Risk factors for bovine periodontal disease - a preliminary study.

The work presented in this pilot study aimed to identify potential risk factors associated with bovine periodontitis development. Bovine periodontitis is a multifactorial polymicrobial infectious disease for which the aetiopathogenesis and risk factors are not fully understood. From cattle slaughtered in an abattoir in Scotland, 35 dental arcades with periodontal lesions and 40 periodontally healthy arcades were selected over seven visits for study. Multivariable logistic regression analysis was used to evaluate the association between periodontitis and the independent variables, gender, age and breed. For every increase in year of age, cattle were 1.5 times more likely to have periodontitis. A graphical analysis indicated that within the limits of this study, we could not detect any major influence of breed on the age-effect. Although logistic regression analysis demonstrated that periodontitis lesions are more prevalent with increasing age of cattle the underlying mechanisms remain unclear. It is likely that periodontitis is an important cause of oral pain in older cattle and can contribute to reduced productivity/performance. Further studies with a larger sample size are necessary to elucidate the associations between potential risk factors and periodontitis in cattle and to define its effects on animal welfare and productivity.

Gingival Toll-like receptor and cytokine messenger RNA levels in equine periodontitis and oral health.

REASONS FOR PERFORMING STUDY: Equine periodontitis is a common and painful condition. However, the disease often goes unnoticed by owners and is thus a major welfare concern. The aetiopathogenesis of the condition remains poorly understood and has been investigated in few studies. The innate immune system is known to play an important role in human periodontitis, but its role in equine periodontitis has not been examined. OBJECTIVES: To quantify the messenger (m)RNA levels of Toll-like receptors (TLRs) and cytokines in gingival tissue from orally healthy horses and those affected by periodontitis. STUDY DESIGN: Observational study. METHODS: Gingival tissue samples were taken post-mortem from 13 horses with no clinical signs of oral disease and 20 horses with periodontitis. mRNA levels of TLR2, TLR4 and TLR9 and cytokines interleukin-1ß (IL-1ß), tumour necrosis factor-α (TNF-α), IL-4, IL-6, IL-10, IL-12, IL-17 and interferon-γ (IFN-γ) were determined using quantitative real-time PCR. The statistical significance of results was assessed using appropriate t tests. RESULTS: mRNA levels of all TLRs and cytokines were upregulated in equine periodontitis. Significant increases in mRNA levels of TLR2, TLR9, IL-4, IL-10, IL-12 (P≤0.05) and IFN-γ (P≤0.01) were observed for both unweighted and age-weighted analyses of diseased gingival tissue samples compared with healthy gingival samples. In comparisons of samples of periodontitis lesions with healthy gingival control samples from the same horse, significant increases in mRNA levels of TLR4, TLR9, IL-10, IFN-γ (P≤0.05), TLR2, IL-1ß and IL-12p35 (P≤0.01) were observed. CONCLUSIONS: This study has provided an initial insight into the involvement of the immune system in equine periodontitis. Increased mRNA levels of TLR2, TLR4 and TLR9 indicate substantial microbial challenge in diseased gingival tissue. A mixed Th1/Th2/Th17 cytokine response is produced in equine periodontitis. Further studies are required to more fully characterise the role of the innate immune system in this disease.

The effect of periodontal scaling and root polishing on serum IL-17E concentrations and the IL-17A:IL-17E ratio.

OBJECTIVES: The serum IL-17A:IL-17E ratio has previously been demonstrated to be a clinical marker of periodontitis. The aim of this study was to determine the effects of non-surgical periodontal treatment on the serum IL-17A:IL-17E ratio. MATERIALS AND METHODS: Forty chronic periodontitis patients completed this study and received periodontal treatment comprising scaling and root planing plus ultrasonic debridement. Clinical data were recorded at baseline, 6 weeks (R1) after treatment completion (full-mouth or quadrant-scaling and root planing) and 25 weeks after baseline (R2). Serum samples were taken at each time point and cytokines concentrations determined by ELISA. RESULTS: Following treatment, statistically significant reductions were noted in clinical parameters. However, IL-17A and IL-17E concentrations were significantly greater than baseline values before- and after-adjusting for smoking. The IL-17A:IL-17E ratio was lower at R1 and R2. Serum IL-6 and TNF levels were significantly lower at R1 only. Also exclusively at R1, serum IL-17A and IL-17E correlated positively with clinical parameters, while the IL-17A:IL-17E ratio correlated negatively with probing pocket depth and clinical attachment. CONCLUSION: Increased serum IL-17E and a reduced IL-17A:IL-17E ratio may be indicative and/or a consequence of periodontal therapy. Therefore, the role of IL-17E in periodontal disease progression and the healing process is worthy of further investigation. CLINICAL RELEVANCE: IL-17E may be a valuable biomarker to monitor the healing process following periodontal treatment as increased IL-17E levels and a reduced IL-17A:IL-17E ratio could reflect clinical improvements post-therapy. Therefore, monitoring serum IL-17E might be useful to identify individuals who require additional periodontal treatment.

