recent approaches in saliva as a credible periodontal ... - Aosr.co.in

RECENT APPROACHES IN SALIVA AS A CREDIBLEPERIODONTAL DIAGNOSTIC AND PROGNOSTICMARKERPriyanka N * , Nitish Kalra * , Namitha Shanbhag † , Kiran Kumar ‡ , Seema ‡ , Biji Brijet ‡ , Uma S.R. † ,A.R. Pradeep *ABSTRACT:Objective: Saliva is used to monitor the general health and the onset of specific diseases. Biomarkers, whether producedby normal healthy individuals or by individuals affected by specific systemic diseases, are tell-tale molecules that could beused to monitor health status, disease onset, treatment response and outcome. It has been a great challenge in periodontologyto determine biomarkers for screening and predicting the early onset of disease (prognostic tests) or evaluating the diseaseactivity and the efficacy of therapy (diagnostic tests).Material and Methods: A Google/Medline search was conducted and relevant literature concerning the applications of salivafor periodontal diagnosis was reviewed. Based on the literature, advanced technologies for measuring salivary biomarkers,that have been studied as potential diagnostic tests for periodontal diseases and various commercially available diagnosticaids and their uses were analyzed and compiled.Conclusion: Traditional diagnostic measures, such as periodontal pocket depth, attachment level, plaque index, bleeding onprobing and radiographic assessment of alveolar bone loss, are not necessarily the most efficient method for early diagnosis.A simple and non-invasive diagnostic tool that allows rapid screening provides accurate predictive information and enablesreliable evaluation of periodontal disease status would be of great value to both dentists and patients. This review presentsan overview of the value of saliva as a credible diagnostic tool in periodontal diseases, and the development of advancedsalivary diagnostic tools.AOSR 2012;2(1):40-46.Key Words: Salivary Biomarkers, Periodontal disease, Saliva.* Department of Periodontics, Government dental College and Research Institute, Bangalore, India.Archives of Oral Sciences & Research† Department of Public Health Dentistry, Government dental College and Research Institute, Bangalore, India.‡ Department of Endodontics & Conservative Dentistry, Government dental College and Research Institute, Bangalore,India.INTRODUCTION:Saliva is said to be a “mirror of the body” because it is anindicator of health not just in the oral cavity but throughoutthe body. 1 The molecular composition of saliva includestherapeutic, hormonal, immunologic, and toxicologicalmolecules, which can provide vital clues to systemichealth. 2 Saliva has been used informally as a clinicaldiagnostic medium for more than 2,000 years. Historically,the viscosity and odor of saliva were used as diagnosticsymptoms for certain diseases. A good diagnostic methodshould have the characteristics of high sensitivity,specificity, and functionality, and meet the requirements ofhigh throughput, portability, and low cost for subsequentclinical application. For salivary diagnostics, many ofthese goals have been met through engaging diversetechnology fields including biology, chemistry, physics,and engineering. Today, the improved efficiency andaccuracy of genomic and proteomic biomarker discoverytechnologies are turning salivary diagnostics into a clinicaland commercial reality.Saliva in comparison with serum:In diagnostics, saliva is an excellent alternative to serumsince it contains sufficient quantities of disease biomarkers,ribonucleic acid (RNA), and deoxyribonucleic acid (DNA),and the collection method is noninvasive, safe, and easy.Saliva is also easier to handle during diagnostic proceduresthan blood because it does not clot, thus reducing the40

