JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1992, p. 2158-21640095-1137/92/082158-07$02.00/0Copyright © 1992, American Society for Microbiology

Vol. 30, No. 8

Retrospective Evaluation of Two Latex Agglutination Testsfor Detection of Circulating Antigens during

Invasive CandidosisPHILIPPE HERENT,1 DIRK STYNEN,2 FERNANDO HERNANDO,3 JEANINE FRUIT,1

AND DANIEL POULAINl*Institut National de la Sante et de la Recherche Medicale, Unite 42, 369 rue Jules Guesdes, F-59650

Villeneuve d'Ascq, and Laboratoire de Parasitologie et Mycologie, Faculte de Me6decine, Place de Verdun,F-59000 Lille, France'; Sanofi Diagnostics Pasteur, B-3600 Genk, Belgium2; and Departamento de

Microbiologia e Inmunologia, Facultad de Ciencias, Universidad del Pais Vasco, E-48080 Bilbao, Spain3

Received 3 February 1992/Accepted 4 May 1992

Two latex agglutination tests for the detection of Candida antigens, Pastorex Candida (Sanofi DiagnosticsPasteur, Marnes-la-Coquette, France) and Cand-Tec (Ramco Laboratories, Inc., Houston, Tex.), were appliedto 79 serum samples from 19 patients who were retrospectively selected on the basis of mycological and clinicalevidence of C. albicans infection and the availability of serial serum samples taken near the date of a positiveculture. The specificity in 60 control individuals was 100% for Pastorex and 98.3% for Cand-Tec. The testsscored positive for 10 (52.6%) and 9 (47.4%) patients, respectively. Pastorex detected antigen in only 3 of 12patients (25%) with positive antibody detection tests, but was positive for all 7 patients (100%Y) who producedno or a low antibody response, suggesting that the test performs better in the absence of antibodies. However,the sensitivity of Pastorex also increased with the number of samples available per patient, which was lower forhigh-antibody-responder patients (2.8 versus 5.7). If the patients who provided only one or two serum sampleswere eliminated, the sensitivity of Pastorex rose to 76.9%. For the Cand-Tec, the sensitivity was not related tothe presence of antibodies, nor was it related to the number of samples per patient. The observed antigenemiawas transient with both Pastorex and Cand-Tec. Only 12.5% of the positive reactions occurred on the sameserum sample, confirming that the two tests react with different antigens. A positive antigen test preceded otherdiagnostic indications for 6 of 10 Pastorex-positive patients and 5 of 9 Cand-Tec-positive patients.

In the past 15 years, the incidence of hospital infectionscaused by yeasts of the genus Candida has shown a steadyincrease (1, 31, 34). Since the clinical symptoms of deepcandidosis are nonspecific (4), the mycological laboratorycan play an essential role by providing assistance to theclinician with the establishment of a diagnosis. Furthermore,this biological diagnosis must be made early, since timelyinitiation of antifungal therapy is essential for a favorableprognosis (1, 31). To reach these goals, three types ofbiological procedures are available, namely, mycologicalexamination and tests for detection of antibodies and anti-gen; each of these has its limitations. The subject has beenextensively discussed in the literature (2, 9, 15, 27). It isgenerally agreed that these three types of tests may comple-ment each other and that mycological and serological sur-veys of high-risk patients, defined according to clinical data,are necessary. The aim of this study was to evaluate thediagnostic value of two commercially available latex agglu-tination tests, i.e., Cand-Tec (Ramco Laboratories, Inc.,Houston, Tex.) which has been available for several years(7, 11, 12), and Pastorex Candida (Sanofi Diagnostics Pas-teur, Marnes-la-Coquette, France), which was introducedmore recently (13, 23). This evaluation was made in thecontext of a routine mycological laboratory to which myco-logical and serological samples are sent at the initiative ofclinicians. It involved 19 patients who were retrospectivelyselected on the basis of mycological and clinical evidence ofdeep C. albicans infection and the availability of several

* Corresponding author.

serial serum samples taken near the date of a positive cultureof samples from normally sterile sites. Our routine serolog-ical procedure involves the detection of antibodies by co-counterimmunoelectrophoresis (Co-CIE) and an indirect im-munofluorescence assay (IFA) (29) plus antigen detection bythe Cand-Tec test in cases of nonsignificant antibody levels(12). The objective of the present analysis was therefore toassess the advantages, if any, of Pastorex Candida com-pared with those of Cand-Tec.

