Journal of Investigative and Clinical Dentistry (2010), 1, 126–132
ORIGINAL ARTICLE Oral Microbiology
Detection rates of presumptive periodontal pathogens in subgingival plaque samples of untreated periodontitis using either four or six pooled samples Martin Wohlfeil, Orhan Tabakci, Rita Arndt, Peter Eickholz & Katrin Nickles Department of Periodontology, Center for Dental, Oral, and Maxillofacial Medicine (Carolinum), Johann Wolfgang Goethe-University, Frankfurt, Germany
Keywords 16S rRNA gene probe, Aggregatibacter actinomycetemcomitans, aggressive periodontitis, chronic periodontitis, generalized, severe, subgingival plaque. Correspondence Prof. Peter Eickholz, Poliklinik fu¨r Parodontologie, ZZMK (Carolinum), Theodor-Stern-Kai 7, Frankfurt am Main, D-60590, Germany. Tel: +49-69-6301-5642 Fax: +49-69-6301-3753 Email: [email protected] Received 12 December 2009; accepted 20 June 2010. doi: 10.1111/j.2041-1626.2010.00026.x
Abstract Aim: A comparison of the detection frequency and number of periodontal pathogens in patients with aggressive or generalized, severe chronic periodontitis using a gene-probe analysis. Methods: In 16 aggressive and 34 generalized, severe chronic periodontitis patients, plaque was sampled from the deepest pockets per quadrant (MT4) and per sextant (MT6). After sampling two paper points simultaneously, one paper point from each pocket was pooled with three paper points of the other pockets (MT4). The remaining four paper points were pooled with two paper points from the deepest pockets from the two remaining sextants (MT6). Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Porphyromonas gingivalis, and Treponema denticola were detected by 16S rRNA gene probes. Results: Log-transformed counts for Aggregatibacter actinomycetemcomitans were statistically significantly higher with MT6 (aggressive: 3.21 ± 2.94; generalized, severe chronic: 2.22 ± 2.70) than MT4 (aggressive: 2.04 ± 2.74; generalized, severe chronic: 1.50 ± 2.37) (P < 0.05). The detection frequency and mean counts were high for Tannerella forsythia, Porphyromonas gingivalis, and Treponema denticola (>95%/>6.0). Conclusion: Aggregatibacter actinomycetemcomitans was detected in higher numbers for MT6 than MT4. For both MT4 and MT6, Tannerella forsythia, Porphyromonas gingivalis, and Treponema denticola were detected in >95% of all patients and with mean log-transformed numbers >6.0.
Introduction From the approximately 400 bacterial species colonizing periodontal pockets, and a further 300 in the rest of the oral cavity,1,2 some are frequently associated with periodontal destruction. Aggregatibacter actinomycetemcomitans,3 Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola 4,5 are considered periodontal pathogens. Further, A. actinomycetemcomitans has been closely associated with the etiology of a severe periodontal disease, aggressive periodontitis (AgP)6–10 and periodontitis as manifestation of Papillon–Lefe`vre syndrome.11 Aggrega126
tibacter actinomycetemcomitans is a micro-aerophilic, facultative anaerobic and Gram-negative coccoid rod belonging to the family of Pasteurellacea.3 Periodontal disease associated with A. actinomycetemcomitans in many cases cannot be treated reliably and predictively by the mechanical removal of the subgingival biofilm alone.6,12–17 Thus, the detection of A. actinomycetemcomitans is a significant factor contributing to the decision as to whether subgingival debridement should be performed in conjunction with systemic antibiotics.18,19 Sampling of the deepest pocket of each quadrant has been demonstrated to quite reliably detect the subgingival ª 2010 Blackwell Publishing Asia Pty Ltd
M. Wohlfeil et al.
presence of periodontal pathogens in untreated patients.15,20–22 Sampling one to six sites per patient increases the probability of detecting the targeted bacteria.23 However, sampling six sites instead of four sites increases the cost of diagnosis. Until now, the comparison of pooled samples from four to six sites has only been investigated for polymerase chain reaction (PCR)-based microbiological analyses.24 Thus, in this study the results of the microbiological 16S rRNA gene probe analyses of pooled subgingival plaque samples from the four deepest sites per quadrant should be compared with the results from the pooled samples from the six deepest sites per sextant. Materials and methods Patients Fifty patients suffering from untreated AgP or generalized, severe, chronic periodontitis (ChP), who were scheduled for systematic periodontal therapy at the Department of Periodontology, Center for Dental, Oral, and Maxillofacial Medicine (Carolinum), Johann Wolfgang GoetheUniversity, Frankfurt, Germany, were recruited for this study between September 2007 and November 2008. To be included in this study the following inclusion criteria had to be fulfilled: (a) a clinical diagnosis of untreated (no non-surgical or surgical subgingival debridement within the last 12 months before microbiological sampling) AgP or ChP; (b) at least 18 years of age; (c) at least five teeth per quadrant; and (d) provided informed, written consent. The following criteria lead to exclusion from the study: (a) a need for antibiotic prophylaxis in advance of periodontal diagnosis or treatment; (b) antibiotic therapy within the last 6 months or subgingival debridement (non-surgical or surgical) within the last 12 months before microbiological sampling; and (c) anti-infective periodontal therapy with adjunctive systemic amoxicillin or ciprofloxacin and metronidazole. For this study, AgP was defined9,25 as a patient who is clinically healthy (i.e. systemic diseases predisposing for periodontitis are not reported; e.g. diabetes mellitus), has a radiographic bone loss ‡50% affecting at least two different teeth, and was aged £35 years at diagnosis. ChP was defined as a patient with attachment loss ‡5 mm at more than 30% of sites, and was aged >35 years. The study conformed to the provisions of the Declaration of Helsinki (as revised in Tokyo 2004) and was approved by the Institutional Review Board for Human Studies of the Medical Faculty of the Johann Wolfgang Goethe-University (study no. 189/07). All patients gave written, informed consent to participate in the study. ª 2010 Blackwell Publishing Asia Pty Ltd
Subgingival plaque sampling strategies
Clinical examinations At six sites per tooth (mesiobuccal, mid-buccal, distobuccal, disto-oral, mid-oral, mesio-oral) probing pocket depths (PD) and vertical clinical attachment levels (CALV) were measured using a manual rigid periodontal probe (PCPUNC15, Hu Friedy, Chicago, IL, USA) to the nearest millimeter. The cemento-enamel junction (CEJ) was used as reference for the CAL-V measurements. If the CEJ was destroyed by restorative treatment, the margin of the restoration was taken as the reference. If the CEJ was located apically of the gingival margin first, the PD was measured. The CEJ was then groped by the probe, and the distance CEJ to the gingival margin was measured. The CAL-V was calculated as PD minus the distance of the CEJ to the gingival margin. Bleeding on probing was recorded 30 sec after probing. Microbiological examination Microbiological sampling was performed within the clinical routine.18,19 For sampling, the deepest site of each of the four quadrants was chosen. The test site was dried by air and held dry by using cotton rolls. Simultaneously, two sterile paper points were inserted into the bottom of the respective pocket.9,24,26 After 20 sec, the paper points were removed. One of each paper point was put into one of two transportation vials. Thus, two pooled samples of the same four sites were created. One of the vials was closed (MT4). The deepest pockets of the two remaining sextants were then selected; plaque samples were taken and pooled with the other remaining vial (MT6). For the analysis, a commercially-available 16S rRNA gene probe test kit (IAI Pado-Test 4.5; Institute for Applied Immunology, Zuchwil, Switzerland) aimed at detecting A. actinomycetemcomitans, P. gingivalis, T. forsythia, and T. denticola was used. This is an oligonucleotide probe test, complementary to conservative regions of the 16S rRNA gene, which encodes the rRNA that forms the small subunit of the bacterial ribosome. The detection limit of this test is 103.3 for A. actinomycetemcomitans and 104 for T. forsythia, P. gingivalis, and T. denticola, respectively. Statistical analysis Two variables were analyzed for each periodontal pathogen separately for AgP and ChP: prevalence (i.e. detection of the pathogen or no detection) and log-transformed bacterial counts. Aggressive periodontitis and ChP were compared according to sex, smoking status, PD, and CAL-V using the chi-squared test and t-test, respectively. The detection 127