Diagnostic and Prognostic Markers in Saliva-Recent Approachessugars, toxins, proteins and antibodies. 7,11-15 Building onthis technology, a second class of miniaturized sensorsystem has been pioneered that contains beads withinetchings of stainless steel plates and utilizes a membranecapture element integrated into a fluidics structure. 16,17These membrane microchip ensembles have been adaptedto service cell, spore, and bacteria separation and biomarkeridentification applications. 18 Importantly, the performancemetrics of these miniaturized sensor systems have beenshown to closely correlate with established macroscopicgold-standard methods, making them suitable for use assubcomponents of highly functional detection systems forthe analysis of complex fluid samples, such as saliva, fora variety of analyte systems. 9,11-17,19-23 The developmentof a point of care (POC) device that contains a modularand miniaturized sensor system, universal analyzer withfunctional integrated mechanical/optical interfaces, andflexible microchip architecture can service the future needsof clinicians and the research communities.In this POC device, saliva (100–300 μl) is placed into thesalivary collection/delivery module, and then deliveredinto the Nano-Biochip. The injection-molded cartridge is`creditcard'size and encloses the array Nano-Biochip where complexfluorescent immunoassays are performed. Here, a networkof fluidic components ensures the complete transfer andprocess of saliva samples to the multiplex bead array toprovide quantitative information of target biomarkers ofdisease. The sample introduction requirements are consistentwith the use of saliva or finger-prick quantities of bloodthat can be directly introduced into the sample introductionport. Detection reagents are stored dry on a conjugatepad embedded within the biochip, and are reconstitutedas needed, through the release of a prepacked buffercontained in biochip-integrated pouches. All processingsteps are conducted within the microfluidic network of thebiochip via actuation inside the analyzer without humanintervention. These features eliminate the need for externalfluidics, such as pumps, tubing and connectors. Therefore,the integrated system has the potential to reduce cost andreduce the risk for leaks and contamination. The assay isprocessed entirely through a 5–15 min sequence that isprogrammed in the main controller board. The flexibilityof the control software allows for modifications to be madethrough an assay builder interface. Control over the flowrate, incubation time and reagent wash, is achieved bythe actuation of stepping motors that direct the fluid flowthrough the depression of the fluid pouches. The sample isdirected to an on-chip waste reservoir, which provides asafe containment of biohazardous fluids. The entire biochipcan be discarded as solid waste after the assay, facilitatingbiohazard waste management. Together, these essentialfeatures serve to facilitate the transition from chips-ina-laboratoryto a lab-on-a-chip, and offer significantopportunities for POC technology needs.Table 2: DEMONSTRATES VARIOUS PRODUCTS AND THEIR USES FOR MEASURING SALIVARY BIOMARKERSPRODUCT NAMEPURPOSEMyPerioIDidentifies the type and concentration of the specific bacteria that cause periodontaldiseases.My PerioPathdetermines the cause of periodontal infections.Oral Fluid NanoSensor Testsimultaneous and precise detection of multiple salivary proteins and nucleic acids.Electronic Taste Chipsdetects multiple biomarkers for early diagnosis of periodontal diseaseOraQuick an antibody test that provides results in 20 minutes, usually detects HIV 1 and HIV 2Integrated MicrofluidicPlatform for Oral Diagnosticsrapidly (3–10 min) measures the concentrations of MMP-8 and other biomarkers in smallamounts (10 ml) of saliva43

Priyanka N et al.Newer aids for measuring salivary biomarkers:Significant advances are in development for the screeningof periodontal diseases. 24 The current method of diagnosingperiodontitis is through assessment of clinical parametersand radiographs, however, this is not necessarily the mostefficient method for early diagnosis. If periodontal diseasesare detected early, treatment can be easier and less painfulfor the patient. Left untreated, periodontal diseases maylead to systemic problems such as cardiovascular diseaseand diabetes. Therefore, early screening for periodontaldiseases is essential during dental examinations.MyPerioID and My PerioPath § :Two DNA-based saliva tests are available. MyPerioPathand MyPerioID. My PerioPath uses a saliva sample toidentify the type and concentration of the specific bacteriathat cause periodontal diseases. MyPerioID test also usessaliva to determine a patient’s genetic susceptibility toperiodontal diseases and which patients are at higher riskof more serious periodontal infections. Both tests requirethe shipping of saliva samples to a laboratory for results.Oral Fluid Nano Sensor Test:The University of California, Los Angeles (UCLA)Collaborative Oral Fluid Diagnostic Research Center,partnered with engineers at the UCLA School ofEngineering, developed a micro electromechanical systembased electrochemical detection platform that is capable ofreal-time, ultrasensitive, ultraspecific multiplex detectionof salivary protein and RNA biomarkers. 