Background. Patients with previous tuberculosis may have residual DNA in sputum that confounds nucleic acid amplification tests such as Xpert MTB/RIF. Little is known about the frequency of Xpert-positive, culture-negative ("false positive") results in retreatment patients, whether these are distinguishable from true positives, and whether Xpert's automated filter-based wash step reduces false positivity by removing residual DNA associated with nonintact cells. Methods. Pretreatment patients (n = 2889) with symptoms of tuberculosis from Cape Town, South Africa, underwent a sputum-based liquid culture and Xpert. We also compared Xpert results from dilutions of intact or heat-lysed and mechanically lysed bacilli. Results. Retreatment cases were more likely to be Xpert false-positive (45/321 Xpert-positive retreatment cases were false-positive) than new cases (40/461) (14% [95% confidence interval {CI}, 10%-18%] vs 8% [95% CI, 6%-12%]; P =. 018). Fewer years since treatment completion (adjusted odds ratio [aOR], 0.85 [95% CI,. 73-.99]), less mycobacterial DNA (aOR, 1.14 [95% CI, 1.03-1.27] per cycle threshold [CT]), and a chest radiograph not suggestive of active tuberculosis (aOR, 0.22 [95% CI,. 06-.82]) were associated with false positivity. CT had suboptimal accuracy for false positivity: 46% of Xpert-positives with CT > 30 would be false positive, although 70% of false positives would be missed. CT's predictive ability (area under the curve, 0.83 [95% CI,. 76-.90]) was not improved by additional variables. Xpert detected nonviable, nonintact bacilli without a change in CT vs controls. Conclusions. One in 7 Xpert-positive retreatment patients were culture negative and potentially false positive. False positivity was associated with recent previous tuberculosis, high CT, and a chest radiograph not suggestive of active tuberculosis. Clinicians may consider awaiting confirmatory testing in retreatment patients with CT > 30; however, most false positives fall below this cut-point. Xpert can detect DNA from nonviable, nonintact bacilli.
