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Editorial papers

Mikol. Lek. 2004, 11 (2): 145-151

ISSN 1232-986X

The role of antigen and antibody testing

in the diagnosis of invasive candidiasis

Znaczenie wykrywania antygenów i przeciwcia³ w diagnostyce inwazyjnej dro¿d¿ycy

Guillermo Quindós

1

, María Dolores Moragues

2

, José Pontón

1

1

Departamento de Inmunología, Microbiología y Parasitología, Facultad de Medicina y Odontología, Bilbao, Spain

2

Departamento de Enfermería I, Universidad del País Vasco-Euskal Herriko Unibertsitatea, Bilbao, Spain

The multiple problems presented by the clinical and microbiological diagnosis of invasive candidiasis have prompted the development of tests based

on detection of Candida antigens or/and antibodies against these antigens. To be valuable, these methods must accomplish the difficult task of diffe-

rentiating normal colonization of human mucous membranes by Candida species, particularly Candida albicans, from tissue invasion and candidemia

requiring antifungal therapy. In this article, we review the current status in diagnosis of invasive candidiasis based on the detection of Candida anti-

gens or/and anti-Candida antibodies.

Key words: candidiasis, diagnosis, antigen, antibody, combination

Liczne problemy zwi¹zane z kliniczn¹ i mikrobiologiczn¹ diagnoz¹ inwazyjnej kandydozy przyczyni³y siê do rozwoju testów opartych na wykrywaniu antyge-

nów Candida lub/i przeciwcia³ skierowanych przeciwko tym antygenom. Aby by³y wartoœciowe, metody te musz¹ spe³niæ trudne zadanie odró¿nienia zwyk³ej

kolonizacji ludzkich b³on œluzowych przez gatunki Candida, szczególnie Candida albicans, od inwazji tkanek i kandydemii wymagaj¹cych terapii przeciwgrzybi-

czej. W artykule zosta³ przedstawiony obecny stan diagnostyki inwazyjnej kandydozy w oparciu o wykrywanie antygenów Candida lub/i przeciwcia³ skierowa-

nych przeciwko Candida.

S³owa kluczowe: kandydoza, diagnoza, antygen, przeciwcia³o, kombinacja testów

145

Streszczenie

Abstract

Introduction

Initial studies on serological methods for the diagnosis of

invasive candidiasis began about 50 years ago, when blood

culture detection techniques often failed to recover Candida

organisms (1, 2) and only antibody detection could provide

useful data for the clinician (3). During the following years,

other techniques for the detection of anti-Candida antibo-

dies, Candida antigens and metabolites were developed,

but none of them have got a widespread clinical use. Today,

the situation has changed; blood culture allows a better de-

tection of candidemia (4, 5) and new and sensitive nucleic

acid-based diagnostic tests are being introduced in the labo-

ratory for diagnosing invasive candidiasis (6, 7). Despite these

advances, the problems associated with the laboratory diag-

nosis of invasive candidiasis are far from solved and it will be

difficult to develop a single test with sufficient sensitivity

and specificity to make a definitive diagnosis. Physicians may

need to rely on cumulative information from serial specimens

and diagnostic techniques to have evidence enough to be-

gin antifungal therapy for invasive candidiasis (4, 8-10) and

serological methods may also be used as markers when mo-

nitoring the efficacy of antifungal therapy (8, 11-13). In this

article, we review the current status in diagnosis of invasive

candidiasis based on the detection of Candida antigens

or/and anti-Candida antibodies.

146

Guillermo Quindós, María Dolores Moragues, José Pontón

Mikol. Lek. 2004, 11 (2)

Detection of antigens

A wide variety of assays have been developed for the de-

tection of circulating Candida antigens, including latex ag-

glutination, ELISA, immunoblotting, dot immunoassay, lipo-

somal immunoassay, and RIA (14). Antigens detected by

these assays include mannan and mannoproteins, glucan,

HSP90, enolase, and other immunodominant cytoplasmic

antigens.

Mannan usually circulates in the form of immune com-

plexes which are rapidly cleared by the liver and kidneys.

