Detection of Aspergillus fumigatus cell-free DNA using Giant Magnetoresistance Technology

Author:

Dr Paschalis Vergidis (UK)

Abstract:

Objectives
The clinical outcomes of invasive pulmonary aspergillosis (IPA) rely on accurate diagnosis and timely initiation of effective antifungal treatment. We aimed to develop a novel fungal pathogen cell-free DNA (cfDNA) detection method using Giant Magnetoresistance (GMR) technology on a Lab-on-a-Chip platform. The technology leverages changes in electrical resistance occurring in the presence of a magnetic field. Magnetic particles are captured by extracted and amplified fungal cfDNA over a GMR sensor. Alterations in magnetoresistance result in a measurable signal.

Methods
The analytical performance of the assay was evaluated in spiked samples. Plasma from healthy humans was used as the sample matrix. For the clinical study, serum was collected from patients diagnosed with IPA and controls diagnosed with invasive candidiasis, cryptococcosis, pneumocystosis and histoplasmosis based on EORTC/MSGERC definitions. Clinical samples were obtained prior to the initiation of antifungal therapy.
Cell-free DNA was extracted and amplified through a multiplex PCR reaction. Amplified and digested single-stranded DNA (ssDNA) was pushed through a microfluidic channel containing a GMR sensor array that can detect 16 different fungal species in duplicates. Each sensor had DNA probes printed and linked to the surface of the GMR sensor for pathogen DNA hybridization. After hybridization, magnetic beads containing streptavidin were bound to the biotin-labeled hybridization complex locking the magnetic bead above the sensor. The magnetic bead linked to the sensor created a change in magnetic resistance, which was measured by the GMR detection system. Change in magnetic resistance was graphed.
To further verify the validity of the signal of specific fungal targets, a melting curve analysis was performed for the hybridized complex after initial signal generation. Mismatched or other nonspecific DNA binding was distinguished from specific binding due to the dissociation from the GMR sensor at a lower temperature.

Results
The assay was highly sensitive and specific. The limit of detection for A. fumigatus and A. terreus was 5 copies/reaction, for A. flavus 25 copies/reaction and for A. niger 50 copies/reaction. Spiked samples with elevated levels of target fungal DNA were analyzed to assess analytical specificity. No false positive signals were detected, indicating no cross-reactivity within the multiplex panel.
In order to evaluate the ability to exclusively detect target DNA without generating false-positive signals, we selected 24 bacterial/fungal microorganisms not included in the panel. No signal was detected for these microorganisms.
For the clinical study, we collected serum samples from 3 patients with IPA and 17 patients with other fungal infections (controls). All IPA cases had growth of A. fumigatus from bronchoalveolar lavage (BAL) fluid and positive BAL Aspergillus galactomannan (index ≥0.5). A. fumigatus DNA was detected in the 3 IPA samples and none of the control samples.

Conclusions
We developed a multiplex PCR assay coupled with GMR technology that can detect fungal cfDNA in serum/plasma. Signals from suboptimal matched probes disappear following thermal stress, leaving only signals from perfectly matched probes to ssDNA. This feature offers an additional verification advantage. The assay demonstrated high sensitivity and specificity and will be further evaluated in future studies.
28.

Abstract Number: 27

Conference Year: 2024

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