Results.
Campylobacter gracilis and C. rectus were, respectively, detected in 21.4 (6 of 28) and 30% (6 of 20) of the root canals associated with chronic asymptomatic periradicular lesions. Campylobacter gracilis was found in16.7% (2 of 12) of the cases diagnosed as acute apical periodontitis, whilst C. rectus was in 33.3% (two of six cases). In the abscessed cases, C. gracilis and C. rectus were detected in 23.5 (4 of17) and11.8% (2 of17) of the cases, respectively. Neither C. gracilis (P = 0.797), nor C. rectus (P = 0.539) was positively associated with clinical symptoms. In general, species-specific nPCR allowed the detection of C. gracilis in 21.1% (12 of 57) and C. rectus in 23.3% (10 of 43) of the samples taken from primary endodontic infections. The species were found together in only two asymptomatic teeth.
Reference DNA and clinical samples that were positive for either C. gracilis or C. rectus showed only one band of the predicted size. Specific primers generated no amplicons with genomic DNA from nontarget bacterial species. The detection limit of then PCR assay used in this study was approximately 10 cells as determined by amplification of serial dilutions of templates prepared from genomic DNA.
All clinical samples contained bacteria as demonstrated after the first round of amplification using universal primers for the 16S rDNA. A product of the appropriate size (1505 bp) was obtained from all samples, revealing that bacteria were present in all cases examined, demonstrating the suitability of the DNA for PCR analysis, and indicating the absence of inhibitors in the reaction mixture.

Discussion.
The specificity of a microbiological diagnostic test is essential to avoid false positive results. In the present study, noevidence of cross-reactivity was observed when checking the C. gracilis- and C. rectus- specific primers against a panel of nontargeted oral species. Nonspecific amplification products were also absent. In addition, nPCR directed to16S rDNA is more sensitive and can still show improved specificity when compared with single PCR by allowing the second species-specific reaction to be performed with reduced background of necrotic tissue, pus debris, eukaryotic DNA and other regions of the bacterial DNA.
Campylobactergracilis have been found in infections of endodontic origin in prevalence values ranging from 1.5-55.6%of cases. Sundqvist et al. (1989) have recovered C. gracilis from 13.6% of canals containing black-pigmented rods. In another study, Sundqvist (1992) investigated the root canal microbiota of 65 teeth with intact pulp chambers and radiographic evidence of periradicular disease and found C. gracilis in only one case (1.5%). Gomes et al. (1996) isolated C. gracilis from 2.9% of 70 infected root canals. Le Goff et al. (1997) reported the highest prevalence value for this bacterial species when evaluating the microbiota of infected root canals in teeth without carious lesions and with intact crowns - 55.6% of cases. Sundqvist et al. (1998) have found this species in12.5% of the canals of teeth with failed endodontic treatment. Some studies evaluating the presence of microbial species in chronic periradicular lesions have also found C. gracilis. Wayman et al. (1992) revealed that C. gracilis was one of the five most commonly isolated bacteria in lesions with no detectable communication with the oral cavity. Recently, C. gracilis was also detected in periradicular lesions of asymptomatic teeth by DNA-DNA hybridization (Sunde et al. 2000).
Studies have revealed that C. rectus may also be present in endodontic infections, in prevalence values ranging from 7.1-27.3%. Ranta et al. (1988) investigated the microbiota of 62 cases of periradicular lesions and found C. rectus in 11.3% of the cases. Sundqvist et al. (1989) observed the occurrence of this bacterial species in 27.3% of 22 root canals. Further, Sundqvist (1992) has reported that C. rectus was isolated from 25% of 65 infected root canals. This species was positively associated with P. endodontalis, Peptostreptococcus micros, Selenomonas sputigena, F. nucleatum, Actinomyces sp. and Eubacterium sp., which may be partly dependent on the production of growth factors, such as formate, by most of these bacteria. Siqueira et al. (2000b) examined the microbiota of infected root canals using whole genomic DNA probes and the checkerboard DNA-DNA hybridization method and found C. rectus in 7.1% of the cases. Using the same method to investigate the microbiota associated with acute periradicular abscesses, Siqueira et al. (2001b) detected C. rectus in 7.4% of the abscessed cases. Other studies using the checkerboard DNA-DNA hybridization method to assess the microbiota present in periradicular lesions have reported the detection of C. rectus in a relatively high prevalence value (Gatti et al. 2000, Sunde et al. 2000).
