, Volume 182, Issue 7–8, pp 625–632 | Cite as

Comparison of DNA Microarray, Loop-Mediated Isothermal Amplification (LAMP) and Real-Time PCR with DNA Sequencing for Identification of Fusarium spp. Obtained from Patients with Hematologic Malignancies

  • Marcela de Souza
  • Tetsuhiro Matsuzawa
  • Kanae Sakai
  • Yasunori Muraosa
  • Luzia Lyra
  • Ariane Fidelis Busso-Lopes
  • Anna Sara Shafferman Levin
  • Angélica Zaninelli Schreiber
  • Yuzuru Mikami
  • Tohoru Gonoi
  • Katsuhiko Kamei
  • Maria Luiza Moretti
  • Plínio TrabassoEmail author


The performance of three molecular biology techniques, i.e., DNA microarray, loop-mediated isothermal amplification (LAMP), and real-time PCR were compared with DNA sequencing for properly identification of 20 isolates of Fusarium spp. obtained from blood stream as etiologic agent of invasive infections in patients with hematologic malignancies. DNA microarray, LAMP and real-time PCR identified 16 (80%) out of 20 samples as Fusarium solani species complex (FSSC) and four (20%) as Fusarium spp. The agreement among the techniques was 100%. LAMP exhibited 100% specificity, while DNA microarray, LAMP and real-time PCR showed 100% sensitivity. The three techniques had 100% agreement with DNA sequencing. Sixteen isolates were identified as FSSC by sequencing, being five Fusarium keratoplasticum, nine Fusarium petroliphilum and two Fusarium solani. On the other hand, sequencing identified four isolates as Fusarium non-solani species complex (FNSSC), being three isolates as Fusarium napiforme and one isolate as Fusarium oxysporum. Finally, LAMP proved to be faster and more accessible than DNA microarray and real-time PCR, since it does not require a thermocycler. Therefore, LAMP signalizes as emerging and promising methodology to be used in routine identification of Fusarium spp. among cases of invasive fungal infections.


Fusarium Molecular methods Fungal infection Filamentous fungi 



This work was supported by JST/JICA and SATREPS, Grant No. Unicamp 02P-29548-09. M.S. has received a master degree scholarship from the São Paulo Research Foundation (FAPESP), Grant No. FAPESP 2011/16205-5.

Limitations of the Study

The DNA microarray, LAMP and real-time PCR techniques utilized in this study only discriminated FSSC from Fusarium spp. However, the real value would be to differentiate FSSC from FOSC, rather than discriminate the components inside the complex. In this respect, the results are still valid and have significance in the decision making for therapeutic purposes.

Nucleotide Sequence Accession Numbers

The sequences determined in this study were deposited in NCBI database with the Accession Numbers: AB817198.1, AB817200.1, AB817204.1, AB817208.1, AB817209.1, AB817210.1, AB817216.1, AB817221.1, AB817222.1, AB817225.1, AB817226.1, AB817227.1, AB817231.1 and KM099396 to KM099400, KU974270, KY020039 and KY020040.


