Diagnosis of pulmonary tuberculosis (PTB) in context of HIV infection remains challenging due 25 to the paucibacillary nature of the disease. Mycobacterium tuberculosis thymidylate kinase 26 (TMKmt) has been validated as a novel biomarker with high detection limits for PTB in sputum. 27 We aimed to clinically test TMKmt as a diagnostic biomarker for pulmonary tuberculosis among 28 HIV positive individuals at Makerere University Joint AIDS Program Immuno Suppression 29 Syndrome clinic. 30 Methods 31 A total of 120 participants with presumptive PTB were enrolled in a cross- sectional study 32 between January and September 2018. Venous blood and expectorated spot sputum was obtained 33 from 116 consenting participants. Tuberculosis culture was performed on sputum as the gold 34 standard to confirm PTB status while direct ELISAs were performed on sputum and serum to 35 determine the level of TMKmt antigen. Sensitivity, specificity and receiver operator 36 characteristic curves were used to assess the precision of TMKmt in diagnosing PTB. 37 Results 38 Of the 116 participants, only 22 (19%) were PTB positive based on either Lowenstein Jensen or 39 Mycobacterial growth inhibition tube `culture. The mean sputum TMKmt levels were 40 significantly higher in PTB positive individuals (4.889 ± 0.135) ng/ml as compared to PTB 41 negative individuals (4.303 ± 0.07295) ng/ml (t-test, P<0.0005). At a cut off value of > 4.683 42 ng/ml, the test had a sensitivity of 73% (95% CI, 49.78% - 89.27%) and a specificity of 72% 43 (95% CI, 62.15% - 81.07%). These results were supported by a receiver operator analysis which 44 showed an area under the curve of 0.75 (95% CI, 0.63 – O.86). 3 45 Conclusion 46 Sputum TMKmt is a potential diagnostic biomarker for active pulmonary tuberculosis. This 47 forms an alternate test that would replace smear microscopy and overcome the current TB 48 diagnostic challenges.