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The aim of this study was to assess the use of qualitative one-tube nested polymerase chain reaction (PCR) for monitoring the treatment response in smear-positive pulmonary tuberculosis, and the factors determining the negative conversion of sputum smear, culture, and PCR during treatment.A total of 53 patients receiving a standard short course of chemotherapy with 24 months follow-up period after treatment cessation were included in the study. Sputum specimens were collected serially for smear, culture, and PCR until the treatment was complete.The conversion rate for sputum culture, smear, and PCR at 8 weeks after treatment were 84.9, 58.5, and 47.1%, and at 16 weeks of treatment were 100, 88.7, and 79.2%, respectively. At the end of the treatment period, there were four PCR persisters, one of whom had disease relapse. Only cavitary disease had an influence over the negative conversion of the smear and PCR at 8 weeks (RR 3.5, 95% CI 1.04–11.95, P = 0.04 for smear; RR 5.06, 95% CI 1.196–21.42, P = 0.03 for PCR).Qualitative PCR was not useful for monitoring therapy in smear-positive pulmonary tuberculosis. Mycobacterium DNA was cleared slowly in cavitary disease. The PCR may be performed at the time of treatment cessation to identify those with potential for disease relapse.