Advertisement

Mycopathologia

, Volume 183, Issue 6, pp 913–920 | Cite as

Determination of Azole Resistance and TR34/L98H Mutations in Isolates of Aspergillus Section Fumigati from Turkish Cystic Fibrosis Patients

  • Özge Güngör
  • Benedita Sampaio-Maia
  • Antonio Amorim
  • Ricardo Araujo
  • Zayre Erturan
Original Paper

Abstract

Background

Aspergillus fumigatus is the species section Fumigati most frequently isolated from the respiratory tract of cystic fibrosis (CF) patients. Recent studies suggest that mutations in the Cyp51 gene, particularly TR34/L98H, are responsible for azole resistance.

Objectives and Methods

The focus of this study was on section Fumigati isolates isolated from the respiratory tract samples of CF patients. More specifically, the goal was to detect A. fumigatus isolates, test their antifungal susceptibility to itraconazole, voriconazole and posaconazole, and finally determine the presence of TR34/L98H and other mutations in the isolates Cyp51A gene.

Results and Conclusions

A set of 31 isolates of Aspergillus section Fumigati were obtained from the sputum samples of 6 CF patients and subsequently identified to species level by microsatellite genotyping. All isolates were determined as A. fumigatus and involved 14 different genotypes. The minimal inhibitory concentrations to the three azoles were determined by the E-test method, and the Cyp51A gene was sequenced. One of the genotypes was found to be resistant to all azoles but no mutations were detected in the Cyp51A gene, especially the TR34/L98H mutation. Therefore, mutations in genes other than Cyp51A or other distinct mechanisms may be responsible for this reported multiazole resistance found in a Turkish CF patient.

Keywords

Cystic fibrosis Section Fumigati Aspergillus fumigatus Azole resistance TR34/L98H mutations Cyp51A 

Notes

Acknowledgements

This work was supported by the Research Fund of Istanbul University, Project No. 47641 and Federation of European Microbiological Societies (FEMS) Research Fellowship 2015-0048. i3S was partially financed by FEDER—Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020—Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT—Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Inovação in the framework of the project “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274). As control isolates, itraconazole sensitive (V089-25) and itraconazole resistant (V089-27) isolates obtained from Paul E. Verweij (Radboud University Nijmegen Medical Center) were used.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical Approval

This article does not contain any studies with human participants or animals performed by any of the authors. This study received approval from Istanbul University, Istanbul Medical Faculty, Clinical Research Ethics Committee to be conducted (15.08.2014/1251).

Informed Consent

For this type of study formal consent is not required.

