Strategy for Overcoming Serum Interferences in Detection of Serum (1,3)-β-d-Glucans

LETTER

The (1,3)-β-d-glucan (BG) is a cell-wall polysaccharide of most fungi. Regular measurement of BG levels in serum is recognized as a useful diagnostic marker for invasive fungal diseases in immunocompromised and intensive care unit (ICU) patients (1). Previous studies have identified several sources of false-positive reactions, such as BG in fungus-derived antibiotics (2, 3) or serum glucans associated with bacteremia caused by Pseudomonas aeruginosa (4). In addition, high serum concentrations of hemoglobulin, bilirubin, and triglycerides may also be mentioned by the manufacturer of detection kits as interfering in the detection of glucanemia (3). We and others have also observed that high levels of proteinemia may prevent proper detection of BG by development of a coagulate in contact with the mix of Fungitell reagent/pyrosol (unpublished observation). Since glucans are thermostable, we explored the effect of the classical heat dissociation procedure initially described for releasing mannans from serum complexes by boiling the serum at 100°C for 3 min in the presence of Na₂-EDTA followed by centrifugation at 10,000 × g (5, 6). Figure 1a shows that serum supernatants after heat treatment of 3 icteric, 3 lipemic, 3 hemolytic, and 3 hyperprotidic sera were depleted in bilirubin, triglycerides, hemoglobin, and proteins, respectively. We then assessed the impact of this procedure on the yield of detected glucanemia in 9 sera drawn from patients with proven invasive fungal infections caused by the 3 major fungal opportunistic pathogens for which BG detection is recommended. These consisted of 3 patients with Candida albicans invasive candidiasis, 3 patients with invasive pulmonary aspergillosis, and 3 patients with pneumocystosis. As shown in Fig. 1b, no significant variation was observed for BG concentrations determined without and with serum treatment. Altogether, our findings show that serum dissociation induced by heating with Na₂-EDTA is a simple and rapid procedure to overcome the interferences previously reported as limitations in the use of the BG detection test.

Fig 1.

(a) Effect of heat treatment (HT) on sera (S) presenting high levels of substances described as interfering in the Fungitell assay. Levels of bilirubin (S1 to S3), triglycerides (S4 to S6), hemoglobin (S7 to S9), and proteins (S10 to S12) measured before and after HT are shown. (b) Effect of heat treatment (HT) on the level of serum (1,3)-β-d-glucans detected by using the Fungitell assay in sera from 3 patients with invasive candidiasis (IC [S1 to S3]), 3 patients with invasive pulmonary aspergillosis (IPA [S4 to S6]), and 3 patients with Pneumocystis pneumonia (PP [S7 to S9]).