Abstract
In this study, we devised a diagnostic platform harnessing a combination of recombinase polymerase amplification (RPA) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system. Notably, this platform obviates the need for intricate equipment and finds utility in diverse settings. Two result display methods were incorporated in this investigation: the RPA-Cas12a-fluorescence method and the RPA-Cas12a-LFS (lateral flow strip). Upon validation, both display platforms exhibited no instances of cross-reactivity, with seven additional types of fungal pathogens responsible for respiratory infections. The established detection limit was ascertained to be as low as 102 copies/µL. In comparison to fluorescence quantitative PCR, the platform demonstrated a sensitivity of 96.7%, a specificity of 100%, and a consistency rate of 98.0%.This platform provides expeditious, precise, and on-site detection capabilities, thereby rendering it a pivotal diagnostic instrument amenable for deployment in primary healthcare facilities and point-of-care settings.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data availability
Data is provided within the manuscript or supplementary information files.
References
Alanio A, Dellière S, Fodil S, Bretagne S, Mégarbane B (2020) Prevalence of putative invasive pulmonary aspergillosis in critically ill patients with COVID-19. The Lancet. Respir Med 8(6):e48–e9. https://doi.org/10.1016/s2213-2600(20)30237-x
Balajee SA, Houbraken J, Verweij PE et al (2007) Aspergillus species identification in the clinical setting. Stud Mycol 59:39–46. https://doi.org/10.3114/sim.2007.59.05
Chen M, Xu Y, Hong N et al (2018) Epidemiology of fungal infections in China. Front Med 12(1):58–75. https://doi.org/10.1007/s11684-017-0601-0
Choi JR, Hu J, Tang R et al (2016) An integrated paper-based sample-to-answer biosensor for nucleic acid testing at the point of care. Lab Chip 16(3):611–621. https://doi.org/10.1039/c5lc01388g
Daher RK, Stewart G, Boissinot M, Bergeron MG (2016) Recombinase polymerase amplification for diagnostic applications. Clin Chem 62(7):947–958. https://doi.org/10.1373/clinchem.2015.245829
Douglas AP, Smibert OC, Bajel A et al (2021) Consensus guidelines for the diagnosis and management of invasive aspergillosis, 2021. Intern Med J 51(Suppl 7):143–176. https://doi.org/10.1111/imj.15591
Henneberg S, Hasenberg A, Maurer A et al (2021) Antibody-guided in vivo imaging of aspergillus fumigatus lung infections during antifungal azole treatment. Nat Commun 12(1):1707. https://doi.org/10.1038/s41467-021-21965-z
Hope WW, Walsh TJ, Denning DW (2005) Laboratory diagnosis of invasive aspergillosis. The Lancet. Infect Dis 5(10):609–622. https://doi.org/10.1016/s1473-3099(05)70238-3
Jiang T, Hu X, Shen J (2023) Establishment of a Novel detection platform for Clostridioides difficile toxin genes based on Orthogonal CRISPR. Microbiol Spectr 11(4):e0188623. https://doi.org/10.1128/spectrum.01886-23
Lamoth F, Calandra T (2022) Pulmonary aspergillosis: diagnosis and treatment. Eur Respiratory Review: Official J Eur Respiratory Soc 31(166). https://doi.org/10.1183/16000617.0114-2022
Ledoux MP, Guffroy B, Nivoix Y, Simand C, Herbrecht R (2020) Invasive pulmonary aspergillosis. Seminars in respiratory and critical care medicine. 41(1):80–98. https://doi.org/10.1055/s-0039-3401990
Lee N, Kym D (2022) Clinical usefulness of serum (1,3)-β-D-glucan to predict invasive candidiasis in patients with severe burn trauma. Journal of microbiology, immunology, and infection = Wei Mian Yu Gan ran Za Zhi. 55(1):138–146. https://doi.org/10.1016/j.jmii.2021.02.004
Lei R, Li L, Wu P et al (2022) RPA/CRISPR/Cas12a-Based On-Site and Rapid Nucleic Acid Detection of Toxoplasma Gondii in the Environment. ACS Synth Biol 11(5):1772–1781. https://doi.org/10.1021/acssynbio.1c00620
Leonardelli F, Macedo D, Dudiuk C, Cabeza MS, Gamarra S, Garcia-Effron G (2016) Aspergillus Fumigatus intrinsic fluconazole resistance is due to the naturally occurring T301I substitution in Cyp51Ap. Antimicrob Agents Chemother 60(9):5420–5426. https://doi.org/10.1128/aac.00905-16
Li J, Macdonald J, von Stetten F (2018) Review: a comprehensive summary of a decade development of the recombinase polymerase amplification. Analyst 144(1):31–67. https://doi.org/10.1039/c8an01621f
Luptáková D, Patil RH, Dobiáš R et al (2023) Siderophore-based noninvasive differentiation of aspergillus fumigatus colonization and Invasion in Pulmonary aspergillosis. Microbiol Spectr 11(2):e0406822. https://doi.org/10.1128/spectrum.04068-22
