Oral-based Diagnostics

Oral diseases and beyond

Antoon J M Ligtenbergn

Antoon J M Ligtenbergn

Assistant Professor Department of Oral Biochemistry Academic Centre for Dentistry Amsterdam (ACTA) The Netherlands.

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With modern proteomic and genomic techniques it is possible to fine-tune diagnostics of oral diseases and monitor other diseases by oral diagnostics.

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In October 2006 the New York Academy of Sciences organised a conference in Atlanta on Oral-based Diagnostics dealing with the diagnostic potential of saliva and its constituents.1 Although blood is still the gold standard for diagnostics of diseases and drugs, oral diagnostics has the same and may be an even larger diagnostic potential. Principally, all substances that are present in blood may be monitored in saliva, since serum components leak from the gingival crevice into the oral cavity. The concentration of serum components in saliva may be enhanced by normal actions like tooth brushing which results in a fourfold increase of serum albumin.2 In addition, not only saliva and serum components, but also oral bacteria and epithelial cells are present in the oral cavity. All these substances have their own specific diagnostic potential, but what they have in common is that samples can be collected non-invasively. This makes collection of saliva safe and patient-friendly. Since no trained staff or sterile equipment is necessary, oral-based diagnostics can be used quite well for point-of-care diagnostics, home testing and road side applications. 3

The current position of oral diagnostics can be illustrated by its role in oral diseases like dental caries and periodontal disease. Dental caries is the demineralisation of teeth caused by acids that are produced by plaque bacteria. Bacteria accumulate as an oral biofilm called dental plaque, particularly at those dental surfaces that cannot be cleaned properly. The frequent consumption of fermentable carbohydrates results in the production of organic acids, primarily lactic acid, that are released into the plaque fluid. This lowers the pH in the oral biofilm, which favours the outgrowth of Streptococcus mutans and Lactobacillus spp., resulting into an ecological shift to a more cariogenic plaque. 4 Saliva secretion rate, buffering capacity and counts of mutans streptococci and lactobacilli, have proven to be sensitive parameters in caries prediction models. High numbers of S.Mutans and Lactobacillus spp. indicate a shift in oral microflora from healthy to more cariogenic. Diagnostic kits for S. mutans and Lactobacillus spp. counting are widely used in dental practice and can be conducted without laboratory facilities. They are based on traditional culturing techniques in selective media but analysis by PCR is also possible.

In a healthy situation, there is no correlation between saliva secretion rate and dental caries. 5 However, when the salivary secretion rate drops below a certain minimum, the amount of dental caries increases dramatically, also at smooth dental surfaces that are normally not prone to caries as well. Salivary secretion rate is easily measured by weighing the saliva volume that is collected by drooling or expectoration divided by the collection time. Low salivary buffering capacity is a risk factor for dental caries and also is indicative for low saliva secretion. Commercial kits are available for determination of the salivary buffering capacity.

Saliva plays an important role in the maintenance of oral health. 6 For that purpose, it contains a large number of different components that kill or inhibit bacteria, prevent their colonisation, act as nutrient for commensal bacteria and promote remineralisation of the teeth. Therefore, numerous studies have aimed at finding a correlation between dental caries and saliva constituents with only weak correlations. 7 Because dental caries is a multifactorial disease, there are several reasons for the weak salivary correlations. First, saliva output and composition are only two links in a whole chain of events that cause dental caries. Second, whole saliva composition doesn’t reflect the composition of the plaque fluid at sites where dental caries develops. Third, salivary proteins show overlap in function and many proteins have more than one function making it difficult to correlate dental caries to a particularly one, or a few salivary components.

Studies that have focused on functional aspects of whole saliva, rather than studying the quantities of individual proteins, have yielded more promising results. For example, high bacterial aggregation activity of saliva has been associated with low caries experience. 8 Since there is a long list of salivary proteins that bind and may aggregate oral bacteria (e.g. S-IgA, mucins, agglutinin/DMBT-1/gp340, lysozyme, lactoferrin, amylase, proline-rich proteins, statherin and histatins) aggregation couldn’t be correlated with a specific salivary protein. It requires modern proteomic techniques to take them all into consideration. NIH is investing millions of dollars in clarifying the human saliva proteome. At the conference on Oral-based Diagnostics in Atlanta Dr David Wong, UCLA, announced that a first version of the human salivary proteome is available at the website of UCLA in 2007 (www.hspp.ucla.edu).