Most studies using PCR methodology have either detected certain bacterial species never previously found in endodontic infections by culture (Conrads et al. 1997, Siqueira et al. 2000a, Jung et al. 2001, Rolph et al. 2001) or detected certain bacterial species usually at higher prevalence when compared with culture (Siqueira et al. 2001a, Hashimura et al. 2001). In the present study, C. gracilis was detected in 21.1% of the samples of all examined samples and C. rectus in 23.3%. These frequency rates as evaluated by highly sensitive nPCR assay were not significantly discrepant from culture studies. This suggests that the prevalence of these bacterial species has not probably been underestimated by culture and confirmed that they may be associated with endodontic infections in a reasonable number of cases. Because both bacterial species were found in asymptomatic as well as symptomatic infections inpractically similar frequencies, no association with symptoms could be detected by statistical analysis.
The mechanisms of pathogenicity of C. gracilis are poorly understood. Its virulence factors probably include lipopolysaccharide (LPS), hydrogen sulphide and succinate. Campylobactergracilis are usuallyless susceptible to antimicrobial agents than other oral Campylobacter species (Johnson et al.1986, Tanner et al.1992, Baron et al.1993, Lee et al.1993). Johnson et al. (1986) evaluated the in vitro activities of 17 antimicrobial agents against 46 clinical isolates of formate/fumarate-requiring anaerobic Gram-negative bacilli. Campylobacter gracilis showed some striking resistance, with penicillin being active against only 67%, the cephalosporins active against 67-89%, and clindamycin active against 67% of the strains tested. Because C. gracilis has been associated with serious deep-tissue infection, coupled with the relatively high frequency of antibiotic resistance, it has been considered as an important human pathogen (Johnson et al. 1985). Supportive evidence for this statement is still lacking.
Whereas the pathogenicity of C. gracilis has not been conclusively demonstrated, there is suggestive evidence that C. rectus is a pathogenic microorganism. Campylobacter rectus possesses some virulence factors that may be involved in the pathogenesis of periradicular diseases. They include an extracellular cytotoxin against polymorphonuclear neutrophils, LPS, a proteinaceous surface structure (S-layer), a native GroEL-like protein, tissue-damaging enzyme arylsulfatase and hydrogen sulphide (Gillespie et al. 1992, 1993, Okuda et al. 1997, Hinode et al. 1998, Zubery et al. 1998). The native GroEL-like protein is able to stimulate production of pro-inflammatory cytokines such as interleukin-6 and -8 (Hinode et al.1998). S-layer possessing C. rectus cells can be resistant to complement-mediated killing and phagocytic killing by leucocytes in the absence of speci- fic antibody (Okuda et al.1997). It has been demonstrated that C. rectus can cause soft tissue destruction following inoculation into subcutaneous tissue of mice (Kesavalu et al.1991). Live and heat-killed cells of C. rectus are also able to stimulate bone resorption in mice, possibly via LPS or other polysaccharide components (Zubery et al. 1998).
Taken together, the findings of this study indicated that C. gracilis and C. rectus participate in infections of endodontic origin. The possible involvement of these species with other human infections, including periodontal diseases and their potential virulence armamentarium might also implicate them in the pathogenesis of periradicular diseases. Nevertheless, whilst a pathogenetic role can be suspected for these species, clear evidence of causation is still lacking. Studies are necessary to elucidate the specific role played by C. gracilis and C. rectus in primary endodontic infections as well as their involvement in the pathogenesis of periradicular diseases.

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