Improved Standardization of the Bio-Rad Platelia Aspergillus Galactomannan Antigen Sandwich Enzyme Immunoassay Using the DS2 (Dynex) Enzyme-Linked Immunosorbent Assay (ELISA) Processing System

Abstract

T he Bio-Rad Platelia Aspergillus galactomannan antigen sandwich enzyme immunoassay (GM-EIA) is widely used as a screening method for prospective surveillance of invasive aspergillosis (IA) in patients at high risk of disease. The assay is well established, as reflected by its recommendation in the European Organization for Research and Treatment of Cancer (EORTC) consensus criteria for defining invasive fungal disease (IFD) (1). Despite this fact, the diagnostic performance of the GM-EIA is variable, with meta-analyses showing combined sensitivities ranging from 0.71 to 0.78 and specificities ranging from 0.81 to 0.89 (2, 3). The false positivity experienced using the GM-EIA has been associated with antimicrobial treatment (e.g., piperacillin-tazobactam), other invasive fungal diseases (e.g., fusariosis), and even ingestion of an ice-pop (4-7). Consequently, the GM-EIA should not be used as a stand-alone diagnostic test but is an important component of the diagnostic strategy for managing IA (1).Although initially the GM-EIA reproducibility was reported to be excellent between laboratories (8), recent reports documented a lack of reproducibility for repeat testing of positive samples (9, 10). In particular, samples with an optical density index (ODI) at or around the positivity threshold (Х0.5) of the assay were regularly found to be negative on repeat testing (8, 11). The storage conditions of specimens appear to have an impact on reproducibility, with a significant decline in the sample ODI reported after storage at Ϫ80°C (11). An IA diagnosis also appears to be important, with nonreproducibility observed more frequently for retesting of false-positive samples from patients without IA (11,12). A significant correlation between the serum albumin concentration and the difference in ODI value on retesting was also recently reported. A larger reduction in indices was observed on retesting sera with increasing albumin concentrations (11).While storage and disease status have been shown to affect the reproducibility of GM-EIA, other factors, such as human error, environmental contamination at the point of testing, and variability in local testing conditions between laboratories, may also have impacts on the assay's performance and reproducibility. The GM-EIA is performed manually by most laboratories and, consequently, is susceptible to fluctuations in the environmental tem-

Comparison of Nonculture Blood-Based Tests for Diagnosing Invasive Aspergillosis in an Animal Model

“…While the cost of multiple tests may seem prohibitive, it can be more than offset by the reduction in unnecessary antifungal therapy driven by the high sensitivity and negative predictive value of combination testing ( 24 – 26 ). From a practical standpoint, serum/plasma PCR and GM can be performed on the same sample and can be automated, reducing pressure on the laboratory ( 27 ). WB and serum PCR can be streamlined by combining serum and WB DNA extraction methods into a single process resulting in a single PCR test to be combined with GM ( 28 ).…”

Developments in Fungal Serology

“…The availability of an automated GM ELISA system is useful for minimizing interassay variability, where manual operational events could be a potential source of non-reproducible GM positivity and have been attributed to 33% of GM positivity [9]. However, the BioRad GM can be automated using the Evolis system or other widely used systems such as the DS2 Dynex platform, and other non-BioRad automated GM systems are available but lack published clinical validation (e.g., ERA FungiXpert) [3,10].…”

Recent Advances and Novel Approaches in Laboratory-Based Diagnostic Mycology

“…The ability to automate GM-EIA testing using the BioRad Evolis instrument limits the impact of the human variable, permits screening of large numbers and allow two different assays (e.g., GM-EIA and Aspergillus IgG) to be run concurrently, although published data specific to the GM-EIA are not currently available. However, it is not necessary to purchase bespoke instrumentation, and more generic platforms (e.g., Dynex DS2 ELISA processing system) have been programmed to perform the GM-EIA assay [ 52 ]. In a multicentre study, the use of the DS2 reduced variability when compared to manual GM-EIA, the detection of positive samples from cases of IA were unaffected while there was a significant decrease in GMI for control-based samples, leading to a reduction in potential false positive results [ 52 ].…”

“…However, it is not necessary to purchase bespoke instrumentation, and more generic platforms (e.g., Dynex DS2 ELISA processing system) have been programmed to perform the GM-EIA assay [ 52 ]. In a multicentre study, the use of the DS2 reduced variability when compared to manual GM-EIA, the detection of positive samples from cases of IA were unaffected while there was a significant decrease in GMI for control-based samples, leading to a reduction in potential false positive results [ 52 ].…”