Salivary Tests

Number: 0608

Table Of Contents

Applicable CPT / HCPCS / ICD-10 Codes


Scope of Policy

This Clinical Policy Bulletin addresses salivary tests.

  1. Medical Necessity

    Aetna considers late night salivary cortisol medically necessary for diagnosing Cushing's syndrome.

  2. Experimental and Investigational

    Aetna considers the following salivary tests experimental and investigational because the effectiveness of these approaches has not been established:

    1. Cordant Health Solutions Comprehensive Oral Fluid Rx Evaluation (CORE) (prescription drug monitoring using oral fluid);
    2. Examination of salivary microbiota patterns for the diagnosis of minimal hepatic encephalopathy;
    3. Measurement of diurnal salivary cortisol patterns for prediction of infant birth weight;
    4. Measurement of salivary levels of cortisol for predicting the efficacy of sleep-promoting treatment in children with postural tachycardia syndrome;
    5. Measurement of salivary levels of creatinine and urea for the determination of uremic state in adults with chronic kidney disease;
    6. Measurement of salivary levels of hemoglobin for screening periodontal disease;
    7. Measurement of salivary levels of IgG antibodies to SARS-CoV-2 for monitoring seroprevalence and vaccine antibody response;
    8. Measurement of salivary levels of immunoglobulins (IgA, IgG, and IgM), interferon-gamma, interleukin-4, interleukin-6, and interleukin-8 for determining diagnostic and therapeutic aims in oral lichen planus;
    9. Measurement of salivary levels of matrix metalloproteinase-8 (MMP-8) for diagnosis of periodontal disease;
    10. Measurement of salivary matrix metalloproteinase 9 (MMP-9) for diagnosis of oral squamous cell carcinoma;
    11. Measurement of salivary levels of osteocalcin for the determination of periodontitis severity;
    12. Measurement of salivary levels of pepsin (Pep-test) for the diagnosis of laryngo-pharyngeal reflux;
    13. Measurement of salivary levels of tumor necrosis factor-alpha for determining diagnostic and therapeutic aims in oral lichen planus;
    14. Salivary antibody testing (IgA, IgG, IgM) for the diagnosis of Sicca syndrome;
    15. Salivary cytokines in the diagnosis and prognosis of oral squamous cell carcinoma (OSCC);
    16. Salivary electrolytes as biomarkers for mouth neoplasms, oral potentially malignant disorders (OPMDs), and oral squamous cell carcinoma;
    17. Salivary hypermethylated DNA biomarkers for diagnosis of oral cancer;
    18. Salivary lactate dehydrogenase as biomarker in OPMDs, and head and neck cancer;
    19. Salivary metabolites for diagnosis of cancer;
    20. Salivary microRNA as a biomarker for traumatic brain injury and for detection of prolonged concussion symptoms;
    21. Salivary testing for anti-tissue transglutaminase for the diagnosis of celiac disease;
    22. Salivary testing of biomarkers for the diagnosis of cancers (e.g., breast cancer, head and neck carcinoma, oral pre-cancer and oral squamous cell carcinoma (e.g.,CYFRA 21-1, interleukin-8, or mRNAs of dual specificity phosphatase 1 (DUSP1), ornithin decarboxylase antizyme 1 (OAZ1), and S100 calcium-binding protein P (S100P));
    23. Salivary tests of cortisol for the screening, diagnosis, or monitoring of menopause or diseases related to aging, or any other indications (e.g., diagnosis of adrenal insufficiency in preterm infants, diagnosis of bipolar disorder, depression, or eating disorders);
    24. Salivary tests of dehydroepiandrosterone (DHEA), estrogen, melatonin, progesterone, or testosterone for the screening, diagnosis, or monitoring of menopause or diseases related to aging, or any other indications;
    25. Salivary testing of mutans streptococci for determining the risk of developing dental caries;
    26. Salivary testing of pepsin for the diagnosis of gastro-esophageal reflux disease;
    27. Salivary testing of telomere length for the diagnosis of depression;
    28. Viome CancerDetect-Oral & Throat Test for diagnosis of oral cancer.

    Note: In addition, laboratory tests are not covered unless they are ordered by a physician or other qualified health professional. Please check benefit plan descriptions.


CPT Codes / HCPCS Codes / ICD-10 Codes

Code Code Description

CPT codes covered if selection criteria are met:

82530 Cortisol; free [other than late night salivary cortisol for diagnosing Cushing's syndrome]
82533     total [other than late night salivary cortisol for diagnosing Cushing's syndrome]

CPT codes not covered for indications listed in the CPB (not all-inclusive):

Measurement of salivary levels of hemoglobin, matrix metalloproteinase-8 (MMP-8) and interleukin-6 / tumor necrosis factor-alpha, matrix metalloproteinase 9 (MMP-9) for diagnosis of oral squamous cell carcinoma, examination of salivary microbiota patterns, salivary immunoglobulins (IgA, IgG, and IgM), salivary hypermethylated DNA biomarkers for the diagnosis of oral cancer, salivary metabolites for the diagnosis of cancer, interferon-gamma, interleukin-4, and interleukin-8 as biomarkers and salivary microRNA as a biomarker, salivary levels urea, salivary levels of pepsin (Pep-test), Measurement of salivary electrolytes of IgG antibodies to SARS-CoV-2 for monitoring seroprevalence and vaccine antibody response, salivary electrolytes as biomarkers for mouth neoplasms and oral squamous cell carcinoma, salivary electrolytes profile - no specific code
0011U Prescription drug monitoring, evaluation of drugs present by LC-MS/MS, using oral fluid, reported as a comparison to an estimated steady-state range, per date of service including all drug compounds and metabolites
0296U Oncology (oral and/or oropharyngeal cancer), gene expression profiling by RNA sequencing of at least 20 molecular features (eg, human and/or microbial mRNA), saliva, algorithm reported as positive or negative for signature associated with malignancy
82530 Cortisol; free
82533     total
82570 Creatinine; other source
82626 Dehydroepiandrosterone (DHEA)
82627 Dehydroepiandrosterone-sulfate (DHEA-S)
82670 Estradiol
82671 Estrogens; fractionated
82672     total
82677 Estriol
82679 Estrone
83516 Immunoassay for analyte other than infectious agent antibody or infectious agent antigen; qualitative or semiquantitative, multiple step method [salivary testing for anti-tissue transglutaminase for the diagnosis of celiac disease]
83520     quantitative, not otherwise specified [not covered for measurement of salivary level of interleukin-8 as biomarkers for oral pre-cancer and oral squamous cell carcinoma] [not covered for salivary antibody testing (IgA, IgG, IgM) for the diagnosis of Sicca syndrome]
83529 Interleukin-6 (IL-6) [Salivary cytokinase]
83615 Lactate dehydrogenase (LD), (LDH) [ Salivary lactate dehydrogenase]
83937 Osteocalcin (bone g1a protein)
84144 Progesterone
84402 Testosterone; free
84403     total
84436 Thyroxine; total
84437     requiring elution (eg, neonatal)
84439     free
84443 Thyroid stimulating hormone (TSH)
84479 Thyroid hormone (T3 or T4) uptake or thyroid hormone binding ratio (THBR)
84480 Triidothyronine T3; total (TT-3)
84481     free
86316 Immunoassay for tumor antigen, other antigen, quantitative (eg, CA 50, 72-4, 549), each [not covered for measurement of salivary level of CYFRA 21-1 as biomarkers for oral pre-cancer and oral squamous cell carcinoma]
88341 Immunohistochemistry or immunocytochemistry, per specimen; each additional single antibody stain procedure (List separately in addition to code for primary procedure)
88342 Immunohistochemistry or immunocytochemistry, per specimen; initial single antibody stain procedure
88344 Immunohistochemistry or immunocytochemistry, per specimen; each multiplex antibody stain procedure

