Performance of a commercial immunoassay for detection and differentiation of periodontal marker bacteria: analysis of immunochemical performance with clinical samples.

BACKGROUND: We employed a commercial immunoassay for simultaneous detection and differentiation of marker bacteria Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia and reassessed the immunochemical performance of the assay. METHODS: We compared the analytical performance of the immunoassay in our study of clinical samples from 249 periodontal patients in 2 private periodontal practices with the previously reported analytical performance of the same immunoassay. We also compared immunoassay measurements of the marker bacteria in clinical samples with values obtained in other studies by direct culture of the same organisms. RESULTS: The assay produced 3 times more high-end readings than reported previously. We also reassessed and revised previously published calibration curves for the immunoassay. The immunoassay provided measurements of the marker bacteria in clinical samples from our patients that were comparable to and consistent with measurements of the same bacteria by direct culture in other studies. CONCLUSIONS: We ascribe the increased sensitivity of the immunoassay in our study to: 1) a more standardized and vigorous sample dispersion that improves release of particulate and soluble antigens from dental plaque biofilm, and 2) better visualization of the reaction product of the enzyme-linked immunoassay. High-technology assays, such as diagnostic immunoassays, have a significant potential for future development in dental diagnosis, because they simplify detection and measurement of biologically important markers such as specific bacteria in clinical samples. Commercial assays also have an important potential for standardization of clinical measurements of biological markers.

Effects of smoking and treatment status on periodontal bacteria: evidence that smoking influences control of periodontal bacteria at the mucosal surface of the gingival crevice.

BACKGROUND: We examined whether smoking status could influence growth of potentially pathogenic bacteria in the periodontal environment of treated and untreated periodontal patients. METHODS: We have previously reported effects of treatment status on marker bacteria in our patients. We established a history of any smoking during 6 months prior to microbiological sampling (F-ME, 16 smokers out of 64; MHM, 70 smokers out of 185). We used a commercial immunoassay to quantitate Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans in paper point samples from periodontal sites. RESULTS: Logistic regression showed that in smokers, neither P. gingivalis nor A. actinomycetemcomitans was quantitatively increased, while P intermedia was somewhat increased. Multiple regression demonstrated that smoking disrupts the positive relationship between increasing probing depth and increasing bacterial growth that is found in non-smokers. In smokers, growth of marker bacteria at shallow sites (< or =5 mm) was significantly increased to the levels found at deeper sites (>5 mm) in both smokers and non-smokers. Supragingival plaque biofilm was identified as a reservoir for marker bacteria; smokers and nonsmokers had equal ranges of oral cleanliness. CONCLUSIONS: Smoking-associated periodontitis is not simply a reflection of oral cleanliness. Smoking extends a favorable habitat for bacteria such as P. gingivalis, P. intermedia, and A. actinomycetemcomitans to shallow sites (< or =5 mm). Molecular byproducts of smoking interfere with mechanisms that normally contain growth of damaging bacteria at the surface of the oral mucosa in gingival crevices. In this way, smoking can promote early development of periodontal lesions.

Antimicrobial resistance: dentistry's role.

The resistance of bacteria, fungi and viruses to antimicrobials is increasing rapidly, with deleterious consequences. Dentistry's role in this development is unclear, because the necessary information has not yet been collected. Nevertheless, dentists should recognize that it is essential to use antimicrobials in an appropriate and responsible manner, both to treat infection effectively, and to minimize the likelihood that the bacteria in the general population will develop resistance to antimicrobials. The purpose of this article is to make dentists aware of the concerns raised by antimicrobial resistance, and how it can be avoided.

Diagnostic utility of specific microbiological markers for periodontal diseases.

Specific detection of marker organisms Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans with an immunoassay provided 2 types of useful information directly into private clinical practice: 1) persistence of P. gingivalis in patients undergoing regular treatment allowed rapid identification of pockets requiring further treatment without waiting for measurable progression of lesions and 2) presence of A. actinomycetemcomitans in adults at any stage of diagnosis or treatment identified patients who may prove to have difficult-to-manage periodontitis. We made these findings in 253 patients (234 in specialist periodontal practices [F-ME 55; MHM 179] and 19 in general dental practice [EWM]). The search for useful diagnostic markers overlaps only partly with the search for periodontal pathogens. The P. gingivalis marker and the A. actinomycetemcomitans marker identify 2 different patterns of infection that appear to reflect 2 different underlying problems. Demonstration of pocket-dependent infection with P. gingivalis in treated patients provides an outcome marker for sites not converting to marker-negative sites at detection levels of the immunoassay. This information facilitates selection of sites and patients requiring adjustment of treatment regimens. Detection of A. actinomycetemcomitans in adult patients is significantly associated with periodontitis characterized as refractory. Positive identification of A. actinomycetemcomitans with the immunoassay supports clinical decision-making by drawing attention to adult patients who require closer monitoring and intensive persistent treatment. Successful application of immunoassay detection of microbiological markers is based on continuous patient monitoring to support clinical decisions; it does not replace careful clinical judgment.

