1 Volume 73, Issue 3, Pages 219-230 (September 2016)Evaluation of cytokine responses against novel Mtb antigens as diagnostic markers for TB disease  Dolapo O. Awoniyi, Andrea Teuchert, Jayne S. Sutherland, Harriet Mayanja-Kizza, Rawleigh Howe, Adane Mihret, Andre G. Loxton, Jacob Sheehama, Desta Kassa, Amelia C. Crampin, Hazel M. Dockrell, Martin Kidd, Ida Rosenkrands, Annemieke Geluk, Tom H.M. Ottenhoff, P.L.A.M. Corstjens, Novel N. Chegou, Gerhard Walzl Gerhard Walzl, Novel N. Chegou, Magdalena Kriel, Gian van der Spuy, Andre G. Loxton, Kim Stanley, Stephanus Malherbe, Shirley Mcanda, Belinda Kriel, Khutso G. Phalane, Paulin Essone, Jayne S. Sutherland, Olumuyiwa Owolabi, Abdou Sillah, Joseph Mendy, Awa Gindeh, Simon Donkor, Toyin Togun, Martin Ota, Amelia C. Crampin, Felanji Simukonda, Alemayehu Amberbir, Femia Chilongo, Rein Houben, Desta Kassa, Atsbeha Gebrezgeabher, Getnet Mesfin, Yohannes Belay, Gebremedhin Gebremichael, Yodit Alemayehu, Marieta van der Vyver, Faustina N. Amutenya, Josefina N. Nelongo, Lidia Monye, Jacob A. Sheehama, Scholastica Iipinge, Harriet Mayanja-Kizza, Ann Ritah Namuganga, Grace Muzanye, Mary Nsereko, Pierre Peters, Rawleigh Howe, Adane Mihret, Yonas Bekele, Bamlak Tessema, Lawrence Yamuah, Tom H.M. Ottenhoff, Annemieke Geluk, Kees Franken, Paul L.A.M. Corstjens, Elisa M. Tjon Kon Fat, Claudia J. de Dood, Jolien J. van der Ploeg-van Schip, Ida Rosenkrands, Claus Aagaard, Stefan H.E. Kaufmann, Maria M. Esterhuyse, Jacqueline M. Cliff, Hazel M. Dockrell Dolapo O. Awoniyi, Andrea Teuchert, Jayne S. Sutherland, Harriet Mayanja-Kizza, Rawleigh Howe, Adane Mihret, Andre G. Loxton, Jacob Sheehama, Desta Kassa, Amelia C. Crampin, Hazel M. Dockrell, Martin Kidd, Ida Rosenkrands, Annemieke Geluk, Tom H.M. Ottenhoff, P.L.A.M. Corstjens, Novel N. Chegou, Gerhard Walzl Gerhard Walzl, Novel N. Chegou, Magdalena Kriel, Gian van der Spuy, Andre G. Loxton, Kim Stanley, Stephanus Malherbe, Shirley Mcanda, Belinda Kriel, Khutso G. Phalane, Paulin Essone, Jayne S. Sutherland, Olumuyiwa Owolabi, Abdou Sillah, Joseph Mendy, Awa Gindeh, Simon Donkor, Toyin Togun, Martin Ota, Amelia C. Crampin, Felanji Simukonda, Alemayehu Amberbir, Femia Chilongo, Rein Houben, Desta Kassa, Atsbeha Gebrezgeabher, Getnet Mesfin, Yohannes Belay, Gebremedhin Gebremichael, Yodit Alemayehu, Marieta van der Vyver, Faustina N. Amutenya, Josefina N. Nelongo, Lidia Monye, Jacob A. Sheehama, Scholastica Iipinge, Harriet Mayanja-Kizza, Ann Ritah Namuganga, Grace Muzanye, Mary Nsereko, Pierre Peters, Rawleigh Howe, Adane Mihret, Yonas Bekele, Bamlak Tessema, Lawrence Yamuah, Tom H.M. Ottenhoff, Annemieke Geluk, Kees Franken, Paul L.A.M. Corstjens, Elisa M. Tjon Kon Fat, Claudia J. de Dood, Jolien J. van der Ploeg-van Schip, Ida Rosenkrands, Claus Aagaard, Stefan H.E. Kaufmann, Maria M. Esterhuyse, Jacqueline M. Cliff, Hazel M. Dockrell  Journal of Infection  Volume 73, Issue 3, Pages (September 2016) DOI: /j.jinf Copyright © 2016 The British Infection Association Terms and Conditions

2 Figure 1 Standards for Reporting of Diagnostic Accuracy (STARD) flow diagram for recruitment of participants with presumed TB, enrolment and exclusion. Three hundred and fifty-six individuals presumed to have pulmonary TB were enrolled from different African field sites and WBA performed. Participants were later classified as definite TB cases, probable TB cases and non TB cases using a pre-established diagnostic algorithm. Thirty-four individuals with inconclusive diagnosis were excluded from the analysis. +ve = positive, −ve = negative. Journal of Infection  , DOI: ( /j.jinf ) Copyright © 2016 The British Infection Association Terms and Conditions

3 Figure 2 Scatter-dot plots of host markers detected in unstimulated and antigen-specific overnight WBA supernatants. Statistical differences in analyte levels were evaluated by the Mann Whitney U test for non-parametric data analysis. Representative plots show the levels of analytes in the overnight whole blood culture supernatants of participants with and without TB disease. Bars in the scatter-dot plots represent the median plus interquartile range of the concentration of analyte. Nil = unstimulated marker levels, Ag = ESAT-6/CFP-10 stimulated marker levels CRP=C reactive protein, SAA=Serum amyloid A, IFN=Interferon gamma, IP-10 = Interferon-inducible protein-10. Journal of Infection  , DOI: ( /j.jinf ) Copyright © 2016 The British Infection Association Terms and Conditions

4 Figure 3 Receiver operating characteristic (ROC) curves of host markers detected in stimulated overnight WBA supernatants. Representative ROC curves showing the accuracy of the markers in discriminating between TB and non TB. All markers had area under the curve (AUC) ≥0.70 except IP-10Ag-Nil, IFN-γAg-Nil, EotaxinRv1284-Nil, IL-2Rv2334-Nil and tPARv0081-Nil. CRP = C reactive protein, SAA = Serum amyloid A, IFN-γ = Interferon gamma, IP-10 = Interferon-inducible protein-10, IL = (Interleukin)-2, tPA = tissue plasminogen activator. Journal of Infection  , DOI: ( /j.jinf ) Copyright © 2016 The British Infection Association Terms and Conditions

5 Figure 4 Frequency of analytes in the top 20 most accurate GDA predictive models for the classification of study participants as TB disease or non TB. Using best subsets method of variable selection, unstimulated and antigen-specific host markers were fitted into general discriminant analysis prediction models. The columns represent the number of times each analyte occurred in the top 20 general discriminatory models. Nil = unstimulated marker levels, Ag = ESAT-6/CFP-10 stimulated marker levels. Journal of Infection  , DOI: ( /j.jinf ) Copyright © 2016 The British Infection Association Terms and Conditions