MATERLILS AND METHODS

Human sera. (i) Sera from patients with C. albicans infec-tion. The following criteria were applied to laboratory andclinical files as retrospective selection rules: (i) selection ofpatients having presented evidence of the presence of C.albicans, which was isolated at least once in large quantitiesfrom normally sterile sites (blood, catheter, drain, wound,peritoneal cavity, gastric or lung biopsy specimens, bile,pancreatic cyst, pericardial fluid); (ii) availability of serialserum samples for the corresponding patients taken near thedate of positive cultures; and (iii) subsequent analysis of theselected patients' clinical files (together with the physiciansin charge of the patients), leading to consideration of candi-dosis as highly probable once it was integrated into theunderlying conditions (e.g., fever resistant to antibacterialagents and response to antifungal therapy). Application ofthis procedure for the period between February 1988 andMay 1989 led to the selection of 73 serum samples from 18patients from the surgery, intensive care, and hematologywards of the Lille University Hospital.

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Details concerning patients with C. albicans infection aregiven in Tables 1 and 2. In Tables 1 and 2, the data are for 19patients, not 18 patients, since 1 patient who underwent twoepisodes of invasive candidosis in a 1-year interval (separat-ed by a total recovery) was treated as two different patients(patients A9 and B5).

(ii) Control sera. Four groups of control individuals (a totalof 60 individuals) were tested in order to determine thespecificity of the methods. The first group consisted of 10members of the laboratory staff, the second group included20 surgery patients without any sign of clinical candidosis,the third group encompassed 10 patients with immunologi-cally confirmed aspergillosis and 2 patients with allergicbronchopulmonary aspergillosis (3), and the fourth groupincluded 18 patients in acute phases of parasitic diseases, asfollows: 13 patients with malaria and 1 patient each withfascioliasis, schistosomiasis, filariasis, echinococciasis, andamebiasis.

Mycological examinations. Cultures were grown on Sab-ouraud glucose agar containing kanamycin (800 ,g/ml). Theisolates were identified as C. albicans on the basis of germtube (20) and chlamydosporulation (32) tests.Antibody detection. Two methods for antibody detection

were used.(i) Co-CIE. Co-CIE (bio Merieux, Marcy l'Etoile, France)

has been described in detail before (29). It essentially con-sists of a counterimmunoelectrophoresis on cellulose acetatemembranes in which three 20-,ul serum deposits, placed atthe anode, are run against a line of antigen consisting of 15 ,ulof a 25-mg/ml solution of somatic antigen from C. albicansVW32 placed at the cathode. Patient sera are run side by sidewith a rabbit anti-VW32 germ tube antiserum to detectprecipitating antibodies that present a line of identity withanti-germ tube antiserum. This line of identity is named thecospecific precipitin line (CSPL). Interpretation of Co-CIEresults refers to the presence or absence of CSPL andquantitative evolution, namely, migration toward the cath-ode and variation in intensity for successive sera from thesame patient run side by side with the anti-germ tubeantiserum.

(ii) IFA. IFA was performed against unfixed blastoconidiaof a C. albicans VW32 serotype A cloned strain (bio-Merieux,) incubated with doubling dilutions (initial dilution,1/100) of patient sera in phosphate-buffered saline (PBS).Antibody binding was revealed by incubation with a fluores-cent goat anti-human immunoglobulin antiserum (1/100 inPBS containing Evans blue at a final dilution of 1/50,000);Sanofi Diagnostics Pasteur). Results were expressed as thereciprocal titer, i.e., the inverse of the highest serum dilutionthat gave strong fluorescence of all blastoconidia. Standard-ization of this test was achieved by the use of a pool of serafrom patients with candidosis (bio-Merieux). A titer equal toor greater than 400 was considered indicative of candidosis.

Relying on the results obtained with Co-CIE and IFA, thefollowing conclusions were assigned to the serum samples:N, no immunological suspicion of candidosis (no CSPL andIFA < 400); S, immunologically suspected candidosis (pres-ence of CSPL and/or IFA > 400); C, immunologicallyconfirmed candidosis (strong CSPL and IFA > 400).

