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| ORIGINAL ARTICLE |
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| Year : 2020 | Volume
: 10
| Issue : 3 | Page : 97-104 |
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Detection and comparison of spirochetes in periodontal health and disease by microscopy and polymerase chain reaction techniques: A microbiological study
Reshma Naik1, SP Pushpa2, Kishore Bhat2, Sanjeevini Hattarki2
1 Consulting Periodontist, Sathyadeep Dental Clinic, BTM Layout 2nd Stage, Bengaluru, Karnataka, India
2 Department of Periodontology, Central Research Laboratory, Maratha Mandal's dental college and research centre, Belgavi, Karnataka, India
| Date of Submission | 13-Feb-2020 |
| Date of Acceptance | 09-Sep-2020 |
| Date of Web Publication | 21-Dec-2020 |
Correspondence Address:
Dr. Sanjeevini Hattarki
Department of Periodontology, Maratha Mandal's Dental College and Research Centre, Bauxite Road, Near APMC Police Station, Belgavi, Karnataka
India
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/jid.jid_5_20
 Abstract | | |
Background: Periodontitis is caused by specific microorganisms or group of specific microorganisms. Spirochetes have been implicated to play a role in the pathogenesis of the periodontal disease. Objectives: The aim is to detect the prevalence of total spirochetes in patients with healthy periodontium, gingivitis, and periodontitis using microscopy and polymerase chain reaction (PCR) techniques. And to compare microscopy (silver nitrate staining) technique and PCR technique in the detection of spirochetes. Subjects and Methods: A total of 150 patients were selected and divided into healthy, gingivitis, and periodontitis groups with 50 patients in each group. Subgingival plaque samples were collected using sterile Gracey curettes. The plaque samples were analyzed for the presence of spirochetes using microscopy and PCR techniques. Statistical analysis for the prevalence of spirochetes was performed using the Chi-square test. The sensitivity and specificity of the microscopy and PCR techniques were compared. Results: The prevalence of spirochetes increased from healthy to gingivitis to periodontitis patients. The prevalence of spirochetes was statistically significantly higher in the periodontitis group as compared to the healthy and gingivitis group. PCR was more sensitive in detecting spirochetes than microscopy, but that microscopy showed slightly greater specificity than PCR. Conclusion: There is a strong association between the presence of spirochetes with periodontal disease as well as its severity. The microscopic method by silver nitrate staining can be a very useful, simple, and rapid screening method for the evaluation of oral spirochetes, but PCR technique may serve as more sensitive indicator of subjects at risk for periodontal disease, thus allowing preventive measures.
Keywords: Microscopy, periodontitis, polymerase chain reaction, spirochetes
How to cite this article:
Naik R, Pushpa S P, Bhat K, Hattarki S. Detection and comparison of spirochetes in periodontal health and disease by microscopy and polymerase chain reaction techniques: A microbiological study. J Interdiscip Dentistry 2020;10:97-104 |
How to cite this URL:
Naik R, Pushpa S P, Bhat K, Hattarki S. Detection and comparison of spirochetes in periodontal health and disease by microscopy and polymerase chain reaction techniques: A microbiological study. J Interdiscip Dentistry [serial online] 2020 [cited 2023 Jun 10];10:97-104. Available from: https://www.jidonline.com/text.asp?2020/10/3/97/304157 |
| Clinical Relevance to Interdisciplinary Dentistry | |  |
Spirochetes are also present in endodontic lesions, and are also associated with periimplantitis.
