|Year : 2016 | Volume
| Issue : 3 | Page : 107-113
Diagnostic kits: An aid to periodontal diagnosis
Amit Mani, Raju Anarthe, PP Marawar, Rachita G Mustilwar, Anuradha Bhosale
Department of Periodontology and Implantology, Rural Dental College, PIMS, Loni, Maharashtra, India
|Date of Web Publication||28-Nov-2016|
Rachita G Mustilwar
Department of Periodontology and Implantology, Rural Dental College, PIMS, Loni, Maharashtra
Source of Support: None, Conflict of Interest: None
Periodontal diseases have been diagnosed by clinical and radiographic examination traditionally, likewise the assessment of plaque using a plaque index, gingival inflammation with the bleeding on probing index, probing pocket depth, and clinical attachment loss. Recently, for more precise molecular diagnostics and treatments, chairside diagnostic kits are used. This review describes chairside diagnostic test kits available in the market that can facilitate the clinical examination and the establishment of a proper diagnosis.
Keywords: Diagnostic chairside test kits, gingival inflammation, periodontal disease
|How to cite this article:|
Mani A, Anarthe R, Marawar P P, Mustilwar RG, Bhosale A. Diagnostic kits: An aid to periodontal diagnosis. J Dent Res Rev 2016;3:107-13
|How to cite this URL:|
Mani A, Anarthe R, Marawar P P, Mustilwar RG, Bhosale A. Diagnostic kits: An aid to periodontal diagnosis. J Dent Res Rev [serial online] 2016 [cited 2019 May 20];3:107-13. Available from: http://www.jdrr.org/text.asp?2016/3/3/107/194837
| Introduction|| |
Periodontitis is a chronic inflammatory disease, results in progressive destruction of the periodontium that supports the teeth and is of bacterial origin.  Studies of the host immune response to pathogenic bacteria have contributed to the understanding of the pathogenesis of periodontal diseases.  The traditional methods for diagnosing periodontal disease have remained virtually unchanged and relies almost exclusively on clinical parameters such as probing depth and clinical attachment level (CAL) and traditional conventional dental radiography particularly intraoral periapical radiograph and ortho pantamogram. The strengths of these traditional tools are their ease of use, their cost-effectiveness, and that they are comparatively noninvasive. However, these diagnostic procedures are limited, in that only disease history can be assessed. Current disease activity cannot be assessed. Hence, there is a need to develop newer diagnostic tests which can detect the presence of active disease as well as predict future disease progression, and evaluate the response to periodontal therapy. This can definitely improve the clinical management of patients with periodontal disease. This article highlights recent advances in the use of biomarker-based disease diagnostics that focus on the identification of active periodontal disease using gingival crevicular fluid (GCF) and saliva.  "A characteristic that can be measured and evaluated as an indicator of normal biological processes, pathological processes or pharmacologic responses to therapeutic interventions" is known as biomarker (NIH Biomarkers Definitions Working Group, 1998).
More than 200 species of microorganisms colonize the oral cavity, but only a few of these are thought to be pathogens.  Among the subgingival bacterial species identified so far, Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), and Aggregatibacter actinomycetemcomitans (Aa) have been often associated as the periodontitis progresses.  Aa is not always found in periodontally healthy individuals.  Whereas Pi has been found in either healthy subjects or patients with gingivitis.  Consequently, elevated levels of these periodontopathogens may be useful indicators of both active periodontitis and increased risk of attachment loss. However, knowledge of how their numbers related to disease progression is still not clear, and for longitudinal studies, accurate assessment of their numbers in clinical samples is needed.
The philosophy behind the emergence of various diagnostic tests is that the earlier the active disease is diagnosed, the less invasive, the less time consuming and therefore less costly the required treatment can be achieved and the better long-term prognosis for patients with the destructive disease. 
