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Toward the Standardization of Mycological Examination of Sputum Samples in Cystic Fibrosis: Results from a French Multicenter Prospective Study

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Abstract

Fungal respiratory colonization of cystic fibrosis (CF) patients emerges as a new concern; however, the heterogeneity of mycological protocols limits investigations. We first aimed at setting up an efficient standardized protocol for mycological analysis of CF sputa that was assessed during a prospective, multicenter study: “MucoFong” program (PHRC-06/1902). Sputa from 243 CF patients from seven centers in France were collected over a 15-month period and submitted to a standardized protocol based on 6 semi-selective media. After mucolytic pretreatment, sputa were plated in parallel on cycloheximide-enriched (ACT37), erythritol-enriched (ERY37), benomyl dichloran–rose bengal (BENO37) and chromogenic (CAN37) media incubated at 37 °C and on Sabouraud–chloramphenicol (SAB27) and erythritol-enriched (ERY27) media incubated at 20–27 °C. Each plate was checked twice a week during 3 weeks. Fungi were conventionally identified; time for detection of fungal growth was noted for each species. Fungal prevalences and media performances were assessed; an optimal combination of media was determined using the Chi-squared automatic interaction detector method. At least one fungal species was isolated from 81% of sputa. Candida albicans was the most prevalent species (58.8%), followed by Aspergillus fumigatus (35.4%). Cultivation on CAN37, SAB27, ACT37 and ERY27 during 16 days provided an optimal combination, detecting C. albicans, A. fumigatus, Scedosporium apiospermum complex and Exophiala spp. with sensitivities of 96.5, 98.8, 100 and 100%. Combination of these four culture media is recommended to ensure the growth of key fungal pathogens in CF respiratory specimens. The use of such consensual protocol is of major interest for merging results from future epidemiological studies.

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References

  1. Cystic Fibrosis Foundation Patient Registry Annual Report 2015. [cited 2016 Nov 18]. https://www.cff.org/Our-Research/CF-Patient-Registry/2015-Patient-Registry-Annual-Data-Report.pdf.

  2. European Cystic Fibrosis Society Patient Registry Annual Report 2014. [cited 2017 Jan 22]. https://www.ecfs.eu/sites/default/files/images/ECFSPR_Annual%20Report%202014_Nov2016.pdf.

  3. Touati K, Nguyen DNL, Delhaes L. The airway colonization by opportunistic filamentous fungi in patients with cystic fibrosis: recent updates. Curr Fungal Infect Rep. 2014;8:302–11.

    Article  Google Scholar 

  4. Rowe SM, Miller S, Sorscher EJ. Cystic fibrosis. N Engl J Med. 2005;352:1992–2001.

    Article  CAS  PubMed  Google Scholar 

  5. Pihet M, Carrère J, Cimon B, et al. Occurrence and relevance of filamentous fungi in respiratory secretions of patients with cystic fibrosis—a review. Med Mycol. 2009;47:387–97.

    Article  PubMed  Google Scholar 

  6. Sudfeld CR, Dasenbrook EC, Merz WG, Carroll KC, Boyle MP. Prevalence and risk factors for recovery of filamentous fungi in individuals with cystic fibrosis. J Cyst Fibros. 2010;9:110–6.

    Article  PubMed  Google Scholar 

  7. Luong ML, Chaparro C, Stephenson A, et al. Pretransplant Aspergillus colonization of cystic fibrosis patients and the incidence of post-lung transplant invasive aspergillosis. Transplantation. 2014;97:351–7.

    Article  CAS  PubMed  Google Scholar 

  8. Symoens F, Knoop C, Schrooyen M, et al. Disseminated Scedosporium apiospermum infection in a cystic fibrosis patient after double-lung transplantation. J Heart Lung Transplant. 2006;25:603–7.

    Article  PubMed  Google Scholar 

  9. Chotirmall SH, O’Donoghue E, Bennett K, et al. Sputum Candida albicans presages FEV1 decline and hospital-treated exacerbations in cystic fibrosis. Chest. 2010;138:1186–95.

