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ORIGINAL RESEARCH Table of Contents   
Year : 2007  |  Volume : 18  |  Issue : 1  |  Page : 6-10
The detection of eight putative periodontal pathogens in adult and rapidly progressive periodontitis patients: An institutional study


1 Dept. of Oral Biology School of Dental Medicine, SUNY at Stony Brook, NY - 11794-8700, USA
2 Dept. of Periodontics, College of Dental Sciences, Davangere, Karnataka, India

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Date of Submission06-Feb-2006
Date of Decision05-Apr-2006
Date of Acceptance12-Apr-2006
 

   Abstract 

Purpose: Periodontal disease is a commonly prevalent problem faced alike by both the developed and third world countries but showing wide variations in prevalence and severity across different geographical areas. The purpose was to identify Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Ekinella corrodens (Ec), Campylobacter rectus (Cr), Bacteroides forsythus (Bf), Treponema denticola (Td) and Fusobacterium nucleatum (Fn) in Indian adult periodontitis and rapidly progressive periodontitis patients. Materials and Methods: Paper points were used to collect the sample from 28 sites in both adult periodontitis and rapidly progressive periodontitis (8 healthy/20 diseased sites) patients and DNA analysis done. The categorical data was analysed by Fishers exact test and difference in the clinical parameters was tested by Mann-Whitney test. Results: In healthy sites of adult and rapidly progressive periodontitis patients, Aa, Ec, Bf and Aa, Pg, Pi, Td, Fn were detected respectively. However, when diseased and healthy sites were compared in both adult periodontitis and rapidly progressive periodontitis patients respectively, only Pg( P =0.004), Cr( P =0.04), Fn( P =0.014) and Pg( P =0.002), Cr( P =0.02), Fn( P =0.008) were statistically significant. Conclusion: The prevalence of the microorganisms correlate with the clinical parameters like probing depth and bleeding on probing as seen in the Japanese and Western periodontitis patients' population.

Keywords: DNA probes, periodontal pathogens, periodontitis

How to cite this article:
Joshi VM, Vandana K L. The detection of eight putative periodontal pathogens in adult and rapidly progressive periodontitis patients: An institutional study. Indian J Dent Res 2007;18:6-10

How to cite this URL:
Joshi VM, Vandana K L. The detection of eight putative periodontal pathogens in adult and rapidly progressive periodontitis patients: An institutional study. Indian J Dent Res [serial online] 2007 [cited 2014 Aug 1];18:6-10. Available from: http://www.ijdr.in/text.asp?2007/18/1/6/30914

   Introduction Top


Periodontal disease is a commonly prevalent oral health problem faced alike by both the developed and third world countries but showing wide variations in prevalence and severity across different geographical areas.

Most information obtained about the micobiota of periodontal disease has emerged from studies carried out in Europe and USA. Only recently have the periodontal microflora been examined in some third world countries and the results indicate some differences both qualitatively and quantitatively when compared to those perceived in the west. This variations in prevalence and severity of the disease may reflect differences in the periodontal microflora in different geographical regions or in different racial groups in the same region.

Also, most of the studies were carried out using conventional cultivation technique and samples being transported to distant laboratories that required greater time. Some studies also reported the periodontal pathogens to be underestimated by cultivation of samples transported from Kenya to Sweden[1] and from Sudan to Norway.[2] A major advantage with the DNA probes are their potential to distinguish between strains of high or low pathogenicity within a given species. Also DNA probes do not require viable microorganisms for detection. Limitations of DNA probes are that only the organisms for which the probes are available can be identified and that information on antimicrobial susceptibility is not provided. Also cross reactivity with related organisms may occur for A. actinomycetemcomitans and other organisms, especially if whole genomic DNA probes are used.

