ORIGINALLima ARTICLE et al
Occurrence of Socransky Red Complex in Pregnant Women With and Without Periodontal Disease Daniela Pereira Limaa/Suzely Adas Saliba Moimazb/Cléa Adas Saliba Garbinc/ Dóris Hissako Sumidad/Elerson Gaetti Jardim Jre/Ana Cláudia Okamotof Purpose: To verify the presence of Socransky Red Complex (Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia) and P. intermedia using polymerase chain reaction (PCR) in periodontally healthy pregnant women and pregnant women with periodontal disease, as well as its relation to arterial blood pressure and capillary glycaemia. Materials and Methods: This case control study included 86 pregnant women, including 50 pregnant women with healthy periodontium, 27 with gingivitis and 9 with periodontitis. Arterial blood pressure and glycaemia were evaluated and recorded. Clinical specimens from the gingival crevice or periodontal pockets were gathered with sterile absorbent paper cones. DNA extraction was accomplished using the Easy-DNA Kit test and the presence of bacteria was detected by PCR with primers and specific probes for each microorganism. Results: The arterial pressure of all pregnant women was found to be within normal levels and 51% presented with hyperglycaemia, these two variables were not associated with periodontal conditions and/or presence of microorganisms. Socransky Red Complex was not present in pregnant women with healthy periodontium; however, it was present in pregnant women with gingivitis (3.7%) and in a higher percentage of pregnant women with periodontitis (33.3%). Conclusion: Socransky Red Complex was found only in cases of periodontal diseases and is not related to blood pressure and/or high levels of blood glucose. Key words: periodontal disease, pregnancy, Socransky Red Complex Oral Health Prev Dent 2015;13:169-176 doi: 10.3290/j.ohpd.a32989
a
Postgraduate Student, Department of Paediatric and Preventive Dentistry, Araçatuba School of Dentistry, UNESP, São Paulo State University, Araçatuba, Brazil.
b
Full Professor, Department of Paediatric and Preventive Dentistry, Araçatuba School of Dentistry, UNESP, São Paulo State University, Araçatuba, Brazil.
c
Associate Professor, Department of Paediatric and Preventive Dentistry, Araçatuba School of Dentistry, UNESP, São Paulo State University, Araçatuba, Brazil.
d
Associate Professor, Department of Physiology and Biophysics, Institute of Science Biomedical, UNESP, São Paulo State University, São Araçatuba, Brazil.
e
Full Professor, Department of Pathology and Propaedeutics Microbiology and Immunology, Araçatuba School of Dentistry, UNESP, São Paulo State University, Araçatuba, Brazil.
f
Associate Professor, Department of Pathology and Propaedeutics Microbiology and Immunology, Araçatuba School of Dentistry, UNESP, São Paulo State University, Araçatuba, Brazil.
Correspondence: Daniela Pereira Lima, Department of Paediatric and Social Dentistry, Araçatuba School of Dentistry, UNESP, São Paulo State University, José Bonifácio Street 1193, Araçatuba, SP, Brazil 16015-050. Tel: +55-18-3636-3249, Fax: +55-18-36363332. Email: [email protected]
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Submitted for publication: 31.07.12; accepted for publication: 26.05.13
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eriodontal diseases are considered the main causes of teeth lost in the adult population and are one of more serious social, economic and public health problems in the world (Albandar, 2002; Brown et al, 2002; Makiura et al, 2008). According to Bodet et al (2007), the development of these diseases depends on the virulence of bacteria involved and the level of immune response of the host. Many authors relate that periodontal diseases are associated with dental plaque, with its evolution depending on intrinsic and extrinsic factors, such as systemic conditions, the use of antibiotic drugs, stress, tobacco use and oral hygiene, and that its progression can accelerate with time (Albandar, 2002; Bodet et al, 2007; Clark and Löe, 1993; Shourie et al, 2012). The presence of dental plaque with a pathogenic potential is important for the development of these diseases and for its evo-
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lution. Some authors have related that periodontal diseases in humans and in other mammals are predominantly associated with gram-negative anaerobic microorganisms (Slot and Genco, 1984; Clark and Löe, 1993; Farsi et al, 2008). Socransky et al (1998) and Socransky and Haffajee (2002) described the colonisation and pathogenesis of six microorganism complexes found in the subgingival plaque of adults. Those authors verified that yellow, blue, green and purple complexes consist of a group of bacteria that can adhere to the dental surface, acting as the base of a plaque pyramid. These complexes are the pioneer colonisers on the dental surface and are not related to periodontal disease, as many of them are considered helpful. However, the same authors found that these initial complexes provide receptors and create beneficial ecologic conditions for the retention of bacteria from the orange complex, which are involved in periodontal diseases and include Prevotella intermedia. The orange complex precedes and creates conditions for the retention of the Red Complex, which is composed of Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia species. The Red Complex is accepted as the aetiological agent of chronic periodontal disease and is related to an increase in pocket depth and the presence of bleeding during probing. During pregnancy, some women can develop pregnancy diabetes, which is a modulating factor of periodontal disease (Clothier et al, 2007; Friedlander et al, 2007; Eldarrat, 2011). According to Xiong et al (2006), pregnancy diabetes can increase the risk of eclampsia, pre-term birth and harm to the baby. It has been verified that pregnant women undergo physiological changes that can favour the colonisation and adherence of Socransky Red Complex and Prevotella intermedia, microorganisms that are associated with many forms of periodontal disease, such as gingivitis and chronic periodontal disease (Griffen et al, 1998; Albandar, 2002; Friedlander et al, 2007; Farsi et al 2008). The polymerase chain reaction (PCR) has been used as a tool to detect microorganisms in Socransky Red Complex, and has been used to characterise and identify these microorganisms (Rosenn et al, 1991). The aim of this study was thus to evaluate the occurence of Socransky Red Complex and the presence of P. intermedia in pregnant women with and without periodontal disease using polymerase chain reaction (PCR) and examine whether
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these organisms were associated with blood glucose level and/or arterial blood pressure.
