The Ability of Quantitative, Specific, and Sensitive Point-of-Care/Chair-Side Oral Fluid Immunotests for aMMP-8 to Detect Periodontal and Peri-Implant Diseases

The analysis of the disease-specific oral and systemic biomarkers in saliva and oral fluids (i.e., mouth rinse, gingival crevicular fluid (GCF), and peri-implantitis fluid (PISF)) is demanding. Several hosts and microbial factors may influence their expression, release, and levels. The type of saliva/oral fluids utilized for the diagnostics affects the analysis. High sensitivity and specificities together with sophisticated methods and techniques are essential for valuable outcome. We describe here recently developed practical, convenient, inexpensive, noninvasive, and quantitative mouth rinse and PISF/GCF/chair-side/point-of-care (PoC) lateral-flow aMMP-8 immunoassays (PerioSafe and ImplantSafe/ORALyser) to detect, predict, and monitor successfully the course, treatment, and prevention of periodontitis and peri-implantitis, respectively. The tests have been independently and successfully validated to differentiate periodontal and peri-implant health and disease in Finland, Germany, Netherland, Sweden, Turkey, Nigeria, Malawi, and USA. The clinical use of salivary/oral fluid biomarkers to identify oral and systemic conditions requires additional studies utilizing these noninvasive screening, diagnostic, and preventive aMMP-8 PoC/chair-side technologies.


Introduction
Diagnosis of periodontal and peri-implant diseases are mostly based on an array of clinical measurements and indices including pocket probing depths, bleeding on probing, and assessment of clinical attachment level together with radiographic findings. Additional information such as medical, hereditary, and specific features and the amount of dental plaque have also been recorded [1,2]. These laborous diagnostic procedures require not only multiple manual recordings but also professional examiners with trained expertise. So far, these clinical and radiographic diagnostic procedures are the best currently available ones for diagnosing and monitoring the disease course, treatment, and maintenance [1]. However, they can only assess the past experience, current extent, and severity of the periodontal and peri-implant diseases. Since these clinical and radiographic analyses of periodontitis and peri-implantitis have low sensitivity and low positive predictive value [1], no reliable information can be obtained regarding the diseases' current activities and their future courses [1]. Also, the episodic progression of periodontitis and peri-implantitis makes the accurate assessment of disease activity and progression difficult and complicated [1]. Neutrophil collagenase, also called matrix metalloproteinase (MMP)-8, polymorphonuclear (PMN) leukocyte collagenase, or collagenase-2, has been identified and characterized as a major collagenolytic enzyme that causes active periodontal and peri-implant degeneration (APD) in periodontitis and peri-implantitis [3][4][5][6][7]. MMP-8 can resolve and regulate inflammatory and immunological cascades by processing nonmatrix bioactive substrates such as chemokines, cytokines, serpins, and complement components. Physiological levels of MMP-8 can exert protective and defensive anti-inflammatory characteristics [5]. Increased levels of especially active MMP-8 (aMMP-8), but not latent, inactive proform, have been found in periodontitis-and peri-implantitis-affected oral fluids (saliva, mouth rinse, gingival crevicular fluid (GCF), and periimplant sulcular fluid (PISF)) [8][9][10]. A key characteristic of active periodontal and peri-implant diseases is the sustained pathological elevation and activation of MMP-8 in periodontal and peri-implant tissues, which are reflected in oral fluids [4]. Consequently, aMMP-8 is a promising biomarker candidate for diagnosing and assessing the progression and course of these episodic oral inflammatory tissue destructive and degenerative diseases [3,4]. More importantly, aMMP-8 in oral fluids can also serve as a predictive and preventive adjunctive biotechnological tool to indicate [4,7,8,11] and time the preventive interventions (secondary prevention or supportive periodontal/periimplant therapy [12,13]) and to inhibit or reduce the conversion of preperiodontitis (formerly gingivitis) and preperi-implantitis (formerly mucositis) to periodontitis and implantitis, respectively.
With this background, we describe the documentation of currently commercially available quantitative reader-based aMMP-8 oral fluid specific point-of-care/chair-side lateralflow reader-equipped immunotests, that is, PerioSafe and ImplantSafe/ORALyser, for periodontal and peri-implant diseases, respectively.

