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

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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.

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Funding

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).

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Authors and Affiliations

  1. Department of Internal Medicine – Hematology and Oncology, University Hospital Brno, Brno, Czech Republic

    Dita Ricna, Martina Lengerova, Matej Bezdicek, Barbora Weinbergerova, Jiri Mayer & Zdenek Racil

  2. Department of Internal Medicine – Hematology and Oncology, Faculty of Medicine, Masaryk University, Brno, Czech Republic

    Dita Ricna, Martina Lengerova, Matej Bezdicek, Barbora Weinbergerova, Jiri Mayer & Zdenek Racil

  3. Center of Molecular Biology and Gene Therapy, Department of Internal Medicine - Hematology and Oncology, University Hospital Brno, Cernopolni 9, 613 00, Brno, Czech Republic

    Martina Lengerova

  4. Department of Clinical Microbiology, University Hospital Brno, Brno, Czech Republic

    Iva Kocmanova

  5. Department of Oncohematology, Comenius University in Bratislava and National Cancer Institute, Bratislava, Slovakia

    Lubos Drgona

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  1. Dita Ricna
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  2. Martina Lengerova
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Correspondence to Martina Lengerova.

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

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Supplementary Table 1

List of sequences obtained in this study (XLSX 25 kb)

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Ricna, D., Lengerova, M., Bezdicek, M. et al. Detection and identification of fungi in bronchoalveolar lavage fluid from immunocompromised patients using panfungal PCR. Folia Microbiol 64, 421–428 (2019). https://doi.org/10.1007/s12223-018-00669-w

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  • DOI: https://doi.org/10.1007/s12223-018-00669-w

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