Clinical Assessment of a Highly Multiplexed Panel Assay for the Diagnosis of Infectious Uveitis.

PURPOSE: Current diagnostic workup for infectious uveitis relies on the use of various immunoassays and polymerase chain reaction (PCR) tests on ocular fluids that are time consuming and not comprehensive, requiring large sample volumes to probe for all causes. To streamline the diagnostic process, improve turnaround time, and target inclusivity, we developed a highly multiplexed pan-ocular-pathogen panel (OcuPan) that can be used for broad-range pathogen detection in only 1 assay, with PCR-level sensitivity and in a timely fashion (12 hours). Here, we assessed the clinical usefulness and performance of this innovative assay as a novel tool for rapid and accurate laboratory diagnosis of infectious uveitis.

DESIGN

Laboratory evaluation of a novel diagnostic test and technology PARTICIPANTS AND CONTROLS: A total of 109 patients from 2 centers were included, including 78 infectious uveitis cases and 31 controls.

METHODS

Intraocular samples (n = 108 from patients and 43 from controls) were processed by PCR and the OcuPan diagnostic assay that detects 46 ocular pathogens plus 2 resistance/virulence markers.

MAIN OUTCOME MEASURES: Clinical sensitivity and specificity, quantification capabilities, and agreement between PCR and the OcuPan assay.

RESULTS

The clinical sensitivity and specificity of the OcuPan assay were 50.9% (95% CI, 41.3-60.5) and 100% (95% CI 89.7%-100%), respectively. Sensitivity varied according to the sample matrix tested, with undiluted vitreous having the highest positivity rates (72.4%, 95% CI 55.1-89.7), followed by dilute vitreous (53.3%, 95% CI 34.4-72.3) and aqueous (36.7%, 95% CI 22.7-50.7). There was excellent overall agreement (93.5%) between the OcuPan assay and PCR (kappa of 0.870 ± 0.047; P < .001) with positive (PPA) and negative (NPA) percentage agreements >92% for all targets. PPA was 100% for herpesviruses and Treponema pallidum, whereas the NPA values ranged from 96.6% to 100% for these pathogens. For Toxoplasma gondii, the PPA was 75% and NPA 100%. We also found significant correlation (Spearman ρ = -0.7506, P < .0001) between the quantitative metric for the OcuPan assay and the real-time PCR cycle threshold values.

CONCLUSIONS

The OcuPan assay offers an all-in-one highly multiplexed detection system for rapid, comprehensive, quantitative, and accurate diagnosis of infectious uveitis.