Abstract
Background/Objectives: Mpox re-emerged in 2022 as a global health concern. Between 2022 and 2025, Portugal experienced three distinct outbreak waves, highlighting the critical role of laboratory surveillance and public health interventions. This study describes the epidemiological trends, diagnostic performance, and key lessons learned to improve outbreak preparedness. Methods: A total of 5610 clinical samples from 2802 suspected cases were analyzed at the National Institute of Health Doutor Ricardo Jorge using real-time PCR methods. Positivity rates and viral loads (Ct values) were assessed across different clinical specimen types, including lesion, anal, oropharyngeal swabs, and urine samples. Results: Mpox was confirmed in 1202 patients. The first outbreak accounted for 79.3% of cases (n = 953), followed by a significant reduction in transmission during subsequent waves. Lesion and rectal swabs provided the highest diagnostic sensitivity (95.1% and 87.9%, respectively). Oropharyngeal swabs contributed to diagnosis in cases without visible lesions, while urine samples showed limited utility. Conclusions: This study underscores the importance of sustained laboratory surveillance and adaptive public health strategies in controlling mpox outbreaks. Optimizing specimen collection enhances diagnostic accuracy, supporting early detection. Continuous monitoring, combined with targeted vaccination and effective risk communication, is essential to prevent resurgence and ensure rapid response in non-endemic regions.