Malhotra S, Kumari R, Chauhan AK et al (2021) Diagnostic value of galactomannan antigen test in serum and bronchoalveolar lavage fluid sample from suspected patients of invasive pulmonary aspergillosis. Indian J Pathol Microbiol 64(4):732–734. https://doi.org/10.4103/ijpm.Ijpm_985_20
Meersseman W, Vandecasteele SJ, Wilmer A, Verbeken E, Peetermans WE, Van Wijngaerden E (2004) Invasive aspergillosis in critically ill patients without malignancy. Am J Respir Crit Care Med 170(6):621–625. https://doi.org/10.1164/rccm.200401-093OC
Moura S, Cerqueira L, Almeida A (2018) Invasive pulmonary aspergillosis: current diagnostic methodologies and a new molecular approach. European journal of clinical microbiology & infectious diseases: official publication of the European Society of Clinical Microbiology. 37(8):1393–1403. https://doi.org/10.1007/s10096-018-3251-5
Pfister J, Summer D, Petrik M et al (2020) Hybrid imaging of Aspergillus Fumigatus Pulmonary infection with fluorescent, (68)Ga-Labelled siderophores. Biomolecules 10(2). https://doi.org/10.3390/biom10020168
Prattes J, Valentin T, Hoenigl M, Talakic E, Reisinger AC, Eller P (2021) Invasive pulmonary aspergillosis complicating COVID-19 in the ICU - a case report. Med Mycol case Rep 31:2–5. https://doi.org/10.1016/j.mmcr.2020.05.001
Slobbe L, Polinder S, Doorduijn JK et al (2008) Outcome and medical costs of patients with invasive aspergillosis and acute myelogenous leukemia-myelodysplastic syndrome treated with intensive chemotherapy: an observational study. Clin Infect Dis 47(12):1507–1512. https://doi.org/10.1086/591531
Soubani AO, Khanchandani G, Ahmed HP (2004) Clinical significance of lower respiratory tract aspergillus culture in elderly hospitalized patients. European journal of clinical microbiology & infectious diseases: official publication of the European Society of Clinical Microbiology. 23(6):491–494. https://doi.org/10.1007/s10096-004-1137-1
Walsh TJ, Anaissie EJ, Denning DW et al (2008) Treatment of aspergillosis: clinical practice guidelines of the Infectious Diseases Society of America. Clin Infect Dis 46(3):327–360. https://doi.org/10.1086/525258
Xiong D, Dai W, Gong J et al (2020) Rapid detection of SARS-CoV-2 with CRISPR-Cas12a. PLoS Biol 18(12):e3000978. https://doi.org/10.1371/journal.pbio.3000978
Zhao Y, Chen D, Xu Z et al (2023) Integrating CRISPR-Cas12a into a Microfluidic Dual-Droplet device enables simultaneous detection of HPV16 and HPV18. Anal Chem 95(6):3476–3485. https://doi.org/10.1021/acs.analchem.2c05320
Funding
This study was supported by 2020 Anhui Provincial University Cooperative Research and Public Health Collaborative Innovation Project of Anhui Provincial Department of Education (Grant No. GXXT-2020-016), 2021 Anhui Provincial Medical and Health Key Specialty Construction Project (Approval number: serial number 95), 2021 Anhui Provincial Key Project of Natural Science Research in Colleges and Universities (Approval number: KJ2021ZD0032), Research Fund of Anhui Institute of translational medicine(Approval number:2022zhyx-C61), Research Fund of Anhui Institute of translational medicine(2023zhyx-C99).
Author information
Authors and Affiliations
Contributions
C.L: Conceptualization, methodology, formal analysis, writing— original draft. J.Z: Investigation, data curation; N.G: Validation ; R.L: Investigation.G.L: Data Curation. J.W:Sample collection; G.L:Funding; J.S: Writing— review and editing, supervision, project administration, funding.All authors reviewed the manuscript.
Corresponding author
Ethics declarations
Ethics approval and consent to participate
The study involved the anonymous use of excess, discarded BAL, clinical fungal isolate samples as part of standard care, and the study did not affect patient health and privacy. All procedures were performed in accordance with the ethical standards of medical ethics in research involving human subjects, the Fourth Affiliated Hospital Committee of Anhui Medical University (reference number: LLSC20210802) and the 1964 Helsinki Declaration and its later amendments or similar ethical standards.
Competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Additional information
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Lin, C., Zhou, J., Gao, N. et al. Establishing a pulmonary aspergillus fumigatus infection diagnostic platform based on RPA-CRISPR-Cas12a. World J Microbiol Biotechnol 40, 116 (2024). https://doi.org/10.1007/s11274-024-03940-0
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s11274-024-03940-0