Background: With 1.4 million deaths in 2019,tuberculosis remains a leading global infectious cause of death, second only to COVID-19 in 2020. Antimicrobial resistant infections are projected to cause over 10 million deaths annually by 2050,in part reflecting non-pathogen directed prescription secondary to limited point-of-care diagnostics and laboratory infrastructure. These two major global public health threats intersect through diagnostic algorithms that have for decades encouraged broad-spectrum antibiotic prescriptions(“trial-of-antibiotics”) during the diagnostic work-up leading to ~5 million people being treated annually for mycobacteriology-negative tuberculosis. However, the underlying assumption that post-treatment symptom improvement “rules out” tuberculosis, had no clear evidence-base. Aims: To use systematic review and a randomised controlled trial to evaluate and address evidence gaps in three key areas:1) diagnostic performance, 2) safety of withheld prescription, and 3) impact on antimicrobial resistance of trial-of-antibiotics. Methods and results: For the systematic review and meta-analysis (CRD42017083915), I searched MEDLINE, Embase and Global Health databases for studies addressing the diagnostic performance of trial-of-antibiotics against mycobacteriology tests in adults with tuberculosis symptoms. Pooled values for sensitivity and specificity of trial-of-antibiotics (index test) versus mycobacteriology tests (reference) were estimated using random-effects bivariate modelling and I2statistic. Only 8/9410 screened studies were eligible, with no randomised trials. Treatment duration, antibiotics used, and definition of response to treatment varied substantially. Pooled sensitivity (67%, 95% CI 42, 85) and specificity (73%, 95% CI 58, 85) of trial-of-antibiotics versus mycobacteriology were below internationally-defined minimum performance profiles for tuberculosis diagnostics with substantial heterogeneity (I296% for sensitivity, 99% for specificity)and low QUADAS-2 quality assessments. My trial (NCT03545373) aimed to strengthen this weak evidence-base. I randomised(1:1:1) Malawian adults attending primary care for illness ≥2 weeks including cough with no immediate indication for hospitalisation, no recent antibiotic or tuberculosis treatment or prevention, to: azithromycin (500mg daily, 3 days) amoxicillin (1g three times/day, 5 days); or standard-of-care (SOC) with no immediate antibiotic. Sputum at enrolment and day 8 was tested using microscopy, Xpert MTB/RIF, and culture. Primary outcomes were day 8 specificity (percentage with symptom improvement among mycobacteriology-negative), and day 29 clinical outcomes (composite: death, hospitalisation or missed tuberculosis diagnosis). The secondary outcome was day 29 risk of resistant Streptococcus pneumoniae identified by culture of nasopharyngeal swabs. Afterscreening5825 adults,1583 were randomised of whom 6.3% (100/1583) had positive baseline mycobacteriology. Compared to SOC (79.1%of 530), trial-of-antibiotics improved tuberculosis specificity: azithromycin (527patients) vs. SOC difference +8.6% (95% confidence interval 3.9%, 13.3%); amoxicillin (526 patients) vs. SOC difference +8.8% (4.0%, 13.6%), but with extremely low sensitivity (10.7% azithromycin, 23.3% amoxicillin). Day 29 composite clinical outcomeswere similar (SOC 1.1%, azithromycin 1.1%, amoxicillin 2.1%). Compared to SOC (5.3%), proportions with day 29 resistant Streptococcus pneumoniaewere higher in azithromycin +2.5% (-0.5, 5.5), but similar in amoxicillin +0.2% (-2.9, 2.5)arms.Using standard decision-analysis tools I compared antibiotic prescription and Xpert MTB/RIFrequirements of 3 algorithms combining Xpert MTB/RIF and trial-of-antibiotics against Xpert alone in 100,000 hypothetical patients with tuberculosis prevalence of 5%. Antibiotic prescriptions (trial-of-antibiotics plus out-of-protocol) needed to identify one tuberculosis patient varied from 22.7 to 220.4 in combination algorithms, with minimal benefit over Xpert alone (NNS =22.5Xpert tests, plus 4.0antibiotic prescriptions). Trial-of-antibiotics exposed patients to considerable misclassification risks. Conclusion: Harms of trial-of-antibiotics likely outweigh benefits, with the poor diagnostic performance ,lack of additional clinical impact, high cost, and likely impact on antimicrobial resistance arguing strongly against routine prescription. National tuberculosis and antimicrobial stewardship programs should limit outpatient prescription of empirical broad-spectrum antibiotics to patients with strong clinical or microbiological indications. Future research should focus on strengthening diagnostics for tuberculosis and other respiratory pathogens, evaluating antibiotic-sparing tuberculosis diagnostic algorithms in less clinically stable patients, and antimicrobial stewardship programs targeting tuberculosis-related prescribing.