Analysis of site specific periodontal bacteria sampling schemes.

A great deal of controversy has existed in the periodontal literature as to whether the site or the subject should be the unit of analysis. Using the site as the unit of analysis assumes that observations of sites within the same subject are independent and ignores between subject variation. The purpose of this report is to evaluate the influence that the unit of analysis has on estimating the number of necessary site specific bacterial samples from each subject. The number of bacterial samples per subject was defined as the number of samples that would insure a clinician at a 95% confidence level that, if the bacteria were present in a subject, it would be discovered. From two data sets in which 20 to 30 bacterial samples were taken from each subject and data generated from a simulation, appropriate within-subject sample size was determined. In one data set the presence of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Bacteroides forsythus, and Prevotella intermedia was determined by indirect immunofluorescence. In the other data set the presence of A. actinomycetemcomitans, P. gingivalis, and P. intermedia was determined using DNA probes. Results of this study demonstrate that there is a large between subject variation in site specific bacterial prevalence, as indicated by an elevated intraclass correlation. Simulated data in this report demonstrated that the number of necessary bacterial samples per subject increased with increasing values of intraclass correlation. The number of necessary within subject samples also increased with decreasing prevalence rate. For A. actinomycetemcomitans, which had a low prevalence rate (0.11 to 0.18), the number of necessary samples per subject was very high (31 to 35).(ABSTRACT TRUNCATED AT 250 WORDS)

Site selection criteria for microbiological testing of periodontal microorganisms.

MICROBIOLOGICAL TESTING IS BECOMING an adjunct to the diagnosis and monitoring of periodontal patients. However, choosing which sites and how many sites among the many available in most patients is difficult. A study of 22 periodontitis patients was undertaken to attempt to provide some guidelines to these issues. All mesiobuccal sites along with sites with 4 mm or greater probing depth were sampled with endodontic points and analyzed by DNA probes for P. gingivalis, P. intermedia, and A. actinomycetemcomitans. The data suggest that sites with the deepest probing depths and sites that bleed on probing were most likely to harbor these pathogenic species. Using these clinical criteria, an approximation of the number of sites required for sampling was suggested.

Distribution of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis by subject age.

The possible associations between periodontitis subject age and the distribution of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis were examined using an extensive data bank of subgingival plaque specimens analyzed using DNA probes. The results suggest that A. actinomycetemcomitans is strongly related to subjects in the youngest age group (10 to 19 years) with decreasing prevalence and concentration levels in older age groups. In contrast, P. gingivalis showed a reverse relationship to subject age with highest prevalence and concentration levels in older subjects (30 years and older) compared to subjects in younger age groups. Statistical testing of these relationships were highly significant. (P less than 0.0001).

DNA probe detection of Eikenella corrodens, Wolinella recta and Fusobacterium nucleatum in subgingival plaque.

This cross-sectional study used species-specific DNA probes to examine subgingival plaque specimens for the presence of Eikenella corrodens, Wolinella recta, and Fusobacterium nucleatum in adults with untreated periodontitis or gingivitis and in healthy controls. W. recta and F. nucleatum were more prevalent in diseased sites from the periodontitis group when compared with the controls (81% vs 22% and 83% vs 20% respectively). E. corrodens was detected in 62% of the control sites and 81% of the periodontitis sites. Because the control sites commonly contained this organism, E. corrodens may not be useful in differentiating between health and disease. In addition, the relationship between the prevalence of W. recta and F. nucleatum and the prevalence of the established periodontal pathogens, Actinobacillus actinomycetemcomitans, Bacteroides intermedius and Bacteroides gingivalis, was examined. Positive detection of W. recta and F. nucleatum correlated closely with the presence of A. actinomycetemcomitans, B. intermedius and B. gingivalis. Therefore, W. recta and F. nucleatum do not appear to be unique indicators of periodontal disease.

DNA probes in the diagnosis of periodontal microorganisms.

