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Saliva testing or Salivaomics is a diagnostic technique that involves
laboratory A laboratory (; ; colloquially lab) is a facility that provides controlled conditions in which scientific or technological research, experiments, and measurement may be performed. Laboratory services are provided in a variety of settings: physicia ...
analysis of
saliva Saliva (commonly referred to as spit) is an extracellular fluid produced and secreted by salivary glands in the mouth. In humans, saliva is around 99% water, plus electrolytes, mucus, white blood cells, epithelial cells (from which DNA can be ...
to identify markers of
endocrine The endocrine system is a messenger system comprising feedback loops of the hormones released by internal glands of an organism directly into the circulatory system, regulating distant target organs. In vertebrates, the hypothalamus is the neu ...
,
immunologic Immunology is a branch of medicineImmunology for Medical Students, Roderick Nairn, Matthew Helbert, Mosby, 2007 and biology that covers the medical study of immune systems in humans, animals, plants and sapient species. In such we can see there ...
, inflammatory, infectious, and other types of conditions. Saliva is a useful biological fluid for assaying steroid hormones such as
cortisol Cortisol is a steroid hormone, in the glucocorticoid class of hormones. When used as a medication, it is known as hydrocortisone. It is produced in many animals, mainly by the ''zona fasciculata'' of the adrenal cortex in the adrenal gland ...
, genetic material like
RNA Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...
, proteins such as
enzyme Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. A ...
s and
antibodies An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the ...
, and a variety of other substances, including natural metabolites, including saliva nitrite, a biomarker for
nitric oxide Nitric oxide (nitrogen oxide or nitrogen monoxide) is a colorless gas with the formula . It is one of the principal oxides of nitrogen. Nitric oxide is a free radical: it has an unpaired electron, which is sometimes denoted by a dot in its che ...
status (see below for Cardiovascular Disease, Nitric Oxide: a salivary biomarker for cardio-protection). Saliva testing is used to screen for or diagnose numerous conditions and disease states, including Cushing's disease, anovulation,
HIV The human immunodeficiency viruses (HIV) are two species of ''Lentivirus'' (a subgroup of retrovirus) that infect humans. Over time, they cause acquired immunodeficiency syndrome (AIDS), a condition in which progressive failure of the immune ...
,
cancer Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. These contrast with benign tumors, which do not spread. Possible signs and symptoms include a lump, abnormal b ...
,
parasite Parasitism is a close relationship between species, where one organism, the parasite, lives on or inside another organism, the host, causing it some harm, and is adapted structurally to this way of life. The entomologist E. O. Wilson has ...
s, hypogonadism, and allergies. Salivary testing has even been used by the U.S. government to assess
circadian rhythm A circadian rhythm (), or circadian cycle, is a natural, internal process that regulates the sleep–wake cycle and repeats roughly every 24 hours. It can refer to any process that originates within an organism (i.e., Endogeny (biology), endogeno ...
shifts in astronauts before flight and to evaluate hormonal profiles of soldiers undergoing military survival training. Proponents of saliva testing cite its ease of collection, safety, non-invasiveness, affordability, accuracy, and capacity to circumvent venipuncture as the primary advantages when compared to blood testing and other types of
diagnostic test A medical test is a medical procedure performed to detect, diagnose, or monitor diseases, disease processes, susceptibility, or to determine a course of treatment. Medical tests such as, physical and visual exams, diagnostic imaging, genetic ...
ing. Additionally, since multiple samples can be readily obtained, saliva testing is particularly useful for performing chronobiological assessments that span hours, days, or weeks. Collecting whole saliva by passive drool has a myriad of advantages. Passive drool collection facilitates large sample size collection. Consequently, this allows the sample to be tested for more than one biomarker. It also gives the researcher the ability to freeze the left over specimen to be used at a later time. Additionally, it lessens the possibility of contamination by eliminating extra collection devices and the need to induce saliva flow. The clinical use of saliva testing occurred at least as early as 1836 in patients with bronchitis. Testing the acidity of saliva occurred at least as early as 1808. The testing of salivation by the use of mercury was performed at least as early as 1685. More recent studies have focused on detection of steroid hormones and antibodies in the saliva. Recent applications emphasize the development of increasingly sophisticated techniques to detect additional proteins, genetic material, and markers of nutritional status. According to Wong, scientists are now viewing saliva as "a valuable biofluid…with the potential to extract more data than is possible currently with other diagnostic methods."


Technique

Most saliva testing is performed using
enzyme-linked immunosorbent assay The enzyme-linked immunosorbent assay (ELISA) (, ) is a commonly used analytical biochemistry assay, first described by Eva Engvall and Peter Perlmann in 1971. The assay uses a solid-phase type of enzyme immunoassay (EIA) to detect the presence ...
(ELISA),
polymerase chain reaction The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete or partial) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) t ...
(PCR), high-resolution mass spectrometry (HRMS), or any number of newer technologies such as fiber-optic-based detection. All of these methods enable detection of specific molecules like
cortisol Cortisol is a steroid hormone, in the glucocorticoid class of hormones. When used as a medication, it is known as hydrocortisone. It is produced in many animals, mainly by the ''zona fasciculata'' of the adrenal cortex in the adrenal gland ...
, C-reactive protein (CRP), or secretory IgA. This type of testing typically involves collection of a small amount of saliva into a sterile tube followed by processing at a remote laboratory. Some methods of testing involve collecting saliva using an absorbent pad, applying a chemical solution, and monitoring for color change to indicate a positive or negative result. This method is commonly used as a point-of-care (POC) technique to screen for HIV. However, using absorbent pads and chemical solutions could very easily skew the results of immunoassays. Research by Dr. Douglas A. Granger and colleagues shows that outcomes for testosterone, DHEA, progesterone, and estradiol biomarkers are elevated when cotton-based collection materials are used as opposed to samples collected by other methods (i.e. passive drool). Researchers are currently examining the expanding role of saliva testing as part of routine dental or medical office examinations where saliva collection is simple to perform.


