kit; false-negative results were uncovered with the use of pronase treatment on 19% of serum specimens but not on CSF specimens. Theseresults demonstrate thatroutine

treat-ment of serum specimens with pronase is essential in order toeliminate false-negative results,regardlessof thekit used.

Rarely, otherstudies havereported false-negative

crypto-coccal antigen test results. In two studies (14, 16) the

false-negative result was resolved by dilution of the speci-men. The specimens (one serum specimen and one CSF specimen) were positive to titers of .1:1,024 when tested after dilution. This prozone-like phenomenon that occurred in the serum specimen was observed with three different kits (16). In the otherstudy (14), all CSF specimens from a single patient tested with commercially unavailable reagents duced the prozone-like phenomenon. The titers of the

pro-zone-like reactions in those two studies were not deter-mined. In the present study the prozone-like reactions

occurredon 9% of the serum specimens, a higher rate than

thatreported previously. It is interesting in the present study that no CSF specimens produced a prozone-like reaction; however, all serum specimens with a prozone-like reaction were from patients who had cryptococcal meningitis. In

addition, prozone-like reactions did not occur on all speci-mens from the same patient, as reported in the one study (14), but couldoccurrandomly among multiple specimens. A newfinding in thepresent study was that pronase treatment eliminated the prozone-like reactions on all specimens.

Apractice ofscreening a diluted and undiluted sample of serumin order to eliminate the potential prozone-like

reac-tions and detect the presence of cryptococcal antigen is questionable. In the present study, 9% of the serum speci-mens tested with the Meridian kit would have required dilution in order to test positive. The optimal dilution would be 1:512 because all of the prozone-like reactions were

l1:256and the antigen titers were .1:1,024. It is interesting that results from other studies of prozone-like reactions support this suggestion (14, 16); however, it is unknown whether all potential combinations of antigen and prozone-like titers could be detected with this dilution. The data from the present study suggest that the treatment of specimens with pronase would be a more reliable method to eliminate false-negative reactions caused by prozone-like phenomenon. Thecause for thefalse-negative results revealed by testing the specimens after dilution or after pronase treatment is not known. Thepresence ofantifungal agents, a resulting met-abolic product from these agents, or repeat testing after freezing and thawing did not account for these findings. Antigen excess is probably not an explanation of the pro-zone-like reaction since the prozone-like reaction occurred overabroad antigen titerrange, and in a previous study (14)

a prozone was not created with the addition of whole organisms. The fact that pronase treatment eliminated the prozone-like reaction with the Meridian kit and the false-negative results obtained with both kits suggests the

possi-bilitythat boundantibody or anonspecific protein masks the antigen. In addition, the fact that the different kits produced variable results on the same specimens suggests the

possi-bilitythat different antigeniccomponents are recognized by

the different kits. These findings may represent a more

universal diagnostic problemwithcryptococcalantigen

test-ing regardless of the kit used.

Different cryptococcalantigenkits have beencompared in

only one other study (16). The IBL kit was

compared

with the Myco-immune kit(AmericanScientific Products) andthe IMMY kit (Immuno-mycologics, Norman, Okla.). One kit

produced negative results on 55% of serum specimens and 17% of CSF specimens that were positive when they were

tested with the IBL kit. Titers with the IBL kit on these falsely negative specimens ranged from 1:2 to 1:16 on CSF specimens and 1:16 to 1:128 on serum specimens. In serum, titers were as much as 8- to 16-fold higher than those produced with either of the other kits, and in CSF titers were as much as 8-fold higher than thoseproducedwith one of the otherkits. Theseresults are consistentwith the resultsfrom

the present study. On specimens that were not treated with pronase, the IBL kit produced titers that rangedfrom 1:2 to 1:4,096 on six ofeight serum specimens that were negative

when they were tested with the Meridian kit. The IBL kit generally produced higher titers than the Meridian kitdidon serum specimens in which discrepant titer results were obtained between the two kits. Titers agreed on only54% of