Molecular detection of transcriptionally active bacteria from failed prosthetic hip joints removed during revision arthroplasty.

The purpose of this study was to use microbiological culture and bacterial 16S rRNA gene sequencing methods to detect transcriptionally active bacteria present on the surface of failed prosthetic hip joints removed during revision arthroplasty. Five failed prosthetic hip joints were sonicated to dislodge adherent bacteria and subjected to microbiological culture. Bacterial RNA was extracted from each sonicate, cDNA prepared by reverse transcription and the 16S rRNA gene amplified using universal primers. Polymerase chain reaction (PCR) products were cloned, assigned to distinct groups by restriction fragment length polymorphism (RFLP) analysis and one representative clone from each group was sequenced. Bacteria were identified by comparison of the obtained 16S rRNA gene sequences with those deposited in public access sequence databases. All five specimens were positive for the presence of bacteria by both culture and PCR. Culture methods identified species from eight genera. Molecular detection of transcriptionally active bacteria identified a wider range of species. A total of 42 phylotypes were identified, of which Lysobacter gummosus was the most abundant (31.6%). Thirty-four clones (14.5%) represented uncultivable phylotypes. No potentially novel species were identified. It is concluded that a diverse range of transcriptionally active bacterial species are present within biofilms on the surface of failed prosthetic hip joints.

Molecular identification of bacteria on the tongue dorsum of subjects with and without halitosis.

AIM: Compare the microbial profiles on the tongue dorsum in patients with halitosis and control subjects in a UK population using culture-independent techniques. MATERIALS AND METHODS: Halitosis patients were screened according to our recently developed recruitment protocol. Scrapings from the tongue dorsum were obtained for 12 control subjects and 20 halitosis patients. Bacteria were identified by PCR amplification, cloning and sequencing of 16S rRNA genes. RESULTS: The predominant species found in the control samples were Lysobacter-type species, Streptococcus salivarius, Veillonella dispar, unidentified oral bacterium, Actinomyces odontolyticus, Atopobium parvulum and Veillonella atypica. In the halitosis samples, Lysobacter-type species, S. salivarius, Prevotella melaninogenica, unidentified oral bacterium, Prevotella veroralis and Prevotella pallens were the most commonly found species. For the control samples, 13-16 (4.7-5.8%) of 276 clones represented uncultured species, whereas in the halitosis samples, this proportion increased to 6.5-9.6% (36-53 of 553 clones). In the control samples, 22 (8.0%) of 276 clones represented potentially novel phylotypes, and in the halitosis samples, this figure was 39 (7.1%) of 553 clones. CONCLUSIONS: The microflora associated with the tongue dorsum is complex in both the control and halitosis groups, but several key species predominate in both groups.

Clinical examination of subjects with halitosis.

OBJECTIVE: To develop and apply a detailed clinical protocol for screening and assessing subjects with a complaint of halitosis. DESIGN: Cross-sectional. SUBJECTS AND METHODS: Several methods were used to recruit subjects with a complaint of halitosis, including a newspaper advertisement. A definition of halitosis arising from within the oral cavity, which is not related to generalized chronic gingivitis, chronic periodontitis or pathology of the oral mucosa was used. An extensive list of exclusion criteria was applied at the initial visit. Eligible subjects were asked to follow strict instructions and complete a questionnaire prior to their second visit for data collection. The clinical examination consisted of an organoleptic assessment, Halimeter reading and periodontal examination. RESULTS: The best method of recruiting subjects was advertising. Of 66 individuals recruited, four failed to attend the screening visit and 25 were excluded. The main reasons for exclusion were poor oral hygiene and existing periodontal disease. Thirty-seven completed the full protocol, resulting in identification of 18 with halitosis and 19 controls. CONCLUSIONS: Application of the exclusion criteria resulted in significant attrition of eligible participants. Our results suggest that organoleptic assessment should be regarded as a useful standard for defining subjects with halitosis.