25 This envisionedproduct has been labeled the Oral Fluid Nano Sensor Test(OFNASET). It is a point-of-care, automated, and easy-touseintegrated system that will enable simultaneous andprecise detection of multiple salivary proteins and nucleicacids. In addition, this system is portable and could be usednot only in the doctor’s office, but also in any other healthcarestation to perform an instant point-of-care diagnosis.The OFNASET technology platform combines cuttingedgetechnologies, such as self-assembled monolayersbionanotechnology, cyclic enzymatic amplification, andmicrofluidics, with several well-established techniquesincluding microinjection molding, hybridization-baseddetection, and molecular purification. The intended use ofthe OFNASET is for the point of care multiplex detectionof salivary biomarkers for oral cancer. It analyzes salivafor the presence of four salivary mRNA biomarkers(SAT, ODZ, IL-8, and IL-1b) and two salivary proteomicbiomarkers (thioredoxin and IL-8).§ Oral DNA ® Labs Inc, Brentwood, Tennessee, USAǁ Ora Sure Technologies, Inc, Bethelem, USAElectronic Taste Chips:Several study groups have reported elevated serum CRPlevels in periodontitis patients. The higher the levels of CRPin periodontitis patients, the more severe the disease, evenwith adjustments for external factors. 7 The biomarker CRPis an acute phase reactant and a well-accepted indicator ofinflammation. Numerous clinical studies have establishedelevated serum CRP as a strong, independent risk factor forthe development of cardiovascular disease (CVD). CVD hasalso been associated with oral infections (i.e. periodontaldiseases) and there is evidence that systemic CRP may bea link between the two. Clinical measurements of CRP inserum are currently performed with "high sensitivity'' CRP(hsCRP) enzyme-linked immunosorbent assay (ELISA)tests that lack the sensitivity for the detection of thisimportant biomarker in saliva. Salivary CRP may representa novel approach for diagnosing and monitoring chronicinflammatory disease, including CVD and periodontaldiseases.Chemically sensitized bead microreactors within the labon-a-chipsystem were recently applied for measurementof CRP and other biomarkers of inflammation in saliva,demonstrating significantly lower detection level (by > 3decade orders of magnitude) for CRP than high-sensitivityCRP ELISA methods, allowing for measurement ofinflammatory biomarkers related to select disease states. 7Currently, researchers at Rice University in Houston,Tex, are developing a lab-on-a-chip system using a newdetection system for measuring analytes in saliva based onan electronic taste chip (ETC). The ETC methodology wascompared with the standard laboratory technology (ELISA)for measuring CRP in saliva, and displayed a 20-fold lowerlimit of detection than the ELISA. With this technique itwas possible to quantitate the difference in CRP levelsbetween healthy individuals and patients with periodontaldiseases. 7 Additional studies confirmed the ability of theETC platform to simultaneously monitor several additionalbiomarkers. 7 The prospect of a commercially available ETClab-on-a-chip platform that can detect multiple biomarkersfor early diagnosis of periodontal disease is promising.Oraquick ǁ :It is an antibody test that provides results in 20 minutes.The blood, plasma or oral fluid is mixed in a vial withdeveloping solution, and the results are read from asticklike testing device. Usually detects HIV 1 and HIV 2.The Oraquick rapid saliva HIV test is definitely the way togo. It is a toothbrush-like device with a fabric swab on thehead and a small plastic stem. The stem naps off to avoidcontamination, while the swab is inserted into a tube oftesting fluid. The testing tube is easily sealed and ready fortransport to a testing laboratory. By using a toothbrush-likedevice that is inserted into the mouth instead of drawingblood, this testing method makes an already high-anxiety44

