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A nested polymerase chain reaction (PCR)-based method was used to directly survey samples taken from primary endodontic infections for the occurrence of Pseudoramibacter alactolyticus. Identification by nested PCR was performed in root-canal samples from teeth associated with asymptomatic periradicular lesions or acute apical periodontitis, and in pus samples from acute periradicular abscesses. DNA was extracted from the samples and initially amplified using universal 16S rDNA primers. A second round of amplification used the first PCR products to detect a specific fragment of P. alactolyticus 16S rDNA. P. alactolyticus was detected in 76% of root-canal samples from teeth showing asymptomatic periradicular lesions, in 60% of samples taken from root canals associated with acute apical periodontitis, and in 32% of pus samples aspirated from acute periradicular abscesses. No significant association of this species with clinical symptoms was observed (p > 0.01). In general, P. alactolyticus occurred in 56% of samples taken from infections of endodontic origin. The high prevalence of P. alactolyticus in infections of endodontic origin as detected by nested PCR in this study, and its apparent pathogenicity, particularly in mixed infections, indicate that this bacterial species is a candidate endodontic pathogen that can participate in the etiology of different forms of periradicular diseases.