Impact of smoking on the clinical, microbiological and immunological parameters of adult patients with periodontitis.

OBJECTIVES: The aim of the current study was to assess the impact of smoking on the clinical indices, the humoral immune response and the detection frequency of putative periodontal pathogens in patients with periodontitis cross-sectionally and following therapy. MATERIAL AND METHODS: Clinical measurements, subgingival plaque samples, gingival crevicular fluid (GCF) and sera were collected from 40 untreated patients with moderate-to-advanced chronic periodontitis before and after treatment over a period of 6 months. The treatment consisted of the initial therapy of scaling and root planing. Smoking status was self-reported and was confirmed by cotinine enzyme inhibition assay (CEIA). Whole-mouth clinical measurements were recorded with a manual periodontal probe at baseline (BAS) and at 6 months (RAS). Selected-site analyses were performed on the deepest site in each quadrant before and after therapy and clinical indices were recorded with an electronic pressure-sensitive probe. GCF sample volume was quantified using the Periotron 6000. Polymerase chain reaction (PCR) was utilized to determine the presence of Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Prevotella intermedia, Treponema denticola and Tanerella forsythensis in subgingival plaque. Enzyme-linked immunosorbent assay examined the systemic antibody titres to these bacteria, and thiocyanate disassociation determined the antibody avidity to these organisms. RESULTS: At baseline, smokers showed significantly less gingival inflammation and lower GCF volume compared with non-smokers. After treatment, a compromised clinical outcome was noted for smokers in terms of pocket depth reduction and gain in attachment levels. No significant differences in the detection of putative periodontal pathogens in subgingival plaque existed between smokers and non-smokers. A consistent trend was noted in that smokers had lower sera immunoglobulin G antibody titres to these organisms before and after treatment (statistically significant for A. actinomycetemcomitans). This pattern was less clear when antibody avidities were considered, revealing only small differences, if any, between the two groups of patients. CONCLUSION: Current data indicate that smokers with periodontal disease have a suppressed inflammatory response, a significantly less favourable clinical outcome and seem to have an altered host antibody response to antigenic challenge than non-smokers. In contrast, the subgingival microflora of smokers appears similar to that of non-smokers.

Microbiological culture analysis of the tongue anaerobic microflora in subjects with and without halitosis.

OBJECTIVE: Determination of the microflora present on the tongue dorsum of subjects with and without halitosis using conventional microbiological culture methods. METHODS: Twenty-one halitosis and 20 control patients were recruited using a strict clinical protocol. Samples were collected from the posterior dorsum of the tongue using a sterile brush. Each sample was vortex mixed for 30 s and serial 10-fold dilutions to 10(-7) were carried out. Samples were plated onto fastidious anaerobe agar (FAA) and FAA enriched with vancomycin. These were incubated under anaerobic conditions for 10 days at 37 degrees C. Strict anaerobes were identified by metronidazole sensitivity and bacteria were identified to genus level by a combination of colony morphology, Gram staining and biochemical and enzymatic tests (rapid ID 32 A). RESULTS: The predominant species in test and control groups were Veillonella sp. and Prevotella sp. Greater species diversity was found in the halitosis samples compared with controls. The halitosis samples contained an increased incidence of unidentifiable Gram-negative rods, Gram-positive rods and Gram-negative coccobacilli. CONCLUSIONS: There was no obvious association between halitosis and any specific bacterial genus. The increased species diversity found in halitosis samples suggests that halitosis may be the result of complex interactions between several bacterial species. The role of uncultivable bacteria may also be important in contributing to this process.

Clinical and microbiological effect of scaling and root planing in smoker and non-smoker chronic and aggressive periodontitis patients.

