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Evaluation of a newly developed down-flow immunoassay for detection of serum mannan antigens in patients with candidaemia

Department of Laboratory Medicine, Graduate School of Medical Science¹ and Department of Clinical Laboratory Science² , Kanazawa University Hospital, School of Medicine, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-8640, Japan

Correspondence
Shin-ichi Fujita
fujita-knz{at}umin.ac.jp

Received 2 September 2005
Accepted 12 January 2006

Abbreviations: BG, (13)-ß-D-glucan.

	INTRODUCTION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Yeasts of the genus Candida are a major cause of invasive infections in hospitalized patients receiving intensive medical care, particularly in neutropenic cancer patients and patients with indwelling intravenous catheters (Hajjeh et al., 2004). The accurate and timely diagnosis of invasive candidiasis is difficult due to nonspecific clinical signs and the natural commensal status of these opportunistic pathogens. In order to overcome these difficulties, non-culture methods have been developed. With these methods, highly sensitive and specific detection of fungal DNA from clinical materials has been demonstrated (Maaroufi et al., 2003; Ahmad et al., 2004). However, technical problems such as the risk of contamination and the inability to distinguish colonized cases from infected patients may need to be solved. Moreover, methods for DNA extraction and DNA detection should be both standardized and simplified for the introduction of a molecular diagnostic method to routine clinical laboratories.

The following findings or tests are used for the diagnosis of invasive candidiasis in clinical laboratories: isolation of Candida species from normally sterile sites, histopathological evidence of Candida invasion, endophthalmitis, hepatosplenic lesions detected by imaging techniques, detection of anti-Candida albicans antibodies (Sendid et al., 1999), and detection of Candida components such as mannan (Yeo & Wong, 2002), D-arabinitol (Walsh et al., 1995) and (13)-ß-D-glucan (BG) (Obayashi et al., 1995). Unfortunately, all of these assays have limitations reflected by their sensitivity and/or specificity. Therefore, a combination of two or more assays is necessary for accurate diagnosis of invasive candidiasis (Sendid et al., 2002; Yeo & Wong, 2002).

Detection of serum mannan has been developed using a microplate enzyme immunoassay, and an assay kit is commercially available as the Platelia Candida Ag test (Bio-Rad). However, the sensitivity of this test has been reported to be 40 % for patients with invasive candidiasis caused by C. albicans (Sendid et al., 1999). Recently a new enzyme immunoassay test for the detection of mannan antigens, the Unimedi Candida monotest (Unitika), has become available commercially. The objective of this study was to clarify the usefulness of the new assay for diagnosing invasive candidiasis by comparison with commercial kits for serum antigens and plasma BG.

	METHODS

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Preparation of antigen extracts. A total of 48 blood culture Candida isolates (5 isolates of C. albicans, 2 of Candida dubliniensis, 13 of Candida glabrata, 5 of Candida guilliermondii, 2 of Candida krusei, 15 of Candida parapsilosis and 6 of Candida tropicalis) and 5 Candida control strains were used to study the cross-reactivity of 2 enzyme immunoassays, the Platelia Candida Ag test and the Unimedi Candida monotest. The Candida control strains were obtained from the NITE Biological and Technological Center, Kisarazu, Japan (NBRC), and included Candida kefyr (NBRC942, NBRC1065), Candida norvegensis (NBRC1020) and Candida rugosa (NBRC1152, NBRC2901). Each strain grown on CHROMagar Candida (Hardy Laboratories) was inoculated into RPMI 1640 medium (Dainippon Pharmaceutical) and incubated at 35 °C for 2–3 days. The culture medium was boiled in a water bath for 7 min. After centrifugation, the supernatant was diluted with the same medium at 1 : 1000, and the diluted solutions were tested for mannan antigens.