For diagnosis of periodontal diseases, the ideal diagnostic test should be:
- Highly sensitive method capable of analyzing a single periodontal site in health as well as disease
- Highly specific
- Simple to perform
- A rapid, one or two stage procedure
- Versatile in terms of sample handling, storage and transport
- Amendable to chairside use
- Dependent on simple and robust instrumentation. 
A given detection method may mistakenly identify nontarget microorganisms and may not detect all strains of the target bacterial species due to strain to strain variability. Sensitivity denotes the percentage of positive test results of a detection method employed to identify different strains of the target microbial species and specificity denotes the percentage of negative results when the detection method is applied to a variety of nontarget bacterial species. The ideal detection method displays sensitivity and specificity of 100%. 
Various technical and clinical difficulties in conventional procedures from taking a sample to dispersion, cultivation, characterization, and identification of isolates of microorganism and problems associated with adequate analysis of microbiologic data makes conventional diagnostic procedures time consuming and inconvenient.  In this context, chairside periodontal diagnostic kits can provide reports in short time.
Advantages of chairside diagnostic kits:
- Simple to use
- Can be read after relatively short periods
- Can be shown to patient and relate to the tooth site affected.
They are categorized as microbiologic kits, biochemical kits, and genetic kits [Table 1].
| Microbiologic Test Kits|| |
The Evalusite test, a product of Eastman Kodak Company, is a polyclonal antibody-based sandwich enzyme immunoassay. It allows for the visual detection and differentiation of antigens from Aa, Pg, and Pi in subgingival paper point samples.
The Evalusite test is well-suited for the simultaneous testing of multiple samples since minimal sample dilution occurs when multiple paper points are placed in a single sample tube.
The likelihood of detecting bacterial colonization with the Evalusite test was shown to be greater in deep pockets as compared to shallow pockets. 
- It is multistage test,
- It has a subjective calorimetric end point.
There is no permanent record of the result and gives the assumption that the three organism are causing the disease.
Evalusite had a sensitivity of 28% and a specificity of 98% for Aa and 52% sensitivity and a specificity of 98% for Pg. 
It is a chairside assay that detects a group of periodontal pathogens Bacteroides forsythus, Pg, Treponema denticola as well as certain Capnocytophaga species that produce trypsin-like enzyme. The presence of this enzyme in a plaque sample can be elicited by the hydrolysis of a synthetic trypsin substrate, N-benzoyl-DL-arginine-2-naphthylamide. The products of this reaction can be demonstrated by a color reaction on a reagent strip producing a blue-black product. This is used as the basis of a chairside test. 
- It cannot identify the presence of other periodontopathogens that do not produce trypsin-like enzyme
- The results are qualitative and rely upon the operator's assessment at the calorimetric end point
- The specific bacteria that are responsible for enzyme production cannot be determined.
Perioscan had a sensitivity of 99% and a specificity of 55% at baseline. 
It is nucleic acid technology genomic probe. Pi, Pg can be detected with the aid of purified DNA fragments, but the same way Aa could not be detected. Aa cloned probe, and Pg whole genomic probe comprises the basis of the commercial DMDx [Figure 1] detection method. For Pg, the DNA probe assay can give false negative data. van Steenbergen et al. found the DMDx detection method to possess 96% sensitivity and 86% specificity for spiked laboratory specimens of Aa and 60% sensitivity and 82% specificity for laboratory specimens of Pg.  For Pg, the sensitivity was 71% and the specificity 53%.
- However, in clinical specimens, the detection of Aa revealed sensitivity as low as 21% and a specificity of 83%.
IAI Pado Test 4.5
With the Pado RNA probe test kit, four periodontal pathogens can be detected: Aa, Pg, Tannerella Forsythia, and T. denticola. This test basically uses oligonucleotide probes complementary to conserved fragments of the 16S rRNA gene that encodes the rRNA, which forms a subunit of the bacterial ribosome. The detection threshold of this test is 103 for Aa and 104 for Pg, T. forsythia and T. denticola. The detection frequencies found with this test indicated a low sensitivity of the Pado Test 4.5 method when compared to the checkerboard method.