The location of the site to be sampled, the level of disease activity and the sensitivity of the detection method often determine the detection frequency of these periodontal bacteria in the oral cavity. DNA probes are highly sensitive in detecting the pathogens than the culture method.[3]

Lopez et al[4] used the Microdentex® DMDx test and found high Porphyromonas gingivalis (Pg) and Prevotella intermedia (Pi) in adult periodontitis patients. The DNA probe was found to be at least equivalent or often superior to culture method, detecting higher levels of Pg, Pi, Fusobacterium nucleatum (Fn) and  Actinobacillus actinomycetemcomitans Scientific Name Search itans (Aa).[2],[5],[6] Information on periodontal microflora in Indian populations are scarce, while no study has yet been carried out to investigate periodontal microflora in any Indian population using DNA probe analysis. Therefore the main aim of the present study was to investigate the presence of Aa, Pg, Pi, Ekinella corrodens (Ec), Campylobacter rectus (Cr), Bacteroides forsythus (Bf) and Treponema denticola (Td), Fusobacterium nucleatum (Fn) in healthy and diseased sites in subgingival samples obtained from adult and rapidly progressive periodontitis patients. Additionally other clinical parameters were also correlated with the presence of microorganisms in adult and rapidly progressive periodontitis patients.


   Materials and Methods Top


Study population

The patients for the following study were selected from the outpatient of Department of Periodontics, College of Dental Sciences, Davangere, Karnataka, India. Both the sexes were included in the study.

The selected patients were clinically diagnosed as having rapidly progressive periodontitis (aggressive periodontitis, AAP, 1999) based on the criteria established by Page and Schroeder[7] and adult periodontitis[8] (World Workshop In Clinical Periodontics 1989). Inclusion criteria for the study were patients who manifested moderate to severe bone loss without any systemic disease that could adversely affect their periodontal status, patients who had not received any antibiotics, steroids or contraceptive drugs or periodontal therapy in the last six months. Exclusion criteria included patients who were smokers/tobacco users, the suppurating sites and third molar teeth.

A total of 56 sites were selected from four patients (three females and one male) of which two were adult periodontitis and two were rapidly progressive periodontitis patients. Of the 56 sites that were selected, 28 sites were from adult periodontitis and 28 sites were from rapidly progressive periodontitis. Out of the 28 sites from each adult periodontitis and rapidly progressive periodontitis patients, eight were healthy sites with probing depth of £ 3 mm and no clinical sign of bleeding on probing present and 20 were diseased sites of moderate to advanced periodontitis, with probing depth of 5 to 7 mm, with clinical sign of bleeding on probing and radiographic evidence of moderate to severe bone loss.

Clinical procedures

The same examiner carried out all the clinical examinations in both patient groups and the bacterial sampling. One week before the sampling, the clinical parameters were recorded. Each selected site were subjected to plaque index,[9] gingival index,[10] gingival bleeding index[11] and pocket depth measurement using William's graduated probe. The dichotomous system was used to register the presence or absence of bleeding.

Bacterial sampling[3]

Subgingival plaque samples were taken from the selected teeth, by means of sterile paper points. The sample site was isolated with cotton rolls and air- dried and the supra gingival dental plaque was removed using a sterile cotton pellet and dried with air. Using a tweezer, a paper point was inserted into the base of the pocket/sulcus. After 10 seconds, the paper point was removed from the selected site and paper point end with collected plaque sample was directed first into the eppendorf vial. The vial was closed and affixed with vial bar code label. An identically numbered form bar code label is affixed to the appropriate area of the test request form. The vial and the text request form were placed into zip-lock plastic bag. All the 56 plastic bags containing the test request form and the plastic vial were properly packed and sent to the laboratory* for DNA probe analysis. A total of 56 DNA probe tests were conducted with a single test for each individual site. Upon receipt in the laboratory, patient and sample information were logged into the computer database and DNA hybridization in a format referred to in the industry as "slot blot" analyzed the samples.

The findings of DNA probe assay was scored as N = Negative (pathogen is less than 0.1% of total or fewer than 10 3 cells), L = Low (pathogen is between 0.1% to 0.9% of total 10 3 cells), M = Moderate (pathogen is between 1% to 9.9% of total 10 4 cells), H = High (pathogen is greater than 10% of total 10 4 cells).

Statistical analysis

Categorical data was analysed by Fisher's exact test and difference in the clinical parameters was tested by Mann-Whitney test. A p level of less than 0.05 was considered for significance.


   Results Top


The results are as expressed in the [Table - 1],[Table - 2],[Table - 3],[Table - 4].