| Introduction | | |
Infectious diseases represent a category of the population–environment interactions involving a host plus a microorganism with the potential for both colonization and pathogenesis.[1] Periodontal diseases are such multifactorial infections elicited by a complex of bacterial species and their interaction with host tissues and cells, leading to the destruction of the periodontal structures. Various spirochetal morphotypes observed in various periodontal diseases possess many of these features needed for adherence, invasion, and damage of the periodontal tissues. However, many of these morphotypes are as yet uncultivable.[2]
Oral spirochetes, inspite of being considered to be dominant bacterial types more often found in diseased periodontal sites, definitive information on the acquisition of oral spirochetes is lacking. This dearth of knowledge relative to oral spirochetes reflects the difficulty in isolating and cultivating them;[3]
- In cultivation methods, spirochetes being relatively fastidious, barely showed up in count data[4]
- As spirochetes are delicate organisms relative to other bacterial types found in dental plaque, this affects their isolation as they are usually lysed.[3]
Hence, they were overlooked, bypassed, and hardly mentioned in studies.[4] They are the least studied and understood members of plaque flora.[3] Furthermore, it is necessary to understand that various factors influence the onset and extent of periodontitis, which include environmental risk factors, genetic variance, geographical distribution, food habits, and susceptibility of an individual/population, etc., The prevalence of microorganisms may also vary depending on these factors.[1],[5] And to the best of our knowledge, no studies have been conducted on the association of spirochetes with periodontal diseases in India.
Hence, the aim of the present study is to detect the prevalence of spirochetes in periodontal health and disease using microscopy and polymerase chain reaction (PCR) techniques and also the comparison of two techniques in detecting these microorganisms.
| Subjects and Methods | | |
Patient selection
Study subjects
The subjects for the study were selected from patients visiting the Department of Periodontology, Maratha Mandal's Nathajirao G Halgekar Institute of Dental Sciences and Research Center, Belgaum. A total of 150 subjects belonging to both sexes, between 30 and 55 years of age, with at least 20 natural teeth remaining, were included in the study. The subjects were divided into three groups of 50 subjects each.
Inclusion criteria
Healthy group
- Probing depth ≤3 mm
- Bleeding on probing absent, and
- No clinical signs of inflammation.
Gingivitis group
- Probing depth ≤3 mm
- Bleeding on probing present and
- Clinical signs of inflammation present.
Periodontitis group
- Clinical attachment loss in the form of periodontal pocket depth of ≥5 mm
- Bleeding on probing present and
- Clinical signs of inflammation present.
Exclusion criteria
- Patients with any systemic diseases/medically compromised patients
- Patients who have received-periodontal therapy, antibiotics/antimicrobials within 3 months before sampling
- Smokers
- Pregnant women and lactating mothers
- Tooth with cervical/proximal/subgingival caries or restorations.
A thorough periodontal examination was carried out, and the parameters selected for the study were carefully recorded. All the recording was done by a single examiner. An ethical clearance to carry out the study was obtained from the Ethical Committee, Maratha Mandal's Nathajirao G Halgekar Institute of Dental Sciences and Research Center, Belgaum. The nature and design of the study were explained to the patients and written consent was obtained for the participation.
Plaque sample collection
The patients were seated comfortably in an upright position on the dental chair, and the light was focused to illuminate the area selected. The clinical parameters, which included the bleeding on probing, gingival index, plaque index, and probing pocket depth, were recorded. Based on the clinical findings, patients were grouped into healthy, gingivitis and periodontitis groups. In periodontitis patients, plaque samples were collected from pockets of >5 mm probing depth. Pooled subgingival plaque samples were collected from each patient. After isolating the selected sites with sterile cotton rolls, supragingival plaque and calculus were removed carefully and air dried to prevent contamination of the samples. A sterile gracey curette was inserted into the selected sites, and the subgingival plaque sample was obtained by scraping along the root surface coronally.[6] From the obtained subgingival plaque samples, first a smear was prepared on the micro slides for staining technique. Subgingival plaque was collected again from the same sites and was then transferred into a sterile vials containing TE buffer and sent to the Microbiological Department for microbiological analysis using PCR technique.
