multicenter evaluation of an enzyme immunoassay (platelia®aspergillus) for the detection of...

Mycopathologia 155: 129–133, 2002.© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

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Multicenter evaluation of an enzyme immunoassay (Platelia� Aspergillus)for the detection of Aspergillus antigen in serum

Gianluigi Lombardi1–2, Claudio Farina1–3, Stefano Andreoni1–4, Domenico D’Antonio5, Elisa-betta Faggi1–6, Esther Manso1–7 & Aldo Mazzoni1–8

1Medical Mycology Committee, Associazione Microbiologi Clinici Italiani (AMCLI); 2Laboratorio di Microbi-ologia, A.O. Ospedale di Circolo, Varese; 3U.O. Microbiologia e Virologia, A.O. Ospedali Riuniti, Bergamo;4Laboratorio di Microbiologia, A.O. Ospedale Maggiore della Carita, Novara; 5Laboratorio U.O. Ematolo-gia, P.O. Ospedale Spirito Santo, Pescara; 6Istituto di Microbiologia, Universita degli Studi, Firenze; 7SezioneMicrobiologia, Laboratorio Analisi Chimico-cliniche, A.O. Ospedale Nuovo di Torrette, Ancona; 8Istituto diMicrobiologia, Policlinico Sant’Orsola, Universita degli Studi, Bologna, Italy

Received 20 November 2001; accepted 3 May 2002

Abstract

Invasive aspergillosis is a serious problem for immunocompromised patients, especially if neutropenic. The dia-gnosis of this infection is complicated, since clinical symptoms are often similar to those of other fungal diseases.The chance of detecting the presence of a specific antigen in the serum could confirm the suspected clinicaldiagnosis and, perhaps, be useful for the follow-up of the patient. The Medical Mycology Committee of theAssociazione Microbiologi Clinici Italiani (AMCLI) decided to evaluate in a multicenter prospective study (from 1November 1998 to 28 February 1999) the performance of the Platelia� Aspergillus Kit (Bio-Rad) for the detectionof Aspergillus galactomannan in human serum. The enrolled patients included various groups of immunosup-pressed patients (mostly neutropenic). Blood samples were drawn at the time of enrollment. This decision wasbased upon a clinical diagnosis of probable aspergillosis (antibiotic non-responsive fever for at least 96 hours,cough, hemophthosis and positive chest X-ray). Additional blood samples were drawn on days 3, 6, 9, 12, 15 and21. Culture and histopathologic examinations were performed according to the individual laboratory workflow. Foreach patient the laboratory filled a form with all the available clinical information, to create a database on whichto evaluate the results of the test. During the study, 187 patients with various kinds of immunosuppression wereenrolled. A total of 256 sera were tested: for 117 patients (62.6%) only the basal sample was tested, whereas for the70 symptomatic patients (37.4%) multiple specimens (range: 1–6) were tested. The results allowed the laboratoriesto exclude (68.6%) or confirm (31.5%: confirmed and/or probable) the clinical diagnosis of invasive aspergillosis;4 cases remained undetermined. Based on the results of this study, it seems that the use of this test should be limitedto those patients with clinical symptoms of aspergillosis.

Key words: enzyme immunoassay, galactomannan, invasive aspergillosis

Introduction

Invasive aspergillosis (IA) is a significant cause oflife-threatening opportunistic infections in immun-osuppressed hosts [1], with a prevalence that variesfrom 0.5 to 25% [2–6]. The reported mortality rangesfrom 50% to nearly 100% [1, 2, 5, 7, 8]. Establish-ing the correct diagnosis is still a major problem for

the clinician since the clinical symptoms of IA arenot pathognomonic of the disease, while histologicaland culture confirmations are often difficult to obtainantemortem [9, 10]. Moreover, efficient imaging tech-niques do not always allow adequate discriminationamong the different etiologies involved in this type ofsymptoms.

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Furthermore, classical serologic tests for the detec-tion of anti-Aspergillus antibodies are not very usefulin this kind of patients.

On the contrary, the detection of Aspergillus anti-gens (galactomannan) in the serum may allow morerapid diagnosis of invasive aspergillosis and bettertherapeutic management of the patient [11].

For this reason the Medical Mycology Commit-tee of the Associazione Microbiologi Clinici Italiani(AMCLI) proposed a multicenter study in order toassess whether an Aspergillus antigen detection testcould be useful for clinical laboratories.