Periodontitis-associated marker bacteria in an urban North American patient population: application of a commercial immunoassay.

We used an immunoassay to demonstrate marker organisms (Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans) in 3 private practice populations (F-ME periodontist, 55 patients; MHM periodontist, 179 patients; and EWM general dentist, 19 patients). Occurrence of the marker organisms involves the whole oral environment, not just individual sites, as shown by close correlation between presence of the marker organisms in 2 independent sites/samples within a single mouth. Presence of the marker P. gingivalis (and P. intermedia) relates closely to periodontal pocketing while presence of A. actinomycetemcomitans does not have this pocket-associated characteristic. There was no significant relationship between presence of the marker organisms and the number of teeth in a mouth, and in the periodontal practice patients there was no significant effect of gender on occurrence of the marker organisms. A. actinomycetemcomitans and the other 2 markers were found over the entire age range (12 to 75) of our patients. Regular periodontal treatment reduced occurrence of all marker organisms and increased the frequency of marker-negative patients and sites. Occurrence of the marker organisms above immunoassay threshold levels appears to represent how receptive a patient is to each individual organism. Most patients appear receptive to the presence of P. intermedia whether treated or not. Significantly fewer patients who underwent regular treatment show the presence of P. gingivalis or A. actinomycetemcomitans when compared to untreated patients. Diagnostic application of microbial markers requires ongoing clinical assessment of patients and careful clinical judgment. 1391.

The pH of gingival crevices and periodontal pockets in children, teenagers and adults.

Gingival crevice and periodontal pocket pH, measured directly with glass micro-electrodes, was near neutral at most sites in most individuals (mean pH 6.92 +/- 0.03 SEM, 69 subjects). Periodontal state ranged from healthy to periodontitis but neither clinical evidence of gingivitis at a site nor pocket depth were associated with crevicular pH different from that at healthy sites. This finding contradicts earlier reports that gingivitis is associated with a crevicular pH as alkaline as pH 9.06. Metallic antimony electrodes as used by earlier investigators were found to give pH readings that were too high by as much as 1.5 pH units in the presence of organic reducing agents of the type produced by oral bacteria within gingival crevices. In contrast, glass micro-electrodes respond only to hydrogen ions and thereby provided accurate measurements of pH even in the presence of organic reducing agents. Loss of CO2 to the atmosphere from biological fluids that are bicarbonate buffered resulted in a shift to alkaline pH by as much as 1 pH unit. As a result, only measurements taken within gingival crevices or periodontal pockets can provide accurate measurements of crevice or pocket pH.

Measurement of neutral sugars in glycoproteins as dansyl derivatives by automated high-performance liquid chromatography.

Automated high-performance liquid chromatography was used to analyse dansylhydrazine derivatives of neutral sugars in unfractionated acid hydrolysates of four well-characterized glycoproteins: fetuin, ovalbumin, alpha-1-acid glycoprotein and bovine submaxillary mucin. After a simple single-step derivatization at 65 degrees C the sugar derivatives in protein hydrolysates chromatographed as single peaks on reversed-phase C18 columns. The isocratic solvent consisted of 20% (v/v) aqueous acetonitrile containing 0.01 M formic acid, 0.04 M acetic acid and 0.001 M triethylamine. The triethylamine significantly increases the sugar peak height at 254 nm. Repeated automatic sample injection without deterioration of column performance or interference from dansyl hydrazine is not possible with published methods, but was achieved by cleaning the column between each analysis with a solvent of 20% (v/v) acetonitrile and 80% (v/v) methanol. Hydrolysis with 2 M trifluoroacetic acid is superior to 2 M hydrochloric acid for both sugar recovery and convenience but must continue for 6-8 h at 105 degrees C to ensure complete sugar release. We confirmed that mannose is present in most preparations of human high-molecular-weight salivary glycoproteins, and also examined purified bovine skin proteodermatan sulphate. p-Nitrophenylhydrazine derivatives of neutral sugars are readily produced, but do not chromatograph as successfully as the dansyl derivatives while phenylhydrazine derivatives are not easily produced at 65 degrees C. Further development of the method should be possible by producing other hydrazine derivatives of neutral sugars.