Subgingival plaque sampling strategies
M. Wohlfeil et al.
rate and log-transformed bacterial counts for MT4 and MT6 were compared using the Wilcoxon signed rank test for paired samples. Agreement of the detection rate of both analyzing strategies was estimated by calculating Cohen’s k.27 The PD and CAL-V were used to describe the clinical status of the sampled sites. Statistical analyses were performed using Systat for Windows (version 12; Systat, Evanston, IL, USA). Results From a total of 50 patients (30 females and 20 males, 47.4 ± 13.6 years of age), clinical and microbiological examinations were obtained. Sixteen patients suffered from untreated AgP and 34 from untreated ChP (Table 1). Detection frequencies and counts for A. actinomycetemcomitans were higher for MT6 (AgP: 56%/3.21 ± 2.94; ChP: 41%/2.22 ± 2.70) than MT4 (AgP: 37%/2.04 ± 2.74; ChP: 29%/1.50 ± 2.37) (Tables 2 and 3). The differences reached statistical significance only for log-transformed counts (AgP: P = 0.047; ChP: P = 0.011) (Table 3). Detection frequencies and mean counts were generally high for P. gingivalis, T. forsythia, and T. denticola (>95%/>6.0). Statistically-significant differences between MT4 and MT6 could not be found (Tables 4–6). For T. forsythia, a small but statistically-significant difference was found between MT4 and MT6 log-transformed counts in ChP only (Table 3). Discussion The clinical relevance of the detection of A. actinomycetemcomitans in periodontal disease is a matter of debate.28,29 A. actinomycetemcomitans, T. forsythia, and P. gingivalis are frequently associated with destructive periodontal Table 1. Patients and clinical parameters (n and mean ± SD) according to diagnosis
n Females Current smokers Age (years) PD (mm) MT4 PD (mm) MT6à CAL-V (mm) MT4 CAL-V (mm) MT6à
Aggressive periodontitis
Generalized, severe, chronic periodontitis
16 11 5 31.7 7.5 7.1 7.5 7.1
34 19 11 54.8 7.9 7.3 8.5 8.0
± ± ± ± ±
6.4 1.4 1.4 1.7 1.5
P-value
0.538 1.000 ± ± ± ± ±
9.0 1.5 1.4 1.3 1.2
0.311 0.630 0.035 0.050
Deepest site per quadrant. àDeepest site per sextant. CAL-V, vertical clinical attachment levels; PD, probing pocket depth.
128
Table 2. Aggregatibacter actinomycetemcomitans detection frequency (n [%]) with the MT4 and MT6à sampling method MT6 Negative Aggressive periodontitis MT4 Negative Positive Total Generalized, severe, chronic MT4 Negative Positive Total
6 (38%) 1 (6%) 7 (44%) periodontitis 19 (56%) 1 (3%) 20 (59%)
Positive
Total
4 (25%) 5 (31%) 9 (56%)
10 (63%) 6 (37%) 16
5 (15%) 9 (26%) 14 (41%)
24 (71%) 10 (29%) 34
Deepest site per quadrant. àDeepest site per sextant. For aggressive periodontitis, there was no statistically-significant difference between MT4 and MT6 (P = 0.180). Cohen’s j: 0.394; standard error: 0.211. For generalized, severe chronic periodontitis, there was no statistically-significant difference between MT4 and MT6 (P = 0.102). Cohen’s j: 0.619; standard error: 0.136.
Table 3. Log-transformed bacterial counts for MT4 and MT6à (mean ± SD) MT4 Aggressive periodontitis Aggregatibacter 2.04 ± 2.74 actinomycetemcomitans Porphyromonas gingivalis 6.11 ± 0.81 Tannerella forsythia 6.47 ± 0.58 Treponema denticola 6.29 ± 0.55 Generalized, severe, chronic periodontitis Aggregatibacter 1.50 ± 2.37 actinomycetemcomitans Porphyromonas gingivalis 6.56 ± 0.53 Tannerella forsythia 6.48 ± 0.43 Treponema denticola 6.19 ± 1.13
MT6
P-value
3.21 ± 2.94
0.047
6.30 ± 0.60 6.55 ± 0.45 6.37 ± 0.45
0.070 0.234 0.070
2.22 ± 2.70
0.011
6.51 ± 1.19 6.58 ± 0.34 6.21 ± 1.13
0.309 0.035 0.714
Deepest site per quadrant. àDeepest site per sextant.
disease.5 In many cases of periodontal disease associated with A. actinomycetemcomitans, the mechanical removal of the subgingival biofilm alone is insufficient to eliminate or at least suppress this microorganism below the detection limit reliably and predictively.6,12–17 Persisting A. actinomycetemcomitans leads to unfavorable clinical results. The systemic use of the combination of amoxicillin and metronidazole or ciprofloxacin and metronidazole adjunctively to mechanical debridement, in most cases, results in the suppression of A. actinomycetemcomitans below detection limits and clinical improvement.30,31 The detection of A. actinomycetemcomitans fails to be a sensitive or specific diagnostic test for AgP.9,28 However, the detection of A. actinomycetemcomitans has a significant influence on ª 2010 Blackwell Publishing Asia Pty Ltd
M. Wohlfeil et al.
Subgingival plaque sampling strategies
Table 4. Porphyromonas gingivalis detection frequency (n [%]) with the MT4 and MT6à sampling method MT6 Negative Aggressive periodontitis MT4 Negative 0 (0%) Positive 0 (0%) Total 0 (0%) Generalized, severe, chronic periodontitis MT4 Negative 0 (0%) Positive 1 (3%) Total 1 (3%)
MT6 Positive
Total
0 (0%) 16 (100%) 16 (100%)
0 (0%) 16 (100%) 16
0 (0%) 33 (97%) 33 (97%)
0 (0%) 34 (100%) 34
Deepest site per quadrant. àDeepest site per sextant. For aggressive periodontitis, there was no statistically-significant difference between MT4 and MT6 (P = 1.000). Cohen’s j: 1.000; standard error: 0.000. For generalized, severe, chronic periodontitis, there was no statistically-significant difference between MT4 and MT6 (P = 0.317).
Table 5. Tannerella forsythia detection frequency (n [%]) with the MT4 and MT6à sampling method MT6 Negative Aggressive periodontitis MT4 Negative 0 (0%) Positive 0 (0%) Total 0 (0%) Generalized, severe, chronic periodontitis MT4 Negative 0 (0%) Positive 0 (0%) Total 0 (0%)
Positive
Total
0 (0%) 16 (100%) 16 (100%)
0 (0%) 16 (100%) 16
0 (0%) 34 (100%) 34 (100%)
0 (0%) 34 (100%) 34
Deepest site per quadrant. àDeepest site per sextant. For aggressive periodontitis, there was no statistically-significant difference between MT4 and MT6 (P = 1.000). Cohen’s j: 1.000; standard error: 0.000. For generalized, severe, chronic periodontitis, there was no statistically-significant difference between MT4 and MT6 (P = 1.000). Cohen’s j: 1.000; standard error: 0.000.
the decision of whether or not mechanical anti-infective therapy should be administered in conjunction with systemic antibiotics. The sampling technique applied in this study has been tested for reproducibility before in 43 patients. For sampling, the deepest pocket of each of the four quadrants was selected, i.e. 4 sites per patient. Simultaneously, two sterile paper points were inserted to the bottom of the respective pocket. After 20 sec, the paper points were removed. One of each paper points was put into one of two transportation vials. Thus, two pooled samples of the same four sites ª 2010 Blackwell Publishing Asia Pty Ltd
Table 6. Treponema denticola detection frequency (n [%]) with the MT4 and MT6à sampling method
Negative Aggressive periodontitis MT4 Negative 0 (0%) Positive 0 (0%) Total 0 (0%) Generalized, severe, chronic periodontitis MT4 Negative 0 (0%) Positive 0 (0%) Total 0 (0%)
Positive
Total
0 (0%) 16 (100%) 16 (100%)
0 (0%) 16 (100%) 16
0 (0%) 34 (100%) 34 (100%)
0 (0%) 34 (100%) 34
Deepest site per quadrant. àDeepest site per sextant. For aggressive periodontitis, there was no statistically significant difference between MT4 and MT6 (P = 1.000). Cohen’s j: 1.000; standard error: 0.000. For generalized, severe, chronic periodontitis, there was no statistically significant difference between MT4 and MT6 (P = 1.000). Cohen’s j: 1.000; standard error: 0.000.
