Clinical Utility of Targeted Third-Generation Sequencing for Pathogen Detection in Community-Acquired Pneumonia: A Real-World Comparative Study of China.

Objective

To evaluate the diagnostic performance and clinical utility of targeted third-generation sequencing (tTGS) for pathogen detection in community-acquired pneumonia (CAP), compared with targeted next-generation sequencing (tNGS) and conventional culture. METHODOLOGY We conducted a real-world, retrospective study including 356 CAP patients who were tested in parallel with culture, tNGS, and tTGS. Diagnostic sensitivity, pathogen spectrum, mixed-infection detection, and antimicrobial resistance (AMR) gene profiling were compared across methods. Clinical relevance was assessed by analyzing treatment adjustments informed by sequencing results.

Results

tTGS demonstrated the highest overall sensitivity (99.16%), surpassing tNGS (96.35%, p < 0.05) and culture (50.27%, p < 0.01). Across all samples, tTGS identified 135 microbial species - substantially more than tNGS (84 species) and culture (27 species) - including fastidious, rare, and slow-growing pathogens. tTGS also showed an improved ability to detect mixed infections (86.24% vs. 87.64% with tNGS; culture detected none). AMR gene detection was significantly higher with tTGS than with tNGS (58.99% vs. 48.88%, p = 0.008), and long-read sequencing enabled the identification of composite resistance patterns that tNGS missed. Among patients without underlying diseases, sequencing-guided therapeutic adjustment occurred in 67.87% of cases, with nearly 80% of AMR-positive patients demonstrating clinical improvement following regimen optimization.

Conclusions

tTGS provides markedly enhanced pathogen detection, greater sensitivity, and more comprehensive AMR profiling than both tNGS and culture. Its strong performance in identifying mixed infections and actionable determinants of resistance supports its incorporation into clinical diagnostic workflows for CAP. Further large-scale, prospective studies are warranted to validate its clinical impact and optimize its integration into routine practice.