|Year : 2014 | Volume
| Issue : 6 | Page : 555-560
|Investigate the correlation between clinical sign and symptoms and the presence of P. gingivalis, T. denticola, and T. forsythia individually or as a "Red complex" by a multiplex PCR method
Tulsi Hasmukhrai Sanghavi1, Nimisha Shah2, Ruchi Rani Shah2, Akta Sanghavi3
1 Private Practice, Junagadh, Gujarat, India
2 Department of Conservative Dentistry, K.M. Shah Dental College, Sumandeep Vidhyapeeth, Piparia, Vadodara, Gujrat, India
3 Department of Conservative Dentistry; Department of Periodontics, K.M. Shah Dental College, Sumandeep Vidhyapeeth, Piparia, Vadodara, Gujrat, India
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|Date of Submission||21-May-2014|
|Date of Decision||28-Aug-2014|
|Date of Acceptance||23-Sep-2014|
|Date of Web Publication||13-Nov-2014|
| Abstract|| |
Aim: The aim of this study was to investigate the correlation between endodontic clinical signs and symptoms and the presence of Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia or their association by Multiplex polymerase chain reaction assay.
Materials and Methods: Microbial samples were taken from 30 cases with necrotic pulp tissues in primary infections. DNA was extracted from the samples, which were analyzed for the presence of three endodontic pathogens by using species-specific primers.
Results: P. gingivalis, T. denticola, T. forsythia, and Red Complex were present in 11, 17, 4, and 2 canals, respectively. Clinical and statistically significant relationships were found between T. forsythia and mobility and between T. denticola and swelling. (P < 0.05). Presence of other Red complex bacteria shows clinical association with presence of signs and symptoms but no statistically significant relationship.
Conclusion: The high prevalence of P. gingivalis, T. denticola, and T. forsythia in the examined samples suggests that these bacteria are related to the etiology of symptomatic periradicular diseases.
Keywords: Multiplex PCR; PCR; red complex
|How to cite this article:|
Sanghavi TH, Shah N, Shah RR, Sanghavi A. Investigate the correlation between clinical sign and symptoms and the presence of P. gingivalis, T. denticola, and T. forsythia individually or as a "Red complex" by a multiplex PCR method
. J Conserv Dent 2014;17:555-60
|How to cite this URL:|
Sanghavi TH, Shah N, Shah RR, Sanghavi A. Investigate the correlation between clinical sign and symptoms and the presence of P. gingivalis, T. denticola, and T. forsythia individually or as a "Red complex" by a multiplex PCR method
. J Conserv Dent [serial online] 2014 [cited 2019 Jul 17];17:555-60. Available from: http://www.jcd.org.in/text.asp?2014/17/6/555/144604
| Introduction|| |
The mouth is a window into the health of the body. It can show signs of nutritional deficiencies or general infection. The human oral cavity is home to approximately 700 identified species of bacteria. Although minute and primordial, bacteria are incredibly versatile and diversified.  Judging by their numbers and biomass, they are arguably the most successful living organisms on earth. They can tolerate environmental extremes and colonize almost every habitat on earth, including human oral cavity. 
The polymicrobial nature of the endodontic microbiota suggests a bacterial interaction that might play an important role for their survival and virulence. A restricted group of anaerobic and facultative microorganisms, especially Prevotella and Porphyromonas spp, Fusobacterium nucleatum, Streptococcus milleri group, and other Gram-positive and Gram-negative species are considered important in the pathogenesis of odontogenic infections in terms of sheer numbers and relevance toward clinical symptoms.
