42. Diagnosis / Indices-Gingival Fluid & Saliva

Classical Periodontal Literature Review

Rapid Search Terms

Gingival Crevicular Fluid – Overview
Association of GCF with health or disease
The composition of gingival fluid
antibiotics in the gingival crevicular fluid
periodontitis disease markers in GCF

Study Questions:

Is the measurement of gingival fluid easy, repeatable, and dependable?
Discuss the composition of gingival fluid.
What is the diagnostic significance of increased amounts of gingival fluid?
What is in composition of gingival crevicular fluid?
What is the diagnostic significance of increased amounts of gingival fluid?
Which antibiotics are concentrated in the gingival crevicular fluid?
Can gingival fluid contents be used to determine severity of periodontal conditions or disease activity?

Gingival Crevicular Fluid – Overview

Griffiths , G. Formation, collection and significance of gingival crevice fluid. Periodontl 2000. 2003; 31: 32-42.
Goodson , J. Gingival crevice fluid flow. Periodontol 2000. 2003; 31:43-54.
Delima , A, Van Dyke, T. Origin and function of the cellualr componenents in gingival crevice fluid. Peridontol 2000. 2003; 31: 55-76.
Uitto V., Overall, C, McCulloch, C. Proteolytic host cell enzymes in gingvial crevice fluid. Periodontol 2000. 2003; 31:77-104.
Eley , G., Cox, S. Proteolytic an hydrolytic enzymes from putative periodotal pathogens: characterization, molecular genetics, effects on host defecses and tissues and detection in gingival crevice fluid. Periodotol 2000. 2003; 31:105-24.
Perinetti G, Di Leonardo B, Di Lenarda R, Contardo L. Repeatability of gingival crevicular fluid collection and quantification, as determined through its alkaline phosphatase activity: implications for diagnostic use. J Periodontal Res. 2013 Feb;48(1):98-104.

Gingival Crevicular Fluid

Orban , J, Stallard, R: Gingival crevicular fluid: A reliable predictor of gingvial health: J Periododontol 40:231-235, 1969.
Hancock , E et al: The relationship between gingival crevicular fluid and gingival inflammation. A Clinical and histologic study. J Periodontol 50: 13-19, 1979.
Offenbacher , S et al: The use of crevicular fluid prostaglandidn E2 levels as a predictor of periodontal attachment loss. J Periodont Res 21:101, 1986.
Lamster , I et al: Development of a risk profile for periodontal disease: Microbial and host response factors. J Periodontol 65:511-520, 1994.
Giannopoulou , C., Kamma J., Mombelli, A. Effect of inflammation, smoking and stress on gingival crevicular fluid cytokine level. J Clinical Periodontol 2003; 30: 145-153.
Safkan-Sppparla , B., Sorsa, T et al: Collagenases in gingival crevicular fluid in type I diabetes mellitus. J Clin Periodontol 2006; 77:189-194.
Bostanciv N. et al. Ginvial crevicular fluidlevels of RANKL and OPG in periodontal diseases: Implications of the relative ratio. J Clin Periodontol 2007; 34:370-376.
Huynh AH, Veith PD, McGregor NR, Adams GG, Chen D, Reynolds EC, Ngo LH, Darby IB. Gingival crevicular fluid proteomes in health, gingivitis and chronic periodontitis. . J Periodontal Res. 2015 Oct;50(5):637-49.
Nowzari H, Phamduong S, Botero JE, Villacres MC, Rich SK. The profile of inflammatory cytokines in gingival crevicular fluid around healthy osseointegrated implants. Clin Implant Dent Relat Res. 2012 Aug;14(4):546-52.

Passage of Antibiotics

Gordon JM, Walker CB, Murphy JC, Goodson JM, Socransky SS. Concentration of tetracycline in human gingival fluid after single doses. J Clin Periodontol8:117-121,1981.
Gordon JM, et al. Tetracycline: Levels achievable and in vitro effect on subgingival organisms. Part I. Concentrations in crevicular fluid after repeated doses. J. Periodontol. 52:609, 1981.
Pascale D, et al: Concentration of doxycycline in human gingival fluid. J. Clin. Periodontol. 13: 841- , 1986
Ciancio S, Mather M, McCullen J : An evaluation of minocycline in patients with periodontal disease. J. Periodontol. 51:530,
1980.
Britt MR, Pohlod DJ. Serum and crevicular fluid concentrations after a single dose of metronidazole. J. Periodontol. 57:104-107, 1986.
Lai PC, Ho W, Jain N, Walters JD. Azithromycin concentrations in blood and gingival crevicular fluid after systemic administration. J Periodontol. 2011 Nov;82(11):1582-6.
Jain N, Lai PC, Walters JD. Effect of gingivitis on azithromycin concentrations in gingival crevicular fluid. J Periodontol. 2012 Sep;83(9):1122-8.
Belibasakis GN, Thurnheer T. Validation of antibiotic efficacy on in vitro subgingival biofilms. J Periodontol. 2014 Feb;85(2):343-8.
Conway TB, Beck FM, Walters JD. Gingival fluid ciprofloxacin levels at healthy and inflammed human periodontal sites. J Periodontol71:1448-1452, 2000.
Needleman IG, Grahn MF, Pandya NV. A rapid spectrophotometric assay for tetracycline in gingival crevicular fluid. J Clin Periodontol. 2001 Jan;28(1):52-6.

Indicators of Disease Activity

Leibur E, Tuhkanen A, Pintson U, Soder P-O. Prostaglandin E2 levels in blood plasma and in crevicular fluid of advanced periodontitis patients before and after surgical therapy. Oral Diseases5:223-228, 1999.
Lamster IB, Oshrain RL, Harper DS, et al : Enzyme activity in crevicular fluid for detection and prediction of clinical attachment loss in patients with chronic adult periodontitis. Six month results. J. Periodontol. 59: 516-523, 1988.
Bader HI, Boyd RL. Long-term monitoring of adult periodontitis patients in supportive therapy: Correlation of gingival crevicular fluid proteases with probing attachment loss. J Clin Perio26:99-105,1999.
Lamster IB, Ahlo JK. Analysis of gingival crevicular fluid as applied to the diagnosis of oral and systemic diseases. Ann N Y Acad Sci. 1098:216-29, 2007. Review.
Guzman Y, Sakellari D, Arsenakis M, Floudas CA. Proteomics for the discovery of biomarkers and diagnosis of periodontitis: a critical review. Expert Rev Proteomics. 2014 Feb;11(1):31-41.

Saliva

Liu J, Duan Y. Saliva: a potential media for disease diagnostics and monitoring. Oral Oncol. 2012 Jul;48(7):569-77.
Fuentes L, Yakob M, Wong DT. Emerging horizons of salivary diagnostics for periodontal disease. Br Dent J. 2014 Nov;217(10):567-73.
Taylor JJ, Preshaw PM. Gingival crevicular fluid and saliva. Periodontol 2000. 2016 Feb;70(1):7-10.23.
Giannobile WV. Salivary diagnostics for periodontal diseases.J Am Dent Assoc. 2012 Oct;143(10 Suppl):6S-11S. Review.
Guentsch A, Preshaw PM, Bremer-Streck S, Klinger G, Glockmann E, Sigusch BW. Lipid peroxidation and antioxidant activity in saliva of periodontitis patients: effect of smoking and periodontal treatment. Clin Oral Investig. 12(4):345-52, 2008. Epub 2008 May 29.
Slots J, Slots H. Bacterial and viral pathogens in saliva: disease relationship and infectious risk. Periodontol 2000. 2011 Feb;55(1):48-69.
Belibasakis GN, Bostanci N. The RANKL-OPG system in clinical periodontology. J Clin Periodontol. 2012 Mar;39(3):239-48.
Reher VG, Zenóbio EG, Costa FO, Reher P, Soares RV. Nitric oxide levels in saliva increase with severity of chronic periodontitis. J Oral Sci. 49(4):271-6. 2007.
Frodge BD, Ebersole JL, Kryscio RJ, Thomas MV, Miller CS. Bone remodeling biomarkers of periodontal disease in saliva. J Periodontol. 79(10):1913-9, 2008.
Tobón-Arroyave SI, Jaramillo-González PE, Isaza-Guzmán DM. Correlation between salivary IL-1beta levels and periodontal clinical status. Arch Oral Biol. 53(4):346-52, 2008.
Rai B, Kharb S, Jain R, Anand SC. Biomarkers of periodontitis in oral fluids. J Oral Sci. 50(1):53-6, 2008.
Miller CS, King CP Jr, Langub MC, Kryscio RJ, Thomas MV. Salivary biomarkers of existing periodontal disease: a cross-sectional study. J Am Dent Assoc. 137(3):322-9, 2006.

Gingival Crevicular Fluid – Overview

Is the measurement of gingival fluid easy, repeatable, and dependable?

Topic:Gingival crevicular fluid (GCF)

Authors:Griffiths, G.

Title: collection and significance of gingival crevice fluid.

Source: Periodontol 2000. 2003; 31: 32-42

DOI: https://doi.org/10.1034/j.1600-0757.2003.03103.x
Type:Discussion

Keywords:gingival crevice fluid

Background: The exact nature of GCF, its origins and composition has been subject of controversy. The principle questions that remain unanswered are whether GCF is a transudate or an exudate, and whether GCF can form in a clinically healthy site. The answers to these questions could yield therapeutic approaches. An increase in GCF flow may have physical protective effects through flushing the pocket, as well as facilitating the passage of immunoglobulins.

Purpose: To discuss the formation, collection, and significance of GCF.

Discussion: How is GCF formed?

GCF is a result of an increase in the permeability of the vessels underlying junctional and sulcular epithelium. Some irritation (chemical or mechanical) is necessary to induce production of GCF, and should therefore be considered a pathological phenomenon, but this stimulation is important for the maintenance of gingival health (e.g. transporting antibiotics, flushing effect).
GCF is an important component of the protective mechanisms of the crevicular regions due to its flushing effect which is capable of removing carbon particles and bacteria introduced into the gingival crevice.
GCF may have an important role in transporting antibacterial substances to the gingival crevice.

Is GCF a transudate of interstitial fluid?

Controversy exists whereas GCF is a transudate or inflammatory exudate.

Studies by Brill and Egelbergsupport that production of GCF was primarily a result of an increase in the permeability of the vessels underlying junctional and sulcular epithelium.
An alternative theory arose from Alfano and Pashleysupporting that the initial fluid produced could simply represent interstitial fluid which appears in the crevice as a result of an osmotic gradient. This initial, pre-inflammatory fluid was considered to be atransudate, and, on stimulation, this changed to become an inflammatory exudate.

Collection Methods:

Several techniques have been employed and the technique chosen will depend upon the objectives of the study as each technique has advantages and disadvantages. Three basic categories:

Gingival washing: the gingival crevice is perfused by isotonic solution. The fluid collected represents a dilution of crevicular fluid and contain both cells and soluble constituents such as plasma proteins. This technique is valuable for harvesting cells from the gingival crevice region. Major disadvantage is that all fluid may not be recovered and accurate quantification of GCF volume or composition is not possible.
Capillary tubing and micropipettes: following isolation and drying of the site capillary tubes are inserted into the entrance of the gingival crevice. GCF from the crevice migrates into the tube and because the internal diameter is known the volume of the fluid collected can be accurately determined by measuring the distance which the GCF has migrated. However, it is difficult to collect an adequate volume of GCF in a short period unless the sites are inflamed and contain large quantities of GCF. It is difficult to conceive that holding a capillary tube at the entrance to the gingival crevice for long time ensures an atraumatic collection. Also, it is difficult to remove the complete sample from the tubing.
Absorbent filter paper strips: quick and easy to use. Can be applied to individual sites and is possibly the least traumatic.

Methods of collection can be broadly divided into intracrevicular and extracrevicular techniques. The former depends on the strip being inserted into the gingival crevice, whereas in the latter the strips are overlaid on the gingival crevice region in an attempt to minimize trauma. The intracrevicular methodis the method used most frequently and can be further subdivided depending upon whether the strip is inserted just at the entrance of the crevice or periodontal pocket or whether the strip is inserted to the base of the pocket or until minimum resistance is felt.

(a) extracrevicular method; (b) intracrevicular method ‘superficial’ (c) intracrevicular method ‘deep’

Collection and quantification of GCF is a very sensitive procedure and all techniques have limitations and flaws (contamination, inconsistent sampling times, volume determination, recovery from strips).
The method of collecting GCF may have a significant effect upon the nature collected and will therefore prejudice results assessing diagnostic markers

Association of GCF with health or disease: The association of an increased volume of GCF with an increase in the severity of inflammation is well supported by evidence from literature.

Topic:Gingival crevicular fluid

Authors:Goodson, J

Title:Gingival crevice fluid flow

Source:J. Periodontol 2000. 2003; 31:43-54

DOI: 10.1034/j.1600-0757.2003.03104.x

Type:Discussion article

Keywords:Gingival fluid

Purpose: To focus attention on the importance of GCF flow and the effect that small streams of fluid flowing out of the periodontal pocket have on the periodontal environment.

Discussion:

GCF flow(or flow rate): is the process of fluid moving into and out of the gingival crevice or pocket.

Fluid flow:is a rate measure of the volume that crosses a defined boundary over a given time.

Resting volumeis the amount of fluid within a given space.

3 Methods of Measurementare available to differentiate fluid flow from the resting GCF volume.

Method 1: Samples are collected(w/ filter paper strips) rapidlyunder strict timing protocol so that the resting volume is removed by the first sampleand the volume of the newly formed GCF can be measured in subsequent samples. The key to this measurement is to select a sampling time that is small enough so that the resting volume of the pocket is not allowed to re-establish. Ideally, one would remove the entire contents of the pocket with the first sample so that all subsequent samples would be repetitive estimates of the GCF volume flow over the sampling time.

Method 2: Collect samples(w/ filter paper strips) after equilibrium has been re-established in the pocket over several different time intervals. In this case, each GCF sample includes both the resting volume (Vr) and the volume of GCF that entered the pocket over the sampling period lends itself directly to linear regression analysis in which the volume and time of each sample contribute to the analysis to determine the slope which is the GCF influx (fi) and the intercept which is the resting volume of the pocket (Vr).

Method 3: Measure the equilibrium concentration of a marker substance pumped into a pocket at a constant rate. By pumping a marker substance (ie Tetracycline) into a periodontal pocket at a constant rate, an equilibrium concentration will be established which is the result of the fluid flow rate and the pump delivery rate. To date, no published study has been conducted to evaluate GCF flow in this manner.