Staining technique
Smear/film of the collected subgingival plaque is prepared on the microslide and is subjected to silver nitrate staining procedure.[7] Fontana's method is employed for staining of the prepared smear. The spirochetes are stained brownish-black on a brownish-yellow background. OLYMPUS BX41 @ ×100microscope was used [Figure 1]. | Figure 1: Spirochetes detected in microscope using silver nitrate staining technique
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Polymerase chain reaction technique
Includes the following laboratory procedures[8],[9]
- DNA extraction [Table 1]
- PCR
- Gel electrophoresis [Figure 2].
 | Table 1: Nucleotide sequences of polymerase chain reaction primers used for identification of Spirochetes[9]
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 | Figure 2: Spirochetes detected using polymerase chain reaction technique. Representative of polymerase chain reaction - generated DNA bands in the gel electrophoresis. Lane 2, 4, 5, 10-represent clinical samples positive for Spirochetes. Lane 15-bp ladder
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Statistical analysis
Software SPSS 19 was used for data analysis. Chi-square test was used for comparison of the prevalence of total spirochetes in patients with healthy periodontium, gingivitis, and periodontitis. The sensitivity and specificity of the microscopy technique and PCR technique was analyzed for the detection of Total Spirochetes. Kruskal–Wallis ANOVA and Mann–Whitney U-test was used for comparison of Plaque scores and Gingival index scores of three groups, i.e., healthy periodontium, gingivitis, and periodontitis. One-way ANOVA and Tukey's multiple post hoc procedures used for comparison of three groups, i.e., healthy periodontium, gingivitis, and periodontitis.
| Results | | |
Observations
[Table 2], [Table 3], [Table 4], [Table 5] describe the comparison of three groups with respect to Plaque scores, gingival index scores, and pocket depth scores, respectively. All the values show significantly higher scores in patients with periodontitis group as compared to gingivitis and healthy groups. | Table 2: Comparison of three group's: Healthy periodontium, gingivitis and periodontitis with respect to plaque scores by Kruskal-Wallis ANOVA and Mann-Whitney U-test
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 | Table 3: Comparison of three group's: Healthy periodontium, gingivitis and periodontitis with respect to gingival index scores by Kruskal-Wallis ANOVA and Mann-Whitney U-test
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 | Table 4: Comparison of healthy periodontium, gingivitis and periodontitis with respect to pocket depth scores by one-way ANOVA
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 | Table 5: Pair wise comparison of three group's i.e., healthy periodontium, gingivitis and periodontitis with respect to pocket depth scores by Tukeys multiple post hoc procedures
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[Table 6] depicts the comparison of prevalence of total spirochetes in the subgingival plaque samples of patients with healthy periodontium [Graph 1], gingivitis and periodontitis sing microscopy technique. In healthy and gingivitis groups, 8 (16%) and 10 (20%) samples respectively showed the presence of spirochetes, while in periodontitis group 30 (60%) samples were positive for spirochetes. The prevalence of total spirochetes was significantly high with P< 0.001 in periodontitis group samples as compared to healthy and gingivitis groups. | Table 6: Comparison of prevalence of total spirochetes in patients with healthy periodontium, gingivitis and periodontitis using microscopy technique
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[Table 7] shows the prevalence of total spirochetes in the subgingival plaque samples of patients with healthy periodontium, gingivitis and periodontitis using PCR technique [Graph 2]. In healthy and gingivitis groups, 14 (28%) and 19 (38%) samples respectively were positive for the presence of spirochetes, in periodontitis group 38 (76%) samples were positive for spirochetes. The prevalence of total spirochetes was significantly high with P< 0.001 in periodontitis group samples as compared to healthy and gingivitis groups. | Table 7: Comparison of prevalence of total spirochetes in patients with healthy periodontium, gingivitis and periodontitis using polymerase chain reaction technique
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[Table 8] shows the comparative results of detection of spirochetes with both microscopy and PCR techniques. Overall, out of 150 samples 42 were positive for spirochetes in both the techniques, 73 were negative in both methods of detection, 29 samples were PCR positive for spirochetes but microscopy negative, while 6 were microscopy positive and PCR negative. | Table 8: Comparison of microscopy and polymerase chain reaction techniques for detection of spirochetes in healthy, gingivitis and periodontitis groups