HCPCS codes not covered for indications listed in the CPB:

S3650 Saliva test, hormone level; during menopause

ICD-10 codes covered if selection criteria are met:

E24.0 - E24.9 Cushing's syndrome

ICD-10 codes not covered for indications listed in the CPB (not all-inclusive):

C00.0 - C10.9 Malignant neoplasm of lip and oral cavity [oral squamous cell carcinoma]
C44.02 Squamous cell carcinoma of skin of lip
C76.0 Malignant neoplasm of head, face and neck
E27.0 - E27.9 Other disorders of adrenal gland
E28.310 - E28.319 Premature menopause
E89.40 - E89.41 Postprocedural ovarian failure
E89.6 Postprocedural adrenocortical (-medullary) hypofunction
F30.10 - F30.9 Manic episode
F31.0 - F31.9 Bipolar disorder
F32.0 - F32.9 Major depressive disorder, single episode
F33.3 Major depressive disorder, recurrent, severe with psychotic symptoms
F34.1 Dysthymic disorder
F50.00 - F50.02 Anorexia nervosa
F50.2 - F50.9 Other eating disorders [bulimia nervosa, Pica, unspecified]
J12.81 Pneumonia due to SARS-associated coronavirus
J12.82 Pneumonia due to coronavirus disease 2019
K05.00 - K05.6 Gingivitis and periodontal diseases
K13.21 Leukoplakia of oral mucosa, including tongue
K13.29 Other disturbances of oral epithelium, including tongue
K21.9 Gastro-esophageal reflux disease without esophagitis
K72.00 - K72.91 Hepatic failure, not elsewhere classified [minimal hepatic encephalopathy]
K90.0 Celiac disease
L43.0 - L43.9 Lichen planus
M35.00 - M35.09 Sicca syndrome [Sjögren]
M80.00x+ - M81.8 Osteoporosis
N19 Unspecified kidney failure
N92.4 Excessive bleeding in the premenopausal period
N95.0 - N95.9 Menopausal and other perimenopausal disorders
O09.00 - O31.8X99 Pregnancy [prediction of infant birth weight]
P07.00 - P0739 Immaturity of newborn
R00.0 Tachycardia, unspecified [postural tachycardia syndrome]
S06.0X0A - S06.A1XS, S06.0XAA - S06.9XAS Intracranial injury
U07.1 COVID-19
Z01.20 Encounter for dental examination and cleaning without abnormal findings
Z01.21 Encounter for dental examination and cleaning with abnormal findings
Z12.0 - Z12.9 Encounter for screening for malignant neoplasms
Z13.810 - Z13.818 Encounter for screening for digestive system disorders [diagnosis of gastro-esophageal reflux disease]
Z13.820 Encounter for screening for osteoporosis
Z13.84 Encounter for screening for dental disorders [periodontal disease]
Z13.850 - Z13.858 Encounter for screening for nervous system disorders [diagnosis of depression]
Z78.0 Asymptomatic menopausal state
Z79.890 Hormone replacement therapy (postmenopausal)


Salivary hormone testing is purported to aid in the diagnosis and treatment of menopause and other diseases related to aging. These tests measure the amount of free hormones (dehydroepiandrosterone [DHEA], estrogen, melatonin, progesterone and/or testosterone) found in the saliva of women. Most hormone tests are now available on the internet and can be obtained without a prescription.

Salivary hormone levels may vary according to the time of day, diet or hydration; therefore, the timing of saliva collection may affect results. Salivary flow rate can also affect the concentration of certain hormones. Different laboratories may require different testing methods, such as obtaining several samples over a couple of weeks at specific times of the day.

Salivary tests of estrogen, progesterone, testosterone, melatonin, cortisol and dehydroepiandrosterone (DHEA) have become available to consumers over the Internet.  Some of these websites include a questionnaire to allow consumers to determine whether they need saliva testing, and a form that allows consumers to order these tests online.  The results of these tests are purportedly used to determine the need prescriptions of DHEA, vitamins, herbs, phytoestrogens, and other anti-aging regimens.

The medical literature on salivary testing correlates salivary levels with serum levels, the gold standard measurement.  However, the medical literature fails to demonstrate that salivary tests are appropriate for screening, diagnosing, or monitoring patients with menopause, osteoporosis, or other consequences of aging.

Evidence-based clinical practice guidelines from the American Association of Clinical Endocrinologists outline the appropriate methods of screening and diagnosing menopause and osteoporosis.  The primary test for menopause screening is serum follicle-stimulating hormone, for thyroid dysfunction serum thyroid-stimulating hormone, and bone density measurement is the primary method of screening for osteoporosis.  None of these guidelines indicates salivary testing as an appropriate method of screening, diagnosing, or monitoring these disorders.

According to available guidelines, primary hypoadrenalism (Addison’s disease) is suggested by a markedly elevated plasma adrenocorticotrophic hormone (ACTH) with low or normal serum cortisol.  The diagnosis of adrenocortical insufficiency is established primarily by use of the rapid ACTH stimulation test, which involves assessment of the response of serum aldosterone and cortisol to ACTH infusion.

