Discrimination of Aspergillus lentulus from Aspergillus fumigatus by Raman spectroscopy and MALDI-TOF MS

  • P. E. B. Verwer
  • W. B. van Leeuwen
  • V. Girard
  • V. Monnin
  • A. van Belkum
  • J. F. Staab
  • H. A. Verbrugh
  • I. A. J. M. Bakker-Woudenberg
  • W. W. J. van de Sande


In 2005, a new sibling species of Aspergillus fumigatus was discovered: Aspergillus lentulus. Both species can cause invasive fungal disease in immune-compromised patients. The species are morphologically very similar. Current techniques for identification are PCR-based or morphology-based. These techniques are labour-intense and not sufficiently discriminatory. Since A. lentulus is less susceptible to several antifungal agents, it is important to correctly identify the causative infectious agent in order to optimize antifungal therapy. In this study we determined whether Raman spectroscopy and/or MALDI-TOF MS were able to differentiate between A. lentulus and A. fumigatus. For 16 isolates of A. lentulus and 16 isolates of A. fumigatus, Raman spectra and peptide profiles were obtained using the Spectracell and MALDI-TOF MS (VITEK MS RUO, bioMérieux) respectively. In order to obtain reliable Raman spectra for A. fumigatus and A. lentulus, the culture medium needed to be adjusted to obtain colourless conidia. Only Raman spectra obtained from colourless conidia were reproducible and correctly identified 25 out of 32 (78 %) of the Aspergillus strains. For VITEK MS RUO, no medium adjustments were necessary. Pigmented conidia resulted in reproducible peptide profiles as well in this case. VITEK MS RUO correctly identified 100 % of the Aspergillus isolates, within a timeframe of approximately 54 h including culture. Of the two techniques studied here, VITEK MS RUO was superior to Raman spectroscopy in the discrimination of A. lentulus from A. fumigatus. VITEK MS RUO seems to be a successful technique in the daily identification of Aspergillus spp. within a limited timeframe.


Voriconazole Fungal Isolate Caspofungin Aspergillus Fumigatus Raman Spectroscopy 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Conflict of interest

AB, VG and VM are employees of BioMérieux. None of them had any influence on the outcome of experiments or on the interpretation of results.