The need for a rapid and sensitive microbiological assay has become necessary for both research and clinical diagnostic purposes. This need has become clear as a result of extensive documentation linking specific bacterial species and periodontal destruction. DNA probe technology provides both a sensitive and specific assay and alleviates the concern for transport of fastidious microorganisms. The DNA probe procedure includes (1) disruption of bacterial cells with denaturation of DNA, (2) immobilization of DNA onto a nitrocellulose filter, (3) blocking unbound nitrocellulose with non-specific DNA, (4) hybridization of the filter with 32P-labelled probe, (5) washing and detection of bound probe. At our laboratory, microbiological analysis with whole-genomic and cloned DNA probes has been used on thousands of plaque specimens in several large-scale research projects. In one study, levels (greater than or equal to 10(5)) of Actinobacillus actinomycetemcomitans were significantly higher in subjects under 21 yr than in subjects with adult periodontitis (over 21 yr old). In another study, the distribution of periodontal pathogens throughout the mouth was examined. High levels were selectively found at sites with probing depths of 5 mm or more and bleeding on probing, directing specimen collection to these sites. In contrast, random selection of sites for sampling was found to be a poor method for detecting high levels of pathogens. These data suggest appropriate sites for specimen collection for further research and diagnostic purposes.

Comparison of serological and DNA probe analyses for detection of suspected periodontal pathogens in subgingival plaque samples.

Several methods are currently being used to identify specific bacteria in dental plaque, namely direct culture, serological techniques and DNA probes. Culture methods are labour-intensive, dependent on the viability of the cells, and require fastidious growth conditions. Serological and DNA probes allow rapid strain-specific identification of periodontal pathogens with limited effort. The purpose of the present study was to compare results from serological and DNA probes in assessing and quantitating the presence of three suspected periodontal pathogens in subgingival plaque: Actinobacillus actinomycetemcomitans, Bacteroides gingivalis and Bact. intermedius. Subgingival bacterial samples were obtained from 4 periodontal sites of each of 13 patients with untreated moderate to advanced adult periodontitis. Samples were taken using 2 paper points that were simultaneously inserted for 10 s to the bottom of the periodontal pockets. The plaque from one paper point was analysed by immunofluorescence using monoclonal antibodies. The other sample was analysed by species-specific cloned DNA probes. The number of bacteria per millilitre was calculated for both methods, and used for comparisons of results obtained with the two techniques. Results from indirect immunofluorescence and DNA hybridization analyses were negative for A. actinomycetemcomitans across all samples. Fluorescence did not detect bacteria at levels lower than 10(4), while the DNA probes identified organisms at levels of 10(3). Similar numbers of samples positive for Bact. gingivalis were obtained with either method (p = 0.227), and the results were not independent. A significantly greater proportion of Bact. intermedius-positive samples was detected by immunofluorescence (p = 0.0039), and the results of immunofluorescence and DNA hybridization were independent.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA probe detection of key periodontal pathogens in juveniles.

Recognition of juvenile forms of periodontitis have been shown to be directly linked with specific Gram-negative rods, primarily Actinobacillus actinomycetemcomitans, Bacteroides gingivalis and Bacteroides intermedius. However, clinical application of these laboratory findings have generally been restricted to the research environment. The purpose of this study was to explore the relationship of these three pathogenic species in children using the reliable and accurate new technology of DNA probes. Results indicated that the pathogenic oral flora did not differ significantly between subjects with and without gingival inflammation. Therefore, tracking of pathogens using clinical parameters of inflammation are unreliable and accurate monitoring requires microbiological testing in order to properly identify the presence or absence of pathogens.

Comparison of cultural methods and DNA probe analyses for the detection of Actinobacillus actinomycetemcomitans, Bacteroides gingivalis, and Bacteroides intermedius in subgingival plaque samples.