Lamster et al:

Described changes in GCF sample volumes taken during experimental gingivitis that are directly amenable to analysis by method 1. In the GCF sampling protocol of this study, a filter paper strip was placed in the sulcus for 30s and removed for volume determination (V1). Thirty seconds was allowed to elapse and the GCF volume was determined by introducing a second filter paper strip into the site for 3s (V2). The results of this study clearly indicated that the two sample volumes collected increased linearly over the period of development of experimental gingivitis. The GCF flow increases as inflammation becomes more severe and vascular permeability increases.

Studies showthat GCF flow as an outcome variable for therapy studies does not have the statistical power of either pocket depth reduction or attachment level gain.

One site from each of 56 systemically healthy subjects with periodontal disease was assigned to each clinical category (Health, gingivitis, mod Periodontitis and advanced periodontitis). The data indicate that broadly defined categories of health and disease can be distinguished by differences in GCF flow.

By this analysis, the GCF flow at healthy sites was significantly less than all other categories.
The GCF flow at sites with gingivitis was significantly less than the GCFflow at advanced periodontitis sites.
Thedifference between the GCF flow at gingivitis sites and sites with moderate periodontitis was not statistically significant.

Conclusion:Given the evidence that change in GCF flow may be a sensitive measure of local inflammation, this type of evaluation could increase the diagnostic potential of this measure being used chair-side. GCF flow measurement could provide added benefit in establishing a diagnosis and monitoring the response to therapy.

Topic:Gingival crevicular fluid

Authors:Delima, A, Van Dyke, T.

Title:Origin and function of the cellualr componenents in gingival crevice fluid.

Source:Peridontol 2000. 2003; 31: 55-76

DOI: 10.1034/j.1600-0757.2003.03105.x

Type:Discussion/Review

Keywords:gingival crevicular fluid

Purpose:To review the origin and function of the cellular components in gingival crevicular fluid

Discussion:Periodontitis is initiated by bacterial colonization, followed by proliferation, and extension of the plaque into the subgingival environment. While diseases of the periodontium are bacterial in origin, the extent and severity of the disease depend upon the interaction between pathologic microbes and host response. The outflow of GCF helps to cleanse the dentogingival space of non-adherent microbes and to reduce the concentrations of toxins and byproducts. GCF contains microbes and antimicrobial compounds and enzymes directed at them. We often think about the flushing mechanism of GCF, but it also serves as an entry point to bacteria. One of the initial responses of the host to bacterial plaque is an increase in the vascular permeability of the subepithelial blood vessels. This leads to edema in the gingival crevice (via the JE) and results in increased crevicular fluid flow. Since this exudate is essentially a growth medium that supports host cells and tissues, the GCF can also act as an excellent source of nutrients for subgingival microbes and may actually contain factors that are necessary for the proliferation of some pathologic bacterial species.

The GCF is rich in cellular elements and also delivers antibodies and the complement system to fight the plaque front. The epithelia of the sulcus are constantly renewing and this rapid turnover appears to aid in the clearance of bacteria that adhere to these cells. One major set of cells found here are the PMN’s – these are the major cellular defense system in the gingival crevice. These cells leave the connective tissue and migrate through the JE into the crevicular space where they accumulate at the interface of the subgingival plaque. PMN’s compromise about 90% of the cells in the GCF, and their defense mechanism includes phagocytosis and release of bactericidal enzymes. It also has been shown that the release of granules from PMN’s that are capable of disengaging bacterial plaque adherence to the tooth. Other cells present in the GCF include monocytes and macrophages. These are usually seen against the oral epithelium, and they function in the cellular defense and acquired immunity of the periodontal tissues.

Conclusion:The nature and extent of bacterial challenge is modulated and attenuated by the host immune response. Inflammatory and immune responses can also contribute to the destruction of host tissues. The narrow balance between periodontal homeostasis and disease depends upon a qualitatively and quantitatively appropriate response of the host defense mechanism to infection of the periodontal tissue. The outflow of GCF helps to cleanse the dentogingival space of non-adherent microbes and to reduce the concentrations of toxins and metabolic byproducts. Constant cellular turnover and the presence of PMNs help to aid in the clearance and protection of the sulcus. Understanding the components of GCF will help to clarify the initial events in the pathogenesis of periodontal disease and aid in the monitoring of the disease process.

Cell Type	Source	Function
Bacteria	Adjacent plaque mass	Etiologic factors of periodontal disease. Initiates the host immune response.
Epithelial cells	Oral sulcular and junctional epithelium	Represents the high turnover rate of the epithelium that comprise the gingival sulcus.
Leukocytes	Gingival plexus of blood vessels	Effector cells of host response. PMNs play a role in innate immunity. Monocytes/macrophages and lymphocytes play roles in cell-mediated immunity.
Erythrocytes	Blood vessels	Incidental finding. Results from damage to the small blood vessels and capillaries of gingival connective tissue.

Topic:GCF

Authors: Uitto V., Overall, C, McCulloch, C

Title:Proteolytic host cell enzymes in gingvial crevice fluid.

Source:Periodontol 2000. 2003; 31:77-104

DOI: 10.1034/j.1600-0757.2003.03106.x.

Type:Discussion

Keywords:GCF

Purpose: This is a review of literature on findings on host cell-derived enzymes that relate to periodontal disease processes. Special emphasis is placed on MMPs. The potential use of tissue-derived GCF in enzymes in clinical periodontology is also discussed.

Discussion: Neutrophils form the first line of defense and are attracted to infected tissues by chemoattractants released from bacteria, host cells or degraded tissue. Over 90% of leukocytes in GCF are neutrophils.

They contain vesicles where molecules used for host defense are stored. These granules are generally classified into azurophilic (primary), specific (secondary) and gelatinase (tertiary) types. Gelatinase granules are released first, then specific and lastly the azurophilic. The major MMPs in neutrophils are MMP-8 and -9.

The serine proteinases (neutrophil elastase, cathepsin G and proteinase 3) are secreted from the azurophilic granules during phagocytosis, stimulation and cell lysis. Stimulation by LPS, TNF-and IL-8 results in increased (20-fold) binding of these proteinases with cell membranes. Their actions is inhibited by the serpins. When there is increased population of neutrophils there is increased concentration of active forms of the enzymes in the tissues, causing degradations of extracellular matrix. Elastase and cathepsin G are capable of activating epithelial cells to produce IL-8, IL-6 and prostaglandin E2, which further increase chemotaxis, immune cell proliferation, and tissue degradation in inflamed tissues.

Epithelial cell proteinases: Although epithelial cells were considered as relatively passive cells whose function was to protect body surfaces, it now clear that they strongly respond to exogenous factors and therefore exhibit different morphotypes. When activated they behave aggressively by migrating, proliferating and producing various cytokines and proteolytic enzymes. They were found to produce collagenases (MMP-13, produced by the basal cells of pocket epithelium) when stimulated in vitro by TNF-TGF-or keratinocyte growth factor. MMPs -2, -9, -7 have also been found to being produced by epithelial cells.

Fibroblast proteinases: Fibroblasts are responsible for the turnover of connective tissue in normally functioning tissues and they have therefore the capacity to degrade all the components of CT. The major MMPs produced are 1, 2, 13, 3, 14. It appears that MMP- 1 is secreted in the crevicular fluid of patients with localized juvenile periodontitis, while neutrophil type collagenase MMP-8 is prevalent in adult periodontitis. Expression of MMPs can be regulated by the protein composition of the extracellular matrix and following ligation of integrin receptors.

Matrix metalloproteinases: MMPs form the most important family of proteinases that participate in the normal turnover of periodontal tissues as well as their degradative aspects during periodontal diseases. As MMPs can potentially destroy tissues, their activity is strictly controlled at different levels. First, specific inhibition of MMPs can be mediated by the four members of the tissue inhibitor of metalloproteinase (TIMP) family, proteins that regulate the extracellular activity of MMPs. Second, MMPs are synthesized as latent zymogens. Activation of MMP zymogens is a critical step for regulating MMP activity and hence the composition, structure, and function of periodontal connective tissue matrices. Third, most MMPs are secreted from cells as a soluble proform. For some soluble MMPs, activation occurs at the cell surface following proteolytic cleavage by MT-MMPs, often in a TIMP dependent pathway. For other MMPs, activation occurs in the extracellular environment in an activation cascade initiated by tissue proteinases, such as plasmin, kallikrein, and tryptase, a process that is often amplified by the activated MMPs functioning as pro-MMP activators.

TIMPs:Inhibition of MMPs by TIMPs is largely interchangeable, except for the MT-MMPs, which are not inhibited by TIMP-1. TIMP-2 also plays a paradoxical role in mediating MMP-2 activation.

Detection methods for GCF MMPs: collagenases (MMP-1, -8, 13), gelatinases (MMP-2, -9), and stromelysin (MMP-3) have all been measured in GCF. Immunological assays, including ELISA and immunoblots, offer highly sensitive and specific testing methods. Immunoblots are time consuming, ELISA using antibodies is accurate and significant numbers of anti-MMPs exist on the market. Although these methods of detection are highly specific, they might not be able to distinguish between active and inactive MMPs, since MMPs can still bind the substrate without cleavage. Immunohistochemical examinations can help identify MMPs but without actually quantifying them. Another possible but untried method would be neo-epitope antibodies that can detect degradation fragments from matrix proteins or MMPs, thus help identify, quantify, and determine activity.

Substrate degradation: measure primarily the ability of MMPs to degrade the substrate, estimating enzyme activity depending on the relative abundance of the substrate degradation products by dot assays.sodium dodecyl sulfate–polyacrylamide gel electrophoresis, radioactivity, zymography, or fluorescence. The rationale for examining enzyme activity compared to the presence or abundance of MMPs (measured typically by immunochemical methods) is that in periodontitis the temporal progression of lesions is more strongly associated with the presence of active MMPs than with the total amount of enzyme. Collagen degradation products: measuring collagen metabolites in GCF can help and more directly quantify MMPs activity

GCF enzymes as indicators of periodontal health:it has been shown that PMNs enzymes detected in the GCF reflect the number of leukocytes rather than tissue destructions, so directing our attention to MMP-13 for example rather than MMP-8 (collagenase-2) appears more rational, since MMP-13 (collagenase-3) can also be produced by fibroblasts and pocket epithelial cells during tissue destruction. Neutrophil elastase, and B-Glucuronidase have been extensively studied as well, both are involved in tissue destruction. A simple mouth rinse assay has determined that total crevicular fluid elastase levels were indicated that there was a good correlation between the oral elastase activity and the number of deep pockets and the average CPITN scores. Longitudinal studies have shown the risk ratio of clinical attachment loss in patients with high B-glucuronidase activity in GCF is about 10-fold, this relation is also high when considering progressive sites vs. non progressive. Another test that has been developed is detection of aspartate aminotransferase activity GCF, this test yielded an OR of 12 for attachment loss.

Topic:Host defense

Author:Eley, G., Cox, S

Title:Proteolytic and hydrolytic enzymes from putative periodontal pathogens: characterization, molecular genetics, effects on host defenses and tissues and detection in gingival crevice fluid.

Source:Periodontology 2000. 2003; 31:105-24

DOI: 10.1034/j.1600-0757.2003.03107.x.

Type:Discussion article

Keywords:proteolytic, hydrolytic, enzymatic reactions, host defense, pathogenicity

Purpose:Discussion article that describes the characteristics of proteolytic and hydrolytic enzymes produced by various bacteria, (Pg, Pi, Aa, Fn, Treponema, Eikenella corrodens, and capnocytophaga).

Discussion:

All of the bacteria discussed produce both proteolytic and hydrolytic enzymes (degrade non-proteinaceous components of connective tissue). Proteases are more critical to bacterial survival since most of their nutrition is obtained from protein sources. P. gingivalisproduces and releases a greater number of proteases with more protease activity than any other bacteria.
The genes encoding for bacterial proteases have been identified for arg-gingipain A and B, lys-gingipain, collagenolytic proteases tripsine and chymotripsine-like protease from T. denticola. These proteases are able to degrade immunoglobulins, clotting factors, proteinase inhibitor and components of host CT. All of these enzymes are released in the GCF.
P. gingivalis appears to produce two cysteine proteinases with trypsin-like activity (cleaves BANA). One of these is arginine specific (arg-gingipains A and B) and the other is lysine specific (lys-gingipain), deriving from rgpA, rgpB and kgp genes respectively.
These enzymes have collagenolytic activities (Type I and IV) increasing host tissue destruction. RgpA degrades IgA1, IgA2 and IgG, reducing the host response. It also degrades complement component C3 reducing opsonization. RgpA and kgp degrade C5 and release C5a, stimulating inflammation. P.g. inhibits PMN migration and diapedesis by degrading Il-8 and intracellular adhesion molecule 1 (ICAM-1).
Furthermore, P.g. cysteine proteinases can degrade the lysozyme that is found in the GCF and saliva.
Gingipains activate pre-kallikrein system, leading to bradykinin formation and subsequent vasodilation. This process increase GCF flow, thus increasing the nutrients in the crevice. P.g. also degrades fibrinogen which increases the local clotting time and as a result leads to gingival bleeding. This provides a rich source of haem and iron required by the bacterium for its survival.
P.g. also releases hydrolytic enzymes, (hyalouronidase and chondroitinase) which hydrolyze the glycozaminoglycan components of proteoglycans.
P.intermedia and P.nigresens, Treponema species, Capnocytophaga species, A.a., F. nucleatum, C. rectus and E.corrodenshave been also shown to have trypsin-like activity, but all is very weak compared to P.g.
They have proteolytic activity for various components of the ECM (laminin, gelatin and fibronectin). Their proteases are able to degrade IgG and fibrinogen. F. nucleatum and E.corrodens are the weakest species. Bacterial proteases are released in the GCF and can be detected on filter paper strips. Selective biochemical assays have been developed to detect and distinguish between those proteases. Cross sectional study showed 93.81% positive prediction and 100% negative prediction for arg-gingipains, and 100% negative prediction for dipeptidylpeptidases for all incidence of rapid episodic CAL loss in a 3-month period. All comparisons of mean patient values in patients with or without CAL loss were SS.

BL:GCF arg-gingipain appears to be an excellent predictor and GCF dipeptidylpeptidase is a moderately good predictor of future progressive CAL loss. A chair-side system has been developed to detect these bacterial proteases.