Physiologic basis

Humans have three major salivary glands: parotid, submandibular, and
sublingual Sublingual (abbreviated SL), from the Latin for "under the tongue", refers to the pharmacological route of administration by which substances diffuse into the blood through tissues under the tongue. The sublingual glands receive their prima ...
. These glands, along with additional minor salivary glands, secrete a rich mixture of biological chemicals,
electrolyte An electrolyte is a medium containing ions that is electrically conducting through the movement of those ions, but not conducting electrons. This includes most soluble salts, acids, and bases dissolved in a polar solvent, such as water. Upon dis ...
s, proteins, genetic material,
polysaccharide Polysaccharides (), or polycarbohydrates, are the most abundant carbohydrates found in food. They are long chain polymeric carbohydrates composed of monosaccharide units bound together by glycosidic linkages. This carbohydrate can react with wa ...
s, and other molecules. Most of these substances enter the salivary gland
acinus An acinus (; plural, acini; adjective, acinar or acinous) refers to any cluster of cells that resembles a many-lobed "berry," such as a raspberry ('' acinus'' is Latin for "berry"). The berry-shaped termination of an exocrine gland, where the ...
and duct system from the surrounding capillaries via the intervening tissue fluid, although some substances are produced within the glands themselves. The level of each salivary component varies considerably depending on the health status of the individual and the presence of disease (oral or systemic). By measuring these components in the saliva, it is possible to screen for a variety of infections, allergies, hormonal disturbances, and
neoplasm A neoplasm () is a type of abnormal and excessive growth of tissue. The process that occurs to form or produce a neoplasm is called neoplasia. The growth of a neoplasm is uncoordinated with that of the normal surrounding tissue, and persists ...
s.


Clinical use

The following conditions are among those that can be detected through saliva testing (list not comprehensive): adrenal conditions (such as Cushing's disease/syndrome and
Addison's disease Addison's disease, also known as primary adrenal insufficiency, is a rare long-term endocrine disorder characterized by inadequate production of the steroid hormones cortisol and aldosterone by the two outer layers of the cells of the adrenal ...
), altered female hormone states (such as
polycystic ovary syndrome Polycystic ovary syndrome, or PCOS, is the most common endocrine disorder in women of reproductive age. The syndrome is named after the characteristic cysts which may form on the ovaries, though it is important to note that this is a sign and no ...
COS
menopause Menopause, also known as the climacteric, is the time in women's lives when menstrual periods stop permanently, and they are no longer able to bear children. Menopause usually occurs between the age of 47 and 54. Medical professionals often d ...
, anovulation, and hormonal alterations in cycling women), altered male hormone states (such as hypogonadism/ andropause and
hyperestrogenic Estrogen or oestrogen is a category of sex hormone responsible for the development and regulation of the female reproductive system and secondary sex characteristics. There are three major endogenous estrogens that have estrogenic hormonal activ ...
states), metabolic disturbances (such as
insulin resistance Insulin resistance (IR) is a pathological condition in which cell (biology), cells fail to respond normally to the hormone insulin. Insulin is a hormone that facilitates the transport of glucose from blood into cells, thereby reducing blood gluco ...
,
diabetes Diabetes, also known as diabetes mellitus, is a group of metabolic disorders characterized by a high blood sugar level ( hyperglycemia) over a prolonged period of time. Symptoms often include frequent urination, increased thirst and increased ap ...
, and
metabolic syndrome Metabolic syndrome is a clustering of at least three of the following five medical conditions: abdominal obesity, high blood pressure, high blood sugar, high serum triglycerides, and low serum high-density lipoprotein (HDL). Metabolic syndrome ...
), benign and metastatic neoplasms (such as
breast cancer Breast cancer is cancer that develops from breast tissue. Signs of breast cancer may include a lump in the breast, a change in breast shape, dimpling of the skin, milk rejection, fluid coming from the nipple, a newly inverted nipple, or a re ...
,
pancreatic cancer Pancreatic cancer arises when cell (biology), cells in the pancreas, a glandular organ behind the stomach, begin to multiply out of control and form a Neoplasm, mass. These cancerous cells have the malignant, ability to invade other parts of t ...
, and oral cancer), infectious conditions (such as HIV, viral hepatitis, amoebiasis, and helicobacter pylori infection), and allergic conditions (such as food allergy).