Discussion
Currently, the practical and quantitative PoC/chair-side lateral-flow oral fluid aMMP-8 immunoassays have been successfully developed and are commercially available. The tests have been independently and successfully validated in Finland, Nigeria, Germany, Holland, Malawi, Turkey, Sweden, and USA [4,5,14,[21][22][23][24]. The tests have diagnostic sensitivity and specificity 76-90% and 96%, respectively, corresponding to odds ratio of >72 [4,5,14,21]. The test results are quantitatively available by the reader in 5 min PoC/chair-side [4]. The tests have been shown to be useful to screen susceptible sites and patients, differentiate active and inactive periodontitis and peri-implantitis sites, predict the future disease progression, and monitor the treatment-with or without different adjunctive medications-response, and maintenance therapy [4,5].
As demonstrated in the present study, the tests excellently differentiated periodontal and peri-implant health and diseases. Additionally, the test can identify initial and alarm or early periodontitis (preperiodontitis) in genetically predisposed adolescents [21]. Thus, the test is effective in both adult and adolescent populations and can act in "a gene"-test manner [14,21]. Pathologically elevated aMMP-8 associates, reflects, and precedes the active phase of periodontal and periimplant diseases, that is, APD [4,7,8]. This forms the basis of predictive value for aMMP-8 oral fluid tests in periodontal and peri-implant diseases; thus, the test is positive ahead or before clinical manifestations and/or radiographic outcomes of APD [4,5,8]. The tests can detect enough early subclinical and silent developing preperiodontitis and preperiimplantitis [4,21]. The tests are very suitable for monitoring the disease's development and progression as well as timing and targeting the preventive and therapeutic interventions (i.e., secondary prevention and/or supportive periodontal/ peri-implant therapy) [12,13]. In comparison to bleeding on probing, aMMP-8 PoC/chair-side test is much more sensitive to distinguish periodontal health and disease [14]. Noteworthy, bleeding on probing always causes bacteraemia. Noninvasive PerioSafe and ImplantSafe testings never cause bacteraemia. Regarding other PoC technologies for periodontitis and peri-implantitis, Ritzer et al. [25] described peri-implantitis/ periodontitis online diagnosis utilizing elegantly the tongue as a 24/7 detector allowing diagnosis by "anyone, anywhere, and anytime." Their clever technology utilizes chewing . Implantitis sites were tested before and after surgical treatment by ImplantSafe (+/−); treatment caused positive sites to be negative (b). Elevated aMMP-8 levels in PISF detected by ImplantSafe positivity and IFMA associated significantly with peri-implantitis (p < 0 0001, Wilcoxon test ( * * * )) and could thus be conveniently PoC detected in 5 min by ImplantSafe. Any forms or total MMP-9 did -not differentiate peri-implantitis and healthy sites (a).
gum-containing peptide sensors as protease cleavable linkers between a bitter taste substance and a microparticle. MMPs upregulated in oral cavity and fluids (i.e., saliva and GCF/ PISF) are presented to be responsible for cleaving the sensor in chewing gum generating the bitter taste consequently to be detected by the tongue as a 24/7 in vivo and online sensor [25]. Nonetheless, their elegant 24/7 tongue sensor assay is not MMP-specific for the main collagenolytic MMP, MMP-8, upregulated and activated in periodontitisand peri-implantitis-affected periodontal and peri-implant tissue, gingiva and oral fluids, including saliva, GCF, PISF, and mouth rinse. The sensor is sensitive also to cleavages by MMP-1, MMP-13, and MMP-9 present and upregulated in the diseased periodontitis and peri-implantitis tissue and oral fluids [25]. Furthermore, no information was provided regarding specificity, sensitivity, and predictability of the 24/7 tongue technology regarding periodontitis and periimplantitis. PerioSafe and ImplantSafe exert 76-90% specificity and >96% sensitivity [4,5]. Also, the susceptibilities of the protease cleavable linker 24/7 sensor to catalytically competent and effective microbial proteases expressed and released by dysbiotic oral periodontopathogens and candidial species were not addressed [26,27]. Recently, a PoC/chairside device, comparable to PCR-detection, for Porphyromonas gingivalis related to chronic periodontitis detection has been developed [28]. A chair-side assay for GCF calprotectin has been described [29]. Calprotectin does not degrade anything [29]. PerioSafe and ImplantSafe are specific and sensitive for aMMP-8, a major destructive and collagenolytic factor for APD [4,5]. Overall, PerioSafe and ImplantSafe, aMMP-8 quantitative oral fluid PoC/chair-side lateral-flow immunotests, are the first clinically validated commercially available diagnostic, prognostic, quantitative, predictive, noninvasive, and preventive PoC/chair-side technologies for periodontal and peri-implant diseases [4,5]; the tests can be used by dental and medical professionals linking these disciplines [4]. Potentially, such aMMP-8 PoC tests can eventually be adapted for other medical disciplines [30,31].

Conflicts of Interest
Professor Timo Sorsa is an inventor of US patents 5652223, 5736341, 5866432, and 6143476. All other authors declare no conflicts of interests.