    Article  PubMed  Google Scholar 

  10. Amin R, Dupuis A, Aaron SD, Ratjen F. The effect of chronic infection with Aspergillus fumigatus on lung function and hospitalization in patients with cystic fibrosis. Chest. 2010;137:171–6.

    Article  PubMed  Google Scholar 

  11. de Vrankrijker AMM, van der Ent CK, van Berkhout FT, et al. Aspergillus fumigatus colonization in cystic fibrosis: implications for lung function? Clin Microbiol Infect. 2011;17:1381–6.

    Article  PubMed  Google Scholar 

  12. Kondori N, Gilljam M, Lindblad A, et al. High rate of Exophiala dermatitidis recovery in the airways of patients with cystic fibrosis is associated with pancreatic insufficiency. J Clin Microbiol. 2011;49:1004–9.

    Article  PubMed  PubMed Central  Google Scholar 

  13. Aaron SD, Vandemheen KL, Freitag A, et al. Treatment of Aspergillus fumigatus in patients with cystic fibrosis: a randomized, placebo-controlled pilot study. PLoS ONE. 2012;7:e36077.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Saunders RV, Modha DE, Claydon A, Gaillard EA. Chronic Aspergillus fumigatus colonization of the pediatric cystic fibrosis airway is common and may be associated with a more rapid decline in lung function. Med Mycol. 2016;54:537–43.

    Article  PubMed  Google Scholar 

  15. Fillaux J, Brémont F, Murris M, et al. Assessment of Aspergillus sensitization or persistent carriage as a factor in lung function impairment in cystic fibrosis patients. Scand J Infect Dis. 2012;44:842–7.

    Article  CAS  PubMed  Google Scholar 

  16. Borman AM, Palmer MD, Delhaes L, et al. Lack of standardization in the procedures for mycological examination of sputum samples from CF patients: a possible cause for variations in the prevalence of filamentous fungi. Med Mycol. 2010;48(Suppl 1):S88–97.

    Article  PubMed  Google Scholar 

  17. Liu JC, Modha DE, Gaillard EA. What is the clinical significance of filamentous fungi positive sputum cultures in patients with cystic fibrosis? J Cyst Fibros. 2013;12:187–93.

    Article  PubMed  Google Scholar 

  18. Blyth CC, Harun A, Middleton PG, et al. 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.

    Article  CAS  PubMed  Google Scholar 

  19. Delhaes L, Monchy S, Fréalle E, et al. The airway microbiota in cystic fibrosis: a complex fungal and bacterial community—implications for therapeutic management. PLoS ONE. 2012;7:e36313.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Rosenstein BJ, Cutting GR. The diagnosis of cystic fibrosis: a consensus statement. Cystic Fibrosis Foundation Consensus Panel. J Pediatr. 1998;132:589–95.

    Article  CAS  PubMed  Google Scholar 

  21. Pashley CH, Fairs A, Morley JP, et al. Routine processing procedures for isolating filamentous fungi from respiratory sputum samples may underestimate fungal prevalence. Med Mycol. 2012;50:433–8.

    Article  PubMed  Google Scholar 

  22. Rainer J, Kaltseis J, de Hoog SG, Summerbell RC. Efficacy of a selective isolation procedure for members of the Pseudallescheria boydii complex. Antonie Van Leeuwenhoek. 2007;93:315–22.

    Article  PubMed  Google Scholar 

  23. Moles DR, Bedi R. A simple technique for data management in general dental practice audit. Prim Dent Care. 1997;4:61–5.

    CAS  PubMed  Google Scholar 

  24. Song Y, Lu Y. Decision tree methods: applications for classification and prediction. Shanghai Arch Psychiatry. 2015;27:130–5.

    PubMed  PubMed Central  Google Scholar 

  25. Nagano Y, Elborn JS, Millar BC, et al. Comparison of techniques to examine the diversity of fungi in adult patients with cystic fibrosis. Med Mycol. 2010;48:166–76.