Furthermore, there is inadequate evidence of the value of measuring salivary components to guide prescription of "anti-aging" regimens.  The clinical value of these tests depends not only on how well the salivary testing corresponds to some gold standard (i.e., a serum test), but also upon the evidence of the effectiveness of the particular intervention (anti-aging regimen) that would be prescribed based on the results of the salivary test.  Meta-analyses of the literature have questioned the value of supplementation with DHEA and melatonin on improving patient outcomes.

According to a committee opinion by the American College of Obstetricians and Gynecologists (ACOG, 2005), there is no scientific evidence to support claims of increased safety or effectiveness for individualized estrogen or progesterone regimens prepared by compounding pharmacies.  Furthermore, hormone therapy does not belong to a class of drugs with an indication for individualized dosing.  The opinion by ACOG also pointed out that salivary hormone level testing used by proponents to "tailor" this therapy isn't meaningful because salivary hormone levels vary within each woman depending on her diet, the time of day, the specific hormone being tested, and other variables.

A National Institutes of Health State-of-the-Art Conference Statement on Management of Menopausal Symptoms (2005) reached the following conclusions about salivary hormone testing and bioidential hormones: "Bioidentical hormones, often called "natural" hormones, are treatments with individually compounded recipes of a variety of steroids in various dosage forms, with the composition and dosages based on a person’s salivary hormone concentration.  These steroids may include estrone, estradiol, estriol, DHEA, progesterone, pregnenolone, and testosterone.  There is a paucity of data on the benefits and adverse effects of these compounds."

An assessment by the Institute for Clinical Systems Improvement (2006) concluded: "Currently, there is insufficient evidence in the published scientific literature to permit conclusions concerning the use of salivary hormone testing for the diagnosis, treatment or monitoring of menopause and aging."

The North American Menopause Society (2005) has concluded: "Salivary testing is not considered to be a reliable measure of testosterone levels."

Flyckt and colleagues (2009) compared salivary versus serum measurements of total testosterone (TT), bioavailable testosterone (BT; consisting of free testosterone [FT] and albumin-bound testosterone), and FT from samples collected simultaneously in women who were either receiving transdermal testosterone patch supplementation (300 microg/d) or a placebo patch.  Naturally and surgically post-menopausal women receiving concomitant hormone therapy were recruited to participate in a 24- to 52-week phase III trial of a 300 microg/day transdermal testosterone patch for the treatment of hypoactive sexual desire disorder.  Initial analysis demonstrated high correlations between TT, BT, and FT levels (r = 0.776 to 0.855).  However, there was no correlation with salivary testosterone levels for any of the serum testosterone subtypes (r = 0.170 to 0.261).  After log transformation, salivary testosterone correlated modestly with BT (r = 0.436, p < 0.001), FT (r = 0.452, p < 0.001), and TT (r = 0.438, p < 0.001).  The authors concluded that although salivary testing of testosterone concentrations is an appealing alternative because it is inexpensive and non-invasive, these findings do not support the routine use of salivary testosterone levels in post-menopausal women.

Salivary hormone tests are purported to predict spontaneous premature labor by measuring salivary estriol, an estrogen hormone. A surge in the levels of salivary estriol typically occurs several weeks prior to the onset of spontaneous labor.  Klebanoff and colleagues (2008) examined if salivary progesterone (P) or estriol (E3) concentration at 16 to 20 weeks' gestation predicts preterm birth or the response to 17alpha-hydroxyprogesterone caproate (17OHPC).  Baseline saliva was assayed for P and E3.  Weekly salivary samples were obtained from 40 women who received 17OHPC and 40 who received placebo.  Both low and high baseline saliva P and E3 were associated with a slightly increased risk of preterm birth.  However, 17OHPC prevented preterm birth comparably, regardless of baseline salivary hormone concentrations.  Thus, salivary P or E3 does not appear to predict preterm birth.

Groschl (2008) provided an overview of the current applications of salivary hormone analysis.  The author noted that although saliva has not yet become a mainstream sample source for hormone analysis, it has proven to be reliable and, in some cases, even superior to other body fluids.  Nevertheless, much effort will be needed for this approach to receive acceptance over the long-term, especially by clinicians.  Such effort entails the development of specific and standardized analytical tools, the establishment of defined reference intervals, and implementation of round-robin trials.  One major obstacle is the lack of compliance sometimes observed in outpatient saliva donors.  Moreover, the author stated that there is a need for standardization of both collection and analysis methods in order to attain better comparability and evaluation of published salivary hormone data.

Late Night Salivary Cortisol

Cushing’s Disease is characterized by abnormal accumulations of facial and trunk fat, fatigue, hypertension and osteoporosis, caused by hyperfunction of the adrenal cortex or administration of adrenal cortical hormones.

Measurement of late-night and/or midnight salivary cortisol currently used in the United States and European countries is a simple and convenient screening test for the initial diagnosis of Cushing's syndrome (CS).  Salivary hormone testing for cortisol may be utilized as part of a two-step process to screen for and diagnose Cushing’s disease. If screening tests are positive, confirmatory tests are then performed. Initial testing could include urine cortisol or late night salivary cortisol. Secondary testing may also include one of these tests or a blood test. 

Carroll et al (2008) stated that making a definite diagnosis of CS is a challenging problem.  Unsuspected CS occurs in 2 to 3 % of patients with poorly controlled diabetes, 0.5 to 1 % with hypertension, 6 to 9 % with incidental adrenal masses, and 11 % with unexplained osteoporosis and vertebral fractures.  The increasing recognition of this syndrome highlights the need for a simple, sensitive, and specific diagnostic test.  Patients with CS consistently do not reach a normal nadir of cortisol secretion at night.  The measurement of late-night salivary cortisol levels might, therefore, provide a new diagnostic approach for this disorder.  Salivary cortisol concentrations reflect those of active free cortisol in plasma and saliva samples can easily be obtained in a non-stressful environment (e.g., at home).  Late-night salivary cortisol measurement yields excellent overall diagnostic accuracy for CS, with a sensitivity of 92 to 100 % and a specificity of 93 to 100 %.  Several factors can, however, make interpretation of results difficult; these factors include disturbed sleep-wake cycles, contamination of samples (particularly by topical corticosteroids), and illnesses known to cause physiologic activation of the pituitary-adrenal axis.