Mannan concentrations in serum from patients with inva-

sive candidiasis are found in the low nanogram per millili-

ter range (9, 15). Moreover, antigenic differences between

the mannans of different species of Candida, can make

that a test devised to detect Candida albicans mannan

may not be appropriate to detect mannan from Candida

krusei or Candida glabrata (16, 17). These facts ma-

ke mannan detection difficult by serological assays, the-

refore dissociation of the complexes by heat, protease,

acid or NaOH treatments and testing of sequential serum

samples are strategies used to improve the sensitivity of as-

says (9). Mannan antigenemia preceded significant rises

in antibodies against mannan and cytoplasmic antigens

by one to three weeks and it has been found to comple-

ment blood culture for the diagnosis of invasive candidia-

sis in neutropenic cancer patients (18, 19). These au-

thors (20) did not detect mannoprotein in the serum spe-

cimens from 15 patients with hematological malignancies

and proven or probable hepatosplenic candidiasis. Howe-

ver, mannan detection has been detected in cerebrospi-

nal fluid from patients with Candida meningitis and this

test may be valuable the diagnosis of meningeal candidia-

sis (21).

Fig. 1. Detection of antibodies to Candida albicans germ

tube by an indirect immunofluorescence assay.

A. Candida albicans IFA IgG kit (Vircell Laborato-

ries, Spain)

B. Microscopic appearance. Only germ tubes are

fluorescent stained

B

A

147

The role of antigen and antibody testing in the diagnosis of invasive candidiasis

Some tests for mannan detection have been commer-

cialized, thus making possible a broad evaluation (22).

LA-Candida Antigen Detection System (ImmunoMycolo-

gics, USA), based on latex particles coated by polyclonal

anti-mannan antibodies to detect the antigen in sera pre-

treated with heat and protease, has been evaluated by

different groups who found it to be insensitive (11, 23,

24). ICON Candida Assay system (Hybritec Inc., USA)

detects mannan with polyclonal antibodies in an ELISA

format, after dissociation of immune complexes with pro-

tease and heat treatment. Pfaller et al. (25) observed for

this test a sensitivity of 86% and a specificity of 92% in

the diagnosis of invasive candidiasis in a mainly immuno-

competent patient population. Detectable mannanemia

preceded diagnosis by other methods, such as blood

culture, histology, etc., in only 36% of patients. However,

the test tended to complement the diagnosis made on

the basis of positive blood culture.

Cand-Tec test (Ramco Laboratories Inc., USA) is a la-

tex agglutination assay to detect an uncharacterized heat-

labile antigen of C. albicans that does not contain man-

nan and is detected without immune complex dissocia-

tion steps. Although there is a vast literature on the

performance of the Cand-Tec test, there is no consensus

about its value for the diagnosis of invasive candidiasis

because variable results have been reported in different

studies (8, 24, 26-30). Misaki et al. (31) compared

a new presentation, Cand-Tec microtiter system (Cand-

-Tec MT, Ramco, Japan) which expresses Candida anti-

gen level as the cutoff index value by a colorimetric analy-

sis, with the original Cand-Tec for the diagnosis of invasi-

ve candidiasis in 25 patients with hematological diseases.

The sensitivity and specificity of Cand-Tec MT were

100% and 80%, respectively. The cutoff index value de-

creased in 75% of patients responding to antifungal the-

rapy.

Directigen (Becton Dickinson, USA) is a liposomal im-

munoassay to detect enolase antigenemia (32, 33) with

a sensitivity in serum spiked with antigen of 0.5 to 1 mg/l.

The assay was positive in cancer patients with invasive

candidiasis by C. albicans and Candida tropicalis, de-

tecting enolase in 85% of patients with deep tissue infec-

tion and in 64% of patients with fungemia (specificity of

96%). The study documented the transient nature of eno-

lase antigenemia and daily sampling was proposed in fe-

brile patients with neutropenia.

An ELISA was developed to detect C. albicans proteina-

ses, which reach serum concentrations of 0.13 mg/ml and

have a half-life of approximately 60 min (34, 35). How-

ever, the assay showed a low sensitivity and specificity

for the diagnosis of invasive candidiasis. The low sensitivi-

ty may be related to the presence of anti-proteinase anti-

bodies in patient’s sera which bind the antigen and facili-

tate clearance. The proteinaceous nature of the antigen

precluded a simple disassociation of immune complexes,

as can be achieved with mannan (22). However, Na and

Song (36) developed a monoclonal antibody-based ELISA

inhibition technique to detect a C. albicans secreted

aspartic proteinase and reported a sensitivity of 93.9%

and a specificity of 96% for the diagnosis of invasive can-

didiasis.

