Laboratory validation of two pathogen detection assays utilizing amplicon sequencing and CRISPR-Cas12a for the diagnosis of lower respiratory tract infections

The underdevelopment of rapid and accurate microbiological tests has contributed to the diagnostic delay and inappropriate antibiotic use in patients with lower respiratory tract infections, which is the fourth leading cause of death globally. While conventional tests cannot fully meet the clinical needs for identifying a broad range of pathogens within an actionable timeframe, culture-independent diagnostic methods have gained traction, such as the unbiased metagenomic Next-Generation Sequencing. However, it is cost-ineffective and may subject to contaminating or colonizing microorganisms. Here we designed two targeted nucleic acid-based assays for the detection of a wide variety of respiratory pathogens from bronchoalveolar lavage fluid, including bacteria, fungi, DNA/RNA viruses, and parasites. One test utilized amplicon Next-Generation Sequencing to identify 363 pathogens while the other used multiplex polymerase chain reaction and CRISPR-Cas12a to detect 174 pathogens. Using mock samples containing spiked-in pathogens and clinical specimen of known microbiology results as reference standards, we evaluated the analytical performances of both tests, which showed high accuracy in detecting various types of pathogens. These assays were relatively easy to implement and suitable for the etiological diagnosis of lower respiratory tract infections.