Elamin et al (2008) summarized the evidence on the accuracy of common tests for diagnosing CS.  These investigators searched electronic databases (Medline, Embase, Web of Science, Scopus, and citation search for key articles) from 1975 through September 2007 and sought additional references from experts.  Eligible studies reported on the accuracy of urinary free cortisol (UFC), dexamethasone suppression test (DST), and midnight cortisol assays versus reference standard in patients suspected of CS.  Reviewers working in duplicate and independently extracted study characteristics and quality and data to estimate the likelihood ratio (LR) and the 95 % confidence interval (CI) for each result.  These researchers found 27 eligible studies, with a high prevalence [794 (9.2 %) of 8,631 patients had CS] and severity of CS.  The tests had similar accuracy: UFC (n = 14 studies; LR+ 10.6, CI: 5.5 to 20.5; LR- 0.16, CI: 0.08 to 0.33), salivary midnight cortisol (n = 4; LR+ 8.8, CI: 3.5 to 21.8; LR- 0.07, CI: 0 to 1.2), and the 1-mg overnight DST (n = 14; LR+ 16.4, CI: 9.3 to 28.8; LR- 0.06, CI: 0.03 to 0.14).  Combined testing strategies (e.g., a positive result in both UFC and 1-mg overnight DST) had similar diagnostic accuracy (n = 3; LR+ 15.4, CI: 0.7 to 358; LR- 0.11, CI: 0.007 to 1.57).  The authors concluded that commonly used tests to diagnose CS appear highly accurate in referral practices with samples enriched with patients with CS.

Doi et al (2008) assessed the usefulness of the measurement of late-night salivary cortisol as a screening test for the diagnosis of CS in Japan.  These investigators studied 27 patients with various causes of CS, consisting of  ACTH-dependent Cushing's disease (n = 5) and ectopic ACTH syndrome (n = 4) and ACTH-independent adrenal CS (n = 11) and subclinical CS (n = 7).  Eleven patients with type 2 diabetes and obesity and 16 normal subjects served as control group.  Saliva samples were collected at late-night (23:00) in a commercially available device and assayed for cortisol by radioimmunoassay.  There were highly significant correlations (p < 0.0001) between late-night serum and salivary cortisol levels in normal subjects (r = 0.861) and in patients with CS (r = 0.788).  Late-night salivary cortisol levels in CS patients (0.975 +/- 1.56 microg/dL) were significantly higher than those in normal subjects (0.124 +/- 0.031 microg/dL) and in obese diabetic patients (0.146 +/- 0.043 microg/dL), respectively.  Twenty-five out of 27 CS patients had late-night salivary cortisol concentrations greater than 0.21 microg/dL, whereas those in control group were less than 0.2 microg/dL.  Receiver operating characteristic curve (ROC) analysis showed that the cut-off point of 0.21 microg/dL provides a sensitivity of 93 % and a specificity of 100 %.  The authors concluded that the measurement of late-night salivary cortisol is an easy and reliable non-invasive screening test for the initial diagnosis of CS, especially useful for large high-risk populations, such as diabetes and obesity.

The Endocrine Society's clinical practice guideline on the diagnosis of CS (Nieman et al, 2008) stated that after excluding exogenous glucocorticoid use, testing for CS in patients with multiple and progressive features compatible with the syndrome, particularly those with a high discriminatory value, and patients with adrenal incidentaloma is recommended.  It recommends the initial use of one test with high diagnostic accuracy such as urine cortisol, late night salivary cortisol, 1 mg overnight or 2 mg 48-hr DST.  The guideline also recommends that patients with an abnormal result see an endocrinologist and undergo a second test, either one of the above or, in some cases, a serum midnight cortisol or dexamethasone-corticotropin-releasing hormone test.  Patients with concordant abnormal results should undergo testing for the cause of Cushing's syndrome.  Patients with concordant normal results should not undergo further evaluation.  The guideline also recommends additional testing in patients with discordant results, normal responses suspected of cyclic hypercortisolism, or initially normal responses who accumulate additional features over time.

Knorr et al (2010) examined if salivary cortisol differs for patients with depression and control persons.  These investigators performed a systematic review with sequential meta-analysis and meta-regression according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) Statement based on comprehensive database searches for studies of depressed patients compared to control persons in whom salivary cortisol was measured.  A total of 20 case-control studies, including 1,354 patients with depression and 1,052 control persons were identified.  In a random-effects meta-analysis salivary cortisol was increased for depressed patients as compared to control persons on average 2.58 nmol/L (95 % CI: 0.95 to 4.21; p = 0.002) in the morning and on average 0.27 nmol/L (95 % CI: 0.03 to 0.51; p=0.03) in the evening.  In a fixed-effects model the mean difference was 0.58 nmol/L (95 % CI).  Study sequential cumulative meta-analyses suggested random error for the finding of this rather small difference between groups.  The reference intervals for morning salivary cortisol in depressed patients (0 to 29 nmol/L) and control persons (1 to 23 nmol/L) showed substantial overlap suggesting lack of discriminative capacity.  These results should be interpreted with caution as the heterogeneity for the morning analysis was large and a funnel plot, suggested presence of bias.  Further, in meta-regression analyses higher intra-assay coefficients of variation in cortisol kits (p = 0.07) and mean age (p = 0.08) were associated with a higher mean difference of morning salivary cortisol between depressed and controls, while gender and depression severity were not.  The authors concluded that based on the available studies, there is not firm evidence for a difference of salivary cortisol in depressed patients and control persons and salivary cortisol is unable to discriminate between persons with and without depression.

Monteleone and colleagues (2011) noted that the stress response involves the activation of the hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic nervous system (SNS).  As a role for stress in determining of the onset and the natural course of eating disorders has been proposed, the study of the psychobiology of the stress response in patients with anorexia nervosa (AN) and bulimia nervosa (BN) should be helpful in understanding the pathophysiology of these disorders.  The 2 neurobiological components of the stress response can be easily explored in humans by the measurement of salivary cortisol and α-amylase response to a stressor.  Thus, these researchers assessed salivary cortisol and α-amylase responses to the Trier Social Stress Test (TSST) in symptomatic patients with AN (n = 7) and BN (n = 8) compared to age-matched healthy females (n = 8).  Subjects underwent the TSST between 1530 and 1700 hr.  Salivary cortisol and α-amylase levels were measured by an enzyme-linked immunosorbent assay (ELISA).  Compared to healthy women, AN patients showed a normal cortisol response to the TSST, although this occurred at significantly increased hormone levels, and an almost complete absence of response of α-amylase.  BN women, however, exhibited enhanced pre-stress levels of salivary α-amylase but a normal response of the enzyme and cortisol to the TSST.  The authors concluded that these findings demonstrated, for the first time, the occurrence of an asymmetry between the HPA axis and SNS components of the stress response in the acute phase of AN but not in BN.  Moreover, they stated that pathophysiological significance of this asymmetry remains to be determined.