Uses in behavioral research

Saliva testing also has specific uses in clinical and experimental psychological settings. Due to its ability to provide insight into human behavior, emotions, and development, it has been used to investigate psychological phenomenon such as
anxiety Anxiety is an emotion which is characterized by an unpleasant state of inner turmoil and includes feelings of dread over anticipated events. Anxiety is different than fear in that the former is defined as the anticipation of a future threat wh ...
, depression, PTSD, and other behavioral disorders. Its primary purpose is to test
cortisol Cortisol is a steroid hormone, in the glucocorticoid class of hormones. When used as a medication, it is known as hydrocortisone. It is produced in many animals, mainly by the ''zona fasciculata'' of the adrenal cortex in the adrenal gland ...
and alpha amylase levels, which are indicative of stress levels. Salivary cortisol is a good stress index, with increased levels of cortisol correlating positively with increased levels of stress. Cortisol levels rise slowly over time and take a while to return to base level, indicating that cortisol is more associated with chronic stress levels. Alpha amylase, on the other hand, spikes quickly when confronted with a stressor and returns to baseline soon after the stress has passed, making salivary amylase measurement a powerful tool for psychological research studying acute stress responses. Samples are usually collected from participants by having them drool through a straw into a collection tube while experiencing a stimulus, with samples taken every few minutes to record the gradual change in stress hormone levels. Because the collection of saliva samples is non-invasive, it has the advantage of not introducing further stress on the participant that may otherwise distort results. In more specific studies looking at the link between cortisol levels and psychological phenomena, it has been found that chronic stressors such as life-threatening situations (example: diseases), depression, and social or economic hardship correlate with significantly higher cortisol levels. In situations where a subject undergoes induced anxiety, high cortisol levels correspond with experiencing more physiological symptoms of nervousness, such as increased heart rate, sweating, and
skin conductance Electrodermal activity (EDA) is the property of the human body that causes continuous variation in the electrical characteristics of the skin. Historically, EDA has also been known as skin conductance, galvanic skin response (GSR), electrodermal ...
. Additionally, a negative correlation was discovered between baseline levels of cortisol and aggression. Salivary cortisol levels can thus provide insight into a number of other psychological processes. Alpha amylase levels in saliva provide a non-invasive way to examine sympathoadrenal medullary (SAM) activity, which can otherwise be measured with
electrophysiological Electrophysiology (from Greek , ''ēlektron'', "amber" etymology of "electron"">Electron#Etymology">etymology of "electron" , ''physis'', "nature, origin"; and , ''-logia'') is the branch of physiology that studies the electrical properties of bi ...
equipment or
blood plasma Blood plasma is a light amber-colored liquid component of blood in which blood cells are absent, but contains proteins and other constituents of whole blood in suspension. It makes up about 55% of the body's total blood volume. It is the intra ...
readings. Salivary alpha amylase levels have been found to correlate with heightened
autonomic nervous system The autonomic nervous system (ANS), formerly referred to as the vegetative nervous system, is a division of the peripheral nervous system that supplies viscera, internal organs, smooth muscle and glands. The autonomic nervous system is a control ...
activity levels, reacting in similar ways to the hormone
norepinephrine Norepinephrine (NE), also called noradrenaline (NA) or noradrenalin, is an organic chemical in the catecholamine family that functions in the brain and body as both a hormone and neurotransmitter. The name "noradrenaline" (from Latin '' ad'', ...
. Subsequent findings reveal a relationship between α-amylase and competition. Results showed that alpha amylase levels changed when reacting to competition, but not when anticipating it. Furthermore, by testing alpha amylase levels, scientists noticed a difference in reactivity behavior among individuals with previous experience in a similar situation. While saliva testing has the promise of becoming a valuable and more widely used tool in psychological research in the future, there are also some disadvantages to the method that must be kept in mind, including the cost of collecting and processing the samples and the reliability of the measure itself. There is a substantial amount of both within-person and between-person variability in cortisol levels that must be taken into account when drawing conclusions from studies. Many studies have been performed to further examine the variables that contribute to these within-person and between-person variances. Analyses of the variables that affect cortisol levels has yielded an extensive list of confounding variables.
Diurnal variation A chronotype is the behavioral manifestation of underlying circadian rhythm's myriad of physical processes. A person's chronotype is the propensity for the individual to sleep at a particular time during a 24-hour period. ''Eveningness'' (delayed ...
is a major factor for within-person variance because baseline cortisol levels have been known to differ based on the time of day. For normally developing individuals who follow a typical day–night schedule, cortisol production peaks during the last few hours of sleep. This peak is thought to aid in preparing the body for action and stimulate the appetite upon waking. Diurnal variation is also affected by psychological conditions. For example, Early morning cortisol levels have been found to be elevated in shy children and late night levels elevated in depressed adolescents, particularly the between the hours of two and four PM. This might be important for understanding emotions and depressive symptoms. Other variables that affect within- and between-person variation are listed below. The list is not meant to be comprehensive and the impact of many of these variables could benefit from further research and discussion. * Age is one of the major factors for between-person variance. Some studies indicate children and adolescents exhibit greater cortisol activity potentially related to development.Kudielka, B., Hellhammer, D. H., & Wust, S. (2009, January). Why do we respond so differently? reviewing determinants of human salivary cortisol responses to challenge. * Gender has been found to impact base line levels of cortisol, contributing to between-person variance. In generally stressful situations, levels of cortisol in males have been found to increase to nearly double the amount when compared to females. In stressful social situations (i.e. social rejection challenge), however, women but not men tend to show significantly higher levels of cortisol. * The
menstrual cycle The menstrual cycle is a series of natural changes in hormone production and the structures of the uterus and ovaries of the female reproductive system that make pregnancy possible. The ovarian cycle controls the production and release of eggs a ...
has been found to impact levels of cortisol in the body, impacting both within- and between-person variance. Women in the luteal phase reportedly have levels of cortisol equal to men, suggesting no sex differences in base levels of cortical when women are not ovulating. Women in the
follicular phase The follicular phase, also known as the preovulatory phase or proliferative phase, is the phase of the estrous cycle (or, in primates for example, the menstrual cycle) during which follicles in the ovary mature from primary follicle to a ful ...
and women taking
oral contraceptives Oral contraceptives, abbreviated OCPs, also known as birth control pills, are medications taken by mouth for the purpose of birth control. Female Two types of female oral contraceptive pill, taken once per day, are widely available: * The combin ...
reportedly have significantly lower levels of cortisol when compared to men and women in the luteal phase. *
Pregnancy Pregnancy is the time during which one or more offspring develops ( gestates) inside a woman's uterus (womb). A multiple pregnancy involves more than one offspring, such as with twins. Pregnancy usually occurs by sexual intercourse, but ca ...
has been found to increase levels of cortisol in the body. In particular, breast-feeding has been found to decrease levels of cortisol in the short-term even if a mother is exposed to a psychosocial stressor. *
Nicotine Nicotine is a natural product, naturally produced alkaloid in the nightshade family of plants (most predominantly in tobacco and ''Duboisia hopwoodii'') and is widely used recreational drug use, recreationally as a stimulant and anxiolytic. As ...
is known to increase levels of cortisol in the body since it stimulates the HPA axis. After at least two cigarettes, smokers show significant elevations of salivary cortisol levels. Furthermore, habitual smokers show blunted salivary cortisol responses to psychological stressors. * Food has been found to affect levels of cortisol. The presence of proteins has been found to increase cortisol. This variable is often affected by diurnal variation, with cortisol being notably higher during lunchtime than dinnertime, and gender, with women having higher levels of cortisol after eating than men. * While some studies examining the effects of alcohol consumption and caffeine intake on base levels of cortisol have found positive correlations, the results are mixed and would benefit from further examination. * Intense or prolonged exercise can result in increased levels of cortisol. Short-term and low-level exercising only mildly increases levels of cortisol. * Repeated exposure to initially stressful stimuli has been found to result in a leveling off of cortisol in the body. * Birth weight has been shown to be inversely related to base levels of cortisol;
low birth weight Low birth weight (LBW) is defined by the World Health Organization as a birth weight of an infant of or less, regardless of gestational age. Infants born with LBW have added health risks which require close management, often in a neonatal intensi ...
is correlated with high levels of cortisol. * Position within a social hierarchy has been found to affect levels of cortisol. One study in particular looked at a sample of 63 army recruits and found that socially dominant subjects showed high salivary cortisol increases compared to only modest elevations in subordinate men after stress exposure and physical exercise. * Some medications (i.e.
glucocorticoid Glucocorticoids (or, less commonly, glucocorticosteroids) are a class of corticosteroids, which are a class of steroid hormones. Glucocorticoids are corticosteroids that bind to the glucocorticoid receptor that is present in almost every vertebr ...
s,
psychoactive drug A psychoactive drug, psychopharmaceutical, psychoactive agent or psychotropic drug is a chemical substance, that changes functions of the nervous system, and results in alterations in perception, mood, consciousness, cognition or behavior. ...
s,
antidepressant Antidepressants are a class of medication used to treat major depressive disorder, anxiety disorders, chronic pain conditions, and to help manage addictions. Common side-effects of antidepressants include dry mouth, weight gain, dizziness, hea ...
s) have been found to affect levels of cortisol in the body but the results from studies examining these affects have been mixed. The impact of medications on cortical levels could benefit from further research.