   Discussion Top


Among the variety of microorganisms identifiable in the subgingival plaque of periodontal pockets, some species of bacteria have been considered to be pathogenic. The most important advantages of DNA probes over conventional culture techniques is that the laboratory technique does not require viable microorganisms for enumeration of specific bacteria. Avoiding the necessity of the live bacteria for enumeration of specific species permits off-site specimen collection, less cumbersome transport and greater reliability of the obtained results as well as improved sensitivity in detecting specific species.

In AP, Aa, Ec and Bf were detected in healthy sites and these findings was similar to the findings of Ali et al,[5] but contrast to Yano Higuchi, et al,[12] who observed that Aa was not detected in healthy sites. In AP patients Aa, Pg, Pi, Ec, Cr, Bf, Td, Fn were detected in diseased sites. These findings were in accordance with culture and DNA probes studies that evaluated the prevalence of subgingival species on sites with different clinical parameters[2],[4],[12],[14],[15] using DNA probe observed significantly high percentage of Pg being detected in bleeding on probing positive sites of AP patients as compared to non bleeding sites.

In RPP patients, Aa, Pg, Pi, Ec, Td, Fn were detected in healthy sites and all eight organisms were detected in diseased sites. Similar findings were shown by other studies, which evaluated subgingival species in sites alongwith clinical parameters. For example, Yano Higuchi et al[12] investigated the subgingival microflora of Japanese adult periodontitis and rapidly progressive periodontitis patients using culture microbiological techniques and found Bf and Pg were detected more frequently from periodontitis patients than from healthy individuals and that total cultivable number of these bacteria were significantly correlated with clinical parameters such as probing pocket depth and bleeding on probing. This evidence indicated that Bf and Pg are closely associated with the process of periodontal breakdown. Kamma et al[13] who used cultural microbiological technique in RPP lesion, found Pg to be predominant followed by Bf, Pi and Fn and Pi to be significantly higher at the bleeding sites.

There are several reasons why bleeding on probing has not had higher degree of sensitivity, yet bleeding on probing has consistently been found to have a higher degree of specificity. Its absence is a good predictor of periodontal health that is sites that do not bleed have a significantly lower risk for attachment loss or less likely to breakdown.[17] The results of the present study showed most of the organisms to be present at higher level in diseased sites with significant higher occurrence of Pg, Cr and Fn.

In AP patients, the detection of organisms in healthy and diseased sites in relation to the probing depth were in accord with culture and DNA probe studies that evaluated presence of subgingival species with different probing depth and found increased detection with increased probing depth.[12],[16],[18] In RPP patients, the detection of organisms in healthy and diseased sites in relation to the probing depth were in agreement with the findings of Yano Higuchi et al[12] who found increase in the detection of Aa, Pg and Bf in diseased sites with increased probing depth as compared to healthy sites.

There was higher percentage of detection of Bf and Td and significant presence of Pg in diseased sites as compared to healthy sites in both AP and RPP patients. A number of studies have indicated that Bf, Pg and Td are found together[19],[20],[21] and were detected more with deeper periodontal pockets.

The diseased sites showed the occurrence of Bf, Pg and Td in higher percentages which form the red complex and also the orange complex like Pi, Fn and Cr of which Pg, Fn and Cr, were significantly detected in diseased sites of both AP and RPP patients.

The relationship of different species in different complexes has been examined. The red complex and individual species in that group were strongly associated with bleeding on probing. Species in red complex exhibited a very strong relationship with pocket depth and similarly species in orange complexes showed a significant association with increasing pocket depth.[21] The species in orange complex were closely associated with one another and this complex appeared closely related to the red complex.[2],[21] This study showed Ec to be significantly present in the diseased sites of AP and RPP patients, which is speculated to be an important bridging species leading to the orange and ultimately at some sites to the red complex.[21]

