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 »  Home  »  Endodontic Articles 4  »  Direct amplification of rRNA gene sequences for identification of selected oral pathogens in root canal infections
Direct amplification of rRNA gene sequences for identification of selected oral pathogens in root canal infections
Discussion - References.



Most oral microorganisms may be considered opportunistic pathogens that cause infections after a change in local environmental conditions. After necrosis, the pulp tissue loses its defence ability and may theoretically be colonized by any oral microorganism. However, the environmental conditions within the root canal system will select the species that will survive and establish an infectious disease. The mere presence of a microorganism in infected root canals does not necessarily assure that it is involved in the pathogenesis of periradicular diseases (Siqueira et al. 1998). Nevertheless, since the species detected in the present study have been associated with infections in diverse oral sites, it is reasonable to suppose that they may participate in the pathogenesis of periradicular diseases.
Four groups of oral streptococci are recognized based on 16S rRNA gene sequence comparisons (Whiley & Beighton 1998). The S. anginosus group includes the species S. anginosus, S. constellatus, and S. intermedius. The primer pair used in this study does not differentiate between these three species (Conrads et al. 1997). The S. anginosus group has been reported to be the most common streptococcal species found in infected root canals (Sundqvist 1994). In the present study, only B. forsythus, F. nucleatum and members of the S. anginosus group were detected in symptomatic cases, including the abscessed teeth. Nonetheless, no correlation could be established since they were also found in a significant number of asymptomatic cases. It is entirely possible that these species were present in higher numbers in symptomatic cases. However, due to the qualitative nature of the PCR assay employed in the present study, any statement in this regard may be considered speculative.
The species A. actinomycetemcomitans has been implicated in the etiology of some forms of periodontal diseases (Meyer & Fives-Taylor 1997). Although this species has been detected by DNA-DNA hybridization in extraradicular infections (Sunde et al. 2000) and occasionally in cases of intraradicular infections (Siqueira et al. 2000a), it was not found in any root canal sample examined by PCR in the present study. This finding corroborates most of the studies regarding the root canal microbiota and suggests that A. actinomycetemcomitans is not a relevant endodontic pathogen. It is possible that some environmental differences between the root canal system and the periodontal pocket may influence colonization by this microorganism. On the other hand, the molecular method used in this study confirmed previous findings that another important periodontal pathogen, B. forsythus, may be also involved in the pathogenesis of periradicular infections (Conrads et al. 1997, Gonçalves & Mouton 1999, Siqueira et al. 2000a).
The fungus-specific primer pair used in the present investigation (ITS3 and ITS4) amplifies a large portion of the 5.8S ribosomal DNA region, the adjacent internal transcribed spacer (ITS) region, and a small portion of the 28S rDNA region, generating PCR products of different lengths depending on the fungal species (Fujita et al. 1995, Shin et al. 1997). It is able to amplify DNA from most, if not all, fungi. PCR assay using this primer pair has been reported to be rapid, reliable and effective in identifying more than one species of yeast in mixed cultures with no cross-reactivity with bacteria or human cells (Fujita et al. 1995, Shin et al. 1997). In the present study, only one clinical case yielded fungi. Precise identification after amplification with ITS3 and ITS4 primers requires use of additional methods, such as DNA sequencing, which was not performed in the present study. The amplicon length of the DNA amplified was greater than that found and reported for most Candida species (Fujita et al. 1995, Shin et al. 1997). Our findings confirmed that fungi are rarely found in primary root canal infections (Möller 1966, Sundqvist 1976). Fungi are considered opportunistic pathogens that cause secondary infections after changes in the ecosystem that permit their overgrowth. Because of this, fungi have been found associated with persistent and/or secondary root canal infections, including teeth refractory to endodontic therapy (Nair et al. 1990, Waltimo et al. 1997).
The rRNA gene directed-PCR assay as used in this study has been reported to possess detection sensitivity of 25–100 colony-forming units (Fujita et al. 1995, Ashimoto et al. 1996, Conrads et al. 1997). Theoretically, an assay with such sensitivity can still fail to detect a few positive cases. This is particularly true when one considers that such a small aliquot of the root canal sample was used to detect each target microorganism. Nonetheless, all the cases having a meaningful number of microorganisms will register a positive result. Moreover, the sensitivity level is still considerably greater when compared with that of culture, which is reported to range from 103 to 104 CFUs (Zambon & Haraszthy 1995).
A question arises when one is using a highly sensitive method such as PCR to assess clinical samples. Since the PCR protocols commonly used for direct microbial detection in a sample are qualitative, what is the number of cells present in a sample? It is well known that microbial virulence associated with a critical number of cells is required for the induction of an infectious disease. It is possible that some microbial species detected in this study were in such low numbers that they were insufficient to cause disease. This could explain why certain cultivable species are found in a higher number of cases when PCR is used, although the fact that some strains within a species are uncultivable should also be taken into account. Quantitative PCR assays are warranted to evaluate the levels of specific microorganisms in endodontic infections and thereby elucidate this question.
Culturing requires at least an 8-h incubation of the sample in culture medium and then biochemical and other tests to identify the microorganism. The time required for identification can be even longer for slowly growing microorganisms or in samples with low microbial counts. The former is especially true for identification of fastidious anaerobic bacteria, which are commonly found in endodontic infections. When traditional culture methods are used, the laboratory can require 7–14 days to identify anaerobic bacteria, whilst some molecular methods (such as PCR) can provide information in only a few hours. As it now stands, the method used in this study can be performed in approximately 5 h and can thus be very useful for rapid clinical diagnosis. This is especially important in cases of life-threatening infections.
The rRNA gene based-PCR is 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 (Rantakokko-Jalava et al. 2000, Jordan & Durso 2000). In addition, it has the potential for excellent sensitivity and a shorter turnaround time than those of culture-based protocols. The PCR assay used in this study does not require prior sample preparation and time-consuming laboratory procedures, and is sensitive, specific, and reproducible (Fujita et al. 1995, Ashimoto et al. 1996, Conrads et al. 1997). Therefore, direct molecular approach appears to be a valuable tool for the rapid and reliable diagnosis of infectious diseases, as well as for research purposes.
Although the impact of molecular genetic methods on the knowledge of the root canal microbiota has not been dramatic, such approaches have allowed the recognition of new putative endodontic pathogens. The well-directed use of these methods will provide additional valuable information regarding the identification and understanding of the causative factors associated with endodontic diseases, helping to elaborate more successful treatment strategies.

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