Rapid and Reliable Detection of Mucormycosis Using Colorimetric Loop-Mediated Isothermal Amplification (LAMP)

Author:

Yiyou Gu,1 Teclegiorgis Gebremariam,1 Sounds Alkhazraji1 Robina Aerts2,3 Katrien Lagrou3,4 Martin Hoenigl5 Ashraf S. Ibrahim.1,6

Author address:

1. Division of Infectious Diseases, The Lundquist Institute for Biomedical Innovation at Harbor-University of California Los Angeles (UCLA) Medical Center, Torrance, California, USA. 2. Department of Internal Medicine, University Hospitals Leuven, Leuven, Belgium. 3. Department of Microbiology, Immunology and Transplantation, KU Leuven, Leuven, Belgium. 4. Department of Laboratory Medicine, National Reference Center for Mycosis, University Hospitals Leuven, Leuven, Belgium. 5. Division of Infectious Diseases, Department of Medicine, Medical University of Graz, Austria. 6. David Geffen School of Medicine at UCLA, Los Angeles, California, USA

Abstract:

Objectives: Mucormycosis is a life-threatening infection caused by Mucorales fungi. The rapid progression of the disease and the current lack of an early and reliable diagnostic assay contribute to high mortality rates of 50%-100%. Loop-mediated isothermal amplification (LAMP), a nucleic acid amplification technique, offers a rapid, accurate, and cost-effective approach to diagnosing infectious diseases. We sought to investigate the potential use of LAMP in diagnosing mucormycosis.

Methods: We used a pH-sensitive LAMP 2X Master Mix. Upon DNA amplification, the reaction mix undergoes a distinctive color change, from vivid pink to yellow without the need for instrumentation. Initially, the sensitivity and specificity of designed LAMP primers were evaluated using various concentrations of Mucorales DNA spike samples. The optimized primers and incubation timepoint were subsequently employed to detect fungal DNA from bronchoalveolar lavage (BAL) samples of intratracheally infected mice. Confirmation of the assay was done using BAL human samples from mucormycosis patients using the Receiver operating characteristic curve (ROC).

Results: Degenerative primers that were designed to amplify DNA samples from several Mucorales fungi detected DNA concentrations of 0.001pg, 0.01pg, 0.01pg, 0.01pg, and 1pg for Rhizopus delemar, Mucor circinelloides, Cunninghamella, Rhizomucor, and Lichtheimia, respectively. An optimized LAMP primer-162 was used to detect fungal DNA in BAL samples collected from mice infected intratracheally with Mucorales fungi (n=48) or uninfected controls (n=15) showing a sensitivity and specificity of 98%, and 100%, respectively. Using BAL samples from mucormycosis patients (n=22) sensitivity was 82%. Importantly, the assay was negative with BAL samples (n=19) collected from patients with invasive pulmonary aspergillosis except for one sample which showed a weak reaction, resulting in a specificity of 95%, and an AUC of 0.8995.

Conclusions: We present a robust LAMP-based approach that offers speed, reliability, and sensitivity for detecting Mucorales genomic DNA in both experimental and clinical samples. The colorimetric LAMP assay could address the critical need for a rapid and accurate diagnostic tool for mucormycosis.

Abstract Number: 23

Conference Year: 2024

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