This evaluation might answer an important ques-tion for the control of aspergillosis in at risk patientsin whom histopathology and culture are difficult toperform. Would the detection of circulating antigenrepresent a reliable confirmation of the clinical dia-gnosis?

The data from this study may allow the issue ofguidelines for the use of a rapid test for a presumptiveetiologic confirmation of the clinical diagnosis.

This prospective study had two major objectives:1. to evaluate the performance of the enzyme im-

munoassay (EIA) test on sera from patients atrisk for invasive aspergillosis, by determining thechance for an early confirmation of the clinical dia-gnosis compared to the results of culture and ofhistopathologic examination;

2. to control the positive patients in order to asses thepredictivity of the clinical outcome compared tothe clinical status.

Materials and methods

Participating laboratories

The study was conducted by selected laboratories from1 November 1998 to 28 February 1999. The parti-cipants were located in different parts of Italy andwere selected on the basis of a convenience sampling,in order to obtain a significant picture of the nationalsituation.

Inclusion criteria

For this study the participating laboratories enrolledall the patients belonging to the following groups:

1. neutropenic (WBC ≤ 200 cells/mm3 for at least5 days), bone marrow transplant patients and pa-tients affected with haematologic malignancies;

2. iatrogenic and/or pathologic immunosuppressedpatients (solid organ transplant, individuals af-fected with diseases associated with immunosup-pression.

For each patient, a form with the following informa-tion was filled in:

– name– underlying disease– presence of a clinical diagnosis of aspergillosis

(fever, cough, hemophthosis), with the date ofonset of the symptoms

– results of imaging techniques (X-ray, CT, NMR)– antifungal therapy/prophylaxis (drugs, length of

treatments)– immunosuppressive therapy– result of antigen detection test– direct microscopic exam– culture– histopathologic exam– autopsy

Serum samples

Consecutive blood samples (5 ml) were collected fromeach patient at different stages:1. on enrollment2. upon clinical diagnosis

– antibiotic un-responsive fever– cough– hemophthosis– positive result of imaging procedures, compat-

ible either with pulmonary aspergillosis or withother organ localization

3. during follow-up (on days 3, 6, 9, 12, 15 and 21).

Microbiology methods

Specimens for culture and histopatholgy were pro-cessed according to the routine protocols in use at eachparticipating laboratory.

The Platelia� Aspergillus Kit (Bio-Rad, cat. n.62797) was used to detect the circulating galactoman-nan of Aspergillus spp.: the kits were kindly suppliedby the Manufacturer and the tests were performed ac-cording to the Manufacturer’s instructions. The test isbased on a monoclonal antibody (EB-A2) which re-cognizes the β-D-galactofuranoside side chain of thegalactomannan molecule.

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A random quality control on 20% of all the spe-cimens was performed by one of the participatinglaboratories.

Diagnostic criteria

On the base of the results, patients were divided intothe following categories:

Group I: confirmed aspergillosis(positive culture, histopathology, ima-ging techniques and clinical symp-toms).

Group II: probable aspergillosis(negative culture and/or histopatho-logy, positive imaging techniques andclinical symptoms).

Group III possible aspergillosis(positive clinical symptoms, negativeculture and/or histopathology and ima-ging techniques).

Group IV unconfirmed aspergillosis(negative control).

Group V colonization(positive clinical symptoms and cul-ture, negative histopathology and ima-ging techniques.

Table 1 presents in detail the adopted diagnosticcriteria.

Results

During the study, 196 patients were enrolled. Their un-derlying immunosuppressive diseases were: haemato-logic malignancies (146, 74.5%), bone marrow trans-plants (23, 11.7%), kidney transplants (12, 6.1%),heart transplants (13, 6.6%), other pathologies (2,1.0%).

From 107 patients (54.6%) only the basal serumwas obtained, whereas 89 patients (45.4%) had mul-tiple specimens taken, since they were eligible forthe study on the base of the above mentioned criteriaof suspected aspergillosis. A total of 322 sera weretested for the detection of circulating galactomannan:the results are summarized in Table 2.

