Enrique Hernández-Jiménez ^1,2* Erika Paola Plata Menchaca ^2,3 Damaris Berbel ² Guillem López de Egea ² Macarena Dastis-Arias ² Laura García-Tejada ² Fabrizio Sbraga ² Pierre Malchair ² Nadia García Muñoz ² Alejandra Larrad Blasco ² Eva Molina Ramírez ² Xose Pérez Fernández ² Joan Sabater Riera ^2* Arnau Ulsamer ²

• ¹ Loop Diagnostics S.L., Barcelona, Spain
• ² Institut d'Investigacio Biomedica de Bellvitge (IDIBELL), Barcelona, Spain
• ³ Vall d'Hebron Research Institute (VHIR), Barcelona, Catalonia, Spain

Bacteremia is a life-threatening condition that can progress to sepsis and septic shock, leading to significant mortality in the emergency department (ED). The standard diagnostic method, blood culture, is time-consuming and prone to false positives and false negatives. Although not widely accepted, several clinical and artificial intelligence-based algorithms have been recently developed to predict bacteremia. However, these strategies require further identification of new variables to improve their diagnostic accuracy. This study proposes a novel strategy to predict positive blood cultures by assessing sepsis-induced immunosuppression status through endotoxin tolerance assessment. Optimal assay conditions have been explored and tested in sepsis-suspected patients meeting the Sepsis-3 criteria. Blood samples were collected at ED admission, and endotoxin (lipopolysaccharide, LPS) challenge was performed to evaluate the innate immune response through cytokine profiling. Clinical variables, immune cell population biomarkers, and cytokine levels (tumor necrosis factor [TNFα], IL-1β, IL-6, IL-8, and IL-10) were measured. Patients with positive blood cultures exhibited significantly lower TNFα production after LPS challenge than did those with negative blood cultures. The study also included a validation cohort to confirm that the response was consistent. The results of this study highlight the innate immune system immunosuppression state as a critical parameter for sepsis diagnosis. Notably, the present study identified a reduction in monocyte populations and specific cytokine profiles as potential predictive markers. This study showed that the LPS challenge can be used to effectively distinguish between patients with bloodstream infection leading to sepsis and those whose blood cultures are negative, providing a rapid and reliable diagnostic tool to predict positive blood cultures. The potential applicability of these findings could enhance clinical practice in terms of the accuracy and promptness of sepsis diagnosis in the ED, improving patient outcomes through timely and appropriate treatment.