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INTRODUCTION
Periodontitis is a multifactorial inflammatory chronic disease associated with dysbiotic biofilms, characterized by the progressive destruction of the periodontal supporting structures of teeth 1 . Its high prevalence, possible functional and esthetic compromise, and negative impact on general health are sufficient reasons to consider it an important public health issue.
In 1999, the American Academy of Periodontology (AAP) established the differences between the chronic and aggressive forms of disease, based on clinical parameters, microbiological composition of subgingival biofilm and host immunological aspects 2 . Aggressive periodontitis (AP) affects the minority of patients with periodontal disease. Nevertheless, its relevance lies in the rapid progression that can lead to early edentulism 3 . There has recently been a change in the classification of periodontal and peri-implant diseases, with the weighting focusing on clinical diagnosis, risk factors, and type of injury, according to which the patients in the current study are classified as stage III or IV and grade C periodontitis 4 .
The search for the specific etiologic agents associated with periodontal disease was begun several decades ago by Slots J. and Socransky S. At present we know that the interaction among bacteria and the balance between pathogenic and beneficial species of the subgingival biofilm affect the progression of disease and the response to treatment 5 , 6 . It is essential to understand the relationships among subgingival microbiota in order to comprehend the biology of the subgingival ecosystem and plan strategies to control it.
Moreover, the microbiologic profile varies according to geographic areas, habits, ethnicities, development level and living conditions 7 . There is currently little available information about prevalence of periodontal pathogens in native patients with AP in Argentina.
Aims: To enhance current knowledge on the periodontal microbiota of aggressive periodontitis in a sample of native Argentine patients, thereby enabling estimation of the microbiological traits in the local population, and to describe the effect of a combined pharmacological-mechanical periodontal treatment on the proportion of the subgingival bacteria found and on the clinical results.
MATERIALS AND METHOD
This was an experimental clinical study with prospective longitudinal design and six-month follow-up. The eligible population included native Argentine patients with clinical-radiological diagnosis of AP. The patients were referred to the Department of Periodontology, School of Dentistry, University of Buenos Aires (FOUBA) between October 2017 and September 2019. Due to the design features and the prevalence of the study disease, a sample of 10 cases/year was calculated, with a drop-out rate of 10%. Patients were invited to participate, and after being informed of the protocol, benefits and possible drawbacks related to the periodontal procedures, they confirmed their voluntary participation by signing the informed consent form (FOUBA EC Exp N 006/2017).
Informed consent
Informed consent was established pursuant to the Declaration of Helsinki. The project and informed consent were accepted in a timely manner by the FOUBA Ethics Committee - 006/2017.
Inclusion criteria
Native Argentines, 18-40 years old, with clinical-radiographic signs of AP (stage III or IV and grade C periodontitis) with PD>5mm, CAL>5 and BOP(+) in at least 4 sites (1 site per quadrant).
Exclusion criteria
Foreigners, pregnant or breast-feeding women, smokers, patients with systemic disease such as diabetes, immuno-compromise and/or other pathologies affecting periodontal tissues 8 (developed prior to or during the study), patients with history of metronidazole and/or penicillin hypersensitivity, patients treated with selective antimicrobials within 6 months before the beginning of the study. Diagnosis was based on the information obtained systematically through: 1. Medical history recorded at the FOUBA. 2. Determination of periodontal clinical parameters (6 sites per tooth): probing depth (PD), clinical attachment level (CAL), bleeding on probing (BOP), mobility, furcation involvement. 3. Routine radiographic assessment using periapical serial radiographs 2 . All patients were evaluated by the same calibrated operator (CP) (k>0.74).
Clinical-microbiological procedures
The protocol consisted of an initial interview during which a complete medical history was prepared, collecting patient’s personal data, medical history and current medication. On the same day, clinical-radiographic screening was performed. Patients who met the inclusion criteria were invited to participate in the protocol and informed of the benefits and potential discomfort they might experience during the course of the protocol. After granting consent, they were summoned to a visit in 7 days to start the protocol.
