Discussion.
Data on microbial morphology provides few clues for the identification of most microorganisms, and physiological traits are often ambiguous (Relman 1999, Chan & McLaughlin 2000). In addition, several microorganisms are difficult or even impossible to grow under laboratory conditions (Relman 1999). These factors are especially true in the case of spirochaetes (Dewhirst et al. 2000). The nested PCR method used in this study was a 16S rDNA-based assay in which practically the entire 16S rDNA was amplified in the first reaction using universal primers followed by a second round of amplification using species-specific nested primers, internal to the first PCR product, which were used to check for the presence of the target species. The advantages of using16S rDNA analysis is that this molecule is found in all bacteria, it appears to lack artifacts of lateral transfer between contemporaneous microorganisms, is large enough to provide a significant number of nucleotides to compare sequences and yet it is small enough to conveniently analyse (Woese1987).The16S rRNA of bacteria has been extensively examined and sequenced and has been used to determine phylogenetic relationships amongst living organisms. As a consequence, data from 16S rDNA sequences have been widely used for accurate and rapid identification of known and even unknown bacterial species, using techniques that do not require microbial cultivation. This makes it possible to rationally design primers covering taxon-specific signatures, which permits microbial identification with high sensitivity and specificity, without the need to cultivate the microorganism. The rRNA gene-based PCR has proved to be superior to culture in clinical situations, such as infections caused by microorganisms with unusual growth requirements that are difficult or even impossible to culture and specimens taken during antimicrobial treatment (Relman 1999, Dewhirst et al. 2000, McPherson & Moller 2000, Sakamoto et al. 2001). PCR methodology is at least10-100timesmoresensitive than the other sensitive microbiological identification methods (Zambon & Haraszthy1995). Nested PCR has increased sensitivity and possibly also increased specificity when compared with single PCR (McPherson & Moller 2000). The assay used in this study had the detection limit of10-100 cells of the target species in the sample. A 5% volume of the original sample (100 mL) was used in individual PCR experiments. Considering sample dilution, the detection limit was approximately 200-2000 cells in the whole sample, which is still more sensitive than practically all other identification methods with regard to fastidious anaerobic bacteria. There is no agreed bacterial load for the induction of a periradicular lesion, and the method of assay should therefore detect the smallest number of possible bacterial cells in root canal samples. Thus, one of the greatest advantages of the PCR methodology as used in the present study.
In the present study, T. denticola was clearly the most prevalent amongst the four species tested. Whereas this bacterial species has been detected in 17.9 and 42.6% of the infected root canals by means of checkerboard DNA-DNA hybridization (Siqueira et al.2000b) and single PCR assay (Siqueira et al. 2001a), respectively, the nested PCR used in this study allowed the detection of T. denticola in 78.1% of the cases examined. Such discrepant findings can be explained by the different detection limits of the methods used. The nested PCR used in the present study is more sensitive than the single PCR assay previously used, which is more sensitive than the checkerboard DNA-DNA hybridization.
Jung et al. (2001) evaluated the occurrence of species of oral treponemes in 79 teeth associated with periradicular lesions by using PCR amplification with bacterial universal primers and subsequent dot-blot hybridization. They found T. maltophilum in 26% and T. socranskii in 2.7%of the cases. No case yielded the species T. denticola, T. pectinovorum and T. vincentii. These findings are diametrally divergent from those reported in this study. Differences between the results could have occurred for a variety of reasons. For instance, the nested PCR used in this study is more sensitive than the dot-blot hybridization used by Jung et al. (2001). In addition, the possibility exists that differences may have also been due to geographical differences in the composition of the oral microbiota and consequently of the root canal microbiota.
Data from the present study revealed that at least one of the four target species was present in 86.4% of the asymptomatic cases and in 80% of the cases diagnosed as acute apical periodontitis. In general, treponemes were found in 84.4% of the cases examined and the possibility exists that this figure could be increased if other species had been also tested. With regard to treponemes, these are the highest figures hitherto reported by identification studies for root canal infections. In addition, it appears that no previous studies had yet detected T. vincentii, a potential periodontal pathogen, in infected root canals.
The four Treponema species evaluated in the present study have been implicated in a variety of oral diseases and have been demonstrated to be pathogenic microorganisms. Kesavalu et al. (1997) investigated the virulence characteristics of T. denticola, T. pectinovorum, T. socranskii and T. vincentii following subcutaneous injection in mice. All species induced well-demarcated, dose-dependent, raised subcutaneous abscesses, which were similar in time of onset, lesion progression and duration of healing. Alterations in the environment may influence the pathogenicity of oral treponemes (Kesavalu et al. 1999). It has been demonstrated that different species of oral treponemes can elicit proinflammatory cytokine production by host cells and this stimulation does not necessarily require live microorganisms. In a study where human gingival fibroblast were challenged by Treponema species, Nixon et al. (2000) observed that dead cells of T. pectinovorum generally induced a twofold greater level of IL-6 and IL-8 than the live bacteria. Several virulence factors have been described and suggested for the four species evaluated, most particularly for T. denticola (the best characterized and more easily cultivated oral spirochaete). They include factors involved in adherence to host cells and tissues, tissue invasion ability, direct and indirect tissue damage and evasion of host defense mechanisms (Sela et al. 1987, Grenier 1991, Klitorinos et al.1993, Umemoto & Namikawa1994, Haapasalo et al. 1996, Scott et al.1996, Fenno & McBride 1998, Yang et al. 1998, Peters et al.1999, Rosen et al.1999,Nixon et al.2000, Grenier & Mayrand 2001, Heuner et al. 2001).

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