MATERIALS AND METHODS This study was approved by the Research Ethics Committee for humans of Araçatuba Dental School, Universidad Estadual Paulista (UNESP), Brazil (process FOA 2007-02032). The patient pool consisted of 105 pregnant women in their 4th to 7th months who were enrolled in prenatal care programmes at Basic Health Units (BHU) in Araçatuba and Birigui, São Paulo State, Brazil. All participants signed the informed consent.
Evaluation of periodontal condition The periodontal status was evaluated using the Periodontal Index (PI) recommended by the World Health Organization (WHO) for oral health epidemiological surveys, which was classified into three categories: absence, mild (presence of gingival bleeding) and severe (presence of periodontal pockets). The exam was performed by two trained researchers (Kappa = 0.91) using a WHO periodontal probe and a clinical mirror, with the pregnant women seated in a chair under natural light. The researchers followed the criteria recognised by the WHO (1987), where the oral cavity is divided into 6 sections: 18–14, 13–23, 24–28, 38–34, 33–43 and 44–48. The presence of two or more teeth without an indication for extraction (for example, furcation involvement, mobility or other problems) was the criterion for the inclusion of a sextant in the evaluation.
Evaluation of blood pressure and blood glucose A sphigmomanometer was used by nurses from the same Basic Health Unit to measure the blood pressure. Blood glucose was evaluated using a blood sample (one drop) from the middle finger, using a disposable lancet (Accu-Chek Softclix Pro; Roche; Mannheim, Germany) to avoid any contamination risk for the patient and the professional that collected the samples. The blood glucose level was measured using a blood glucose level monitor (Ac-
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Table 1 Specifics primers used in conventional PCR analysis Specific primers
Oligonucleotides
Annealing temperature*
P. gingivalis
5’-TGTAGATGATCATCTGATGGTGAAACC-3’ 5’-ACGTCATCGGCTACCCACCTTCCTC-3’
60°C
P. intermedia
5’-TTTGTTGGGTGACCCAGTAAAGCGGG-3’ 5’-TTCAACCGATCTCTGTATCCCCTGCGT-3’
55°C
T. denticola
5’-TAACCGACTTCGTTATGTGCTCATTTACAT-3’ 5’-CAAAGAAGCATAATCGCCTCTTCTTCTTA-3’
55°C
T. forsythia
5’-GCGTATGTAAATCGGAACCTGCCCGCA-3’ 5’-TGCACTACGTTTCAGTGTCAGTTATACCT-3’
60°C
cu-Chek Advantage II, Roche). Pregnant women with blood glucose levels over 85 mg/dl were considered to have unusual values (Passini, 2007).
Clinical specimen collection The bacterial samples were collected from the crevicular or periodontal pocket. First, chlorhexidine (0.12%) was used to clean the tooth. Then the tooth was isolated using cotton rolls. The clinical specimens (bacterial samples) were collected using three sterile absorbent paper cones (No 30, Endopoints; Rio de Janiero, RJ, Brazil), which were inserted into the crevicular or periodontal pocket and left there for 10 s (Ashimoto, 1996). The cones were then transferred to cryovials containing 0.3 ml of ultra-pure water. The tubes were maintained in liquid nitrogen until processing in the laboratory.
Detection of microorganisms The DNA from the studied species was extracted using the Easy-DNA kit (Invitrogen; Sao Paulo, SP, Brazil). Detection was performed with PCR and specific primers. The DNA was amplified in volumes of 25 ml, containing 2.5 ml of 10x buffer PCR, 1.25 ml of MgCl2 (50 mM), 2.0 ml of dNTP (10 mM) (Introgen), 0.25 ml of Platinum Taq DNA polymerase (0.5 U; Invitrogen), 1.0 ml of each primer (0.4 mM), 7 ml of sterilised ultra-pure water Milli-Q and 10 ml of DNA (ng). The amplification was performed in a thermocycler (Perkin Elmer, GeneAmp PCR System 2400; Branchburg, NJ, USA) programmed for 1 cycle at 72°C for 5 min. The primers and annealing temperature used, as described by Ashimoto et al (1996), are presented in Table 1.
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Strains of ATCC 25611 were used as a positive control; the negative control was prepared by mixing the reagents with ultra-pure water. The PCR products underwent electrophoresis on 1% agarose gel, were stained with ethidium bromide (0.5 mg/ml) and photographed on a UV transilluminator using a Kodak camera (Electrophoresis Documentation and Analysis System 120, EastmanKodak; Rochester, NY, USA).
Statistical analysis Considering the nature of the studied variables, non-parametric tests were used to analyse the results. In all tests, statistical significance was set at 0.05 (5%). Fisher’s Exact Test was used to examine whether blood glucose level and/or blood pressure differed significantly among the pregnant women and their different periodontal conditions. The presence of Socransky Red Complex and Prevotella intermedia was examined according to periodontal status.
RESULTS Among the 86 subjects, 50 (58.1%) showed no signs of periodontal disease, 27 (31.4%) had gingivitis and 9 (10.5%) had periodontitis. (Table 2). Figure 1 depicts the blood glucose levels among the pregnant women who participated in the current study. Among the 50 pregnant women with a healthy periodontium, 23 (46%) had a blood glucose level within the normal range, and 6 of the 9 pregnant women with periodontitis (66.7%) did as well. A statistically significant association between abnormal blood glucose levels and periodontal disease was not found.
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Table 2 Numerical and percent distribution of pregant women Araçatuba/Birigui cities, São Paulo State, Brazil, 2010, according to the presence of periodontal disease n
%
None
50
58.1
Mild/presence of gingival bleeding
27
31.4
Severe/presence of periodontal pockets
09
10.5
Total
86
100.0
Normal blood glucose level Abnormal blood glucose level
25 Number of patients
Periodontal disease
30
20
15
10
5
Fig 1 Glycaemic status among pregnant women from three periodontal status groups (healthy, gingivitis and periodontitis) in Araçatuba/Birigui cities, São Paulo State, Brazil, 2010.
