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Detection and identification of fungi in bronchoalveolar lavage fluid from immunocompromised patients using panfungal PCR

  • Dita Ricna
  • Martina Lengerova
  • Matej Bezdicek
  • Iva Kocmanova
  • Lubos Drgona
  • Barbora Weinbergerova
  • Jiri Mayer
  • Zdenek Racil
Original Article

Abstract

Rapid diagnostics of fungal pneumonia and initiation of appropriate therapy are still challenging. In this study, we used two panfungal assays to test bronchoalveolar lavage fluid (BALF) samples to prove their ability to confirm invasive fungal disease diagnosis and identify causative agents. Two methods targeting different fungal rDNA regions were used, and the obtained PCR products were sequenced directly or after cloning. In total, 106 BALF samples from 104 patients were tested. After sequencing, we obtained 578 sequences. Four hundred thirty-seven sequences were excluded from further analysis due to duplication (n = 335) or similarity with sequences detected in the extraction control sample (n = 102); 141 unique sequences were analyzed. Altogether, 23/141 (16%) of the fungi detected belonged to pathogenic species, and 63/141 (45%) were identified as various yeasts; a variety of environmental or very rare fungal human pathogens represented 29/141 (21%) of the total and 26/141 (18%) were described as uncultured fungus. Panfungal PCR detected fungal species that would be missed by specific methods in only one case (probable cryptococcosis). Panfungal PCR followed by sequencing has limited use for testing BALF samples due to frequent commensal or environmental fungal species pickup.

Notes

Funding information

This study was supported by the Ministry of Health, Czech Republic—conceptual development of research organization (FNBr, 65269705), Grant Agency of the Masaryk University (MUNI/A/1105/2018), and Technology Agency of the Czech Republic (TE02000058).

Compliance with ethical standards

Conflict of interests

ZR is a consultant and speaker for Astellas Pharma and Pfizer. The other authors declare that they have no relevant conflict of interest.

Supplementary material

12223_2018_669_MOESM1_ESM.xlsx (26 kb)
Supplementary Table 1 List of sequences obtained in this study (XLSX 25 kb)

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Copyright information

© Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2018

Authors and Affiliations

  • Dita Ricna
    • 1
    • 2
  • Martina Lengerova
    • 1
    • 2
    • 3
  • Matej Bezdicek
    • 1
    • 2
  • Iva Kocmanova
    • 4
  • Lubos Drgona
    • 5
  • Barbora Weinbergerova
    • 1
    • 2
  • Jiri Mayer
    • 1
    • 2
  • Zdenek Racil
    • 1
    • 2
  1. 1.Department of Internal Medicine – Hematology and OncologyUniversity Hospital BrnoBrnoCzech Republic
  2. 2.Department of Internal Medicine – Hematology and Oncology, Faculty of MedicineMasaryk UniversityBrnoCzech Republic
  3. 3.Center of Molecular Biology and Gene Therapy, Department of Internal Medicine - Hematology and OncologyUniversity Hospital BrnoBrnoCzech Republic
  4. 4.Department of Clinical MicrobiologyUniversity Hospital BrnoBrnoCzech Republic
  5. 5.Department of OncohematologyComenius University in Bratislava and National Cancer InstituteBratislavaSlovakia

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