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Optic neuritis (ON) may be the initial manifestation of neuromyelitis optica spectrum disorder (NMOSD). Aquaporin-4 antibody (AQP4 Ab) is used to diagnose NMOSD. This has implications on prognosis and is important for optimal management. We aim to evaluate if clinical features can distinguish AQP4 Ab seropositive and seronegative ON patients.We reviewed patients with first episode of isolated ON from Tan Tock Seng Hospital and Singapore National Eye Centre who tested for AQP4 Ab from 2008 to 2017. Demographic and clinical data were compared between seropositive and seronegative patients.Among 106 patients (120 eyes) with first episode of isolated ON, 23 (26 eyes; 22%) were AQP4 Ab positive and 83 (94 eyes; 78%) were AQP4 Ab negative. At presentation, AQP4 Ab positive patients had older mean onset age (47.9 ± 13.6 vs 36.8 ± 12.6 years, Plt; 0.001), worse nadir VA (OR 1.714; 95% CI, 1.36 to 2.16; Plt; 0.001), less optic disc swelling (OR 5.04; 95% CI, 1.682 to 15.073; p = 0.004), and higher proportions of concomitant anti-Ro antibody (17% vs 4%, p = 0.038) and anti-La antibody (17% vs 1%, p = 0.008). More AQP4 Ab positive patients received steroid-sparing immunosuppressants (74% vs 19%, plt; 0.001) and plasma exchange (13% vs 0%, p = 0.009). AQP4 Ab positive patients had worse mean logMAR VA (visual acuity) at 12 months (0.70 ± 0.3 vs 0.29 ± 0.5, p = 0.051) and 36 months (0.37±0.4 vs 0.14 ± 0.2, p = 0.048) follow-up.Other than older onset age and retrobulbar optic neuritis, clinical features are non-discriminatory for NMOSD. We propose a low threshold for AQP4 Ab serology testing in inflammatory ON patients, particularly in high NMOSD prevalence populations, to minimize diagnostic and treatment delays.