Kamali and associates (2012) compared HPA axis activity in bipolar individuals with and without suicidal behavior and unaffected healthy controls through measurement of salivary cortisol.  Salivary cortisol was collected for 3 consecutive days in 29 controls, 80 bipolar individuals without a history of suicide and 56 bipolar individuals with a past history of suicide.  Clinical factors that affect salivary cortisol were also examined.  A past history of suicide was associated with a 7.4 % higher bedtime salivary cortisol level in bipolar individuals.  There was no statistical difference between non-suicidal bipolar individuals and controls in bedtime salivary cortisol, and awakening salivary cortisol was not different between the 3 groups.  The authors concluded that bipolar individuals with a past history of suicidal behavior exhibit hyperactivity in the HPA axis.  This biological marker remains significant regardless of demographic factors, mood state, severity and course of illness.  This finding in bipolar disorder is consistent with the evidence for altered HPA axis functioning in suicide and mood disorders and is associated with a clinical subgroup of bipolar patients at elevated risk for suicide based on their history, and in need of further attention and study.  The drawbacks of this study were
  1. measure of salivary cortisol was a home-based collection by the study subjects, and
  2. the retrospective clinical data was primarily based on their historical account.

Salivary Test for Bioidentical Hormone Therapy

The American Association of Clinical Endocrinologists (AACE) Reproductive Medicine Committee’s position statement on bioidentical hormones (2007) noted that “Salivary hormone level testing is recommended by many BH proponents as a way of providing patients with “individualized” therapy.  Such tests are available to consumers over the Internet.  Some of the websites include elaborate questionnaires supposedly designed to establish the type of saliva testing needed.  The results of these tests are subsequently used to determine the type and dosage of compounded formulations.  Only a few types of salivary hormone testing methods are FDA/CLIA approved.  In fact, the vast majority of the salivary hormone tests results contain the disclaimer that those tests are not FDA/CLIA approved and should be used only for research purposes.  Yet such tests are still utilized to support clinical decisions by some supporters of BH …. the limited research, although interesting, does not prove that salivary testing can be used as reliable ancillary tests for clinical purposes …. the evidence often quoted by Salivary Test promoters simply do not pass the muster of the level 1 or even 2 of the Level of Evidences (LOE) as endorsed by AACE ”.

The North American Menopause Society’s position statement on “Hormone Therapy” (2012) stated that “Use of BHT (bioidentical hormone therapy) has escalated in recent years, along with the use of salivary hormone testing, which has been proven to be inaccurate and unreliable …. The Food and Drug Administration also states that there is no scientific basis for using saliva testing to adjust hormone levels”.

Salivary Testing for Anti-Tissue Transglutaminase

Bonamico et al (2011) stated that the high prevalence of celiac disease (CD) prompted them to evaluate a new, non-invasive disease screening strategy.  The aim was to identify CD in 6- to 8-year old children for a timely diagnosis, start gluten-free diet (GFD) in compliant subjects, achieve the growth target, and prevent CD complications.  A total of 5,0000 subjects were invited to participate in the study; 4,048 saliva samples were tested for anti-tissue transglutaminase (tTG) immunoglobulin (Ig)A using a fluid-phase radioimmunoprecipitation method.  Positive children were tested for serum radioimmunoassay tTG IgA, ELISA tTG IgA, and anti-endomysium IgA.  Children confirmed as positive by serum assays underwent endoscopy with duodenal biopsies and, at the diagnosis of CD, were suggested to start GFD.  Consent was obtained from 4,242 parents (84.8 %) for the screening to be performed, and adequate saliva samples were collected from 4,048 children (95.4 %).  Thirty-two children were found to be salivary tTG IgA positive and 9 with borderline autoantibody levels; 31 of the 32 and 3 of the 9 subjects were also serum positive.  Twenty-eight children showed villous atrophy when undergoing intestinal biopsy, whereas 1 had Marsh 1 lesions; 3 children were suggested to start GFD without performing endoscopy.  Celiac disease prevalence in the population investigated (including 19 CD known cases) was 1.16 %.  The ratio between screening-detected patients and those diagnosed before the screening was 3:2.  The ratio between symptomatic and asymptomatic patients was 1:1.6.  The authors concluded that it is possible to perform a simple and sensitive CD screening using saliva.

However, the American College of Gastroenterology’s clinical guideline on “Diagnosis and management of celiac disease” (Rubio-Tapia et al, 2013) stated that “Stool studies or salivary tests are neither validated nor recommended for use in the diagnosis of CD”

Salivary Tests of Cortisol for Diagnosis of Adrenal Insufficiency in Preterm Infants

Maas and colleagues (2014)
  1. examined the relationship of salivary and plasma cortisol levels in preterm infants with a focus on the usability of salivary cortisol in diagnostic work-up of infants at risk of adrenal insufficiency, and
  2. performed a systematic review addressing this question.

These researchers conducted a prospective observational single-center study in preterm infants.  They analyzed plasma and saliva cortisol concentrations by enzyme immunoassay.  Correlation analysis was used to determine the relation between salivary and plasma cortisol levels and the agreement of the measurement methods was analyzed according to Bland-Altman.  Furthermore, these investigators performed a systematic literature search (PubMed and Embase) on the relationship of salivary and plasma cortisol levels in neonates.  These researchers enrolled 58 preterm infants (median (inter-quartile range [IQR]) gestational age at birth was 31.4 (28.1 to 32.7) weeks, birth weight 1,340 (974 to 1,745) g, respectively).  Correlation analyses revealed a relationship of plasma cortisol and salivary cortisol levels.  Rank correlation coefficient was 0.6.  Estimating plasma cortisol levels based on measured salivary cortisol levels showed poor agreement of the 2 methods for determining plasma cortisol levels (direct and via salivary cortisol).  Sensitivity and specificity of salivary cortisol for the detection of adrenal insufficiency were 0.66 and 0.62, respectively.  A total of 6 studies in preterm infants and term neonates depicting the correlation of salivary and plasma cortisol were identified with a range of saliva-plasma correlation coefficients from 0.44 to 0.83.  The authors concluded that substitution of plasma cortisol by salivary cortisol determination cannot be recommended in preterm infants because of unsatisfactory agreement between methods.

