An innovative fungal-specific targeted next-generation sequencing method: analytical performance and a single-center prospective clinical study

Invasive pulmonary fungal infections (IPFIs) pose significant diagnostic challenges, particularly in immunocompromised patients. Accurate and timely diagnosis is crucial to improve outcomes. While metagenomic next-generation sequencing (mNGS) is widely utilized, it is expensive and affected by host DNA interference. Targeted next-generation sequencing (tNGS) offers a cost-effective and efficient alternative for fungal pathogen detection. We developed the Fi-tNGS assay, a targeted next-generation sequencing method, specifically designed to detect 64 fungal species. Analytical performance was validated by assessing its limit of detection (LoD), reproducibility, and resistance to host DNA interference. Subsequently, a prospective clinical study was conducted, enrolling 104 patients with suspected IPFIs. Clinical diagnostic performance was evaluated by comparing Fi-tNGS, mNGS, and conventional microbial culture against a comprehensive diagnostic standard. Fi-tNGS detected 109 pathogens, compared to 110 for mNGS and 77 for culture. The sensitivity and specificity of tNGS were 89.7% and 94.2%, respectively, outperforming culture (65.8% sensitivity, 100% specificity). Combining culture with tNGS or mNGS significantly improved sensitivity to 94.8% and 94.0%, respectively. These findings demonstrate the added diagnostic value of NGS methods for IPFIs. tNGS provides accurate and efficient fungal pathogen detection, with sensitivity comparable to mNGS and superior to culture. Its cost-effectiveness and shorter turnaround time highlight its potential as a practical tool for the rapid and precise diagnosis of IPFIs in clinical settings.