Topic: GCF

Author: Perinetti G, Di Leonardo B, Di Lenarda R, Contardo L

Title: Repeatability of gingival crevicular fluid collection and quantification, as determined through its alkaline phosphatase activity: implications for diagnostic use

Source: J Periodontal Res. 2013 Feb;48(1):98-104

DOI: https://doi.org/10.1111/j.1600-0765.2012.01508.x

Type: Clinical study

Keywords: Alkaline phosphatase, diagnosis, method error, repeatability, resting/flow gingival crevicular fluid

Purpose: To identify a threshold value above which a variation in gingival crevicular fluid activity can be considered as indicative of metabolic changes in periodontal tissues in individual subjects.

Methods: 27 periodontally healthy subjects had clinical measurements taken and gingival crevicular fluid (GCF) collected at baseline, 1 day, 1 week, and 3 months. PerioPaper strips were used to collect the GCF during rest, flow, and overall. Patients were instructed to rinse with 0.12% chlorhexidine mouthwash twice daily throughout the study. Alkaline phosphatase (ALP) activity was measured in samples.

Results: Clinical conditions of the subjects were excellent throughout the study and were not significantly different at any time point. No significant differences in ALP were seen over time for any of the resting, flow, or overall GCF samples. However, the flow GCF ALP activity was significantly lower compared with the corresponding resting GCF at 1 day and 1 week time points.

Discussion: Reliable use of GCF collection and quantification should take into account relevant errors. For ALP activity, variations are to be considered as genuine only above relevant thresholds, which are at least 40%.

Gingival Crevicular Fluid

Discuss the composition of gingival fluid.

What is the diagnostic significance of increased amounts of gingival fluid?

Topic:Gingival crevicular fluid

Authors:Orban, J, Stallard, R

Title: Gingival crevicular fluid: A reliable predictor of gingvial health

Source:J Periododontol 40:231-235, 1969

DOI: 10.1902/jop.1969.40.4.231

Type:Clinical study

Keywords: gingival crevicular fluid, hyaluronidase, biopsy

Purpose: To evaluate the accuracy of GCF as an indicator of gingival inflammation.

Method: Dental school patients were chosen at random and evaluated by using Ramfjord’s criteria for Perio Dx Index (PDI) & Greene & Vermillion’s Oral Hygiene Index-Simplified (OHI-S). GCF measurements were made using filter strips 1mm wide and several mm long, placed intracrevicularly for 3 minutes, removed and allowed to dry. The area that absorbed crevicular fluid was stained by 2% ninhydrin solution. The area stained was then measured and mean scores were calculated. Calculus was scored for OHI-S and plaque for Ramfjord’s Index using disclosing tablets. Biopsies taken from distal half of each area evaluated. Each biopsy was evaluated according to amount and extent of inflammatory infiltrate and scored on a 0-10 scale.

Results: From the data obtained it was apparent that GCF scores were not directly related to biopsy scores. Thus, crevicular fluid did not prove to be an accurate predictor of gingival inflammation. Plaque scores revealed a high degree of correlation on both an individual and overall evaluation when compared to biopsy scores of inflamed gingiva.

Discussion:Enzymes commonly found in plaque, primarily hyaluronidase, cause an increase in crevicular fluid flow without altering the inflammatory condition of the gingiva. Apparently, the intercellular cementing substance is modified, resulting in an increased permeability of the crevicular epithelium. Other factors including chewing, brushing, gingival massage, circadian rhythm and hormones all affect crevicular fluid flow without necessarily altering the inflammatory condition of the gingival tissues. However, it is a measure of the intactness and permeability of the gingival tissuesdue to previously mentioned factors. Further, as a single measurement on a patient, crevicular fluid recordings may have little significant value. Consecutive measurements, however, are of value in relating the response of the gingival tissues to environmental, physiologic and pathologic conditions and changes.

Conclusion: Dental plaque is a better indicator of the inflammatory status of the gingival tissues than GCF levels.

Topic:gingival fluid

Authors: Hancock EB, Cray RJ, O’Leary TJ

Title:The relationship between gingival crevicular fluid and gingival inflammation. A clinical and histologic study.

Source:J Periodontol. 1979Jan;50(1):13-9.

DOI: 10.1902/jop.1979.50.1.13

Type:clinical

Keywords:GCF, gingival crevicular fluid, gingival inflammation,

Background:Past research reported that the flow of GCF begins a few days before other clinical signs of inflammation are apparent. Therefore, it is suggested that its flow might be measured to evaluate gingival inflammation.

Purpose: To evaluate the relationship between GI, GCF flow, and histology in order to see if GCF could be an indicator of disease severity in form of gingival inflammation.

Methods: 60 patients (26M, 34F, 18-72 years old); considered themselves in good health without known systemic diseases. No periodontal therapy within the last 90 days. Teeth #5-#12 were examined in each patient and evaluated for selection by the following criteria:

1) Presence of an adequate zone of gingiva, 2) No cervical or interproximal carious lesions. 3) No defective interproximal restorations. 4) No facial cervical restorations. 5) No acute gingivitis or periodontitis condition.

Data was collected on 57 sites; amount of GCF flow, gingival health status, GI, histologic evaluation, and histologic evaluation. After isolation with cotton rolls, GCF collected from mid-facial with filter paper in the opening of the gingival crevice for 3 min. A second sample was taken to verify fluid flow rate. Gingiva at these areas was evaluated for inflammation. GI assessed using modified GI by Löe. One tooth from each patient had gingival biopsy, trying to take equal numbers of healthy and inflamed tissues. Histology looked for inflammatory cell density. Histologic analysis looked at extent of inflammatory infiltrate using a gravimetric method

Results: results show that GCF flow tended to increase as the degree of inflammation became more severe. The quantity of GCF, by itself, was a poor indicator of the severity of gingival inflammation.The highest correlation was seen between GI and the gingival status (mainly BOP based). A high correlation was seen between clinical and histologic scores, but a very weak correlation was seen between GCF and either of these factors.

Conclusion: GCF was weakly correlated with GI and histologic evidence of inflammation; it did, however, tend to increase when inflammation increased.

Topic:Indices

Author: Offenbacher S.

Title:The use of Crevicular fluid prostaglandin E2 levels as a predictor of periodontal attachment loss

Source:J Periodont Res 21:101, 1986

DOI: 10.1111/j.1600-0765.1986.tb01443.x.

Type:Longitudinal study

Keywords:Indices; prostaglandin E2; periodontal disease

Purpose:To report results of a three-year longitudinal study, which demonstrate that crevicular fluid PGE levels can be used to reliably indicate ongoing tissue destruction, and may also be used to predict future acute periodontal attachment loss.

Methods:7 healthy patients, 41 adult periodontitis patients and 12 juvenile periodontitis patients were assessed. All patients had a negative history of major systemic illness. All patients had at least 20 remaining teeth. Data was obtained over a period of 18 months up to 3 years for analysis. CF fluid was collected and clinical measurements (redness, edema, suppuration, BOP, pain on probing, Attach loss) were obtained at baseline and repeated one week later. SRP was performed on all diseased patients. CF collection and clinical measurements were repeated one month following initial therapy. Patients were placed on a three-month recall and CF collection was repeated at each maintenance. When a site demonstrated a statistically significant loss of periodontal attachment, after the data collection (CF and clinical parameters), local curettage and root planning was performed. One month after therapy data was again collected, and then patients were exited from the study and referred to periodontist for additional therapy. During the visit at which AL was identified, 6 consecutive CF samples were obtained at attachment loss site and 6 CF samples at the contralateral control site. Mean CF-PGE level was determined.

Results:The mean CF-PGE2 of the healthy individuals(26.9 ng/mL) was significantly lower than the one of the adult periodontitis patients (56.6± 5.4 ng/mL) and the juvenile periodontitis patients(139.4±15.3 ng/mL). The CF- PGE2 was also elevated at the time of detectable ALcomparing to the cross-sectional adult periodontitis MCF-PGE.

It was observed that CF-PGE levels were increased prior to the development of the acute lesion (immediately preceding the AL episode). There was a high degree of overall and specific agreement between high CF-PGE2 levels and attachment loss. The elevated levels of CF-PGE are highly specific (0.94) and sensitive (0.76) of ongoing AL.

Conclusion: PGE levels in GCF reliably indicate ongoing disease activity and could predict upcoming attachment loss.

Topic:risk factors

Authors: Lamster I, et al

Title:Development of a risk profile for periodontal disease: Microbial and host response factors

Source:J Periodontol 65:511-520, 1994

Type:report

Rating: Good

Keywords:periodontitis/microbiology; periodontal diseases/microbiology; risk factors, host response, gingival crevicular fluid, glucuronidase

Purpose:To list risk factors associated with the host-microbial interaction in periodontal disease

Conclusion:4 factors and their association with active chronic periodontal disease were explored in this review: Elevated GCF levels of β-glucuronidase (a lysosomal hydrolase that can serve as a marker for primary granule release from PMNs), elevated levels of periodontal pathogens, reduced levels of IgA in GCF, and reduced levels of serum IgG antibody to putative periodontal pathogens. These pathogen associated risk factors, as well as patient- associated risk factors (DM, smoking), can provide diagnostic information to aid in the treatment of patients with periodontal disease.

Topic:Smoking on gingival crevicular fluid

Authors:Giannopoulou, C., Kamma J., Mombelli

Title: A. Effect of inflammation, smoking and stress on gingival crevicular fluid cytokine level.

Source: J Clinical Periodontol 2003; 30: 145-153

DOI: 10.1034/j.1600-051x.2003.300201.x

Type:Clinical study

Keywords:smoking, interleukins, gingival crevicular fluid

Purpose:To determine the levels of interleukin (IL)-1β, IL-4, IL-6 and IL-8 in gingival crevicular fluid of periodontally healthy and diseased individuals and to study their association to environmental factors such as smoking and stress.

Material and method:

A total of 80 patients from a private periodontal practice were included in the study:
20 patients per disease category: aggressive periodontitis (EOP), chronic adult periodontitis (AP), gingivitis (G) healthy periodontium (H).
PD, AL, Pl, BOP, suppuration, and smoking and stress information were recorded at initial visit and after GCF sampling. Full mouth standardized periapical radiographs were taken and bone loss was assessed.
GCF was collected from four sites per patient, randomly selected in each quadrant by means of durapore filter membranes. After isolation of the test sites from saliva, a first Durapore strip was inserted 1mm into the sulcus or pocket and left in place for 15s. Three minutes after removal of the first strip, a second Durapore strip was similarly inserted in the same site for 15s. The two strips were then placed into a microcentrifuge tube and immediately frozen until the day of the analysis. In case of visible contamination with blood, the strips were discarded.
The contents of IL- 1β, IL-4, IL-6 and IL-8 were measured in 320 samples by use of commercially available sandwich enzyme-linked immune adsorbent assays.

Results:In subjects with periodontitis, the total amounts of IL-1β, IL-6 and IL-8were significantly elevated compared to healthy subjects. IL-4showed an inverse relationship to periodontal status and higher amounts were found in the healthy group. The amounts of all four cytokines were positively correlated with probing depths. IL-4, IL-6 and IL-8 were significantly correlated to smoking while stress was associated withIL-1β, IL-6 and IL-8levels.

Conclusion:IL-1β, IL-6 and IL-8 reflect the activity of periodontal destruction, whereas IL-4 shows an inverse correlation to it.

Topic: Gingival Crevicular Fluid

Authors:Safkan-Sppparla, B., Sorsa, T

Title:Collagenases in gingival crevicular fluid in type I diabetes mellitus.

Source:Implant Dent. 2008 Mar;17(1):16-23

DOI: 10.1902/jop.2006.040322

Type:Clinical study

Reviewer:Laura Porras

Rating: Good

Keywords:

Purpose: To analyze GCF collagenases of poorly and well-controlled type 1 DM and chronic periodontitis subjects compared to non-diabetic with chronic periodontitis and periodontally healthy control subjects

Method: Collagenase activity was studied in human GCF. 22 pts with chronic periodontitis, poorly controlled diabetes(with history of problems related to the control of their diabetes, such as hypo-, or hyperglycemias, recurrent infections, ketoacidosis, diabetic coma, glucosuria, nephropathies, neuropathies – average HbA1c 9.6%) and 5 pts with chronic periodontitis, well controlled diabetic pts(average HbA1c 8.4%) were compared to 6 chronic periodontitis, non-diabetic ptsand 5 periodontally healthy, non-diabetic controls. PI, bleeding index, loss of attachment, PD, bone loss (approximal loss of marginal alveolar bone was measured from panoramic radiographs. The distance from the cemento-enamel junction to the alveolar bone crest was measured mesially and distally on all teeth to the nearest millimeter using a transparent scale. A mean score based on these measurements was calculated for each individual.were evaluated).

GCF was collected. Sites (two to five sites per subject) were cleaned and dried gently and supragingivally with sterile curettes and kept dry with cotton wool rolls. Visible plaque was carefully removed. One to two filter paper strips were placed at the opening of the gingival margin 0 to 1 mm into the sulcus for 3 minutes for gingival crevicular fluid collection. Strips were placed into Eppendorf tubes. Collagenase activity against type 1 collagen was measured using gel electrophoresis.

Results:

Poorly controlled diabetic patients had more alveolar bone loss (3.7 mm) than the well-controlled diabetic patients (2.4 mm) and controls.
Same PD between poorly and well controlled.
Collagenase activity in GCF was higher in poorly controlled diabetic patients than in well-controlled subjects or controls.
NS correlations between collagenase activity values and clinical parameters when diabetic group was analyzed alone.
Poorly controlled vs. well-controlled diabetic patients showed significant correlations between bone loss and loss of attachment; BOP and loss of attachment; bone loss and BOP.
NSSD between well controlled diabetic patients and systemic healthy controls.

Conclusion:Poorly controlled diabetes is strongly related to periodontal tissue destruction, and collagenases in GCF may mediate and reflect this effect.

Topic:RANKL and OPG

Authors:Bostanci N et al

Title:Ginvial crevicular fluidlevels of RANKL and OPG in periodontal diseases: Implications of the relative ratio.

Source:J Clin Periodontol 2007; 34:370-376

DOI: 10.1111/j.1600-051X.2007.01061.x

Type:Retrospective study

Keywords:bone resorption; ELISA; gingival crevicular fluid; OPG; RANKL

Background:RANKL is expressed predominantly as a membrane-bound ligand on osteoblasts, fibroblasts and activated T and B cells, and its osteoclastogenic action can be blocked by its soluble decoy receptor OPG.

Purpose: To investigate RANKL and OPG levels, as well as their relative ratio in GCF of patients with gingivitis, chronic periodontitis, generalized aggressive periodontitis, and chronic periodontitis under immunosuppressive treatment, as well as healthy subjects.