Salivary Testing of Mutans Streptococci for Risk Assessment of Dental Caries

Senneby et al (2015) evaluated the accuracy of different methods used to identify individuals with increased risk of developing dental coronal caries. Studies on following methods were included: Previous caries experience, tests using microbiota, buffering capacity, salivary flow rate, oral hygiene, dietary habits and socio-demographic variables.  QUADAS-2 was used to assess risk of bias.  Sensitivity, specificity, predictive values, and likelihood ratios (LR) were calculated.  Quality of evidence based on greater than or equal to3 studies of a method was rated according to Grading of Recommendation, Assessment, Development, and Evaluation (GRADE).  PubMed, Cochrane Library, Web of Science and reference lists of included publications were searched up to January 2015.  From 5,776 identified articles, 18 were included.  Assessment of study quality identified methodological limitations concerning study design, test technology and reporting.  No study presented low risk of bias in all domains; 3  or more studies were found only for previous caries experience and salivary mutans streptococci and quality of evidence for these methods was low.  Evidence regarding other methods was lacking.  For previous caries experience, sensitivity ranged between 0.21 and 0.94 and specificity between 0.20 and 1.  Tests using salivary mutans streptococci resulted in low sensitivity and high specificity.  For children with primary teeth at baseline, pooled LR for a positive test was 3 for previous caries experience and 4 for salivary mutans streptococci, given a threshold greater than or equal to 10(5) colony forming unit (CFU)/ml.  The authors concluded that the evidence on the validity of analyzed methods used for caries risk assessment is limited.  As methodological quality was low, there is a need to improve study design.

Measurement of Diurnal Salivary Cortisol Patterns for Prediction of Infant Birth Weight

Guardino and colleagues (2016) noted that elevated maternal psychosocial stress during pregnancy and accompanying changes in stress hormones may contribute to risk of adverse birth outcomes such as low birth weight and preterm birth.  Relatedly, research on fetal programming showed intriguing associations between maternal stress processes during pregnancy and outcomes in offspring that extend into adulthood.  These researchers  examined if HPA patterns in mothers during the period between 2 pregnancies (i.e., the inter-pregnancy interval) and during the subsequent pregnancy predict infant birth weight.  This study sampled salivary cortisol before and during pregnancy in a diverse community sample of 142 women enrolled in the Community Child Health Network study.  Using multi-level modeling, these investigators found that flatter diurnal cortisol slopes in mothers during the interval between one birth and a subsequent pregnancy predicted lower infant birth weight of the subsequent child.  This inter-pregnancy cortisol pattern in mothers also correlated with significantly shorter inter-pregnancy intervals, such that women with flatter cortisol slopes had more closely spaced pregnancies.  After adding demographic co-variates of household income, cohabitation with partner, and maternal race to the model, these results were unchanged.  For subjects who provided both inter-pregnancy and pregnancy cortisol data (n = 73), these researchers found that inter-pregnancy cortisol slopes predicted infant birth weight independent of pregnancy cortisol slopes.  The authors concluded that the these novel findings on inter-pregnancy HPA axis function and subsequent pregnancy outcomes supported life course health approaches and underscored the importance of maternal stress physiology between pregnancies.  The clinical value of measurement of diurnal salivary cortisol patterns for prediction of infant birth weight needs to be validated by well-designed studies.

Salivary Testing of Biomarkers for the Diagnosis of Cancers

Breast Cancer

Porto-Mascarenhas and colleagues (2017) assessed the capability of salivary biomarkers in the diagnosis and monitoring of breast cancer (BC).  Studies were eligible for inclusion if they evaluated the potential diagnostic value or other discriminatory properties of biomarkers in saliva of patients with BC.  The search was performed in 6 electronic databases (Cochrane, LILACS, PubMed, Science Direct, Scopus, Web of Science).  In addition, the biomarkers were classified according to their potential clinical application.  These researchers identified 567 pertinent studies, of which 13 met the inclusion criteria.  Combined biomarker approaches demonstrated better ability to predict BC patients than individual biomarkers.  As single biomarker, namely proline, reported great capacity in both early and late stage BC diagnosis; taurine showed interesting capability to identify early BC individuals.  Furthermore, valine also demonstrated excellent diagnostic test accuracy for advanced stages of BC.  Only 7 studies reported sensitivity and specificity, which varied considerably from 50 % to 100 %, and from 51 % to 97 %, respectively.  In general, salivary biomarkers identified advanced stages BC better than early stages.  The authors concluded that there is currently limited evidence to confirm the putative implementation of salivary biomarkers as diagnostic tools for BC; however, this review provided new research directions.

Furthermore, National Comprehensive Cancer Network’s clinical practice guideline on “Breast cancer” (Version 2.2017) does not mention testing of salivary biomarkers as a management tool.

Koopaie et al (2022) noted that salivary diagnostics and their use as a non-aggressive approach for BC diagnosis have been extensively studied in recent years.  In a meta-analysis, these researchers examined the diagnostic value of salivary biomarkers in differentiating between patients with breast cancer and controls.  They carried out a meta-analysis and systematic review of studies related to salivary diagnostics published in PubMed, Embase, Scopus, Ovid, Science Direct, Web of Science (WOS), and Google Scholar.  The studies were chosen by means of inclusion and exclusion criteria, as well as assessing their quality.  Specificity and sensitivity, along with negative and positive likelihood ratios (NLR and PLR) and diagnostic odds ratio (DOR), were calculated based on random- or fixed-effects model.  AUC and summary ROC (SROC) were plotted and evaluated, and Fagan's Nomogram was evaluated for clinical utility.  This systematic review and meta-analysis included 14 articles containing 121 study units with 8,639 adult subjects (4,149 BC patients and 4,490 controls without cancer).  The pooled specificity and sensitivity were 0.727 (95 % CI: 0.713 to 0.740) and 0.717 (95 % CI: 0.703 to 0.730), respectively.  The pooled NLR and PLR were 0.396 (95 % CI: 0.364 to 0.432) and 2.597 (95 % CI: 2.389 to 2.824), respectively.  The pooled DOR was 7.837 (95 % CI: 6.624 to 9.277), with the AUC equal to 0.801.  The Fagan's nomogram showed post-test probabilities of 28 % and 72 % for negative and positive outcomes, respectively.  These investigators also carried out subgroup analyses to determine specificity, sensitivity, DOR, PLR, and NLR based on the mean age of patients (less than or equal to 52 or greater than 52 years of age), saliva type (stimulated and unstimulated saliva), biomarker measurement method (mass spectrometry [MS] and non-MS measurement methods), sample size (less than or equal to 55 or greater than 55), biomarker type (proteomics, metabolomics, transcriptomics and proteomics, and reagent-free biophotonic), and nations.  The authors concluded that saliva, as a non-invasive biomarker, has the potential to accurately differentiate BC patients from healthy controls.  Moreover, these researchers stated that future large-scale studies that appropriately account for various potential confounders such as age, race, tobacco, and alcohol consumption, type of BC disease stage, biomarker type, methodology, and defined threshold values could confirm and fine-tune the effectiveness of salivary biomarkers before clinical implementation in BC detection.