Patients. Candidaemic patients admitted to Kanazawa University Hospital during the period January 1995 to July 2005 were included in the present study, provided that at least two serum samples taken during the fever episode were available. Candidaemia was diagnosed on the basis of at least one blood culture positive for Candida species, together with a temperature of more than 38 °C, which was refractory to antibiotics for more than 2 days. Antifungal drugs were given intravenously to all candidaemic patients. A total of 105 patients (37 females and 68 males, mean age 52·8 years) were selected, and these included 49 (47 %) patients infected with C. albicans, 11 with C. glabrata, 11 with C. guilliermondii, 20 with C. parapsilosis, 9 with C. tropicalis and 5 with other Candida species. When two or more episodes of candidaemia were observed in one patient, the first episode was included. Blood cultures and identification of yeast isolates were performed as described previously (Fujita et al., 2001). The clinical and mycological findings are summarized in Table 1. Of the 105 candidaemic patients, 69 (66 %) had a solid tumour, 6 (6 %) had cardiovascular disease, 5 (5 %) had leukaemia, 3 (3 %) had malignant lymphoma and 22 (20 %) had other disorders. The intravenous catheter tip cultures grew Candida belonging to the same species as that of blood isolate in 49 (47 %) candidaemic patients. Control febrile patients who had no clinical evidence of fungal findings and were treated successfully without systemic antifungal drugs included 105 bacteraemic patients and 70 non-bacteraemic patients. None of the control patients also showed clinical evidence of pneumocytosis. The 105 blood cultures included 37 (35 %) Gram-negative bacteria (7 were identified as Escherichia coli, 9 as Klebsiella spp., 4 as Enterobacter spp., 9 as Pseudomonas aeruginosa and 8 as other Gram-negative bacteria). A total of 68 (65 %) of the 105 blood cultures contained Gram-positive bacteria (19 were identified as Staphylococcus aureus, 18 as Staphylococcus epidermidis, 12 as coagulase-negative staphylococci, 11 as Enterococcus spp. and 8 as other Gram-positive bacteria).

For the Cand-Tec assay, sera were diluted 1 : 4 with the diluent supplied in the kit. Twenty microlitres of the diluted specimen was mixed with the same volume of latex particles coated with anti-Candida antibody. The mixture was rotated at 140 r.p.m. for 10 min then checked immediately for agglutination.

The Platelia Candida Ag test, which employs mAb that recognizes the alpha-linked mannopentose of C. albicans mannan, was performed as described by Sendid et al. (1999). Each experiment included a calibration curve, which was made with a pool of normal human serum supplemented with known mannan antigen concentrations (0·25, 0·5, 1·0 and 2·0 ng ml^–1). Although the test detected as little as 0·25 ng serum mannan ml^–1, mannan concentrations greater than 0·5 ng ml^–1 were considered a positive result, according to the manufacturer's recommendations.

The Unimedi Candida test involves affinity-purified antibodies. Antisera were prepared by immunizing Japanese white rabbits with intravenous injections of 0·5 mg (1 mg ml^–1 in PBS) of formalin-killed and acetone-dried cells of C. albicans YD1124 twice a week. After eight inoculations, blood was collected, and sera were treated using a mannan-fixed formyl-cellulofine column. Mannan antigens were prepared by the method reported by Reiss et al. (1986). A total of 150 µl serum sample was mixed with 150 µl of treatment solution (20 mM NaH₂PO₄), boiled for 4 min, and then centrifuged at 10 000 r.p.m. for 10 min. One hundred microlitres of supernatant was added to antibody-coated microtitre plate wells. After incubation at room temperature for 2 h, the plates were washed thoroughly and 100 µl alkaline phosphatase-conjugated antibodies was added to the wells. After further incubation at room temperature for 1 h, the plates were washed. The reaction was revealed by incubation with 100 µl substrate solution (50 mM Tris(hydroxymethyl)aminomethane, 2·2 mM p-iodonitrotetrazolium violet, 0·3 mM NADPH) for 20 min at room temperature. After addition of stopping solution (1 M phosphate) to the wells, the A₄₉₀ was read on a microplate reader. In the preliminary study, the test detected as little as 0·25 ng mannan ml^–1 when the antigen was seeded into pooled human serum.

The Unimedi Candida monotest (a down-flow immunoassay) was used according to the manufacturer's instructions. Briefly, aliquots of 200 µl serum sample were denatured with 200 µl treatment solution, and the mixture was boiled for 4 min and then centrifuged at 10 000 r.p.m. for 10 min. A total of 200 µl supernatant were mixed in microcentrifuge tubes with 100 µl alkaline phosphatase-conjugated antibodies, which were the same antibodies as used in the Unimedi Candida kit. Following incubation for 30 min at room temperature, 250 µl of the mixture was applied to the loading window of the cassettes. After drying, the cassettes were washed with 10 drops of wash solution, and then 4 drops of the substrate solution (0·5 mM 5-bromo-4-chloro-3-indolyl phosphate, 0·5 mM nitrotetrazolium blue) was added to the loading window. After 10 min of incubation at room temperature, four drops of stopping solution (0·1 M phosphate) were added, and the reactions (purple colour) appeared on the anti-mannan antibodies coated nitrocellulose membrane were classified visually into four categories according to the photographs of the standards; the intensity of reactions obtained with pooled serum samples spiked with mannan at concentrations of 0, 0·125, 0·25 and 0·5 ng ml^–1 were scored as – (negative), 1+, 2+ and 3+, respectively. The sensitivity of this method in heat-treated sera was 0·125 ng ml^–1.