A number of earlier culture studies showed that microbial composition of primary endodontic lesion includes predominantly Peptostreptococcus, Fusobacterium, and Bacteroides species. Out of these black-pigmented Bacteroides are now classified as Prevotella and Porphyromonas species, and Fusobacterium species have been associated with symptomatic teeth, indicating a more specific pathological potential. The predominance of Peptostreptococcus species such as Micromonas (formerly Peptostreptococcus) micros, Fusobacterium such as F. nucleatum, Porphyromonas species such as P. gingivalis and P. endodontalis, and Prevotella species such as P. intermedia in endodontics is further confirmed in some recent culture studies. ,
It has been well established that Gram-negative anaerobic bacteria are the predominant microorganisms in endodontic infections and are often associated with signs and symptoms of some forms of periradicular diseases. However, it is still unclear that the same species is responsible for asymptomatic periradicular lesions. 
Studies have shown that molecular techniques can detect the presence of bacteria in endodontic infections when culture techniques yield a negative result. These techniques can also be used to identify a wider range of endodontic infection-related bacteria including the presence of previously unidentified or unculturable species. ,
Gomes et al. detected a higher prevalence of black-pigmented species in symptomatic teeth by polymerase chain reaction (PCR) analysis, and P. gingivalis was present in nearly half of the samples. Siqueira et al. detected Treponema denticola in the root canals of teeth that were tender to percussion and diagnosed as acute periradicular abscesses, which suggests that this microorganism could also participate in the pathogenesis of periradicular diseases. 
Different molecular methods are available for microbial identification such as DNA-DNA hybridization, Macroarrays, Microarrays, PCR, etc.
PCR is a procedure that can generate millions of copies of any specific DNA sequence in vitro. ,
It has been revealed that Gram-negative anaerobic bacteria are predominant microorganism in endodontic infection.  Current evidence suggests that some Gram-negative anaerobic bacteria are closely associated with the etiology of symptomatic periradicular lesions, including cases of acute periradicular abscess. In general, primary infections are mixed and predominated by anaerobic bacteria. 
P. gingivalis, T. denticola, and Tannerella forsythia together form "Red complex". This complex is of particular interest because it is associated with bleeding on probing, which is an important clinical parameter of destructive periodontal diseases. 
Rôças et al. assess the occurrence of the Red complex in root canal infections by using a sensitive technique - the 16S rDNA-directed PCR. At least 1 member of the red complex was found in 33 of 50 cases. T. denticola, P. gingivalis, and B. forsythus were detected in 44%, 30%, and 26% of the cases, respectively. The red complex was found in 4 of 50 cases. 
Individually, P. gingivalis, T. denticola, and T. forsythia are found in more than 70% cases of endodontic infection. However, as "Red complex" they are found only in 16% cases. Nevertheless, whenever they are present, there is some significant association found between the presence of bacteria and clinical sign and symptoms such as spontaneous pain, tenderness to percussion, pain on palpation, swelling, and periapical lesions. 
"Red complex" bacteria are difficult to isolate from root canal by culture method, as they are dependent upon growth media and conditions; their loss of black pigmentation was associated with capability of hemolysis of the blood agar. 
The purpose of this study was to investigate the correlation between clinical sign and symptoms and the presence of P. gingivalis, T. denticola, and T. forsythia present individually or as a "Red complex" by a Multiplex PCR method.
| Materials and methods|| |
Total sample size was 40. Out of this, 30 samples were obtained from patients and 10 as a negative control group. Ethical clearance has been obtained from ethical research and cell in advance. All patients were explained about the procedure and consent was obtained prior to the commencement of the procedure. Patients with prior endodontic treatment, with vital pulp, on regular medication, on antibiotic since last 1 month, with acute or chronic irreversible pulpitis, with calcified canal, having periodontal disease (such as chronic generalized periodontitis and aggressive periodontitis), and who were pregnant were excluded from study.
