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

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Supplementary Information: The online version contains supplementary material available at 10.1186/s12967-025-06784-w.