  1. 1.
    Al-Hatmi AM, Meis JF, de Hoog GS. Fusarium: molecular diversity and intrinsic drug resistance. PLoS Pathog. 2016;12:e1005464.CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Van Diepeningen AD, Brankovics B, Iltes J, van der Lee TA, Waalwijk C. Diagnosis of Fusarium Infections: approaches to identification by the clinical mycology laboratory. Curr Fungal Infect Rep. 2015;9:135–43.CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Guarro J. Fusariosis, a complex infection caused by a high diversity of fungal species refractory to treatment. Eur J Clin Microbiol Infect Dis. 2013;32:1491–500.CrossRefPubMedGoogle Scholar
  4. 4.
    O’Donnell K, Sarver BA, Brandt M, Chang DC, Noble-Wang J, Park BJ, et al. Phylogenetic diversity and microsphere array-based genotyping of human pathogenic Fusarium, including isolates from the multistate contact lens-associated U.S. keratitis outbreaks of 2005 and 2006. J Clin Microbiol. 2007;45:2235–48.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Schroers HJ, Samuels GJ, Zhang N, Short DP, Juba J, Geiser DM. Epitypification of Fusisporium (Fusarium) solani and its assignment to a common phylogenetic species in the Fusarium solani species complex. Mycologia. 2016;108:806–19.CrossRefPubMedGoogle Scholar
  6. 6.
    Lombard L, van der Merwe NA, Groenewald JZ, Crous PW. Generic concepts in Nectriaceae. Stud Mycol. 2015;80:189–245.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Geiser DM, Aoki T, Bacon CW, Baker SE, Bhattacharyya MK, Brandt ME, Brown DW, et al. One fungus, one name: defining the genus Fusarium in a scientifically robust way that preserves longstanding use. Phytopathology. 2013;103:400–8.CrossRefPubMedGoogle Scholar
  8. 8.
    Al-Hatmi AM, van Diepeningen AD, Curfs-Breuker I, de Hoog GS, Meis JF. Specific antifungal susceptibility profiles of opportunists in the Fusarium fujikuroi complex. J Antimicrob Chemother. 2015;70:1068–71.PubMedGoogle Scholar
  9. 9.
    Liao MH, Lin JF, Li SY. Application of a multiplex suspension array for rapid and simultaneous identification of clinically important mold pathogens. Mol Cell Probes. 2012;26:188–93.CrossRefPubMedGoogle Scholar
  10. 10.
    Tortorano AM, Prigitano A, Esposto MC, Arsic Arsenijevic V, Kolarovic J, Ivanovic D, et al. European Confederation of Medical Mycology (ECMM) epidemiological survey on invasive infections due to Fusarium species in Europe. Eur J Clin Microbiol Infect Dis. 2014;33:1623–30.CrossRefPubMedGoogle Scholar
  11. 11.
    Buelow DR, Gu Z, Walsh TJ, Hayden RT. Evaluation of multiplexed PCR and liquid-phase array for identification of respiratory fungal pathogens. Med Mycol. 2012;50:775–80.CrossRefPubMedGoogle Scholar
  12. 12.
    Campa D, Tavanti A, Gemignani F, Mogavero CS, Bellini I, Bottari F, et al. DNA microarray based on arrayed-primer extension technique for identification of pathogenic fungi responsible for invasive and superficial mycoses. J Clin Microbiol. 2008;46:909–15.CrossRefPubMedGoogle Scholar
  13. 13.
    Spiess B, Seifarth W, Hummel M, Frank O, Fabarius A, Zheng C, Mörz H, et al. DNA microarray-based detection and identification of fungal pathogens in clinical samples from neutropenic patients. J Clin Microbiol. 2007;45:3743–53.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Niessen L. Loop-mediated isothermal amplification-based detection of Fusarium graminearum. Methods Mol Biol. 2013;968:177–93.CrossRefPubMedGoogle Scholar
  15. 15.
    Niessen L, Gräfenhan T, Vogel RF. ATP citrate lyase 1 (acl1) gene-based loop-mediated amplification assay for the detection of the Fusarium tricinctum species complex in pure cultures and in cereal samples. Int J Food Microbiol. 2012;158:171–85.CrossRefPubMedGoogle Scholar
  16. 16.
    Niessen L, Vogel RF. Detection of Fusarium graminearum DNA using a loop-mediated isothermal amplification (LAMP) assay. Int J Food Microbiol. 2010;140:183–91.CrossRefPubMedGoogle Scholar
  17. 17.
    Muraosa Y, Schreiber AZ, Trabasso P, Matsuzawa T, Taguchi H, Moretti ML, et al. Development of cycling probe-based real-time PCR system to detect Fusarium species and Fusarium solani species complex (FSSC). Int J Med Microbiol. 2014;304:505–11.CrossRefPubMedGoogle Scholar
  18. 18.
    O’Donnell K, Rooney AP, Proctor RH, Brown DW, McCormick SP, Ward TJ, Frandsen RJ, Lysøe E, Rehner SA, Aoki T, Robert VA, Crous PW, Groenewald JZ, Kang S, Geiser DM. Phylogenetic analyses of RPB1 and RPB2 support a middle Cretaceous origin for a clade comprising all agriculturally and medically important fusaria. Fungal Genet Biol. 2013;52:20–31.CrossRefPubMedGoogle Scholar
  19. 19.
    Al-Hatmi AM, Van Den Ende AH, Stielow JB, Van Diepeningen AD, Seifert KA, McCormick W, Assabgui R, Gräfenhan T, De Hoog GS, Levesque CA. Evaluation of two novel barcodes for species recognition of opportunistic pathogens in Fusarium. Fungal Biol. 2016;120:231–45.CrossRefPubMedGoogle Scholar
  20. 20.
    Verweij PE, Brandt ME. Aspergillus, Fusarium, and other opportunistic moniliaceous fungi. In: Murray PR, Baron EJ, Jorgensen JH, Landry ML, Pfaller MA, editors. Manual of clinical microbiology. Washington, DC: ASM Press; 2007. p. 1802–38.Google Scholar
  21. 21.
    Sakai K, Trabasso P, Moretti ML, Mikami Y, Kamei K, Gonoi T. Identification of fungal pathogens by visible microarray system in combination with isothermal gene amplification. Mycopathologia. 2014;178:11–26.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Matsuzawa T, Tanaka R, Horie Y, Gonoi T, Yaguchi T. Development of rapid and specific molecular discrimination methods for pathogenic emericella species. Nihon Ishinkin Gakkai Zasshi. 2010;51:109–16.CrossRefPubMedGoogle Scholar
  23. 23.
    Moreira-Oliveira MS, Mikami Y, Miyaji M, Imai T, Schreiber AZ, Moretti ML. Diagnosis of candidemia by polymerase chain reaction and blood culture: prospective study in a high-risk population and identification of variables associated with development of candidemia. Eur J Clin Microbiol Infect Dis. 2005;24:721–6.CrossRefPubMedGoogle Scholar
  24. 24.
    Hung WT, Su SL, Shiu LY, Chang TC. Rapid identification of allergenic and pathogenic molds in environmental air by an oligonucleotide array. BMC Infect Dis. 2011;11:91.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Hsiao CR, Huang L, Bouchara JP, Barton R, Li HC, Chang TC. Identification of medically important molds by an oligonucleotide array. J Clin Microbiol. 2005;43:3760–8.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Denschlag C, Vogel RF, Niessen L. Hyd5 gene-based detection of the major gushing-inducing Fusarium spp. in a loop-mediated isothermal amplification (LAMP) assay. Int J Food Microbiol. 2012;156:189–96.CrossRefPubMedGoogle Scholar
  27. 27.
    Atoui A, El Khoury A, Kallassy M, Lebrihi A. Quantification of Fusarium graminearum and Fusarium culmorum by real-time PCR system and zearalenone assessment in maize. Int J Food Microbiol. 2012;154:59–65.CrossRefPubMedGoogle Scholar
  28. 28.
    Haegi A, Catalano V, Luongo L, Vitale S, Scotton M, Ficcadenti N, Belisario A. A newly developed real-time PCR assay for detection and quantification of Fusarium oxysporum and its use in compatible and incompatible interactions with grafted melon genotypes. Phytopathology. 2013;103:802–10.CrossRefPubMedGoogle Scholar
  29. 29.
    Bernal-Martínez L, Buitrago MJ, Castelli MV, Rodríguez-Tudela JL, Cuenca-Estrella M. Detection of invasive infection caused by Fusarium solani and non-Fusarium solani species using a duplex quantitative PCR-based assay in a murine model of fusariosis. Med Mycol. 2012;50:270–5.CrossRefPubMedGoogle Scholar
  30. 30.
    Scauflaire J, Godet M, Gourgue M, Liénard C, Munaut F. A multiplex real-time PCR method using hybridization probes for the detection and the quantification of Fusarium proliferatum, F. subglutinans, F. temperatum, and F. verticillioides. Fungal Biol. 2012;116:1073–80.CrossRefPubMedGoogle Scholar
  31. 31.
    Reischer GH, Lemmens M, Farnleitner A, Adler A, Mach RL. Quantification of Fusarium graminearum in infected wheat by species specific real-time PCR applying a TaqMan Probe. J Microbiol Methods. 2004;59:141–6.CrossRefPubMedGoogle Scholar
  32. 32.
    Galimberti R, Torre AC, Baztán MC, Rodriguez-Chiappetta F. Emerging systemic fungal infections. Clin Dermatol. 2012;30:633–50.CrossRefPubMedGoogle Scholar
  33. 33.
    Azor M, Gené J, Cano J, Manikandan P, Venkatapathy N, Guarro J. Less-frequent Fusarium species of clinical interest: correlation between morphological and molecular identification and antifungal susceptibility. J Clin Microbiol. 2009;47:1463–8.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2017

