The addition of the (1→3)-β-d-glucan (BDG) assay to the capabilities of most mycology laboratories has improved the diagnostic approach to patients with suspected invasive fungal disease (IFD) [1]. As a component of the fungal cell wall, BDG is considered an almost pan-fungal marker, with the reported exceptions of mucormycosis, cryptococcosis and blastomycosis [2]. The detection of serum BDG has been consistently proven to yield an excellent sensitivity and negative predictive value for the non-invasive diagnosis of Pneumocystis jirovecii pneumonia (PCP) [3,4]. On the other hand, meta-analyses have revealed considerable heterogeneity in the accuracy of this biomarker for the diagnosis of invasive candidiasis, invasive aspergillosis (IA) or fusariosis [[5], [6], [7], [8], [9]].

A commonly perceived limitation of the serum BDG assay is the occurrence of false-positive (FP) results due to the cross-reactivity with bacteria and other fungi or to the presence of fungal antigens in dietary and blood-derived products [10]. As summarized in Table 1, previous studies have reported a highly variable rate of FP results for serum BDG levels among bacteremic patients [[11], [12], [13], [14]]. In contrast, some authors found no cases of significant BDG positivity in this clinical setting [15,16]. The majority of these studies [[11], [12], [13], [14], [15],17] were performed with the Fungitell test (Associates of Cape Cod, East Falmouth, MA), which has been widespread used in Europe and North America. The Fungitell test is the only BDG assay endorsed by the European Organization for Research and Treatment of Cancer and the Mycoses Study Group (EORTC/MSG) in the latest revision of the diagnostic criteria of IFD [18].

The Wako BDG assay (Wako Pure Chemical Industries, Osaka, Japan) has been used in Japan since two decades ago [19] and has received its Conformité Européenne (CE) marking. Although both assays are based on the ability of BDG to activate factor G —a serine protease zymogen— of the horseshoe crab (Limulus polyphemus) coagulation cascade, the Fungitell and Wako kits differ in a number of technical features. In detail, both assays utilize different BDG standards (pachyman and lentinan, respectively) and optical measurement techniques. The Fungitell test is a colorimetric assay based on the assessment of the rate of optical density increase produced by the sample upon cleavage of p-nitroaniline from a chromogenic peptide substrate, which is interpreted against a standard curve to estimate the BDG concentration. In contrast, the Wako test measures the turbidity change caused by the gelation reaction initiated by factor G. The time interval taken for the transmittance to reach a given threshold (gelation time) correlates with BDG concentration. As a result, both the recommended cut-off values (≥80 pg/mL for the Fungitell test and initially ≥11 pg/mL for the Wako test [latter optimized at ≥7 pg/mL]) and in their diagnostic performance vary [20,21]. From a practical perspective, the Fungitell test is performed more economically when 42 individual serum samples are run in a single batch, whereas the Wako test has been designed for multiple- or single-sample use, allowing to sequentially process individual samples on demand in a shorter turnaround time [20]. This advantage in terms of laboratory workflow likely explains the growing popularity of this kit, despite the relative paucity of clinical data and the lack of direct comparability with the results of the colorimetric Fungitell assay.

In addition to previous studies that have investigated the degree of qualitative and quantitative agreement between the Fungitell and Wako tests [[22], [23], [24]], it is also necessary to characterize the potential effect of bacteremia in the cross-reactivity of the turbidimetric assay. Therefore, we investigated the probability and clinical profile of FP results with the Wako BDG test in the presence of concurrent bacteremia due to a diverse collection of Gram-positive and Gram-negative pathogens in patients not suspected to have IFD.