The purpose of this study was to compare DNA probe analyses to cultural methods for detecting three periodontal pathogens, Actinobacillus actinomycetemcomitans, Bacteroides gingivalis, and Bacteroides intermedius, in human subgingival plaque. Subgingival sites from patients diagnosed as either healthy or showing evidence of gingivitis or juvenile or adult periodontitis were sampled using two paper points. The number of these pathogens from one paper point was determined using microbiologic media and speciated by biochemical tests. Results were then compared to bacterial numbers obtained from the other paper point using species-specific DNA probes. In 60 samples from the disease group, DNA probe analysis demonstrated 100% effectiveness in detecting A. actinomycetemcomitans and B. intermedius and 91% effectiveness in detecting B. gingivalis at culture positive levels (greater than or equal to 10(3) cells). In addition, probe assays frequently identified these pathogens in samples that were culture negative. Probe analysis revealed a better correlation between presence of a pathogen and clinical evidence of disease on an individual patient basis. In contrast, most samples taken from sites of healthy individuals showed undetectable levels of all three pathogens as determined by both techniques. These results suggest that DNA probe technology is at least equivalent and often superior to cultural methods for detecting A. actinomycetemcomitans, B. gingivalis, and B. intermedius in human subgingival plaque samples.

A cross-reactivity study of whole genomic DNA probes for Haemophilus actinomycetemcomitans, Bacteroides intermedius, and Bacteroides gingivalis.

In this study, we evaluated the sensitivity and specificity of whole genomic DNA probes for the periodontal pathogens Haemophilus actinomycetemcomitans, Bacteroides intermedius, and Bacteroides gingivalis. By means of these probes, DNA hybridizations were performed against other organisms found in the oral cavity and organisms previously determined to be genetically similar. All three probes were sensitive to 10(3) cells for their respective organism. The H. actinomycetemcomitans probe cross-reacted with several haemophilus strains, Wolinella, and Campylobacter, indicating that H. actinomycetemcomitans-specific sequences would have to be identified and cloned for accurate detection of this organism in heterogeneous patient samples. Only very low levels of cross-reactivity were observed between the B. intermedius probe and representative black-pigmented Bacteroides. This low level of cross-reactivity did not interfere with the accurate identification of B. intermedius in sample evaluations. The B. gingivalis probe showed no cross-reactivity. Whole genomic probes will be used for the detection of B. intermedius and B. gingivalis in patient samples.

Evaluation in reconstructive implantology.

Acceptance of implants by dentist, readiness for use, and general indications are described. Individual evaluation for implants is detailed and success of implants defined. Indications for removal are cited and appropriate statistic methodology presented.

The effect of treatment on Actinobacillus actinomycetemcomitans in localized juvenile periodontitis.

Three treatment regimens including local tetracycline delivery, systemic doxycycline and surgery plus systemic doxycycline were investigated in a localized juvenile periodontitis (LJP) population. Of the investigated treatments only surgery plus systemic doxycycline for 14 days was effective in eliminating or suppressing Actinobacillus actinomycetemcomitans, an organism strongly associated with LJP lesions. While surgery plus antibiotics was the superior treatment, it appears that the possibility of reinfection or incomplete elimination of the organism exists. Careful long-term follow-up, including clinical and microbiological monitoring, is highly recommended in this periodontal population.

Capnocytophaga: new genus of gram-negative gliding bacteria. III. Physiological characterization.

Sixty-eight strains of capnophilic fusiform Gram-negative rods from the human oral cavity were subjected to extensive physiologic characterization, tested for susceptibility to various antibiotics, and the mol-percent guanine plus cytosine of each isolate determined. The characteristics of the isolates were compared with 10 fresh and 2 stock isolates of Fusobacterium nucleatum. The isolates clearly differed from the Fusobacterium species on the basis of mol-percent guanine plus cytosine, end products, growth in a capnophilic environment and fermentation of carbohydrates. All of the gliding isolates required CO2 and formed acetate and succinate, but not H2S, indole or acetylmethylcarbinol. All fermented glucose, sucrose, maltose and mannose. The organisms may be differentiated on the basis of fermentation of additional carbohydrates, hydrolysis of polymers and reduction nitrate. Three species are proposed: Capnocytophaga ochracea, Capnocytophaga sputigena and Capnocytophaga gingivalis. Ten isolates did not fit into the proposed species.