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Development and Analysis of qPCR for the Identification of Arthroconidial Yeasts of the Genus Magnusiomyces

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Abstract

The arthroconidial yeasts Magnusiomyces capitatus and M. clavatus are emerging opportunistic pulmonary pathogens. They are closely related and difficult to distinguish based on morphological and physiological traits. We applied an SYBR® green-based quantitative PCR (qPCR) assay to identify the species. We analyzed 30 reference strains originating from clinical and environmental sources by targeting the Rpb2 gene encoding the second largest subunit of RNA polymerase II. The qPCR assays were tested by direct identification of M. capitatus and M. clavatus in spiked sputum and household dishwasher swabs, respectively, as models for clinical and environmental samples. The assays were proved to be reliable for species-level identification of both species, with 100% sensitivity and 100% specificity, lowest inter-assay deviations (RSDr ≤ 1.65%, R2 values >0.99), detection limit of 10 theoretical copy number of target DNA, and detection cell limit of ≥5000 yeast cells from spiked sputum samples. The developed qPCR assay is a practical molecular approach for the detection of M. capitatus and M. clavatus that can be used as a stand-alone assay or in conjunction with culture-dependent approaches.

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References

  1. de Hoog GS, Smith MT. The ribosomal gene phylogeny and species delimitation in Geotrichum and its teleomorphs. Stud Mycol. 2004;50:489–515.

    Google Scholar 

  2. de Hoog GS, Smith MT, Guého E. A revision of the genus Geotrichum and its teleomorphs. Stud Mycol. 1986;29:1–131.

    Google Scholar 

  3. Kaplan E, Al-Hatmi AMS, Ilkit M, et al. Molecular diagnostics of arthroconidial yeasts, frequent pulmonary opportunists. J Clin Microbiol. 2018;56(1):e01427-e1517.

    CAS  PubMed  Google Scholar 

  4. García-Ruiz JC, López-Soria L, Olazábal I, et al. Invasive infections caused by Saprochaete capitata in patients with haematological malignancies: report of five cases and review of the antifungal therapy. Rev Iberoam Micol. 2013;30(4):248–55.

    Article  Google Scholar 

  5. Vaux S, Criscuolo A, Desnos-Ollivier M, et al. Multicenter outbreak of infections by Saprochaete clavata, an unrecognized opportunistic fungal pathogen. mBio. 2014;5:e02309-14.

    Article  Google Scholar 

  6. Mazzocato S, Marchionni E, Fothergill AW, et al. Epidemiology and outcome of systemic infections due to Saprochaete capitata: case report and review of the literature. Infection. 2015;43(2):211–5.

    Article  CAS  Google Scholar 

  7. Schuermans C, van Bergen M, Coorevits L, et al. Breakthrough Saprochaete capitata infections in patients receiving echinocandins: case report and review of the literature. Med Mycol. 2011;49(4):414–8.

    Article  CAS  Google Scholar 

  8. Birrenbach T, Bertschy S, Aebersold F, et al. Emergence of Blastoschizomyces capitatus yeast infections, Central Europe. Emerg Infect Dis. 2012;18(1):98–101.

    Article  Google Scholar 

  9. Zalar P, Novak M, de Hoog GS, Gunde-Cimerman N. Dishwashers: a man-made ecological niche accommodating human opportunistic fungal pathogens. Fungal Biol. 2011;115(10):997–1007.

    Article  CAS  Google Scholar 

  10. Döğen A, Kaplan E, Öksüz Z, Serin MS, Ilkit M, de Hoog GS. Dishwashers are a major source of human opportunistic yeast-like fungi in indoor environments in Mersin. Turkey Med Mycol. 2013;51(5):493–8.

    Article  Google Scholar 

  11. Gümral R, Özhak-Baysan B, Tümgör A, et al. Dishwashers provide a selective extreme environment for human-opportunistic yeast-like fungi. Fungal Divers. 2016;76(1):1–9.

    Article  Google Scholar 

  12. Zupančič J, Novak Babic M, Zalar P, Gunde-Cimerman N. The black yeast Exophiala dermatitidis and other selected opportunistic human fungal pathogens spread from dishwashers to kitchens. PLoS ONE. 2016;11(2):e0148166.

    Article  Google Scholar 

  13. Tanuskova D, Horakova J, Svec P, et al. First case of invasive Magnusiomyces capitatus infection in Slovakia. Med Mycol Case Rep. 2017;16:12–5.