Table 3 reports the time elapsed before a positiveantigen test confirmed the suspicion of aspergillosis.Table 4 indicates how long before the EIA test was

Table 1. Diagnostic criteria

Confirmed aspergillosis

Culture Positive

Histopathology Positive

Imaging techniques Positive

Clinical symptoms Positive

Antigen detection Positive

Probable aspergillosis

Culture Negative

Histopathology Negative

Imaging techniques Positive



Possible aspergillosis

Culture Negative


Imaging techniques Negative



Unconfirmed aspergillosis

Culture Negative



Clinical symptoms Positive/negative

Antigen detection Negative

Colonization

Culture (nasal swab) Positive



Clinical symptoms Positive/negative

Antigen detection Negative

Table 2. Summary of results

N %

Unconfirmed aspergillosis 58 65.2

Confirmed aspergillosis 7 7.9

Probable aspergillosis 12 13.5

Possible aspergillosis 9 10.1

Colonization 3 3.3

Total 89 100

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Table 3. Time elapsed (days) between the clinical diagnosis andthe first positive antigen detection

Days from clinical diagnosis

<2 3–7 >7

Confirmed aspergillosis (7) 5 1 1

Probable aspergillosis (12) 7 2 3

Possible aspergillosis (9) 7 1 1

Table 4. Diagnostic precocity of antigenemia versus cul-ture/histopathology

Days from pos. culture/histo.

<2 5 9


positive compared to routine culture and/or histopath-ology.

Finally, the EIA test was very useful for the follow-up of patients with confirmed aspergillosis who weretreated with either intravenous Amphotericin B (6cases) or Itraconazole (1 case). Following appropri-ate therapy, antigen levels decreased and eventuallybecame negative after different lengths of time (Table5).

Discussion

Invasive aspergillosis is a major clinical problem,whose incidence and prevalence have been increas-ing over the last years among immunocompromisedpatients, with very high associated mortality.

A correct diagnosis is sometimes difficult for theclinician to achieve, since confirmation of clinical as-pergillosis, by imaging techniques and/or histologicaland microbiological identification of the agent, often

Table 5. Reduction/negativization of antigenemia following in-travenous antifungal therapy

Red./neg.∗ of antigenemia

(days of systemic therapy∗∗)

<2 3–7 >7


∗Red./neg. = reduction/negativization.∗∗Amphotericin B = 6 cases; itraconazole = 1 case.

requires several weeks. Moreover, the techniques in-volved in the collection of reliable specimens are quiteinvasive and often not suitable for such debilitatedpatients [12].

The classic serological tests for the detection ofanti-Aspergillus antibodies are not very useful in thesesubjects; on the contrary, a method for the detection ofa specific circulating antigen (galactomannan) seemsto be more reliable in order to establish an earlier dia-gnosis and to begin appropriate antifungal therapy [13,14].

The results of this study confirmed that the detec-tion of Aspergillus antigens is very helpful to excludeor to confirm a clinical diagnosis of invasive aspergil-losis (68.5% excluded, 31.5% confirmed [confirmed,probable, possible]) at the onset of clinical symptoms.

It is very important that a basal serum sample beobtained from each at risk patient (neutropenic) andthen as soon as possible at the onset of the clinicaldiagnosis, in order to achieve early confirmation of IA(19/28 cases [67.9%] within 2 days after the clinicaldiagnosis).

Moreover, since in vivo animal models demon-strated that the circulating antigen usually disappearedwithin 7 day of systemic therapy [15], the test mustbe performed before the beginning of antifungal treat-ment. One of the patients included in the study re-peatedly resulted negative for circulating antigen eventhough he had an A. flavus positive BAL culture: thespecimen was obtained after 6 days of treatment withliposomal Amphotericin B.

However, antigen detection should not induce theclinician to neglect traditional methods (histopatholo-gic exam and culture) to confirm a clinical diagnosis.

Furthermore, from the data of this study, it seemsthat the test for the detection of circulating galacto-mannan should be performed only at the onset of aclinical diagnosis.

Acknowledgements

The Authors would like to thank Dr. Libero Ajello(Centers for Disease Control and Prevention, Atlanta)for his suggestions and for revising the text.

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Address for correspondence: Dr. Gianluigi Lombardi, Laboratoriodi Microbiologia, Ospedale di Circolo e Fondazione Macchi, VialeBorri, 57, 21100 Varese, Italy.Phone: +39 0332 278435; Fax: +39 0332 260017;E-mail: [email protected]

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