Evidence and current research


Cortisol and melatonin aberrations

In 2008 the
Endocrine Society The Endocrine Society is a professional, international medical organization in the field of endocrinology and metabolism, founded in 1916 as The Association for the Study of Internal Secretions. The official name of the organization was changed ...
published diagnostic guidelines for Cushing's syndrome, wherein they recommended midnight salivary cortisol testing on two consecutive days as one possible initial screening tool. A 2009 review concluded that late-night salivary cortisol testing is a suitable alternative to serum cortisol testing for diagnosing Cushing's syndrome, reporting that both sensitivity and specificity exceeded ninety percent. In 2010 Sakihara, ''et al''., evaluated the usefulness and accuracy of salivary, plasma, and urinary cortisol levels and determined salivary cortisol to be the "method of choice" for Cushing's syndrome screening. In 2008 Restituto, ''et al''., found early morning salivary cortisol to be "as good as serum" as an
Addison's disease Addison's disease, also known as primary adrenal insufficiency, is a rare long-term endocrine disorder characterized by inadequate production of the steroid hormones cortisol and aldosterone by the two outer layers of the cells of the adrenal ...
screening technique. In 2010 Bagcim ''et al''., determined that saliva
melatonin Melatonin is a natural product found in plants and animals. It is primarily known in animals as a hormone released by the pineal gland in the brain at night, and has long been associated with control of the sleep–wake cycle. In vertebrates ...
levels "reflect those in serum at any time of the day" and are a reliable alternative to serum melatonin to study the pineal physiology in newborns. A 2008 review article described saliva melatonin testing as a "practical and reliable method for field, clinical, and research trials".


Reproductive hormone irregularities

A 2009 study examined the use of saliva testing to measure estradiol,
progesterone Progesterone (P4) is an endogenous steroid and progestogen sex hormone involved in the menstrual cycle, pregnancy, and embryogenesis of humans and other species. It belongs to a group of steroid hormones called the progestogens and is the m ...
,
dehydroepiandrosterone Dehydroepiandrosterone (DHEA), also known as androstenolone, is an endogenous steroid hormone precursor. It is one of the most abundant circulating steroids in humans. DHEA is produced in the adrenal glands, the gonads, and the brain. It functio ...
(DHEA), and
testosterone Testosterone is the primary sex hormone and anabolic steroid in males. In humans, testosterone plays a key role in the development of Male reproductive system, male reproductive tissues such as testes and prostate, as well as promoting secondar ...
levels in 2,722 individuals (male and female). The researchers confirmed the "good validity of alivarysex hormone measurements" and concluded that salivary testing was a good method for testing older adults due to the ease of in-home collection. However, other studies suggest that such tests do not represent either the amount of hormones in the
blood Blood is a body fluid in the circulatory system of humans and other vertebrates that delivers necessary substances such as nutrients and oxygen to the cells, and transports metabolic waste products away from those same cells. Blood in the c ...
, or their
biological activity In pharmacology, biological activity or pharmacological activity describes the beneficial or adverse effects of a drug on living matter. When a drug is a complex chemical mixture, this activity is exerted by the substance's active ingredient or ...
. Saliva testing is often used as part of
bioidentical hormone replacement therapy Bioidentical hormone replacement therapy (BHRT), also known as bioidentical hormone therapy (BHT) or natural hormone therapy, is the use of hormones that are identical on a molecular level with endogenous hormones in hormone replacement therapy ...
, though it has been criticized for being expensive, unnecessary and meaningless.


Female

In 2010 a study identified luteinizing hormone (LH) as an accurate salivary biomarker of ovulation in females. Researchers measured various hormones in the saliva throughout the menstrual cycle and found that salivary luteinizing hormone was reliably elevated during the ovulatory period and, for that reason, "salivary LH level is a reliable way to determine ovulation." A 1983 study of various salivary steroid assays showed that daily salivary progesterone measurements "provides a valuable means of assessing ovarian function". A 2001 study involved performing daily saliva collection from healthy subjects and plotting these over the entire menstrual cycle. The researchers determined that salivary estradiol and progesterone curves corresponded to the daily profiles normally observed in blood, although of lesser amplitude. In 1999 researchers determined that ELISA-based saliva testing "can serve as a reliable ethodfor estriol determination." A 2007 article reported that the free testosterone measurement, including via saliva assay, represents "the most sensitive biochemical marker supporting the diagnosis of PCOS." In 1990 Vuorento, et al., found that luteal phase defects, wherein progesterone levels decline prematurely within the menstrual cycle, were identified with high frequency using salivary progesterone testing among women with unexplained infertility.


Male

In 2009 Shibayama, et al., examined the accuracy of salivary androgen measurement for diagnosing late-onset hypogonadism (age-related decline in androgens, often called "andropause"). Researchers determined that the accuracy of saliva testosterone and DHEA measurement exceeded 98.5% and that this method "has satisfactory applicability" in the diagnosis of late-onset hypogonadism. A 2007 study reported a sensitivity and specificity of 100% for salivary testosterone in ruling out hypogonadism and concluded that salivary testosterone is a useful biomarker in the diagnosis of male androgen deficiency. The use of salivary testosterone to screen for hypogonadism has been validated by other studies, including one involving 1454 individuals. Those researchers concluded that salivary testosterone is "an acceptable assay for screening for hypogonadism."