0 Healthy
Gingivitis
Periodontitis
Table 3 Presence of P. gingivalis, T. denticola, T. forsythia, P. intermedia and Socransky Red Complex in pregnant women with and without periodontal disease in Araçatuba/Birigui cities, São Paulo State, Brazil, 2010 Periodontal condition Bacteria
Healthy (n = 50)
Gingivitis (n = 27)
Periodontitis (n = 9)
P. gingivalis
7 (14.0%)
6 (22.2%)
6 (66.7%)
T. denticola
6 (12.0%)
8 (29.6%)
5 (55.5%)
T. forsythia
6 (12.0%)
8 (29.6%)
6 (66.7%)
P. intermedia
11 (22.0%)
6 (22.2%)
7 (77.8%)
Red Complex
0 (0%)
1 (3.7%)
3 (33.3%)
There was no statistically significant association between abnormal glucose levels and the presence of Porphyromonas gingivalis in healthy periodontium or in gingivitis and periodontitis (p = 0.545; p = 0.22, p = 0.75, respectively). The same was observed for Treponema denticola (p = 0.16, p = 0.39 and p = 0.51, respectively), Tannerella forsythia (p = 0.41, p = 0.5 and p = 0.66, respectively), Prevotella intermedia (p = 0.56, p = 0.56 and p = 0.58, respectivly) and Socransky Red Complex (p = 1.0, p = 1.0 and p = 0.5). P. gingivalis was present in 7 (14%) pregnant women without periodontal disease, in 6 (22.2%) with gingivitis and in 6 with periodontal disease. Similarly, 12% of periodonally healthy pregnant women had T. denticola and another 12% had T. forsythia, while these were found in 29.7% and 29.6%, respectively, of pregnant women with gingivitis and in 55.5% and 66.7%, respectively, of pregnant women with periodontitis. P. intermedia was found in 11
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(22%) pregnant women with a healthy periodontium, in 6 (22.2%) pregnant women with gingivitis and in 7 (77.8%) with periodontitis (Table 3). Socransky Red Complex was not found in any pregnant women with a healthy periodontium and was present in only one (3.7%) pregnant woman with gingivitis and in 3 (33.3%) with periodontitis. There was no statistically significant difference between the presence of P. gingivalis, T. denticola, T. forsythia, P. intermedia and Socransky Red Complex among pregnant women with healthy periodontia and those with gingivitis (p = 0.27, p = 0.05, p = 0.05, p = 0.59 and p = 0.35, respectively). On the other hand, there was a highly significant difference when comparing the presence of P. gingivalis, T. denticola, T. forsythia, P. intermedia and Socransky Red Complex between pregnant women with healthy periodontia and those with periodontitis (p = 0.002, p = 0.008, p = 0.001, p = 0.002 and p = 0.003, respectively).
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No statistically significant difference existed between pregnant women with healthy periodontia and those with gingivitis in terms of T. denticola and T. forsythia (p = 0.15 and p = 0.06, respectively). However, a statistically significant difference was found between the presence of P. intermedia in pregnant women with gingivitis vs those with periodontitis (p = 0.005). Further, a significant difference was observed between pregnant women with gingivitis and those with periodontitis in terms of P. gingivalis (p = 0.02) and Socransky Red Complex (p = 0.04). All pregnant women that presented with Socransky Red Complex also presented with P. intermedia.
DISCUSSION Systemic conditions, including pregnancy, can exacerbate periodontal diseases. Some authors have suggested that the reverse can also occur, i.e. that periodontal disease can have systemic effects, with a possible link to eclampsia during pregnancy (Offenbacher et al, 1996; Ruma et al, 2008). Taani et al (2003) studied periodontal conditions of pregnant women and related them to sociodemographic conditions and clinical variables, such as dental plaque index, gingival index and depth of the periodontal pocket. Those authors verified that the symptoms of periodontal diseases worsen during pregnancy and were associated with a low educational level. Some authors affirm that periodontal disease is a significant risk factor for premature birth and low birth weight (Beck et al, 1996; Radnai et al, 2006; Santos-Pereira et al, 2007). It has also been identified as a risk factor for cardiovascular and respiratory diseases, indicating the importance of preventive and educational programmes for pregnant women (Farsi et al, 2008; Pitiphat et al, 2008). In this study, it was found that 50 (58.1%) pregnant women did not show symptoms of periodontal disease, 27 (31.4%) presented with gingivitis and 9 (10.5%) with periodontitis, disagreeing with the results of Louro et al (2001) and Kunnen et al (2007). Those studies found higher values for the occurence of periodontitis, 61.7% to 97.8%, when compared to the values that were found here (10.5%). However, Pitiphat et al (2008) showed that only 3.7% of pregnant women who participated in the ‘Viva Project’ presented with periodontitis. It is possible that this low prevalence of periodontitis occurred due to the fact that pregnant women were
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enrolled in a health awareness and education programme and no clinical study was performed to verify the occurence of gingivitis. Albandar et al (1998) related that over 82% of the North American population had gingivitis; however, the values found in the present study were much lower (31.4%) for a cohort of pregnant women, who should be more susceptible to these diseases. A possible explanation for this would be the participation of pregnant women in prenatal programmes. In contrast to Pitiphat et al (2008), who found a low occurence of periodontitis (3.8%), the current study observed a higher percentage (10.5%), which could be related to the host immune response and patients’ ethnicity. Albandar and Rams (2002) noted that African, Hispanic and Asiatic populations are more susceptible than Caucasians to periodontal disease; the Brazilian population has a variety of ethnic heritages, but it is predominantly African. In this study, the low occurrence and progression of periodontal diseases can be explained by the fact that the Basic Health Units (BHUs), a participant in research, offer preventive programmes such as orientation lectures on oral health and dental care during pregnancy, which are important for controlling periodontal diseases (Taani et al, 2003). These BHUs are part of the Programme for Integrated Women’s Health Care (PAISM), which aims to promote the health of women during pregnancy and in newborns, as well as to establish ideal behaviors for prevention, diagnostics and clinical treatment of obstetric problems that have already occurred or could occur in the future. This programme is favoured by the low-income population (Coutinho et al, 2003). The present study did not observe a relationship between blood pressure and periodontal health, gingivitis and/or periodontitis, because all pregnant women had normal blood pressures. This can be attributed to the prenatal programme, because all pregnant women who participated in this research received prenatal monitoring in a BHU. In terms of the occurence of periodontal diseases and pregnancy diabetes (intolerance to glucose during pregnancy), it was verified that this disease occurred in a majority of patients (51.2%) in our study. This result is in accordance with Friedlander et al (2007). Those authors indicated that pregnancy diabetes occurred in 7% of North American pregnant women, that this condition is increasing due to obesity, is highly related to diabetes and has reached epidemic proportions in the United States.