Monoclonal antibody EBCA1 coated on latex beads in

the Pastorex Candida test (Bio-Rad, France) has been

used to detect systemic candidiasis. This antibody reacts

with mannans from most common Candida species, such

as C. albicans, C. tropicalis, Candida dubliniensis,

Candida famata, C. glabrata, Candida guilliermondii,

Candida lusitaniae, and Candida parapsilosis (37,38),

but not with mannan from C. krusei (37). Several authors

(30, 33, 39, 40) have reported a sensitivity range of 26-

-60% and a specificity of 100% for the diagnosis of invasive

candidiasis in a mainly immunocompetent adults and chil-

dren. Although sera were treated to dissociate the immu-

nocomplexes with EDTA and heat, the test showed a low

sensitivity in patients who had high titers of anti-mannan an-

tibodies. In an attempt to increase the sensitivity of mannan

detection, an ELISA was developed using EBCA1 (Platelia

Candida Ag, Bio-Rad). Mannanemia was detected on an

average of 6 days before isolation of C. albicans. How-

ever, false-positive reactions have been observed in diffe-

rent groups of patients, as in intensive care patients treated

with hydroxyethyl starch (14).

There is a wide consensus that antigen detection may

be useful for the diagnosis of invasive candidiasis in im-

munodeficient patients as antigen concentration is inde-

pendent of the immunological status of the patient, and

may give an early indication of infection. However, anti-

gen detection may not automatically be the serological

Table I: Cell wall antigens of Candida albicans. Modified from Pontón et al. (14)

Antigen described

Antigen type ()

11C11

II

12B12

I

155 kDa

I

15C9

II

16B1F10

I

180 kDa

I

200 kDa

I

21E6

IV

260

I

2G8

I

3B7

IV

3D9

I

3G6

IV

3H8

III

4E1

I

AF-1

IV

Ag 1.183

II

B9E

III

C3d receptor

II

C6

IV

G3B

IV

Hwp1

I

Mannans

III

Mp58

III

N3B

III

PA10F

II

D9F

IV

14.8

IV

Enolase

IV

148

Guillermo Quindós, María Dolores Moragues, José Pontón

Mikol. Lek. 2004, 11 (2)

test of choice in immunodeficient patients as it may de-

pend on the type of antigen and the mechanisms needed

for its release in infected tissues. Common features ob-

served in all antigen detection assays are low levels of cir-

culating antigens and a transient nature of antigenemia

(10, 18, 19, 41-44). Therefore, antigen detection tests

for the diagnosis of invasive candidiasis would require the

use of sensitive assays and frequent sampling, possibly

on a daily basis in patients at high risk.

Detection of antibodies

Antibody detection-based diagnostic methods must ac-

complish the difficult task of differentiating Candida coloni-

zation of mucous membranes or superficial infection from

candidemia and invasive candidiasis requiring antifungal

therapy (14). Two main limitations are evident, the specifici-

ty of tests may be low because antibody titers can be high in

colonized patients, and tests may present a low sensitivity

because the antibody response may be delayed, reduced

or absent. These limitations can be overcome by improving

the specificity of the tests through the appropriate selection

of antigens (purified molecules, recombinant antigens, etc.)

and sensitivity can be increased with more sensitive techni-

ques, such as the ELISA. A useful test must combine impro-

vements in both sensitivity and specificity, since nothing

is gained from a more sensitive test unless a more specific

antigen is also used (45, 46). There is a general belief that

antibody tests are both insensitive and non-specific (9, 46),

but there is an important evidence suggesting that detection

of antibodies in highly immunodeficient patients, such as

neutropenic bone marrow transplant recipients or liver

transplant recipients, is possible and useful for the diagno-

sis of invasive candidiasis (47-50).

Six different types of antigens have been described in

the C. albicans cell wall (51). Type I antigens are truly

germ tube-specific and are expressed on the germ tube

cell wall surface only. Type II antigens are expressed on

both the germ tube cell surface and within the blastoconi-

dium cell wall. Presence of type II antigens within the bla-

stoconidium cell wall would explain the induction of anti-

bodies to germ tubes in both patients and animals infec-

ted with Candida species unable to produce germ tubes

in serum (52-54). Type III antigens are expressed on

both the blastoconidium and germ tube surface. Type IV

antigens are expressed within the cell wall of both blasto-

conidia and germ tubes (tab. I). Type V antigens are

expressed on the blastoconidium cell surface and within

the germ tube cell wall and type VI antigens are expres-

sed on the blastoconidium surface only.

Mannan is an abundant and highly immunogenic type

III antigen located on the Candida cell wall surface (55)

and anti-mannan antibodies have been detected in many

studies (14). A sensitivity of 64% and a specificity of 97%

in the diagnosis of invasive candidiasis in immunocompe-

tent patients, have been reported for anti-mannan antibo-

dy detection by using a commercially available ELISA

(Platelia Candida Ab, Bio-Rad) in sequential serum sam-

ples (42, 43, 56). Other studies have shown lower dia-

gnostic values (sensitivity, 59% to 91%; specificity, 18%

to 63%) using this and other commercial tests (such as

Candiquant, Biomerica, USA; and Candida albicans ELISA,

Virotech GmbH, Germany) (57).