    Article  Google Scholar 

  14. Leoni M, Riccardi N, Rotulo GA, et al. Magnusiomyces clavatus infection in a child after allogeneic hematotopoetic stem cell transplantation: diagnostic and therapeutic implications. Med Mycol Case Rep. 2018;23:65–7.

    Article  Google Scholar 

  15. Buchta V, Bolehovská R, Hovorková E, Cornely OA, Seidel D, Žák P. Saprochaete clavata invasive infections: a new threat to hematological-oncological patients. Front Microbiol. 2019;10:2196.

    Article  Google Scholar 

  16. Pamidimukkala U, Kancharla A, Sudhaharan S, et al. Isolation of the rare opportunistic yeast Saprochaete capitata from clinical samples-experience from a tertiary care hospital in southern India and a brief review of the literature. J Clin Diagn Res. 2017;11(9):36–42.

    Google Scholar 

  17. Esposto MC, Prigitano A, Lo Cascio G, et al. Yeast-like filamentous fungi: molecular identification and in vitro susceptibility study. Med Mycol. 2019;57(7):909–13.

    Article  CAS  Google Scholar 

  18. Desnos-Ollivier M, Blanc C, Garcia-Hermoso D, Hoinard D, Alanio A, Dromer F. Misidentification of Saprochaete clavata as Magnusiomyces capitatus in clinical isolates: utility of internal transcribed spacer sequencing and matrix-assisted laser desorption ionization: time of flight mass spectrometry and importance of reliable databases. J Clin Microbiol. 2014;52(6):2196–8.

    Article  CAS  Google Scholar 

  19. Koç AN, Atalay MA, Timur D, Demir G, Kaynar L. Molecular epidemiology and antifungal susceptibility of Saprochaete capitata (Blastoschizomyces capitatus) isolates causing nosocomial infection in Kayseri/Turkey. Infect Dis (Lond). 2016;48(8):596–603.

    Article  Google Scholar 

  20. Springer J, McCormick Smith I, Hartmann S, et al. Identification of Aspergillus and Mucorales in formalin-fixed, paraffin-embedded tissue samples: comparison of specific and broad-range fungal qPCR assays. Med Mycol. 2019;57(3):308–13.

    Article  CAS  Google Scholar 

  21. Salehi E, Hedayati MT, Zoll J, et al. Discrimination of aspergillosis, mucormycosis, fusariosis, and scedosporiosis in formalin-fixed paraffin-embedded tissue specimens by use of multiple real-time quantitative PCR assays. J Clin Microbiol. 2016;54(11):2798–803.

    Article  CAS  Google Scholar 

  22. Krohn S, Zeller K, Böhm S, et al. Molecular quantification and differentiation of Candida species in biological specimens of patients with liver cirrhosis. PLoS ONE. 2018;13(6):e0197319.

    Article  Google Scholar 

  23. Fidler G, Leiter E, Kocsube S, Biro S, Paholcsek M. Validation of a simplex PCR assay enabling reliable identification of clinically relevant Candida species. BMC Infect Dis. 2018;18(1):393.

    Article  Google Scholar 

  24. Springer J, Lackner M, Ensinger C, et al. Clinical evaluation of a Mucorales-specific real-time PCR assay in tissue and serum samples. J Med Microbiol. 2016;65(12):1414–21.

    Article  CAS  Google Scholar 

  25. Al-Hatmi AM, Bonifaz A, de Hoog GS, et al. Keratitis by Fusarium temperatum, a novel opportunist. BMC Infect Dis. 2014;14:588.

    Article  Google Scholar 

  26. Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016;33(7):1870–4.

    Article  CAS  Google Scholar 

  27. Owczarzy R, Tataurov AV, Wu Y, et al. IDT SciTools: a suite for analysis and design of nucleic acid oligomers. Nucleic Acids Res. 2008;36:163–9.

    Article  Google Scholar 

  28. Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden T. Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics. 2012;13:134.

    Article  CAS  Google Scholar 

  29. Bustin SA, Benes V, Garson JA, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009;55(4):611–22.

    Article  CAS  Google Scholar 

  30. European Network of GMO Laboratories. Verification of analytical methods for GMO testing when implementing interlaboratory validated methods. Luxembourg: Joint Research Centre, Luxembourg; 2011.

    Google Scholar 

  31. Blakely T, Salmond C. Probabilistic record linkage and a method to calculate the positive predictive value. Int J Epidemiol. 2002;31(6):1246–52.