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A study by Dasanayake et al (2008) showed a prevalence of 8.3% patients with pregnancy diabetes among examined patients. Friedlader et al (2007) verified that this disease frequently presented in older women with an elevated body mass index (BMI) before pregnancy. Chapper et al (2005), demonstrated that patients with pregnancy diabetes and pre-pregnancy obesity showed more gingivitis and less periodontal disease than those with normal pre-pregnancy BMI, pointing out that periodontal treatment should be considered based on future recommendations of metabolic control for this group of patients, because there is evidence that the levels of maternal and perinatal mortality are influenced by life conditions and the quality of obstetric and prenatal care. In this study, pregnancy diabetes showed a higher occurrence than that in a study by Friedlander et al (2007). However, those authors reported that diabetes has been increasing in recent years. The discrepancy between the results of Friedlander et al (2007) and the current study could be explained by many factors, such as decreased levels of glucose tolerance, the absence of population information and the difficulty of access to ideal health-care services. No relationship was found between pregnancy diabetes and periodontal conditions (periodontal health, gingivitis and periodontitis) in the current study, possibly because the pregnant women were being monitored and underwent a prenatal programme, which included dental treatment. In a study by Makiura et al (2008), patients with periodontitis and diabetes mellitus showed a high prevalence of P. gingivalis (53.3%), T. forsythia (86.7%), T. denticola (63.3%) and P. intermedia (33.3%). A strain of P. gingivalis which has type II fimbriae was associated with glucose level. In the present study, no association was observed between abnormal glucose levels (>85 mg/ dl) and the presence of Porphyromonas gingivalis in patients with a healthy periodontium, gingivitis or periodontitis (p = 0.55, p = 0.22 and p = 0.75, respectively). This absence of an association was also found for Treponema denticola (p = 0.16, p = 0.39 and p = 0.51, respectively), Tannerella forsythia (p = 0.41, p = 0.50 and p = 0.66, respectively), Prevotella intermedia (p = 0.56, p = 0.56 and p = 0.58, respectively) and Socransky Red Complex (p = 1.0, p = 1.0 and p = 0.5), which is in agreement with the data of Dasanayake et al (2008). It is interesting to note that Makiura et al (2008) found an association between high levels of blood
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glucose and the presence of these microorganisms in pregnant women with periodontitis. In contrast, no such association was found in the current study. More pregnant women presented with gingivitis than with periodontitis; the latter was the group with the lowest number of volunteers. The present study detected Socransky Red Complex (Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia) in 66.7% of pregnant women with periodontitis, T. denticola in 55.5% and P. intermedia in 77.9% using PCR, from samples collected with absorbent papers cones, indicating that this is an acceptable method of collecting crevicular microorganisms (Passini et al, 2007). Therefore, the results of this study are similar to those found by Makiura et al (2008), who collected material from the periodontal pocket using a curette and detected microorganisms using PCR; although the collection method was different, the results were similar. Although there was no relation between hypertension and high levels of blood glucose either with periodontal status or the presence of microorganisms, it was verified that the occurence of these periodontopathogens was related with the periodontal conditions of the pregnant women. P. gingivalis was present in 7 (14%) pregnant women without periodontal disease, in 6 (22.2%) with gingivitis and in 6 (66.7%) pregnant women with periodontal disease. T. denticola was present in similar proportions (12% without periodontal disease, 29.7% with gingivitis and 55.5% with periodontitis), as were T. forsythia (12% with a healthy periodontium, 29.6% with gingivitis and 66.7% with periodontitis) and P. intermedia (22% with healthy periodontal conditions, 22.2% with gingivitis and 77.8% with periodontitis). These results showed that these microorganisms increased the severity of periodontal disease, whereas Socransky Red Complex was detected in only one pregnant woman with gingivitis (3.7%) and in 3 (33.3%) pregnant women with periodontitis. Therefore, the microbiological results of the current study is in agreement with the results of Griffen et al (1998), Socransky et al (1998), Socransky and Haffajee (2002), Holt and Ebersole (2005) and Bodet et al (2008), all of whom observed an increased occurence of these microorganisms in periodontal diseases. These authors suggested that the Red Complex would be present in higher proportions and severities in areas with active periodontitis, indicating that these bacteria form a potentially highly pathogenic group.
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CONCLUSIONS
• There was no association between periodontal diseases and/or blood pressure and high levels of blood glucose. • There was no association between detected microorganisms and blood pressure and/or high levels of blood glucose. • Socransky Red Complex was found only in cases of periodontal diseases and is not related to blood pressure and/or high levels of blood glucose. • A higher number of P. intermedia was found in patients with gingivitis and periodontitis.
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Lima et al 31. Santos-Pereira SA, Giraldo PC, Saba-Chujfi E, Amaral RL, Morais SS, Fachini AM, et al. Chronic periodontitis and pre-term labour in Brazilian pregnant women: an association to be analysed. J Clin Periodontol 2007;34:208–213. 32. Shourie V, Dwarakanath CD, Prashanth GV, Alampalli RV, Padmanabhan S, Bali S. The effect of menstrual cycle on periodontal health – a clinical and microbiological study. Oral Health Prev Dent 2012;10:185–192. 33. Slots J, Genco RJ. Black pigmented Bacteroides species and Capnocytophaga species and Actinobacillus actinomycetemcomitans in human periodontal disease;virulence factors in colonization, survival and tissue destruction. J Dent Res 1984;63:412–421. 34. Socransky SS, Haffajee AD, Cugini MA, Smith C, Kent RL Jr. Microbial complexes in subgingival plaque. J Clin Periodontol 1998;25:134–144.