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[Table 9] describes sensitivity and specificity of microscopy technique using PCR technique as standard reference for the detection of spirochetes. For the detection of spirochetes, the sensitivity of microscopy technique in healthy group was 50%, while the specificity was 97.22%. In gingivitis group, sensitivity was 47.36%, and the specificity was 96.77%. In periodontitis patients, the sensitivity was 68.42%, while the specificity was 66.67%. For overall combined samples, the sensitivity was 59.15%, specificity was 92.41%. | Table 9: Sensitivity and specificity of microscopy technique using polymerase chain reaction technique as standard reference for the detection of spirochetes
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[Table 10] describes sensitivity and specificity of PCR technique using microscopy technique as standard reference for the detection of spirochetes. For detection of spirochetes, in healthy group the sensitivity and specificity of PCR technique was 87.5% and 83.33%, respectively. In gingivitis group, sensitivity was 90%, and the specificity was 75%. In periodontitis patients, the sensitivity was 86.67%, while the specificity was 40%. For overall combined samples, the sensitivity and specificity was 87.5% and 71.57%, respectively. | Table 10: Sensitivity and specificity of polymerase chain reaction technique using microscopy technique as standard reference for the detection of spirochetes
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| Discussion | | |
Periodontal diseases are multifactorial infections elicited by a complex of bacterial species that interact with host tissues and cells causing the release of a broad array of inflammatory mediators which lead to destruction of the periodontal structures, including the tooth supporting tissues, alveolar bone and periodontal ligament.[10]
It has been recognized for decades that the chief insults/triggers for the initiation of disease is the presence of complex microbial biofilms that colonize the tooth surface.[10] Yet, the promulgation of the idea that only a small and specific subset of the bacterial microflora is of etiological significance has always been evidenced through various studies.[4] A large body of evidence accumulated over the decades has categorized oral spirochetes, as periodontal pathogens. Also, early in the history of Periodontal microbiology, when dentists and dental scientists used microscopes as the principal research tool to identify oral bacteria, spirochetes were considered key players among the types of bacteria observed in subgingival plaque removed from diseased periodontal sites.[4] Yet they are one of the least-studied and understood members of the plaque flora because of the difficulty in isolating them, cultivating them and in maintaining them in vitro once isolated.[3] They were overlooked, bypassed, and hardly mentioned in studies. Only recently, with the advent of various molecular techniques, have spirochetes re-emerged among a growing list of putative periodontal pathogens.[4] There is a diverse body of evidence present regarding the role of spirochetes in periodontal diseases. Facilitating the detection of these pathogens would be a major benefit in periodontal assessment.
Thus, the present study was conducted to detect the prevalence of spirochetes in the subgingival plaque samples of patients with healthy periodontium, gingivitis and periodontitis using microscopy technique and PCR technique and also to compare the sensitivity and specificity of both the techniques in detection of spirochetes. To our knowledge this is the first study conducted on Indian population, to detect the prevalence of spirochetes in periodontal health and disease using microscopy and PCR technique and comparison of two techniques in detection of these microorganisms. In the present study, a total of 150 subjects were included, 50 subjects in healthy group, 50 in gingivitis group, and 50 in periodontitis group. Subgingival plaque samples were obtained from each patient and analyzed using both microscopic technique and PCR technique for the presence of spirochetes.
Spirochetes are delicate organisms relative to the other bacterial types that are found in dental plaque. This affects their isolation, since procedures used to disperse the plaque, usually lyse the spirochetes. Also due to their fastidious nature and stringent requirements for anaerobic atmosphere and complex nutritional supplements, cultivation of these microorganisms is difficult.[3]
Thus in the present study, microscopic technique using silver nitrate staining method was used for detection of spirochetes. The results of the study for detection of spirochetes using microscopy showed that the prevalence of spirochetes in healthy periodontium was 16%, in gingivitis was 20%, and while in periodontitis was 60%. The results were statistically significant (P < 0.001) with a higher prevalence of spirochetes in the periodontitis group as compared to healthy and gingivitis groups. The results were in accordance with the results obtained by Armitage et al.[11] They used darkfield microscopy to determine the percentage of spirochetes and found clinically healthy sites harbored much lower percentage of spirochetes as compared to diseased sites, and significant relationship was demonstrated between disease-state category and the percentage of spirochetes, with the percentage of spirochetes increasing with greater severity of the periodontal disease.