In AP patients, the mean gingival index of healthy sites was 1.0 ± 0.10 (mild inflammation) where in Aa, Bf were found and at mean gingival index of 0.75 ± 0.50 (mild inflammation) Ec was found. In RPP patients, the mean gingival index of healthy sites was 1.0 ± 0.0 (mild inflammation) where in Pg, Pi, Td and Fn were found, at 0.75 ± 0.05 (mild inflammation) where in Aa and Ec were found. The mean gingival index of diseased sites was 2.0 ± 0.0 (moderate inflammation) wherein all eight organisms were found in these sites in both AP and RPP patients. The organisms detected in the present study were similar to the results of the studies conducted in periodontitis patients of developing countries.[2],[14] The prevalence of the microorganisms correlate with the clinical parameters like probing depth and bleeding on probing as seen in the Japanese and Western periodontitis patients population. The gingival index have also been correlated with the presence of microorganisms as the inflammatory response results in an environmental change subgingivally, which produces a shift in the balance of the resident microflora. Such a shift predisposes a site to disease.[22] In conclusion, this study reports occurrence of eight (Aa, Pg, Pi, Ec, Cr, Bf, Td, Fn) important periodontal pathogens in Indian periodontitis population using advanced diagnostic aid such as DNA probe analysis unlike several other studies reporting DNA probe analysis of only three periodontal pathogens (Aa, Pg, Pi).

This study being a site-specific study it cannot be compared with results of studies where AP and RPP patients and healthy individuals are compared.[2],[21],[23] There is prevalence of different species or different percent of same microorganisms in the healthy and diseased sites of AP and RPP patients.[23],[24],[25] Even a minor shift in the percent of prevalence in healthy and diseased sites of same patients could strengthen the findings of previous studies of specific organisms for periodontal pathogenesis.[2],[21],[23] DNA probe has proved to be a useful chair side test to detect the organisms, avoiding the necessity of live bacteria with greater sensitivity and specificity. Unfortunately, DNA probe analysis cannot be included as a routine diagnostic aid in India due to its non-availability and the cost factor. Considering the scarce data on the microbial population in Indian sub continent further DNA probe analysis may be considered for assessment of microbial profile in various forms of periodontitis and also the effect of inflammatory response on the specific microflora.


   Acknowledgments Top


This study was supported in part by a sponsor from Microdentex® DMDx, FL, USA. The authors are grateful to Mr. Kelvin Thomas for the help extended to carry out the study. Sincere thanks expressed to Mr. Sangam for advice in statistical analysis.

