saliva diagnostics

Saliva as a diagnostic fluid

DENT 5302

Topics in Dental BiochemistryDr. Joel Rudney

Why use saliva?

Collection is relatively non-invasive - no needles! Range of potential analytes as broad as plasma

Bacteria in saliva Salivary ions Secreted salivary proteins Inflammatory mediators Proteins, metabolites, from other parts of the body Nucleic acids (from the mouth and other regions)

This is considered a high-priority research area The vision - chairside diagnostic testing

The search for biomarkers

A big buzzword in diagnostics today Any sort of analyte can be a biomarker The keys to the definition

Changes in a biomarker must be associated with the active presence of a disease, or future risk of a disease

There doesn’t have to be a direct etiological relationship

A marker for risk or diagnosis Multiple analytes can improve sensitivity/specificity

Earlier efforts - caries risk

Bacteria in saliva as biomarkers - chairside tests Dentocult SM® "Strip mutans", S. mutans in saliva Dentocult LB®, Lactobacilli in saliva

Dentobuff® test strips, salivary buffering capacity (HCO3-)

General problem - high sensitivity, but low specificity Combination testing (all three plus flow rate)

Not much improvement These products not widely used in the U.S.

Other oral diseases

Detect candidal infection - Oricult N® Not widely used in the U.S.

Periodontitis The major focus is on biomarkers in gingival fluid Periodontists want to predict which sites will lose

attachment Saliva can only provide indirect indications

Saliva drug testing

Main focus is drugs of abuse Much less invasive than observed urine testing

Harder to fake, no oral equivalent of the “whizzinator” Products for “roadside” forensic testing

Rapiscan ®, Oral-Screen™, 4-5 drugs simultaneouslyCannabinoids, opiates, meth, cocaine, diazepinesSome published validation of these methods

Largely unregulated online offers for home testing Matched by online offers of products to beat the tests No real validation of either

Saliva and steroids Passively carried into saliva with water from plasma Accurately correlated with plasma levels Monitoring of female reproductive hormones

Convenient for monitoring therapeutic usesPost-menopausal HRTMenstrual cycle timing

Saliva and androgens Can be used to monitor testosterone levels

HRT, but also by bodybuilders Steroids of athletic abuse (with the right test system)

Also lots of online activity

Saliva and HIV testing

Extremely low levels of virus relative to other body fluids Assumed to be due to anti-viral salivary proteins

SLIPI, MUC7 Antibodies to HIV (protective or not?) are present

The basis for HIV testing in saliva Saliva-based home HIV test on track for approval

Controversial issuesPro - more people will get tested, seek treatmentCon - no counseling, outside the public health system

Sjogren’s Syndrome proteome Early detection of salivary gland changes Previous studies of individual proteins

Differences, but sensitivity/specificity inadequate Can this be improved by comparing proteomes? Recent data from NIDCR

Compared to controls, patients show:Incr. inflammatory proteins: lysozyme, lactoferrin,

cystatin C, 2-microglobulin, Ig -light chain, polymeric Ig receptor (component of S-IgA)

Decr. acinar proteins: presumed PRPs, amylase, carbonic anhydrase

Key question: How early do these changes begin?

Cancer antigens in saliva

Breast cancer biomarkers Salivary c-erbB-2 protein - an oncogene product Elevated in breast cancer patients

Sensitivity = 87%, specificity = 65% Proteomic studies in progress

Not useful for prostate-specific antigen (PSA)

Saliva and oral cancer

The earlier the diagnosis, the better the prognosis Dentists are the first line of defense Oral cancer is hard to distinguish from transitory lesions

When should you biopsy? Can pre-cancerous conditions be detected? Can salivary biomarkers provide an earlier warning?

An area of active research at U of M

Many potential biomarkers

Elevated levels of certain bacteria in saliva Three very common commensal species

Capnocytophaga gingivalis, Prevotella melaninogenica, and Streptococcus mitis

Probably effect rather than causeSensitivity = 80%, specificity = 82%

Proteomes, genomes, and transcriptomes Dr. David Wong at UCLA School of Dentistry

Collaborators at U of M• Drs. Nelson Rhodus and Patrick Gaffney• Dr. Kathy Moser (Sjogren’s)

Findings from the Wong lab

Oral cancer biomarkers in the salivary proteome Elevated levels of the proinflammatory cytokine IL8

A general marker of inflammationOther sources of oral inflammation - confounders?Additional protein biomarkers may be needed

Oral cancer biomarkers in the salivary transcriptome Wong lab discovered that saliva contains “intact” mRNA Marker of changes in gene expression due to cancer? Seven genes upregulated in patients relative to controls

Includes two proinflammatory cytokines, IL8 and IL1

Chairside testing

Wong lab is collaborating with engineers Objective: “lab on a chip”

Miniaturized multi-analyte detection and quantification Intended for use in a dental office Results at chairside

Application: Identify patients to watch or biopsy Detect oral cancer while it is still treatable

The same concept can be applied to other diseases Oral and systemic diseases both People see dentists more often than physicians The hunt for biomarkers is ongoing

Format of the saliva midterm

Short-answer questions How Dr. Rudney defines a short answer:

When I say short, I mean short Some of them can be answered with a single word.

Respond with words, phrases, or diagrams, as seems appropriateDon’t mess around with complete sentences or essays.Use only the space provided for each question.

In the case of questions worth more than one point:The number of points given will be based on the number of

correct answers you provide for that questionWhen I ask for examples, it means that the number of potentially

correct answers is greater than the number of examples that I’m asking for.

There are no trick questions (at least, not intentionally)

A sample one-point question

Q. Identify the component of the water secretion system that requires energy to function. (1 pt.)

A sample one-point question

Q. Identify the component of the water secretion system that requires energy to function. (1 pt.)

A. Na+-K+-ATPase

You get one point

A sample multi-point question

Q. Give three examples of proteins found in pellicle (3 pts.)

A sample multi-point question

Q. Give three examples of proteins found in pellicle (3 pts.) A. Statherin, acidic proline-rich proteins (aPRP), histatins

You get three points

A sample multi-point question

Q. Give three examples of proteins found in pellicle (3 pts.) Acceptable answers:

Statherin, acidic proline-rich proteins, amylase, histatins, cystatins, MUC7 mucin, lysozyme, albumin, carbonic anhydrase

Use any three, and you get three points

A sample multi-point question

Q. Give three examples of proteins found in pellicle (3 pts.) Acceptable answers:

Statherin, acidic proline-rich proteins, amylase, histatins, cystatins, MUC7 mucin, lysozyme, albumin, carbonic anhydrase

Your answer: Statherin, aPRP, basic PRP

You get two points