The explanation for the higher prevalence of spirochetes in the periodontitis group is presumably because spirochetes show a predilection for subgingival plaque in deeper periodontal pockets, as, in this ecosystem these organisms are not at as great a risk of being swept away by saliva and masticatory forces. Furthermore, these microorganisms require anaerobic environment and stringent nutritional supplements; thus in the periodontal pockets presence of lower oxygen tension, combined with the availability of nutritional requirements, contribute to the establishment of spirochetes in these sites.[3] Similar findings were also observed in studies by Riviere et al.[12] where they showed that spirochetes were found at more sites of periodontitis (20%–40%) than gingivitis (6%–20%) and were only infrequently found at sites of periodontal health (4%–10%). In a study by Loesche, the spirochetes were the overwhelming microbial type in the plaques of adult periodontitis patients, averaging about >45% of the microscopic count. The cultivation and identification of oral spirochetes are one of the most challenging and difficult tasks; they are difficult to isolate, grow, and identify. Hence, laboratory identification of oral spirochetes is largely dependent on different molecular methods of detection.[13] Thus, in the present study, the prevalence of spirochetes in the healthy group, gingivitis group, and periodontitis group was also detected using the PCR technique.
The prevalence of spirochetes in the healthy group was 28%, gingivitis group 38% and in the periodontitis group was 76%, using PCR technique, which was statistically significantly higher than in the healthy and gingivitis group with P< 0.0001. The prevalence rate of spirochetes in the present study was similar to the results found in the study by Loesche et al.[14] using the BANA technique. Spirochetes averaged 43% of the plaque samples removed from periodontitis patients, whereas only 8% in healthy patients who were BANA positive. In a study by Riviere et al.[15] the spirochete morph group was identified by phase-contrast microscopy, and results showed the presence of Pathogen - related oral spirochetes, and some unidentified spirochetes in health associated plaque were associated with increased susceptibility to periodontitis. As the sensitivity and specificity of bacterial identification differ among different assays or techniques used for detection of microorganisms, this affects the detection frequency. To the best of our knowledge, the present study is the first attempt to examine the prevalence of total spirochetes in periodontal health and disease using spirochete-specific primers for the PCR technique. Since decades, the analysis of spirochetes has primarily relied on morphological observation using various microscopic techniques. Microscopes have been used as a primary research tool to identify spirochetes. Also a study by Loesche et al.[16] showed that the microscopic approach was comparable to DNA probes and the immunological reagents in the detection of Treponema denticola. Recently, technological advances in microbial identification and classification based on the introduction of nonculture methods such as DNA hybridization, PCR, etc., have allowed the detection of microorganisms that are difficult or even impossible to culture, like spirochetes.[6] The identification of oral spirochetes is largely dependent on either various microscopic techniques or different molecular methods such as PCR and hybridization.[6] Thus one of the objectives of the present study was to compare Microscopy and PCR techniques for the detection of spirochetes by evaluating the sensitivity and specificity of the two techniques.
In a study, Coffey et al.[17] compared silver staining with the darkfield technique, which is considered the gold standard for morphotype differential counting of plaque. The silver staining technique provided a quick and convenient method. The counts made by silver stain were closely associated with counts made under darkfield microscopy and had the additional benefit of requiring no special microscopic setup, and permanent records can be maintained, making it easier to evaluate the prognosis after therapy in patients. The silver stain technique was particularly suitable for the enumeration of the proportion of Spirochetes in plaque. Furthermore, a study by Choudhary et al.[13] described silver nitrate staining as a useful method for the detection of spirochetes. It can also be used to assess the cell size, density, and posttherapy evaluation. Hence, in the present study, direct light microscopy using silver nitrate stain was used.
