Related Experiment Video
Updated: Mar 21, 2026
Detection of Invasive Pulmonary Aspergillosis in Haematological Malignancy Patients by using Lateral-flow Technology
Published on: March 22, 2012
Rapid detection of fungal pathogens in bronchoalveolar lavage samples using panfungal PCR combined with high
Matej Bezdicek1, Martina Lengerova2, Dita Ricna1
1Department 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.
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.
Insights
A new panfungal polymerase chain reaction (PCR) with high resolution melting analysis (HRM) offers rapid and accurate identification of fungal pathogens in bronchoalveolar lavage (BAL) samples for invasive fungal diseases (IFD). This cost-effective screening method aids in early diagnosis for at-risk patients.
Area of Science:
- Medical Microbiology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Invasive fungal diseases (IFD) have high mortality rates despite treatment advances.
- Rapid and accurate identification of diverse fungal pathogens is crucial for effective IFD management.
- Bronchoalveolar lavage (BAL) is a common sample type, but diagnostic tools for IFD from BAL are limited.
Purpose of the Study:
- To develop and evaluate a panfungal polymerase chain reaction (PCR) assay combined with high resolution melting analysis (HRM) for the detection and identification of fungal pathogens in BAL samples.
- To assess the diagnostic performance of this assay in immunocompromised patients at risk for IFD.
Main Methods:
- A panfungal PCR targeting the ITS2 region of rDNA genes was designed.
- High resolution melting analysis (HRM) was employed for species identification based on divergent melting peaks.
- The assay was validated on a broad spectrum of clinically important fungal pathogens and tested on 104 BAL samples from immunocompromised patients.
Main Results:
- The panfungal PCR-HRM assay successfully differentiated various fungal species, including Aspergillus spp., Candida spp., and mucormycetes.
- In clinical evaluation, 13 out of 18 patients with proven or probable IFD tested positive.
- The assay demonstrated high specificity (100%), positive predictive value (100%), and negative predictive value (94%), with a sensitivity of 67%.
Conclusions:
- The developed panfungal PCR-HRM assay is a sensitive, specific, and rapid method for identifying fungal pathogens in BAL samples.
- This assay eliminates the need for costly and time-consuming sequencing, offering a low-price alternative for routine diagnostics.
- The assay is suitable as a quick screening tool for early detection of IFD in immunocompromised patients, potentially improving patient outcomes.