Method: 107 subjects: 21 healthy group, 22 gingivitis group, 26 G-AgP (GAP), 28 chronic periodontitis group, and 11 patients who were taking immunosuppressive drugs for renal transplants (cyclosporin-A) that had significant gingival enlargement. GCF samples were collected from mesio-buccal single-rooted teeth with PD 6-8 mm, in the gingivitis group the GCF was taken from teeth with 3 mm or less PD with BOP, in healthy GCF was taken from areas of no BOP. Commercially available ELISA kits used.

Results:

Healthy and gingivitis groups showed low levels of RANKL, some patients had no detectable RANKL.
RANKL was always detected and significantly elevated in all 3 periodontitis groups, NSSD between G-AgP and chronic, but the immunosuppressed group had lower RANKL levels than either periodontitis forms (2x lower).
In contrast, OPG was detected in all patients at all sites, highest in healthy patients, lowest in chronic and G-AgP patients, whereas it was mildly lowered in gingivitis patients.
RANKL/OPG ratio was lowest in healthy and gingivitis subjects, and elevated in all three diseased groups, with no SSD between them.
RANKL positively correlated with PPD, CAL, PI
OPG negatively correlated with PPD, CAL, PI and PBI (papilla bleeding index)
RANKL/OPG ratio positively correlated with PPD< CAL, and PI

Discussion: GCF RANKL and OPG levels were oppositely regulated in periodontitis but no gingivitis, resulting in an enhanced RANKL/OPG ratio.

Conclusion: GCF concentrations of RANKL and OPG may be indicative of disease occurrence.

Topic: GCF

Authors: Huynh AH, Veith PD, McGregor NR, Adams GG, Chen D, Reynolds EC, Ngo LH, Darby IB.

Title: Gingival crevicular fluid proteomes in health, gingivitis and chronic periodontitis.

Source: J Periodontal Res. 2015 Oct;50(5):637-49.

DOI: 10.1111/jre.12244. Epub 2014 Nov 29.

Type: clinical

Keywords: gingival crevicular fluid; liquid chromatography mass spectrometry; periodontitis; proteome

Background: In gingivitis, enzymes such as collagenase and b-glucuronidase, as well as cytoskeletal elements were all found to increase in abundance significantly relative to healthy conditions. Alkaline phosphatase, collagenase, osteocalcin, prostaglandin, antigenic elastase and alpha2-macroglobulin, are examples of proteins that increase during periodontitis. Important enzymes such as neutrophil elastase, cathepsin G and proteinase 3 are also elevated due to the phagocytosis and cell lysis that occurs with inflammation. dipeptidyl peptidase II, cathepsin B, aspartate aminotransferase and MMP8 have also been confirmed as biomarkers for periodontal disease

Purpose: to compare the proteome composition of gingival crevicular fluid obtained from healthy periodontium, gingivitis and chronic periodontitis affected sites.

Methods: 15 males for each of three different groups, healthy periodontium, gingivitis and chronic periodontitis. They were categorized based on clinical measurements including probing depth, bleeding on probing, plaque index, radiographic bone level, modified gingival index and smoking status. Gingival crevicular fluid was collected from each patient, pooled into healthy, gingivitis and chronic periodontitis groups and their proteome analyzed by gel electrophoresis and liquid chromatography electrospray ionization ion trap tandem mass spectrometry.

Results:

121 proteins in total were identified, and two-thirds of these were identified in all three conditions.
42 proteins were considered to have changed in abundance.
Cystatin B and cystatin S decreased in abundance from health to gingivitis and further in chronic periodontitis.
Complement proteins demonstrated an increase from health to gingivitis followed by a decrease in chronic periodontitis.
Fibronectin levels were 7.2- and 5.1-fold higher in gingivitis relative to the healthy and chronic periodontitis groups respectively. Its apparent downregulation in chronic periodontitis is in agreement with other studies. This is possibly due to enzymaticdestruction by bacteria such as Aggregatibacter actinomycetemcomitans protease and increased degradation with heightened inflammation
Immunoglobulins, keratin proteins, lactotransferrin precursor, 14-3-3 protein zeta/delta, neutrophil defensin 3 and alpha-actinin exhibited fluctuations in levels.

Conclusion: 121 proteins in total were found, and 42 were considered to have changed in amount. The gingival crevicular fluid proteome in each clinical condition was different and its analysis may assist us in understanding periodontal pathogenesis.

Topic: Gingival crevicular fluid

Author: Nowzari H, Phamduong S, Botero JE, Villacres MC, Rich SK

Title: The profile of inflammatory cytokines in gingival crevicular fluid around healthy osseointegrated implants

Source: Clin Implant Dent Relat Res. 2012 Aug;14(4):546-52

DOI: 10.1111/j.1708-8208.2010.00299.x. Epub 2010 Jul 17.

Type: Clinical

Keywords: Cytokines, gingival crevicular fluid, osseointegrated implants, peri-implant crevicular

Purpose: The aim of the present study was to describe levels of inflammatory cytokines and subgingival microbiology in clinically healthy implant sites as compared with sites in clinically healthy teeth.

Methods:

Subgingival plaque and gingival crevicular fluid (GCF) were obtained from 28 clinically healthy implants and 26 teeth selected from 24 patients
Microbial composition was determined by selective anaerobic culture techniques.
Pro-inflammatory cytokines were quantified by flow cytometry analysis of GCF.
The concentration between implants and teeth were compared with the independent t-test.

Results:

There was an overall trend for higher values of cytokines observed around implants.
The highest value corresponded to IL-8 in implants compared with teeth (SS)
Concentration of TNF-alpha was two-fold higher around implants than teeth (SS)
IL-6 was four times higher around implants compared to teeth (NSSD)
The frequency detection of important periodontopathic bacteria (P. gingivalis, T. forsythia, Fusobacterium sp.) was higher in teeth than implants, but cultivable microbiota were similar.
NSSD for the subgingival microbiota were observed between groups

Bottom Line: The subgingival microbiota around clinically healthy implants presented similar composition to teeth sites. In contrast, the frequency detection of periodontopathic bacteria was higher in teeth as compared with implants. Regardless of gingival tissue health and scare plaque accumulation, the profile of inflammatory cytokines in implant crevicular fluid was distinctive or an innate immune response and in higher concentration than in teeth.

Passage of Antibiotics

Which antibiotics are concentrated in the gingival crevicular fluid?

Topic:Passage of Antibiotics

Authors: Gordon JM, Walker CB, Murphy JC, Goodson JM, Socransky SS

Title:Concentration of tetracycline in human gingival fluid after single doses.

Source:J Clin Periodontol 8:117-121,1981

Type:Clinical

Rating: Good

Keywords:gingival fluid, antibiotics, tetracycline

Purpose:To measure the concentration of tetracycline (TTC) in the GCF following oral administration of single doses.

Materials and methods:6 volunteers were administered single doses of 250-500mg of TTC. GCF was sampled every 15 minutes for the first 2 hours, every 30 mins for the next 2 hours and at 5, 6, and 7 hours. Blood was sampled at 0, 3.5 and 7 hours. A second group of 4 volunteers were administered a single-dose of 250 or 500mg TTC and GCF was sampled every hour for 24 hours. Sample sites were the mesial sites of the first molars. Blood samples were obtained every hour for the first 6 hours and at 9,12,16,20 and 24 hours. Samples were taken with filter paper strips and GCF was measured with GCF meter. The concentration of TTC was determined by comparing the inhibition zones on the paper strips with standard strips containing known concentrations of TTC.

Results:The concentration of TTC in GCF after a single oral dose of 250 or 500mg peaked at 3.5-7 hours achieving average peak levels of 5-12µm/ml. Blood levels peaked at 3-4 hours and had concentrations of 1.0-2.6 µm/ml. After a brief lag period TTC persisted at higher levels in the GCF than in blood for at least 19 hours after administration however by 24 hours was rarely detectable. GCF levels varied from between the individuals and between each site in the same individual.

Conclusion:TTC in GCF was typically 2-10 times the amount in blood levels after a single dose. The presented investigation opens alternative means for the clinician and research worker to optimize therapeutic benefit derived from a given antimicrobial agent. Rational selection of the antibiotic clearly depends on knowledge of levels typically attained in the periodontal pocket and the intrinsic antibiotic susceptibility of pathogens present in the individual patient.

Topic:Host defense

Author:Gordon JM, et al

Title:Tetracycline: Levels achievable and in vitro effect on subgingival organisms. Part I. Concentrations in crevicular fluid after repeated doses.

Source: J. Periodontol. 52:609, 1981

Type:Clinical trial

Keywords:gingival crevicular fluid, tetracycline, antibiotic, biomodulation

Purpose:To measure the concentration of tetracycline in gingival fluid following the oral administration of multiple doses of 250 mg.

Materials and methods:

2 volunteers received doses of 250 mg of TCN every 12h, & 2 volunteers received it every 6h for 5d.
GCF was sampled at intervals from hours 0 to 15, 21 -36, 48-60, and 96-102.
Blood was sampled at 3-hour intervals to compare with the GCF. All pts had minimal or no clinically evident signs of inflammation.

Results:

Volunteers given 250mg every 6h had an average GCF concentrations between 4-8μg/ml and blood concentrations between 2-2.5μg/ml at 48h; these were approximately double the levels achieved by pts given TCN every 12h were the GCF was 2 to 4 μg/ml and the blood concentrations were 0.3-1.4μg/ml. These differences were SS.

Discussion:

TCN is rarely detectable in GCF 19h after a single dose and are unlikely to be inhibitory at 24 h. The reason for this concentration of TCN in the GCF is not clear, but it may be related to the elevated calcium content of the GCF.

BL:After repeated doses of TCN, the GCF is typically 2-4 times blood levels.

Topic:Gingival crevicular fluid

Authors:Pascale D et al

Title:Concentration of doxycycline in human gingival fluid.

Source:J. Clin. Periodontol. 13: 841, 1986

Type:Clinical study

Keywords: doxycycline, tetracycline, antibiotics, gingival fluid

Background: The recognition that periodontal disease is caused by specific microrganisms has led to increased interest in the use of antibiotics as adjunctive treatment. To be effective in the treatment of periodontal disease, an antibiotic must penetrate to the site of infection. Tetracycline hydrochloride and minocycline have been shown to have gingival fluid levels in excess of blood levels. Doxycycline is a synthetic tetracycline compound with advantages over tetracycline hydrochloride, increased oral absorption, prolonged serum half-life and decreased GI side effects.

Purpose: To measure the concentration of doxycycline in gingival fluid and blood after oral administration.

Methods: 4 volunteers were given doses of 100 mg doxycycline q 12 h on 1^st day of antibiotic regimen, then a maintenance dose of 100 mg per day for an additional 4 days. 3 of these volunteers were also given 250 mg tetracycline hydrochloride q 6 h for 5 days either 1 month before or after doxycycline to compare gingival fluid levels of these 2 tetracycline compounds. Gingival fluid was sampled from 4 gingival sites (MB sites of 1^st molars) in each volunteer at hours 0 – 6 then 9, 24, 27, 48 – 54, 57, 72, 75, 96 – 102 and 105. Blood was sampled (finger puncture) at hours 0, 3, 6, 24, 48, 54, 72, 96 and 102. Gingival fluid levels from the 4 sites (mesiobuccal surface of first molars) were averaged for each time interval. Antibiotic levels in GCF and blood were measured using an agar diffusion assay method.

Results: Gingival fluid levels of doxycycline were higher than blood levels at all times sampled and were significantly higher at 48, 72, 96 and 102 hrs. Overall, tetracycline achieved slightly higher gingival fluid levels than doxycycline. The difference was not stastistically significant. There was also no SSD between blood levels of the two antibiotics. Doxycycline levels in gingival fluid ranged between 1.2 µg/ml and 8.1 µg /ml in the first 24 h and generally achieved 3-10 µg /ml after 48 h. Blood levels after 48 h ranged between 2.1 µg /ml and 2.9 µg /ml. Tetracycline levels in gingival fluid after 48 h were generally in the range of 4 µg /ml-10 micrograms/ml with blood levels between 2.2 µg /ml and 3.4 µg /ml.

Conclusion: Doxycycline will achieve higher levels in the GCF than in the blood. The levels detected in gingival fluid for doxycycline were comparable to tetracycline hydrochloride gingival fluid levels. Due to diminished side effects and efficacious levels in GCF, doxycycline may be favorably considered as an alternative to tetracycline in periodontal therapy.

Topic:gingival fluid and saliva

Authors: Ciancio SG, Mather ML, McMullen JA.

Title:An evaluation of minocycline in patients with periodontal disease.

Source:J Periodontol. 1980 Sep;51(9):530-4. DOI: 10.1902/jop.1980.51.9.530

Type:clinical

Keywords:gingival crevicular fluid, minocycline, saliva, drug concentration in saliva and GCF,

Purpose:To determine the passage into and concentration of minocycline in gingival crevicular fluid (GCF) and the relationship between its concentration in saliva, GCF, serum and changes in periodontal health.

Methods:20 adults with gingivitis and/or periodontitis were included. Over an 8-day period, 10 subjects received 100 mg minocycline twice a day (200mg) orally (Group 1), and 10 subjects received 50 mg minocycline in the A.M and 100 mg minocycline in P.M (150mg). Patients were told to take medication 1 hr prior to morning appointments. The parameters evaluated: DMF, GI, PI, crevice/pocket depth, soft tissue, SMA-12 (a blood chemistry screen test), CBC, prothrombin times, and concentrations of minocycline in serum, saliva and GCF (by modified Bennet method) from 1 to 7 hrs after taking the medication. DMF score, PD, CBC and prothrombin time were determined on days 1 and 8. All other parameters were evaluated on days 1, 2, 3, 5 and 8.

Results:Minocycline administration resulted in no significant changes in blood chemistry or blood counts. Prothrombin time was not altered (but diet was not considered). Minocycline was present in GCF>serum>saliva.The antibiotic levels in serum and GCF reached bacteriostatic concentrations on day 1 and remained bacteriostatic throughout the study. GI scores were markedly reduced in this study. PI was also reduced. Significant reduction in PD in group 1 but not in group 2. The remaining data showed no significant difference between groups 1 and 2. 4 people in group 1 reported vertigo by the 3rd day. A dose of 150 mg/day should be adequate. GCF concentration of minocycline was 500% of that of serum and its concentration in saliva was approximately 6%.

Conclusion: Minocycline is effective against oral microorganism. It is present in serum at therapeutic levels with either 150 or 200 mgs/day, is concentrated in GCF 5x higher than serum and produces an improvement in gingival health.