According to the present study, the sensitivity of PCR for the detection of spirochetes in the healthy group, gingivitis group, periodontitis group, and combined samples was 87.5%, 90%, 86.6%, and 87.5%, respectively. The sensitivity of microscopy for the detection of spirochetes in the healthy group, gingivitis group, periodontitis group, and combined samples was 50%, 47.36%, 68.42%, and 59.15%, respectively. While the specificity of PCR for the detection of spirochetes in the healthy group, gingivitis group, periodontitis group, and combined samples was 83.33%, 75%, 40%, and 71.5%, respectively. as compared to the microscopy technique where the specificity of microscopy for the detection of spirochetes in the healthy group, gingivitis group, periodontitis group and combined samples was 97.22%, 96.77%, 66.67%, and 92.41%, respectively. The results of the present study showed that PCR was more sensitive in detecting spirochetes than microscopy, but that microscopy showed slightly greater specificity than PCR. In a total of 150 samples, for the detection of spirochetes, PCR positive, but microscopy negative samples were 29, while microscopy positive and PCR negative were only 6. Similar observations were also made in the study by Claude Bayingana et al.[18] where PCR was found to be more sensitive than BANA in detecting red complex organisms, but BANA showed greater specificity. Furthermore, in a study by Eick and Pfister[19] commercial PCR based method was shown to be highly sensitive and specific than microbial cultivation for the detection of periodontopathogenic species in subgingival plaque samples. However, the comparison of our microbiological results with those of other studies is complicated by the fact that different methods and cutoffs were applied and also different groups of organisms were analyzed in each study.[19]
However, in the present study, the higher sensitivity of PCR as compared to microscopy may be largely attributed to the fact that PCR may detect as little as one organism in a sample. It has the ability to detect and identify specific species even when present in low numbers, while microscopic technique could not detect.[18] The detection of spirochetes may be difficult when various forms and groups of microorganisms are present in clusters or clumps in a prepared smear where individual organisms cannot be clearly differentiated and identified using microscopy, thus giving false-negative results.
The low specificity of PCR may be due to a large number of positive samples with PCR technique as compared to microscopy. This could be first, due to cross reactions with related species or contamination due to error during DNA extraction, DNA amplification or gel electrophoresis procedures that can give rise to false-positive results, second, due to deficiency of microscopy procedure in its ability to detect target organisms,[16] especially T. denticola because of its smaller size.
| Conclusion | | |
Thus, to conclude, in the present study, the prevalence of spirochetes increased from healthy to gingivitis to periodontitis patients, thus ascertaining their correlation to the severity of the periodontal disease. Furthermore, in the present study, a comparison of microscopy and PCR techniques for the detection of spirochetes is made. PCR is found to be more sensitive but slightly less specific than the microscopy technique.
Some of the limitations of the present study could be, conventional PCR technique was used in the present study to detect just the mere presence or absence of spirochetes rather than quantification of the spirochetal load in each sample using quantitative real-time PCR and correlating it with the severity of periodontal destruction. Microscopy technique was used in the present study. Although microscopic method by silver nitrate staining can be a very useful screening method for the evaluation of oral spirochetes, one must not forget that using silver nitrate microscopic technique, and the final outcome is heavily dependent on the quality of reagents used and the experience of the personnel involved in staining and microscopic analysis and also there could be a high amount of variability among different microscopists reporting same slide or even same microscopist reporting the same slide at different times. Thus a more definitive and reliable method can be tried in future studies. Furthermore, the effect of periodontal treatment on the incidence and prevalence of these organisms was not studied. Thus longitudinal studies and clinical trials are needed to be carried out to know the exact association of spirochetes in periodontal health and disease.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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| 13. | Choudhary V, Ingalagi P, Bogar C, Rao S, Bhat K. Utility of silver nitrate staining in detection of spirochetes from oral cavity of periodontally healthy and diseased individuals. Int J Pharm Bio Sci 2012;3:980-6. |
| 14. | Loesche WJ, Syed SA, Stoll J. Trypsin-like activity in subgingival plaque. A diagnostic marker for spirochetes and periodontal disease? J Periodontol 1987;58:266-73. |
| 15. | Riviere GR, DeRouen TA, Kay SL, Avera SP, Stouffer VK, Hawkins NR. Association of oral spirochetes from sites of periodontal health with development of periodontitis. J Periodontol 1997;68:1210-4. |
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9], [Table 10]
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