Identification of Aspergillus Species by Matrix-Assisted Laser Desorption Ionisation-Time-of-Flight Mass Spectrometry

Abstract

Matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometry (MALDI-TOF MS) has already been used routinely in clinical laboratories for rapid bacterial and yeast identification. However, its routine use for Aspergillus identification is still controversial because of a lack of unification of the culture method and a suboptimal database. In this study, we first examined the difference in using broth and agar plate cultures for growing the aspergilli before protein extraction and found that agar culture was much superior to broth culture in generating interpretable protein mass spectra. Then, based on the solid culture method, we examined the accuracy, with benA and/or CaM sequencing as the identification gold standard, in using MALDI-TOF MS for Aspergillus identification. Overall, the original Bruker Library could only identify 38.2% and 22.8% of the strains characterised at the genus/section and species level, respectively. When the Bruker Library was expanded with reference mass spectra generated from reference Aspergillus species in-house, the identification rates were improved to 61.0% and 48.0%, respectively. The MSI database, freely available online, outperformed the former two libraries for Aspergillus identification at all levels (genus: 95.1%, section: 92.7%, species: 74.8%). Agar plate culture and the MSI database should be used for MALDI-TOF MS identification of aspergilli in clinical laboratories.

Keywords

Aspergillus / rare species / cryptic species / MALDI-TOF MS / identification

Supplementary Materials

The additional data and information can be downloaded at: https://media.sciltp.com/articles/others/2604011410043077/eMicrobe-25110126-SI.pdf. Table S1. Aspergillus strains characterised in this study. Table S2. Detailed MALDI-TOF MS identification results for the Aspergillus strains characterised in this study using different mass spectral libraries/databases.

Author Contributions

Conceptualisation, C.-C.T. and P.C.Y.W.; Data curation, C.-C.T., W.C. and J.Y.M.T.; Formal analysis, C.-C.T., W.C. and J.Y.M.T.; Funding acquisition, C.-C.T., S.K.P.L. and P.C.Y.W.; Investigation, W.C., J.Y.M.T., O.H.Y.C., Y.-W.L., T.W.S.H., T.Z., C.W.H.L., C.Y., Y.-N.T., A.H.Y.N., J.H.K.C., H.Y., F.X., S.K.F.L., C.X., L.H., D.M.W.T., C.K.C.L., A.K.L.W. and T.-L.Q.; Methodology, C.-C.T., W.C. and J.Y.M.T.; Project administration, S.K.P.L. and P.C.Y.W.; Resources, S.K.P.L. and P.C.Y.W.; Supervision, C.-C.T., S.K.P.L. and P.C.Y.W.; Validation, C.-C.T., W.C. and J.Y.M.T.; Visualization, C.-C.T., W.C., J.Y.M.T., S.K.P.L. and P.C.Y.W.; Writing—original draft, C.-C.T., W.C., J.Y.M.T., S.K.P.L. and P.C.Y.W.; Writing—review & editing, C.-C.T., W.C., J.Y.M.T., O.H.Y.C., Y.-W.L., T.W.S.H., T.Z., C.W.H.L., C.L., C.Y., Y.-N.T., A.H.Y.N., J.H.K.C., H.Y., F.X., S.K.F.L., C.X., L.H., D.M.W.T., C.K.C.L., A.K.L.W., T.-L.Q., S.K.P.L. and P.C.Y.W. All authors have read and agreed to the published version of the manuscript.

Funding

This work was partly supported by the Feature Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE-114-S-0023-A) in Taiwan; the Seed Fund for Basic Research, The University of Hong Kong, Hong Kong; as well as the Early Career Researcher Award (2022/2023), Tung Wah College, Hong Kong.

Institutional Review Board Statement

This study was approved by the Institutional Review Board (IRB) of The University of Hong Kong/Hospital Authority Hong Kong West Cluster, Hong Kong East Cluster Research Ethics Committee, the IRB of The University of Hong Kong-Gleaneagles Hospital Hong Kong, the Medical Ethics Committee of The University of Hong Kong-Shenzhen Hospital as well as the Shanghai Jiaotong University School of Medicine Ruijin Hospital Ethics Committee.

Informed Consent Statement

Patient consent was waived since only leftover microorganisms isolated from clinical specimens were included in this study. No patient intervention was involved, and no patient information was collected.

Data Availability Statement

Nucleotide sequences generated in this study were deposited to the DDBJ/ENA/GenBank databases with nucleotide accession numbers listed in Table S1.

Acknowledgements

We thank the curators, especially Travis W. Adkins, of NRRL for providing the NRRL reference strains for free.

Conflicts of Interest

Patrick C.Y. Woo has provided scientific advisory/laboratory services for Gilead Sciences, Incorporated; International Health Management Associates, Incorporated; Merck & Corporation, Incorporated, Micología Molecular S.L. and Pfizer, Incorporated. Christopher K. C. Lai has provided scientific advisory services from GenMark Diagnostics. The other authors report no conflicts of interest. The funding sources had no role in study design, data collection, analysis, interpretation, or writing of the report.

Given the role as Editor-in-Chief, Patrick C.Y. Woo had no involvement in the peer review of this paper and had no access to information regarding its peer-review process. Full responsibility for the editorial process of this paper was delegated to another editor of the journal. The authors alone are responsible for the content and the writing of the manuscript.

Use of AI and AI-Assisted Technologies

No AI tools were utilised for this paper.

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