    Article  Google Scholar 

  32. Brejová B, Lichancová H, Brázdovič F, et al. Genome sequence of the opportunistic human pathogen Magnusiomyces capitatus. Curr Genet. 2019;65(2):539–60.

    Article  Google Scholar 

  33. Turin L, Riva F, Galbiati G, Cainelli T. Fast, simple and highly sensitive double-rounded polymerase chain reaction assay to detect medically relevant fungi in dermatological specimens. Eur J Clin Invest. 2000;30(6):511–8.

    Article  CAS  Google Scholar 

  34. Everaerts S, Lagrou K, Vermeersch K, Dupont LJ, Vanaudenaerde BM, Janssens W. Aspergillus fumigatus detection and risk factors in patients with COPD-Bronchiectasis overlap. Int J Mol Sci. 2018;19(2):e523.

    Article  Google Scholar 

  35. Liu CM, Kachur S, Dwan MG, et al. FungiQuant: a broad-coverage fungal quantitative real-time PCR assay. BMC Microbiol. 2012;12:255.

    Article  Google Scholar 

  36. Kaplan E, Ilkit M, de Hoog GS. Comparison of the rolling circle amplification and ligase-dependent reaction methods for the identification of opportunistic Exophiala species. Med Mycol. 2018;56(6):759–69.

    Article  CAS  Google Scholar 

  37. Prévost-Bouré NC, Christen R, Dequiedt S, et al. Validation and application of a PCR primer set to quantify fungal communities in the soil environment by real-time quantitative PCR. PLoS ONE. 2011;6(9):e24166.

    Article  Google Scholar 

  38. Fredricks DN, Smith C, Meier A. Comparison of six DNA extraction methods for recovery of fungal DNA as assessed by quantitative PCR. J Clin Microbiol. 2005;43(10):5122–8.

    Article  CAS  Google Scholar 

  39. Dalla-Costa LM, Morello LG, Conte D, et al. Comparison of DNA extraction methods used to detect bacterial and yeast DNA from spiked whole blood by real-time PCR. J Microbiol Methods. 2017;140:61–6.

    Article  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Pharmaceutical Microbiology, Faculty of Pharmacy, University of Zonguldak Bülent Ecevit, Zonguldak, Turkey

    Engin Kaplan

  2. Department of Pharmaceutical Microbiology, Faculty of Pharmacy, University of Mersin, Mersin, Turkey

    Deniz Aktaş & Aylin Döğen

  3. Department of Microbiology, Faculty of Medicine, University of Ege, Izmir, Turkey

    Süleyha Hilmioğlu-Polat

  4. Department of Microbiology, Gülhane Military Medical Academy, Ankara, Turkey

    Ramazan Gümral

  5. Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands

    Ferry Hagen

  6. Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands

    Ferry Hagen

  7. Laboratory of Medical Mycology, Jining No. 1, People’s Hospital, Jining, Shandong, P.R. China

    Ferry Hagen

  8. Division of Mycology, Department of Microbiology, Faculty of Medicine, University of Çukurova, Adana, Turkey

    Macit Ilkit

  9. Centre of Expertise in Mycology, Radboud University Medical Centre/Canisius Wilhelmina Hospital, Nijmegen, The Netherlands

    G. Sybren de Hoog

Authors
  1. Engin Kaplan
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  2. Deniz Aktaş
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  8. G. Sybren de Hoog
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Contributions

Conceived and designed the experiments: EK, AD, SHP, MI, GSH. Performed the experiments: EK, DA, AD, SHP, RG. Analyzed the data: EK, DA, AD, SHP, RG, FH, MI, GSH. Contributed reagents/materials/analysis tools: EK, DA, AD, SHP, RG, FH, MI, GSH. Wrote the paper: EK, DA, AD, SHP, RG, FH, MI, GSH.

Corresponding authors

Correspondence to Engin Kaplan or Macit Ilkit.

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Conflict of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of this paper.

Ethics Statement

Ethical approval and patient consensus were not necessary because the study design was largely based on reference strains. Non-reference clinical isolates originated from anonymized clinical samples obtained during routine laboratory activity.

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Handling Editor: Vishnu Chaturvedi

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Kaplan, E., Aktaş, D., Döğen, A. et al. Development and Analysis of qPCR for the Identification of Arthroconidial Yeasts of the Genus Magnusiomyces. Mycopathologia 186, 41–51 (2021). https://doi.org/10.1007/s11046-020-00510-4

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  • DOI: https://doi.org/10.1007/s11046-020-00510-4

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