Saliva testing
Encyclopedia
Saliva testing is a diagnostic technique
that involves laboratory analysis of saliva to identify markers of endocrine, immunologic, inflammatory, infectious, and other types of conditions. Saliva is a useful biological fluid for assaying steroid hormones such as cortisol, genetic material like RNA, proteins such as enzymes and antibodies, and a variety of other substances. Saliva testing is used to screen for or diagnose numerous conditions and disease states, including Cushing’s disease, anovulation, HIV, cancer, parasites, hypogonadism, and allergies. Notably, salivary testing has even been used by the U.S. government to assess circadian rhythm
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 testing. Additionally, since multiple samples can be readily obtained, saliva testing is particularly useful for performing chronobiological assessments that span hours, days, or weeks.
Saliva testing is not just the subject of immense research, but it also serves as a valuable diagnostic tool evidenced by three decades’ use in clinical practice. Initial studies focused primarily 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.”
(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
, 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. 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.
with high sensitivity and specificity
(90.0% and 95.0%, respectively) by screening saliva for four specific mRNA 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.”
patients to that from healthy individuals and observed, notably, that breast cancer patients’ samples contained dysplastic cells and reduced lipid peroxides. 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 was reduced in the saliva of women with breast cancer compared to healthy controls and women with benign breast lesions.
had elevated levels transferrin
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 (increased compared to controls) and Maspin (decreased compared to controls), had sensitivities and specificities of 100% for oral cancer detection when measured in saliva. Saliva testing for specific mRNAs 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 (ROC) value being 95% for saliva but only 88% for serum.
diagnosis in dyspeptic patients using salivary anti-H. pylori IgG 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.
, hydroxyproline
and calcium
) were significantly increased in the saliva of patients with periodontitis. 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.
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, 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.
Medical test
A diagnostic test is any kind of medical test performed to aid in the diagnosis or detection of disease. For example:* to diagnose diseases, and preferably sub-classify it regarding, for example, severity and treatability...
that involves laboratory analysis of saliva to identify markers of endocrine, immunologic, inflammatory, infectious, and other types of conditions. Saliva is a useful biological fluid for assaying steroid hormones such as cortisol, genetic material like RNA, proteins such as enzymes and antibodies, and a variety of other substances. Saliva testing is used to screen for or diagnose numerous conditions and disease states, including Cushing’s disease, anovulation, HIV, cancer, parasites, hypogonadism, and allergies. Notably, salivary testing has even been used by the U.S. government to assess circadian rhythm
Circadian rhythm
A circadian rhythm, popularly referred to as body clock, is an endogenously driven , roughly 24-hour cycle in biochemical, physiological, or behavioural processes. Circadian rhythms have been widely observed in plants, animals, fungi and cyanobacteria...
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 testing. Additionally, since multiple samples can be readily obtained, saliva testing is particularly useful for performing chronobiological assessments that span hours, days, or weeks.
Saliva testing is not just the subject of immense research, but it also serves as a valuable diagnostic tool evidenced by three decades’ use in clinical practice. Initial studies focused primarily 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 (ELISA), polymerase chain reactionPolymerase chain reaction
The polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....
(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
Cortisol is a steroid hormone, more specifically a glucocorticoid, produced by the adrenal gland. It is released in response to stress and a low level of blood glucocorticoids. Its primary functions are to increase blood sugar through gluconeogenesis; suppress the immune system; and aid in fat,...
, C-reactive protein
C-reactive protein
C-reactive protein is a protein found in the blood, the levels of which rise in response to inflammation...
(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. 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. These glands, along with additional minor salivary glands, secrete a rich mixture of biological chemicals, electrolytes, proteins, genetic material, polysaccharides, and other molecules. Most of these substances enter the salivary gland acinus 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 neoplasms.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), altered female hormone states (such as polycystic ovary syndrome [PCOS], menopause, anovulation, and hormonal alterations in cycling women), altered male hormone states (such as hypogonadism/andropause and hyperestrogenic states), metabolic disturbances (such as insulin resistance, diabetes, and metabolic syndrome), benign and metastatic neoplasms (such as breast cancer, pancreatic cancer, and oral cancer), infectious conditions (such as HIV, viral hepatitis, amoebiasis, and helicobacter pylori infection), and allergic conditions (such as food allergy).Cortisol and Melatonin Aberrations
In 2008 the Endocrine Society 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 screening technique. In 2010 Bagcim, et al., determined that saliva melatonin 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, dehydroepiandrosterone (DHEA), and testosterone levels in 2,722 individuals (male and female). The researchers confirmed the “good validity of [salivary] sex hormone measurements” and concluded that salivary testing was a good method for testing older adults due to the ease of in-home collection.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 [method] for 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 suffering from unexplained fertility.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.”Pancreatic cancer
A 2010 study by Zhang, et al., demonstrated that researchers were able to detect pancreatic cancerPancreatic cancer
Pancreatic cancer refers to a malignant neoplasm of the pancreas. The most common type of pancreatic cancer, accounting for 95% of these tumors is adenocarcinoma, which arises within the exocrine component of the pancreas. A minority arises from the islet cells and is classified as a...