The authors stated that one of the main drawbacks of this meta-analysis was dissemination bias because studies with positive diagnostic results were more accessible than those that reported negative findings.  Another drawback was the risk of bias in small, unmatched studies, which could not be controlled.  Another drawback was the inclusion of studies with small sample size that could have resulted in higher bias risk.  Furthermore, this analysis was potentially subject to a variety of confounders because many of the articles reviewed did not provide adequate information (e.g., the majority of studies did not specify the type of BC).  Moreover, only a limited number of studies provided information regarding tumor, node, metastasis (TNM) stage, or tobacco and alcohol use history.  Many studies did not report correlation analysis between TNM stages and salivary biomarkers either.  Thus, controlled studies with larger sample sizes with clinical and demographical details of BC and matching are needed to confirm and provide additional evidence for clinical application of salivary biomarkers in BC diagnosis.

Head and Neck Carcinoma

In a systematic review and meta-analysis, Guerra and associates (2015) evaluated the diagnostic value of salivary biomarkers in the diagnosis of head and neck carcinoma (HNC).  Studies were gathered by searching Cochrane, Embase, LILACS, Medline, and PubMed.  The references were also cross-checked and a partial grey literature search was undertaken using Google Scholar.  The methodology of selected studies was evaluated using the 14-item Quality Assessment Tool for Diagnostic Accuracy Studies.  After a 2-step selection process, 15 articles were identified and subjected to qualitative and quantitative analyses.  The studies were homogeneous, and all had high methodological quality.  Combined biomarkers demonstrated better accuracy with higher sensitivity and specificity than those tested individually.  Furthermore, the salivary biomarkers reviewed predicted the early stages of HNC better than the advanced stages.  A restricted set of 5 single biomarkers (interleukin-8 [IL-8], choline, pipecolinic acid, l-phenylalanine, and S-carboxymethyl-l-cysteine) as well as combined biomarkers demonstrated excellent diagnostic test accuracy.  The authors concluded that the present systematic review confirmed the potential value of a selected set of salivary biomarkers as diagnostic tools for HNC.

Furthermore, National Comprehensive Cancer Network’s clinical practice guideline on “Head and neck cancers” (Version 2.2017) does not mention testing of salivary biomarkers as a management tool.

Oral Pre-Cancer and Oral Squamous Cell Carcinoma

Punyani and SathawaPe (2013) noted that due to the pro-angiogenic characteristic of interleukin 8 (IL-8), it may play a vital role in tumor angiogenesis and progression.  These researchers estimated the levels of salivary IL-8 in oral pre-cancer and oral squamous cell carcinoma (OSCC) patients and compared them with healthy controls.  The aim was to evaluate its effectiveness as a potential biomarker for these diseases.  Each group comprised 25 individuals.  The salivary IL-8 levels were determined by ELISA.  The levels of salivary IL-8 were found to be significantly elevated in patients with OSCC as compared to the pre-cancer group (p < 0.0001) and healthy controls (p < 0.0001).  However, the difference in salivary IL-8 concentrations among the pre-cancer group and controls was statistically non-significant (p = 0.738).  The authors concluded that these findings suggested that salivary IL-8 can be utilized as a potential biomarker for OSCC.  Salivary IL-8 was found to be non-conclusive for oral pre-malignancy in this preliminary study.  Hence, its possible role in transition from pre-malignancy to malignancy needs further research with larger sample sizes.  They stated that the role of IL-8 in oral cancer if validated further by future research can provide an easy diagnostic test as well as a prognostic indicator for patients undergoing treatment.  Thus, if the role of Il-8 in tumor genesis can be sufficiently assessed, it could open up new avenues to find out novel treatment modalities for oral cancer.

Rajkumar et al (2015) stated that CYFRA 21-1, a constituent of the intermediate filament proteins of epithelial cells, is known to be increased in many cancers.  These investigators estimated the levels of salivary and serum CYFRA 21-1 in patients with oral pre-cancer and OSCC and compared them with healthy controls.  Each group comprised of 100 subjects.  Saliva and blood samples were collected from patients with OSCC, pre-malignant (PML) subjects, and normal healthy subjects.  Serum and salivary CYFRA 21-1 levels were measured by ELISA.  Appropriate statistical tests were employed to assess diagnostic potency of CYFRA 21-1.  These researchers found a significant increase in CYFRA 21-1 level in OSCC compared with PML and healthy subjects.  Salivary CYFRA 21-1 levels in OSCC was 3-fold higher when compared to serum levels.  Pre-malignant group showed increased salivary CYFRA 21-1 when compared to control subjects, but it was significantly lower compared with OSCC.  Receiver operator characteristic curve analysis showed salivary CYFRA 21-1 to have superior sensitivity in detecting OSCC compared with serum CYFRA 21-1.  The authors concluded that the outcome of this study suggested that salivary CYFRA 21-1 can be utilized as a biomarker in early detection of oral cancer.  These findings need to be validated by well-designed studies.

Cheng and colleagues (2014) gathered preliminary data concerning the feasibility of using 7 salivary mRNAs: IL-8; IL-1β; dual specificity phosphatase 1 (DUSP1); H3 histone family 3A (H3F3A); ornithin decarboxylase antizyme 1 (OAZ1); S100 calcium-binding protein P (S100P); and spermidine/spermine N1-acetyltransferase 1 (SAT1) for detecting development of OSCC in oral lichen planus (OLP) patients and OSCC patients whose disease was in remission.  Saliva samples were collected from 5 study groups (25 subjects per group): newly diagnosed OSCC, OSCC-in-remission, disease-active OLP, disease-inactive OLP, and normal controls.  The salivary mRNA levels were determined by a pre-amplification reverse transcription quantitative polymerase chain reaction (RT-qPCR) approach with nested gene-specific primers.  Mean fold changes between each pair of study groups were analyzed by the Mann-Whitney U test.  Salivary levels of OAZ1, S100P, and DUSP1 mRNAs were significantly higher in newly diagnosed OSCC patients, compared to:
  1. normal controls (p = 0.003; p = 0.003; and p < 0.001, respectively);
  2. OSCC-in-remission (p < 0.001; p = 0.001; and p < 0.001, respectively);
  3. disease-active OLP (p < 0.001; p = 0.016; and p < 0.001, respectively); and
  4. disease-inactive OLP (p = 0.043; p < 0.001; and p < 0.001, respectively).