Topic:Pharmacological efficacy

Author:Britt MR, Pohlod DJ

Title:Serum and Crevicular fluid concentrations after a single dose of metronidazole

Source:J. Periodontol. 57:104-107, 1985

Type:Clinical trial

Keywords:Metronidazole; GCF concentrations; Serum concentrations

Background:No study at that time analyzed the concentrations of metronidazole in serum or GCF for an extended period of time, greater than 4 hours, with dose levels of 100-150mg p.o. as used in clinical trials

Purpose:To demonstrate the effectiveness of metronidazole as a site-specific therapeutic agent in treating periodontal disease over 18 hours.

Methods:6 female patients (age 22 to 47) with no clinical or radiographic evidence of advancing periodontitis, each with14 maxillary teeth. To increase the flow rate of gingival fluid they were told to stop brushing the upper teeth for 14 days. One 250mg tablet of metronidazole was taken at 5am on test day without eating after midnight. Samples of GCF and serum were taken at 1, 2, 3, 4, 5, 6, 7, 8, 12, 18 hours after the single oral dose. Metronidazole and its metabolites were assayed and statistical analysis was performed.

Results:Drug levels were detectable by the first hour in both serum and GCF. In serum the mean concentration peaked by the 2^ndhours. In GCF, the mean concentration peaked at the 2^ndhour and again at 7^thhour. Mean drug concentrations in GCF never exceeded those of serum (NSSD). In both fluids drug levels were detectable for up to 18 hours after the single dose.

Discussion:The second peak observed at 7 hours, has been shown in other drugs also. This second peak is possibly due to enterohepatic recycling of the drug.

Conclusion:Metronidazole can be found in sufficient concentrations in both serum and GCFto inhibit a wide range of suspected periodontopathogens. Metronidazole demonstrates site specificity as a potential chemotherapeutic agentin treating periodontal infections caused by obligate anaerobes.

Topic: Antibiotics in GCF

Author: Lai PC, Ho W, Jain N, Walters JD

Title: Azithromycin concentrations in blood and gingival crevicular fluid after systemic administration

Source: J Periodontol. 2011 Nov;82(11):1582-6

DOI: 10.1902/jop.2011.110012

Keywords: Anti-infective agents, periodontitis, pharmacokinetics

Purpose: To determine if after systemic administration, steady-state azithromycin concentrations in GCF are higher and more sustained and the corresponding concentrations in blood.

Methods: 4 healthy adult volunteers with no attachment loss were recruited. Patients were 500 mg of azithromycin initially, followed by 250 mg doses at 24 and 48 hours to obtain steady state levels. GCF samples were collected immediately before the first dose, 2 hours after the last dose on day 2 and on days 4 and 7 after the initial dose. Paper strips were used to collect samples. Blood samples were collected on days 2, 4, and 7. Samples were evaluated for azithromycin content and compared.

Results: A reduction in pooled volume of GCF was seen after azithromycin administration. Mean concentrations of GCF were significantly higher than those in serum on days 2, 4, and 7. The perfusion rate of azithromycin into the sulcus also did not change significantly at any time point.

Discussion: Results demonstrate that systemic administration of azithromycin produces relatively hight and sustained levels in GCF and provide a rationale for further clinical evaluation of its adjunctive benefits in the treatment of periodontitis.

Topic: antibiotics and GCF

Authors: Jain N, Lai PC, Walters JD

Title: Effects of gingivitis on azithromycin concentrations in gingival crevicular fluid

Source: J Periododontol 2012; 83(9):1122-8

Type: clinical study

Keywords: gingival crevicular fluid, gingivitis, systemic antibiotics, azithromycin, split mouth study

Purpose: To assess the influence of gingivitis on azithromycin in GCF through a prospective split mouth study.

Methods: 9 students from Ohio State University were recruited, pts were systemically healthy with no periodontal disease. Baseline measurements and GCF were taken from experimental and control sites. Experimental gingivitis was induced using a splint to cover one randomly selected sextant, contralateral sextants were used as controls. After 3 weeks of gingivitis induction, patients received 500mg of azithromycin followed by 250mg dose 24 hours later. 4 hours after the second dose, plaque was removed from the experimental sites. GCF was collected from 8 surfaces on both the experimental and control sites and pooled separately. GCF samples were collected on the 2^nd, 3^rd, 8^th and 15^th days and azithromycin content was determined by agar diffusion bioassay.

Results:

At baseline, there were no significant differences between control and experimental sites.
Within 21 days, GI and PI increased to a mean of 1.5 at the experimental sites.
After initiation of azithromycin regimen PI and GI decreased. GI was back to baseline 7 days after plaque was removed from experimental sites.
GCF Volume
- Control sites: No change in pooled GCF volume
- Experimental sites: GCF volume increased 2-fold. And was SS higher than control. By Day 3 after azithromycin GCF volumes did not differ.
Total Azithromycin levels
- Total azithromycin was consistently higher than control from day 1-3, it was SS higher on day 2 and 3 and then no further differences were seen.
Azithromycin concentration
- Azithromycin concentration was higher in control GCF on day 1 and thereafter there was no difference. Azithromycin concentration in GCF was higher than serum.

Conclusion: Azithromycin concentration are similar in GCF from gingivitis sites and healthy sites, suggesting that the processes that regulate GCF azithromycin concentration can compensate for local inflammatory changes.

Topic: Antibiotic efficacy

Author: Belibasakis GN, Thurnheer T.

Title: Validation of antibiotic efficacy on in vitro subgingival biofilms.

Source: J Periodontol. 2014 Feb;85(2):343-8.

DOI: 10.1902/jop.2013.130167

Type: In Vitro

Purpose: To test the antimicrobial efficacy of five common antibiotic schemes at physiologically relevant concentrations on a multispecies in vitro biofilm.

Materials and methods:

10 species biofilm consisiting of Steptococcus oralis, Streptococcus anginosus, Actinomyces oris, Fusobacterium nucleatum, Veilloelaa dispar, Campylobacter recturs, Prevotella intermedia, P.gingivalis, T. Forsythia, and T. denticola.
Exposed to 15 ug/ml metronidazole, 15 ug/ml amocicillin, metronidazole and ampxicillin combination, 2ug/ml doxycycline, and a\10 ug/ml azithromycin. For 24 hours
Species-specific bacterial numbers were determined by culture on selective agar media or by epifluorescence microscopy

Results:

Metronidazole alone did not have any significant effect on the total bacterial counts or any of the individual 10 bacterial species of the biofilm
Amoxicillin alone caused a significant reduction in total bacterial numbers , P. gingivalis , F. nucleatum , and S. anginosus. P. intermedia numbers also exhibited a reduction.
metronidazole and amoxicillin caused significant reductions in all the species affected by amoxicillin alone, and additionally on C. rectus. It was found that the reduction in numbers of this species was more pronounced than when amoxicillin was administered alone.
Doxycycline and azithromycin caused a significant reduction in the same species as the combination of amoxicillin and metronidazole, albeit numerically to a lower extent.

Conclusion: The findings validate that the commonly used systemic antibiotic regimens in periodontal treatment do not dramatically reduce total bacterial loads, but they exert a species-specific reduction in targeted subgingival biofilms. This constitutes biologic proof of the clinical principle that antibiotics alone do not have an overall effect on the biofilms and justifies further the necessity of mechanical debridement in periodontal treatment.

Topic:drug therapy

Authors: Conway TB, Beck FM, Walters JD

Title:Gingival fluid ciprofloxacin levels at healthy and inflammed human periodontal sites

Source:J Periodontol 71:1448-1452, 2000

DOI: 10.1902/jop.2000.71.9.1448

Type:cross-sectional study

Keywords:gingival crevicular fluid/analysis; ciprofloxacin/therapeutic use; serum/analysis; neutrophils; periodontal diseases/drug therapy; follow-up studies; cross-sectional studies

Purpose:PMNs take up and accumulate ciprofloxacin. This may allow them to enhance drug delivery in the inflamed periodontium. The purpose of this study was to determine if systemic ciprofloxacin attains higher concentrations in GCF from inflamed periodontal tissues than it does in the blood stream or GCF from healthy sites. The effect of SRP on GCF ciprofloxacin levels was also investigated.

Methods:2 groups were recruited: 7 subjects without periodontal disease and good OH and 8 subjects with untreated adult periodontitis (PD ≥5 mm, mod-adv bone loss in at least 2 quads). Three doses of 500 mg of ciprofloxacin were given to both groups to establish steady state tissue levels of the drug and samples were taken 28 hours after the first dose of Cipro. The diseased group continued Cipro (500 mg bid) for another 7 days. GCF was collected from 12 interproximal sites with paper strips in healthy patients. In diseased subjects, 2 quads with the most severe disease were selected and 12 paper strip samples were obtained. Venous blood was drawn on all subjects. After initial collection, 1 quadrant in the disease group was selected for SRP (split-mouth design). These patients returned 196 hours after the first Cipro dose for GCF and serum samples. GCF and serum Cipro content was measured with liquid chromatography

Results:In both healthy and diseased patients, GCF Cipro levels were significantly higher than the serum levels. There was no difference in GCF Cipro levels in the healthy and diseased groups. At 196 hours, the mean pooled GCF volume at root planed sites was 16% lower than at untreated control sites.

Conclusion: The results of this study suggest that inflammation has no significant impact on GCF levels of Cipro. Mean levels of GCF Cipro were 4-5 fold higher than in serum, regardless of periodontal status. The increased availability of this drug in GCF should enhance its antimicrobial effects against susceptible subgingival microorganisms. The total amount of ciprofloxacin delivered to inflamed tissues was greater than at healthy sites because GF flow was higher.

Topic: GCF

Authors: Needleman IG, Grahn MF, Pandya NV

Title: A rapid spectrophotometric assay for tetracycline in gingival crevicular fluid

Source: J Clin Periodontol. 2001 Jan;28(1):52-6.

DOI: 10.1034/j.1600-051x.2001.280108.x

Type: Clinical

Keywords: periodontal diseases; tetracycline; gingival crevicular fluid; assay

Purpose: The aim of this study was to investigate rapid spectrophotometric assay for its potential to measure tetracycline levels in gingivalcrevicular fluid (GCF).

Material and methods: The technique involves complexation of tetracycline with molybdenum in order to shift the absorbance spectrum away from that region where interference with plasma proteins is a problem. The sensitivity of the assay and reproducibility of elution were examined together with an assessment of the effect of plasma proteins. The assay was also tested in a small pilot clinical project, measuring tetracycline levels in GCF following placement of a test gel formulation in 25 periodontal pockets in 5 patients.

Results: The in vitro results showed good sensitivity of the assay over the concentration range tested (0.5-200 microg tetracycline) and with little effect of plasma proteins. Elution from the paper strips was reproducible with a good linear correlation between direct and filter absorbed assays. The pilot clinical study indicated a mean half-time of tetracycline in GCF of 28 min with confidence intervals of 21 to 34 min, although wide variation between the drug levels of individual periodontal pockets was seen.
Conclusion: The results indicate good sensitivity for this assay to measure tetracycline hydrochloride in vivo. The potential for rapidly processing large numbers of samples contrasts with the assay time and limited sample throughput of other methods such as high pressure liquid chromatography (HPLC) and suggests that the technique may be a useful addition to current techniques for measuring tetracycline hydrochloride in vivo.

Indicators of Disease Activity

Can gingival fluid contents be used to determine severity of periodontal conditions or disease activity?

Topic:Prostaglandin E2 in crevicular fluid

Authors:Leibur E, Tuhkanen A, Pintson U, Soder P-O

Title: Prostaglandin E2 levels in blood plasma and in crevicular fluid of advanced periodontitis patients before and after surgical therapy.

Source: Oral Diseases5:223-228, 1999.

DOI: 1354-523X/99
Type:clinical study

Keywords:prostaglandin E2, blood plasma, gingival crevicular fluid, periodontitis surgical treatment.

Purpose:To determine PGE2 levels in venous blood plasma (VBP), gingival blood plasma (GBP) and GCF in advanced periodontal patients before and after surgical treatment.

Material and methods:12 periodontal patients, 28-45 years old and 7 periodontally healthy patients, same age, were used as controls. During the pre-study period of 7 days patients in both groups received professional cleaning. Clinical parameters (PI, GI, BI, PD, AL) and radiographic (panoramic) bone height were measured before and 6 months after surgical therapy (surgery involved autogenous bone graft in periodontal defects). PGE2 were determined by radioimmunoassay, and measured prior to surgery in venous blood plasma, gingival blood plasma, and GCF, and after surgery as well.

Before treatment

After Treatment

Diseased sites of periodontal patients had higher plaque levels and more severe gingival inflammation than healthy sites.
Diseased sites had significantly deeper mean PD and lower BH (bone height) % than that in control subjects. At baseline means of PI, GI, BI had higher values than that of control.
The mean VBP, GBP and GCF PGE₂levels in periodontal patients were higher than healthy controls.

The values of clinical parameters as PI, GI, BI, PD and AL diminished and there was no statistically significant differences between the data of clinical parameters of the control subjects and the patients after treatment.
After 6 months treatment there was no significant differences between the means of BH% as compared with the same parameter of the control.
6 months after Treatment, the mean levels of PGE₂in VBP, GBP and GCF were significantly reduced in patients with improvement of clinical and radiographic parameters

The mean levels of PGE₂in GBP and GCF before treatment were significantly higher compared to the mean level of PGE₂after treatment.

C: The present study showed that PGE₂is involved in the pathogenesis of periodontal disease. The inflamed periodontal tissues may produce significant amounts of PGE₂and the degree of inflammation might be determined by the ratios of PGE₂.

BL: The lower levels of PGE₂in blood plasma and in gingival crevicular fluid after treatment are signs of improvement of periodontal disease.

Topic:Antibiotics

AuthorsLamster IB, Oshrain RL, Harper DS

Title:Enzyme activity in crevicular fluid for detection and prediction of clinical attachment loss in patients with chronic adult periodontitis. Six month results.

Source: J. Periodontol. 59: 516-523, 1988.

DOI: 10.1902/jop.1988.59.8.516.

Type:Clinical study

Rating: Good

Keywords:Antibiotics, GCF

Purpose: To study the relationship of changes in GCF levels of the vertebrate lysosomal enzymes B-glucuronidase, arylsulfatase and the cytoplasmic enzyme lactate dehydrogenase in reference to attachment loss in patients with existing periodontitis.