with high sensitivity and specificity
Sensitivity and specificity
Sensitivity and specificity are statistical measures of the performance of a binary classification test, also known in statistics as classification function. Sensitivity measures the proportion of actual positives which are correctly identified as such Sensitivity and specificity are statistical...
(90.0% and 95.0%, respectively) by screening saliva for four specific mRNA 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 cancerBreast cancer
Breast cancer is cancer originating from breast tissue, most commonly from the inner lining of milk ducts or the lobules that supply the ducts with milk. Cancers originating from ducts are known as ductal carcinomas; those originating from lobules are known as lobular carcinomas...
patients to that from healthy individuals and observed, notably, that breast cancer patients’ samples contained dysplastic cells and reduced lipid peroxides. 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 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 carcinomaSquamous cell carcinoma
Squamous cell carcinoma , occasionally rendered as "squamous-cell carcinoma", is a histologically distinct form of cancer. It arises from the uncontrolled multiplication of malignant cells deriving from epithelium, or showing particular cytological or tissue architectural characteristics of...
had elevated levels transferrin
Transferrin
Transferrins are iron-binding blood plasma glycoproteins that control the level of free iron in biological fluids. In humans, it is encoded by the TF gene.Transferrin is a glycoprotein that binds iron very tightly but reversibly...
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 (increased compared to controls) and Maspin (decreased compared to controls), had sensitivities and specificities of 100% for oral cancer detection when measured in saliva. Saliva testing for specific mRNAs has been found to possess significant potential for oral cancer
Oral cancer
Oral cancer is a subtype of head and neck cancer, is any cancerous tissue growth located in the oral cavity. It may arise as a primary lesion originating in any of the oral tissues, by metastasis from a distant site of origin, or by extension from a neighboring anatomic structure, such as the...
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 (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 [glucose] concentration 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.”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 and IgG) was an effective method to identify HAV-infected individuals. Hepatitis C 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. In 2004 El Hamshary and Arafa found that salivary anti-E. histolytica IgA 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 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 secondary to Taenia solium.Helicobacter pylori infection
In a 2005 study, researchers investigated the accuracy of Helicobacter pyloriHelicobacter pylori
Helicobacter pylori , previously named Campylobacter pyloridis, is a Gram-negative, microaerophilic bacterium found in the stomach. It was identified in 1982 by Barry Marshall and Robin Warren, who found that it was present in patients with chronic gastritis and gastric ulcers, conditions that were...
diagnosis in dyspeptic patients using salivary anti-H. pylori IgG 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 (peroxidasePeroxidase
Peroxidases are a large family of enzymes that typically catalyze a reaction of the form:For many of these enzymes the optimal substrate is hydrogen peroxide, but others are more active with organic hydroperoxides such as lipid peroxides...
, hydroxyproline
Hydroxyproline
-4-Hydroxyproline, or L-hydroxyproline , is a common non-proteinogenic amino acid, abbreviated as HYP, e.g., in Protein Data Bank.-Structure and discovery:...
and calcium
Calcium
Calcium is the chemical element with the symbol Ca and atomic number 20. It has an atomic mass of 40.078 amu. Calcium is a soft gray alkaline earth metal, and is the fifth-most-abundant element by mass in the Earth's crust...
) were significantly increased in the saliva of patients with periodontitis. 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
In 2011 Punyadeera, et al., studied “the clinical utility of salivary [C-reactive protein] levels in assessing coronary events [such as myocardial infarction] 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.”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 and three of its metabolites (methanol, ethylene glycol, 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…[and] shows 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, amphetamine, methamphetamine, 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 benzodiazepines, cannabis, and cocaine.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, 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.