  1. 1.
    Fera MT, La Camera E, De Sarro A (2009) New triazoles and echinocandins: mode of action, in vitro activity and mechanisms of resistance. Expert Rev Anti Infect Ther 7:981–998PubMedCrossRefGoogle Scholar
  2. 2.
    Ameen M (2010) Epidemiology of superficial fungal infections. Clin Dermatol 28:197–201PubMedCrossRefGoogle Scholar
  3. 3.
    Pfaller MA, Diekema DJ (2010) Epidemiology of invasive mycoses in North America. Crit Rev Microbiol 36:1–53PubMedCrossRefGoogle Scholar
  4. 4.
    Vazquez JA (2010) Invasive fungal infections in the intensive care unit. Semin Respir Crit Care Med 31:79–86PubMedCrossRefGoogle Scholar
  5. 5.
    De Hoog GS, Cuarro GJ, Figueras MJ (2000) Atlas of clinical fungi, 2nd edn. ASM Press, Washington DCGoogle Scholar
  6. 6.
    Balajee SA, Nickle D, Varga J, Marr KA (2006) Molecular studies reveal frequent misidentification of Aspergillus fumigatus by morphotyping. Eukaryot Cell 5:1705–1712PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Balajee SA, Gribskov JL, Hanley E, Nickle D, Marr KA (2005) Aspergillus lentulus sp. nov., a new sibling species of A. fumigatus. Eukaryot Cell 4:625–632PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Mellado E, Alcazar-Fuoli L, Cuenca-Estrella M, Rodriguez-Tudela JL (2011) Role of Aspergillus lentulus 14-alpha sterol demethylase (Cyp51A) in azole drug susceptibility. Antimicrob Agents Chemother 55:5459–5468PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Balajee SA, Weaver M, Imhof A, Gribskov J, Marr KA (2004) Aspergillus fumigatus variant with decreased susceptibility to multiple antifungals. Antimicrob Agents Chemother 48:1197–1203PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Alcazar-Fuoli L, Mellado E, Alastruey-Izquierdo A, Cuenca-Estrella M, Rodriguez-Tudela JL (2008) Aspergillus section Fumigati: antifungal susceptibility patterns and sequence-based identification. Antimicrob Agents Chemother 52:1244–1251PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Van de Sande WW, Mathot RA, ten Kate MT, van Vianen W, Tavakol M, Rijnders BJ, Bakker-Woudenberg IA (2005) Combination therapy of advanced invasive pulmonary aspergillosis in transiently neutropenic rats using human pharmacokinetic equivalent doses of voriconazole and anidulafungin. Antimicrob Agents Chemother 53:2005–2013CrossRefGoogle Scholar
  12. 12.
    Willemse-Erix DF, Scholtes-Timmerman MJ, Jachtenberg JW, van Leeuwen WB, Horst-Kreft D, Bakker Schut TC, Deurenberg RH, Puppels GJ, van Belkum A, Vos MC, Maquelin K (2009) Optical fingerprinting in bacterial epidemiology: Raman spectroscopy as a real-time typing method. J Clin Microbiol 47:652–659PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Buijtels PC, Willemse-Erix HF, Petit PL, Endtz HP, Puppels GJ, Verbrugh HA, van Belkum A, van Soolingen D, Maquelin K (2008) Rapid identification of mycobacteria by Raman spectroscopy. J Clin Microbiol 46:961–965PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Hettick JM, Green BJ, Buskirk AD, Kashon ML, Slaven JE, Janotka E, Blachere FM, Schmechel D, Beezhold DH (2008) Discrimination of Aspergillus isolates at the species and strain level by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry fingerprinting. Anal Biochem 380:276–281PubMedCrossRefGoogle Scholar
  15. 15.
    Bille E, Dauphin B, Leto J, Bougnoux ME, Beretti JL, Lotz A, Suarez S, Meyer J, Join-Lambert O, Descamps P, Grall N, Mory F, Dubreuil L, Berche P, Nassif X, Ferroni A (2012) MALDI-TOF MS Andromas strategy for the routine identification of bacteria, mycobacteria, yeasts. Aspergillus spp. and positive blood cultures. Clin Microbiol Infect 18:1117–1125PubMedCrossRefGoogle Scholar
  16. 16.
    Del Chierico F, Masotti A, Onori M, Fiscarelli E, Mancinelli L, Ricciotti G, Alghisi F, Dimiziani L, Manetti C, Urbani A, Muraca M, Putignani L (2012) MALDI-TOF MS proteomic phenotyping of filamentous and other fungi from clinical origin. J Proteomics 75:3314–3330PubMedCrossRefGoogle Scholar
  17. 17.
    Ray AC, Eakin RE (1975) Studies on the biosynthesis of aspergillin by Aspergillus niger. Appl Microbiol 30:909–915PubMedCentralPubMedGoogle Scholar
  18. 18.
    Buskirk AD, Hettick JM, Chipinda I, Law BF, Siegel PD, Slaven JE, Green BJ, Beezhold DH (2011) Fungal pigments inhibit the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of darkly pigmented fungi. Anal Biochem 411:122–128PubMedCrossRefGoogle Scholar
  19. 19.
    Bernardo K, Pakulat N, Macht M, Krut O, Seifert H, Fleer S, Hünger F, Krönke M (2002) Identification and discrimination of Staphylococcus aureus strains using matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Proteomics 2:747–753PubMedCrossRefGoogle Scholar
  20. 20.
    Arnold RJ, Karty JA, Ellington AD, Reilly JP (1999) Monitoring the growth of a bacteria culture by MALDI-MS of whole cells. Anal Chem 71:1990–1996PubMedCrossRefGoogle Scholar
  21. 21.
    Alanio A, Beretti JL, Dauphin B, Mellado E, Quesne G, Lacroix C, Amara A, Berche P, Nassif X, Bougnoux ME (2011) Matrix-assisted laser desorption ionization time-of-flight mass spectrometry for fast and accurate identification of clinically relevant Aspergillus species. Clin Microbiol Infect 17:750–755PubMedCrossRefGoogle Scholar
  22. 22.
    Pinel C, Arlotto M, Issartel JP, Berger F, Pelloux H, Grillot R, Symoens F (2011) Comparative proteomic profiles of Aspergillus fumigatus and Aspergillus lentulus strains by surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS). BMC Microbiol 11:172PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Araujo R, Amorim A, Gusmao L (2012) Diversity and specificity of microsatellites within Aspergillus section Fumigati. BMC microbiol 12:154Google Scholar
  24. 24.
    Serrano R, Gusmao L, Amorim A, Araujo R (2011) Rapid identification of Aspergillus fumigatus within the section Fumigati. BMC microbiol 11:82Google Scholar
  25. 25.
    Etienne KA, Kano R, Balajee SA (2009) Development and validation of a microsphere-based Luminex assay for rapid identification of clinically relevant aspergilli. J Clin Microbiol 47:1096–100Google Scholar
  26. 26.
    Balajee SA, Kano R, Baddley JW, Moser SA, Marr KA, Alexander BD Andes D, Kontoyiannis DP, Perrone G, Peterson S, Brandt ME, Pappas PG and Chiller T (2009) Molecular identification of Aspergillus species: Transplant Associated Infection Surveillance Network (TRANSNET). J Clin Microbiol 47:3138–3141Google Scholar
  27. 27.
    Buess M, Cathomas G, Halter J, Junker L, Grendelmeier P, Tamm M, Stolz D (2012) Aspergillus-PCR in bronchoalveolar lavage for detection of invasive pulmonary aspergillosis in immunocompromised patients. BMC Infect Dis 12:237Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • P. E. B. Verwer
    • 1
  • W. B. van Leeuwen
    • 1
    • 2
  • V. Girard
    • 3
  • V. Monnin
    • 3
  • A. van Belkum
    • 3
  • J. F. Staab
    • 4
  • H. A. Verbrugh
    • 1
  • I. A. J. M. Bakker-Woudenberg
    • 1
  • W. W. J. van de Sande
    • 1
  1. 1.Department of Medical Microbiology and Infectious Diseases, Erasmus MCUniversity Medical Centre RotterdamRotterdamThe Netherlands
  2. 2.TopLab University of Applied Sciences LeidenLeidenThe Netherlands
  3. 3.Microbiologie R&DbioMérieuxLa Balme les GrottesFrance
  4. 4.Johns Hopkins University School of MedicineBaltimoreUSA

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