The initial sample comprised twenty patients, but only eleven completed the study. (Fig. 1). Clinical parameters were recorded at baseline (T0); 45 days (T1); 90 days (T2) and 180 days (T3) after treatment. Six sites per tooth were evaluated by the same calibrated investigator (k>0.74) with appropriate lighting, employing standardized instruments (North Carolina periodontal probe and dental mirror). After initial periodontal recording and radiographic evaluation, 4 sites (1 per quadrant) were selected according to the inclusion criteria (PD>5 mm, CAL>5 and BOP) for the microbiological study.
Fig. 1 Conformation of the population sample. Fourteen patients were excluded for any of the following reasons: allergic to penicillin and its derivatives, did not attend maintenance sessions, selected periodontal pockets belonged to hopeless prognosis teeth, mistakes in taking prescribed antibiotics, did not complete periodontal therapy, or did not conform to the established protocol times.
Seven days after the instrumental diagnosis, microbiological samples of subgingival biofilm were obtained by inserting 4 paper points (No. 30-35) per site, having previously removed the supragingival biofilm with a curette. Paper points were consecutively inserted with an absorption time of 20 seconds each. Samples were transported to the laboratory in accordance with the biosecurity rules of the institution, together with two slides obtained from the periodontal pocket soft wall. Study design is shown in Fig. 2.
Periodontal therapy
All patients were instructed in oral hygiene and received full mouth scaling and root planing, systemic administration of antibiotics and ecologic control through caries inactivation and tooth extraction, if needed. Local anesthesia was used as necessary. Full mouth periodontal therapy was accomplished in no more than 2 appointments within a maximum period of 7 days during antibiotic treatment. Patients were administered 250 mg metronidazole (half a tablet of Ovufem® 500 mg, Laboratorios Bernabó, Buenos Aires, Argentina) and 500 mg amoxicillin (1 tablet of Amixen® 500 mg, Laboratorios Bernabó, Buenos Aires, Argentina) every 8 hours for 7 days, starting 48 hours before instrumentation.
Molecular procedure
Samples were obtained and processed from the selected sites at baseline (T0), before periodontal therapy, and 180 days (T3), after protocolized periodontal treatment. Samples were homogenized for molecular processing. Genomic DNA was extracted by rupture and purified in affinity columns (Presto™ Mini gDNA Bacteria Kit, Geneaid). The endpoint PCR technique was employed to detect six periodontopathic species. The specific primers for Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Tannerella forsythia (Tf), Treponema denticola (Td), Prevotella intermedia (Pi) and Fusobacterium nucleatum (Fn) were synthetized as designed by Ashimoto et al. 9 . Amplified sequences of the uncultivable or special nutritional species T. denticola and T. forsythia were confirmed by sequencing, while in the other cases, pattern strains of ATCC standard collection were used ( P. gingivalis ATCC 33277; F. nucleatum ATCC 25586; P. intermedia ATCC 25611). Products of amplification were evidenced by electrophoresis in 2% agarose gel in TAE buffer, using GelRed® Nucleic Acid Gel Stain as intercalated fluorophore and visualized with The Gel-Doc XR - Gel Imaging System from BIO-RAD®.
Statistical analysis
Quantitative variables were described by mean, standard deviation, median, minimal and maximal confidence interval. Nonparametric Friedman’s ANOVA p<0.01, Bonferroni post-hoc was used for múltiple comparisons, giving significantly different probing depth and clinical attachment loss measurements at baseline (T0) compared to the other evaluation times (T1, T2, T3).
To compare periodontal pathogens PCR results between baseline (T0) and final time (T3), the McNemar test was applied. The difference was considered significant when p<0.05. All statistical analyses were performed using the SPSS software (version 28), MS Excel and Epidata 4.0.