    Article  CAS  PubMed  Google Scholar 

  26. Güngör O, Tamay Z, Güler N, Erturan Z. Frequency of fungi in respiratory samples from Turkish cystic fibrosis patients. Mycoses. 2013;56:123–9.

    Article  PubMed  Google Scholar 

  27. 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.

    Article  PubMed  PubMed Central  Google Scholar 

  28. Paugam A, Baixench MT, Demazes-Dufeu N, et al. Characteristics and consequences of airway colonization by filamentous fungi in 201 adult patients with cystic fibrosis in France. Med Mycol. 2010;48(Suppl 1):S32–6.

    Article  PubMed  Google Scholar 

  29. Valenza G, Tappe D, Turnwald D, et al. Prevalence and antimicrobial susceptibility of microorganisms isolated from sputa of patients with cystic fibrosis. J Cyst Fibros. 2008;7:123–7.

    Article  CAS  PubMed  Google Scholar 

  30. Masoud-Landgraf L, Badura A, Eber E, et al. Modified culture method detects a high diversity of fungal species in cystic fibrosis patients. Med Mycol. 2014;52:179–86.

    PubMed  Google Scholar 

  31. Nielsen SM, Kristensen L, Søndergaard A, et al. Increased prevalence and altered species composition of filamentous fungi in respiratory specimens from cystic fibrosis patients. APMIS. 2014;122:1007–12.

    Article  PubMed  Google Scholar 

  32. Ren CL, Pasta DJ, Rasouliyan L, et al. Relationship between inhaled corticosteroid therapy and rate of lung function decline in children with cystic fibrosis. J Pediatr. 2008;153:746–51.

    Article  CAS  PubMed  Google Scholar 

  33. Burgel PR, Paugam A, Hubert D, Martin C. Aspergillus fumigatus in the cystic fibrosis lung: pros and cons of azole therapy. Infect Drug Resist. 2016;9:229–38.

    Article  PubMed  PubMed Central  Google Scholar 

  34. Chotirmall SH, McElvaney NG. Fungi in the cystic fibrosis lung: bystanders or pathogens? Int J Biochem Cell Biol. 2014;52:161–73.

    Article  CAS  PubMed  Google Scholar 

  35. Whiteson KL, Bailey B, Bergkessel M, et al. The upper respiratory tract as a microbial source for pulmonary infections in cystic fibrosis. Parallels from island biogeography. Am J Respir Crit Care Med. 2014;189:1309–15.

    Article  PubMed  PubMed Central  Google Scholar 

  36. Nagano Y, Millar BC, Goldsmith CE, et al. Development of selective media for the isolation of yeasts and filamentous fungi from the sputum of adult patients with cystic fibrosis (CF). J Cyst Fibros. 2008;7:566–72.

    Article  PubMed  Google Scholar 

  37. Kerr J. Inhibition of fungal growth by Pseudomonas aeruginosa and Pseudomonas cepacia isolated from patients with cystic fibrosis. J Infect. 1994;28:305–10.

    Article  CAS  PubMed  Google Scholar 

  38. Horré R, Marklein G, Siekmeier R, Nidermajer S, Reiffert SM. Selective isolation of Pseudallescheria and Scedosporium species from respiratory tract specimens of cystic fibrosis patients. Respiration. 2009;77:320–4.

    Article  PubMed  Google Scholar 

  39. Cortez KJ, Roilides E, Quiroz-Telles F, et al. Infections caused by Scedosporium spp. Clin Microbiol Rev. 2008;21:157–97.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Horré R, Marklein G, Siekmeier R, Reiffert S-M. Detection of hyphomycetes in the upper respiratory tract of patients with cystic fibrosis. Mycoses. 2011;54:514–22.

    Article  PubMed  Google Scholar 

  41. Lebecque P, Leonard A, Huang D, et al. Exophiala (Wangiella) dermatitidis and cystic fibrosis—prevalence and risk factors. Med Mycol. 2010;48(Suppl 1):S4–9.