Neoplastic conditions


Pancreatic cancer

A 2010 study by Zhang, et al., demonstrated that researchers were able to detect
pancreatic cancer Pancreatic cancer arises when cell (biology), cells in the pancreas, a glandular organ behind the stomach, begin to multiply out of control and form a Neoplasm, mass. These cancerous cells have the malignant, ability to invade other parts of t ...
with high
sensitivity and specificity ''Sensitivity'' and ''specificity'' mathematically describe the accuracy of a test which reports the presence or absence of a condition. Individuals for which the condition is satisfied are considered "positive" and those for which it is not are ...
(90.0% and 95.0%, respectively) by screening saliva for four specific
mRNA In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of Protein biosynthesis, synthesizing a protein. mRNA is ...
biomarkers. In a 2011 review article that examined pancreatic cancer biomarkers, Hamade and Shimosegawa concluded that clinical application of saliva biomarker testing is "beneficial for the screening and early detection of pancreatic cancer."


Breast cancer

In 2008 Emekli-Alturfan, et al., compared saliva from
breast cancer Breast cancer is cancer that develops from breast tissue. Signs of breast cancer may include a lump in the breast, a change in breast shape, dimpling of the skin, milk rejection, fluid coming from the nipple, a newly inverted nipple, or a re ...
patients to that from healthy individuals and observed, notably, that breast cancer patients' samples contained
dysplastic Dysplasia is any of various types of abnormal growth or development of cells (microscopic scale) or organs (macroscopic scale), and the abnormal histology or anatomical structure(s) resulting from such growth. Dysplasias on a mainly microscopic ...
cells and reduced
lipid peroxide Lipid peroxidation is the chain of reactions of oxidative degradation of lipids. It is the process in which free radicals "steal" electrons from the lipids in cell membranes, resulting in cell damage. This process proceeds by a free radical chain ...
s. A 2000 study compared the salivary levels of a breast cancer marker (HER2/neu) in healthy women, women with benign breast lesions, and women with breast cancer. Researchers found that the salivary (as well as serum) level of this marker was significantly higher in women with breast cancer than in healthy women and women with benign breast lesions; they went on to state that the marker may have potential as a tool for diagnosing breast cancer or detecting its recurrence. A separate study corroborated these findings and further demonstrated that another breast cancer marker (CA15-3) was elevated while the
tumor suppressor protein p53 p53, also known as Tumor protein P53, cellular tumor antigen p53 (UniProt name), or transformation-related protein 53 (TRP53) is a regulatory protein that is often mutated in human cancers. The p53 proteins (originally thought to be, and often s ...
was reduced in the saliva of women with breast cancer compared to healthy controls and women with benign breast lesions.


Oral cancer

In 2010 Jou, et al., found that patients diagnosed with oral
squamous cell carcinoma Squamous-cell carcinomas (SCCs), also known as epidermoid carcinomas, comprise a number of different types of cancer that begin in squamous cells. These cells form on the surface of the skin, on the lining of hollow organs in the body, and on the ...
had elevated levels
transferrin Transferrins are glycoproteins found in vertebrates which bind to and consequently mediate the transport of iron (Fe) through blood plasma. They are produced in the liver and contain binding sites for two Fe3+ ions. Human transferrin is encode ...
in saliva compared to healthy controls and, moreover, that salivary transferrin measurement using ELISA technique was "highly specific, sensitive, and accurate for the early detection of oral cancer." A 2009 study reported that the levels of two biomarkers,
Cyclin D1 Cyclin D1 is a protein that in humans is encoded by the ''CCND1'' gene. Gene expression The CCND1 gene encodes the cyclin D1 protein. The human CCND1 gene is located on the long arm of chromosome 11 (band 11q13). It is 13,388 base pairs lon ...
(increased compared to controls) and
Maspin Maspin (mammary serine protease inhibitor) is a protein that in humans is encoded by the ''SERPINB5'' gene. This protein belongs to the serpin (serine protease inhibitor) superfamily. ''SERPINB5'' was originally reported to function as a tumor su ...
(decreased compared to controls), had sensitivities and specificities of 100% for oral cancer detection when measured in saliva. Saliva testing for specific
mRNA In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of Protein biosynthesis, synthesizing a protein. mRNA is ...
s has been found to possess significant potential for oral cancer diagnosis. In fact, there is evidence to suggest that saliva RNA diagnostics are slightly superior to serum RNA diagnostics, with the comparative
receiver operating characteristic A receiver operating characteristic curve, or ROC curve, is a graphical plot that illustrates the diagnostic ability of a binary classifier system as its discrimination threshold is varied. The method was originally developed for operators of ...
(ROC) value being 95% for saliva but only 88% for serum.


Glucose dysregulation

A 2009 study compared the saliva glucose levels of diabetic patients to those of non-diabetic controls. The authors reported that "salivary lucoseconcentration and excretion were much higher in diabetic patients than in control subjects." In 2009 Rao, et al., investigated salivary biomarkers that could aid identification of type-2 diabetic individuals. Researchers found that sixty-five proteins, the majority of which are involved in regulating metabolism and immune response, were significantly altered in type-2 diabetics. They further observed that the relative increase of these specific proteins was directly proportional to the severity of disease (i.e., they were somewhat elevated in pre-diabetics and significantly elevated in diabetics). In 2010 Soell, et al., determined that one particular salivary biomarker (chromogranin A) was over-expressed in 100% of diabetic patients when compared to controls. In 2010 Qvarnstrom, et al., conducted a cross-sectional analysis of 500 individuals and found that an increase in salivary lysozyme was "significantly associated with metabolic syndrome."


Infectious conditions


Human immunodeficiency virus

The accuracy of saliva anti-HIV antibody testing has been demonstrated in numerous studies; two recent large-scale studies found both sensitivity and specificity to be 100%. The first of these was published in 2008 by Zelin, et al., and compared saliva antibody testing and serum antibody testing using ELISA technique in 820 individuals. The second study, conducted by Pascoe, et al., compared saliva antibody testing to serum antibody testing using ELISA followed by confirmatory Western Blot analysis in 591 individuals. The accuracy of saliva anti-HIV antibody testing has been confirmed by many additional studies, leading to approval of this method by the U.S. Food & Drug Administration in 2004.


