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Updated: Jun 12, 2025
Author Spotlight: Advancements in Multiplex Detection of Respiratory Viruses
Published on: November 10, 2023
Development of a Multiplex Real-Time PCR Assay for the Simultaneous Detection of Two Fungal Pathogens Causing
Ho-Jae Lim1, Seojin Ahn1, Jee-Hyun No1
1Department of Molecular Diagnostics, Seegene Medical Foundation, Seoul 04805, Republic of Korea.
Abstract:
Infectious diseases caused by fungal sources are of great interest owing to their increasing prevalence. Invasive fungal infections, including invasive pulmonary aspergillosis caused by Aspergillus fumigatus, and Pneumocystis pneumonia caused by Pneumocystis jirovecii, are significant causes of morbidity and mortality among immunocompromised patients. The accurate and timely detection of these pathogens in this high-risk population is crucial for effective patient management. We developed a multiplex real-time polymerase chain reaction (PCR) assay, RF2 mRT-PCR, specifically designed to detect two respiratory fungi, P. jirovecii and A. fumigatus, and evaluated its performance in specimens of patients with lower respiratory tract infection. The performance was evaluated using 731 clinical samples, 55 reference species, and one synthetic DNA. The reproducibility test yielded a probit curve with a lower limit of detection of 19.82 copies/reaction for P. jirovecii and 64.20 copies/reaction for A. fumigatus. The RF2 mRT-PCR assay did not cross-react with non-A. fumigatus Aspergillus species or other common bacterial and viral species, and showed 100% in vitro sensitivity and specificity with reference assays. Additionally, it simultaneously detected A. fumigatus and P. jirovecii in co-infected samples. Therefore, the RF2 mRT-PCR assay is an efficient and reliable tool for in vitro diagnosis of A. fumigatus and P. jirovecii pulmonary infections.
Insights
A new multiplex real-time PCR assay, RF2 mRT-PCR, accurately detects fungal pathogens Aspergillus fumigatus and Pneumocystis jirovecii. This tool aids in the rapid diagnosis of invasive pulmonary infections in immunocompromised patients.
Area of Science:
- Medical Mycology
- Clinical Microbiology
- Molecular Diagnostics
Background:
- Invasive fungal infections, particularly invasive pulmonary aspergillosis (Aspergillus fumigatus) and Pneumocystis pneumonia (Pneumocystis jirovecii), pose significant threats to immunocompromised individuals.
- Timely and accurate pathogen detection is critical for managing these life-threatening conditions.
Purpose of the Study:
- To develop and evaluate a novel multiplex real-time polymerase chain reaction (PCR) assay, termed RF2 mRT-PCR.
- The assay is designed for the simultaneous detection of two key respiratory fungal pathogens: P. jirovecii and A. fumigatus.
Main Methods:
- The RF2 mRT-PCR assay was developed and validated using 731 clinical samples, 55 reference species, and synthetic DNA.
- Performance evaluation included assessing reproducibility, limit of detection, and cross-reactivity with other microorganisms.
- Comparison with existing reference assays was conducted to determine in vitro sensitivity and specificity.
Main Results:
- The assay demonstrated high reproducibility with a low limit of detection (19.82 copies/reaction for P. jirovecii, 64.20 copies/reaction for A. fumigatus).
- The RF2 mRT-PCR assay exhibited 100% in vitro sensitivity and specificity, showing no cross-reactivity with other Aspergillus species or common bacterial/viral pathogens.
- Simultaneous detection of co-infections with both A. fumigatus and P. jirovecii was achieved.
Conclusions:
- The RF2 mRT-PCR assay is an efficient and reliable diagnostic tool for the in vitro detection of Aspergillus fumigatus and Pneumocystis jirovecii.
- This assay facilitates accurate diagnosis of pulmonary fungal infections, particularly in high-risk immunocompromised patient populations.
- The multiplex capability allows for the simultaneous identification of common co-infections, improving patient management strategies.