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35. Socransky S, Haffajee AD. Dental biofilms: difficult therapeutic targets. Periodontol 2000 2002;28:12–55. 36. Taani DQ, Habashneh R, Hammad MM, Batieha A. The periodontal status of pregnant women and its relationship with socio-demographic and clinical variables. J Oral Rehabil 2003;30:440–445. 37. Tomazinho LF, Avila-Campos MJ. Detection of Porphyromonas gingivalis Porphyromonas endodontalis Prevotella intermediaPrevotella nigrescens in chronic endodontic infection. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007;103:285–288. 38. Xiong X, Buekens P, Vastardis S, Pridjian G. Periodontal disease and gestational diabetes mellitus. Am J Obstet Gynecol 2006;195:1086–1089.
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Occurrence of Socransky Red Complex in Pregnant Women With and Without Periodontal Disease Daniela Pereira Limaa/Suzely Adas Saliba Moimazb/Cléa Adas Saliba Garbinc/ Dóris Hissako Sumidad/Elerson Gaetti Jardim Jre/Ana Cláudia Okamotof Purpose: To verify the presence of Socransky Red Complex (Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia) and P. intermedia using polymerase chain reaction (PCR) in periodontally healthy pregnant women and pregnant women with periodontal disease, as well as its relation to arterial blood pressure and capillary glycaemia. Materials and Methods: This case control study included 86 pregnant women, including 50 pregnant women with healthy periodontium, 27 with gingivitis and 9 with periodontitis. Arterial blood pressure and glycaemia were evaluated and recorded. Clinical specimens from the gingival crevice or periodontal pockets were gathered with sterile absorbent paper cones. DNA extraction was accomplished using the Easy-DNA Kit test and the presence of bacteria was detected by PCR with primers and specific probes for each microorganism. Results: The arterial pressure of all pregnant women was found to be within normal levels and 51% presented with hyperglycaemia, these two variables were not associated with periodontal conditions and/or presence of microorganisms. Socransky Red Complex was not present in pregnant women with healthy periodontium; however, it was present in pregnant women with gingivitis (3.7%) and in a higher percentage of pregnant women with periodontitis (33.3%). Conclusion: Socransky Red Complex was found only in cases of periodontal diseases and is not related to blood pressure and/or high levels of blood glucose. Key words: periodontal disease, pregnancy, Socransky Red Complex Oral Health Prev Dent 2015;13:169-176 doi: 10.3290/j.ohpd.a32989
a
Postgraduate Student, Department of Paediatric and Preventive Dentistry, Araçatuba School of Dentistry, UNESP, São Paulo State University, Araçatuba, Brazil.
b
Full Professor, Department of Paediatric and Preventive Dentistry, Araçatuba School of Dentistry, UNESP, São Paulo State University, Araçatuba, Brazil.
c
Associate Professor, Department of Paediatric and Preventive Dentistry, Araçatuba School of Dentistry, UNESP, São Paulo State University, Araçatuba, Brazil.
d
Associate Professor, Department of Physiology and Biophysics, Institute of Science Biomedical, UNESP, São Paulo State University, São Araçatuba, Brazil.
e
Full Professor, Department of Pathology and Propaedeutics Microbiology and Immunology, Araçatuba School of Dentistry, UNESP, São Paulo State University, Araçatuba, Brazil.
f
Associate Professor, Department of Pathology and Propaedeutics Microbiology and Immunology, Araçatuba School of Dentistry, UNESP, São Paulo State University, Araçatuba, Brazil.
Correspondence: Daniela Pereira Lima, Department of Paediatric and Social Dentistry, Araçatuba School of Dentistry, UNESP, São Paulo State University, José Bonifácio Street 1193, Araçatuba, SP, Brazil 16015-050. Tel: +55-18-3636-3249, Fax: +55-18-36363332. Email: [email protected]
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Submitted for publication: 31.07.12; accepted for publication: 26.05.13
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eriodontal diseases are considered the main causes of teeth lost in the adult population and are one of more serious social, economic and public health problems in the world (Albandar, 2002; Brown et al, 2002; Makiura et al, 2008). According to Bodet et al (2007), the development of these diseases depends on the virulence of bacteria involved and the level of immune response of the host. Many authors relate that periodontal diseases are associated with dental plaque, with its evolution depending on intrinsic and extrinsic factors, such as systemic conditions, the use of antibiotic drugs, stress, tobacco use and oral hygiene, and that its progression can accelerate with time (Albandar, 2002; Bodet et al, 2007; Clark and Löe, 1993; Shourie et al, 2012). The presence of dental plaque with a pathogenic potential is important for the development of these diseases and for its evo-
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lution. Some authors have related that periodontal diseases in humans and in other mammals are predominantly associated with gram-negative anaerobic microorganisms (Slot and Genco, 1984; Clark and Löe, 1993; Farsi et al, 2008). Socransky et al (1998) and Socransky and Haffajee (2002) described the colonisation and pathogenesis of six microorganism complexes found in the subgingival plaque of adults. Those authors verified that yellow, blue, green and purple complexes consist of a group of bacteria that can adhere to the dental surface, acting as the base of a plaque pyramid. These complexes are the pioneer colonisers on the dental surface and are not related to periodontal disease, as many of them are considered helpful. However, the same authors found that these initial complexes provide receptors and create beneficial ecologic conditions for the retention of bacteria from the orange complex, which are involved in periodontal diseases and include Prevotella intermedia. The orange complex precedes and creates conditions for the retention of the Red Complex, which is composed of Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia species. The Red Complex is accepted as the aetiological agent of chronic periodontal disease and is related to an increase in pocket depth and the presence of bleeding during probing. During pregnancy, some women can develop pregnancy diabetes, which is a modulating factor of periodontal disease (Clothier et al, 2007; Friedlander et al, 2007; Eldarrat, 2011). According to Xiong et al (2006), pregnancy diabetes can increase the risk of eclampsia, pre-term birth and harm to the baby. It has been verified that pregnant women undergo physiological changes that can favour the colonisation and adherence of Socransky Red Complex and Prevotella intermedia, microorganisms that are associated with many forms of periodontal disease, such as gingivitis and chronic periodontal disease (Griffen et al, 1998; Albandar, 2002; Friedlander et al, 2007; Farsi et al 2008). The polymerase chain reaction (PCR) has been used as a tool to detect microorganisms in Socransky Red Complex, and has been used to characterise and identify these microorganisms (Rosenn et al, 1991). The aim of this study was thus to evaluate the occurence of Socransky Red Complex and the presence of P. intermedia in pregnant women with and without periodontal disease using polymerase chain reaction (PCR) and examine whether
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these organisms were associated with blood glucose level and/or arterial blood pressure.