Viral hepatitis

Several studies have demonstrated diagnostic potential for salivary hepatitis testing. A 2011 study demonstrated that HBV surface antigen saliva testing using ELISA had a sensitivity and specificity of 93.6% and 92.6%, respectively. Other studies found that saliva assay for anti-HAV antibodies (
IgM Immunoglobulin M (IgM) is one of several isotypes of antibody (also known as immunoglobulin) that are produced by vertebrates. IgM is the largest antibody, and it is the first antibody to appear in the response to initial exposure to an antig ...
and
IgG Immunoglobulin G (Ig G) is a type of antibody. Representing approximately 75% of serum antibodies in humans, IgG is the most common type of antibody found in blood circulation. IgG molecules are created and released by plasma B cells. Each IgG a ...
) was an effective method to identify HAV-infected individuals.
Hepatitis C Hepatitis C is an infectious disease caused by the hepatitis C virus (HCV) that primarily affects the liver; it is a type of viral hepatitis. During the initial infection people often have mild or no symptoms. Occasionally a fever, dark urine, a ...
has also been identified using salivary detection methods. Yaari, et al., reported in 2006 that saliva testing for anti-HCV antibodies yielded a sensitivity of 100% and a specificity that was "similar or better" when compared to serum testing.


Parasitic infection

A 2010 study found that saliva-based detection of the parasite '' Entamoeba histolytica'' was superior to existing fecal detection methods for patients with E. histolytica-associated
liver abscess A liver abscess is a mass filled with pus inside the liver. Common causes are abdominal conditions such as appendicitis or diverticulitis due to haematogenous spread through the portal vein. It can also develop as a complication of a liver injury. ...
. In 2004 El Hamshary and Arafa found that salivary anti-E. histolytica
IgA Iga may refer to: Arts and entertainment * Ambush at Iga Pass, a 1958 Japanese film * Iga no Kagemaru, Japanese manga series * Iga, a set of characters from the Japanese novel '' The Kouga Ninja Scrolls'' Biology * ''Iga'' (beetle), a gen ...
concentration had "predictive diagnostic value of intestinal amoebiasis…as well as in tissue amoebiasis." A 1990 study that involved saliva testing for E. histolytica in 223 school children demonstrated a sensitivity and specificity of 85% and 98%, respectively. In 2005 Stroehle, et al., determined that saliva detection of IgG antibodies against ''
Toxoplasma gondii ''Toxoplasma gondii'' () is an obligate intracellular parasitic protozoan (specifically an apicomplexan) that causes toxoplasmosis. Found worldwide, ''T. gondii'' is capable of infecting virtually all warm-blooded animals, but felids, such as d ...
'' had a sensitivity and specificity of 98.5% and 100%, respectively. A study published in 1990 demonstrated the diagnostic utility of saliva IgG testing in identifying
neurocysticercosis Neurocysticercosis is a specific form of the infectious parasitic disease cysticercosis that is caused by the infection with ''Taenia solium'', a tapeworm found in pigs. Neurocysticercosis occurs when cysts formed by the infection take hold with ...
secondary to '' Taenia solium''.


''Helicobacter pylori'' infection

In a 2005 study, researchers investigated the accuracy of '' Helicobacter pylori'' diagnosis in dyspeptic patients using salivary anti-''H. pylori''
IgG Immunoglobulin G (Ig G) is a type of antibody. Representing approximately 75% of serum antibodies in humans, IgG is the most common type of antibody found in blood circulation. IgG molecules are created and released by plasma B cells. Each IgG a ...
levels. They determined that saliva testing for ''H. pylori'' antibodies "could be used reliably for screening dyspeptic patients in general practice." That same year Tiwari, et al., examined the accuracy of testing saliva for ''H. pylori'' DNA and how well this correlated with presence of ''H. pylori'' detected via gastric biopsy. Based on their results, researchers concluded that saliva testing could serve as a reliable non-invasive detection method for ''H. pylori'' infection.


Periodontitis

A 2009 study conducted by Koss, et al., studied salivary biomarkers of periodontal disease; their findings revealed that three substances ( peroxidase,
hydroxyproline (2''S'',4''R'')-4-Hydroxyproline, or L-hydroxyproline ( C5 H9 O3 N), is an amino acid, abbreviated as Hyp or O, ''e.g.'', in Protein Data Bank. Structure and discovery In 1902, Hermann Emil Fischer isolated hydroxyproline from hydrolyzed gelatin. ...
and
calcium Calcium is a chemical element with the symbol Ca and atomic number 20. As an alkaline earth metal, calcium is a reactive metal that forms a dark oxide-nitride layer when exposed to air. Its physical and chemical properties are most similar to ...
) were significantly increased in the saliva of patients with
periodontitis Periodontal disease, also known as gum disease, is a set of inflammatory conditions affecting the tissues surrounding the teeth. In its early stage, called gingivitis, the gums become swollen and red and may bleed. It is considered the main caus ...
. A 2010 study found that elevation of three saliva biomarkers (MMP-8, TIMP-1, and ICTP), particularly when analyzed using time-resolved immunofluorometric assay, was suggestive of periodontitis.


Cardiovascular disease


CRP: a salivary biomarker for cardiovascular risk

In 2011 Punyadeera, et al., studied "the clinical utility of salivary C-reactive protein levels in assessing coronary events such as
myocardial infarction A myocardial infarction (MI), commonly known as a heart attack, occurs when blood flow decreases or stops to the coronary artery of the heart, causing damage to the heart muscle. The most common symptom is chest pain or discomfort which may ...
in a primary health care setting." Researchers found that saliva CRP levels in cardiac patients were significantly higher when compared to healthy controls. Furthermore, they found that saliva CRP correlated with serum CRP in cardiac patients and, thus, could be a useful tool for "large patient screening studies for risk assessment of coronary events."


