Aim To evaluate the effect of an enhanced infection control protocol on root canal treatment outcomes and on microbial load within root canals after chemomechanical preparation. Methodology A total of 144 molar teeth from 139 healthy patients receiving primary root canal treatment were block randomized to a standard protocol (StP) or an enhanced infection control protocol (EnP). Both treatment arms adhered to current best practice recommendations, while the EnP comprised additional steps that included replacing rubber dams, gloves, files, all instruments and surface barriers at the time of canal filling to reduce the chances of iatrogenic contamination. Patients and radiographic examiners were blinded to the protocol used. Intracanal microbial samples were taken at baseline (S1) and after completion of chemomechanical preparation (S2). Microbial 16S rDNA copy numbers were enumerated by quantitative polymerase chain reaction (qPCR). Cone beam computed tomography (CBCT) scans were taken before treatment and at one‐year follow‐up. The outcome was assessed clinically and radiographically using CBCT by logistic regression modelling. Results At one‐year follow‐up, 115 teeth were analysed (54 in StP and 61 in EnP). The percentage of favourable outcomes assessed by CBCT was 85.2% in the EnP and 66.7% in the StP. The odds of 12‐month success was three times higher in the EnP group compared with the StP group (OR=2.89; p=0.022, CI: 1.17 – 7.15). The median bacterial reads were reduced from 8.1×103 in S1 samples to 3.5×103 in the StP group and from 8.6×103 to 1.3×103 in the EnP group. The enhanced protocol significantly reduced bacterial counts in pre‐canal filling samples when compared to the standard protocol (p=0.009). Conclusions The implementation of a facile, enhanced infection control protocol in primary root canal treatment resulted in less detectable bacterial DNA before canal filling and significantly more successful outcomes at one year.

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