A mannoprotein of 230-250 kDa located on the germ

tube cell wall surface is recognized by sera from patients

with invasive candidiasis. Our group (14, 58, 59) has de-

veloped an indirect immunofluorescence assay to detect

antibodies (CAGTA) against this antigen present in C. al-

bicans germ tubes that has been useful in the diagnosis

of invasive candidiasis in different groups of patients, inc-

luding intravenous heroin users (60-62), bone marrow

transplant recipients (63), patients with hematological dis-

orders and intensive care patients (12, 63-67). The test

has shown an overall sensitivity of 77-89% and a specifi-

city of 91-100% (48, 58, 59, 68). These results are in

contrast to those obtained when antibodies to C. albi-

cans blastoconidia were detected, since detection of an-

tibodies to C. albicans blastoconidia, which are mainly

directed against mannans, is more sensitive than detec-

tion of CAGTA, but less specific. Sera sequentially drawn

from patients at risk of developing invasive candidiasis

showed CAGTA before the microbiological diagnosis was

made, especially in patients with tissue proven invasive

candidiasis and detection of CAGTA seemed to comple-

ment and even anticipate blood culture (63). Detection of

CAGTA in patients with invasive infections caused by

Candida species other than C. albicans (C. tropicalis,

C. parapsilosis, C. glabrata, C. dubliniensis, C. guil-

liermondii and C. krusei) may also be positive, although

titers are lower than in candidiasis by C. albicans (12,

52, 54, 59, 63, 64, 67, 69, 70). In addition, detection

of CAGTA may be useful for the therapeutic monitoring of

patients with invasive candidiasis, since the administra-

tion of antifungal therapy usually results in decreasing ti-

ters of CAGTA (12, 61, 67, 70).

The test Candida albicans IFA IgG (Vircell Laborato-

ries, Spain) has been recently commercialized for CAGTA

detection (fig. 1). This test has been compared in a retro-

spective study to the standard test in 172 sera from 51

hematological and intensive care patients (123 sera from

32 patients with invasive candidiasis and 49 sera from 19

patients without evidence of infection by Candida). Can-

dida albicans IFA IgG test showed a sensitivity of 84%

and a specificity of 95%, while the standard test showed

a sensitivity of 78% and a specificity of 100%. Results

with both techniques presented a high correlation (R2=

0.951 by patients, R2=0.899 by sera). The commercially

available Candida albicans IFA IgG test was similar to the

test generally used for the detection of CAGTA and provi-

ded faster and easier diagnosis of invasive candidiasis in

the clinical microbiology laboratory (67).

CAGTA present in patients with invasive candidiasis

are likely to be directed against both C. albicans type

I and II antigens (59). Since the germ tube cell wall also

contains mannan, to detect CAGTA the sera have to be

adsorbed, a process that is time consuming and requires

large amounts of heat-killed blastoconidia. A test based

on the detection of antibodies against type I antigens may

not require the adsorption of the sera and would therefo-

re facilitate serodiagnosis of invasive candidiasis. In this

regard, by using an ELISA and purified C. albicans type

I and II antigens, Bikandi et al. (14) detected antibodies

in sera from patients with invasive candidiasis with a sen-

sitivity of 78% and a specificity of 68% without removing

the anti-mannan antibodies from the sera. In a similar stu-

dy, Berdin et al. (71) developed an ELISA to detect IgG

149

The role of antigen and antibody testing in the diagnosis of invasive candidiasis

antibodies against a C. albicans type I antigen in patients

with invasive candidiasis and reported a sensitivity of 78%

and a specificity of 82%.

Candida antigens with enzymatic activity (enolase,

aspartic proteinase and metallopeptidase) have also been

used as targets for antibody detection with controversial re-

sults. Antibody response against enolase has shown a sen-

sitivity of 50 to 92% and a specificity of 86-95% for the dia-

gnosis of invasive candidiasis in immunocompetent patients

and a sensitivity of 53% and a specificity of 78% in immuno-

deficient patients (30, 72, 73). Detection of antibodies aga-

inst a C. albicans secreted aspartic proteinase in patients

with invasive candidiasis had a sensitivity of 70% and a spe-

cificity of 76% (35, 36). Antibodies against a C. albicans

metallopeptidase detected by ELISA showed a sensitivity of

83% and a specificity of 97% (74).