- The Pado Test 4.5 seems to underestimate the number of positive sites/individuals suggested by a high number of false negatives. 
MyPerioPath is DNA test used to identify the type and concentration of the perticular bacteria that cause periodontal diseases using saliva sample. This test requires the shipping of saliva samples to a laboratory for results.
- Detects (from bacterial DNA) the specific bacteria known to cause periodontal inflammation and destruction
- Eleven species fall within this group
- Sub-divides these bacteria into "risk" groups based on known risk/virulence properties: high, moderate, low
- Determines concentration/bacterial load (e.g., inflammatory burden)
- Helps to determine therapy options based on bacterial risk assessment. 
- Very specific and determine phenotypic markers
- Has great specificity and sensitivity
- Not affected by transport conditions
- Do not require anaerobic conditions to be maintained
- Can be done in dead bacteria and they do not depend on bacterial viability. 
- Very expensive
- The minimal detection limits for particular species are 103-105 cells
- The chairside diagnosis is not possible
- The cross reactivity by oligonucleotide probes can occur
- Antibiotic sensitivity is not possible. 
| Biochemical Test Kits|| |
Perio-Check (Ac Tech)
Perio-Check has Food and Drug Administration approval in the US. It is the most rapid chairside diagnostic test for neutral proteases such as collagenases, elastases, and proteinases in GCF. The GCF sample strip is placed on a gel containing insoluble dye-labeled collagen fibrils (remazobrilliant blue-collagen substrate powder) and incubated. In the presence of neutral proteases (which diffuse from the strip into the gel), the insoluble collagen-dye complex is digested to release soluble dye-labeled fragments, which diffuse back into the strip, turning it blue. With the development of gingivitis as well as sites of established periodontitis the levels of these enzymes in GCF have been noted to increase. Not specific for Polymorphonuclear Leukocytes collagenase which is thought to be the dominant collagenase at active sites.  Interproximal sites cannot be sampled due to the risk of saliva contamination. Perio-Check had a sensitivity of 88% and a specificity of 61% at baseline. 
This test kit was released in the year 1993. This system detects the presence of serine proteinase elastase in GCF sample.  The GCF is collected onto the filter paper strip impregnated with a known amount of buffered elastase substrate labeled with a fluorescent indicator. Elastase on the test strip cleaves the substrate during the reaction time of 4-6 min and releases the indicator, visible under fluorescent light. Elastase is released from the lysosomes of polymorphonuclear leukocytes which is accumulated at sites of gingival inflammation. The presence of elevated levels of elastase in GCF may thus be indicative of active disease sites.  Although a relationship between elastase levels in GCF and periodontal disease activity has been reported, the position is still far from clear. Further, clinical trials are needed before the value of this test kit in clinical practice can be ascertained.
PerioGard is based on the measurement of levels of enzyme aspartate aminotransferase (AST) in GCF sample. AST is a soluble intracellular cytoplasmic enzyme that is released from within the cell on its death. Since cell death is an important part of periodontal pathogenesis, AST levels in GCF have great potential as markers of early periodontal tissue destruction. Elevated total AST levels in a 30-s sample have been positively associated with disease-active sites in contrast to inactive sites. , This commercial test consists of a tray with two test wells for each tooth, and appropriate reagent for conducting the test. The test involves the collection of GCF with the filter paper strip which is then placed in tromethamine hydrochloride buffer. A substrate reaction mixture containing 1-aspartic and α-ketoglutaric acid is added to the sample and allowed to react for ten minutes. In the presence of AST, the aspartate, and α-ketoglutaric acid are catalyzed to oxaloacetate and glutamate. The addition of a dye such as fast red results in a color product, the intensity of which is proportional to the AST activity in the GCF sample.  The test is designed to be positive at >800 μIU of AST activity and negative at values <800 μIU. It cannot discriminate between sites with severe inflammation but with no attachment loss from sites with attachment loss. 