were created and analyzed using the 16S rRNA probe test. With regard to detection frequency per patient of A. actinomycetemcomitans, for both samples (MT4/MT6) substantial agreement (Cohen’s j: 0.694) was observed. For the other periodontal pathogens, excellent agreement32 was observed (Cohen’s j: P. gingivalis: 0.758; T. forsythia: 0.845; T. denticola: 1.0).33 In the present study, paper points were used for the microbiological sampling. This is standard for commercially-available gene probe tests, and was also used in many microbiological studies.9,15,20,21,26,34,35 Thus, after investigating a commercially-available 16S rRNA gene probe test, we used paper points for sampling subgingival plaque. However, paper points have some disadvantages: if they become wet by gingival crevicular fluid, they lose stiffness, making it difficult to move them to the bottom of the pocket. Another disadvantage is that different paper points placed in the same pocket at the same time and for the same period of time might not sample the same microorganisms.26 Subgingival plaque might also be sampled using curettes.23 Curettes stay stiff, even when they become wet, and can sample subgingival plaque from a larger area than a paper point. Thus, sampling plaque with a curette might overcome some of the disadvantages of paper points. However, whereas the paper point sampling detects free floating bacteria rather than adhering bacteria, it does not remove all biofilm with sampling. Thus, paper point sampling might influence subgingival plaque less than curetting, sampling which removes most of the adhering biofilm in one stroke. Jervøe-Storm et al. compared the curette and paper point sampling technique using quantitative real-time PCR and 129
Subgingival plaque sampling strategies
demonstrated that the plaque composition, with regard to total target pathogens, was similar for both sampling techniques.36 Sixou et al. compared four different sampling techniques and drew the conclusion that curette sampling is efficient both quantitatively and qualitatively. However, difficulties in standardizing this method were encountered with the failure to achieve reproducible results. The paper point technique was found to be more reliable and reproducible in each of the three groups of patients sampled.37 Renvert et al. also considered the paper point sampling technique to be the best alternative; they found significantly higher numbers of colony-forming units and spirochetes with this method.38 Using paper points of standardized size provides standardized samples that are preferred, particularly by laboratories. Aggregatibacter actinomycetemcomitans might not be present at all oral sites in a patient suffering from untreated periodontitis.15,22 Taking subgingival samples from all teeth would be the most reliable way to detect A. actinomycetemcomitans. However, this method is too time consuming and expensive to be used in daily practice. Sampling of the deepest pocket of each quadrant has been demonstrated to quite reliably detect the subgingival presence of periodontal pathogens in untreated patients.15,20,22 Mombelli et al. sampled and microbiologically analyzed all sites separately, and theoretically evaluated different sampling strategies.15,20,21 Yet their strategies were based on separate analyses of the samples, whereas in daily practice, the different samples taken from the deepest sites per quadrant are pooled prior to analysis for economic reasons. Haffajee et al.22 analyzed the effect of sampling strategy on the false-negative rate for detection of selected subgingival species. They compared plaque samples obtained from one site per tooth with samples taken from one maxillary first molar, the maxillary first molars, four-first molars, six Ramfjord teeth,39 the deepest pocket, and the four deepest pockets. The lowest rates of undetected species were found for the four deepest pocket sampling strategy.22 Thus, for microbiological analyses in daily practice, sampling the deepest site per quadrant is generally recommended. After sampling one to six sites per patient, and with a separate analysis per site, Beikler et al. reported increasing the probability to detect the targeted bacteria with an increasing number of sampled sites.23 However, other authors reported contradicting observations.40 These differences might be explained by significant differences in methodology: sampling with curettes23 or paper points40 and separate23 or pooled analyses40. Thus, at least for pooled analyses, up until now it has not been clear whether sampling the deepest site per sextant (MT6) instead of the deepest per quadrant (MT4) increases the probability of detection. For the 16S rRNA gene probe test kit that was 130
M. Wohlfeil et al.
investigated in this study, the MT6 sampling strategy demonstrated advantages over MT4, particularly in the detection of A. actinomycetemcomitans. It has been recently demonstrated that there are statistically significantly higher log-transformed bacterial counts after pooled analyses than after separate analyses with the 16S rRNA gene probe (used in this study) after sampling from the deepest sites of three quadrants (MT3).26 After sampling from the deepest site of four quadrants (MT4), only minor differences were reported for P. gingivalis, T. forsythia, and T. denticola.9 For the detection rates, sampling of three sites revealed higher rates for pooled analyses (MT3) than for separate analyses only for T. denticola, but not for A. actinomycetemcomitans, P. gingivalis, and T. forsythia.26 After sampling four sites, one study failed to reveal any differences between detection rates after separate and pooled analyses (MT4).9 With increasing numbers of sampled sites, the differences between separate and pooled analyses decrease. The studied population corresponds to the populations analyzed in previous studies9,26 regarding patient parameters (age, sex) and clinical parameters (PD, CAL-V). Krigar et al. reported an A. actinomycetemcomitans detection frequency after a pooled analysis (MT3) of 26% for a population with a proportion of 27% patients with untreated aggressive periodontitis.26 For a population consisting of 50% patients with aggressive periodontitis, 38% of all patients were observed to be A. actinomycetemcomitans positive after the pooled analysis (MT4).9 In the present study on a sample with 32% of patients suffering from aggressive periodontitis, A. actinomycetemcomitans was detected in 32% (MT4) and 46% (MT6) of all patients. Porphyromonas gingivalis, T. forsythia, and T. denticola were detected in almost all patients (P. gingivalis: 98–100%, T. forsythia: 100%, and T. denticola: 98%). These detection frequencies are in accordance with observations previously made by others in patients with untreated AgP and ChP.9,26,29 Thus, in untreated AgP and ChP, the detection of P. gingivalis, T. forsythia, and T. denticola is not considered to be essential information because it could be expected in more than 95% of patients. However, in individuals with minimal periodontal disease, but no history of periodontal prophylaxis, the microbiological results are heterogeneous. In a group of 10 Arabs, P. gingivalis was detected in 100%,35 whereas in 30 individuals from different developing countries, the detection frequency was 67% for P. gingivalis and 29% for T. forsythia.34 In the present study, P. gingivalis was detected in 33 of 34 (97%) chronic periodontitis patients by MT4 and MT6; in one ChP patient, P. gingivalis was detected by MT4, but not by MT6. The ChP detection frequency and bacterial counts of P. gingivalis were not statistically significantly different between MT4 and MT6. The bacterial ª 2010 Blackwell Publishing Asia Pty Ltd
M. Wohlfeil et al.
Subgingival plaque sampling strategies
count for MT4 in the respective ChP patient was 50 000, which was the lowest count for P. gingivalis in the whole study and close to the detection limit. It is plausible to assume that the bacterial count of the MT6 sample might have been just below the detection limit, and as a result, failed to detect P. gingivalis. Within the limitations of the present study, the current data show that sampling six pooled samples from the deepest sites per sextant (MT6) might provide higher bacterial counts for A. actinomycetemcomitans than sampling four pooled samples from these sites (MT4) by using 16S
References 1 Paster BJ, Boches SK, Galvin JL et al. Bacterial diversity in human subgingival plaque. J Bacteriol 2001; 183: 3770–83. 2 Paster BJ, Olsen I, Aas JA, Dewhirst FE. The breadth of bacterial diversity in the human periodontal pocket and other oral sites. Periodontol 2000 2006; 42: 80–7. 3 Nørskov-Lauritsen N, Kilian M. Reclassification of Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, Haemophilus paraphrophilus and Haemophilus segnis as Aggregatibacter actinomycetemcomitans gen. nov., comb. nov., Aggregatibacter aphrophilus comb. nov. and Aggregatibacter segnis comb. nov., and emended description of Aggregatibacter aphrophilus to include V factor-dependent and V factor-independent isolates. Int J Syst Evolutionary Microbiol 2006; 56: 2135–46. 4 Socransky SS, Haffajee AD, Cugini MA, Smith C, Kent RL Jr. Microbial complexes in subgingival plaque. J Clin Periodontol 1998; 25: 134–44. 5 van Winkelhoff AJ, Loos BG, van der Reijden WA, van der Velden U. Porphyromonas gingivalis and Bacteroides forsythus and other putative periodontal pathogens in subjects with and without periodontal destruction. J Clin Periodontol 2002; 29: 1023–8. 6 Bragd L, Dahlen G, Wikstro¨m M, Slots J. The capability of Actinobacillus actinomycetemcomitans, Bacte-
ª 2010 Blackwell Publishing Asia Pty Ltd
7
8
9
10
11
12
13
rRNA gene probes. For both MT4 and MT6 P. gingivalis, T. forsythia, and T. denticola were detected in more than 95% of all patients, and with mean log-transformed numbers greater than 6.0 in untreated AgP and ChP. Acknowledgments This study was funded by the authors and their institutions, with the exception of the microbiological analyses, which were provided by the Institute for Applied Immunology, Zuchwil, Switzerland.
