Hygiene and SanitationHygiene and SanitationГигиена и санитария0016-99002412-0650Federal Scientific Center of Hygiene named after F.F. Erisman68060410.47470/0016-9900-2025-104-4-503-509rmxyecMETHODS OF HYGIENIC AND EXPERIMENTAL INVESTIGATIONSМЕТОДЫ ГИГИЕНИЧЕСКИХ ИССЛЕДОВАНИЙReview ArticleIdentification of filamentous fungi with MALDI-TOF: methods and perspectives (literature review)Идентификация плесневых грибов методом MALDI-TOF: подходы и перспективы (обзор литературы)KurbatovaIrina V.КурбатоваИрина ВалентиновнаIKurbatova@cspfmba.ruRakitinaDaria V.РакитинаДарья ВикторовнаRakitina@cspmz.ruCentre for Strategic Planning of the Federal medical and biological agencyФГБУ «Центр стратегического планирования и управления медико-биологическими рисками здоровью» Федерального медико-биологического агентства150420251044VOL 104, NO4 (2025)ТОМ 104, №4 (2025)503509260520252025https://journals.eco-vector.com/0016-9900/article/view/680604

This review addresses various attempts of adaptation of MALDI-TOF method for identification of filamentous fungi. This method has become a golden standard in laboratory diagnostics. Accuracy, simplicity, and low cost per analysis promote making it a widespread technology. However, attempts of applying it to filamentous fungi face many problems. Solid cell wall of fungi mycelium is an obstacle to cell lysis and protein ionization. Heterogeneity of cells in culture – old and young hyphs, conidia etc, provides variety in ion patterns and prevents forming single reproducible spectra for the analyzed isolate. These issues are addressed by various optimizations in methods of cultivation, sampling, protein extraction, spectra recording, and data analysis. Commercial systems exist, offering ready solutions of the problem; however, these systems also require adjustments to be effective in the clinical laboratory practice.Contribution of the authors: Kurbatova I.V. – concept and design of the study, editing; Rakitina D.V. – literature search, writing. All authors are responsible for the integrity of all parts of the manuscript and approval of the manuscript final versionConflict of interest. The authors declare no conflict of interest.Acknowledgment. The research was carried out within the framework of the research work “Development of unified methods, including sampling, for the determination of microbiological and parasitological contamination of wastewater” (code “Wastewater”).Received: August 27, 2024 / Accepted: December 3, 2024 / Published: April 30, 2025

В обзоре систематизированы представленные в литературе сведения об адаптации метода MALDI-TOF к идентификации плесневых грибов. Масс-спектрометрическая идентификация бактерий методом MALDI-TOF является «золотым стандартом» в современной лабораторной диагностике. Точность, скорость, простота и невысокая стоимость одного анализа обеспечивают всё более широкое распространение этого метода. Однако применение технологии MALDI-TOF к другим микроорганизмам, в том числе плесневым грибам, затруднено, поскольку прочная стенка клеток грибного мицелия препятствует клеточному лизису и ионизации белков, а разнообразие клеток в культуре (гифы, споры, органы размножения) приводит к разнообразию ионов белков и усложняет получение единого воспроизводимого спектра для изолята. Для преодоления этих трудностей применяются различные методы культивирования, подготовки образцов, получения и обработки спектров. На рынке присутствуют готовые решения в этой области в виде коммерческих систем, однако и они требуют доработки.Участие авторов: Курбатова И.В. – концепция обзора, редактирование; Ракитина Д.В. – подбор литературы, написание текста. Все соавторы – утверждение окончательного варианта статьи и ответственность за целостность всех её частей.Конфликт интересов. Авторы декларируют отсутствие явных и потенциальных конфликтов интересов в связи с публикацией данной статьи.Финансирование. Исследование проведено в рамках НИР по теме «Разработка унифицированных методов, включающих отбор 8807 проб, для осуществления определения микробиологического и паразитологического загрязнения сточных вод» (шифр «Сточные воды»).Поступила: 27.08.2024 / Принята к печати: 03.12.2024 / Опубликована: 30.04.2025