Although in most studies only anti-Candida IgG anti-

bodies were detected, other immunoglobulin classes have

also been investigated in some studies. Aubert et al. (75)

used an immunocapture technique to detect IgM, IgA

and IgE anti-Candida antibodies against somatic antigens

in immunocompetent patients and found that IgA was

a particularly valuable marker of invasive candidiasis. De-

tection of IgA CAGTA showed a higher sensitivity than IgG

or IgM detection in the diagnosis of invasive candidiasis

(58, 60). Gutierrez et al. (33) detected IgM antibodies

to C. albicans whole cells by indirect immunofluorescen-

ce in patients with first time candidemia and reported

a 100% sensitivity and specificity. Kostiala et al. (76) de-

tected rises in titers of IgG and IgA antibodies by ELISA in

sera sequentially drawn from patients with candidemia.

Since the main limitations of antibody detection (low sen-

sitivity and specificity) are especially manifested when only

a single serum sample is studied, detection of antibodies by

commercially available test kits with multiple sequential sera

from the patient at risk for developing invasive candidiasis

should be performed. This will allow a kinetic study of the anti-

body response in the patient who will provide a more reliable

reflection of the development of the infection (12, 42, 77).

Combinations of tests

Combinations of tests for detection of antibodies and an-

tigens (29,44,56,78), antigens and non antigenic compo-

nents – such as D-arabinitol, mannose, (1®3)-b-D-glucan,

etc. (8, 30, 79), and antibodies, antigens and non antigenic

components (11, 80, 81) may be useful to overcome the

deficiencies of individual tests. The need for a combination

of tests is particularly evident when a specific antigen and an-

tibodies to this antigen are detected in the same patient, sin-

ce the antibodies may facilitate the clearance of the antigen.

Interestingly, detection of mannan seemed to complement

anti-mannan antibody detection, since patients with antige-

nemia did not have significant levels of anti-mannan antibo-

dies or vice versa, and detection of anti-mannan antibodies

was inversely correlated to the antigenemia. For combined

results of both tests, a sensitivity of 80% to 95% and a speci-

ficity of 53% to 93% have been reported (42-44, 56, 82).

The accuracy of diagnosis of candidemia was increased

when several combinations of tests were used, including

(1®3)-b-D-glucan and mannan detection (8, 30) and enola-

se and (1®3)-b-D-glucan detection (30). Platenkamp et al.

(80) reported a sensitivity of 77% and a specificity of 100%

when a combination of antibody, antigen and D-arabinitol de-

tection was used to differentiate invasive candidiasis from

Candida colonization in immunodeficient patients. However,

Bougnoux et al. (81) did not find the combination of anti-

body, antigen and D-arabinitol assays useful to differentiate

disseminated from peripheral candidiasis.

Conclusions

Substantial progress has been made in diagnosis of in-

vasive candidiasis with the development of a variety of me-

thods for the detection of antibodies and antigens. How-

ever, no single test has found widespread clinical use due

to the difficulties in obtaining consistently reliable serological

diagnosis in all patients with invasive candidiasis and due to

the limited number of commercial assays available. There

is a consensus in the literature that diagnosis based on

a single specimen lacks sensitivity. Therefore, it is neces-

sary to test sequential samples taken while the patient is at

greatest risk for developing invasive candidiasis to optimize

the diagnosis. Testing of serum samples should be started

at patient admission to obtain baseline data and considera-

tion should be given to the fact that different assays may be

needed for the diagnosis of invasive candidiasis. Further-

more, patients should be screened taking into account the

different expected kinetics of each assay (weekly for anti-

body and (1®3)-b-D-glucan detection, and at least twice

a week for antigen and D-arabinitol detection). Results ob-

tained from a panel of diagnostic tests in association with

blood culture findings and clinical aspects of the patient,

will likely be the most useful strategy for early diagnosis of

patients with invasive candidiasis and monitoring of thera-

peutic response.

Authors have been financed by the project IE019 (Diamolfun subproject)

from the Departamento de Industria, Comercio y Turismo del Gobierno Va-

sco-Eusko Jaurlaritza.

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Address for correspondence:

Guillermo Quindós

Departamento de Immunología

Microbiología y Parasitología

Fascultad de Medicina y Odontología

Universidad del País Vasco-Euskal Herriko Unibertsitatea

Apartado 699, E-48080 Bilbao, Spain

Tel.: [phone]

Fax: [phone]

e-mail: [e-mail]

Received: 31-03-2004

Approved: 12-05-2004