BG assay. Of 105 patients with candidaemia, 76 patients (90 sera) were studied for plasma BG levels. The kinetic turbidimetric Limulus method (BG test; Wako) was used prospectively for quantification of plasma BG as described by Mori et al. (1997). Briefly, blood samples collected using a syringe, the inside wall of which was coated with a small amount of heparin, were centrifuged at 3000 r.p.m. for 40 s. Aliquots of 0·1 ml plasma samples were added to 0·9 ml pretreatment solution, and heated at 70 °C for 10 min. Then, 0·2 ml aliquots of the pretreated samples were added to the Limulus amebocyte lysate reagent, and the turbidities of the resultant mixtures were measured using a Toxinometer MT-358 (Wako). The cut-off value was 11 pg BG ml^–1.

Statistical analysis. The chi-square test and Fisher's exact probability test were employed by use of computer software (Statview statistical software package; Abacus Concepts). Statistical significance was assured when the P value was less than 0·05.

	RESULTS AND DISCUSSION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Both the Platelia Candida Ag test and the Unimedi Candida monotest revealed strongly positive reactions with the strains of C. albicans, C. dubliniensis, C. rugosa and C. tropicalis, compared to negative or weakly positive reactions with culture fluids of C. kefyr and C. norvegensis. Concentrations of antigens obtained from C. guilliermondii, C. krusei and C. parapsilosis differed between the two assays: the Unimedi Candida monotest showed stronger reactions than the Platelia Candida Ag test. Interestingly, 9 (60 %) of 15 C. parapsilosis strains and 2 of 5 C. guilliermondii strains showed negative reactions with the Platelia Candida Ag test, while none of these strains tested showed negative reactions with the Unimedi Candida monotest. Furthermore, the reactions obtained with each assay varied from weak to strong when culture fluids of C. glabrata strains were tested. Table 2 shows the antigen levels in 64 sera obtained from 27 patients who had two or more positive blood cultures and in culture fluids of the blood culture isolate. All patients infected with Candida strains that showed negative or weakly positive reactions with the Platelia Candida Ag test or the Unimedi Candida monotest did not develop mannanaemia. In addition, three patients infected with C. glabrata isolates that showed strong reactions (>2·0 ng ml^–1) with the Platelia Candida Ag test and four patients infected with Candida strains (three of C. glabrata and one of C. parapsilosis) that showed strong reactions (scored as 3+) using the Unimedi Candida monotest did not have mannanaemia, suggesting incomplete dissociation of mannan-serum complexes.

Of 74 patients with candidaemia whose sera were available before or on positive blood culture sampling, 29 (39 %), 38 (51 %) and 48 (65 %) patients showed mannanaemia with the Platelia Candida Ag test, the Unimedi Candida and the Unimedi Candida monotest, respectively. A total of 17 (81 %) of 21 patients had elevated BG levels before or on positive blood culture sampling.

The Cand-Tec was the first commercially available antigen test. As shown by other investigators (Yeo & Wong, 2002), the usefulness of this test seems to be limited because of the low sensitivity (38 % in our study) for diagnosing invasive candidiasis. Therefore, attempts to improve the immunological detection of mannan, involving the use of a more sensitive test format, and the use of monoclonal antibodies that react with defined epitopes, have been made (Yeo & Wong, 2002).

