Evaluation of a multiplex PCR assay using barcoded magnetic bead technology for detection of fungal pathogens in bronchoalveolar lavage fluid.

Objectives: Invasive fungal infections (IFIs) are increasingly prevalent, particularly in immunocompromised patients. Traditional culture-based diagnostic methods have limitations, including low sensitivity and long turnaround times. The Applied BioCode (ABC) assay, utilizing barcoded magnetic bead technology, enables simultaneous detection of 22 fungal pathogens directly in bronchoalveolar lavage (BAL) fluids in 5 hours. This study aimed to assess the ABC's diagnostic performance in combination with other methods for diagnosis of IFIs.

Methods: A prospective study was conducted from August 2023 to March 2024 in patients with suspected pulmonary IFIs. BAL samples were tested simultaneously using the ABC assay, fungal microscopy, culture, BAL galactomannan (GM), and an in-house Pneumocystis jirovecii PCR. The diagnostic performance of the ABC assay was evaluated individually and in combination with other methods.

Results: Among 323 patients (44 proven/probable IFIs, 279 non-IFIs), the ABC assay demonstrated a sensitivity of 43.2% (95% CI: 28-59%) and a specificity of 98.5% (95% CI: 96-99%). When combined with BAL GM (≥0.5), the ABC assay was able to detect 70.4% (95% CI: 54-83%) of cases with proven/probable IFIs in 5 hours. When the ABC assay, fungal culture, and BAL GM (≥0.5) were combined and IFIs were considered to be detected if either of the combined tests was positive, sensitivity increased to 88.6% (95% CI: 75-96%), with a specificity of 93.5% (95% CI: 90-96%). The ABC assay' sensitivity was not affected by antifungal therapy (46.1% [95% CI: 19-74%] with antifungal therapy vs. 42.0% [95% CI: 24-60%] without antifungal therapy, p 0.77), unlike fungal culture (23.0% [95% CI: 0-53%] with antifungal therapy vs. 64.0% [95% CI: 45%-80%] without antifungal therapy, p = 0.01).

Discussion: The ABC assay, when combined with BAL GM and fungal culture, significantly improved diagnostic accuracy for IFIs. Further validation of the ABC assay is warranted to optimize its clinical use.