Reaction of human colostral and early milk antibodies with oral streptococci.

Colostrum or early breast milk or both from each of 16 healthy women contained agglutinating antibodies for all normal streptococcal inhabitants of the human oral cavity (S. mutans, S. sanguis, S. mitis, and S. salivarius), including those which colonize the neonatal oral cavity in significant numbers. Agglutination correlated with the amount of immunoglobulin A (IgA) binding to bacterial surfaces as measured by mixed reverse passive antiglobulin hemagglutination. Surprisingly, colostral IgA agglutinated our control organism, Brucella abortus. Low levels of colostral or milk IgM and IgG antibodies also reacted with all of the test bacteria. Absorption studies with an enzyme-linked immunosorbent assay showed that a proportion of antibodies in colostrum and early milk is specific for each of the different oral streptococci. Fractionation on Sepharose 4B indicated that 11S secretory IgA is the predominant form of colostral and milk antibody for all of the test bacteria, including B. abortus. No evidence was found that reactions other than antigen-antibody reactions resulted in binding of colostral immunoglobulins by any of the test bacteria.

Staining of demineralized cartilage. I. Alcoholic versus aqueous demineralization at neutral and acidic pH.

Demineralization of cartilage with alcoholic EDTA provides cartilage staining that is no better, as measured by scanning microdensitometry, than that of adequately fixed specimens demineralized with aqueous EDTA. Aqueous EDTA is a faster demineralizing agent than alcoholic EDTA. Certain fixatives can preserve maximal proteoglycan staining in articular cartilage even with subsequent rapid demineralization in formate buffer at pH 3.3. Although alcoholic formalin fixation provided optimum quantitative cartilage staining, cetylpyridinium chloride (CPC) in aqueous buffered formalin improved cellular detail, but CPC partially suppressed matrix staining.

Staining of demineralized cartilage. II. Quantitation of articular cartilage proteoglycan after fixation and rapid demineralization.

Safranin O in the orthochromatic form stains articular cartilage proteoglycan quantitatively in histological sections of demineralized cartilage. This was shown by scanning microdensitometry of stained sections of undemineralized and demineralized articular cartilage and by biochemical analysis of 35S labelled cartilage subjected to demineralization. In contrast, Alcian Blue staining is affected by unknown factors other than simply the amount of proteoglycan present. Alcoholic formalin fixes articular cartilage proteoglycan more successfully than formol Zenker for subsequent rapid demineralization. Alcoholic formalin does not preserve cellular appearance as well as formol Zenker. Staining of articular cartilage with PAS appears unaffected by demineralization.

The nature of secretory agglutinins and aggregating factors. I. Secretory conglutinin-like factor, secretory bacterial aggregating factors and secretory IgA antibody in human saliva and amniotic fluid.

Secretory conglutinin-like factor (SKF), secretory bacterial aggregating factors (SBAF) and 11S secretory IgA antibodies of human saliva and full term amniotic fluid were quantitated by microtitration and partially characterized. The SBAF of both saliva and amniotic fluid aggregated a variety of oral streptococci, but no secretory IgA antibodies were found in amniotic fluid. The SKF and SBAF are distinguished from 11S IgA antibodies by being inhibited with EDTA and by being more susceptible to inhibition with reducing agents. These active factors are also distinguished from submaxillary mucins. Components of normal serum inhibit both SKF and SBAF, but blood-group reactive sugars cannot block either SKF or SBAF.

The nature of secretory agglutinins and aggregating factors. III. Secretory conglutinin-like factor SKF detects a cross-reaction between bacteria and complement component C3.

Secretory conglutinin-like factor (SKF) reacts directly with an unknown surface component of some, but not all, oral gram-positive organisms. The absorption of SKF by bacteria is EDTA-sensitive and cannot be blocked with immunoglobulins. High levels of SKF in EDTA extracts of washed salivary sediment reveal the direct in vivo reaction of SKF with oral bacteria. Mixed aggregation with alexinated erythrocytes showed the SKF corresponds to the secretory bacterial aggregating factor (SBAF) for Streptococcus mutans serotype c and also that for Streptococcus mitis. These reactions represent a cross-reaction between bacteria and complement component C3. SKF/SBAF non-mucin glycoproteins and immunoglobulins possess receptors for bacterial components while mucins are passive carriers of blood group determinants.

Stable acid phosphatase: I. Demonstration and distribution.