MALDI-TOFmass-spectrometryMALDI-TOFfilamentous fungireviewмасс-спектрометрияплесневые грибыобзорNenoff P., Erhard M., Simon J.C., Muylowa G.K., Herrmann J., Rataj W., et al. MALDI-TOF mass spectrometry – a rapid method for the identification of dermatophyte species. Med. Mycol. 2013; 51(1): 17–24. https://doi.org/10.3109/13693786.2012.685186Becker P., Normand A.C., Vanantwerpen G., Vanrobaeys M., Haesendonck R., Vercammen F., et al. Identification of fungal isolates by MALDI-TOF mass spectrometry in veterinary practice: validation of a web application. J. Vet. Diagn. Invest. 2019; 31(3): 471–4. https://doi.org/10.1177/1040638719835577Giordano A.L.P.L., Pontes L., Beraquet C.A.G., Lyra L., Schreiber A.Z. Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry azole susceptibility assessment in Candida and Aspergillus species. Mem. Inst. Oswaldo Cruz. 2023; 118: e220213. https://doi.org/10.1590/0074-02760220213Reeve M.A., Buddie A.G., Pollard K.M., Varia S., Seier M.K., Offord L.C., et al. A highly-simplified and inexpensive MALDI-TOF mass spectrometry sample-preparation method with broad applicability to microorganisms, plants, and insects. J. Biol. Methods. 2018; 5(4): e103. https://doi.org/10.14440/jbm.2018.261Sanguinetti M., Posteraro B. Identification of molds by matrix-assisted laser desorption ionization-time of flight mass spectrometry. J. Clin. Microbiol. 2017; 55(2): 369–79. https://doi.org/10.1128/JCM.01640-16Coulibaly O., Marinach-Patrice C., Cassagne C., Piarroux R., Mazier D., Ranque S. Pseudallescheria/Scedosporium complex species identification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Med. Mycol. 2011; 49(6): 621–6. https://doi.org/10.3109/13693786.2011.555424Reeve M.A., Bachmann D. MALDI-TOF MS protein fingerprinting of mixed samples. Biol. Methods. Protoc. 2019; 4(1): bpz013. https://doi.org/10.1093/biomethods/bpz013Buskirk A.D., Hettick J.M., Chipinda I., Law B.F., Siegel P.D., Slaven J.E., et al. Fungal pigments inhibit the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of darkly pigmented fungi. Anal. Biochem. 2011; 411(1): 122–8. https://doi.org/10.1016/j.ab.2010.11.025Kondori N., Erhard M., Welinder-Olsson C., Groenewald M., Verkley G., Moore E.R. Analyses of black fungi by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS): species-level identification of clinical isolates of Exophiala dermatitidis. FEMS Microbiol. Lett. 2015; 362(1): 1–6. https://doi.org/10.1093/femsle/fnu016Lau A.F., Drake S.K., Calhoun L.B., Henderson C.M., Zelazny A.M. Development of a clinically comprehensive database and a simple procedure for identification of molds from solid media by matrix-assisted laser desorption ionization-time of flight mass spectrometry. J. Clin. Microbiol. 2013; 51(3): 828–34. https://doi.org/10.1128/JCM.02852-12Honsig C., Selitsch B., Hollenstein M., Vossen M.G., Spettel K., Willinger B. Identification of filamentous fungi by MALDI-TOF mass spectrometry: evaluation of three different sample preparation methods and validation of an in-house species cutoff. J. Fungi (Basel). 2022; 8(4): 383. https://doi.org/10.3390/jof8040383Ao K., Li X., Zhang W., Chen Z., Liu Y., Shu L., et al. Evaluation of the Autof ms1000 mass spectrometry for rapid clinical identification of filamentous fungi. BMC Microbiol. 2023; 23(1): 228. https://doi.org/10.1186/s12866-023-02968-wBarker K.R., Kus J.V., Normand A.C., Gharabaghi F., McTaggart L., Rotstein C., et al. A practical workflow for the identification of Aspergillus, Fusarium, Mucorales by MALDI-TOF MS: database, medium, and incubation optimization. J. Clin. Microbiol. 2022; 60(12): e0103222. https://doi.org/10.1128/jcm.01032-22Nellessen C.M., Nehl D.B. An easy adjustment of instrument settings (‘Peak MALDI’) improves identification of organisms by MALDI-TOF mass spectrometry. Sci. Rep. 2023; 13(1): 15018. https://doi.org/10.1038/s41598-023-42328-2Fissel J.A., Holdren-Serrell C.K., Memon W., Zhang S.X. Performance of a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry testing algorithm for the rapid identification of clinical filamentous molds. Front. Cell. Infect. Microbiol. 