Detection of circulating antigens. Both antigen detectionkits were used according to the manufacturer's instructions.The Cand-Tec test (Ramco) uses latex beads coated with a

polyclonal rabbit anti-Candida serum against an unknownantigen (12). The test is positive when serum, which isdiluted 1/4, agglutinates the latex beads. Serial dilutions ofserum were tested to determine the antigen titer, which is

defined as the reciprocal of the highest serum dilution thatstill causes agglutination.

In the Pastorex Candida test (Sanofi Diagnostics Pasteur),the latex beads were sensitized with a monoclonal anti-mannan antibody (23). Serum samples were incubated with aserum treatment solution at 100°C. After centrifugation, thesupematants were incubated with the latex beads. A serumsample scored positive when undiluted supernatant aggluti-nated the latex beads. Serial dilutions of the supernatantwere tested so that we could obtain semiquantitative results.

RESULTS

The patients were divided into two groups according totheir antibody responses as analyzed by Co-CIE and IFA.Group A consisted of 12 patients with immunologicallyconfirmed candidosis, as established by these routine proce-dures. The average number of serum samples per patient inthis group was 2.8 + 1.3 (Table 1). Group B included sevenpatients who failed to produce an antibody response, whichwas considered indicative of infection, but they were judgedto suffer from an invasive candidosis on clinical and myco-logical grounds. The average number of serum samples perpatient in this group was 5.7 ± 3.1 (Table 2).For group A patients (Table 1), Cand-Tec was positive at

a significant titer for 5 of 12 patients, whereas PastorexCandida was positive for 3 patients. For group B patients(Table 2), four of seven patients were positive by Cand-Tec;Pastorex Candida yielded positive results for all sevenpatients. When the results for both patient groups werecombined, 9 of 19 cases scored positive by Cand-Tec, asopposed to 10 of 19 by Pastorex Candida.

Patients were often positive by only one test (Tables 1 and2). Five patients (patients A6, All, B3, B4, and B6) werenegative by Cand-Tec but positive by Pastorex Candida,while four patients (patients A3, A4, A9, and A12) remainednegative by Pastorex Candida but were positive by Cand-Tec. Both tests failed to detect antigen in five patients(patients Al, A2, A5, A8, and A10). A remarkable featureshared by both tests in both groups of patients was that whenmore than one serum sample was available per patient,antigenemia was always transient. For the serum samplestested and sent at the initiative of clinicians, the minimumdelay observed for the appearance of detectable antigen byCand-Tec and Pastorex were, respectively, 4 days (patientB7) and 1 day (patient A6); conversely, it was possible toobserve the disappearance of detectable antigens in bothtests within 48 h (patient B5, Cand-Tec; patient B7, Pas-torex). The longest antigenemia period detected without anyintervening negative test was of 47 days and concerned thePastorex Candida in patient B5.

Figure 1 shows the sensitivities of both tests as a functionof the number of serum samples tested per patient. Thesensitivity of the Pastorex Candida increased when therewere increasing numbers of serum samples available foreach patient. In the entire patient population, the sensitivityof Pastorex was 52.6%. When data for the patients whoprovided only one or two samples were omitted, the sensi-tivity increased to 76.9%, eventually attaining 100% inpatients who provided five or more serum samples. TheCand-Tec test did not show such a correlation.Both tests were negative for samples from the healthy

controls and from surgery patients (Table 3). In patients withdifferent fungal and parasite infections, one sample waspositive by Cand-Tec. This sample came from a patient withaspergillosis. For this patient, the Candida serology tests,

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TABLE 1. Patient information, culture data, and results of antibody and antigen testing in patients with an antibody responseto C. albicans (group A)

Patient Sex' Age (yr) Underlying condition Site ofn' sera were Co-CIEdyFo Conclusionfcollectedc IFCCand-Tec Pastorex

Al M 63 Pancreas carcinoma Bile -2 4 1,600 C 2 (NS) -

8 3 400 C _

A2 M 86 Catheter

A3 M 27 IleostomyJejunostomy

A4 M 51 Oesophagal neoplasm

Blood (day 0)Drain (day 6)

Drain

-10 015 3

1 4 1,600-7 3 400

N 2 (NS)800 C -

CC

1 3 400 C12 4 400 C18 4 800 C

4

4

AS M 62 Duodenal perforation

A6 M 18 Liver transplant

A7 M 43 Non-Hodgkin's malignantlymphoma

Pneumopathy

Peritonealcavity

Gastric biopsy

Lung biopsy

-1 0 10013 4 1,600

NC

2 (NS) -

-5 2 200 S 2 (NS)-4 3 200 S -16 4 800 C 2 (NS)