RESULTS
Initially, 20 patients (80 periodontal sites) were included in the study. The analyzed sample consisted of eleven native Argentine patients, 9 women and 2 men, with clinical-radiological diagnosis of AP, and mean age 28.4 ± 7.9 years (16-39). Forty-two periodontal sites were studied clinically, microbiologically and molecularly (Fig.1).
Microbiological parameters
The baseline (T0) detection frequencies were the following: A. actinomycetemcomitans 14.3%,
P. intermedia 61.9%, P gingivalis 71.4%, T. forsythia 81.0%, F. nucleatum 95.2% and T. denticola 97.6%. In the initial samples, the prevalence of P gingivalis over A. actinomycetemcomitans was significant (p=0.012). At 180 days after the end of the treatment protocol (T3), A. actinomycetemcomitans could no longer be detected by this methodology at any of the study sites. T. denticola, F. nucleatum, T. forsythia and P intermedia decreased significantly during the same interval. F. nucleatum remained detectable in all the residual periodontal pockets with PD> 5mm (p=0.053). P gingivalis was the only species that showed no statistically significant difference between evaluation times (p=0.052) (Table 1; Fig. 3).
Fig. 3 Molecular parameters. Baseline and final time samplesMcNemar Test *: p<0.05; **: p≤0.001. Pg: Porphyromona gingivalis; Fn: Fusobacterium nucleatum; Td: Treponema denticola; Tf: Tannerella Forsythia; Pi: Prevotella intermedia; Aa: Aggregatibacter actinomycetemcomitans.
Table 1 Molecular parameters
| Count | % | Ci 95.0% LL | Ci 95.0% UL | p value | |
|---|---|---|---|---|---|
| 30 | PCR Pg inicial | 71.4% | 56.7% | 83.3% | 0.052 |
| PCR Pg final | 20 | 47.6% | 33.1% | 62.5% | |
| PCR Fn inicial | 40 | 95.2% | 85.6% | 99.0% | 0.039* |
| PCR Fn final | 32 | 76.2% | 61.9% | 87.1% | |
| PCR Td inicial | 41 | 97.6% | 89.4% | 99.7% | 0.001** |
| PCR Td final | 16 | 38.1% | 24.6% | 53.2% | |
| PCR Tf inicial | 34 | 81.0% | 67.3% | 90.6% | 0.004* |
| PCR Tf final | 22 | 52.4% | 37.5% | 66.9% | |
| PCR Pi inicial | 26 | 61.9% | 46.8% | 75.4% | 0.001** |
| PCR Pi final | 5 | 11.0% | 4.7% | 24.1% | |
| PCR Aa inicial | 6 | 14.3% | 6.2% | 27.1% | 0.031* |
| PCR Aa final | 0 | 0.0% |
Clinical parameters
Bleeding on probing (BOP): Initially, BOP was present at 100% of the 42 sites. It declined to 19.5% at 45 days, 11.9% at 90 days, and 9.5% at 180 days. Reduction of BOP was statistically significant (p<0.001) (Fig. 4).
Fig. 4 Bleeding on probing. BOP 0 baseline, BOP 1 45 days, BOP 2 90 days, BOP 3 180 days. McNemar Test.
Probing depth (PD): Median PD at the selected sites was initially 8 mm (5-12mm). Final median PD was 3 mm (2-10 mm). The difference between the initial value and each instance of re-evaluation was statistically significant (p<0.001) (Fig. 5 A; Table 2). Clinical attachment level (CAL): Median CAL was 7 mm (5-12 mm) at baseline, and 4 mm (1-10 mm) at 180 days. The difference between the baseline value and each instance of re-evaluation was statistically significant (p<0.001) (Fig. 5B; Table 2).
