Development of a recombinase-aided amplification assay for rapid and sensitive detection of Pneumocystis jirovecii infection: A single-center molecular study.

Pneumocystis jirovecii (PJ) is a major opportunistic pathogen that causes severe pneumonia in immunocompromised individuals, leading to significant morbidity and mortality. Early and accurate diagnosis is critical for effective clinical management. However, existing diagnostic methods have limitations such as low sensitivity, long turnaround time and high equipment requirements. Recombinase-aided amplification (RAA) is an isothermal nucleic acid amplification technology with advantages of rapidity, simplicity and high sensitivity. The aim of this study was to develop and validate an RAA assay for the rapid detection of PJ. Clinical bronchoalveolar lavage fluid samples were collected from 44 PJ-positive patients confirmed by targeted next-generation sequencing (tNGS) and extracted nucleic acid. A recombinant PJ plasmid (pUC57-PJ) was constructed as the standard positive template. Specific primers and probes for PJ were designed and the RAA reaction conditions were optimized. The analytical performance (sensitivity, specificity) of the RAA method was evaluated using pUC57-PJ and clinical sample nucleic acids. Additionally, the clinical performance of RAA was validated by comparing with tNGS and RAA results. In conclusion, a rapid, sensitive and specific RAA assay for PJ detection was successfully developed. This assay exhibits excellent analytical and clinical performance, and is suitable for rapid diagnosis of PJ infection, particularly in resource-limited settings. It provides a valuable new tool for the early diagnosis and management of PJ pneumonia.