roides gingivalis and Bacteroides intermedius to indicate progressive periodontitis; retrospective study. J Clin Periodontol 1985; 14: 95–9. Newman MG, Socransky SS, Savitt ED, Propas DA, Crawford A. Studies of the microbiology of periodontosis. J Periodontol 1976; 47: 373–9. Tonetti MS, Mombelli A. Earlyonset periodontitis. Ann Periodontol 1999; 4: 39–52. Schacher B, Baron F, Rossberg M, Wohlfeil M, Arndt R, Eickholz P. Aggregatibacter actinomycetemcomitans as indicator for aggressive periodontitis by two analysing strategies. J Clin Periodontol 2007; 34: 566–73. Haubek D, Ennibi O-K, Væth M, Poulsen S, Kilian M. Risk of aggressive periodontitis in adolescent carriers of the JP2 clone of Aggregatibacter (Actinobacillus) actinomycetemcomitans in Morocco: a prospective longitudinal cohort study. Lancet 2008; 371: 237–42. Schacher B, Baron F, Ludwig B, Valesky E, Noack B, Eickholz P. Periodontal therapy in siblings with Papillon–Lefe`vre syndrome and tinea capitis: a report of two cases. J Clin Periodontol 2006; 33: 829–36. Christersson L, Fransson C, Dunford R, Zambon J. Microbiological and clinical effects of surgical treatment of localized juvenile periodontitis. J Peridontol 1992; 63: 465–76. Kornman K, Robertson P. Clinical and microbiological evaluation of therapy of juvenile peridontitis. J Periodontol 1985; 56: 443–6.
14 Mu¨ller H-P, Lange DE, Mu¨ller RF. Failure of adjunctive minocycline-HCl to eliminate oral Actinobacillus actinomycetemcomitans. J Clin Periodontol 1993; 20: 498–504. 15 Mombelli A, Gmu¨r R, Gobbi C, Lang NP. Actinobacillus actinomycetemcomitans in adult periodontitis. I. Topographic distribution before and after treatment. J Periodontol 1994; 65: 820–6. 16 Takamatsu N, Yano K, He T, Umeda M, Ishikawa I. Effect of initial periodontal therapy on the frequency of detecting Bacteroides forsythus, Porphyromonas gingivalis, and Actinobacillus actinomycetemcomitans. J Periodontol 1999; 70: 574–80. 17 Ehmke B, Moter A, Beikler T, Milian E, Flemmig TF. Adjunctive antimicrobial therapy of periodontitis: longterm effects on disease progression and oral colonization. J Periodontol 2005; 76: 749–59. 18 American Academy of Periodontology. Parameter on progressive periodontitis. J Periodontol 2000; 71: 867–9. 19 American Academy of Periodontology. Treatment of plaque-induced gingivitis, chronic periodontitis, and other clinical conditions. J Periodontol 2001; 72: 1790–800. 20 Mombelli A, McNabb H, Lang NP. Black-pigmenting Gram-negative bacteria in periodontal disease. I. Topographic distribution in the human dentition. J Periodontal Res 1991; 26: 301–7. 21 Mombelli A, McNabb H, Lang NP. Distribution patterns of black-pigmenting Gram-negative bacteria in
131
Subgingival plaque sampling strategies
22
23
24
25
26
27
28
periodontitis patients. FEMS Immunol Med Microbiol 1993; 6: 199. Haffajee AD, Socransky SS. Effect of sampling strategy on the false-negative rate for detection of selected subgingival species. Oral Microbiol Immunol 1992; 7: 57–9. Beikler T, Schnitzer S, Abdeen G, Ehmke B, Eisenacher M, Flemmig TF. Sampling strategy for intraoral detection of periodontal pathogens before and following periodontal therapy. J Periodontol 2006; 77: 1323–32. Himmer K, Mayer M, Mayer I, Eickholz P. Detection of periodontal pathogens with PCR from subgingival plaque––comparison of two sampling strategies. J Clin Periodontol 2009; 9(suppl): 158. Kim C-K, Choi S-H, Kim T-S, Kaltschmitt J, Eickholz P. The infrabony defect and its determinants. J Periodont Res 2006; 41: 498–502. Krigar D-M, Kaltschmitt J, Krieger JK, Eickholz P. Two subgingival plaque sampling strategies used with RNA-probes. J Periodontol 2007; 78: 72–8. Fleiss JL Statistical methods for rates and proportions. New York: John Wiley & Sons (2nd edition); 1981: 223–5. Mombelli A, Casagni F, Madianos PN. Can presence or absence of periodontal pathogens distinguish between subjects with chronic and aggressive periodontitis? A system-
132
M. Wohlfeil et al.
29
30
31
32
33
34
atic review. J Clin Periodontol 2002; 29(Suppl 3): 10–21. discussion 37-8. Cionca N, Giannopoulou C, Ugolotti G, Mombelli A. Microbiologic testing and outcomes of full-mouth scaling and root planing with or without amoxicillin/metronidazole in chronic periodontitis. J Periodontol 2010; 81: 15–23. van Winkelhoff AJ, Rodenburg JP, Goene´ RJ, Abbas EG, Winkel EG, de Graaff J. Metronidazole plus amoxicillin in the treatment of Actinobacillus actinomycetemcomitans associated periodontitis. J Clin Periodontol 1989; 16: 128–31. Dannewitz B, Pohl S, Eickholz P, Kim T-S. Clinical and microbiological effects of a combined mechanicantibiotic therapy in patients with Actinobacillus actinomycetemcomitans-associated periodontitis. Am J Dent 2007; 20: 153–6. Landis JR, Koch GG. The measurement of observer agreement for categorical data. Biometrics 1977; 33: 159–174. Eickholz P, Kaltschmitt J, Krieger J, Dannewitz B, Kim T-S, Krigar D-M. Variability of detection of periodontal pathogens from subgingival plaque. J Dent Res 2009; 88 (Special Issue A): Abstract no. 2162. [cited 12 Sep 2010] Available from: http:// iadr.confex.com/iadr/2009miami/ webprogram/Paper116846.html McNabb H, Mombelli A, Gmu¨r R, Mathey-Dinc¸ S, Lang NP. Periodon-
35
36
37
38
39
40
tal pathogens in the shallow pockets of immigrants from developing countries. Oral Microbiol Immunol 1992; 7: 267–72. Al-Yahfoufi Z, Mombelli A, Wicki A, Lang NP. The effect of plaque control in subjects with shallow pockets and high prevalence of periodontal pathogens. J Clin Periodontol 1995; 22: 78–84. Jervøe-Storm P-M, AlAhdab H, Koltzscher M, Fimmers R, Jepsen S. Comparison of curet and paper point sampling of subgingival bacteria as analyzed by real-time polymerase chain reaction. J Periodontol 2007; 78: 909–17. Sixou M, Duffaut-Lagarrigue D, Lodter JP. In French (A comparison between 4 different subgingival bacteriologic sampling technics). J Biol Buccale 1991; 19: 16–21. Renvert S, Wikstro¨m M, Helmersson M, Dahle´n G, Claffey N. Comparative study of subgingival microbiological sampling techniques. J Periodontol 1992; 63: 797–801. Ramfjord SP. The Periodontal Disease Index (PDI). J Periodontol 1967; 38(Suppl): 602–10. Casas A, Herrera D, Martin-Carnes J, Gonza´les I, O’Connor A, Sanz M. Influence of sampling strategy on microbiologic results before and after periodontal therapy. J Periodontol 2007; 78: 1103–12.