References

  1. 1.
    Müller FM, Seidler M. Characteristics of pathogenic fungi and antifungal therapy in cystic fibrosis. Expert Rev Anti Infect Ther. 2010;8:957–64.CrossRefGoogle Scholar
  2. 2.
    Bauernfeind A, Bertele RM, Harms K. Qualitative and quantitative microbiological analysis of sputa of 102 patients with cystic fibrosis. Infection. 1987;15:270–7.CrossRefGoogle Scholar
  3. 3.
    Blyth CC, Harun A, Middleton PG. Detection of occult Scedosporium species in respiratory tract specimens from patients with cystic fibrosis by use of selective media. J Clin Microbiol. 2010;48:314–6.CrossRefGoogle Scholar
  4. 4.
    Speirs JJ, van der Ent CK, Beekman JM. Effects of Aspergillus fumigatus colonization on lung function in cystic fibrosis. Curr Opin Pulm Med. 2012;18:632–8.CrossRefGoogle Scholar
  5. 5.
    Alcazar-Fuoli L, Mellado E, Alastruey-Izquierdo A, et al. Aspergillus section Fumigati: antifungal susceptibility patterns and sequence-based identification. Antimicrob Agents Chemother. 2008;52(4):1244–51.CrossRefGoogle Scholar
  6. 6.
    Yaguchi T, Horie Y, Tanaka R, et al. Molecular phylogenetics of multiple genes on Aspergillus section Fumigati isolated from clinical specimens in Japan. J Med Mycol. 2007;48:37–46.CrossRefGoogle Scholar
  7. 7.
    Ullmann AJ, Aguado JM, Arikan-Akdagli S, et al. Diagnosis and management of Aspergillus diseases: executive summary of the 2017 ESCMID-ECMM-ERS guideline. Clin Microbiol Infect. 2018;24:e1–38.CrossRefGoogle Scholar
  8. 8.
    Balajee SA, Gribskov J, Brandt M, et al. Mistaken identity: Neosartorya pseudofisheri and its anamorph masquerading as Aspergillus fumigatus. J Clin Microbiol. 2005;43:5996–9.CrossRefGoogle Scholar
  9. 9.
    Khare R, Gupta S, Arif S, et al. Misidentification of Neosartorya pseudofischeri as Aspergillus fumigatus in a lung transplant patient. J Clin Microbiol. 2004;52:2722–5.CrossRefGoogle Scholar
  10. 10.
    Denning DW, Perlin DS. Azole resistance in Aspergillus: a growing public health menace. Future Microbiol. 2011;6(11):1229–32.CrossRefGoogle Scholar
  11. 11.
    Howard SJ, Cerar D, Anderson MJ, et al. Frequency and evolution of Azole resistance in Aspergillus fumigatus associated with treatment failure. Emerg Infect Dis. 2009;15(7):1068–76.CrossRefGoogle Scholar
  12. 12.
    Morio F, Aubin GG, Danner-Boucher I, et al. High prevalence of triazole resistance in Aspergillus fumigatus, especially mediated by TR/L98H, in a French cohort of patients with cystic fibrosis. J Antimicrob Chemother. 2012;67:1870–3.CrossRefGoogle Scholar
  13. 13.
    Snelders E, van der Lee HA, Kuijpers J, et al. Emergence of azole resistance in Aspergillus fumigatus and spread of a single resistance mechanism. PLoS Med. 2008;5(11):e219.CrossRefGoogle Scholar
  14. 14.
    Amorim A, Guedes-Vaz L, Araujo R. Susceptibility to five antifungals of Aspergillus fumigatus strains isolated from chronically colonised cystic fibrosis patients receiving azole therapy. Int J Antimicrob Agents. 2010;35:396–9.CrossRefGoogle Scholar
  15. 15.
    Terpstra PD, van WEstreenen MJ, Croughs P, et al. Filamentous fungi in the Netherlands among CF patients. Trends in medical mycology, 5th ed. 2011.Google Scholar
  16. 16.
    Burgel PR, Baixench MT, Amsellem M, et al. High prevalence of azole-resistant Aspergillus fumigatus in adults with cystic fibrosis exposed to itraconazole. Antimicrob Agents Chemother. 2012;56(2):869–74.CrossRefGoogle Scholar
  17. 17.
    Hamprecht A, Morio F, Bader O, et al. Azole resistance in Aspergillus fumigatus in patients with cystic fibrosis: a matter of concern? Mycopathologia. 2018;183:151–60.CrossRefGoogle Scholar
  18. 18.