Nitric Oxide: a salivary biomarker for cardio-protection

Cardio-protective nitric oxide is generated in the body by a family of specific enzymes,
nitric oxide synthase Nitric oxide synthases () (NOSs) are a family of enzymes catalyzing the production of nitric oxide (NO) from L-arginine. NO is an important cellular signaling molecule. It helps modulate vascular tone, insulin secretion, airway tone, and perista ...
. An alternative pathway for the generation of
nitric oxide Nitric oxide (nitrogen oxide or nitrogen monoxide) is a colorless gas with the formula . It is one of the principal oxides of nitrogen. Nitric oxide is a free radical: it has an unpaired electron, which is sometimes denoted by a dot in its che ...
is the nitrate-nitrite-nitric oxide pathway in which dietary inorganic nitrate is sequentially reduced to
nitric oxide Nitric oxide (nitrogen oxide or nitrogen monoxide) is a colorless gas with the formula . It is one of the principal oxides of nitrogen. Nitric oxide is a free radical: it has an unpaired electron, which is sometimes denoted by a dot in its che ...
. A necessary and obligatory step in the generation of
nitric oxide Nitric oxide (nitrogen oxide or nitrogen monoxide) is a colorless gas with the formula . It is one of the principal oxides of nitrogen. Nitric oxide is a free radical: it has an unpaired electron, which is sometimes denoted by a dot in its che ...
by the non-nitric oxide synthase or alternative pathway involves the uptake of nitrate by the salivary gland, excretion in saliva, and subsequent reduction to nitrite by oral commensal bacteria in the mouth. Salivary nitrite is then further chemically reduced in blood and tissue to
nitric oxide Nitric oxide (nitrogen oxide or nitrogen monoxide) is a colorless gas with the formula . It is one of the principal oxides of nitrogen. Nitric oxide is a free radical: it has an unpaired electron, which is sometimes denoted by a dot in its che ...
resulting in the lowering of
blood pressure Blood pressure (BP) is the pressure of circulating blood against the walls of blood vessels. Most of this pressure results from the heart pumping blood through the circulatory system. When used without qualification, the term "blood pressure" r ...
, inhibition of platelet aggregation, increasing cerebral blood flow and flow-mediated dilation, and decreasing oxygen cost during exercise. A principal source of dietary inorganic nitrate, which is reduced to nitric oxide in the body, is from leafy green vegetables. The blood pressure lowering effects of leafy green vegetables, in particular,
spinach Spinach (''Spinacia oleracea'') is a leafy green flowering plant native to central and western Asia. It is of the order Caryophyllales, family Amaranthaceae, subfamily Chenopodioideae. Its leaves are a common edible vegetable consumed either f ...
and
arugula Arugula (American English) or rocket (Commonwealth English) (''Eruca vesicaria''; syns. ''Eruca sativa'' Mill., ''E. vesicaria'' subsp. ''sativa'' (Miller) Thell., ''Brassica eruca'' L.) is an edible annual plant in the family Brassicaceae used a ...
, are abundant in anti-hypertensive diets such as the DASH diet. Several papers have shown saliva nitrite levels correlate with
blood Blood is a body fluid in the circulatory system of humans and other vertebrates that delivers necessary substances such as nutrients and oxygen to the cells, and transports metabolic waste products away from those same cells. Blood in the c ...
nitrite levels which both serve as meaningful surrogates for blood pressure lowering effects. Sobko et al. shows that Japanese traditional diets rich in
leafy vegetables Leaf vegetables, also called leafy greens, pot herbs, vegetable greens, or simply greens, are plant leaves eaten as a vegetable, sometimes accompanied by tender petioles and shoots. Leaf vegetables eaten raw in a salad can be called salad gre ...
elevated both plasma and saliva nitrite levels with a corresponding decrease in
blood pressure Blood pressure (BP) is the pressure of circulating blood against the walls of blood vessels. Most of this pressure results from the heart pumping blood through the circulatory system. When used without qualification, the term "blood pressure" r ...
. Webb et al. in 2008 reinforced the obligatory role of saliva in humans to generate
nitric oxide Nitric oxide (nitrogen oxide or nitrogen monoxide) is a colorless gas with the formula . It is one of the principal oxides of nitrogen. Nitric oxide is a free radical: it has an unpaired electron, which is sometimes denoted by a dot in its che ...
. Here, they showed ingestion of beet juice, a nitrate-rich food, by healthy volunteers markedly reduced blood pressure and by disrupting saliva, either by spitting or interrupting the bioconversion of dietary nitrate to nitrite in the mouth with anti-bacterial mouthwash, the chemical reduction of nitrate to nitrite to nitric oxide with an associated decease in blood pressure was abated. By blocking saliva from recirculating or preventing salivary nitrate from being chemically reduced to nitrite, it prevented a rise in plasma nitrite levels, and blocked a decrease in blood pressure as well as abolished nitric oxide-mediated inhibition of
platelets Platelets, also called thrombocytes (from Greek θρόμβος, "clot" and κύτος, "cell"), are a component of blood whose function (along with the coagulation factors) is to react to bleeding from blood vessel injury by clumping, thereby ini ...
aggregation confirming the cardio-protective effects were attributable to nitric oxide via the conversion of nitrate to nitrite in saliva. In a series of reports by Ahluwalia and colleagues, they showed in a cross over protocol of 14 volunteers who ingested inorganic nitrates, plasma and saliva nitrite level increased 3 hours post ingestion with a significant reduction of
blood pressure Blood pressure (BP) is the pressure of circulating blood against the walls of blood vessels. Most of this pressure results from the heart pumping blood through the circulatory system. When used without qualification, the term "blood pressure" r ...
. Nitrate extracted from blood by the salivary gland, accumulates in saliva, which is then reduced to nitric oxide to have a direct blood pressure lowering effect. Decreasing saliva nitrite in volunteers that already had elevated levels, a rise in
systolic Systole ( ) is the part of the cardiac cycle during which some chambers of the heart contract after refilling with blood. The term originates, via New Latin, from Ancient Greek (''sustolē''), from (''sustéllein'' 'to contract'; from ''sun ...
and diastolic blood pressure resulted. Furthermore, pre-hypertensives may be more sensitive to the blood pressure lowering effects of the dietary nitrate-nitrite-nitric oxide pathway. Monitoring the bioconversion of plant-derived nitrate into salivary nitrite serves as a surrogate biomarker for total body nitric oxide status.


Allergic states

A 2002 study explored the relationship between allergies and salivary immunoglobulin levels in eighty subjects. Researchers demonstrated an association between development of allergies and disturbances in saliva allergen-specific IgA levels (elevated compared to controls) and total secretory IgA (reduced compared to controls). In 2011 Peeters, et al., identified characteristic aberrations in certain salivary metabolites that were associated with peanut-allergic individuals when compared to peanut-tolerant controls. In 2003 Vojdani, et al., found that individuals exposed to various allergenic molds and mycotoxins showed "significantly higher levels of salivary IgA antibodies against one or more mold species."