OBJECTIVES: To compare the effects of scaling and root planing (SRP) on clinical and microbiological parameters at selected sites in smoker and non-smoker chronic and generalized aggressive periodontitis patients. MATERIALS AND METHODS: Clinical parameters including probing depth (PD), relative attachment level (RAL), and bleeding upon probing (BOP), and subgingival plaque samples were taken from four sites in 28 chronic periodontitis (CP) and 17 generalized aggressive periodontitis (GAgP) patients before and after SRP. Polymerase chain reaction assays were used to determine the presence of A. actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythensis, Prevotella intermedia and Treponema denticola. RESULTS: Both CP and GAgP non-smokers had significantly greater reduction in pocket depth (1.0+/-1.3 mm in CP smokers versus 1.7+/-1.4 mm in non-smokers, p=0.007 and 1.3+/-1.0 in GAgP smokers versus 2.4+/-1.2 mm in GAgP non-smokers, p<0.001) than respective non-smokers, with a significant decrease in Tannerella forsythensis in CP sites (smokers 25% increase and non-smokers 36.3% decrease, p<0.001) and Prevotella intermedia at GAgP sites (smokers 25% reduction versus 46.9% in non-smokers, p=0.028). CONCLUSION: SRP was effective in reducing clinical parameters in both groups. The inferior improvement in PD following therapy for smokers may reflect the systemic effects of smoking on the host response and the healing process. The lesser reduction in microflora and greater post-therapy prevalence of organisms may reflect the deeper pockets seen in smokers and poorer clearance of the organisms. These detrimental consequences for smokers appear consistent in both aggressive and CP.

A novel species-specific PCR assay for identifying Lactobacillus fermentum.

Lactobacillus fermentum is a Gram-positive bacterium that is associated with active caries lesions. Methods for identifying Lactobacillus species traditionally have been based upon culture methods coupled with biochemical tests, which are generally unreliable. The aim of this study was to develop a species-specific PCR assay for the direct detection of L. fermentum in oral clinical samples. PCR primers specific for L. fermentum were identified by alignment of bacterial 16S rRNA genes and selection of sequences specific for L. fermentum at their 3' ends. PCR positivity for L. fermentum DNA was indicated by amplification of a 337 bp product. The primers were shown to be specific for L. fermentum DNA, since no PCR product was obtained when genomic DNA from a wide range of other oral bacteria, including closely related Lactobacillus species, were used as test species. The PCR assay was then used in an attempt to identify L. fermentum DNA in supragingival plaque samples and in pus aspirates from subjects with acute dento-alveolar abscesses. Four out of 70 (5.7 %) supragingival plaque samples analysed were positive for the presence of L. fermentum DNA while none of the 19 pus samples analysed was positive for L. fermentum DNA. This PCR assay provides a more rapid, specific and sensitive alternative to conventional culture methods for the identification of L. fermentum in clinical specimens.

Quadrant root planing versus same-day full-mouth root planing. II. Microbiological findings.

OBJECTIVES: The aim of this study was to test the hypothesis that over a period of 6 months, same-day full-mouth scaling and root planing (FM-SRP) resulted in greater reductions in the detection frequency of five putative periodontal pathogens compared with quadrant scaling and root planing (Q-SRP) in chronic periodontitis patients. MATERIALS AND METHODS: Forty patients were recruited into this study. Subjects were randomised into two groups. The FM-SRP group received full-mouth scaling and root planing completed within the same day, while the Q-SRP group received quadrant root planing at 2-weekly intervals over four consecutive sessions. Selected-site analyses were performed on the deepest site in each quadrant before and after therapy, at approximately 3 and 6 months from baseline (R1 and R2) and clinical indices were recorded with an electronic pressure-sensitive probe. In addition, subgingival plaque samples were collected from these sites at baseline (BAS), at reassessment 1 (R1), approximately 6 weeks after the completion of therapy and at reassessment 2 (R2), 6 months from baseline. Polymerase chain reaction (PCR) was used to determine the presence of Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Prevotella intermedia, Treponema denticola and Bacteroides forsythus in plaque. RESULTS: Both therapies resulted in significant improvements in all clinical indices both at R1 and R2. A marked reduction in the presence of all candidate periodontal pathogens was noted after both treatment modalities, reaching statistical significance for the majority of the test organisms. These improvements were maintained over a period of 6 months. When the two treatment groups were compared, a significantly higher percentage of Q-SRP patients was positive for P. intermedia at R1 compared with FM-SRP patients (p<0.05). In addition, a greater reduction in the patient prevalence for T. denticola was found for the FM-SRP group than the Q-SRP group at R1 and R2 from baseline (p<0.005), but the significance of this is questionable given the skewed detection frequency of this organism at baseline between the two treatments (p<0.01). CONCLUSION: This study failed to confirm that same-day FM-SRP resulted in greater microbiological improvements compared with Q-SRP at 2-weekly intervals over a 6-month period, as determined by PCR.