ª 2010 Blackwell Publishing Asia Pty Ltd
ORIGINAL ARTICLE Oral Microbiology
Detection rates of presumptive periodontal pathogens in subgingival plaque samples of untreated periodontitis using either four or six pooled samples Martin Wohlfeil, Orhan Tabakci, Rita Arndt, Peter Eickholz & Katrin Nickles Department of Periodontology, Center for Dental, Oral, and Maxillofacial Medicine (Carolinum), Johann Wolfgang Goethe-University, Frankfurt, Germany
Keywords 16S rRNA gene probe, Aggregatibacter actinomycetemcomitans, aggressive periodontitis, chronic periodontitis, generalized, severe, subgingival plaque. Correspondence Prof. Peter Eickholz, Poliklinik fu¨r Parodontologie, ZZMK (Carolinum), Theodor-Stern-Kai 7, Frankfurt am Main, D-60590, Germany. Tel: +49-69-6301-5642 Fax: +49-69-6301-3753 Email: [email protected] Received 12 December 2009; accepted 20 June 2010. doi: 10.1111/j.2041-1626.2010.00026.x
Abstract Aim: A comparison of the detection frequency and number of periodontal pathogens in patients with aggressive or generalized, severe chronic periodontitis using a gene-probe analysis. Methods: In 16 aggressive and 34 generalized, severe chronic periodontitis patients, plaque was sampled from the deepest pockets per quadrant (MT4) and per sextant (MT6). After sampling two paper points simultaneously, one paper point from each pocket was pooled with three paper points of the other pockets (MT4). The remaining four paper points were pooled with two paper points from the deepest pockets from the two remaining sextants (MT6). Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Porphyromonas gingivalis, and Treponema denticola were detected by 16S rRNA gene probes. Results: Log-transformed counts for Aggregatibacter actinomycetemcomitans were statistically significantly higher with MT6 (aggressive: 3.21 ± 2.94; generalized, severe chronic: 2.22 ± 2.70) than MT4 (aggressive: 2.04 ± 2.74; generalized, severe chronic: 1.50 ± 2.37) (P < 0.05). The detection frequency and mean counts were high for Tannerella forsythia, Porphyromonas gingivalis, and Treponema denticola (>95%/>6.0). Conclusion: Aggregatibacter actinomycetemcomitans was detected in higher numbers for MT6 than MT4. For both MT4 and MT6, Tannerella forsythia, Porphyromonas gingivalis, and Treponema denticola were detected in >95% of all patients and with mean log-transformed numbers >6.0.
Introduction From the approximately 400 bacterial species colonizing periodontal pockets, and a further 300 in the rest of the oral cavity,1,2 some are frequently associated with periodontal destruction. Aggregatibacter actinomycetemcomitans,3 Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola 4,5 are considered periodontal pathogens. Further, A. actinomycetemcomitans has been closely associated with the etiology of a severe periodontal disease, aggressive periodontitis (AgP)6–10 and periodontitis as manifestation of Papillon–Lefe`vre syndrome.11 Aggrega126
tibacter actinomycetemcomitans is a micro-aerophilic, facultative anaerobic and Gram-negative coccoid rod belonging to the family of Pasteurellacea.3 Periodontal disease associated with A. actinomycetemcomitans in many cases cannot be treated reliably and predictively by the mechanical removal of the subgingival biofilm alone.6,12–17 Thus, the detection of A. actinomycetemcomitans is a significant factor contributing to the decision as to whether subgingival debridement should be performed in conjunction with systemic antibiotics.18,19 Sampling of the deepest pocket of each quadrant has been demonstrated to quite reliably detect the subgingival ª 2010 Blackwell Publishing Asia Pty Ltd
M. Wohlfeil et al.
presence of periodontal pathogens in untreated patients.15,20–22 Sampling one to six sites per patient increases the probability of detecting the targeted bacteria.23 However, sampling six sites instead of four sites increases the cost of diagnosis. Until now, the comparison of pooled samples from four to six sites has only been investigated for polymerase chain reaction (PCR)-based microbiological analyses.24 Thus, in this study the results of the microbiological 16S rRNA gene probe analyses of pooled subgingival plaque samples from the four deepest sites per quadrant should be compared with the results from the pooled samples from the six deepest sites per sextant. Materials and methods Patients Fifty patients suffering from untreated AgP or generalized, severe, chronic periodontitis (ChP), who were scheduled for systematic periodontal therapy at the Department of Periodontology, Center for Dental, Oral, and Maxillofacial Medicine (Carolinum), Johann Wolfgang GoetheUniversity, Frankfurt, Germany, were recruited for this study between September 2007 and November 2008. To be included in this study the following inclusion criteria had to be fulfilled: (a) a clinical diagnosis of untreated (no non-surgical or surgical subgingival debridement within the last 12 months before microbiological sampling) AgP or ChP; (b) at least 18 years of age; (c) at least five teeth per quadrant; and (d) provided informed, written consent. The following criteria lead to exclusion from the study: (a) a need for antibiotic prophylaxis in advance of periodontal diagnosis or treatment; (b) antibiotic therapy within the last 6 months or subgingival debridement (non-surgical or surgical) within the last 12 months before microbiological sampling; and (c) anti-infective periodontal therapy with adjunctive systemic amoxicillin or ciprofloxacin and metronidazole. For this study, AgP was defined9,25 as a patient who is clinically healthy (i.e. systemic diseases predisposing for periodontitis are not reported; e.g. diabetes mellitus), has a radiographic bone loss ‡50% affecting at least two different teeth, and was aged £35 years at diagnosis. ChP was defined as a patient with attachment loss ‡5 mm at more than 30% of sites, and was aged >35 years. The study conformed to the provisions of the Declaration of Helsinki (as revised in Tokyo 2004) and was approved by the Institutional Review Board for Human Studies of the Medical Faculty of the Johann Wolfgang Goethe-University (study no. 189/07). All patients gave written, informed consent to participate in the study. ª 2010 Blackwell Publishing Asia Pty Ltd
Subgingival plaque sampling strategies
Clinical examinations At six sites per tooth (mesiobuccal, mid-buccal, distobuccal, disto-oral, mid-oral, mesio-oral) probing pocket depths (PD) and vertical clinical attachment levels (CALV) were measured using a manual rigid periodontal probe (PCPUNC15, Hu Friedy, Chicago, IL, USA) to the nearest millimeter. The cemento-enamel junction (CEJ) was used as reference for the CAL-V measurements. If the CEJ was destroyed by restorative treatment, the margin of the restoration was taken as the reference. If the CEJ was located apically of the gingival margin first, the PD was measured. The CEJ was then groped by the probe, and the distance CEJ to the gingival margin was measured. The CAL-V was calculated as PD minus the distance of the CEJ to the gingival margin. Bleeding on probing was recorded 30 sec after probing. Microbiological examination Microbiological sampling was performed within the clinical routine.18,19 For sampling, the deepest site of each of the four quadrants was chosen. The test site was dried by air and held dry by using cotton rolls. Simultaneously, two sterile paper points were inserted into the bottom of the respective pocket.9,24,26 After 20 sec, the paper points were removed. One of each paper point was put into one of two transportation vials. Thus, two pooled samples of the same four sites were created. One of the vials was closed (MT4). The deepest pockets of the two remaining sextants were then selected; plaque samples were taken and pooled with the other remaining vial (MT6). For the analysis, a commercially-available 16S rRNA gene probe test kit (IAI Pado-Test 4.5; Institute for Applied Immunology, Zuchwil, Switzerland) aimed at detecting A. actinomycetemcomitans, P. gingivalis, T. forsythia, and T. denticola was used. This is an oligonucleotide probe test, complementary to conservative regions of the 16S rRNA gene, which encodes the rRNA that forms the small subunit of the bacterial ribosome. The detection limit of this test is 103.3 for A. actinomycetemcomitans and 104 for T. forsythia, P. gingivalis, and T. denticola, respectively. Statistical analysis Two variables were analyzed for each periodontal pathogen separately for AgP and ChP: prevalence (i.e. detection of the pathogen or no detection) and log-transformed bacterial counts. Aggressive periodontitis and ChP were compared according to sex, smoking status, PD, and CAL-V using the chi-squared test and t-test, respectively. The detection 127