Method: Longitudinal study 36 patients were followed up for 6 months. Patients had existing chronic adult periodontitis (minimal 20 natural teeth, and at least two teeth in each quadrant ≥5mm attachment loss). CAL (baseline-3m-6m), GCF (baseline-3m) were recorded. GCF was collected using filter paper strips, from mesial surface of the 4-5 most distal teeth. 681 samples analyzed at baseline and 3 months. Three groups of patients were identified based on disease progression:

Group I (5 pts) – generalized form of disease activity (CAL >2 mm at least 3 sites)

Group II (4 pts) – localized form (CAL >2.5 mm at 1-2 sites)

Group III (27 pts) – did not display CAL

Enzyme analysis was evaluated as a whole mouth score (the percent of samples from a patient in which enzyme activity was at least twice the population mean) and at individual samples.

Results:

Group I: elevated whole mouth scores for B-glucuronidase

Group II: could not be identified by whole mouth scores for any of the enzymes, but individual site of B-glucuronidase
B-glucuronidasewas found to be a predictable indicator of attachment loss. It is associated with PMN granule releaseà clinical attachment loss can be related to exuberant PMN response. B-glucuronidase is a common marker for attachment loss.
Arylsulfatase and lactate dehydrogenase had NSSD in measurement of localized clinical attachment loss in patients with at least 2.5 mm attachment loss in two sites before samples were taken.

Conclusion:An exuberant PMN response is related to clinical attachment loss in patients with existing chronic periodontitis.

Topic:Gingival crevicular fluid proteases

Authors:Bader HI, Boyd RL

Title:Long-term monitoring of adult periodontitis patients in supportive therapy: Correlation of gingival crevicular fluid proteases with probing attachment loss.

Source:J Clin Perio26:99-105,1999.

DOI: 10.1034/j.1600-051x.1999.260206.x

Type:Longitudinal retrospective study

Rating:Fair

Keywords:periodontits; proteases; probing attachment loss; disease activity; longitudinal studies

Purpose:To determine if a chair-side assay for neutral protease activity in GCF could provide an early indication of site-specific disease activity as defined by probing attachment.

Method:The cohorts in this longitudinal retrospective study were selected from one private practice limited to periodontics. 38 subjects that had undergone periodontal therapy and were selected from a maintenance program with 3 or 6 months recall intervals. All sites selected had ≥4mm CAL loss. Samples of GCF were collected from 71 selected sites using paper strips. The assay is designed to detect only the active and not the total amount of enzyme present in the GCF sample. A score of 0 and 1 was considered negative and a score of 2 was positive. Positive BOP and positive neutral protease activity (NPA) scores were classified as true positives if sites subsequently lost at least 1mm of CAL over the next 12 months. Sites were monitored at 6-month intervals for 24-36 months.

Result: As a predictor of breakdown the NPA assay had an accuracy of 94% and a risk ratio of 37.6 as compared to values of 58% and 1.5 for BOP. When only the subset of sites ≥7mm were considered the NPA assay had a calculated accuracy of 92% vs. a value of 50% for BOP.

Conclusion: The results indicate that the NPA assay appears to differentiate between bleeding at sites exhibiting only chronic inflammation with no CAL loss and bleeding at sites undergoing active CAL loss.

Topic:Indicators of Disease Activity

Authors: Lamster IB, Ahlo JK

Title:Analysis of gingival crevicular fluid as applied to the diagnosis of oral and systemic diseases.

Source:Ann N Y Acad Sci. 1098:216-29, 2007

DOI: 10.1196/annals.1384.027

Type:Discussion

Keywords:gingival crevicular fluid; diagnosis; periodontal disease

Purpose:A review of GCF and its applications in diagnosing oral and systemic diseases.

Discussion:GCF can be either a serum transudate or more commonly as an inflammatory exudate. It reflects the constituents of blood, cells and tissues of the peridontium. As such, it has been studied to identify active periodontitis.

Collection of GCF includes using micropipettes, appliances to isolate and collect fluid, and filter paper strips (methylcellulose). Attention must be given during paper strip placement in order not to be contaminated with plaque, blood or saliva. The strip must remain in the sulcus long enough to obtain adequate amount of fluid. The volume of GCF may be related to inflammation and permeability/ulceration of crevicular epithelium. 65 GCF components have been examined as potential diagnostic markers of disease progression which are classified as enzymes, inflammatory mediators and tissue breakdown by-products. Loos and Tjoa identified 8 possibly valuable markers: alkaline phosphatase, -glucuronidase, cathepsin B, MMP-8, MMP-9, DPP II and III, and elastase. The predominance of enzymes associated with tissue breakdown clearly emphasizes the importance of the inflammatory response to the pathogenesis of periodontitis. Limitations of application of using this for diagnostic testing include needing to collect samples from multiple sites on a patient, isolation of GCF is difficult, and laboratory tests are not routinely used.

-Systemic Diseases

Diabetes: PGE2 and IL-1B have been found in higher concentrations in type 1 DM patients, regardless of severity of the disease. Higher IL-6 levels have been seen in type 2 DM compared to adult periodontitis and healthy controls.

Smoking: Smokers demonstrate elevated levels of IL-8 and lower levels of IL-4. IL-6 was elevated in with aggressive periodontitis that smoked. IL-1α was lower in smokers.

CVD: Elevated leukotrienes were found in GCF of patients with atherosclerosis (with or without periodontitis).

HIV: High levels of IL-1B, IL-6 and TNF-A and IFN-gamma have been found in HIV patients with periodontitis suggesting the elevated inflammatory mediators may play a role in the bony destruction in these patients.

Conclusion:Analysis of GCF has contributed to understanding the role of inflammation in the periodontal disease process but sampling is impractical in the clinical setting. Saliva collection is a less technique sensitive and more amenable to chairside utilization.

Topic: Biomarkers

Author: Guzman YA, Sakellari D, Arsenakis M, Floudas CA

Title: Proteomics for the discovery of biomarkers and diagnosis of periodontitis: a critical review

Source: Expert Rev Proteomics. 2014 Feb;11(1):31-41

DOI: 10.1586/14789450.2014.864953

Type: Review

Keywords: 2D gel electrophoresis, aggressive periodontitis, biomarkers, chronic periodontitis, gingivitis, gingival crevicular fluid, shotgun proteomics, whole saliva

Purpose: To discuss the experimental design/clinical application and benefit of diagnostic biomarkers.

Methods: 17 proteomics studies that utilize different biological mediums and mass spectrometry platforms were analyzed in this review study.

Selection of sample medium
Saliva, serum, subgingival plaque, gingival tissue and gingival crevicular fluid
GCF contributes a small percentage of the total protein content of saliva
GCF sampling is more technically demanding, and whole-mouth evaluations are difficult
Experimental workflow
By-protein analyses with separation by gel electrophoresis
Digestion with high throughput liquid chromatography

Results:

MMPs were reported as upregulated in periodontitis
Exposure to red complex group bacteria induced actin rearrangement gingival fibroblast detachment
HDL downregulated and LDL upregulated in periodontitis
Vitamin D binding proteins upregulated in periodontitis

Conclusion: Due to their sensitivity and global scale, proteomics studies offer the opportunity to uncover critical host and pathogen activity indicators and can elucidate clinically applicable biomarkers for improved diagnosis and treatment of the disease. This review summarizes the literature of proteomics studies on periodontitis and comprehensively discusses commonly found candidate biomarkers. Key considerations in the design of an experimental proteomics platform are also outlined. The applicability of protein biomarkers across the progression of periodontitis and unexplored areas of research are highlighted.

Saliva

How does saliva play a role in a patient’s susceptibility or resistance to periodontal disease ?

Topic: saliva diagnostics

Authors: Liu J, Duan Y

Title: Saliva: a potential media for disease diagnostics and monitoring

Source: Oral Oncol. 2012; 48(7): 569-77

Type: review

Keywords: saliva, biomarkers, disease diagnosis, periodontal disease, oral SCC, cancer, sjogren’s syndrome

Purpose: Review of the properties of saliva, the salivary analysis for biomarker discovery, and the diagnostic potentials of salivary biomarkers in diagnosing and monitoring periodontal disease, oral and breast cancers and Sjogren’s syndrome.

Discussion:

Saliva Profile
- 99% water, inorganic and organic compounds
Saliva is a potential source for diagnosis because of exchange with substances in serum via thin epithelial lining of the salivary ducts
Biomarker: characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes or pharmaceutical responses to a therapeutic intervention
Salivary Biomarkers
- Oral squamous cell carcinoma (OSCC)
  - Common malignant tumor which is increasing in frequency
  - Detection currently based on expert clinical examination and histological analysis
  - Biomarkers may help screen high-risk patients
  - Salivary biomarkers have been seen to be altered in OSCC
- Periodontal disease
  - Salivary biomarkers could aid in diagnosis and treatment planning
  - MMP-8 -candidate for diagnosing disease progression
  - Fibronectin -reduced in periodontitis patients
  - IgA associated with aggressive periodontitis
  - No biomarker for periodontal disease has been validated
- Cancer
  - Prostate cancer, ongoing research into potential biomarkers
  - Breast cancer, potential biomarkers in saliva include VEGF, EGF, CEA, c-erbB-2
  - SCC of the tongue, salivary levels of IL-1a, IL-6, IL-8, VEGF-a, TNF-a could serve as potential biomarkers for cancer screening and early detection
- Sjogren’s Syndrome
  - Characterized by dry mouth and dry eye, one of the most common autoimmune disorders
  - Changes in saliva include increased Na, Cl, IgG, lysozyme, MMP-2, MMP-9

Conclusions: Since saliva collection is less invasive than that of blood, it is a convenient diagnostic fluid. Many salivary biomarkers need to be further validated and do not currently meet sensitivity or specificity goals to be diagnostic.

Topic: salivary diagnostics

Authors: Fuentes L, Yakob M, Wong DT.

Title: Emerging horizons of salivary diagnostics for periodontal disease.

Source: Br Dent J. 2014 Nov;217(10):567-73.

DOI: 10.1038/sj.bdj.2014.1005.

Type: Brief

Introduction: Periodontitis is a multifactorial disease with complex pathogenesis. Although microorganisms are the main etiological agents, genetic predisposition and environmental factors, such as smoking, can alter the host immune-inflammatory response. Gingivitis is common in the United States with reports indicating upwards of 50% of the adult population had gingivitis. A survey of adults in the United Kingdom estimated that 42% of 35–44 years old and 70% of 55–64 years showed evidence of per- iodontitis,21 and similar results were found in American adults. The field of salivary diagnostics to allow risk determination for both oral and systemic diseases is advancing. Researchers are looking into the use of saliva as a diagnostic medium that would be able to aid clinicians in risk determination, diagnosis, and treatment planning for periodontal diseases.

Saliva as a diagnostic medium: Biomarkers for the detection of diseases, such as caries, oral cancer and periodontal disease, as well as systemic disease such as hepatitis, Sjögren’s syndrome, breast cancer, pancreatic cancer and HIV have been shown to be present in saliva. Whole saliva contains a mixture of fluids from the major and minor salivary glands, gingival crevicular fluid (GCF), serum, immune and epithelial cells, and many microbes.53 A large body of scientific research has focused on GCF biomarkers that communicate site- specific periodontal disease progression; however, it is more difficult to implement clinically due to the possibility for salivary contamination, difficulty to probe all tooth sites and potentially statistical method error.

Current Detected Biomarkers for periodontal disease: Matrix metalloproteinases (MMPs) are zinc-dependent proteases that are known to be associated in diseases such as arthritis, atherosclerosis, as well as periodontitis, because they are involved in the degradation of various extracellular, pericellular and non-matrix substrates. A study conducted at the University of Kentucky reported that MMP-8 was higher in chronic adult periodontitis patients than healthy controls (AUC: 0.92).74 The study also showed this same high detection capabilities in IL-1β and interleukin-6 (IL-6) (AUC: 0.95 for both) and when these three protein markers were combined the AUC elevated to 0.984 with a sensitivity of 0.94 and specificity of 0.966. MMP-9, previously shown to increase with periodontal disease severity. Other protein biomarkers in saliva for periodontal disease have recently been studied. Quantitative proteomics was analyzed to determine the alterations in the salivary proteome before and after periodontal treatment was administered. The prominent findings were for proteins S100A6, S100A8, and S100A9 where abundance increased by fold changes of 1.64, 2.31, and 1.99, respectively. Levels of S100A8/A9 have previously been shown in GCF and saliva to correlate with periodontitis likely due to active secretion by gingival keratinocytes and neutrophils that are in ltrating.

8-Hydroxydeoxyguanosine (8-OHdG) is an oxidized nucleoside that is commonly used as a marker for oxidative DNA damage in inflammatory diseases. 8-OHdG was further studied in saliva to determine its potential as a marker for periodontitis disease activity in a Turkish population. Results found that 8-OHdG levels of the chronic periodontitis group were statistically higher than healthy and chronic gingivitis subjects.

Point of care devices: The goal of salivary diagnostics is to be able to provide information regarding a number of oral and systemic disease status results to clinicians and patients during the time of a regular check-up. A main hindrance has been that many biomarkers are available in very low quantities in saliva, therefore making detection sensitivity a challenge.

Topic: Saliva

Authors: Taylor JJ, Preshaw PM

Title: Gingival crevicular fluid and saliva

Source: Periodontol 2000. 2016 Feb;70(1):7-10

DOI: 10.1111/prd.12118

Type: review

Keywords: gingival crevicular fluid; saliva; physiologic function; systemic health

Purpose: To discuss the role of gingiva crevicular fluid and saliva in physiological function, maintenance of oral tissues and defense

Discussion: Saliva from major/ minor salivary glands is critical for physiological function and host defense against infection. Understanding physiologic and pharmacologic regulation of salivary excretion is critical in clinical management of xerostomia. Saliva biomarkers for periodontal disease diagnosis and monitoring has gained popularity due to easy collection and contains a large proportion of total gingival crevicular fluid. Due to limited understanding of molecular details of periodontal disease pathogenesis restricts identification of possible biomarkers. Zhang et al. describe approaches to biomarker discovery using global analysis of RNA and proteins in the saliva. These approaches are beginning to yield valuable information about molecular biology of oral diseases.

Gingival crevicular fluid function and analysis:

GCF is a serum exudate that carries all key molecular and cellular components of the immune response that are needed to prevent tissue invasion by subgingival plaque bacteria. Collection and analysis has been a popular approach to investigating localized inflammatory processes but has clinical and technical challenges.

Control of host/ microbiome interactions in the soft/ hard tissues of the mouth is central to maintaining oral health. Saliva has many molecular elements that modulate pathogenic microorganisms. Consequences of salivary hypofunction include candidiasis, burning mouth syndrome and lichen planus.

Matrix metalloproteinases have been studied as biomarkers for periodontitis and have been highlighted for application in the analysis in the management of periodontitis and periimplatitis.