This test is indicated in:
- Assistance or aid to diagnose in identification of active sites at the time of periodontal disease
- To appreciate the results of a periodontal treatment
- In monitoring of high risk sites.
The PocketWatch™ method analyzes aspartate transaminase at the chair side. The principle of this test is that, in the presence of pyridoxal phosphate, AST catalyzes the transfer of an amino group of cysteine sulfuric acid by α-ketoglutaric acid to yield β-sulfinyl pyruvate. Glutamate β-sulfinyl pyruvate spontaneously and rapidly decomposes and releases inorganic sulfite. The sulfite ion instantaneously reacts with malachite green (MG), simultaneously causing MG to convert from a green dye to its colorless form, thereby allowing the pink-colored rhodamine B dye to show through. The rate of conversion of MG is directly proportional to AST concentration. However, components of the extracellular matrix and its dissolved products are present in GCF of destructive pockets, and they may release sulfide ions. It is possible to distinguish between active and inactive sites with PocketWatch™. AST activity determined by PocketWatch™ provides not only an index of cell death but also the extent of the destructive pockets. 
Perio 2000 System/Diamond Probe
Various pathogenic microorganisms like Pg, Pi and T. forsythia produce sulfates, thereby significant levels of volatile sulfide compounds (VSCs) by degradation of serum proteins: Cysteine and methionine. Since these VSCs can directly degrade periodontal structures aggravating periodontitis, their evaluation can indicate the subgingival microbial load. Perio 2000 system [Figure 2] is designed in such a way that it combines the features of a periodontal probe with the detection of volatile sulfur compounds in the periodontal pocket.  Its handpiece has the shape, depth markings, and feel of typical periodontal probes, but it has unique microsensor in its tip which measures the level of bacterial activity at individual tooth sites, even before the gums are bleeding.
| Dip Stick Test|| |
The matrix metalloproteinase-8 (MMP-8) test stick is based on the immunochromatography principle that uses two monoclonal antibodies specific for different epitopes of MMP-8. The test stick results can be detected in 5 min. The antibody detects both neutrophils and non-PMN-type MMP-8 isoforms.  The GCF sample collected will be placed in a test tube containing 0.5 ml of a buffer at pH 7.4. When the dip area of dipstick is placed in the extracted sample; the dipstick absorbs liquid, which starts to flow up the dipstick. When the sample contains MMP-8, it binds to the antibody attached to the latex particles. The particles are carried by the liquid flow if MMP-8 is bound to them; they bind to the catching antibody. If the concentration of MMP-8 in the sample exceeds the cutoff value for the test, a positive line will appear in the result area. 
| Toxicity Prescreening Assay|| |
Toxicity Prescreening Assay [Figure 3] detects the indirect presence of bacteria by two markers of gingival infection which are bacterial toxins and bacterial proteins. This test can be associated with the severity of inflammation and with the evolution of destructive process. It makes the difference between an active and an inactive periodontal disease. 
| Integrated Microfluidic Platform for Oral Diagnostics|| |
A clinical point of care diagnostic test that enables rapid quantification of an oral disease biomarker in human saliva using a monolithic disposable cartridge designed to operate in a compact, handy analytical instrument. Microfluidic method facilitates hands-free saliva analysis by integrating sample pretreatment such as filtering, enrichment, mixing with electrophoretic immunoassays to quickly measure concentrations in minimally pretreated saliva samples. It rapidly (<10 min) measures MMP-8 in saliva from healthy and periodontally diseased subjects. It requires low sample volume (10 L). 