2022; 12: 915049. https://doi.org/10.3389/fcimb.2022.915049Gómez-Velásquez J.C., Loaiza-Díaz N., Norela Hernández G., Lima N., Mesa-Arango A.C. Development and validation of an in-house library for filamentous fungi identification by MALDI-TOF MS in a clinical laboratory in Medellin (Colombia). Microorganisms. 2020; 8(9): 1362. https://doi.org/10.3390/microorganisms8091362Stein M., Tran V., Nichol K.A., Lagacé-Wiens P., Pieroni P., Adam H.J., et al. Evaluation of three MALDI-TOF mass spectrometry libraries for the identification of filamentous fungi in three clinical microbiology laboratories in Manitoba, Canada. Mycoses. 2018; 61(10): 743–53. https://doi.org/10.1111/myc.12800Zvezdanova M.E., Escribano P., Ruiz A., Martínez-Jiménez M.C., Peláez T., Collazos A., et al. Increased species-assignment of filamentous fungi using MALDI-TOF MS coupled with a simplified sample processing and an in-house library. Med. Mycol. 2019; 57(1): 63–70. https://doi.org/10.1093/mmy/myx154Normand A.C., Becker P., Gabriel F., Cassagne C., Accoceberry I., Gari-Toussaint M., et al. Validation of a new web application for identification of fungi by use of matrix-assisted laser desorption ionization-time of flight mass spectrometry. J. Clin. Microbiol. 2017; 55(9): 2661–70. https://doi.org/10.1128/JCM.00263-17Heireman L., Patteet S., Steyaert S. Performance of the new ID-fungi plate using two types of reference libraries (Bruker and MSI) to identify fungi with the Bruker MALDI Biotyper. Med. Mycol. 2020; 58(7): 946–57. https://doi.org/10.1093/mmy/myz138Jeraldine V.M., Wim L., Ellen V.E. A comparative study for optimization of MALDI-TOF MS identification of filamentous fungi. Eur. J. Clin. Microbiol. Infect. Dis. 2023; 42(10): 1153–61. https://doi.org/10.1007/s10096-023-04652-3Robert M.G., Romero C., Dard C., Garnaud C., Cognet O., Girard T., et al. Evaluation of ID fungi plates medium for identification of molds by MALDI Biotyper. J. Clin. Microbiol. 2020; 58(5): e01687-19. https://doi.org/10.1128/JCM.01687-19Becker P.T., de Bel A., Martiny D., Ranque S., Piarroux R., Cassagne C., et al. Identification of filamentous fungi isolates by MALDI-TOF mass spectrometry: clinical evaluation of an extended reference spectra library. Med. Mycol. 2014; 52(8): 826–34. https://doi.org/10.1093/mmy/myu064Riat A., Hinrikson H., Barras V., Fernandez J., Schrenzel J. Confident identification of filamentous fungi by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry without subculture-based sample preparation. Int. J. Infect. Dis. 2015; 35: 43–5. https://doi.org/10.1016/j.ijid.2015.04.013Wilkendorf L.S., Bowles E., Buil J.B., van der Lee H.A.L., Posteraro B., Sanguinetti M., et al. Update on matrix-assisted laser desorption ionization-time of flight mass spectrometry identification of filamentous fungi. J. Clin. Microbiol. 2020; 58(12): e01263-20. https://doi.org/10.1128/JCM.01263-20Schulthess B., Ledermann R., Mouttet F., Zbinden A., Bloemberg G.V., Böttger E.C., et al. Use of the Bruker MALDI Biotyper for identification of molds in the clinical mycology laboratory. J. Clin. Microbiol. 2014; 52(8): 2797–803. https://doi.org/10.1128/JCM.00049-14Vidal-Acuña M.R., Ruiz-Pérez de Pipaón M., Torres-Sánchez M.J., Aznar J. Identification of clinical isolates of Aspergillus, including cryptic species, by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Med. Mycol. 2018; 56(7): 838–46. https://doi.org/10.1093/mmy/myx115Paul S., Singh P., Rudramurthy S.M., Chakrabarti A., Ghosh A.K. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry: protocol standardization and database expansion for rapid identification of clinically important molds. Future Microbiol. 2017; 12: 1457–66. https://doi.org/10.2217/fmb-2017-0105Li Y., Wang H., Hou X., Huang J.J., Wang P.C., Xu Y.C. Identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry and antifungal susceptibility testing of non-aspergillus molds. Front. Microbiol. 