Rapid Laboratory Confirmation of the 2025 Ebola Sudan Virus Disease (Suvd) Index Case in Uganda

Background On January 30, 2025, the Uganda Ministry of Health declared an outbreak of Sudan ebolavirus ( SUDV ) disease following laboratory confirmation from postmortem samples of a suspected case identified through routine mortality surveillance for viral hemorrhagic fevers (VHFs) at Mulago National Referral Hospital, Kampala. This report describes the laboratory procedures used to confirm the 2025 SUDV index case, emphasizing the vital role of rapid diagnostics in containing VHFs. Methods We leveraged existing surveillance infrastructure to collect, package, transport, test, and report results for the index-confirmed case. Testing was performed within the framework of the established laboratory quality and bio risk management system at the Central Emergency Response and Surveillance Laboratory. The workflow consisted of sample delivery, reception, preparation, nucleic acid extraction, master mix preparation, real-time polymerase chain Reaction (RT-PCR) differential diagnosis for VHFs, parallel PCR testing and typing for SUDV confirmation, result validation, and reporting. Results The sample tested negative for Marburg fever, Yellow fever, Rift Valley fever, and Crimean-Congo Hemorrhagic Fever viruses. However, it tested positive for SUDV . Four parallel tests utilizing the Real Star® Filovirus Screen RT-PCR Kit 1.0 (Altona Diagnostics GmbH, Hamburg, Germany) on CFX96 Bio-Rad platforms, with varying sample volumes (140 µL, 90 µL, 50 µL, and 30 µL diluted with nuclease-free water), confirmed the presence of SUDV . The 50 µL sample had the lowest Ct values for the Ebolavirus target, while the 30 µL sample had the lowest Ct values for the internal control target. The turnaround time, from sample reception to results reporting to the Ministry of Health leadership, was less than 12 hours. Conclusion Mortality surveillance of VHFs is essential for rapid detection of Filovirus outbreaks in high-risk regions, and performing comprehensive VHFs panel testing is crucial for thorough pathogen detection in suspected cases. Sample dilution significantly impacts diagnostic sensitivity; therefore, optimizing testing protocols is necessary for accurate molecular diagnostics. A short turnaround time is pivotal in outbreak scenarios, as it facilitates prompt decision-making for public health responses, such as case isolation, contact tracing, and resource mobilization.