Clinical and radiographic examination
The 30 teeth selected had no prior endodontic treatment and exhibited a necrotic pulp (primary infection), periradicular periodontitis, or periradicular abscess/cyst. Age, gender, tooth type, and pulp status were recorded for each patient. Clinical symptoms and signs included history of previous pain, tenderness to percussion, pain on palpation, mobility, presence of a sinus tract and its origin (endodontic or periodontal), presence of swelling of the periodontal tissues (i.e., acute abscess), probing depth of any periodontal pockets, history of previous and present antibiotic therapy, and any other relevant medication. The internal status of the canal, such as a dry canal or the presence of clear, hemorrhagic, or purulent exudates, was detected as a distinct dampening or stain on the sampling paper points. Each type of exudates was analyzed separately and grouped with the other types under the denomination "wet canal."
Aseptic techniques were used throughout the endodontic sample acquisition. Briefly, after caries removal without the exposure of the canals, the teeth were individually isolated from the oral cavity with a rubber dam. Teeth and rubber dam were disinfected with 30% hydrogen peroxide followed by 2.5% sodium hypochlorite. Thereafter, the canals were exposed under manual irrigation with sterile saline solution using sterile burs. Sampling included a single root canal. In multi-rooted teeth, only the largest canal in the root with the periapical radioluscency was sampled to confine the microbial evaluation to a single ecological environment.
A sterile #15 file was used to check patency of canal. For microbial sampling, a sterile paper point was introduced into the full length of the root canal, as determined in a preoperative radiograph, and kept in place for 60 seconds. In the case of a dry root canal, a second paper point, moistened in sterile saline solution, was used to ensure adequate sample acquisition. In the case of a wet root canal, as many paper points as needed were used to absorb all fluid inside the canal.
For negative control group, 10 samples were obtain by moistened the paper point in sterile normal saline and transferred it into Eppendorf tubes containing TE buffer.
The paper points were transferred to a test tube containing TE buffer transport medium and stored at −70°C for further PCR analyses.
• Multiplex PCR was done to evaluated present bacteria P. gingivalis, T. denticola, and T. forsythia individually or as a "Red Complex".
Steps for multiplex PCR are as follows:
DNA extraction method
Sample tubes containing TE buffer were transferred to the centrifugation machine and centrifuged at 10,000 rpm for 5 minutes. The supernatant was discarded and 500 μl of fresh TE buffer was added with the help of sterile micropipette and centrifuge for 3-4 minutes. The above procedure was repeated 3-4 times in a laminar flow chamber with fresh TE buffer.
Thereafter, the supernatant was discarded again and 100 μl of lysis buffer II was added for lysis of bacteria, and 10-μl proteinase-K (100 ug/ml) was added for digestion of protein and removal of contamination from preparations of nucleic acid. Eppendorf tubes were kept in water bath for 2 hrs followed by immersing them in boiling water bath for 10 minutes. Extracted DNA were stored in Eppendorf tubes at −20°C.
Primer mix preparation
The following primers were used [Table 1]
Primer mix contains water, Taq buffer, dNTP, Taq polymerase Enzyme and primer (specific for particular bacteria).
Total 22 μl of mixture was prepared for each sample.
Buffer-2.5 μl* per sample
dNTP-2 μl* per sample
Taq Polymerase −0.5 μl per sample
Remaining count adjust with water.
The 22-μl primer mixture and 3-μl extracted DNA mixture were added to sterile test tube and this mixture was used for further analysis.
Multiplex PCR method was used to evaluate the presence of "Red Complex" bacteria. PCR machine (Veriti® 96-Well Fast Thermal Cycler) was adjusted for Multiplex PCR cycles.
95°-3 min (for initial denaturation)
94°-1 min Denaturation
42°-2 min Anneling (22 cycles)
72°-2 min Extension
72°-5 min (final extension)
10° - (storage)
Agarose gel electrophoresis
At the end of polymerase cycles, we will have chains of targeted bacteria. To visualize them, agarose gel electrophoresis is used. For Agarose gel electrophoresis, 50 gm agarose and running buffer Tris-acetate EDTA (TAE) were used.
The gel was removed from electrophoresis and viewed under UV Transillumination to identify P. gingivalis, T. denticola, and T. forsynthia individually or together as a "Red complex".