2020; 11: 922. https://doi.org/10.3389/fmicb.2020.00922Park J.H., Shin J.H., Choi M.J., Choi J.U., Park Y.J., Jang S.J., et al. Evaluation of matrix-assisted laser desorption/ionization time-of-fight mass spectrometry for identification of 345 clinical isolates of Aspergillus species from 11 Korean hospitals: comparison with molecular identification. Diagn. Microbiol. Infect. Dis. 2017; 87(1): 28–31. https://doi.org/10.1016/j.diagmicrobio.2016.10.012Peng Y., Zhang Q., Xu C., Shi W. MALDI-TOF MS for the rapid identification and drug susceptibility testing of filamentous fungi. Exp. Ther. Med. 2019; 18(6): 4865–73. https://doi.org/10.3892/etm.2019.8118Lecerf P., De Paepe R., Jazaeri Y., Normand A.C., Martiny D., Packeu A. Evaluation of a Liquid Media MALDI-TOF MS Protocol for the Identification of Dermatophytes Isolated from Tinea capitis Infections. J. Fungi (Basel). 2022; 8(12): 1248. https://doi.org/10.3390/jof8121248Рябинин И.А., Васильева Н.В., Борзова Ю.В. Характеристика сериновой протеазы Sуnсерhаlаstrum rасеmоsum Cohn, 1886. Проблемы медицинской микологии. 2020; 22(2): 50–5. https://doi.org/10.24412/1999-6780-2020-2-50-55 https://elibrary.ru/ojvpisCassagne C., Normand A.C., L’Ollivier C., Ranque S., Piarroux R. Performance of MALDI-TOF MS platforms for fungal identification. Mycoses. 2016; 59(11): 678–90. https://doi.org/10.1111/myc.12506Calderaro A., Motta F., Montecchini S., Gorrini C., Piccolo G., Piergianni M., et al. Identification of Dermatophyte species after implementation of the in-house MALDI-TOF MS database. Int. J. Mol. Sci. 2014; 15(9): 16012–24. https://doi.org/10.3390/ijms150916012Wilson D.A., Young S., Timm K., Novak-Weekley S., Marlowe E.M., Madisen N., et al. Multicenter evaluation of the Bruker MALDI Biotyper CA system for the identification of clinically important bacteria and yeasts. Am. J. Clin. Pathol. 2017; 147(6): 623–31. https://doi.org/10.1093/ajcp/aqw225Lopes R.B., Faria M., Souza D.A., Bloch C. Jr., Silva L.P., Humber R.A. MALDI-TOF mass spectrometry applied to identifying species of insect-pathogenic fungi from the Metarhizium anisopliae complex. Mycologia. 2014; 106(4): 865–78. https://doi.org/10.3852/13-401Ning Y.T., Yang W.H., Zhang W., Xiao M., Wang Y., Zhang J.J., et al. Developing two rapid protein extraction methods using focused-ultrasonication and zirconia-silica beads for filamentous fungi identification by MALDI-TOF MS. Front. Cell. Infect. Microbiol. 2021; 11: 687240. https://doi.org/10.3389/fcimb.2021.687240Rychert J., Slechta E.S., Barker A.P., Miranda E., Babady N.E., Tang Y.W., et al. Multicenter evaluation of the Vitek MS v3.0 system for the identification of filamentous fungi. J. Clin. Microbiol. 2018; 56(2): e01353–17. https://doi.org/10.1128/JCM.01353-17Huang Y., Zhang M., Zhu M., Wang M., Sun Y., Gu H., et al. Comparison of two matrix-assisted laser desorption ionization-time of flight mass spectrometry systems for the identification of clinical filamentous fungi. World J. Microbiol. Biotechnol. 2017; 33(7): 142. https://doi.org/10.1007/s11274-017-2297-3McMullen A.R., Wallace M.A., Pincus D.H., Wilkey K., Burnham C.A. Evaluation of the Vitek MS matrix-assisted laser desorption ionization-time of flight mass spectrometry system for identification of clinically relevant filamentous fungi. J. Clin. Microbiol. 2016; 54(8): 2068–73. https://doi.org/10.1128/JCM.00825-16Pinheiro D., Monteiro C., Faria M.A., Pinto E. Vitek® MS v3.0 system in the identification of filamentous fungi. Mycopathologia. 2019; 184(5): 645–51. https://doi.org/10.1007/s11046-019-00377-0Bille E., Dauphin B., Leto J., Bougnoux M.E., Beretti J.L., Lotz A., et al. MALDI-TOF MS Andromas strategy for the routine identification of bacteria, mycobacteria, yeasts, Aspergillus spp. and positive blood cultures. Clin. Microbiol. Infect. 2012; 18(11): 1117–25. https://doi.org/10.1111/j.1469-0691.2011.03688.xSingh A., Singh P.K., Kumar A., Chander J., Khanna G., Roy P., et al. Molecular and matrix-assisted laser desorption ionization-time of flight mass spectrometry-based characterization of clinically significant melanized fungi in India. J. Clin. Microbiol. 