Introduction: Attributing a causative agent to any Acute Febrile Illness (AFI) in a timely manner remains challenging, yet an important task towards clinical care and public health response. Therefore, the need for a diagnostic assay with capacity to detect a wide array of pathogen targets. A multi-pathogen detection assay could reduce time taken for an etiology to be identified therefore enhancing timely diagnosis and precise patient care and management.  
Methods: This was a cross-sectional retrospective study where VHF negative blood and plasma samples archived at the Uganda Virus Research Institute-Viral Hemorrhagic Fevers (UVRI-VHF) Laboratory (August 2018 to March 2019) from patients presenting with a temperature of ≥37.5 ºC, bleeding and any other febrile symptom were tested on AFI-TAC for 35 potential pathogens. Positive samples were used for assay verification. Epidemiological correlation including demographic and epidemiological correlates to establish causation.
Results: To verify the assay, positive samples, Crimean Congo Hemorrhagic Fever (CCHF) (n= 1) and Rift Valley Virus (RVF) (n=5) were tested on the card and there was 100% agreement between the individual PCR assays specific for CCHF and RVF and the assays on the TAC for the same pathogens. A total of 182 were included of which 152 (81.7%) were from Uganda, 22 (11.8%) from Democratic Republic of Congo, 7 (3.76%) from South Sudan, and 1 (0.54%) from Kenya. The median age was of 27 years, the majority being adults, 145 (80%) and 37 (20%) children. TAC detected seven pathogen targets the most prevalent being Plasmodium 50 (26.9%) of which 34 were Plasmodium falciparum, Yellow fever virus 2 (1.07%), non-typhus salmonella 3 (1.6%), Salmonella typhi (1.07%), Leptospira 1 (0.54%), Streptococcus pneumonia (0.54%), and Rickettsia (0.54%). The epidemiological correlation revealed statistically significant relationship between being dead or alive (status) and disease while age, occupation, sex and disease were not statistically significant. Cough was found to be the only clinical symptom associated with plasmodial infections.
Conclusion: This is the first study in Uganda to employ a multi-pathogen detection assay for etiologies of febrile illness. The use of TAC is feasible, readily adoptable in the Ugandan setting and may be incorporated in the national testing algorithm for routine differential diagnostics of AFIs. The TAC can be customised for application in veterinary diagnostics especially for diseases of economic and public health importance.