MATERIALS AND METHODS This study was approved by the Research Ethics Committee for humans of Araçatuba Dental School, Universidad Estadual Paulista (UNESP), Brazil (process FOA 2007-02032). The patient pool consisted of 105 pregnant women in their 4th to 7th months who were enrolled in prenatal care programmes at Basic Health Units (BHU) in Araçatuba and Birigui, São Paulo State, Brazil. All participants signed the informed consent.
Evaluation of periodontal condition The periodontal status was evaluated using the Periodontal Index (PI) recommended by the World Health Organization (WHO) for oral health epidemiological surveys, which was classified into three categories: absence, mild (presence of gingival bleeding) and severe (presence of periodontal pockets). The exam was performed by two trained researchers (Kappa = 0.91) using a WHO periodontal probe and a clinical mirror, with the pregnant women seated in a chair under natural light. The researchers followed the criteria recognised by the WHO (1987), where the oral cavity is divided into 6 sections: 18–14, 13–23, 24–28, 38–34, 33–43 and 44–48. The presence of two or more teeth without an indication for extraction (for example, furcation involvement, mobility or other problems) was the criterion for the inclusion of a sextant in the evaluation.
Evaluation of blood pressure and blood glucose A sphigmomanometer was used by nurses from the same Basic Health Unit to measure the blood pressure. Blood glucose was evaluated using a blood sample (one drop) from the middle finger, using a disposable lancet (Accu-Chek Softclix Pro; Roche; Mannheim, Germany) to avoid any contamination risk for the patient and the professional that collected the samples. The blood glucose level was measured using a blood glucose level monitor (Ac-
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Table 1 Specifics primers used in conventional PCR analysis Specific primers
Oligonucleotides
Annealing temperature*
P. gingivalis
5’-TGTAGATGATCATCTGATGGTGAAACC-3’ 5’-ACGTCATCGGCTACCCACCTTCCTC-3’
60°C
P. intermedia
5’-TTTGTTGGGTGACCCAGTAAAGCGGG-3’ 5’-TTCAACCGATCTCTGTATCCCCTGCGT-3’
55°C
T. denticola
5’-TAACCGACTTCGTTATGTGCTCATTTACAT-3’ 5’-CAAAGAAGCATAATCGCCTCTTCTTCTTA-3’
55°C
T. forsythia
5’-GCGTATGTAAATCGGAACCTGCCCGCA-3’ 5’-TGCACTACGTTTCAGTGTCAGTTATACCT-3’
60°C
cu-Chek Advantage II, Roche). Pregnant women with blood glucose levels over 85 mg/dl were considered to have unusual values (Passini, 2007).
Clinical specimen collection The bacterial samples were collected from the crevicular or periodontal pocket. First, chlorhexidine (0.12%) was used to clean the tooth. Then the tooth was isolated using cotton rolls. The clinical specimens (bacterial samples) were collected using three sterile absorbent paper cones (No 30, Endopoints; Rio de Janiero, RJ, Brazil), which were inserted into the crevicular or periodontal pocket and left there for 10 s (Ashimoto, 1996). The cones were then transferred to cryovials containing 0.3 ml of ultra-pure water. The tubes were maintained in liquid nitrogen until processing in the laboratory.
Detection of microorganisms The DNA from the studied species was extracted using the Easy-DNA kit (Invitrogen; Sao Paulo, SP, Brazil). Detection was performed with PCR and specific primers. The DNA was amplified in volumes of 25 ml, containing 2.5 ml of 10x buffer PCR, 1.25 ml of MgCl2 (50 mM), 2.0 ml of dNTP (10 mM) (Introgen), 0.25 ml of Platinum Taq DNA polymerase (0.5 U; Invitrogen), 1.0 ml of each primer (0.4 mM), 7 ml of sterilised ultra-pure water Milli-Q and 10 ml of DNA (ng). The amplification was performed in a thermocycler (Perkin Elmer, GeneAmp PCR System 2400; Branchburg, NJ, USA) programmed for 1 cycle at 72°C for 5 min. The primers and annealing temperature used, as described by Ashimoto et al (1996), are presented in Table 1.
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Strains of ATCC 25611 were used as a positive control; the negative control was prepared by mixing the reagents with ultra-pure water. The PCR products underwent electrophoresis on 1% agarose gel, were stained with ethidium bromide (0.5 mg/ml) and photographed on a UV transilluminator using a Kodak camera (Electrophoresis Documentation and Analysis System 120, EastmanKodak; Rochester, NY, USA).
Statistical analysis Considering the nature of the studied variables, non-parametric tests were used to analyse the results. In all tests, statistical significance was set at 0.05 (5%). Fisher’s Exact Test was used to examine whether blood glucose level and/or blood pressure differed significantly among the pregnant women and their different periodontal conditions. The presence of Socransky Red Complex and Prevotella intermedia was examined according to periodontal status.
RESULTS Among the 86 subjects, 50 (58.1%) showed no signs of periodontal disease, 27 (31.4%) had gingivitis and 9 (10.5%) had periodontitis. (Table 2). Figure 1 depicts the blood glucose levels among the pregnant women who participated in the current study. Among the 50 pregnant women with a healthy periodontium, 23 (46%) had a blood glucose level within the normal range, and 6 of the 9 pregnant women with periodontitis (66.7%) did as well. A statistically significant association between abnormal blood glucose levels and periodontal disease was not found.