Fig. 5 Probing depth and clinical attachment levelA. Probing depth: PD 0 baseline, PD 1 45 days, PD 2 90 days, PD 3 180 days. B. Clinical attachment level: CAL 0 baseline, CAL1 45 days, CAL2, 90 days, CAL3 180 days. Nonparametric Friedman’s ANOVA p<0.01, Bonferroni post-hoc was used for múltiple comparisons, giving significantly different measurements at baseline (0) compared to the other evaluation times (1,2,3).
Table 2 Probing depth and clinical attachment loss
| Mean | Standard Deviation | CI 95.0% LL | CI 95.0% UL | Percentile 25 | Median | Percentile 75 | p value | |
|---|---|---|---|---|---|---|---|---|
| Probing Depth | ||||||||
| PD T0 | 7.57 | 1.4 | 7.14 | 8.01 | 7 | 8 | 8 | <0.001** |
| PD T1 | 4.33 | 1.65 | 3.82 | 4.85 | 3 | 4 | 5 | |
| PD T2 | 4.31 | 1.7 | 3.78 | 4.84 | 3 | 4 | 5 | |
| PD T3 | 4 | 1.74 | 3.46 | 4.54 | 3 | 3 | 5 | |
| Clinical Attachment loss | ||||||||
| CAL T0 | 7.29 | 1.93 | 6.69 | 7.89 | 6 | 7 | 9 | <0.001** |
| CAL T1 | 5.21 | 2.03 | 4.58 | 5.85 | 4 | 5 | 6 | |
| CAL T2 | 5.1 | 1.82 | 4.53 | 5.66 | 4 | 5 | 6 | |
| CAL T3 | 4.71 | 1.76 | 4.17 | 5.26 | 4 | 4 | 6 |
At 180 days after treatment, pocket closure (PD<4mm) was achieved in 73.8% of the study sites (Fig. 6). Eleven sites from 6 patients remained with PD>5 mm, 8 of which were in premolar or molar areas. All these residual pockets showed presence of F nucleatum (p=0.033), and in 10 sites, at least 2 of the study species were found. P gingivalis was detected in 6 residual pockets from 3 patients. 3:11 sites with final PD>5 mm showed BOP, and P gingivalis was detectable in 2 of these 3 sites.
DISCUSSION
Most of the information on the prevalent microorganisms in AP comes from other regions. However, the microbiologic profile of periodontitis in different populations seems to differ in frequency and composition.
In Argentina, little is known about the microbiological composition or relative abundance in subgingival biofilm of native patients with this infrequent disease. According to data collected in the department of Periodontology at the FOUBA between 1999 and 2005, only 7% (n=365) of the treated diseases were diagnosed as AP 10 . Two local reports evaluated the prevalence of periodontal pathogens in chronic periodontitis (CP) 11 , 12 . A pilot study was recently conducted on patients with AP 13 at the Fundación Independencia, Universidad Nacional de Cuyo, which analyzed 30 sites in 5 patients, finding significant prevalence of P gingivalis, T. forsythia, and T. denticola, over P. intermedia and A. actinomycetemcomitans . Even though the results of the present study were similar, the sample showed a 61.9% prevalence for P intermedia and 14.3% for A. actinomycetemcomitans, slightly higher levels than reported by Usin et al. 13 . Another study on a 2011-2015 cohort at FOUBA 14 considered 18 patients with clinical-radiological diagnosis of AP, and 32 with CP. Prevalence of P gingivalis in subgingival biofilm was 50% in patients with AP and 54.2% in patients with CP, while prevalence of A. actinomycetemcomitans was 32.8% and 19.7%, respectively. Although P gingivalis was 1.6 and 2.7 times higher than A. actinomycetemcomitans, the latter was significantly associated to AP (p=0.039) in relation to CP 14 .