Subgingival plaque sampling strategies
M. Wohlfeil et al.
rate and log-transformed bacterial counts for MT4 and MT6 were compared using the Wilcoxon signed rank test for paired samples. Agreement of the detection rate of both analyzing strategies was estimated by calculating Cohen’s k.27 The PD and CAL-V were used to describe the clinical status of the sampled sites. Statistical analyses were performed using Systat for Windows (version 12; Systat, Evanston, IL, USA). Results From a total of 50 patients (30 females and 20 males, 47.4 ± 13.6 years of age), clinical and microbiological examinations were obtained. Sixteen patients suffered from untreated AgP and 34 from untreated ChP (Table 1). Detection frequencies and counts for A. actinomycetemcomitans were higher for MT6 (AgP: 56%/3.21 ± 2.94; ChP: 41%/2.22 ± 2.70) than MT4 (AgP: 37%/2.04 ± 2.74; ChP: 29%/1.50 ± 2.37) (Tables 2 and 3). The differences reached statistical significance only for log-transformed counts (AgP: P = 0.047; ChP: P = 0.011) (Table 3). Detection frequencies and mean counts were generally high for P. gingivalis, T. forsythia, and T. denticola (>95%/>6.0). Statistically-significant differences between MT4 and MT6 could not be found (Tables 4–6). For T. forsythia, a small but statistically-significant difference was found between MT4 and MT6 log-transformed counts in ChP only (Table 3). Discussion The clinical relevance of the detection of A. actinomycetemcomitans in periodontal disease is a matter of debate.28,29 A. actinomycetemcomitans, T. forsythia, and P. gingivalis are frequently associated with destructive periodontal Table 1. Patients and clinical parameters (n and mean ± SD) according to diagnosis
n Females Current smokers Age (years) PD (mm) MT4 PD (mm) MT6à CAL-V (mm) MT4 CAL-V (mm) MT6à
Aggressive periodontitis
Generalized, severe, chronic periodontitis
16 11 5 31.7 7.5 7.1 7.5 7.1
34 19 11 54.8 7.9 7.3 8.5 8.0
± ± ± ± ±
6.4 1.4 1.4 1.7 1.5
P-value
0.538 1.000 ± ± ± ± ±
9.0 1.5 1.4 1.3 1.2
0.311 0.630 0.035 0.050
Deepest site per quadrant. àDeepest site per sextant. CAL-V, vertical clinical attachment levels; PD, probing pocket depth.
128
Table 2. Aggregatibacter actinomycetemcomitans detection frequency (n [%]) with the MT4 and MT6à sampling method MT6 Negative Aggressive periodontitis MT4 Negative Positive Total Generalized, severe, chronic MT4 Negative Positive Total
6 (38%) 1 (6%) 7 (44%) periodontitis 19 (56%) 1 (3%) 20 (59%)
Positive
Total
4 (25%) 5 (31%) 9 (56%)
10 (63%) 6 (37%) 16
5 (15%) 9 (26%) 14 (41%)
24 (71%) 10 (29%) 34
Deepest site per quadrant. àDeepest site per sextant. For aggressive periodontitis, there was no statistically-significant difference between MT4 and MT6 (P = 0.180). Cohen’s j: 0.394; standard error: 0.211. For generalized, severe chronic periodontitis, there was no statistically-significant difference between MT4 and MT6 (P = 0.102). Cohen’s j: 0.619; standard error: 0.136.
Table 3. Log-transformed bacterial counts for MT4 and MT6à (mean ± SD) MT4 Aggressive periodontitis Aggregatibacter 2.04 ± 2.74 actinomycetemcomitans Porphyromonas gingivalis 6.11 ± 0.81 Tannerella forsythia 6.47 ± 0.58 Treponema denticola 6.29 ± 0.55 Generalized, severe, chronic periodontitis Aggregatibacter 1.50 ± 2.37 actinomycetemcomitans Porphyromonas gingivalis 6.56 ± 0.53 Tannerella forsythia 6.48 ± 0.43 Treponema denticola 6.19 ± 1.13
MT6
P-value
3.21 ± 2.94
0.047
6.30 ± 0.60 6.55 ± 0.45 6.37 ± 0.45
0.070 0.234 0.070
2.22 ± 2.70
0.011
6.51 ± 1.19 6.58 ± 0.34 6.21 ± 1.13
0.309 0.035 0.714
Deepest site per quadrant. àDeepest site per sextant.
disease.5 In many cases of periodontal disease associated with A. actinomycetemcomitans, the mechanical removal of the subgingival biofilm alone is insufficient to eliminate or at least suppress this microorganism below the detection limit reliably and predictively.6,12–17 Persisting A. actinomycetemcomitans leads to unfavorable clinical results. The systemic use of the combination of amoxicillin and metronidazole or ciprofloxacin and metronidazole adjunctively to mechanical debridement, in most cases, results in the suppression of A. actinomycetemcomitans below detection limits and clinical improvement.30,31 The detection of A. actinomycetemcomitans fails to be a sensitive or specific diagnostic test for AgP.9,28 However, the detection of A. actinomycetemcomitans has a significant influence on ª 2010 Blackwell Publishing Asia Pty Ltd
M. Wohlfeil et al.
Subgingival plaque sampling strategies
Table 4. Porphyromonas gingivalis detection frequency (n [%]) with the MT4 and MT6à sampling method MT6 Negative Aggressive periodontitis MT4 Negative 0 (0%) Positive 0 (0%) Total 0 (0%) Generalized, severe, chronic periodontitis MT4 Negative 0 (0%) Positive 1 (3%) Total 1 (3%)
MT6 Positive
Total
0 (0%) 16 (100%) 16 (100%)
0 (0%) 16 (100%) 16
0 (0%) 33 (97%) 33 (97%)
0 (0%) 34 (100%) 34
Deepest site per quadrant. àDeepest site per sextant. For aggressive periodontitis, there was no statistically-significant difference between MT4 and MT6 (P = 1.000). Cohen’s j: 1.000; standard error: 0.000. For generalized, severe, chronic periodontitis, there was no statistically-significant difference between MT4 and MT6 (P = 0.317).
Table 5. Tannerella forsythia detection frequency (n [%]) with the MT4 and MT6à sampling method MT6 Negative Aggressive periodontitis MT4 Negative 0 (0%) Positive 0 (0%) Total 0 (0%) Generalized, severe, chronic periodontitis MT4 Negative 0 (0%) Positive 0 (0%) Total 0 (0%)
Positive
Total
0 (0%) 16 (100%) 16 (100%)
0 (0%) 16 (100%) 16
0 (0%) 34 (100%) 34 (100%)
0 (0%) 34 (100%) 34
Deepest site per quadrant. àDeepest site per sextant. For aggressive periodontitis, there was no statistically-significant difference between MT4 and MT6 (P = 1.000). Cohen’s j: 1.000; standard error: 0.000. For generalized, severe, chronic periodontitis, there was no statistically-significant difference between MT4 and MT6 (P = 1.000). Cohen’s j: 1.000; standard error: 0.000.
the decision of whether or not mechanical anti-infective therapy should be administered in conjunction with systemic antibiotics. The sampling technique applied in this study has been tested for reproducibility before in 43 patients. For sampling, the deepest pocket of each of the four quadrants was selected, i.e. 4 sites per patient. Simultaneously, two sterile paper points were inserted to the bottom of the respective pocket. After 20 sec, the paper points were removed. One of each paper points was put into one of two transportation vials. Thus, two pooled samples of the same four sites ª 2010 Blackwell Publishing Asia Pty Ltd
Table 6. Treponema denticola detection frequency (n [%]) with the MT4 and MT6à sampling method
Negative Aggressive periodontitis MT4 Negative 0 (0%) Positive 0 (0%) Total 0 (0%) Generalized, severe, chronic periodontitis MT4 Negative 0 (0%) Positive 0 (0%) Total 0 (0%)
Positive
Total
0 (0%) 16 (100%) 16 (100%)
0 (0%) 16 (100%) 16
0 (0%) 34 (100%) 34 (100%)
0 (0%) 34 (100%) 34
Deepest site per quadrant. àDeepest site per sextant. For aggressive periodontitis, there was no statistically significant difference between MT4 and MT6 (P = 1.000). Cohen’s j: 1.000; standard error: 0.000. For generalized, severe, chronic periodontitis, there was no statistically significant difference between MT4 and MT6 (P = 1.000). Cohen’s j: 1.000; standard error: 0.000.