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Table 2 Numerical and percent distribution of pregant women Araçatuba/Birigui cities, São Paulo State, Brazil, 2010, according to the presence of periodontal disease n
%
None
50
58.1
Mild/presence of gingival bleeding
27
31.4
Severe/presence of periodontal pockets
09
10.5
Total
86
100.0
Normal blood glucose level Abnormal blood glucose level
25 Number of patients
Periodontal disease
30
20
15
10
5
Fig 1 Glycaemic status among pregnant women from three periodontal status groups (healthy, gingivitis and periodontitis) in Araçatuba/Birigui cities, São Paulo State, Brazil, 2010.
0 Healthy
Gingivitis
Periodontitis
Table 3 Presence of P. gingivalis, T. denticola, T. forsythia, P. intermedia and Socransky Red Complex in pregnant women with and without periodontal disease in Araçatuba/Birigui cities, São Paulo State, Brazil, 2010 Periodontal condition Bacteria
Healthy (n = 50)
Gingivitis (n = 27)
Periodontitis (n = 9)
P. gingivalis
7 (14.0%)
6 (22.2%)
6 (66.7%)
T. denticola
6 (12.0%)
8 (29.6%)
5 (55.5%)
T. forsythia
6 (12.0%)
8 (29.6%)
6 (66.7%)
P. intermedia
11 (22.0%)
6 (22.2%)
7 (77.8%)
Red Complex
0 (0%)
1 (3.7%)
3 (33.3%)
There was no statistically significant association between abnormal glucose levels and the presence of Porphyromonas gingivalis in healthy periodontium or in gingivitis and periodontitis (p = 0.545; p = 0.22, p = 0.75, respectively). The same was observed for Treponema denticola (p = 0.16, p = 0.39 and p = 0.51, respectively), Tannerella forsythia (p = 0.41, p = 0.5 and p = 0.66, respectively), Prevotella intermedia (p = 0.56, p = 0.56 and p = 0.58, respectivly) and Socransky Red Complex (p = 1.0, p = 1.0 and p = 0.5). P. gingivalis was present in 7 (14%) pregnant women without periodontal disease, in 6 (22.2%) with gingivitis and in 6 with periodontal disease. Similarly, 12% of periodonally healthy pregnant women had T. denticola and another 12% had T. forsythia, while these were found in 29.7% and 29.6%, respectively, of pregnant women with gingivitis and in 55.5% and 66.7%, respectively, of pregnant women with periodontitis. P. intermedia was found in 11
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(22%) pregnant women with a healthy periodontium, in 6 (22.2%) pregnant women with gingivitis and in 7 (77.8%) with periodontitis (Table 3). Socransky Red Complex was not found in any pregnant women with a healthy periodontium and was present in only one (3.7%) pregnant woman with gingivitis and in 3 (33.3%) with periodontitis. There was no statistically significant difference between the presence of P. gingivalis, T. denticola, T. forsythia, P. intermedia and Socransky Red Complex among pregnant women with healthy periodontia and those with gingivitis (p = 0.27, p = 0.05, p = 0.05, p = 0.59 and p = 0.35, respectively). On the other hand, there was a highly significant difference when comparing the presence of P. gingivalis, T. denticola, T. forsythia, P. intermedia and Socransky Red Complex between pregnant women with healthy periodontia and those with periodontitis (p = 0.002, p = 0.008, p = 0.001, p = 0.002 and p = 0.003, respectively).
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No statistically significant difference existed between pregnant women with healthy periodontia and those with gingivitis in terms of T. denticola and T. forsythia (p = 0.15 and p = 0.06, respectively). However, a statistically significant difference was found between the presence of P. intermedia in pregnant women with gingivitis vs those with periodontitis (p = 0.005). Further, a significant difference was observed between pregnant women with gingivitis and those with periodontitis in terms of P. gingivalis (p = 0.02) and Socransky Red Complex (p = 0.04). All pregnant women that presented with Socransky Red Complex also presented with P. intermedia.
DISCUSSION Systemic conditions, including pregnancy, can exacerbate periodontal diseases. Some authors have suggested that the reverse can also occur, i.e. that periodontal disease can have systemic effects, with a possible link to eclampsia during pregnancy (Offenbacher et al, 1996; Ruma et al, 2008). Taani et al (2003) studied periodontal conditions of pregnant women and related them to sociodemographic conditions and clinical variables, such as dental plaque index, gingival index and depth of the periodontal pocket. Those authors verified that the symptoms of periodontal diseases worsen during pregnancy and were associated with a low educational level. Some authors affirm that periodontal disease is a significant risk factor for premature birth and low birth weight (Beck et al, 1996; Radnai et al, 2006; Santos-Pereira et al, 2007). It has also been identified as a risk factor for cardiovascular and respiratory diseases, indicating the importance of preventive and educational programmes for pregnant women (Farsi et al, 2008; Pitiphat et al, 2008). In this study, it was found that 50 (58.1%) pregnant women did not show symptoms of periodontal disease, 27 (31.4%) presented with gingivitis and 9 (10.5%) with periodontitis, disagreeing with the results of Louro et al (2001) and Kunnen et al (2007). Those studies found higher values for the occurence of periodontitis, 61.7% to 97.8%, when compared to the values that were found here (10.5%). However, Pitiphat et al (2008) showed that only 3.7% of pregnant women who participated in the ‘Viva Project’ presented with periodontitis. It is possible that this low prevalence of periodontitis occurred due to the fact that pregnant women were
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enrolled in a health awareness and education programme and no clinical study was performed to verify the occurence of gingivitis. Albandar et al (1998) related that over 82% of the North American population had gingivitis; however, the values found in the present study were much lower (31.4%) for a cohort of pregnant women, who should be more susceptible to these diseases. A possible explanation for this would be the participation of pregnant women in prenatal programmes. In contrast to Pitiphat et al (2008), who found a low occurence of periodontitis (3.8%), the current study observed a higher percentage (10.5%), which could be related to the host immune response and patients’ ethnicity. Albandar and Rams (2002) noted that African, Hispanic and Asiatic populations are more susceptible than Caucasians to periodontal disease; the Brazilian population has a variety of ethnic heritages, but it is predominantly African. In this study, the low occurrence and progression of periodontal diseases can be explained by the fact that the Basic Health Units (BHUs), a participant in research, offer preventive programmes such as orientation lectures on oral health and dental care during pregnancy, which are important for controlling periodontal diseases (Taani et al, 2003). These BHUs are part of the Programme for Integrated Women’s Health Care (PAISM), which aims to promote the health of women during pregnancy and in newborns, as well as to establish ideal behaviors for prevention, diagnostics and clinical treatment of obstetric problems that have already occurred or could occur in the future. This programme is favoured by the low-income population (Coutinho et al, 2003). The present study did not observe a relationship between blood pressure and periodontal health, gingivitis and/or periodontitis, because all pregnant women had normal blood pressures. This can be attributed to the prenatal programme, because all pregnant women who participated in this research received prenatal monitoring in a BHU. In terms of the occurence of periodontal diseases and pregnancy diabetes (intolerance to glucose during pregnancy), it was verified that this disease occurred in a majority of patients (51.2%) in our study. This result is in accordance with Friedlander et al (2007). Those authors indicated that pregnancy diabetes occurred in 7% of North American pregnant women, that this condition is increasing due to obesity, is highly related to diabetes and has reached epidemic proportions in the United States.