41 serum specimens and on 86% of 14 CSF specimens before pronase treatment. Even after pronase treatment, agreement between the kits increased only to68% on serum specimens

and 93% on CSF specimens. The best titer correlation of 83% between the two kits was observed with 24 serum specimens that on previous testing showed no titer change after pronase treatment when tested with the Meridian kit. On specimens that were falsely negative, titersafter pronase treatment obtained with the two kits were similar, except for one specimen. That specimen was tested with the IBL kit and was negative before and after pronase treatment. It appears from the results of these studies that the IBL kit more characteristically produces higher titers compared with those produced by other kits, especially when specimens are not treated with pronase before testing. There are uncontrol-lable and unpredictable differences in the results between the IBL and Meridian kits, even with the use of pronase, to the degree that the different kits cannot be used interchangeably. This is especially important in comparing serial changes in titer, as in following the response to treatment. Comparison of results from one laboratory to another is also problematic when one considers the variables in kit sensitivity, the differences in patient populations and disease conditions that mayexist, and the use of pronase or other reagents, as well as the differences produced by subjective interpretation of some agglutination reactions.

Agglutination was difficult to interpret without pronase treatment with both kits because of the minimal degree of agglutination that occurred on many specimens. In a previ-ous study (11), the 1+ reactive serum and CSF specimens wereshown to correlate only 86 and33%, respectively, with

cryptococcal infection. The results of the present study on serum specimens parallel those findings somewhat, in which the 1+ reactions were indicators of a positive reaction that markedly changed to 3+ to 4+ reactivity after treatment of thespecimen with pronase.

Itwas necessary to differentiate between a 1+ and a2+ reactivity with the Meridian kit. This was difficult to do consistently, and as a result it led to variable endpoint interpretations in which the 1+ reactivity could be inter-preted aspositive or negative and, at times, the 2+ reactivity could be interpreted as negative. A similar problem occurred with the IBL kit in determining whether a reaction was

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discernible, since any agglutination was interpreted as

pos-itive. These problems in endpoint interpretation may have accounted for some of the higher titers with the IBL kit in the present study. For some of the specimens in which the IBLkitproduced titers that were 2 or 3 dilutions higher than those produced by the Meridian kit before pronase treat-ment, the Meridian kit produced 1+ reactivity to the

end-pointof the IBL kit reaction.

We also found that results from a titration could vary

considerably, especially withaweaklyreactive specimen,in which it was tempting to continue to manipulate the glass

plate to "better" resolve the questionable reaction. The longer the reaction was observed with manual tilting and

rotating,the more difficultitbecame toresolve an endpoint. The titer could increase tremendously during this interval. As an example of this phenomenon, a specimen of weak

reactivity (1+ to 2+) wasgivento severaltechnologists for

titration; titers ranged from 1:1,024 to 1:65,536. Pronase treatment of the specimeneliminatedthediscrepant readings andproduced titers that were the same or that varied by only 1 dilution. It is suspected that one serum specimen with a reported titer of 1:1,000,000 from another laboratory

re-ferred to our laboratory for testing was a result of this discrepant reading phenomenon. Our result on that

speci-men before pronase treatment was difficult to determine because ofthe weakreactivity (1+ to 2+), and with

contin-ued manipulation of the agglutination glassplate, a titerof

>1:262,144 wasproduced.Afterpronasetreatment, atiter of

1:16,384 wasproduced withavery clearendpoint reading of

3+ to 4+ reactivity.

We recommend pronase treatment of serum specimens

but notCSFspecimens fortesting withtheMeridiankit. We do not recommend that kits from differentmanufacturersbe used interchangeably for detection or titration of antigen.

With the use of pronase treatment of serum specimens, consistentresults areobtainedby eliminatingthesubjective interpretations ofthe agglutinationreactions,and the

detec-tion of antigen is enhanced by eliminating false-negative results and prozones. These findings have important impli-cations for thediagnosis of cryptococcal

meningitis

and for

monitoring patient responsesto therapy.

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