title: Rapid detection of fungal pathogens in bronchoalveolar lavage samples using panfungal PCR combined with high resolution melting analysis.
abstract: Despite advances in the treatment of invasive fungal diseases (IFD), mortality rates remain high. Moreover, due to the expanding spectrum of causative agents, fast and accurate pathogen identification is necessary. We designed a panfungal polymerase chain reaction (PCR), which targets the highly variable ITS2 region of rDNA genes and uses high resolution melting analysis (HRM) for subsequent species identification. The sensitivity and specificity of this method was tested on a broad spectrum of the most clinically important fungal pathogens including Aspergillus spp., Candida spp. and mucormycetes. Despite the fact that fluid from bronchoalveolar lavage (BAL) is one of the most frequently tested materials there is a lack of literature sources aimed at panfungal PCR as an IFD diagnostic tool from BAL samples. The applicability of this method in routine practice was evaluated on 104 BAL samples from immunocompromised patients. Due to high ITS region variability, we obtained divergent melting peaks for different fungal species. Thirteen out of 18 patients with proven or probable IFD were positive. Therefore, the sensitivity, specificity, positive predictive value and negative predictive value of our method were 67%, 100%, 100%, and 94%, respectively. In our assay, fungal pathogens identification is based on HRM, therefore omitting the expensive and time consuming sequencing step. With the high specificity, positive and negative predictive values, short time needed to obtain a result, and low price, the presented assay is intended to be used as a quick screening method for patients at risk of IFD.
authors.affiliation: Department of Internal Medicine - Hematology and Oncology, University Hospital Brno, Brno, Czech Republic Department of Internal Medicine - Hematology and Oncology, Faculty of Medicine, Masaryk University, Brno, Czech Republic.
authors.forename: Matej
authors.initials: M
authors.lastname: Bezdicek
authors.affiliation: Department of Internal Medicine - Hematology and Oncology, University Hospital Brno, Brno, Czech Republic Department of Internal Medicine - Hematology and Oncology, Faculty of Medicine, Masaryk University, Brno, Czech Republic CEITEC - Central European Institute of Technology, Masaryk University, Brno, Czech Republic mlengerova@fnbrno.cz.
authors.forename: Martina
authors.initials: M
authors.lastname: Lengerova
authors.affiliation: Department of Internal Medicine - Hematology and Oncology, University Hospital Brno, Brno, Czech Republic Department of Internal Medicine - Hematology and Oncology, Faculty of Medicine, Masaryk University, Brno, Czech Republic.
authors.forename: Dita
authors.initials: D
authors.lastname: Ricna
authors.affiliation: Department of Internal Medicine - Hematology and Oncology, University Hospital Brno, Brno, Czech Republic Department of Internal Medicine - Hematology and Oncology, Faculty of Medicine, Masaryk University, Brno, Czech Republic.
authors.forename: Barbora
authors.initials: B
authors.lastname: Weinbergerova
authors.affiliation: Department of Clinical Microbiology, University Hospital Brno, Brno, Czech Republic.
authors.forename: Iva
authors.initials: I
authors.lastname: Kocmanova
authors.affiliation: Department of Internal Medicine - Hematology and Oncology, University Hospital Brno, Brno, Czech Republic.
authors.forename: Pavlina
authors.initials: P
authors.lastname: Volfova
authors.affiliation: Department of Oncohematology, Comenius University in Bratislava and National Cancer Institute, Bratislava, Slovakia.
authors.forename: Lubos
authors.initials: L
authors.lastname: Drgona
authors.affiliation: Department of Mycology, HPL Ltd., Bratislava, Slovakia.
authors.forename: Miroslava
authors.initials: M
authors.lastname: Poczova
authors.affiliation: Department of Internal Medicine - Hematology and Oncology, University Hospital Brno, Brno, Czech Republic Department of Internal Medicine - Hematology and Oncology, Faculty of Medicine, Masaryk University, Brno, Czech Republic CEITEC - Central European Institute of Technology, Masaryk University, Brno, Czech Republic.
authors.forename: Jiri
authors.initials: J
authors.lastname: Mayer
authors.affiliation: Department of Internal Medicine - Hematology and Oncology, University Hospital Brno, Brno, Czech Republic Department of Internal Medicine - Hematology and Oncology, Faculty of Medicine, Masaryk University, Brno, Czech Republic CEITEC - Central European Institute of Technology, Masaryk University, Brno, Czech Republic.
authors.forename: Zdenek
authors.initials: Z
authors.lastname: Racil
mesh_terms: D001992:Bronchoalveolar Lavage Fluid / Q000382:microbiology*; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics*; D021903:DNA, Ribosomal Spacer / Q000737:chemistry / Q000235:genetics; D005260:Female; D005658:Fungi / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008172:Lung Diseases, Fungal / Q000175:diagnosis* / Q000382:microbiology; D008297:Male; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011237:Predictive Value of Tests; D012680:Sensitivity and Specificity; D013997:Time Factors; D044366:Transition Temperature
chemical_list: D004271:DNA, Fungal; D021903:DNA, Ribosomal Spacer
keywords: Bronchoalveolar lavage fluid; High resolution melting analysis; Immunocompromised patients; Panfungal PCR