Validation and verification of predictive salivary biomarkers for oral health

Abstract

Oral health is important not only due to the diseases emerging in the oral cavity but also due to the direct relation to systemic health. Thus, early and accurate characterization of the oral health status is of utmost importance. There are several salivary biomarkers as candidates for gingivitis and periodontitis, which are major oral health threats, affecting the gums. These need to be verified and validated for their potential use as differentiators of health, gingivitis and periodontitis status, before they are translated to chair-side for diagnostics and personalized monitoring. We aimed to measure 10 candidates using high sensitivity ELISAs in a well-controlled cohort of 127 individuals from three groups: periodontitis (60), gingivitis (31) and healthy (36). The statistical approaches included univariate statistical tests, receiver operating characteristic curves (ROC) with the corresponding Area Under the Curve (AUC) and Classification and Regression Tree (CART) analysis. The main outcomes were that the combination of multiple biomarker assays, rather than the use of single ones, can offer a predictive accuracy of > 90% for gingivitis versus health groups; and 100% for periodontitis versus health and periodontitis versus gingivitis groups. Furthermore, ratios of biomarkers MMP-8, MMP-9 and TIMP-1 were also proven to be powerful differentiating values compared to the single biomarkers.

Figure 1

Box plots of the selected markers (and combinations thereof) showing levels of each in saliva from individuals in a comparison between health, gingivitis and periodontitis groups: (A) MMP-2; (B) MMP-3; (C) MMP-8; (D) MMP-9; (E) TIMP-1; (F) MMP-8/TIMP-1; (G) MMP-9/TIMP-1; (H) MMP-8 + MMP-9; (I) (MMP-8 + MMP-9)/TIMP-1; (J) LBP; (K) OPG; (L) IL-1b; (M) IL-8; (N) HGF. The boxes represent the values from the 25^th to the 75^th percentile. The middle lines represent the medians. The vertical lines extend from the minimal to the maximal values. (*) for p < 0.05; (**) for p < 0.01; (***) for p < 0.001; (****) for p < 0.0001.

Figure 1

Box plots of the selected markers (and combinations thereof) showing levels of each in saliva from individuals in a comparison between health, gingivitis and periodontitis groups: (A) MMP-2; (B) MMP-3; (C) MMP-8; (D) MMP-9; (E) TIMP-1; (F) MMP-8/TIMP-1; (G) MMP-9/TIMP-1; (H) MMP-8 + MMP-9; (I) (MMP-8 + MMP-9)/TIMP-1; (J) LBP; (K) OPG; (L) IL-1b; (M) IL-8; (N) HGF. The boxes represent the values from the 25^th to the 75^th percentile. The middle lines represent the medians. The vertical lines extend from the minimal to the maximal values. (*) for p < 0.05; (**) for p < 0.01; (***) for p < 0.001; (****) for p < 0.0001.

Figure 2

Classification and diagnostic accuracy mode for distinguishing different stages of periodontal disease. ROC (receiver-operating characteristic analysis) and AUC (the area under ROC curve) as described ealrier. (A) health versus gingivitis (AUC ≥ 0.80); (B,C) health versus periodontitis (AUC ≥ 0.95); and (D,E) gingivitis versus periodontitis (AUC ≥ 0.85). (B,C and D,E were split in two graphs due to graphical software limitations).