    Seufert R, Sedlacek L, Kahl B, et al. Prevalence and characterization of azole-resistant Aspergillus fumigatus in patients with cystic fibrosis: a prospective multicentre study in Germany. J Antimicrob Chemother.  https://doi.org/10.1093/jac/dky147.
  19. 19.
    Ozmerdiven GE, Ak S, Ener B, et al. First determination of azole resistance in Aspergillus fumigatus strains carrying the TR34/L98H mutations in Turkey. J Infect Chemoter. 2015;21:581–6.CrossRefGoogle Scholar
  20. 20.
    Larone DH. Medically important fungi: a guide to identification. 5th ed. Washington, DC: ASM Press; 2011.CrossRefGoogle Scholar
  21. 21.
    De Hoog GS, Guarro J, Gene J. Atlas of clinical fungi. 2nd ed. Washington, DC: ASM Press; 2001.Google Scholar
  22. 22.
    Aspergillus & aspergillosis website. Accessed January 2018. http://www.aspergillus.org.uk/content/dna-extraction-%E2%80%93-miniprep-spores-pcr.
  23. 23.
    Araujo R, Pina-Vaz C, Rodrigues AG, et al. Simple and highly discriminatory microsatellite-based multiplex PCR for Aspergillus fumigatus strain typing. Clin Microbiol Infect. 2009;15:260–6.CrossRefGoogle Scholar
  24. 24.
    Araujo R, Gungor O, Amorim A. Single-tube PCR coupled with mini-sequencing assay for the detection of cyp51A and cyp51B polymorphisms in Aspergillus fumigatus. Future Microbiol. 2015;10(11):1797–804.CrossRefGoogle Scholar
  25. 25.
    Clinical and Laboratory Standards Institute (CLSI). Reference method for broth dilution antifungal susceptibility testing of filamentous fungi; approved standard. 2nd ed. Document M38-A2 Wayne, PA: CLSI; 2008.Google Scholar
  26. 26.
    Mortensen KL, Jensen RH, Johansen HK, et al. Aspergillus species and other molds in respiratory samples from patients with cystic fibrosis: a laboratory-based study with focus on Aspergillus fumigatus azole resistance. J Clin Microbiol. 2011;49:2243–51.CrossRefGoogle Scholar
  27. 27.
    Gungor O, Tamay Z, Guler N, et al. Frequency of fungi in respiratory samples from Turkish cystic fibrosis patients. Mycoses. 2013;56:123–9.CrossRefGoogle Scholar
  28. 28.
    Fischer J, van Koningsbruggen-Rietschel S, Rietschel E, et al. Prevalence and molecular characterization of azole resistance in Aspergillus spp. isolates from German cystic fibrosis patients. J Antimicrob Chemother. 2014;69:1533–6.CrossRefGoogle Scholar
  29. 29.
    Pasquoletto AC. Aspergillosis: from diagnosis to prevention. Berlin: Springer; 2010.CrossRefGoogle Scholar
  30. 30.
    Symoens F, Haase G, Pihet M, et al. Unusual Aspergillus species in patients with cystic fibrosis. Med Mycol. 2010;48(Suppl 1):10–6.CrossRefGoogle Scholar
  31. 31.
    Lestrade PP, van der Velden WJFM, Bouwman F, et al. Epidemiology of invasive aspergillosis and triazole-resistant Aspergillus fumigatus in patients with haematological malignancies: a single-centre retrospective cohort study. J Antimicrob Chemother. 2018;73(5):1389–94.CrossRefGoogle Scholar
  32. 32.
    De Valk HA, Klaassen CHW, Yntema JB, et al. Molecular typing and colonization patterns of Aspergillus fumigatus in patients with cystic fibrosis. J Cyst Fibros. 2009;8:110–4.CrossRefGoogle Scholar
  33. 33.
    Vanhee LME, Symoens F, Bouchara JP, et al. High-resolution genotyping of Aspergillus fumigatus isolates recovered from chronically colonized patients with cystic fibrosis. Eur J Clin Microbiol Infect Dis. 2008;27:1005–7.CrossRefGoogle Scholar
  34. 34.
    Chazalet V, Debeaupuis JP, Sarfati J, et al. Molecular typing of environmental and patient isolates of Aspergillus fumigatus from various hospital settings. J Clin Microbiol. 1998;36:1494–500.PubMedPubMedCentralGoogle Scholar
  35. 35.
    Araujo R, Amorim A, Gusmão L. Genetic diversity of Aspergillus fumigatus in indoor hospital environments. Med Mycol. 2010;48:832–8.CrossRefGoogle Scholar
  36. 36.