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A study by Dasanayake et al (2008) showed a prevalence of 8.3% patients with pregnancy diabetes among examined patients. Friedlader et al (2007) verified that this disease frequently presented in older women with an elevated body mass index (BMI) before pregnancy. Chapper et al (2005), demonstrated that patients with pregnancy diabetes and pre-pregnancy obesity showed more gingivitis and less periodontal disease than those with normal pre-pregnancy BMI, pointing out that periodontal treatment should be considered based on future recommendations of metabolic control for this group of patients, because there is evidence that the levels of maternal and perinatal mortality are influenced by life conditions and the quality of obstetric and prenatal care. In this study, pregnancy diabetes showed a higher occurrence than that in a study by Friedlander et al (2007). However, those authors reported that diabetes has been increasing in recent years. The discrepancy between the results of Friedlander et al (2007) and the current study could be explained by many factors, such as decreased levels of glucose tolerance, the absence of population information and the difficulty of access to ideal health-care services. No relationship was found between pregnancy diabetes and periodontal conditions (periodontal health, gingivitis and periodontitis) in the current study, possibly because the pregnant women were being monitored and underwent a prenatal programme, which included dental treatment. In a study by Makiura et al (2008), patients with periodontitis and diabetes mellitus showed a high prevalence of P. gingivalis (53.3%), T. forsythia (86.7%), T. denticola (63.3%) and P. intermedia (33.3%). A strain of P. gingivalis which has type II fimbriae was associated with glucose level. In the present study, no association was observed between abnormal glucose levels (>85 mg/ dl) and the presence of Porphyromonas gingivalis in patients with a healthy periodontium, gingivitis or periodontitis (p = 0.55, p = 0.22 and p = 0.75, respectively). This absence of an association was also found for Treponema denticola (p = 0.16, p = 0.39 and p = 0.51, respectively), Tannerella forsythia (p = 0.41, p = 0.50 and p = 0.66, respectively), Prevotella intermedia (p = 0.56, p = 0.56 and p = 0.58, respectively) and Socransky Red Complex (p = 1.0, p = 1.0 and p = 0.5), which is in agreement with the data of Dasanayake et al (2008). It is interesting to note that Makiura et al (2008) found an association between high levels of blood
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glucose and the presence of these microorganisms in pregnant women with periodontitis. In contrast, no such association was found in the current study. More pregnant women presented with gingivitis than with periodontitis; the latter was the group with the lowest number of volunteers. The present study detected Socransky Red Complex (Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia) in 66.7% of pregnant women with periodontitis, T. denticola in 55.5% and P. intermedia in 77.9% using PCR, from samples collected with absorbent papers cones, indicating that this is an acceptable method of collecting crevicular microorganisms (Passini et al, 2007). Therefore, the results of this study are similar to those found by Makiura et al (2008), who collected material from the periodontal pocket using a curette and detected microorganisms using PCR; although the collection method was different, the results were similar. Although there was no relation between hypertension and high levels of blood glucose either with periodontal status or the presence of microorganisms, it was verified that the occurence of these periodontopathogens was related with the periodontal conditions of the pregnant women. P. gingivalis was present in 7 (14%) pregnant women without periodontal disease, in 6 (22.2%) with gingivitis and in 6 (66.7%) pregnant women with periodontal disease. T. denticola was present in similar proportions (12% without periodontal disease, 29.7% with gingivitis and 55.5% with periodontitis), as were T. forsythia (12% with a healthy periodontium, 29.6% with gingivitis and 66.7% with periodontitis) and P. intermedia (22% with healthy periodontal conditions, 22.2% with gingivitis and 77.8% with periodontitis). These results showed that these microorganisms increased the severity of periodontal disease, whereas Socransky Red Complex was detected in only one pregnant woman with gingivitis (3.7%) and in 3 (33.3%) pregnant women with periodontitis. Therefore, the microbiological results of the current study is in agreement with the results of Griffen et al (1998), Socransky et al (1998), Socransky and Haffajee (2002), Holt and Ebersole (2005) and Bodet et al (2008), all of whom observed an increased occurence of these microorganisms in periodontal diseases. These authors suggested that the Red Complex would be present in higher proportions and severities in areas with active periodontitis, indicating that these bacteria form a potentially highly pathogenic group.
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CONCLUSIONS
• There was no association between periodontal diseases and/or blood pressure and high levels of blood glucose. • There was no association between detected microorganisms and blood pressure and/or high levels of blood glucose. • Socransky Red Complex was found only in cases of periodontal diseases and is not related to blood pressure and/or high levels of blood glucose. • A higher number of P. intermedia was found in patients with gingivitis and periodontitis.
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