Despite the small sample size, the results of the current study suggest that P gingivalis is the prevalent keystone pathogen, and A. actinomycetemcomitans is a marker of this particularly aggressive disease. This local information agrees with the results reported by other research groups such as Kamma et al. 15 , Mombelli et al. 16 , Dahlen et al. 17 , Mullally et al. 18 , Mayorga-Fayad et al. 19 , Cortelli et al. 20 and Gajardo et al. 21 Nevertheless, other scientific evidence reports A. actinomycetemcomitans as the most prevalent pathogen in in the etiopathogenesis of rapidly progressive severe periodontal disease 22 . Recent studies on subgingival microbiome outlinenew bacterial combinations with periodontopathogenic profile, such as Cryptobacterium curtum, Dialister pneumosintes, Filifactor alocis, Mitsuokella dentalis, Slackia exigua, Selenomonas sputigena, Solobacterium moorei, Treponema lecithinolyticum and Synergistes sp. 22 , 23 .
Mechanical instrumentation by means of scaling and root planing remains the basis of non-surgical periodontal therapy. However, results are not always predictable and depend on multiple factors, which is why the use of systemic antibiotics as an adjuvant to mechanical therapy has been studied for many years 24 . In a systematic review, the use of systemic antibiotic therapy together with scaling and root planing reduced the risk of progression of loss of attachment and showed statistically significant differences in the gain of clinical attachment level in deep pockets over mechanical therapy alone 25 . These results agree with a meta-analysis by Rabelo et al. 26 .
Within possible antibiotic regimens, the combination of amoxicillin and metronidazole has been indicated in the treatment of AP. This strategy assumes that multiple species can be eliminated or inhibited simultaneously during periodontal treatment, achieving reestablishment of the commensal microbiota and a healing response from the host 27 . A recent systematic review and meta-analysis by Teughels et al. 28 assessed the effect of the adjunctive use of different systemic antimicrobial schemes in the active phase of periodontal treatment. The best outcomes were observed with amoxicillin plus metronidazole, especially in AP, showing statistically significant benefits in all the clinical parameters studied 28 .
According to the evidence obtained in the current study, and as Faveri et al. 29 have published recently, fUll mouth treatment with adjuvant administration of the antibiotic scheme (amoxicillin and metronidazole) may be an effective protocol for the treatment of AP in the young study population. There was a significant improvement of the periodontal parameters in terms of reduction of bleeding on probing, pocket closure and gain on clinical attachment level. It should be highlighted that most of the sites with residual pockets were in molar or premolar areas. Tooth type has been shown to be an important variable that influences the results of periodontal treatment, since non-surgical periodontal therapy is often less effective in deep pockets in multi-rooted teeth 30 . The implementation of the chemical-mechanical protocol produced a shift in the bacterial composition of the subgingival biofilm, with a tendency to restitution of the homeostasis of the subgingival ecosystem. Proof supporting the ecological changes achieved by the combination therapy is provided by the marked decrease in T. denticola 31 , which is a sensitive marker of periodontal activity.
Within the limitations of this study, the microbio-logical results showed significant prevalence of P. gingivalis over A. actinomycetemcomitans in a ratio of up to 5 to 1. The prevalence of A. actinomycetemcomitans was lower than reported for other Latin American populations 15-22 , and it was the species most susceptible to treatment. Kulik et al. 32 demonstrated the broad susceptibility of A. actinomycetemcomitans to Amoxicillin/clavulanic acid and tetracycline and non-susceptibility to clindamycin, metronidazole or phenoxymethylpenicillin. More recent studies 33 showed changes in the susceptibility of A. actinomycetemcomitans and P. gingivalis , pointing to moxifloxacin as a useful therapeutic option.
Despite the apparent ecological reestablishment after combined pharmacological-mechanical periodontal treatment and standard maintenance appointments, P gingivalis appeared to have a moderate rate of persistence at the end of the study. Although the data from this study do not evaluate recolonization at 45-days, persistence in Pg detection could be more associated with dysbiosis as a result of ecological conditions than resistance to treatment 34 . The persistence of F. nucleatum in residual pockets and P. gingivalis in most of the treated sites characterized the study sample. Further studies with a larger sample size are needed to enhance local epidemiological data.