Specific PCR detection of Peptostreptococcus magnus.

Peptostreptococcus magnus is the most pathogenic and one of the most common Gram-positive anaerobic cocci found in human clinical specimens. The organism has been isolated in pure culture from a range of serious infections, including meningitis and endocarditis. However, isolation of Peptostreptococcus magnus from the oral cavity has rarely been attempted. Identification of Peptostreptococcus magnus in clinical specimens is reliant upon microbiological culture and biochemical methods, which often give ambiguous results. The aim of this study was to develop a PCR assay for the specific detection of Peptostreptococcus magnus in oral clinical specimens. PCR primers specific for Peptostreptococcus magnus DNA were derived by comparison of 16S rRNA gene sequences and selection of primers that demonstrated specificity at their 3' ends for Peptostreptococcus magnus. PCR positivity for Peptostreptococcus magnus DNA was indicated by the amplification of a 553 bp product. The PCR assay was then used to attempt detection of Peptostreptococcus magnus DNA in subgingival plaque samples from adult periodontitis patients and pus aspirates from subjects with acute dento-alveolar abscesses. The PCR assay was demonstrated to be highly specific for Peptostreptococcus magnus DNA, since no PCR products were obtained when genomic DNA from a wide range of other oral bacteria, including closely related Peptostreptococcus species, was used in the PCR assay. Confirmation of specific amplification of Peptostreptococcus magnus DNA was obtained by digestion of PCR products with the restriction endonuclease RsaI, which gives a unique restriction profile for Peptostreptococcus magnus. Of the 33 subgingival plaque samples analysed, 2 (6 %) were positive for Peptostreptococcus magnus DNA. None of the 60 pus aspirates analysed was positive for Peptostreptococcus magnus DNA. It is concluded that Peptostreptococcus magnus is not a major pathogen in adult periodontitis or dento-alveolar abscesses. The PCR assay provides a more rapid, specific and sensitive alternative to conventional methods for identification of Peptostreptococcus magnus in clinical specimens.

Development of a PCR assay specific for Peptostreptococcus anaerobius.

Peptostreptococcus anaerobius is a gram-positive anaerobic coccus that is widely distributed in the normal human flora. The organism has also been implicated as a causative agent of several systemic infections, including endocarditis and infections of the genitourinary and gastrointestinal tracts. Its role in oral disease is less well defined, although it has been implicated in periodontal disease, gingivitis and root canal infections. Identification of P. anaerobius in clinical samples is currently reliant upon traditional culture and biochemical methods. The aim of this study was to develop a novel PCR assay for the detection of P. anaerobius and to attempt detection of this organism in oral samples. PCR primers specific for P. anaerobius DNA were developed by alignment of bacterial 16S ribosomal RNA gene sequences and selection of sequences specific at their 3' ends for P. anaerobius. When used in a PCR assay, positivity for P. anaerobius DNA was indicated by the amplification of a 943-bp product. The primers were shown to be specific for P. anaerobius DNA, as no PCR products were obtained when genomic DNA from a wide range of other Peptostreptococcus species and other oral bacteria were used as templates. The PCR assay was then applied to the detection of P. anaerobius DNA in subgingival plaque samples from adult periodontitis patients and pus aspirates from subjects with acute dento-alveolar abscesses. All of 60 subgingival plaque samples from 16 patients were negative for P. anaerobius DNA. None of the 43 pus samples analysed contained P. anaerobius DNA. These results suggest that P. anaerobius is not a major pathogen in adult periodontitis and dento-alveolar abscesses. The PCR assay is a more rapid, sensitive and specific alternative to culture-based methods for identification of P. anaerobius in clinical samples.