After a pH-dependent reactivation a highly stable form of acid phosphatase (SAPhase) could be demonstrated in active cells of the macrophage/giant cell/osteoclast series and also in epiphyseal chondrocytes, in cells lining bone undergoing resorption and in hamster eosinophils. Because acid phosphatases of epithelial cells in rat, hamster and Macaca sp. tissues did not possess this stability, SAPhase served as a useful cell marker for the above mesenchymal cell types in paraffin and glycol-methacylate sections even after rapid demineralization in acidic buffers. Conformational alterations appear to occur in the enzyme during formaldehyde fixation, embedding, and reactivation. The granular staining of SAPhase and the successful use of a non-aqueous fixative suggest an association of SAPhase with lysosomes and their membranes. Cells of mesencymal origin that are actively engaged in intra- and/or extracellular digestion contain high levels of SAPhase. The distribution and properties of SAPhase indicate an interrelationship between mononuclear and mutinuclear cell types actively engaged in such digestive processes.

Stable acid phosphatase: II. Effects of pH and inhibitors.

Microdensitometry demonstrated that stable acid phosphatase (SAPhase) in rat and hamster osteoclasts, chondroclasts, and chondrocytes has very similar properties. The differences that were observed suggest that conformational alterations in the enzymes may be responsible for inhibition by some agents such as tartrate. These differences in response to inhibitors depend on the method of embedding as well as on species differences. SAPhase appears to correspond to acid nitrophenyl posphatase, as shown by its pH dependent re-activation, resistance to fluoride inhibition at near-neutral pH, and the inverse effect of pH on inhibition by zinc versus aluminium ions. That proportion of SAPhase resistant to fluoride is an acid phosphatase with activity at near-neutral pH rather than a strict neutral phosphatase. The difference between fluoride sensitive and fluoride resistant SAPhase may relate to the varying association of a single enzyme with cell or lysosomal membranes. The close similarity of acid and neutral SAPhase suggests that both may represent a single enzyme in two forms rather than two distinct enzymes.

The gingival epithelium of rodent molars with limited eruption.

We have re-examined the arrangement of epithelia surrounding molar teeth with limited eruption in the rat, mouse and hamster. Our methods permitted enamel to be retained in paraffin and glycolmethacrylate sections, so providing optimal preservation of epithelial relationships. Three basic patterns of epithelial arrangement were observed on the various aspects of molars. Non-keratinized junctional epithelium covers the interdental septum and the base of all gingival crevices even in areas of gingival recession. In all areas other than the interdental septum, a fold of keratinized gingival epithelium lies beneath the lateral aspects of junctional epithelium. The buccal and lingual aspects of interdental septa show a zone of transition between these two basic types of epithelial arrangement. In intact gingiva the junctional epithelium can be seen to extend a considerable distance on to enamel surfaces, with the result that actual gingival crevice depths are even less than previously assumed. Artefacts resulting from loss of enamel during processing are discussed in the light of previous attempts to explain epithelial arrangements in rodent gingivae.

The nature of secretory agglutinins and aggregating factors. IV. Complexing between non-mucin glycoproteins, immunoglobulins and mucins in human saliva and amniotic fluid.

By themselves the non-mucin glycoproteins of saliva and amniotic fluid produce secretory conglutinin-like factor (SKF) and secretory bacterial aggregating factor (SBAF) activity. These glycoproteins also bind immunoglobulins; a secretory binding factor for immunoglobulins (SBFI) with EDTA-reversible activity is found in amniotic fluid. Agglutination of particles by very small amounts of secretory antibody is facilitated by reversible and irreversible binding of antibody to SKF/SBAF active glycoproteins. EDTA effects on the carrier glycoproteins make the bound antibody activity EDTA-sensitive. The SKF/SBAF glycoproteins also have EDTA-sensitive interactions with each other and possibly with mucins. These complex interactions of secretory glycoproteins and immunoglobulins are of importance in mucosal protection.

The nature of secretory agglutinins and aggregating factors. II. Biochemical and immunochemical properties of factors in human saliva and amniotic fluid.

Secretory conglutinin-like factor (SKF) and secretory bacterial aggregating factors (SBAF) of saliva and amniotic fluid were characterized as high molecular weight non-mucin glycoproteins. Most biochemical tests or procedures used in previous investigations did not permit an unequivocal distinction among immunoglobulins, non-mucin glycoprotein, or mucin factors. Partitioning with hot phenol enabled the separation of a high molecular weight non-mucin glycoprotein fraction from saliva and amniotic fluid with SKF and SBAF activity that was distinct from mucins and antibodies. Non-mucin glycoproteins, immunoglobulins and mucins represent three classes of secreted molecules capable of clumping bacteria.