were created and analyzed using the 16S rRNA probe test. With regard to detection frequency per patient of A. actinomycetemcomitans, for both samples (MT4/MT6) substantial agreement (Cohen’s j: 0.694) was observed. For the other periodontal pathogens, excellent agreement32 was observed (Cohen’s j: P. gingivalis: 0.758; T. forsythia: 0.845; T. denticola: 1.0).33 In the present study, paper points were used for the microbiological sampling. This is standard for commercially-available gene probe tests, and was also used in many microbiological studies.9,15,20,21,26,34,35 Thus, after investigating a commercially-available 16S rRNA gene probe test, we used paper points for sampling subgingival plaque. However, paper points have some disadvantages: if they become wet by gingival crevicular fluid, they lose stiffness, making it difficult to move them to the bottom of the pocket. Another disadvantage is that different paper points placed in the same pocket at the same time and for the same period of time might not sample the same microorganisms.26 Subgingival plaque might also be sampled using curettes.23 Curettes stay stiff, even when they become wet, and can sample subgingival plaque from a larger area than a paper point. Thus, sampling plaque with a curette might overcome some of the disadvantages of paper points. However, whereas the paper point sampling detects free floating bacteria rather than adhering bacteria, it does not remove all biofilm with sampling. Thus, paper point sampling might influence subgingival plaque less than curetting, sampling which removes most of the adhering biofilm in one stroke. Jervøe-Storm et al. compared the curette and paper point sampling technique using quantitative real-time PCR and 129
Subgingival plaque sampling strategies
demonstrated that the plaque composition, with regard to total target pathogens, was similar for both sampling techniques.36 Sixou et al. compared four different sampling techniques and drew the conclusion that curette sampling is efficient both quantitatively and qualitatively. However, difficulties in standardizing this method were encountered with the failure to achieve reproducible results. The paper point technique was found to be more reliable and reproducible in each of the three groups of patients sampled.37 Renvert et al. also considered the paper point sampling technique to be the best alternative; they found significantly higher numbers of colony-forming units and spirochetes with this method.38 Using paper points of standardized size provides standardized samples that are preferred, particularly by laboratories. Aggregatibacter actinomycetemcomitans might not be present at all oral sites in a patient suffering from untreated periodontitis.15,22 Taking subgingival samples from all teeth would be the most reliable way to detect A. actinomycetemcomitans. However, this method is too time consuming and expensive to be used in daily practice. Sampling of the deepest pocket of each quadrant has been demonstrated to quite reliably detect the subgingival presence of periodontal pathogens in untreated patients.15,20,22 Mombelli et al. sampled and microbiologically analyzed all sites separately, and theoretically evaluated different sampling strategies.15,20,21 Yet their strategies were based on separate analyses of the samples, whereas in daily practice, the different samples taken from the deepest sites per quadrant are pooled prior to analysis for economic reasons. Haffajee et al.22 analyzed the effect of sampling strategy on the false-negative rate for detection of selected subgingival species. They compared plaque samples obtained from one site per tooth with samples taken from one maxillary first molar, the maxillary first molars, four-first molars, six Ramfjord teeth,39 the deepest pocket, and the four deepest pockets. The lowest rates of undetected species were found for the four deepest pocket sampling strategy.22 Thus, for microbiological analyses in daily practice, sampling the deepest site per quadrant is generally recommended. After sampling one to six sites per patient, and with a separate analysis per site, Beikler et al. reported increasing the probability to detect the targeted bacteria with an increasing number of sampled sites.23 However, other authors reported contradicting observations.40 These differences might be explained by significant differences in methodology: sampling with curettes23 or paper points40 and separate23 or pooled analyses40. Thus, at least for pooled analyses, up until now it has not been clear whether sampling the deepest site per sextant (MT6) instead of the deepest per quadrant (MT4) increases the probability of detection. For the 16S rRNA gene probe test kit that was 130
M. Wohlfeil et al.
investigated in this study, the MT6 sampling strategy demonstrated advantages over MT4, particularly in the detection of A. actinomycetemcomitans. It has been recently demonstrated that there are statistically significantly higher log-transformed bacterial counts after pooled analyses than after separate analyses with the 16S rRNA gene probe (used in this study) after sampling from the deepest sites of three quadrants (MT3).26 After sampling from the deepest site of four quadrants (MT4), only minor differences were reported for P. gingivalis, T. forsythia, and T. denticola.9 For the detection rates, sampling of three sites revealed higher rates for pooled analyses (MT3) than for separate analyses only for T. denticola, but not for A. actinomycetemcomitans, P. gingivalis, and T. forsythia.26 After sampling four sites, one study failed to reveal any differences between detection rates after separate and pooled analyses (MT4).9 With increasing numbers of sampled sites, the differences between separate and pooled analyses decrease. The studied population corresponds to the populations analyzed in previous studies9,26 regarding patient parameters (age, sex) and clinical parameters (PD, CAL-V). Krigar et al. reported an A. actinomycetemcomitans detection frequency after a pooled analysis (MT3) of 26% for a population with a proportion of 27% patients with untreated aggressive periodontitis.26 For a population consisting of 50% patients with aggressive periodontitis, 38% of all patients were observed to be A. actinomycetemcomitans positive after the pooled analysis (MT4).9 In the present study on a sample with 32% of patients suffering from aggressive periodontitis, A. actinomycetemcomitans was detected in 32% (MT4) and 46% (MT6) of all patients. Porphyromonas gingivalis, T. forsythia, and T. denticola were detected in almost all patients (P. gingivalis: 98–100%, T. forsythia: 100%, and T. denticola: 98%). These detection frequencies are in accordance with observations previously made by others in patients with untreated AgP and ChP.9,26,29 Thus, in untreated AgP and ChP, the detection of P. gingivalis, T. forsythia, and T. denticola is not considered to be essential information because it could be expected in more than 95% of patients. However, in individuals with minimal periodontal disease, but no history of periodontal prophylaxis, the microbiological results are heterogeneous. In a group of 10 Arabs, P. gingivalis was detected in 100%,35 whereas in 30 individuals from different developing countries, the detection frequency was 67% for P. gingivalis and 29% for T. forsythia.34 In the present study, P. gingivalis was detected in 33 of 34 (97%) chronic periodontitis patients by MT4 and MT6; in one ChP patient, P. gingivalis was detected by MT4, but not by MT6. The ChP detection frequency and bacterial counts of P. gingivalis were not statistically significantly different between MT4 and MT6. The bacterial ª 2010 Blackwell Publishing Asia Pty Ltd
M. Wohlfeil et al.
Subgingival plaque sampling strategies
count for MT4 in the respective ChP patient was 50 000, which was the lowest count for P. gingivalis in the whole study and close to the detection limit. It is plausible to assume that the bacterial count of the MT6 sample might have been just below the detection limit, and as a result, failed to detect P. gingivalis. Within the limitations of the present study, the current data show that sampling six pooled samples from the deepest sites per sextant (MT6) might provide higher bacterial counts for A. actinomycetemcomitans than sampling four pooled samples from these sites (MT4) by using 16S
References 1 Paster BJ, Boches SK, Galvin JL et al. Bacterial diversity in human subgingival plaque. J Bacteriol 2001; 183: 3770–83. 2 Paster BJ, Olsen I, Aas JA, Dewhirst FE. The breadth of bacterial diversity in the human periodontal pocket and other oral sites. Periodontol 2000 2006; 42: 80–7. 3 Nørskov-Lauritsen N, Kilian M. Reclassification of Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, Haemophilus paraphrophilus and Haemophilus segnis as Aggregatibacter actinomycetemcomitans gen. nov., comb. nov., Aggregatibacter aphrophilus comb. nov. and Aggregatibacter segnis comb. nov., and emended description of Aggregatibacter aphrophilus to include V factor-dependent and V factor-independent isolates. Int J Syst Evolutionary Microbiol 2006; 56: 2135–46. 4 Socransky SS, Haffajee AD, Cugini MA, Smith C, Kent RL Jr. Microbial complexes in subgingival plaque. J Clin Periodontol 1998; 25: 134–44. 5 van Winkelhoff AJ, Loos BG, van der Reijden WA, van der Velden U. Porphyromonas gingivalis and Bacteroides forsythus and other putative periodontal pathogens in subjects with and without periodontal destruction. J Clin Periodontol 2002; 29: 1023–8. 6 Bragd L, Dahlen G, Wikstro¨m M, Slots J. The capability of Actinobacillus actinomycetemcomitans, Bacte-
ª 2010 Blackwell Publishing Asia Pty Ltd
7
8
9
10
11
12
13
rRNA gene probes. For both MT4 and MT6 P. gingivalis, T. forsythia, and T. denticola were detected in more than 95% of all patients, and with mean log-transformed numbers greater than 6.0 in untreated AgP and ChP. Acknowledgments This study was funded by the authors and their institutions, with the exception of the microbiological analyses, which were provided by the Institute for Applied Immunology, Zuchwil, Switzerland.