    Article  PubMed  Google Scholar 

  42. Cimon B, Carrère J, Chazalette JP, et al. Fungal colonization and immune response to fungi in cystic fibrosis. J Mycol Méd. 1995;5:211–6.

    Google Scholar 

  43. Bakare N, Rickerts V, Bargon J, Just-Nübling G. Prevalence of Aspergillus fumigatus and other fungal species in the sputum of adult patients with cystic fibrosis. Mycoses. 2003;46:19–23.

    Article  CAS  PubMed  Google Scholar 

  44. 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.

    Article  CAS  PubMed  Google Scholar 

  45. Baxter CG, Dunn G, Jones AM, et al. Novel immunologic classification of aspergillosis in adult cystic fibrosis. J Allergy Clin Immunol. 2013;132(560–566):e10.

    Google Scholar 

  46. Rougeron A, Giraud S, Razafimandimby B, et al. Different colonization patterns of Aspergillus terreus in patients with cystic fibrosis. Clin Microbiol Infect. 2014;20:327–33.

    Article  CAS  PubMed  Google Scholar 

  47. Cimon B, Carrère J, Vinatier JF, et al. Clinical significance of Scedosporium apiospermum in patients with cystic fibrosis. Eur J Clin Microbiol Infect Dis. 2000;19:53–6.

    Article  CAS  PubMed  Google Scholar 

  48. Russell GK, Gadhok R, Simmonds NJ. The destructive combination of Scedosporium apiospermum lung disease and exuberant inflammation in cystic fibrosis. Paediatr Respir Rev. 2013;14(Suppl 1):22–5.

    Article  PubMed  Google Scholar 

  49. Guarro J, Kantarcioglu AS, Horré R, et al. Scedosporium apiospermum: changing clinical spectrum of a therapy-refractory opportunist. Med Mycol. 2006;44:295–327.

    Article  PubMed  Google Scholar 

  50. Rodriguez-Tudela JL, Berenguer J, Guarro J, et al. Epidemiology and outcome of Scedosporium prolificans infection, a review of 162 cases. Med Mycol. 2009;47:359–70.

    Article  PubMed  Google Scholar 

  51. Muthig M, Hebestreit A, Ziegler U, Seidler M. Müller F-MC. Persistence of Candida species in the respiratory tract of cystic fibrosis patients. Med Mycol. 2010;48:56–63.

    Article  CAS  PubMed  Google Scholar 

  52. LiPuma JJ. The changing microbial epidemiology in cystic fibrosis. Clin Microbiol Rev. 2010;23:299–323.

    Article  PubMed  PubMed Central  Google Scholar 

  53. Chotirmall SH, Greene CM, McElvaney NG. Candida species in cystic fibrosis: a road less travelled. Med Mycol. 2010;48(Suppl 1):S114–24.

    Article  PubMed  Google Scholar 

  54. Packeu A, Lebecque P, Rodriguez-Villalobos H, et al. Molecular typing and antifungal susceptibility of Exophiala isolates from patients with cystic fibrosis. J Med Microbiol. 2012;61:1226–33.

    Article  CAS  PubMed  Google Scholar 

  55. Kondori N, Lindblad A, Welinder-Olsson C, Wennerås C, Gilljam M. Development of IgG antibodies to Exophiala dermatitidis is associated with inflammatory responses in patients with cystic fibrosis. J Cyst Fibros. 2014;13:391–9.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

L. Delhaes has received research grants from the French Ministry of Health and Research (PHRC N°2006/1902), Lille Hospital, the association “Vaincre la Mucoviscidose” (Defeat Cystic Fibrosis) (MucoFong and Mucofong-ATF N8 2006/351) and the Pharmaceutical Division of Pfizer France (Nu 2006/158). The authors would like to thank Arnaud Hautecoeur for his assistance in organizing the data set.