Chemical substances

In 2009 Pink, et al., reported that saliva testing had become so widespread that it had begun to replace urine testing as the standard for detecting illicit drugs and prescription medications. Shin, et al., reported in 2008 that salivary detection of
ethanol Ethanol (abbr. EtOH; also called ethyl alcohol, grain alcohol, drinking alcohol, or simply alcohol) is an organic compound. It is an Alcohol (chemistry), alcohol with the chemical formula . Its formula can be also written as or (an ethyl ...
and three of its metabolites (
methanol Methanol (also called methyl alcohol and wood spirit, amongst other names) is an organic chemical and the simplest aliphatic alcohol, with the formula C H3 O H (a methyl group linked to a hydroxyl group, often abbreviated as MeOH). It is a ...
,
ethylene glycol Ethylene glycol (IUPAC name: ethane-1,2-diol) is an organic compound (a vicinal diol) with the formula . It is mainly used for two purposes, as a raw material in the manufacture of polyester fibers and for antifreeze formulations. It is an odo ...
, and diethylene glycol) had "relatively high sensitivity and specificity" and that such testing facilitates rapid diagnosis of alcohol intoxication. A 2002 study demonstrated that there was good agreement between saliva and breath ethanol analysis, and that chromatographic saliva ethanol assay is "specific… ndshows good accuracy and precision." In 2011 Vindenes, et al., investigated the viability of drug abuse monitoring using saliva, comparing this method to urine drug detection. Researchers found that several drug metabolites were detected more frequently in saliva than in urine; this was true for
6-monoacetylmorphine 6-Monoacetylmorphine (6-MAM, 6-acetylmorphine, or 6-AM) is one of three active metabolites of heroin (diacetylmorphine), the others being morphine and the much less active 3-monoacetylmorphine (3-MAM). Pharmacology 6-MAM occurs as a metabolite o ...
,
amphetamine Amphetamine (contracted from alpha- methylphenethylamine) is a strong central nervous system (CNS) stimulant that is used in the treatment of attention deficit hyperactivity disorder (ADHD), narcolepsy, and obesity. It is also commonly used ...
,
methamphetamine Methamphetamine (contracted from ) is a potent central nervous system (CNS) stimulant that is mainly used as a recreational drug and less commonly as a second-line treatment for attention deficit hyperactivity disorder and obesity. Methamph ...
, and N-desmethyldiazepam. This same study showed that saliva testing could detect other drug metabolites, as well, although not as frequently as urine testing; this was the case for morphine, other
benzodiazepine Benzodiazepines (BZD, BDZ, BZs), sometimes called "benzos", are a class of depressant drugs whose core chemical structure is the fusion of a benzene ring and a diazepine ring. They are prescribed to treat conditions such as anxiety disorders, ...
s,
cannabis ''Cannabis'' () is a genus of flowering plants in the family Cannabaceae. The number of species within the genus is disputed. Three species may be recognized: ''Cannabis sativa'', '' C. indica'', and '' C. ruderalis''. Alternatively ...
, and
cocaine Cocaine (from , from , ultimately from Quechuan languages, Quechua: ''kúka'') is a central nervous system (CNS) stimulant mainly recreational drug use, used recreationally for its euphoria, euphoric effects. It is primarily obtained from t ...
.


Selected criticism


Sensitivity and specificity

One often cited criticism of using saliva as a diagnostic fluid is that biomarkers are present in amounts that are too low to be detected reliably. As Wong points out, however, this "is no longer a limitation" due to the development of increasingly sensitive detection techniques. Advances in ELISA and mass spectrometry, in addition to the emergence of novel detection methods that take advantage of nanotechnology and other technologies, are enabling scientists and practitioners to achieve high analyte sensitivity. Biomarker specificity is another consideration with saliva testing, much as it is with blood or urine testing. Many biomarkers are nonspecific (for example, CRP is a nonspecific inflammatory marker), and thus they can not be used alone to diagnose any particular disease. This issue is currently being addressed through identification of multiple biomarkers that are correlative of a disease; these can then be screened concomitantly to create a comprehensive panel of tests that significantly increases diagnostic specificity. Of note, certain types of saliva testing are considered by many to be more specific than blood testing; this is particularly true for steroid hormones. Since salivary hormone tests measure only those hormones that are not bound to sex hormone-binding globulin (SHBG) or
albumin Albumin is a family of globular proteins, the most common of which are the serum albumins. All the proteins of the albumin family are water-soluble, moderately soluble in concentrated salt solutions, and experience heat denaturation. Albumins ...
, they are regarded as reflecting only the bioactive ("free") fraction. With continued research into the field of salivary testing, accuracy parameters such as sensitivity and specificity will continue to improve.


Standardization

As with other diagnostic testing methods, one drawback of saliva testing is the variability that exists among diagnostic devices and laboratory analysis techniques, especially for measuring hormones. Consequently, although a test result may be accurate and reliable within a particular assay method or laboratory, it may not be comparative to a test result obtained using a different method or laboratory. As the research community continues to validate and refine test methods and establish standard diagnostic ranges for various saliva biomarkers, this issue should be resolved. Recently, the U.S. National Institute of Health and Public Health Service each granted significant funding to further advancements in salivary testing, including the continued development of diagnostic standards.


See also

*
YES and YAS assay Yeast estrogen screen (YES) and Yeast androgen screen (YAS) are ''in vitro'' screens that have been developed in order to detect estrogenic and androgenic activities, respectively, of natural and synthetic compounds, mixtures and environmental samp ...


References


Further reading

*


External links


Salivaomics

International Conference on Frontiers of Dental and Craniofacial
{dead link, date=May 2018 , bot=InternetArchiveBot , fix-attempted=yes
OralDNA Labs, Innovations in Salivary Diagnostics

clincancerres.aacrjournals.org

ada.org
Medical tests
Testing An examination (exam or evaluation) or test is an educational assessment intended to measure a test-taker's knowledge, skill, aptitude, physical fitness, or classification in many other topics (e.g., beliefs). A test may be administered verba ...