Clinical performance of the multiplex solid-phase "Direct Strip PCR" for infectious uveitis: a multicenter diagnostic accuracy study.

Abstract

Purpose: To evaluate the clinical performance of "Direct Strip PCR," a multiplex solid-phase real-time polymerase chain reaction (PCR) kit, for diagnosing infectious uveitis.

Study design: Multicenter, prospective, diagnostic accuracy study METHODS: We analyzed 475 ocular-fluid samples (336 aqueous humor and 139 vitreous fluid) from 29 sites in Japan. Direct Strip PCR, an IVD-grade multiplex real-time PCR kit targeting HSV-1, HSV-2, VZV, EBV, CMV, HHV-6, HTLV-1, Toxoplasma gondii, and Treponema pallidum requiring no DNA extraction and incorporating internal controls, was compared with quantitative PCR (qPCR) for concordance and DNA copy-number correlation.

Results: All 9 of the target pathogens were detected. Direct Strip PCR showed excellent agreement with qPCR, with percent positive agreement (PPA), percent negative agreement (PNA), and percent overall agreement (POA) in aqueous humor of 98.0%, 99.2%, and 98.5%, respectively, and in vitreous fluid of 95.7%, 97.1%, and 96.4%, respectively, indicating high concordance for positive and negative results. All the values exceeded predefined 90% thresholds agreed upon with the Pharmaceuticals and Medical Devices Agency, meeting the required performance criteria. Discordant results were infrequent (10/4275 targets) and involved low-copy samples near the detection limit. The DNA copy numbers correlated strongly between the methods (r = 0.948-0.996). No adverse events were reported.

Conclusion: Direct Strip PCR demonstrated high concordance with qPCR, reliably detected major pathogens of infectious uveitis, and yielded quantitative results that correlated with the qPCR results, supporting disease monitoring. Its solid-phase, extraction-free, and per-run calibration-free format provides a practical basis for regulatory submissions and global dissemination of multiplex PCR.