-18 0 200 N 8-12 2 400 S 2 (NS)-4 3 400 C 44 3 400 C 2 (NS)

A8 M 49 Chronic pancreatitis Pancreatic cyst 0 2 400 S -

7 3 800 C -

A9 F 69 Liver cancer Blood -17 2 100 S 2 (NS)-1 2 400 S 2 (NS)4 3 400 C 4

A10 M 64 Sternal resynthesis

All M 46 Bronchopneumopathy

A12 M 70 DuodenostomyGastrostomy

Pericardial fluid(day 0)

Median wound(day 27)

Blood

Peritonealcavity

10 2 20021 2 40034 2 400

>57 4 1,600

S 2 (NS)S

S

C

-5 2 400 C - -

0 3 800 C - 13 3 800 C - -

5 3 800 C - -

14 3 800 C - -

0 2 400 C 4

a M, male; F, female.b Unless otherwise indicated, yeasts were isolated on day 0.c Indicated as the days before (negative numbers) or after the first isolation of C. albicans.d 0, absence of a precipitin line; 1, nonspecific precipitin line; 2, cospecific precipitin line; 3, intense cospecific precipitin line; 4, very intense cospecific

precipitin line.e Reciprocal titer. NS, not significant. -, negative.f N, no immunological suspicion of candidosis; S, immunologically suspected candidosis; C, immunologically confirmed candidosis.

however, also yielded results compatible with immunologi-cally confirmed candidosis. The other serum samples wereall negative by the Pastorex Candida and Cand-Tec tests.

DISCUSSION

The presence of Candida antigens in serum appears to bea specific indicator of invasive disease (2, 9, 15, 22, 24, 28,33). However, the low and varying antigen concentrations,which often fall below the detection limits of the available

methods (5, 26, 33), also lead to controversy about theclinical usefulness of antigen detection tests. In this study,we compared the results of culture and of antibody andantigen detection in a group of 19 patients with or without an

antibody response. Special emphasis was placed on the roleof antigen detection and, more particularly, on the compar-ison of two commercially available test kits. We found an

overall sensitivity of 47.5% for Cand-Tec and 52.6% forPastorex Candida (Tables 1 and 2). The Cand-Tec test hasbeen the subject of numerous studies which have yielded

1

2

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ANTIGENEMIA IN CANDIDOSIS 2161

TABLE 2. Patient information, culture data, and results of antibody and antigen testing in patients with an antibody responseto C albicans (group B)

Patient Sex° Age (yr) Underlying condition Site of yeast sera wereCoyCIE IF ConclusiocollectedIlAl Cand-Tec Pastorex

Bi F 23 Acute lymphoblastic Blood -14 1 100 N - ---ia ---

leukemia -11-10

3

211

100 N100 N

N 4 4

B2 F 46 Gastrectomy Catheter -1 0 100 N5 2 200 S14 2 200 S

B3 M 21 Acute lymphobasticleukemia

Blood -115

>30

0 1000 2002 200

N 2 (NS) 1N - -

S - -

B4 M 65 Ileostomy Blood (day 0) -21Blood (day 1) -16

-13-8-5-2-108

111228

1 200 N0 200 N1 200 N2 200 S2 200 S2 200 S2 200 S2 200 S2 200 S2 200 S1 200 S0 100 N

B5 F 69 Liver cancer Bile

B6 M 43 Acute myelomonocytic Bloodleukemia

B7 F 50

-3 1 100 N2 2 200 S4 0 200 N10 1 100 N48 2 200 S51 1 200 S56 0 100 N

-30-13

1814

>30

Drain

0

111

0

0

100 N100 N100 N100 N

NN

-9 1 400 S-7 1 400 S8 1 200 S

10 0 200 N14 0 200 N

I F, female; M, male.b Unless otherwise indicated, yeasts were isolated on day 0.I Indicated as the days before (negative numbers) or after the first isolation of C. albicans.d 0, absence of precipitin line; 1, nonspecific precipitin line; 2, cospecific precipitin line; 3, intense cospecific precipitin line; 4: very intense cospecific precipitin

line.I Reciprocal titer. NS, not significant. -, negative.f N, no immunological suspicion of candidosis; S, immunologically suspected candidosis; C, immunologically confirmed candidosis.