The data collected for each case (clinical features) were typed onto a spreadsheet and statistically analyzed with the help of SPSS14. The Pearson chi-square test or the one-sided Fisher exact test, as appropriate, was chosen to test the null hypothesis that there was a relationship between endodontic clinical symptoms and signs and the presence of P. gingivalis, T. forsythia, and T. denticola.
| Results|| |
Out of 30 samples, some signs and symptoms were present as follows: 28 with pain, 4 with swelling, 6 with pus discharge, 7 with discoloration, and 4 with mobility. P. gingivalis, T. denticola, T. forsythia, and Red Complex were present in 11, 17, 4, and 2 canals, respectively.
The pair T. forsythia/P. gingivalis was detected in 3 cases, including in 1 tooth that was tender to percussion and in 1 pus sample. The pair T. forsythia/T. denticola was found in 3 symptomatic cases. P. gingivalis and T. denticola were found in 12 cases, including 4 were with pain, 3 with pain on percussion, and 2 with periapical lesion.
However, clinical and statistically significant relationships were found between T. forsythia and mobility, and between Red complex and swelling. (P < 0.05).
Other bacteria did not show any significant relation with clinical finding. No bacterial DNA was found in case of negative control group [Table 2].
|Table 2: Clinical sign and symptoms and the presence of P. gingivalis, T. denticola, and T. forsythia individually or as a "Red complex"|
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| Discussion|| |
The development of acute endodontic signs and symptoms might depend on the synergy between black-pigmented bacteria and other bacterial species. P. gingivalis, T. forsythia, and T. denticola as having the highest association with the severity of periodontal disease, as measured by pocket depth and bleeding on probing. The authors named the microbial consortium the Red complex. The bacteria have been demonstrated to produce virulence factors that can contribute to the pathogenesis of primary endodontic lesion. ,
P. gingivalis is a non-spore-forming Gram-negative anaerobic rod mainly found in lesion directly associated with severity of periodontal disease. Virulence factors such as lipopolysaccharide, fimbriae, proteases, phospholipase, alkaline and acid phosphatases, DNase and RNase, hemolysins, and cytotoxic metabolites (such as hydrogen sulfide, methylmercaptan, dimethyl disulfide, butyrate, propionate, indole, and ammonia), which helps in colonization of a niche in the host (this includes adhesion to cells). ,,
T. forsythia, a non-spore-forming Gram-negative anaerobic fusiform rod, virulence factors probably include gingival pain, (a trypsin-like enzyme), lipopolysaccharide, alkaline phosphatase, acid phosphatase, propionate, isovalerate, phenylacetate, and butyrate helps in immunoevasion (evasion of the host's immune response) and immunosuppression (inhibition of the host's immune response). ,
The main virulence factors of T. denticola, a Gram-negative, obligate anaerobic, motile, and highly proteolytic bacterium, include surface-expressed proteins with cytotoxic activities, such as the major surface protein and the chymotrypsin-like protease complex, extracellular or membrane-associated proteolytic and hydrolytic enzymes, and metabolites. These factors help in entry and exit from cells (if the pathogen is an intracellular one) and in obtaining nutrition from the host. ,
Cavrini et al. investigated the presence of T. denticola in primary and secondary root-infected canal systems with periapical pathology and correlations with clinical signs and symptoms. The presence of T. denticola in root canal system in association with specific clinical signs and symptoms of endodontic disease strongly suggests that this spirochete might play a critical role in the pathogenesis of the acute infection and rapid bone tissue alterations in both primary and secondary endodontic infections. 
Selcuk M. Ozbek and Ahmet Ozbek investigate the presence of "Red complex" in acute periradicular abscesses by using real-time PCR method. At least 1 member of the red complex was found in 84% of the cases. In general, T. denticola, P. gingivalis, and T. forsythia were detected in 65.6%, 43.7%, and 40.6% of the cases, respectively. Red complex was detected in 15.6% of samples taken from acute periradicular abscesses. Our findings suggest that "Red complex" can participate in the pathogenesis of acute periradicular abscesses. 