An emerging principle is the wider role of cytokines and molecules with cytokine-like activity in other physiologic processes such as development, tissue homeostasis and repair. However, few molecules can yet be described as biomarkers for periodontitis.

Conclusion: Saliva collection is a simple, painless procedure that does not require skilled personnel; therefore, if saliva collection can be coupled with suitable analytic protocols, salivary analysis not only has great potential in large clinical trials, in self-management of disease and in epidemiologic studies.

Both GCF and saliva with application of biomarkers could have oral health benefits. It is critical that this field have strong microbiology backing to understand the complexity of the oral microbiome as well as cell biology/ immunology.

Topic:Host defense

Author:Giannobile WV.

Title:Salivary diagnostics for periodontal diseases.

Source:J Am Dent Assoc. 2012 Oct;143(10 Suppl):6S-11S.

DOI: 10.14219/jada.archive.2012.0341

Type:Review

Keywords:gingival crevicular fluid, tetracycline, antibiotic, biomodulation

Purpose:To review the literature with respect to currently available salivary diagnostics used to identify bacteria prevalent in periodontal disease, and to focus on the future development and use of a variety of rapid detection platforms, such as lab-on-a-chip, as a point-of-care (POC) device for identification of a pt’s risk.

Discussion:

Recent data pertaining to the use of genetic, microbial, and protein saliva-based byproducts support the predictive value to forecast gingival inflammation or periodontal bone destruction in the clinical setting.
New methodological approaches allow researchers to evaluate multiple salivary biomarkers including MMP-8, microbial factors, viruses, and proinflammatory cytokines such as IL-1Beta or IL-17 from a single saliva sample to predict disease.
When investigators consider MMP-8 or MMP-9 with the red complex, they are better able to predict periodontal status. To date, there is no single biomarker that is specific for perio dz.
Therefore, there is strong potential for the use of microbial (initiator) and host-response (responder) biomarkers in combination to enhance identification of the dz process, given the multifactorial nature of the dz.
Although viruses are not strongly implicated in perio dz, the use of multi-analytic techniques that consider both viral and microbial infectious agents in saliva may be beneficial.
With readily accessible and robust salivary diagnostics for the identification of DNA, genome-wide association studies offer significant potential for the discovery of gene expression indicating susceptibility to perio dz modulated by microbial infection (tests for genotype IL-1 already exists).
This type of technology may be used in the future for screening pts for perio dz in a community setting. One of the greatest challenges is not going from bench to chairside, but in going from chairside to clinical practice.

BL:Saliva-based diagnostics offers a promising future for diagnosing perio dz and monitoring tx outcomes.

Topic:Saliva

Authors: Guentsch A, Preshaw PM, Bremer-Streck S, Klinger G, Glockmann E, Sigusch BW.

Title:Lipid peroxidation and antioxidant activity in saliva of periodontitis patients: effect of smoking and periodontal treatment.

Source:Clin Oral Investig. 12(4):345-52, 2008. Epub 2008 May 29.

DOI: 10.1007/s00784-008-0202-z

Type:Clinical study

Keywords: Periodontitis, Oxidative stress, Lipid peroxidation, Antioxidant capacity, Smoking, Saliva

Background: PMNs constitute the first line of cellular host defense against bacteria in the gingival sulcus. PMNs produce reactive oxygen species (ROS), molecules which are capable of initiating periodontal tissue destruction. ROS can cause tissue damage via DNA damage, lipid peroxidation, protein damage, and enzyme oxidation. Several ROS and lipid peroxidation products are produced in physiological quantities in the human body, but an overproduction of ROS occurs at sites of chronic inflammation. During gingival inflammation, GCF flow increases, and components of the inflammatory response are detectable in saliva, including lipid peroxidation products.

Purpose:To examine lipid peroxidation (as an end product of oxidative stress) and corresponding antioxidant activity in patients with periodontitis and assess the influence of smoking and periodontal treatment on these parameters.

Methods: 30 subjects with generalized chronic periodontitis were included. Patients did not have perio tx, abx, or immunosuppressive agents in the preceding 6 months. Periodontally healthy control subjects (n=30) with no evidence of periodontal disease were also recruited. Test and control groups each contained the same proportion of smokers and non-smokers. Subjects with significant systemic dz (diabetes, cancer, coronary heart dz), pregnant or lactating females were excluded. Clinical data were recorded after sample collection (saliva and blood). PDs, BOP were assessed. Non-surgical tx (full mouth sc/rp, maintenance, monitor OH) given in the periodontitis patients. Sample collection and clinical data was recorded following hygiene phase and at 6 months. Malondialdehyde (MDA) (final end product of lipid breakdown caused by oxidative stress), glutathione peroxidase (GSHPx) (antioxidant parameter), and total antioxidant capacity (TAOC) were measured.

Results: Patients with periodontal dz demonstrated significantly higher mean PDs and BOP. There were no SSD in mean PDs between smokers and non-smokers. Mean PDs significantly reduced after periodontal treatment in the periodontitis patients.

MDA: The level of MDA increased progressively from the non-smokers to the smokersof the periodontally healthy controls and then to the non-smokers and smokers of the periodontitis group. Lowest in saliva of the non-smoking periodontally healthy subjects (0.065±0.05 μmol/l). Significantly higher in periodontitis patients who smoked (0.123±0.08 μmol/l). Periodontal tx led to a significant reduction in lipid peroxidation products.

GSHPx: Patients with periodontitis (smokers and non-smokers) demonstrated significantly elevated glutathione peroxidase activitycompared to periodontally health controlled groups. GSHPx activity was significantly reduced after periodontal tx.

TAOC flow rate: Significantly lower in patients with periodontitis (0.34±0.26 μmol/ml) in comparison to the controls (0.62±0.24 μmol/ml; p<0.05). TAOC flow rated did not increase in perio patients after treatment.

– Patients with periodontitis demonstrated more lipid peroxidationthan healthy subjects and smoking enhanced this effect.

Conclusion: Patients who smoke and have periodontal dz demonstrate more lipid peroxidation in saliva than healthy subjects. There was an increase in glutathione peroxidase activity in periodontal dz and an additive effect of smoking was identified. A reduced antioxidant capacity in periodontitis patients was noticed. Successful periodontal therapy had an effect on MDA and GSHPx activity in saliva, but not the antioxidant flow rate.

Topic: Saliva

Authors: Slots J, Slots H.

Title: Bacterial and viral pathogens in saliva: disease relationship and infectious risk.

Source: Periodontol 2000. 2011 Feb;55(1):48-69.

DOI: 10.1111/j.1600-0757.2010.00361.x

Type: Discussion

Keywords: gingival crevicular fluid, bacteria

Purpose: To presents evidence that pathogenic bacteria and viruses can be present in saliva at levels that pose a disease risk for individuals with whom saliva is exchanged.

Discussion:

Periodontogenic Bacteria

Umeda et al. compared the presence of six species of periodontopathic bacteria in whole saliva and subgingival plaque from 202 subjects. Each study subject contributed a whole saliva sample and a paper point sample pooled from the deepest periodontal pocket in each quadrant of the dentition, and the test bacteria were identified using a 16Sribosomal RNA-based PCR assay. A statistical relationship was found between the presence of Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens and Treponema denticola in whole saliva and in periodontal pocket samples, and in the event of disagreement, the organisms were more frequently present in whole saliva than in periodontal pockets (P < 0.01). The oral presence of Aggregatibacter actinomycetemcomitans and Tannerella forsythia was not reliably detected by sampling either whole saliva or periodontal pockets. Other studies also found that a salivary sample alone did not identify all individuals infected with A. actinomycetemcomitans. Taken together, a sample of whole saliva seems to be superior to a pooled periodontal pocket sample for detecting oral P. gingivalis, P. intermedia, P. nigrescens and T. denticola, but samples of both whole saliva and periodontal pockets may be needed in order to detect oral A. actinomycetemcomitans and T. forsythia with reasonably good accuracy. The reason for this is that A. actinomycetemcomitans and T. forsythia can persist in nondental sites, as best demonstrated in fully edentulous individuals. Umeda et al. also investigated risk factors for harboring A. actinomycetemcomitans, P. gingivalis,T. forsythia, P. intermedia, P. nigrescens and T. denticola in periodontal pockets, in whole saliva, or inboth sites (i.e. orally). The study subjects included 49 African–Americans, 48 Asian–Americans, 50 Hispanics and 52 Caucasians living in Los Angeles. Periodontal probing depth was positively associated with all six study bacteria. African–Americans were at in-creased risk (compared with Caucasians) for harboring P. gingivalis in saliva [odds ratio (OR) 2.95]and orally (OR 2.66), and at reduced risk for harboring T. denticola orally (OR 0.34). Asian–Americans showed an increased risk for harboring A. actinomycetemcomitans in periodontal pockets (OR 6.63) and for harboring P. gingivalis in periodontal pockets (OR5.39), in saliva (OR 5.74) and orally (OR 5.81). His-panics demonstrated an increased risk for harboring A. actinomycetemcomitans in periodontal pockets(OR 12.27), and for harboring P. gingivalis in periodontal pockets (OR 6.07), in saliva (OR 8.72) and orally (OR 7.98). Age was positively associated with the prevalence of A. actinomycetemcomitans orally(OR 1.18), and with P. gingivalis in saliva (OR 1.20)and orally (OR 1.20). The male gender was a risk factor for harboring P. intermedia in periodontal pockets (OR 2.40), in saliva (OR 3.31) and orally (OR 4.25), and for harboring P. nigrescens in saliva (OR2.85). The longer the subjects had resided in the USA, the greater the decrease in detection of A. actinomycetemcomitans orally (OR 0.82). Former smokers demonstrated a decreased risk for harboring A. actinomycetemcomitans in saliva (OR 0.23), and current smokers displayed an increased risk for harboring T. denticola in periodontal pockets (OR4.61). Current and passive smokers revealed less salivary P. nigrescens than nonsmokers (127). In sum, the study found a relationship between the presence of periodontopathic bacteria in whole saliva and in periodontal pockets, and pointed to the importance of genetic or environmental factors in the colonization of these pathogens. Salivary tests for periodontitis may show increased accuracy if supplementing infectious disease variables with ethnic and social factors and with smoking habit.

Cariogenic bacteria

The major cariogenic bacteria are mutans strepto-cocci in incipient dental caries and lactobacilli in advanced caries lesions, perhaps in combination with other bacteria of the dental biofilm. After adjusting for age and ethnicity, 6- to 36-month-oldchildren with high levels of Streptococcus mutans were found to be five times more likely to have dental caries than children with low levels of the bacterium. Recent large-scale microbiological studies have linked S. mutans to crown caries in children and adolescents and to root caries in elderly patients. Herpesviruses have been statistically associated with severe dental caries, but their role, if any, in the caries process remains obscure. The finding of a relatively unique cariogenic mi-croflora has a practical implication. Routine testing for elevated caries risk, based on the salivary level of mutans streptococci (>1,000,000 per ml saliva) and lactobacilli (>100,000 per ml saliva), has been per-formed in Sweden for more than 30 years. Repeat swabbing of teeth of young children with 10%povidone-iodine can reduce the number of mutans streptococci and the incidence of caries. Suppression of high levels of S. mutans in the mother may delay or prevent the establishment of the organism in her child.

Medical bacteria

Streptococcus pyogenes (beta-hemolytic group A Streptococcus) is the cause of a variety of human diseases ranging from mild illnesses of the skin or throat (pharyngitis or strep. throat) to severe invasive infections, including necrotizing fasciitis (flesh-eating disease), septicemia, toxic shock syndrome, erysipelas, cellulitis, acute post infectious glomerulonephritis, rheumatic fever and scarlet fever. S. pyogenes normally resides in the throat and is one of the most common medical pathogens in the saliva. An asymptomatic carriage stage of S. pyogenes was detected in approximately 10% of adults and 25% of children, and in as many as 60% of subjects during large outbreaks of streptococcal pharyngotonsillitis. Beta-hemolytic group A streptococci were found in 20% of pharyngeal samples and in 5% of saliva samples of young schoolchildren in New Zealand, with a suggestion of a child-to-child trans-mission of the organism.

Haemophilus influenzae can cause acute bronchitis and exacerbations of chronic obstructive pulmonary disease, as well as meningitis in children and other serious diseases.

Staphylococcus spp., Pseudomonas spp. and Acinetobacter spp. are also potential pathogens in

respiratory (and other) diseases. These bacteria were detected in the oral cavity of 85% of hospitalized

patients in Brazil (216) and in subgingival sites of periodontitis patients in the USA Staphylococcus spp., Pseudomonas spp. and Acinetobacter spp. are also potential pathogens in respiratory (and other) diseases. These bacteria were detected in the oral cavity of 85% of hospitalized patients in Brazil and in subgingival sites of periodontitis patients in the USA.

Meningococcal invasive disease (septicemia and ⁄or meningitis in association with hemorrhagic rash) is a life-threatening condition that primarily affects young children. Meningococcal disease can also occur in teenagers, and is more common in collage ⁄ university students than in the general population (OR 3.4).

Neisseria gonorrhoeae (which causes gonorrhea) and Treponema pallidum (which causes syphilis) can produce acute and chronic oral infections. Gonorrhea is a widespread disease worldwide, with an estimated 600,000 new cases each year in the USA.

Tuberculosis remains a serious disease worldwide. In 2005, there were an estimated 8.8 million new cases of tuberculosis, with 7.4 million occurring in Asia and sub-Saharan Africa, and 1.6 million people died of tuberculosis, including 195,000 with HIV infection.

Viruses

Herpesviruses:

The most prevalent viral family in human saliva that establish a lifelong infection
HSV-1, HSV-2, Varicella-Zoster virus, Epstein-Barr virus, Human Cytomegalovirus, HHV-6, HHV-7, HHV-8.
Herpesvirus infections in the oral cavity can give rise to asymptomatic and unrecognized shedding of virions into the saliva, oral mucosa or periodontium.
Biphasic infection cycle: a lytic, replicative, phase; and a latent, nonproductive, phase.
Conversion from one phase to the other can be caused by environmental stimuli, chemical agents, physical and psychosocial events, or immune suppression from medications/disease.
Systemically healthy adults continually shed many different strains of herpesvirus DNA into saliva.
Periodontal dual infection of herpesviruses and pathogenic bacteria give rise to enhanced cytokine release and immune signaling dysregulation and is shown with more severe periodontitis.