Building on technologies introduced above, our group is actively developing an integrated microfluidic platform for oral diagnostics (IMPOD). An image of an early hardware version of the portable diagnostic is based on that developed at Sandia previously.  IMPOD and quantitative, portable instrumentation like it, has the potential to be translated to clinical settings for use in rapid, chairside analysis of human saliva and oral fluids. The methods and technologies also have applicability to nonoral diagnostic fluids, as well as other local and systemic diseases.
| Oral Fluid NanoSensor Test|| |
It is a handheld, automated, easy-to-use integrated system. It enables simultaneous and rapid detection of multiple salivary proteins and nucleic acid targets. Oral Fluid Nano Sensor Test (OFNASET) [Figure 4] is a microelectromechanical system based on electrochemical detection platform that is capable of real-time, very sensitive, ultraspecific multiplex detection of salivary protein, and RNA biomarkers. OFNASET is used for the point of care multiplex detection of salivary biomarkers for oral cancer. It analyzes saliva for the presence of four salivary mRNA biomarkers (SAT, ODZ, interleukin [IL]-8, and IL-1β) and two salivary proteomic biomarkers (thioredoxin and IL-8) with high specificity and sensitivity. Simultaneous detection of multiple salivary proteins and nucleic acids is possible. 
| Electronic Taste Chips|| |
They are chemically sensitized bead microreactors within the lab-on-a-chip system and were applied for measurement of C reactive protein and other biomarkers of inflammation in saliva. The electronic taste chips methodology was compared with the standard laboratory technology (ELISA) for measuring C reactive protein in saliva and displayed a 20-fold lower limit of detection than the ELISA. With this technique, it is possible to differentiate in C-reactive protein levels between healthy individuals and patients with periodontal diseases quantitatively and can simultaneously monitor several biomarkers. 
| Genetic Test Kits|| |
Periodontitis susceptibility trait test
The periodontitis susceptibility trait test is the first genetic susceptibility test for severe periodontitis. It is commercially available. It evaluates the simultaneous occurrence of allele 2 at the IL-1α +4845 and 1β +3954 loci.  IL-1 genetic susceptibility may not initiate or cause the disease but rather may lead to earlier or more severe disease. The IL-1 genetic test can be used to differentiate certain IL-1 genotypes associated with varying inflammatory responses to identify individuals at risk for severe periodontal disease even before the age of 60.
MyPerioID test uses saliva to determine a patient's genetic susceptibility to periodontal diseases. It assesses patients which are at higher risk of more serious periodontal infections. This test requires the transportation of saliva samples to a laboratory for results. 
- Detects (from human DNA) genetic variation/polymorphism within the IL-1 gene
- IL-1 is a major inflammatory mediator
- 30-35% of the US population has this genetic variation
- IL-1 positive individuals tend to have more aggressive and more severe infections
- Determines patients that are most susceptible to severe disease, especially if the patients smoke
- This genetic variation can increase risk for severe disease or tooth loss by 2-7 times when present.
| Conclusion|| |
Chairside diagnostic kits offer rapid, reproducible mode of diagnosis and the results can be used for patient motivation as well. They are useful especially in making out the active site and monitoring patients posttreatment for evaluating the response to therapy and disease recurrence. Integrating new salivary diagnostic methods to clinical practice is important to help dental professionals in making health-related decisions for patients.
In periodontology, the success of any treatment is based on the accuracy of the initial diagnosis. At present, the majority of chronic periodontitis cases can be sufficiently managed using existing diagnostic methodology, although it is always more desirable to be able to diagnose "active disease" as it occurs, rather than months later.
Many of the biochemical chairside diagnostic test kits have been marketed. The newly commercially available chairside tests for host and bacterial markers of periodontal disease offer prospects which would make the monitoring of specific sites possible. However, the clinician must ensure that the use of such tests will benefit the patients in terms of both the value of diagnostic data obtained and the cost in time and money.
Validation of such novel periodontal diagnostics needs to be benchmarked with existing gold standards of the disease, such as alveolar bone levels and clinical attachment levels, in large population.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
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