Rapid detection of fungal pathogens in bronchoalveolar lavage samples using panfungal PCR combined with high resolution melting analysis

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Authors

BEZDÍČEK Matěj LENGEROVÁ Martina ŘÍČNÁ Dita WEINBERGEROVÁ Barbora KOCMANOVA Iva VOLFOVA Pavlina DRGONA Lubos POCZOVA Miroslava MAYER Jiří RÁČIL Zdeněk

Year of publication 2016
Type Article in Periodical
Magazine / Source Medical Mycology
MU Faculty or unit

Faculty of Medicine

Citation
Doi http://dx.doi.org/10.1093/mmy/myw032
Field Epidemiology, infectious diseases and clinical immunology
Keywords Panfungal PCR; High resolution melting analysis; Bronchoalveolar lavage fluid; Immunocompromised patients
Attached files
Description 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.
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