Comparison of Endotoxin Levels Found in Primary and Secondary Endodontic Infections Brenda P.F.A. Gomes, DDS, MSc, PhD, Marcos S. Endo, DDS, MSc, and Frederico C. Martinho, DDS, MSc, PhD Abstract Introduction: This clinical study was conducted to compare the levels of endotoxins (lipopolysaccharides [LPSs]) found in primary and secondary endodontic infections with apical periodontitis by correlating LPS contents with clinical/radiographic findings. In addition, the presence of target gram-negative anaerobic bacteria was also investigated. Methods: Samples were taken from 15 root canals with primary infections and 15 with secondary infections by using paper points. The limulus amebocyte lysate assay was used to quantify endotoxins, and the polymerase chain reaction tech- nique (16S rDNA) was used for bacterial investigation. Results: Endotoxins were detected in 100% of the root canal samples collected from primary (15/15) and secondary (15/15) infections with median values of 7.49 EU/mL and 3.96 EU/mL, respectively (P < .05). The median value of endotoxins found in the presence of clinical symptoms was significantly higher than in asymptomatic teeth with primary infections (P < .05). A positive correlation was found between endotoxin contents and a larger size of the radiolucent area (>3 mm) (P < .05). Prevotella nigrescens (10/15, 4/15), Fusobacterium nucleatum (5/15, 1/15), Treponema denticola (3/15, 1/15), and Trepo- nema socranskii (5/15, 1/15) were detected in teeth with primary and secondary infections, respectively. P. endodontalis was present only in teeth with primary infections (5/15). Conclusions: Teeth with primary endodontic infections had higher contents of endotoxins and a more complex gram-negative bacterial community than teeth with secondary infections. More- over, the levels of endotoxins were related to the severity of bone destruction in periapical tissues as well as the development of clinical features in teeth with primary infections. (J Endod 2012;38:1082–1086) Key Words Endodontic infection, endotoxin, limulus amebocyte lysate assay, root canal L ipopolysaccharide (LPS), or endotoxin, is a major constituent of the outer cell wall of gram-negative bacteria (1) secreted in vesicles by growing organisms or released during disintegrations of bacteria after death (2). Endotoxin is one of the most impor- tant virulent factors involved in the development of periapical inflammation (3–5), activating immune-competent cells and leading to the release of a variety of proinflam- matory mediators (5–8). A primary endodontic infection is a polymicrobial infection caused predominantly by gram-negative anaerobic bacteria, especially Prevotella, Porphyromonas, Trepo- nema, and Fusobacterium spp (6, 8–13). Clinical investigations of primary infections have found a correlation between endotoxins and the presence of apical periodontitis (3, 6, 14–18). Higher contents of endotoxins in root canals have been associated with the development of clinical signs/symptoms and a larger area of bone destruction (3, 6, 8, 14, 15). Endodontic treatment failure is characterized by the presence of signs and/or symptoms of persistent or emergent apical periodontitis after treatment. Culture methods revealed the bacterial etiology of post-treatment apical periodontitis as being a gram-positive bacterial infection (19, 20). However, with molecular techniques, studies have also indicated the presence of Porphyromonas, Prevotella, and Treponema spp in post-treatment apical periodontitis (21–23). Currently, little information is provided in the endodontic literature regarding endotoxins in teeth with secondary/persistent endodontic infections (24). Moreover, no study compared the levels of endotoxins found in primary and secondary/persistent infections. There- fore, this clinical study was conducted to compare the levels of endotoxins found in primary and secondary/persistent endodontic infections with apical periodontitis by correlating their LPS contents with clinical/radiographic findings and to investigate the presence of target gram-negative anaerobic bacteria by using the polymerase chain reaction (PCR) (16S rDNA). Materials and Methods Patient Selection A total of 30 patients who attended the Piracicaba Dental School, S ~ ao Paulo, Brazil, were included in the present study, 15 needing primary endodontic treatment and 15 needing nonsurgical endodontic retreatment because of persistent or emergent apical periodontitis. A detailed dental history was obtained from each patient. Those who had received antibiotic treatment during the last 3 months or who had any general disease From the Department of Restorative Dentistry, Endodontics Division, Piracicaba Dental School, State University of Campinas, S~ ao Paulo, S~ ao Paulo, Brazil. Supported by the Brazilian agencies FAPESP (08/57954-8, 08/57551-0, 10/19136-1, 10/17877-4, 11/50051-5, 11/50510-0) and CNPq (302575/2009-0, 150557/ 2011-6). Address requests for reprints to Dr Brenda P.F.A. Gomes, Piracicaba Dental School, State University of Campinas-UNICAMP, Department of Restorative Dentistry, Endodontics Division, Av Limeira 901, Bairro Areiao, Piracicaba, S~ ao Paulo, Brazil CEP 13414-903. E-mail address: bpgomes@fop.unicamp.br 0099-2399/$ - see front matter Copyright ª 2012 American Association of Endodontists. doi:10.1016/j.joen.2012.04.021 Clinical Research 1082 Gomes et al. JOE — Volume 38, Number 8, August 2012