Anne Ahrens Østergaard ^1,2* Søren Feddersen ^3,4 Mike B. Barnkob ⁵ Rasmus B. Lynggaard ⁴ Amanda C. Karstoft ⁶ Maria Borup ⁷ Ingrid L. Titlestad ^7,8 Torben T. Jensen ⁹ Ole Hilberg ¹⁰ Christian Wejse ^11,12 Stephanie Bjerrum ^13,6 Morten Blaabjerg ^14,15 Kristian Assing ^15,5 Isik S. Johansen ^2,6

• ¹ Research Unit of Infectious Diseases, Department of Clinical Research, University of Southern Denmark, Odense, Denmark
• ² Department of Infetious Diseases, Odense University Hospital, Odense, Denmark
• ³ Research unit of Clinical Biochemistry, Department of Clinical Research, Faculty of Health Sciences, University of Southern Denmark, Odense, Denmark
• ⁴ Department of Clinical Biochemistry and Pharmacology, Odense University Hospital, Odense, Denmark
• ⁵ Department of Clinical Immunology, Odense University Hospital, Odense, Denmark
• ⁶ Research unit of Infectious Diseases, Department of Clinical Research, Faculty of Health Sciences, University of Southern Denmark, Odense, Denmark
• ⁷ Department of Respiratory Medicine, Odense University Hospital, Odense, Denmark
• ⁸ Odense Respiratory Research Unit (ODIN), Department of Clinical Research, Faculty of Health Sciences, University of Southern Denmark, Odense, Denmark
• ⁹ Department for Pulmonary Diseases, Esbjerg Hospital, Esbjerg, Denmark
• ¹⁰ Department of Medicine, Vejle Hospital, Vejle, Denmark
• ¹¹ Department of Infectious Diseases, Aarhus University Hospital, Aarhus, Denmark
• ¹² Center for Global Health, Department of Public Health, Faculty of Health, Aarhus University, Aarhus, Capital Region of Denmark, Denmark
• ¹³ Department of Infectious Diseases, Copenhagen University Hospital, Copenhagen, Capital Region of Denmark, Denmark
• ¹⁴ Department of Neurology, Odense University Hospital, Odense, Denmark
• ¹⁵ Research unit of Clinical Immunology, Department of Clinical Research, Faculty of Health Sciences, University of Southern Denmark, Odense, Denmark

The diagnosis of tuberculosis (TB) disease and TB infection (TBI) remains a challenge, and there is a need for non-invasive and blood-based methods to differentiate TB from conditions mimicking TB (CMTB), TBI, and healthy controls (HC). We aimed to determine whether combination of cytokines and established biomarkers can discriminate between 1) TB and CMTB 2) TB and TBI 3) TBI and HC. We used hemoglobin, total white blood cell count, neutrophils, monocytes, C-reactive protein, and ten Meso Scale Discovery analyzed cytokines (interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, interferon (IFN)-ɣ, and tumor necrosis factor (TNF)-α) in TruCulture whole blood tubes stimulated by lipopolysaccharides (LPS), zymosan (ZYM), anti-CD3/28 (CD3), and unstimulated (Null) to develop three index tests able to differentiate TB from CMTB and TBI, and TBI from HC. In 52 persons with CMTB (n=9), TB (n=23), TBI (n=10), and HC (n=10), a combination of cytokines (LPS-IFN-ɣ, ZYM-IFN-ɣ, ZYM-TNF-α, ZYM-IL-1β, LPS-IL-4, and ZYM-IL-6) and neutrophil count could differentiate TB from CMTB with a sensitivity of 52.2% (95%CI: 30.9 – 73.4) and a specificity of 100 % (66.4-100). Null- IFN-ɣ, Null-IL-8, CD3-IL-6, CD3-IL-8, CD3-IL-13, and ZYM IL-1b discriminated TB from TBI with a sensitivity of 73.9% (56.5 - 91.3) and a specificity of 100% (69.2-100). Cytokines and established biomarkers failed to differentiate TBI from HC with ≥ 98% specificity. In summary, selected cytokines may serve as blood-based add-on tests to detect TB in a low-endemic setting, although these results need to be validated.