B-253 Development and Validation of a Multiplex Real-Time PCR Assay for Simultaneously Detection of Aspergillus, Cryptococcus neoformans, and Pneumocystis jirovecii

Background Approximately 6.5 million new cases of invasive fungal infections (IFI) and 3.8 million deaths are reported annually, affecting over 1 billion people worldwide. Aspergillus, Cryptococcus, and Pneumocystis are the common pathogens leading to IFI. In this study, a multiple real-time polymerase chain reaction (PCR) assay was developed to simultaneously detect and identify three of the most frequent pathogens in sputum and bronchoalveolar lavage fluid (BALF) samples, including Aspergillus, Cryptococcus neoformans, and Pneumocystis jirovecii, and to evaluate its performance on clinical samples. Methods Sputum samples (n=244) and BALF samples (n=299) from patients with suspected IFI were collected from three centres. The diagnostic sensitivity and specificity of the multiplex real-time PCR assay were analysed. DNA sequencing was used as the reference method, and the performance of the multiplex real-time assay was evaluated by determining the positive percent agreement (PPA), negative percent agreement (NPA), and overall coincidence rate. The reproducibility, limit of detection (LoD), and interference experiment of the multiplex real-time PCR assay were also evaluated. Results From the clinical evaluation results, the sensitivity and specificity of the multiplex real-time PCR assay were greater than 90% for Aspergillus, Cryptococcus neoformans, and Pneumocystis jirovecii. The consistency of the multiplex real-time PCR assay and DNA sequencing was almost perfect: the overall coincidence rate of the two methods was close to 100%. The percent coefficient variations (CVs%) of reproducibility was less than 5%, meeting requirements, and the LoD of the kit for each of the three species less than or equal to 1000 copies/mL. No cross-reactivity was detected for any bacteria or fungi. Conclusion The multiplex real-time PCR assay was welestablished with high sensitivity and specificity for rapid and simultaneous detection on three common pathogens in respiratory tract samples.