roides gingivalis and Bacteroides intermedius to indicate progressive periodontitis; retrospective study. J Clin Periodontol 1985; 14: 95–9. Newman MG, Socransky SS, Savitt ED, Propas DA, Crawford A. Studies of the microbiology of periodontosis. J Periodontol 1976; 47: 373–9. Tonetti MS, Mombelli A. Earlyonset periodontitis. Ann Periodontol 1999; 4: 39–52. Schacher B, Baron F, Rossberg M, Wohlfeil M, Arndt R, Eickholz P. Aggregatibacter actinomycetemcomitans as indicator for aggressive periodontitis by two analysing strategies. J Clin Periodontol 2007; 34: 566–73. Haubek D, Ennibi O-K, Væth M, Poulsen S, Kilian M. Risk of aggressive periodontitis in adolescent carriers of the JP2 clone of Aggregatibacter (Actinobacillus) actinomycetemcomitans in Morocco: a prospective longitudinal cohort study. Lancet 2008; 371: 237–42. Schacher B, Baron F, Ludwig B, Valesky E, Noack B, Eickholz P. Periodontal therapy in siblings with Papillon–Lefe`vre syndrome and tinea capitis: a report of two cases. J Clin Periodontol 2006; 33: 829–36. Christersson L, Fransson C, Dunford R, Zambon J. Microbiological and clinical effects of surgical treatment of localized juvenile periodontitis. J Peridontol 1992; 63: 465–76. Kornman K, Robertson P. Clinical and microbiological evaluation of therapy of juvenile peridontitis. J Periodontol 1985; 56: 443–6.
14 Mu¨ller H-P, Lange DE, Mu¨ller RF. Failure of adjunctive minocycline-HCl to eliminate oral Actinobacillus actinomycetemcomitans. J Clin Periodontol 1993; 20: 498–504. 15 Mombelli A, Gmu¨r R, Gobbi C, Lang NP. Actinobacillus actinomycetemcomitans in adult periodontitis. I. Topographic distribution before and after treatment. J Periodontol 1994; 65: 820–6. 16 Takamatsu N, Yano K, He T, Umeda M, Ishikawa I. Effect of initial periodontal therapy on the frequency of detecting Bacteroides forsythus, Porphyromonas gingivalis, and Actinobacillus actinomycetemcomitans. J Periodontol 1999; 70: 574–80. 17 Ehmke B, Moter A, Beikler T, Milian E, Flemmig TF. Adjunctive antimicrobial therapy of periodontitis: longterm effects on disease progression and oral colonization. J Periodontol 2005; 76: 749–59. 18 American Academy of Periodontology. Parameter on progressive periodontitis. J Periodontol 2000; 71: 867–9. 19 American Academy of Periodontology. Treatment of plaque-induced gingivitis, chronic periodontitis, and other clinical conditions. J Periodontol 2001; 72: 1790–800. 20 Mombelli A, McNabb H, Lang NP. Black-pigmenting Gram-negative bacteria in periodontal disease. I. Topographic distribution in the human dentition. J Periodontal Res 1991; 26: 301–7. 21 Mombelli A, McNabb H, Lang NP. Distribution patterns of black-pigmenting Gram-negative bacteria in
131
Subgingival plaque sampling strategies
22
23
24
25
26
27
28
periodontitis patients. FEMS Immunol Med Microbiol 1993; 6: 199. Haffajee AD, Socransky SS. Effect of sampling strategy on the false-negative rate for detection of selected subgingival species. Oral Microbiol Immunol 1992; 7: 57–9. Beikler T, Schnitzer S, Abdeen G, Ehmke B, Eisenacher M, Flemmig TF. Sampling strategy for intraoral detection of periodontal pathogens before and following periodontal therapy. J Periodontol 2006; 77: 1323–32. Himmer K, Mayer M, Mayer I, Eickholz P. Detection of periodontal pathogens with PCR from subgingival plaque––comparison of two sampling strategies. J Clin Periodontol 2009; 9(suppl): 158. Kim C-K, Choi S-H, Kim T-S, Kaltschmitt J, Eickholz P. The infrabony defect and its determinants. J Periodont Res 2006; 41: 498–502. Krigar D-M, Kaltschmitt J, Krieger JK, Eickholz P. Two subgingival plaque sampling strategies used with RNA-probes. J Periodontol 2007; 78: 72–8. Fleiss JL Statistical methods for rates and proportions. New York: John Wiley & Sons (2nd edition); 1981: 223–5. Mombelli A, Casagni F, Madianos PN. Can presence or absence of periodontal pathogens distinguish between subjects with chronic and aggressive periodontitis? A system-
132
M. Wohlfeil et al.
29
30
31
32
33
34
atic review. J Clin Periodontol 2002; 29(Suppl 3): 10–21. discussion 37-8. Cionca N, Giannopoulou C, Ugolotti G, Mombelli A. Microbiologic testing and outcomes of full-mouth scaling and root planing with or without amoxicillin/metronidazole in chronic periodontitis. J Periodontol 2010; 81: 15–23. van Winkelhoff AJ, Rodenburg JP, Goene´ RJ, Abbas EG, Winkel EG, de Graaff J. Metronidazole plus amoxicillin in the treatment of Actinobacillus actinomycetemcomitans associated periodontitis. J Clin Periodontol 1989; 16: 128–31. Dannewitz B, Pohl S, Eickholz P, Kim T-S. Clinical and microbiological effects of a combined mechanicantibiotic therapy in patients with Actinobacillus actinomycetemcomitans-associated periodontitis. Am J Dent 2007; 20: 153–6. Landis JR, Koch GG. The measurement of observer agreement for categorical data. Biometrics 1977; 33: 159–174. Eickholz P, Kaltschmitt J, Krieger J, Dannewitz B, Kim T-S, Krigar D-M. Variability of detection of periodontal pathogens from subgingival plaque. J Dent Res 2009; 88 (Special Issue A): Abstract no. 2162. [cited 12 Sep 2010] Available from: http:// iadr.confex.com/iadr/2009miami/ webprogram/Paper116846.html McNabb H, Mombelli A, Gmu¨r R, Mathey-Dinc¸ S, Lang NP. Periodon-
35
36
37
38
39
40
tal pathogens in the shallow pockets of immigrants from developing countries. Oral Microbiol Immunol 1992; 7: 267–72. Al-Yahfoufi Z, Mombelli A, Wicki A, Lang NP. The effect of plaque control in subjects with shallow pockets and high prevalence of periodontal pathogens. J Clin Periodontol 1995; 22: 78–84. Jervøe-Storm P-M, AlAhdab H, Koltzscher M, Fimmers R, Jepsen S. Comparison of curet and paper point sampling of subgingival bacteria as analyzed by real-time polymerase chain reaction. J Periodontol 2007; 78: 909–17. Sixou M, Duffaut-Lagarrigue D, Lodter JP. In French (A comparison between 4 different subgingival bacteriologic sampling technics). J Biol Buccale 1991; 19: 16–21. Renvert S, Wikstro¨m M, Helmersson M, Dahle´n G, Claffey N. Comparative study of subgingival microbiological sampling techniques. J Periodontol 1992; 63: 797–801. Ramfjord SP. The Periodontal Disease Index (PDI). J Periodontol 1967; 38(Suppl): 602–10. Casas A, Herrera D, Martin-Carnes J, Gonza´les I, O’Connor A, Sanz M. Influence of sampling strategy on microbiologic results before and after periodontal therapy. J Periodontol 2007; 78: 1103–12.
ª 2010 Blackwell Publishing Asia Pty Ltd