Many oral bacteria bind carbohydrate chains on salivary glycoproteins. Therefore, the salivary ‘glycome’ might be an important pre-determinant for oral disease. This is illustrated by research of Paul Denny from the University of Southern California, who showed a difference in carbohydrate composition between children at low and high caries risk. 1 Another oral disease for which several aspects of oral-based diagnostics are evaluated, is periodontal disease. Due to poor oral hygiene the dental plaque accumulates at the gingival margin and the composition of the plaque changes, inducing gingival inflammation (gingivitis). This progresses to periodontal disease characterised by breakdown of alveolar bone and connective tissue fibers, resulting in loss of attachment and deepening of the periodontal pocket. Progress from gingivitis to periodontal disease is determined by genetic susceptibility, environmental factors like smoking, and the presence of pathogenic bacteria.

Diagnosis of periodontal disease is primarily based on radiographic analysis and measurement of the pocket depth with a sonde. Though efficient, such clinical methods do not provide adequate information for identifying people at risk, disease activity, causative agents, and treatment outcome. This information could be provided by oral-based diagnostics. There is a large, genetically determined, variation in susceptibility for periodontal disease. 9 Mutations in the cathepsin C gene have been identified as causal for the Papillon-Lefèvre syndrome, including severe forms of prepubertal periodontitis. In addition, multiple genes have been associated with less severe forms of periodontal disease. People at high risk for periodontal disease might be determined therefore by genetic screening. DNA can easily be isolated from oral epithelial cells, collected by use of a buccal swab, one of the most common oral diagnostics.

The loss of attachment and deepening of the periodontal pocket leads to increased leakage of a serum-like fluid, designated gingival crevicular fluid, into the oral cavity. Since serum has a 50 to 70 fold higher protein concentration the average protein concentrations in oral fluid increases dramatically and the concentration of a typical serum component like albumin shows an 8-fold increase. 10 At the conference on Oral-based diagnostics Dr Christoph Ramseier from the University of Michigan, and Dr Ira Lamster from the Columbia University, New York, showed that during active periods of the disease increased levels of inflammatory markers, like interleukins, can be demonstrated both in gingival crevicular fluid and in saliva. Several important marker bacteria have been associated with periodontal disease such as Porphyromonas gingivalis, Prevotella intermedia and Acinobacillus actinomycetemcomitans. Eradication of these bacteria significantly enhances the chance of a positive outcome of treatment. As part of the therapy, antibiotics are applied frequently. However, different periodontopathogens are susceptible to different antibiotics. Therefore, prior to antibiotic treatment pathogens should be determined by culturing or PCR techniques. Oral fluid may be used for that, but since the bacterial numbers in saliva may be too low, small methylcellulose paper strips are used to collect fluid from the gingival crevice. Next to the salivary proteome, there is a salivary ‘transcriptome’ represented by RNA in saliva, as was shown by Dr David Wong. Approximately 3,000 different mRNAs have been found in saliva of which ~200 are commonly present in all people. Upon exploring the clinical utility of the salivary transcriptome in human oral cancer subjects it was found that 4 salivary mRNAs (OAZ, SAT, IL8 and IL1b) collectively have a 91% sensitivity and specificity for detection of oral cancer.

Going beyond oral diseases, oral-based diagnostics finds its way to other applications. A widely used test that is approved by the FDA is an oral test for HIV that detects antibodies against the p24 antigen of HIV. The applicator swab is gently rubbed along the outer gums­­—both upper and lower—and inserted into a vial containing the developer solution that detects the antibody to p24 antigen of HIV. In about 20 minutes, an indicator shows that it is working. A second signal appears if it detects the presence of the p24 antigen; those individuals are given a confirmatory test. Oral samples are also used for testing of illegal street drugs such as marijuana, cocaine, XTC and heroin. Unlike urine samples where switching may be possible, oral samples allow for observed, controlled sample collection.

Conclusively, it can be said that oral-based diagnostics already plays its own specific role in caries prediction and periodontal disease classification. Since blood components are leaking into the oral cavity, we expect that oral-based diagnostics will replace more and more of the current serum-based tests. In addition, microchips for multiple saliva analytes will become available in the near future, bringing proteomics, transcriptomics and genomics within reach of point-of-care diagnostics.

References

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