Authors and Affiliations

  • Marcela de Souza
    • 1
    • 5
  • Tetsuhiro Matsuzawa
    • 2
    • 6
  • Kanae Sakai
    • 2
  • Yasunori Muraosa
    • 2
  • Luzia Lyra
    • 3
  • Ariane Fidelis Busso-Lopes
    • 1
  • Anna Sara Shafferman Levin
    • 4
  • Angélica Zaninelli Schreiber
    • 3
  • Yuzuru Mikami
    • 2
  • Tohoru Gonoi
    • 2
  • Katsuhiko Kamei
    • 2
  • Maria Luiza Moretti
    • 1
  • Plínio Trabasso
    • 1
    Email author
  1. 1.Department of Internal Medicine, School of MedicineUniversity of CampinasCampinasBrazil
  2. 2.Medical Mycology Research CenterChiba UniversityChibaJapan
  3. 3.Department of Clinical Pathology, School of MedicineUniversity of CampinasCampinasBrazil
  4. 4.Division of Infectious Diseases, School of MedicineUniversity of São PauloSão PauloBrazil
  5. 5.LIM 46 – Laboratory of ParasitologyHC/FMUSPSão PauloBrazil
  6. 6.Tetsuhiro MatsuzawaUniversity of NagasakiNagasakiJapan

Personalised recommendations