Molecular identification of microorganisms from endodontic infections.

A relatively wide range of bacteria have been isolated from root canals using standard culture techniques. However, only 50% of the bacteria in the oral cavity are cultivable (S. S. Socransky et al., Arch. Oral Biol. 8:278-280, 1963); hence, bacterial diversity in endodontic infections is underestimated. This study used a PCR-based 16S rRNA gene assay, followed by cloning and sequencing of 16S rRNA amplicons from a small subset of samples to assess the diversity of bacteria present in infected root canals. A total of 41 clinical samples from 15 de novo and 26 refractory cases of endodontic infections were assessed. Of these samples, 44% were positive by culture and 68% were positive by PCR. Eight samples were selected for further analysis. Of these, the two de novo cases yielded sequences related to those of the genera Enterococcus, Lactobacillus, Propionibacterium, and Streptococcus and two clones were related to previously uncultivated bacteria, while the sinus-associated, de novo case yielded sequences related to those of the genera Lactobacillus, Pantoea, Prevotella, and Selenomonas. The five refractory cases produced clones which were related to the genera Capnocytophaga, Cytophaga, Dialister, Eubacterium, Fusobacterium, Gemella, Mogibacterium, Peptostreptococcus, Prevotella, Propionibacterium, Selenomonas, Solobacterium, Streptococcus, and Veillonella and two clones representing previously uncultivated bacteria. The phylogenetic positions of several clones associated with the Clostridiaceae and Sporomusa subgroups of the Firmicutes grouping are also shown. This study demonstrates that molecular techniques can detect the presence of bacteria in endodontic infections when culture techniques yield a negative result and can be used to identify a wider range of endodontic-infection-related bacteria including the presence of previously unidentified or unculturable bacteria.

Failure to detect an association with IL1 genotypes in European Caucasians with generalised early onset periodontitis.

OBJECTIVES: In order to elucidate the genetic background to EOP it is useful to investigate associations with genetic polymorphisms of immune response genes, whose products play a r le in the inflammatory process. The aim of this study was to examine IL1A and IL1B genetic polymorphisms in unrelated European white Caucasian patients with generalised early-onset periodontitis (GEOP). MATERIAL AND METHODS: Polymerase chain reaction (PCR) amplification of the ILIA (-889) gene and IL1B (+3953) gene (56 patients, 56 controls) was carried out and PCR products subjected to restriction fragment length polymorphism (RFLP) analysis. RESULTS: There were no significant differences between patients and controls for any of the genotype or allele frequencies investigated (p = 1.0). Smoking status was also included as a covariate but this did not alter the results. Furthermore, expression of the composite genotype described by Kornman and coworkers was also investigated in these subjects. No significant differences were found between patients and controls whether smoking was included as a covariate or not. CONCLUSION: The lack of any association between the IL1 polymorphisms and GEOP, in the population presented here, brings into doubt the usefulness of these candidate genes as markers of susceptibility to this form of periodontitis.

Detection of peptostreptococcus micros DNA in clinical samples by PCR.

Peptostreptococcus micros is a gram-positive anaerobic coccus which, although considered to be a natural commensal of the human oral cavity, is associated with periodontal, endodontal and peritonsillar infections. Identification of the organism has to date relied upon conventional culture methods and biochemical analyses. The purpose of this study was to develop a PCR method for rapid and specific identification of this organism in clinical samples. A pair of primers was selected, each of which was specific at the 3' end for P. micros DNA; they were used in the PCR assay, resulting in a 1074-bp product. The primers were shown to be specific for P. micros DNA as no PCR products were obtained when genomic DNA extracts from a wide range of other Peptostreptococcus species and other oral bacteria were used as templates. The PCR assay was then applied to the identification of P. micros DNA in subgingival plaque samples from adult periodontitis patients and pus samples from subjects with acute dento-alveolar abscesses. Confirmation of specific amplification of P. micros DNA was obtained by digestion of PCR products with the restriction endonuclease RsaI, which gives a unique restriction profile for P. micros, and DNA sequencing. Sixty-eight subgingival plaque samples from 18 patients were analysed, of which 19 (28%) were positive for P. micros DNA; the proportion of patients carrying P. micros DNA in at least one sampled site was 11 (61%) of 18. Twenty (71%) of 28 pus samples analysed by PCR contained P. micros DNA. These results confirm that P. micros may be involved in the aetiology of acute dentoalveolar abscesses and adult periodontitis. The PCR assay provides a more rapid and reliable alternative to conventional methods for identification of P. micros in clinical samples.