"Red complex" is directly associated with the severity of periodontal disease, but limited information is available on role of Red complex in endodontic lesion. Therefore, we have taken Red complex as an experimental group.
Traditionally, endodontic infections have been studied only by culture-dependent methods. Curiously, all previous cultural studies have demonstrated that most of the putative periodontopathogens have not commonly been isolated from endodontic infections.
To overcome these limitations, novel culture-independent methods for microbial identification that involve DNA amplification of 16S rDNA followed by cloning and sequencing have recently been used to determine the bacterial diversity within diverse environments. A significant contribution of molecular methods to medical microbiology relates to the identification of previously unknown human pathogens. In addition, molecular studies have revealed that about 40-50% of the bacterial clones in the oral cavity represent unknown and as-yet uncultivable species. ,
The PCR method is based on the in vitro replication of DNA through repetitive cycles of denaturation, primer annealing, and extension steps. The target DNA serving as template melts at temperatures high enough to break the hydrogen bonds holding the strands together, thus liberating single strands of DNA. Two short oligonucleotides (primers) are annealed to complementary sequences on opposite strands of the target DNA. Primers are selected to encompass the desired genetic material, defining the two ends of the amplified stretch of DNA. In sequence, a complementary second strand of new DNA is synthesized through the extension of each annealed primer by a thermostable DNA polymerase in the presence of excess deoxyribonucleoside triphosphates. All previously synthesized products act as templates for new primer extension reactions in each ensuing cycle. The result is the exponential amplification of new DNA products, which confers extraordinary sensitivity in detecting the target DNA. In fact, PCR has unrivaled sensitivity - it is at least 10-100 times more sensitive than the other more sensitive identification method. ,,,
There are nine types of PCR such as Nested PCR, Reverse Transcriptase PCR (RT-PCR), PCR-Based Microbial Typing, Real-Time PCR, Broad-Range PCR, Multiplex PCR, etc.  Variations in PCR technology can also be used to type microbial strains.
Most PCR assays have concentrated on the detection of a single microbial species by means of individual reactions. In multiplex PCR, two or more sets of primers specific for different targets are introduced in the same reaction tube. Since more than one unique target sequence in a clinical specimen can be amplified at the same time, multiplex PCR assays permit the simultaneous detection of different microbial species. Multiplex PCR assays have been used to minimize the time and expenditure needed for detection approaches. Primers used in multiplex assays must be designed carefully to have similar annealing temperatures and to lack complementarity. ,
Bacteria have to live and survive under continuously changing environmental conditions and therefore are forced to adapt, which might be accomplished by their association in complexes. In the periodontal tissues, this intrinsic dependence is a determinant to the microorganism survival to avoid host defenses. On the other hand, in the apical third of the root canal, which is considered a critical territory because of its anatomical complexity, the host tissues can only prevent periradicular damage because the defense system is unable to act inside the root canal system during pulp necrosis. Therefore, the bacterial associations in stratified complexes forming biofilms might not be a critical factor for their survival inside the root canal. However, their interactions are the key for the development of pulpal and periradicular pathosis. 
| Conclusions|| |
Because of the high prevalence of P. gingivalis, T. denticola, and T. forsythia in the samples examined in the present study and considering their virulence factors and pathogenicity, the results suggest that these bacteria are related to the etiology of periapical abscesses. The "red complex" was detected in few cases and was associated with specific signs and symptoms of endodontic origin. However, further studies are required to stratify the microbiota of root canals and determine its relationship with the development of periradicular diseases.
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Tulsi Hasmukhrai Sanghavi
K. M. Shah Dental College, Piparia, Vadodara - 391 760, Gujarat
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2]
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