2017; 55(4): 1090–103. https://doi.org/10.1128/JCM.02413-16Paul S., Singh P., Sharma S., Prasad G.S., Rudramurthy S.M., Chakrabarti A., et al. MALDI-TOF MS-based identification of melanized fungi is faster and reliable after the expansion of in-house database. Proteomics. Clin. Appl. 2019; 13(3): e1800070. https://doi.org/10.1002/prca.201800070Lévesque S., Dufresne P.J., Soualhine H., Domingo M.C., Bekal S., Lefebvre B., et al. A side by side comparison of Bruker Biotyper and Vitek MS: Utility of MALDI-TOF MS technology for microorganism identification in a public health reference laboratory. PLoS One. 2015; 10(12): e0144878. https://doi.org/10.1371/journal.pone.0144878Hankins J.D., Amerson-Brown M.H., Brown C.A., Riegler A.N., Muldrew K.L., Dunn J.J. Comparison of Bruker Biotyper® and Vitek® MS matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry platforms for the identification of filamentous fungi. Future Microbiol. 2023; 18: 553–61. https://doi.org/10.2217/fmb-2023-0084Lau A.F., Walchak R.C., Miller H.B., Slechta E.S., Kamboj K., Riebe K., et al. Multicenter study demonstrates standardization requirements for mold identification by MALDI-TOF MS. Front. Microbiol. 2019; 10: 2098. https://doi.org/10.3389/fmicb.2019.02098Quéro L., Girard V., Pawtowski A., Tréguer S., Weill A., Arend S., et al. Development and application of MALDI-TOF MS for identification of food spoilage fungi. Food Microbiol. 2019; 81: 76–88. https://doi.org/10.1016/j.fm.2018.05.001Américo M.F., Machado Siqueira P.L., Del Negro B.G.M., Favero Gimenes M.V., Trindade S.M.R., Motta L.A., et al. Evaluating VITEK MS for the identification of clinically relevant Aspergillus species. Med. Mycol. 2020; 58(3): 322–7. https://doi.org/10.1093/mmy/myz066De Respinis S., Monnin V., Girard V., Welker M., Arsac M., Cellière B., et al. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry using the Vitek MS system for rapid and accurate identification of dermatophytes on solid cultures. J. Clin. Microbiol. 2014; 52(12): 4286–92. https://doi.org/10.1128/JCM.02199-14Karabıçak N., Karatuna O., İlkit M., Akyar I. Evaluation of the Bruker matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) system for the identification of clinically important dermatophyte species. Mycopathologia. 2015; 180(3–4): 165–71. https://doi.org/10.1007/s11046-015-9898-xda Cunha K.C., Riat A., Normand A.C., Bosshard P.P., de Almeida M.T.G., Piarroux R., et al. Fast identification of dermatophytes by MALDI-TOF/MS using direct transfer of fungal cells on ground steel target plates. Mycoses. 2018; 61(9): 691–7. https://doi.org/10.1111/myc.12793Triest D., Stubbe D., De Cremer K., Piérard D., Normand A.C., Piarroux R., et al. Use of matrix-assisted laser desorption ionization-time of flight mass spectrometry for identification of molds of the Fusarium genus. J. Clin. Microbiol. 2015; 53(2): 465–76. https://doi.org/10.1128/JCM.02213-14Dolatabadi S., Kolecka A., Versteeg M., de Hoog S.G., Boekhout T. Differentiation of clinically relevant Mucorales Rhizopus microsporus and R. arrhizus by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). J. Med. Microbiol. 2015; 64(7): 694–701. https://doi.org/10.1099/jmm.0.000091Packeu A., De Bel A., l’Ollivier C., Ranque S., Detandt M., Hendrickx M. Fast and accurate identification of dermatophytes by matrix-assisted laser desorption ionization-time of flight mass spectrometry: validation in the clinical laboratory. J. Clin. Microbiol. 2014; 52(9): 3440–3. https://doi.org/10.1128/JCM.01428-14Shao J., Wan Z., Li R., Yu J. Species identification and delineation of pathogenic mucorales by matrix-assisted laser desorption ionization-time of flight mass spectrometry. J. Clin. Microbiol. 2018; 56(4): e01886-17. https://doi.org/10.1128/JCM.01886-17Sleiman S., Halliday C.L., Chapman B., Brown M., Nitschke J., Lau A.F., et al. Performance of matrix-assisted laser desorption ionization-time of flight mass spectrometry for identification of Aspergillus, Scedosporium, and Fusarium spp. in the Australian Clinical Setting. J. Clin. Microbiol. 2016; 54(8): 2182–6. https://doi.org/10.1128/JCM.00906-16