Diagnostic and Prognostic Markers in Saliva-Recent Approachesscreening a little more comfortable.Integrated Microfluidic Platform for Oral Diagnostics(IMPOD):A clinical point-of care diagnostic test that enables rapidquantification of an oral disease biomarker in human salivaby using a monolithic disposable cartridge designed tooperate in a compact analytical instrument was reported. 26,27This microfluidic method facilitates hands-free salivaanalysis by integrating sample pretreatment (filtering,enrichment, mixing) with electrophoretic immunoassaysto quickly measure analyte concentrations in minimallypretreated saliva samples. Rapid (< 10 min) measurementof levels of the collagen cleaving enzyme MMP-8 in salivafrom healthy and periodontally diseased subjects can beachieved using 20 µl of saliva. 26 Based on this, a portablediagnostic device called the IMPOD was developed. Anearly clinical study in which the hand-held IMPOD wasused to rapidly (3–10 min) measure the concentrations ofMMP-8 and other biomarkers in small amounts (10 ml) ofsaliva has been reported. 27CONCLUSION:Saliva is being accepted as an excellent diagnostic mediumand an indispensable tool in the field of diagnostics. It islikely that the development of saliva based methods willimpact and expand the role of dentists. Integrating thesenew salivary diagnostics methods into clinical practice isimportant to aid dental professionals in making essentialhealth-related decisions for patients. In the near future,taking a saliva sampling in a dental clinic will become asroutine as obtaining a urine or serum sample at a physician’soffice.REFERENCES:1. Mandel ID. Salivary diagnosis: promises, promises.Ann N Y Acad Sci 1993;694:1-10.2. Zhang L, Xiao H, Wong DT. Salivary biomarkers forclinical applications. Mol Diagn Ther 2009;13:245-259.3. Johnson LR. Salivary Secretion. GastrointestinalPhysiology. 6th Edition. Mosby; St. Louis, MO, USA:2001; 65-74.4. Oberg SG, Izutsu KT, Truelove EL. Human parotidsaliva protein composition: dependence on physiologicalfactors. Am J Physiol 1982;242:231–236.5. Rudney JD, Kajander KC, Smith QT. Correlationsbetween human salivary levels of lysozyme, lactoferrin,salivary peroxidase and secretory immunoglobulin Awith different stimulatory states and over time. ArchOral Biol 1985;30:765–771.6. Tabak LA. Point-of-care diagnostics enter the mouth.Ann NY Aad Sci 2007;1098:7-14.7. Christodoulides N, Mohanty S, Miller CS etal. Application of microchip assay system for themeasurement of C-reactive protein in human saliva.Lab Chip 2005;5:261–269.8. Floriano PN, Christodoulides N, Romanovicz D et al.Membrane-based on-line optical ana lysis system forrapid detection of bacteria and spores. BiosensBioelectron 2005; 20:2079–2088.9. Floriano PN, Christodoulides N, Miller CS et al. Useof saliva-based nano-biochip tests for acute myocardialinfarction at the point of care: a feasibility study. ClinChem 2009; 55:1530–1538.10. Wong DT. Salivary diagnostics for oral cancer. J CalifDent Assoc 2006;34;303-308.11. Christodoulides N, Tran M, Floriano PN et al. Amicrochip-based multianalyte assay system for theassessment of cardiac risk. Anal Chem2002;74:3030–3036.12. Curey TE, Goodey A, Tsao A et al. Characterization ofmulticomponent monosaccharide solutionsusing an enzyme-based sensor array. Anal Biochem2001;293:178–184.13. Goodey A, Lavigne JJ, Savoy SM et al.Development of multianalyte sensor arrays composedof chemically derivatized polymeric microsphereslocalized in micromachined cavities. J Am Chem Soc2001;123:2559–2570.14. McCleskey SC, Griffin MJ, Schneider SE, McDevitt JT,Anslyn EV. Differential receptors create patternsdiagnostic for ATP and GTP. J Am Chem Soc2003;125: 1114–1115.15. Ali MF, Kirby R, Goodey AP et al. DNA hybridizationand discrimination of single-nucleotide mismatchesusing chip-based microbead arrays. Anal Chem 2003;75:4732–4739.16. Rodriguez WR, Christodoulides N, Floriano PN et al.A microchip CD4 counting method for HIV monitoringin resource-poor settings. PLoS Med 2005;2:e182.17. Christodoulides N, Floriano PN, Acosta SA et al.Toward the development of a lab-on-a-chip dualfunctionleukocyte and C-reactive protein analysismethod for the assessment of inflammation and cardiacrisk. Clin Chem 2005;51:2391–2395.18. Cohen J. Monitoring treatment: at what cost? Science2004;304(5679):1936.45