Detection of Helicobacter pylori DNA in recurrent aphthous stomatitis tissue by PCR.

Helicobacter pylori is recognised as being an aetiological agent of chronic active gastritis and peptic ulcer disease and has been associated with an increased risk of gastric cancer. The natural reservoir for H. pylori is unknown, although the oral cavity has been the focus of much attention in this respect. Given the histological similarities between gastric and oral ulceration, it seemed prudent to investigate a possible association between H. pylori and recurrent aphthous stomatitis (RAS). In this study, the potential involvement of H. pylori in the aetiology of RAS was investigated using the polymerase chain reaction (PCR). Biopsies from 28 RAS patients were analysed, in addition to 20 oral lichen planus (OLP) and 13 normal biopsies that were used as controls. Genomic DNA was extracted from biopsies, and confirmation of successful extraction of PCR-amplifiable DNA was achieved by carrying out PCR on each DNA sample with nested primers specific for the human beta-haemoglobin gene. PCR identification of H. pylori was carried out using a primer pair specific for the H. pylori 16S ribosomal RNA (rRNA) gene. Two rounds of PCR were carried out to amplify a 295-bp product, and the identity of amplified products was confirmed by DNA sequencing. H. pylori DNA was detected in 3 of 28 (11%) RAS samples but not in any of 20 OLP and 13 normal samples. These results do not support a definitive aetiological role for H. pylori in RAS, although the possibility that H. pylori may be involved in a small proportion of RAS cases cannot be excluded.

Microbial comparison of smoker and non-smoker adult and early-onset periodontitis patients by polymerase chain reaction.

A number of bacterial species are involved in the aetiology of periodontitis and include Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Bacteroides forsythus and Treponema denticola. Several studies have shown differences in the microflora between the various forms of periodontal disease. It is recognised that smoking is a risk factor for periodontal disease, but there are conflicting reports on whether or not smoking has an effect on the periodontal microflora. We utilised the polymerase chain reaction to determine the presence of A. actinomycetemcomitans, P. gingivalis, P. intermedia, B. forsythus and T. denticola in subgingival plaque samples in 33 adult periodontitis (AP) patients and 24 generalized early-onset periodontitis (GEOP) patients prior to treatment. When GEOP and AP patients were compared there were significant differences in the number of positive patients and sites for both A. actinomycetemcomitans and B. forsythus (p=0.0023 and 0.00001, respectively). No statistically significant differences in the prevalence of these organisms were found between smoker and non-smoker groups. These results confirm that AP and GEOP sites harbour varied microflora, but show that B. forsythus and A. actinomycetemcomitans were detected to a significantly greater extent in this group of GEOP than in the AP patients investigated. Our findings do not support the hypothesis that smokers have significant differences in the prevalence of periodontal pathogens from non-smokers.

Lack of association between Streptococcus oralis and recurrent aphthous stomatitis.

In the present study, the potential involvement of Streptococcus oralis in the aetiology of recurrent aphthous stomatitis (RAS) was investigated using the polymerase chain reaction (PCR). Biopsies from 28 RAS patients were analysed, in addition to 20 oral lichen planus (OLP) and 13 normal biopsies that were used as controls. PCR was carried out using a primer pair that targets the D-alanine:D-alanine ligase gene and detects DNA from both S. oralis and the closely related species Streptococcus mitis. Discrimination between these two species was achieved by digestion of PCR products with the restriction endonucleases HaeIII and HindIII, which both give distinct restriction profiles for each species. S. oralis DNA was detected in 8 of 28 (29%) RAS samples, 10 of 20 (50%) OLP samples and 6 of 13 (46%) normal samples. These results suggest that S. oralis is not of primary aetiological significance in RAS.