very variable results; the sensitivity of the test has rangedfrom 19 to 91% (7, 8, 10-12, 16, 28). In an early study ofPastorex Candida, Meulemans et al. (23) found 5 positivepatients of 10 autopsy-proven cases, but the number ofserum samples per patient tested in that study was only oneto three. The number of serum samples per patient is veryimportant, since our data showed that both tests are liable tobecome positive or negative within 24 to 48 h. In animalmodels, mannan clearance from the blood has been provento be very rapid. The antigen accumulated in the liver and

the spleen and was also excreted into the urine (18). Becauseof the efficiency of the clearance mechanism, mannan con-centrations remain low and may drop below the detectionlimit of a reverse passive latex agglutination test. Patients atrisk or patients suspected of being at risk should therefore betested as frequently as possible. Our results with PastorexCandida confirm that the sensitivity of the test increaseswith the number of serum samples available per patient. Theaverage number of serum samples provided by the Pastorex-negative patients was 2.3 versus an average of 5.2 serum

4 4

2 (NS)

2 (NS)2 (NS)2 (NS)2 (NS)2 (NS)2 (NS)

2 (NS)

2 (NS)

1

442 (NS)2 (NS)

4

2 (NS)2 (NS)

2 (NS)

4

1412

2

14

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2162 HERENT ET AL.

100 T

80 +

60 +

40 +

20 +

0

.1 .2 .3 .4 .5 .6(19) (17) (13) (9) (5) (3)

n samples/patient(n patients/category)

FIG. 1. Sensitivity of Pastorex Candida (0) and Cand-Tec (0)

antigen detection tests in relation to the number of serum samplesfrom each patient tested; the number of patients entering eachcategory was defined as the minimal number of serum samplestested.

samples provided by the Pastorex-positive patients. Whenpatients who provided only one or two serum samples were

omitted from the analysis, the sensitivity of Pastorex rose to76.9%. This correlation between sensitivity and number ofserum samples could not be observed with Cand-Tec, but ithas been described by Burnie and Williams (7). In theirstudy, the average number of samples per patient was 3.1 forCand-Tec-negative patients and 9.7 for Cand-Tec-positiveones. In the same context, the time interval between samplesis important. It is noteworthy that the average intervalbetween the time that serum samples were obtained fromfour of five patients who were negative by both tests was 10days or more.We observed discordant results between the two tests in

nine patients: five patients were negative by Cand-Tec andpositive by Pastorex, while four patients were negative byPastorex and positive by Cand-Tec. Only 12.5% of the serumsamples were positive when they were tested by using bothtests. This observation can be explained by the fact that thetwo latex agglutination tests detect different antigens. Therat monoclonal antibody EB-CA1, which is used in thePastorex test, recognizes mannan, a major cell wall polysac-charide, from C. albicans, C. tropicalis, C. glabrata, C.guilliermondii, and to a lesser extent, C. pseudotropicalisand C. parapsilosis (14). The antigen detected by Cand-Tec,however, has not been identified, but it is certainly differentfrom mannan (12, 30). The high degree of discord betweenthe two tests suggests that the detection of more than one

antigen may improve the sensitivity.The prognosis of the patient improves when a diagnosis

can be established in an early phase of the infection (31). Itis therefore important to see whether the antigen tests wouldhave allowed an earlier diagnosis. In the nine positivepatients, the Cand-Tec test turned positive in five patientsbefore cultures became positive or antibody levels consid-ered as significative of candidosis were detected (29). In the10 Pastorex-positive patients, Pastorex Candida was the firstindication of invasive disease in six of them. Both latex

TABLE 3. Result of antibody and antigen testing incontrol populations

AntigenPopulation (no. of Patient Co-Ciea Serology detection

subjects) no. by IFAb by Cand-Tec"

Healthy 1 0 200 -

individuals (10)2 0 100 -3 0 100 -4-10 0

Surgery patients 1 2 200 -

(20)2-3 1 200 -4-6 1 100 2 (NS)7-8 0 200 -9-15 0 100 -16-20 0

Aspergillosis (10) 1 1 100 -

2 23 2 100 -4 2 200 -5 06 3 400 47 3 200 -8 2 100 -9 010 0 -

Allergic 2 0bronchopulmonaryaspergillosis (2)

