Current developments in salivary diagnostics

Abstract

Salivary diagnostics is an emerging field that has progressed through several important developments in the past decade, including the publication of the human salivary proteome and the infusion of federal funds to integrate nanotechnologies and microfluidic engineering concepts into developing compact point-of-care devices for rapid analysis of this secretion. In this article, we discuss some of these developments and their relevance to the prognosis, diagnosis and management of periodontitis, as an oral target, and cardiovascular disease, as a systemic example for the potential of these biodiagnostics. Our findings suggest that several biomarkers are associated with distinct biological stages of these diseases and demonstrate promise as practical biomarkers in identifying and managing periodontal disease, and acute myocardial infarction. The majority of these studies have progressed through biomarker discovery, with the identified molecules requiring more robust clinical studies to enable substantive validation for disease diagnosis. It is predicted that with continued advances in this field the use of a combination of biomarkers in multiplex panels is likely to yield accurate screening tools for these diagnoses in the near future.

Figure 1. Potential disease targets for salivary diagnosis

BNP: B-type natriuretic peptide; CK-MB: Creatine kinase-MB; CRP: C-reactive protein; FFA: Free fatty acid; IMA: Ischemia modified albumin; LDL: Low-density lipoprotein; MMP: Matrix metalloproteinase; MPO: Myeloperoxidase; MYO: Myoglobin; sCD40L: Soluble CD-40 ligand; TF: Tissue factor; TnI: Troponin I; TnT: Troponin T.

Figure 2. Relative abundance of biomarkers in unstimulated whole saliva shown as median values in saliva compared with median values in serum from 45 healthy adults

The ratio of median value in unstimulated/stimulated saliva is shown in parenthesis. All samples analyzed by standard enzyme immunoassays. BNP: B-type natriuretic peptide; CK-MB: Creatine kinase-MB; CRP: C-reactive protein;ENAP: Epithelial neutrophil-activating peptide; Gro-a: Growth-regulated oncogene; MCP: Monocyte chemotactic protein; MMP: Matrix metalloproteinase; MPO: Myeloperoxidase; MYO: myoglobin; sCD-40: soluble CD-40; sICAM: Soluble ICAM; sVCAM: Soluble VCAM; TnI: Troponin I.

Figure 3. LabNow analyzer and Nano-Biochip

(A) Analyzer, by contrast to the actual production configuration, is shown here with transparent outer covering to allow inner features to be viewed. (B) A representation of the multiple biochip functions performed within the credit-card-sized Nano-Biochip, which serve to eliminate constraints imposed by traditional laboratory-confined methods.

Figure 4. Nano-Biochip elements

(A) Fully integrated laminate structure. (B) Exploded view for individual modules: (1) the microchip array, (2) microfluidics platform, (3) fluidic interfaces and (4) saliva collection–delivery module. (C) Computational fluid dynamic studies aided our micro-engineers to adjust structures for optimal fluid flow to the beads. (D) Profile of a salivary sample of a heart attack victim from assay performed in the Cardiac Arrest Rapid Diagnostic Information Using Saliva (CARDIUS) study. Here, signals generated on CRP, IL-1β, MPO and MYO-sensitized bead sensors are shown. Also shown are calibrator beads, as well as negative control beads conjugated to an antibody irrelevant to the targets. CAL: Calibrated; CRP: C-reactive protein; MPO: Myeloperoxidase; MYO: Myoglobin; NEG: Negative.

Figure 5. Mediator levels of six putative biomarkers of periodontal disease in whole expectorated saliva analyzed by enzyme immunosorbent assays and Luminex© technology

Bars denote mean levels in 35 healthy and 18 periodontitis patients. Severity of periodontitis was categorized based on increasing clinical disease severity (i.e., H < PD-1 < PD-2 < PD-3) using frequency of sites with bleeding on probing, pocket depths of at least 5 mm and clinical attachment levels of at least 2 mm. *Significantly greater than H and PD-1. ^‡Significantly greater than other categories at least at p < 0.01. ^§Significantly less than other categories at least at p < 0.01 using Kruskal–Wallis ANOVA on ranks with post-hoc Dunn's test for pairwise comparisons. CRP: C-reactive protein; H: Healthy; L: Luminex; MMP: Matrix metalloproteinase; OPG: Osteoprotegerin; PD: Periodontitis.

Figure 6. Diagnostic thresholds used in combination predict a screening diagnosis of periodontal disease

(A) Levels of IL-1β above the threshold (red line) demonstrate a sensitivity of 66%, specificity of 98.3%, positive predictive value of 91.7% and negative predictive value of 91.2% for the diagnosis of periodontal disease. (B) Levels of matrix metalloproteinase-8 above the threshold (red line) demonstrate a sensitivity of 40%, specificity of 98.3%, positive predictive value of 90% and negative predictive value of 85.5%. Samples elevated above both thresholds have a positive predictive value of 96% or higher. All samples analyzed by standard enzyme immunoassays. SD: Standard deviation.

Figure 7. Median analyte levels expressed as log values in unstimulated whole saliva from 45 acute myocardial infarction patients and 40 non-acute myocardial infarction adult controls

Samples were obtained within 48 h of chest-pain onset and analyzed in duplicate by Luminex© and Beckman Access instruments. P values shown above the bars indicate significant differences between the groups. AMI: Acute myocardial infarction; Cont: Control; CRP: C-reactive protein; MMP: Matrix metalloproteinase; MPO: Myeloperoxidase; MYO: myoglobin; s: Soluble; sICAM: Soluble ICAM.

Figure 8. Correlations between serum and salivary biomarkers of acute myocardial infarction demonstrate C-reactive protein and myoglobin potential utility

Log concentrations in the serum (X-axis) and saliva (Y-axis) of: (A) CRP from 64 acute myocardial infarction patients, (B) MYO from 55 acute myocardial infarction patients, and (C) TnI from 53 acute myocardial infarction patients. Samples were analyzed using Luminex and Beckman Access. CRP: C-reactive protein; MYO: Myoglobin; TnI: Troponin I.