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Author notes

  1. Sylvie Leroy

    Present address: Centre Hospitalier Universitaire de Nice - Hôpital Pasteur, Nice, France

  2. Laurence Delhaes

    Present address: Laboratoire de Parasitologie-Mycologie, Centre Hospitalier Universitaire, INSERM U1045, Université de Bordeaux, Bordeaux, France

Authors and Affiliations

  1. Laboratoire de Parasitologie-Mycologie, Centre Hospitalier Universitaire, Angers, France

    Noémie Coron, Marc Pihet & Jean-Philippe Bouchara

  2. Laboratoire de Parasitologie-Mycologie, Centre Hospitalier Universitaire de Lille, Université de Lille 2, Lille, France

    Emilie Fréalle & Laurence Delhaes

  3. Service de Parasitologie-Mycologie, Pôle de Biologie et Pathologie, CHU Grenoble-Alpes, Université Grenoble Alpes, Grenoble, France

    Yolande Lemeille, Claudine Pinel & Hervé Pelloux

  4. Laboratoire de Parasitologie-Mycologie, Centre Hospitalier Universitaire Charles Nicolle, Rouen, France

    Gilles Gargala & Loic Favennec

  5. Laboratoire de Parasitologie-Mycologie, Centre Hospitalier Universitaire, Université de Bordeaux, Bordeaux, France

    Isabelle Accoceberry

  6. Hygiène hospitalière, Centre Hospitalier de Dunkerque, Dunkerque, France

    Isabelle Durand-Joly

  7. Laboratoire de Parasitologie-Mycologie, Service Microbiologie Agents Transmissibles, Centre Hospitalier Universitaire, Dijon, France

    Frédéric Dalle

  8. CRCM mixte, Hôpital d’Enfants, Centre Hospitalier Universitaire, Dijon, France

    Frédéric Huet & Annlyse Fanton

  9. CRCM, Centre Hospitalier de Dunkerque, Dunkerque, France

    Amale Boldron & Guy-André Loeuille

  10. CRCM Adulte, Centre Hospitalier Universitaire, Bordeaux, France

    Philippe Domblides

  11. CRCM Adulte, Centre Hospitalier Universitaire, Grenoble, France

    Bérengère Coltey

  12. Pédiatrie, Centre Hospitalier Universitaire de Grenoble Alpes, Grenoble, France

    Isabelle Pin & Catherine Llerena

  13. CRCM mixte, Centre Hospitalier Universitaire, Angers, France

    Françoise Troussier & Christine Person

  14. CRCM mixte, Normandie Univ, UNIROUEN, EA2656/Inserm U1404, département de Pediatrie, Centre Hospitalier Universitaire de Rouen, 76000, Rouen, France

    Christophe Marguet

  15. CRCM de pédiatrie, Centre Hospitalier Universitaire de Lille, Université de Lille 2, Lille, France

    Nathalie Wizla, Caroline Thumerelle & Dominique Turck

  16. CRCM de pédiatrie, CIC 1401, Centre Hospitalier Universitaire de Bordeaux, Université de Bordeaux, Bordeaux, France

    Stéphanie Bui & Michael Fayon

  17. CERIM, EA2694, Faculté de Médecine, Lille, France

    Alain Duhamel

  18. CRCM adulte, Centre Hospitalier Universitaire de Lille, Université de Lille 2, Lille, France

    Anne Prévotat, Benoit Wallaert & Sylvie Leroy

  19. Laboratoire de Parasitologie-Mycologie, Centre Hospitalier Universitaire, INSERM U1045, Université de Bordeaux, Bordeaux, France

    Noémie Coron

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  1. Noémie Coron
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  2. Marc Pihet
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  33. Laurence Delhaes
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Correspondence to Laurence Delhaes.

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Coron, N., Pihet, M., Fréalle, E. et al. Toward the Standardization of Mycological Examination of Sputum Samples in Cystic Fibrosis: Results from a French Multicenter Prospective Study. Mycopathologia 183, 101–117 (2018). https://doi.org/10.1007/s11046-017-0173-1

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