Malaria (13)C 1 0 200 -

2-13 0 -

Fascioliasis (1) 1 0Schistosomiasis (1) 1 0Filariasis (1) 1 0Echinococcosis (1) 1 0 200 -

Amebiasis (1) 1 0

a 0, absence of precipitin line; 1, nonspecific precipitin line; 2, weakcospecific precipitin line; 3, intense cospecific precipitin line; 4, very intensecospecific precipitin line.

b Reciprocal titer. NS, not significant. -, negative. No antigen was de-tected by Pastorex.

c Six of these patients presented with a Cand-Tec titer of 2.

agglutination tests were preceded by positive cultures inthree cases and by positive serological tests in one case. Weconsidered antigenemia as positive before culture if theantigen test was positive on day 1 at the latest, sincemycological cultures take, on average, 72 h of incubationbefore they can be interpreted. Our results confirm thatcirculating antigens appear early in the course of the infec-tion, and therefore, they often provide the clinician withinformation that allows for the earlier initiation of treatment.Both tests yielded comparable results in this respect.Although the number of patients examined in this study

was too small to allow firm conclusions to be made, Pastorexseemed to be a good test for use on immunocompromisedpatients, because it was positive for all seven patients with aweak serological response, while Cand-Tec was positive forfour patients (Table 1). Several other investigators also failedto obtain very good results by Cand-Tec in patients withleukemia (5, 10, 25, 28). On the other hand, Pastorex seemedto be more sensitive to the presence of specific antibodies inthe patient's serum (17) (Table 2). This is a surprisingobservation, because unlike Cand-Tec, the Pastorex Can-dida protocol includes a serum treatment to dissociateimmune complexes. In the four patients whose sera were

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ANTIGENEMIA IN CANDIDOSIS 2163

positive only by Cand-Tec, the antibody levels were high.Moreover, the majority of Pastorex-positive samples be-longed to patients who failed to produce an antibody re-sponse to C. albicans (group B). Conversely, in the antibodyresponder group (group A), only three patients were posi-tive. Among those three patients, only one exhibited highantibody levels at the date of positive antigenemia. Thisraises the possibility that the patients who respond to theCandida infections immunologically are able to clear man-nan from their blood more efficiently (18), leading to a lowerincidence of detectable antigenemia. However, since theaverage number of serum samples per patient was lower inthe antibody responder group (2.8 versus 5.7), it cannot beexcluded that the lower sensitivity of Pastorex Candida inthis group was due to this lower number of serum samples.

In this study, both tests were very specific; all 60 controlsamples were negative by Pastorex Candida (100% specific-ity), and only 1 control sample was positive by Cand-Tec(98.3% specificity) (Table 3). This positive sample, whichwas from a patient with aspergillosis, may have been atrue-positive result, since serological results were consistentwith immunologically confirmed candidosis. For PastorexCandida, the high specificity is in agreement with earlierfindings (23). The specificity of Cand-Tec reported in otherstudies varied considerably (6, 8, 12, 16, 25, 26, 28).

Results of our study suggest that the laboratory diagnosisof invasive candidosis benefits from an integrated, kineticapproach in which patients suspected of having candidosisor, preferably, patients at risk of candidosis are submitted tomycological surveillance, serological tests, and antigen de-tection at regular time intervals. This approach allows phy-sicians to follow kinetics of antibody fluctuations (29) whichcomplements or relates with (28) results from antigen test-ing. Depending on the antigen detected, immunoassays forthe detection of circulating fungal components may consti-tute specific markers for invasive candidosis (15, 21, 22, 24,33). Provided that testing is not restricted to isolated serumsamples and that patients are regularly screened, antigentesting is also a sensitive method and is especially useful inimmunocompromised patients for whom antibody detectiontests remain negative. Moreover, a positive antigen test isoften the earliest indication of invasive infection. Althoughthe results of this retrospective study suggest that PastorexCandida is a useful new tool for the diagnosis of invasivecandidosis, more studies are necessary to confirm theseresults. Further investigations, including prospective stud-ies, are needed to firmly establish the clinical value of thistest and to assess its reproducibility with different produc-tion batches.

ACKNOWLEDGMENTSWe thank Laurence Richard, Nadine Francois, and Gilbert Lep-

age for valuable technical assistance.

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