 
   References Top

1.Dahlen G, Manji F, Baelum V, Fejerskov O. Black-pigmented bacteroides species and actinobacillus actinomycetemcomitans in subgingival plaque of adult Kenyans. J Clin Periodontol 1989;16:305-10.  Back to cited text no. 1  [PUBMED]  
2.Ali RW, Bakken V, Nilsen R, Skaug N. Comparative detection frequency of 6 putative periodontal pathogens in Sudanese and Norwegian adult periodontitis patients. J Periodontol 1994;65:1046-52.  Back to cited text no. 2  [PUBMED]  
3.Noraian KW, Towner MD. O'Brien J. Providing optimal care to the patients today: the importance of monitoring levels of periodontal pathogens; The Microdentex Dmdx® DNA Probe Test for periodontal pathogens. DNA Testing Report.  Back to cited text no. 3    
4.Lopez NJ. Occurrence of actinobacillus actinomycetemcomitans, porphyromonas gingivalis and prevotella intermedia in progressive adult periodontitis. J Periodontol 2000;71:948-55.  Back to cited text no. 4    
5.Ali RW, Johannessesn AC, Dahlen G, Socransky SS, Skaug N. Comparison of the subgingival microbiota of periodontally healthy and diseased adults in Northern Cameroon. J Clin Periodontol 1997;24:830-5.  Back to cited text no. 5    
6.Savitt ED, Strzempko MN, Vaccaro KK, Peros WJ, French CK. Comparison of cultural methods and DNA probe analysis for the detection of actinobacillus actinomycetemcomitans, bacteroides gingivalis and bacteroides intermedius in subgingival plaque samples. J Periodontol 1988;59:431-8.  Back to cited text no. 6    
7.Roy C, Schroeder HE. Periodontitis in man and other animals: A comparative review. Karger: New York; 1982. p. 54.  Back to cited text no. 7    
8.Carranza FA Jr. Classification of diseases of the periodontium. In : Carranza FA Jr, Newman MG. Clinical Periodontology. 8th ed. Prism Books Pvt Ltd: 1996. p. 58-61.  Back to cited text no. 8    
9.Silleness J, Loe H. Periodontal disease in pregnancy. II. Correlation between oral hygiene and periodontal condition. Acta Odontologica Scandinavica 1964;22:121-35.  Back to cited text no. 9    
10.Loe H. and Sillenes J. The gingival index, the plaque index and the retention index system. J Periodontol 1967;38:610-16.  Back to cited text no. 10    
11.Valdmir WS. Epidemiology of gingival and periodontal disease. In : Carranza FA Jr, Newman MG. Clinical Periodontology. 8th ed. Prism Books Pvt Ltd: 1996. p. 61-79.  Back to cited text no. 11    
12.Yano-Higuchi K, Takamatsu N, He T, Umeda M, Ishikawa I. Prevalence of bacteroides forsythus, porphyromonas gingivalis and actinobacillus actinomycetemcomitans in subgingival microflora of Japanese patients with adult and rapidly progressive periodontitis. J Clin Periodontol 2000;27:597-602.  Back to cited text no. 12    
13.Kamma JJ, Nakou M, Manti FA. Microbiota of rapidly progressive periodontitis lesions in association with clinical parameters. J Periodontol 1994;65:1073-8.  Back to cited text no. 13    
14.Ali RW, Velcescu G, Jivanescu MC, Lofthus B, Skaug N. Prevalence of six putative periodontal pathogens in subgingival plaque sample from Romanian adult periodontitis patients. J Clin Periodontol 1996;23:133-9.  Back to cited text no. 14    
15.Ximenez-Fyvie LA, Haffajee AD, Socransky SS. Microbial composition of supra and subgingival plaque in subjects with adult periodontitis. J Clin Periodontol 2000;27:722-32.   Back to cited text no. 15    
16.Kojima T, Yasui S, Ishikawa I. Distribution of porphyromonas gingivalis in adult periodontitis patients. J Periodontol 1993;64:1231-7.   Back to cited text no. 16    
17.Joss A, Adler R, Lang N, Bleeding on probing. A parameter for maintaining periodontal conditions in clinical practice, J Clin Periodontal 1994;21:402-08.  Back to cited text no. 17    
18.Socransky SS, Haffajee AD, Smith C, Dibart S. Relation of counts of microbial species to clinical status at the sampled site. J Clin Periodontol 1991;18:766-75.  Back to cited text no. 18    
19.Simonson LG, McMahon KT, Childers DW, Morton HE. Bacterial synergy of Treponema denticola and Porphyromonas gingivalis in a multinational population. Oral Microbiol Immunol 1992;7:111-2.  Back to cited text no. 19    
20.Haffajee AD, Socransky SS. Microbial etiological agents of destructive periodontal disease. Periodontology 2000-1994;5:78-111.  Back to cited text no. 20    
21.Socransky SS, Haffajee AD, Cugini MA, Smith C, Kent RL Jr. Microbial complexes in subgingival plaque. J Clin Periodontol 1998;25:134-44.  Back to cited text no. 21    
22.Marsh PD. Microbial ecology of dental plaque and its significance in health and disease. Adv Dent Res 1994;8:263-71.  Back to cited text no. 22    
23.Haffajee AD, Cugini MA, Tanner A, Pollack RP, Smith C, Kent RL Jr, et al . Sub gingival microbiota in healthy, well maintained elder and periodontal subjects. J Clin Periodontol 1998;25:346-53.  Back to cited text no. 23    
24.Socransky SS, Haffajee AD. The bacterial etiology of destructive periodontal disease: Current concepts. J Periodontol 1992;63:322-31.  Back to cited text no. 24    
25.Socransky SS, Haffajee AD. Evidence of bacterial etiology; a Historical perspective. Periodontology 2000-1994;5:7-25.   Back to cited text no. 25    

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Correspondence Address:
K L Vandana
Dept. of Periodontics, College of Dental Sciences, Davangere, Karnataka
India
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DOI: 10.4103/0970-9290.30914

PMID: 17347537

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    Tables

[Table - 1], [Table - 2], [Table - 3], [Table - 4]

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