    Araujo R, Amorim A, Gusmão L. Microbial forensics: Do Aspergillus fumigatus strains present local or regional differentiation? Forensic Sci Int Genet Suppl Series. 2009;2:297–9.CrossRefGoogle Scholar
  37. 37.
    Prigitano A, Esposto MC, Biffi A, et al. Triazole resistance in Aspergillus fumigatus isolates from patients with cystic fibrosis in Italy. J Cyst Fibros. 2017;16(1):64–9.CrossRefGoogle Scholar
  38. 38.
    Rath PM, Ratjen F, Ansorg R. Genetic diversity among isolates of Aspergillus fumigatus in patients with cystic fibrosis. Zentralbl Bakteriol. 1997;285(3):450–5.CrossRefGoogle Scholar
  39. 39.
    Rivero-Menendez O, Alastruey-Izquierdo A, Mellado E, et al. Triazole resistance in Aspergillus spp.: A worldwide problem? J Fungi (Basel). 2016;2(3):21.CrossRefGoogle Scholar
  40. 40.
    Stensvold CR, Jorgensen LN, Arendrup MC. Azole-resistant invasive aspergillosis: relationship to agriculture. Curr Fungal Infect Rep. 2012;6:178–91.CrossRefGoogle Scholar
  41. 41.
    Bader O, Weig M, Reichard U, et al. Cyp51A-based mechanisms of Aspergillus fumigatus azole drug resistance present in clinical samples from Germany. Antimicrob Agents Chemother. 2013;57:3513–7.CrossRefGoogle Scholar
  42. 42.
    Garczewska B, Jarzynka S, Kus J, et al. Fungal infection of cystic fibrosis-single center experience. Pneumonol Alergol Pol. 2016;84:151–9.CrossRefGoogle Scholar
  43. 43.
    Guegan H, Chevrier S, Belleguic C, et al. Performance of molecular approaches for Aspergillus detection and azole resistance surveillance in cystic fibrosis. Front Microbiol. 2018;27(9):531.CrossRefGoogle Scholar
  44. 44.
    Arendrup MC, Mavridou E, Mortensen KL, et al. Development of azole resistance in Aspergillus fumigatus during azole therapy associated with change in virulence. PLoS ONE. 2010;5:e10080.  https://doi.org/10.1371/journal.pone.0050034.CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Camps SM, Dutilh BE, Arendrup MC, et al. Discovery of a hapE mutation that causes azole resistance in Aspergillus fumigatus through whole genome sequencing and sexual crossing. PLoS ONE. 2012;7:e50034.  https://doi.org/10.1371/journal.pone.0050034.CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Albarrag AM, Anderson MJ, Howard SJ, et al. Interrogation of related clinical pan-azole-resistant Aspergillus fumigatus strains: G138C, Y431C, and G434C single nucleotide polymorphisms in cyp51A, upregulation of cyp51A, and integration and activation of transposon Atf1 in the cyp51A promoter. Antimicrob Agents Chemother. 2011;55:5113–21.CrossRefGoogle Scholar
  47. 47.
    Slaven JW, Anderson MJ, Sanglard D, et al. Increased expression of a novel Aspergillus fumigatus ABC transporter gene, atrF, in the presence of itraconazole in an itraconazole resistant clinical isolate. Fungal Genet Biol. 2002;36:199–206.CrossRefGoogle Scholar
  48. 48.
    Bowyer P, Mosquera J, Anderson M, et al. Identification of novel genes conferring altered azole susceptibility in Aspergillus fumigatus. FEMS Microbiol Lett. 2012;332:10–9.CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.Istanbul Medical Faculty, Department of Medical MicrobiologyIstanbul UniversityCapa, IstanbulTurkey
  2. 2.Faculty of Dental MedicineUniversity of PortoPortoPortugal
  3. 3.INEB, Instituto Nacional de Engenharia Biomédica da Universidade do PortoPortoPortugal
  4. 4.i3S, Instituto de Investigação e Inovação em Saúde da Universidade do PortoPortoPortugal
  5. 5.Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP)PortoPortugal
  6. 6.Faculdade de Ciências, Universidade do PortoPortoPortugal
  7. 7.Department of Medical Biotechnology, College of Medicine and Public HealthFlinders UniversityAdelaideAustralia

Personalised recommendations