Development of a Multiplex Real-Time PCR Assay for the Simultaneous Detection of Two Fungal Pathogens Causing Pneumonia

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.

Keywords: Aspergillus fumigatus; Pneumocystis jirovecii; diagnostic accuracy study; fungal respiratory infection; molecular diagnostics; multiplex real-time PCR.

Figure 1

The limit of detection (LOD) of the RF2 mRT-PCR assay. The LOD of the RF2 mRT-PCR for both (A) Pneumocystis jirovecii and (B) Aspergillus fumigatus was calculated using a probit curve. A ten-fold dilution series of synthetic DNA ranging from 1 to 10⁴ copies/reaction was tested for 40 replicates each. The LOD at 95% was extrapolated from the sigmoid curve.

Figure 2

The distribution of A. fumigatus detected using Aspergillus species sequencing with P. jirovecii results. A total of 2332 samples were tested using Aspergillus spp. primers with Sanger sequencing.