The Platelia Candida Ag test is based on the use of a monoclonal antibody, EB-CA1, which recognizes a mannopentose epitope of C. albicans. This epitope has also been found at high levels in C. glabrata and C. tropicalis, but has only been found to a lesser extent in C. krusei, C. kefyr and C. parapsilosis (Jacquinot et al., 1998; Rimek et al., 2003). With the exception of the results with C. glabrata, these previous reports support our results of antigen detection from serum samples of candidaemic patients and from culture fluids of Candida isolates. In contrast to the positive reactions of EB-CA1 with C. guilliermondii (Rimek et al., 2003), we observed negative reactions with two strains and slightly or moderately positive reactions (<1·0 ng ml^–1) with the remaining three C. guilliermondii strains. As a result, only 3 (27 %) of 11 patients with candidaemia due to C. guilliermondii showed mannanaemia in the present study. According to the reported results (Sendid et al., 2002), the sensitivity for the detection of mannanaemia with the Platelia Candida Ag test was 58–70 % for infections caused by C. albicans, C. glabrata and C. tropicalis, while it was 25–30 % for infections caused by C. parapsilosis and C. krusei. As for C. glabrata, the amounts of epitopes recognized by the antibodies used by two assays (the Platelia Candida Ag test and the Unimedi Candida monotest) differed greatly from strain to strain, and may be less than for other clinically relevant Candida species, resulting in a low sensitivity for diagnosing C. glabrata infections. According to the reported results of mannan detection from 34 serum samples from 12 candidaemic patients (Yoshida et al., 2002), the sensitivity of the Unimedi Candida has been higher than that achieved with the Platelia Candida Ag test (82·1 vs 40·0 %). This may has been caused by a difference in the reactivities of the respective kits against different species of Candida. Therefore, an increased proportion of patients with invasive candidiasis due to C. parapsilosis and C. glabrata, which are common non-C. albicans Candida species causing yeast blood stream infections (Hajjeh et al., 2004; Pfaller et al., 1998, 1999), may lower the sensitivity of the Platelia Candida Ag test.

The mannan assay employing affinity-purified polyclonal antibodies against C. albicans mannan has been marketed as the Unimedi Candida (a microplate enzyme immunoassay) since 2000. Recently, the Unimedi Candida monotest, which uses the same antibody as the Unimedi Candida assay, has become commercially available.

In the present study, the Unimedi Candida monotest was more sensitive and specific than the Unimedi Candida assay: the sensitivity improved from 69 to 82 %, and the specificity from 89 to 96 %. Moreover, the Unimedi Candida monotest has the advantage over previous mannan assays in that semi-quantitative results can be obtained within 1 h, and it requires no specialized instruments, such as microplate washer and microplate reader. In addition, the test is available for testing a single serum sample.

BG is a cell wall polysaccharide component specific for fungi except for Mucor spp., and to a lesser extent, cryptococci (Miyazaki et al., 1995). Measurement of plasma BG is used widely in Japan as a test to screen a febrile patient for fungal infections, because quantitative results can be obtained within the same day of blood collection. Two methods are currently available for the sensitive detection of BG: a chromogenic assay and a kinetic turbidimetric assay. Elevated BG levels have been detected in >90 % of patients with candidaemia, with a specificity of >90 % (Mori et al., 1997; Odabasi et al., 2004). Although the sensitivity of the BG assay was high (95 %), the specificity was lower than those of the mannan antigen assays. In our series of patients, none of the control patients received haemodialysis and/or administration of antitumour BG products. Both of these are known to be factors resulting in false-positive BG measurements (Kanda et al., 2001; Ishizuka et al., 2004). There are two things that may contribute to false-positive results in the BG assay. First, administration of albumin products (Usami et al., 2002) and/or immunoglobulin products (Ikemura et al., 1989) before or during fever episodes may result in false-positive BG elevation. Second, BG may reach the circulation through damaged intestinal or respiratory mucosa and cause false-positive results in tests to detect BG. Because the BG assay had a high sensitivity of 95 % (94 % per serum), the assay seems to be most useful for excluding candidiasis.

Serological tests to detect C. albicans mannan and C. albicans antibodies have been positive in 73 % of patients with candidiasis at least 2 days before blood cultures became positive (Yera et al., 2001). The present study showed that both mannan detection by the Unimedi Candida monotest and BG measurement by the kinetic turbidimetric assay were useful for the early diagnosis of invasive candidiasis. However, further studies of rapid and efficient methods for dissociating mannan from antibodies in serum, a major blocking factor (Lew et al., 1982; Sendid et al., 2002), are mandatory to improve the sensitivity of the assay.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Fujita, S.-i. Articles by Hashimoto, T. Search for Related Content PubMed PubMed Citation Articles by Fujita, S.-i. Articles by Hashimoto, T. Agricola Articles by Fujita, S.-i. Articles by Hashimoto, T.