Other Viruses:

Human papillomaviruses are frequent inhibitors of oral mucosa of normal adults and have been found in 25% of healthy individuals, 26% of periodontitis lesions, 92% of cyclosporin induced gingival hyperplasia from renal transplant recipients.
HIV has shown transmission between parents/infants but is thought to be due to chewing food with bleeding gums and not through direct salivary transmission.
Human T-cell lymphotropic virus type 1 was detected in 77% of whole saliva from patients with viral myelopathy.
Hepatitis viruses are also highly transmissible with Hep B having high concentrations in 15% of diagnosed patients, which may be enough to cause horizontal transmission and could be the source of patients with unknown origin of infection.
Hep C virus RNA was present in 59% of GCF of viremic patients.
Influenza virus has been seen in 74% of oral specimens
RSV has been seen in 76% of saliva

Perspectives:

Several oral and medical pathogens occur in saliva sufficient to transmit to close individuals and that saliva may be more infectious than previously realized
Virtually all periodontal patients can benefit from treatment with antiseptics against bacteria and herpesviruses like sodium hypochlorite or povidone-iodine, and some patients may benefit from systemic medication.
Most chronic infectious diseases such as periodontitis and caries are only able to be defeated by mass scale vaccines, which need further studies to make effective in the dental practice.

Topic: RANKL/OPG

Authors: Belibasakis GN, Bostanci N.

Title: The RANKL-OPG system in clinical periodontology

Source: J Clin Periodontol. 2012 Mar;39(3):239-48.

DOI: 10.1111/j.1600-051X.2011.01810.x. Epub 2011 Oct 24

Type: review

Keywords: diagnostics; gingival crevicular fluid; osteoprotegerin; periodontal disease; receptor activator of NF-jB ligand; receptor activator of NF-jB ligand/osteoprotegerin ratio

Background: The receptor activator of NF-κB ligand-osteoprotegerin (RANKL-OPG) bi-molecular system is the “bottle-neck” regulator of osteoclastogenesis and bone resorption, both in physiological and pathological conditions.

Purpose: to elaborate the current knowledge on RANKL and OPG in periodontal disease, and to evaluate their diagnostic and prognostic potential as biomarkers of the disease.

Methods: electronic and manual searches were performed for identifying clinical and in vivo studies on RANKL and OPG in gingival tissue, gingival crevicular fluid, saliva and blood. Smoking and diabetes mellitus were also considered for their potential effects.

Cell sources of RANKL and OPG

RANK: produced mainly by Th1 or Th17 and B cells
Released due to tooth eruptions or orthodontic tooth movements
OPG: produced by connective tissue fibroblast and endothelial cells or t cells

Periodontitis affected tissue

Higher levels of RANKL and lower OPG
RANKL 2.7-15.8 fold higher in chronic perio then in healthy (CP and AP similar levels)

Detection of RANKL and OPG in GCF

RANKL was found highest concentration in GCF of mild periodontitis, not as much moderate/severe
OPG was found higher levels in healthy

RANKL and OPG in Saliva

Not enough research has been done for the detection of RANKL and OPG in saliva

Bacteria and RANKL activation

Subgingival bacteria have correlation with RANKL expression increase (p.ging and t.denticola)

Smoking

Smokers with Chronic perio had lower levels of OPG and higher RANKL (enhanced RANKL/OPG ratio)
Increased exposure to smoking changes RANKL/OPG ratio (20 pack years)

Osteoporosis

Chronic perio with osteoporosis RANKL/OPG ratio was the same however overall had higher levels of RANKL and OPG

Diabetes

Altered RANKL/OPG ratio in periodontium by having lower OPG levels

Perio treatment effect on RANKL and OPG

No decrease in RANKL/OPG ratio however lower overall levels of both
Not a good biomarker for predicting clinical success of treatment

Adjunct and perio treatment

Potassium channel blocker Kaliotoxin: decrease RANKL expression
SRP, Omega-3 fatty acids and aspirin showed reduction of RANKL in saliva

Conclusion: The increased RANKL/OPG ratio may serve as a biomarker that denotes the occurrence of periodontitis but may not necessarily predict on-going disease activity. Its steadily elevated levels post treatment may indicate that the molecular mechanisms of bone resorption are still active, holding an imminent risk for relapse of the disease. Additional adjunct treatment modalities that would “switch-off” the RANKL/OPG ratio may therefore be required.

Topic:gingival fluid and saliva

Authors:Reher VG, Zenóbio EG, Costa FO, Reher P, Soares RV.

Title:Nitric oxide levels in saliva increase with severity of chronic periodontitis.

Source:J Oral Sci. 2007Dec;49(4):271-6.

Type:clinical

Keywords:chronic periodontitis; saliva; nitric oxide; biological marker; diagnostic.

Background:Nitricoxide (NO) has been linked to the etiopathogenesis of periodontal disease. Cytokines and other bacterial products stimulate the expression of inducible NO synthase (iNOS) and interfere with periodontal disease progression.

Purpose:To measure and compare salivary NO levels in patients with and without generalized chronic periodontitis (GCP); to evaluate correlations between these levels and probing depth as a clinical diagnostic parameter; and to determine usefulness of NO measurements as biological markers of perio disease.

Methods:30 individuals; exclusion criteria: current smokers, pregnant, recent use of Abs or recent trauma. Inclusion: periodontitis with no perio tx in past year. Subjects were divided into three groups: control group (GC) of subjects without periodontitis; group (GM) with moderate generalized chronic periodontitis, and group (GA) with advanced generalized chronic perio. Salivary samples were collected and NO levels measured.

Results:

GCP group NO mean concentrations (GM: 7.78 μM; GA: 15.79 μM) were higher than in the control group (GC: 5.86 μM).
NO levels in the GA group were significantly higher than in the GC group
Positive correlations between NO level and the number of teeth with a probing depth of ≥ 4 mm and ≥ 7 mm were observed.

Conclusion:NO levels are elevated in individuals with GCP, and are associated with periodontal severity. NO may serve as a potential biological marker for detection and monitoring of GCP.

Topic:Saliva Indices

Author: Frodge BD, Ebersole JL, et al.

Title:Bone remodeling biomarkers of periodontal disease in saliva

Source:J Periodontol. 79(10):1913-9, 2008

Type:Clinical Trial

Keywords:C-telopeptide pyridinoline; periodontal diseases; receptor activator of nuclear factor-kappa B ligand; tumor necrosis factor-alpha

Background:Tumor necrosis factor-alpha (TNF-a), C-telo-peptide pyridinoline cross-links of type I collagen (ICTP), and receptor activator of nuclear factor-kappa B ligand (RANKL) have been associated with bone remodeling and periodontal tissue destruction.

Purpose:To evaluate the hypothesis that biomolecules involved in bone remodeling (TNF-RANKL, ICTP) are increased in the saliva of patients with periodontal disease compared to control subjects.

Methods:

Experimental group: 35 patients with moderate to severe periodontitis

>30% BOP
>20% of sites with PD >4mm
>10% sites with interproximal AL >2mm
Evidence of alveolar crestal bone loss >2mm at >30% of sites in vertical BWX

Control group:39 healthy adults of similar age, race and gender.

<10% sites with BOP
<2% of sites with PD >5mm, no sites with PD >6mm
<1% of sites with CAL >2mm
No radiographic evidence of bone loss

Un-stimulated saliva was collected from each subject. One examiner recorded clinical indices for each patient: 1-PD (measured at six locations per tooth); 2-BOP; 3-AL (interproximal sites only).

Concentrations of TNF-a were determined in all 74 adults. Salivary levels of RANKL and ICTP of a subset of 21 subjects and 21 matched controls were examined (levels were below the limit of detection in >80% of subjects in both groups).

Results: RANKL and ICPT were detected only in a minority of patients. The small numbers of subjects with these two markers detected did not allow meaningful comparisons of these analyses between groups.

TNF-αwas detected in all samples. Mean levels of TNF-αwere significantly higher in subjects with periodontal disease than in controls. Subjects with salivary TNF-a levels above a threshold of 5.75 pg/ml (i.e., two standard deviations above the mean of the controls) had significantly more sites with BOP, PD ≥4 mm, and AL ≥2 mm.

Bottom Line:Salivary levels of TNF-α above a threshold might help to identify patients who have periodontal disease.

Topic:saliva immunology/IL-1β

Authors: Tobón-Arroyave SI, Jaramillo-González PE, Isaza-Guzmán DM

Title:Correlation between salivary IL-1beta levels and periodontal clinical status

Source:Arch Oral Biol. 53(4):346-52, 2008

Type:clinical study

Keywords:aggressive periodontitis, chronic periodontitis, IL-1β, immunology, saliva

Purpose:To assess the concentration of proinflammatory cytokine IL-1β in saliva of periodontally diseased and healthy patients and their relationship with periodontal status.

Methods:66 patients were recruited for this study. Clinical parameters recorded included PD and CAL. These values were used to classify the extent of periodontal disease. 30 patients had chronic periodontitis, 18 had aggressive, and 18 were used as healthy controls. About 10-ml of unstimulated whole saliva was collected from each subject. ELISA was used to analyze the amount of IL-1β in the samples.

Results:No differences were noted between men and women. The chronic group was statistically higher in age than the aggressive and healthy groups. Detectable levels of IL-1β were found in all groups. Aggressive and chronic periodontitis groups had significantly higher amounts of IL-1β in their saliva, however no statistical difference was noted between the chronic and aggressive groups.

Conclusion: The study suggests that whole saliva sampling could play an important role in terms of immunological purposes in periodontal disease and that elevated IL-1β concentration may be one of the host-response components associated with the clinical manifestation of periodontal disease.

Topic:saliva

Authors:Rai B, Kharb S, Jain R, Anand SC.

Title: Biomarkers of periodontitis in oral fluids.

Source:J Oral Sci. 50(1):53-6, 2008

Type:clinical study

Keywords:Matrix metalloproteinases, gingivitis, periodontitis, healthy.

Background: MMP’s (Matrix metalloproteinases) are the major group of enzymes responsible for degradation of extracellular matrix (ECM). The onset of collagen destruction is caused by the action of collagenases, a subgroup of MMPs. MMP-1,2,3,8 and 9 have been found in biopsy specimens of human inflammatory periodontal tissues whereas healthy gingiva contains only pro-MMP-2. MMP-2is secreted by gingival fibroblasts and MMP-9is mainly secreted by PMNs and they degrade type IV collagen present in gingival tissues (basement membrane). MMP-8degrades type I and type III collagen which is critical for periodontal destruction.

Purpose:The aim of the present study was to compare the levels of MMP-2 and MMP-9 in GCF and MMP-8 in saliva samples from healthy subjects, and patients with gingivitis and periodontitis.

Materials and methods:

20 patients withperiodontitis, with at least 18 teeth present and moderate to advanced disease with PPD ≥6mm, absence of systemic disease, no history of medication and no previous treatment.
18 patients withgingivitis, generalized inflammation and BOP, no CAL loss, no previous history of SCRP and no history of systemic disease.
15 healthy controls were included in this study.
CAL loss was measured only in inter-proximal sites. Non-stimulated saliva was collected from each subject. One to four sites per patient were selected for GCF collection. After collecting GCF, the levels of MMP-2, MMP-9 and MMP-8 were measured by ELIZA,

Results:Elevated salivary levels of MMP-8 and crevicular levels of MMP-9 were observed in the periodontitis and gingivitis patients compared to the controls. Crevicular MMP-2 levels were lower in gingivitis and periodontitis as compared to healthy controls. PPD, CAL and BOP were correlated with elevated levels of MMP-8. CAL loss and BOP were significantly correlated with elevated levels of MMP-2 and MMP-8.

Discussion:MMP-9 levels in GCF were higher in patients with chronic periodontitis than in patients with gingivitis and healthy subjects, while MMP-2 levels in GCF were lower in patients with chronic periodontitis than in patients with gingivitis and healthy subjects. Salivary MMP-8 levels were higher in patients with chronic periodontitis than in patients with gingivitis and healthy subjects.

BL:MMP-2, MMP-8 and MMP-9 levels were highly correlated with the CAL loss and PDs.

Topic: Salivary biomarkers

Authors:Miller CS, King CP Jr, Langub MC, Kryscio RJ, Thomas MV

Title:Salivary biomarkers of existing periodontal disease: a cross-sectional study.

Source: J Am Dent Assoc. 137(3):322-9, 2006.

DOI: 10.14219/jada.archive.2006.0181

Type: Cross-sectional study

Keywords: GCF flow, Salivary biomarkers, saliva

Background: Interleukin 1 beta (IL-1β)– a proinflammatory cytokine that stimulates the induction of adhesion molecules and other mediators that facilitate and amplify the inflammatory response that occurs in periodontal disease.

Matrixmetalloproteinase (MMP)-8- a key enzyme in extracellular collagen matrix degradation derived predominantly from PMNs during acute stages of periodontal disease.

OPG-a glycoprotein that acts as an osteoblast- secreted decoy receptor and competitively inhibits osteoclast differentiation and activity by preventing osteoclast differentiation factor or the receptor activator of NF-κB ligand (RANKL) from binding to osteoclast precursors and promoting the formation of bone-resorbing osteoclasts.

Purpose: To test the hypothesis that levels of salivary biomarkers specific for three aspects of periodontitis: inflammation, collagen degradation and bone turnover correlate with the clinical features of the disease.

Method: The experimental group constisted 28 pts (12 M, 16 F). Inclusion criteria: generalizedBOP, at least 20% of sites with ≥4mm PD, at least 5% of sites with interproximal CAL loss >2mm, and evident radiographic bone loss. 29 healthy subjects of similar age were used as controls.

Unstimulated whole expectorated saliva was collected and was immediately frozen and analyzed within 6 months. Subjects rinsed their mouths with tap water, and then expectorated whole saliva into sterile tubes.

PD, BOP, and CAL were recorded immediately after saliva sampling. Saliva samples were analyzed for the concentration of IL-1b, MMP-8 and OPG using ELISA or EIA. Statistical analysis was done.

Results:

The mean salivary levels of IL-1b was 3.5 times higher and MMP-8 was 4.3 times higher in case subjects and were highly correlated with BOP and PD ≥4mm.

OPG levels were 1.4 times higher, but after adjustments for cofounding factors there was NSSD.
The probability of having existing periodontitis was 11.3 times more likely with elevated MMP-8, 15.4 times with elevated IL-1b and 45.5 times when both markers are elevated.
When OPG was elevated there was NSSD between the case and control groups in terms of the likelihood of having periodontitis.
Cofounding factors (age, sex, smoking, race, etc.) were not significantly correlated with elevated salivary biomarkers.

Conclusion:Levels of salivary biomarkers, for inflammation, collagen degradation and bone turnover correlate with the clinical features of periodontal disease and suggest that elevated salivary levels of MMP- 8 and IL-1b are candidate biomarkers of periodontal disease.

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