No significant differences were found in the levels of salivary IL-8, IL-1β, H3F3A, and SAT1 mRNAs between newly diagnosed OSCC patients and the normal controls (p = 0.093, 0.327, 0.764, and 0.560, respectively).  The authors concluded that salivary OAZ1, S100P, and DUSP1 mRNAs are candidate biomarkers for detecting OSCC development in OSCC patients in remission and in OLP patients.  Moreover, they state that these findings serve as the basis for a further large-scale study that may lead to a non-invasive screening method for early detection of OSCC.

Furthermore, an UpToDate review on “Recognition and management of high-risk (aggressive) cutaneous squamous cell carcinoma” (DeSimone et al, 2014) does not mention salivary testing of CYFRA 21-1, IL-8, or mRNAs of DUSP1, OAZ1, and S100P, as a management tool.

Novy et al (2014) noted that the use of saliva as a diagnostic fluid has the potential to shape the role of oral health care professionals in the health care system.  While more than a handful of chair-side diagnostic tests are available for use by private practitioners, the evidence supporting their use continues to emerge.  These investigators performed an electronic search of the literature indexed on the PubMed electronic database to identify human clinical trials utilizing commercially available salivary diagnostics.  Papers meeting the inclusion criteria, and any applicable references were critically appraised following Strength of Recommendation Taxonomy (SORT) guidelines.  The authors concluded that while the literature concerning salivary analysis is continuously growing, the limited literature that is available doesn't focus on patient oriented health outcomes.  This “infant” literature is focused on validating metrics and identifying biomarkers with diagnostic potential.  As such, the evidence level of the literature is graded as level 3.  The authors stated that despite the lower grade, the research in this area showed consistent results, coherent conclusions, and research identifying new biomarkers will provide additional dimensions to salivary diagnostics.

Macey et al (2015) stated that oral squamous cell carcinoma is the most common form of malignancy of the lip and oral cavity, often being preceded by potentially malignant disorders (PMD).  Early detection can reduce the malignant transformation of PMD and can improve the survival rate for oral cancer.  The current standard of scalpel biopsy with histology is painful for patients and involves a delay while histology is completed; other tests are available that are unobtrusive and provide immediate results.  Ina Cochrane review, these investigators estimated the diagnostic accuracy of index tests for the detection of oral cancer and PMD of the lip and oral cavity, in people presenting with clinically evident lesions.  They also estimated the relative accuracy of the different index tests.  The electronic databases were searched on April 30, 2013.  These investigators searched MEDLINE (OVID) (1946 to April 2013) and 4 other electronic databases (the Cochrane Diagnostic Test Accuracy Studies Register, the Cochrane Oral Health Group's Trials Register, EMBASE (OVID) and MEDION (Ovid)).  There were no restrictions on language in the searches of the electronic databases.  They conducted citation searches and screened reference lists of included studies for additional references.  These researchers selected studies that reported the diagnostic test accuracy of the following index tests when used as an adjunct to conventional oral examination in detecting PMD or oral squamous cell carcinoma of the lip or oral cavity: vital staining, oral cytology, light-based detection and oral spectroscopy, blood or saliva analysis (which test for the presence of biomarkers in blood or saliva).  Two review authors independently screened titles and abstracts for relevance.  Eligibility, data extraction and quality assessment were carried out by at least 2 authors, independently and in duplicate.  Studies were assessed for methodological quality using QUADAS-2.  Meta-analysis was used to combine the results of studies for each index test using the bivariate approach to estimate the expected values of sensitivity and specificity.  The authors included 41 studies, recruiting 4,002 participants, in this review.  These studies evaluated the diagnostic accuracy of conventional oral examination with: vital staining (14 studies), oral cytology (13 studies), light-based detection or oral spectroscopy (13 studies).  Six studies assessed 2 combined index tests.  There were no eligible diagnostic accuracy studies evaluating blood or salivary sample analysis.  The summary estimates for vital staining obtained from the meta-analysis were sensitivity of 0.84 (95 % CI: 0.74 to 0.90) with specificity of 0.70 (95 5 CI: 0.59 to 0.79), with 14 studies were included in the meta-analysis.  For cytology, sensitivity was 0.91 (95 % CI: 0.81 to 0.96) and specificity was 0.91 (95 5 CI: 0.81 to 0.95) with 12 studies included in the meta-analysis.  For light-based detection, sensitivity was 0.91 (95 5 CI: 0.77 to 0.97) and specificity was 0.58 (95 5 CI: 0.22 to 0.87) with 11 studies included in the meta-analysis.  The relative test accuracy was assessed by adding covariates to the bivariate analysis, no difference in model fit was observed.  The authors concluded that the overall quality of the included studies was poor.  None of the adjunctive tests can be recommended as a replacement for the currently used standard of a scalpel biopsy and histological assessment.  They stated that given the relatively high values of the summary estimates of sensitivity and specificity for cytology, this would appear to offer the most potential; and combined adjunctive tests involving cytology warrant further investigation.

In a systematic review, Gualtero and Suarez Castillo (2016) examined the capacity of salivary biomarkers in the early diagnosis of oral squamous cell carcinoma (SCC).  These investigators performed a systematic review of the literature based on the English titles listed in the PubMed, EBSCO, Cochrane, Science Direct, ISI web Science and SciELO databases using the following search descriptors: Oral cancer, diagnosis, biomarkers, saliva and oral squamous cell carcinoma.  Abstracts and full-text articles were assessed independently by 2 reviewers.  International check-lists for assessment of methodological quality were used.  Levels of evidence and grades of recommendation through the Scottish Intercollegiate Guidelines Network (SIGN) template were recognized.  The units of analysis were identified through a reference matrix.  Through the research strategy and after application of different filters and considering choosing criteria, a total of 6 studies were obtained for analysis.  Salivary biomarkers for oral cancer most frequently found were mRNA and proteins for IL-8, CD44 (a cell-surface glycoprotein), matrix metalloproteinase-1 (MMP-1) and MMP-3.  New peptide-biomarkers such as Cyfra 21-1 and zinc finger protein 510 (ZNF510) were found; ZNF 510 was the only biomarker that increased in the population with tumor stage T1 + T2 and T3 + T4.  Only 1 study showed a sensitivity and specificity of 96 % when the biomarker ZNF 510 was employed to discriminate early and late tumor stages.  The authors concluded that there is insufficient evidence to support the use of the identified salivary biomarkers for the early diagnosis of oral cancer (sub-clinical stages of the pathogenic period before cancer phenotypes are manifested).

AlAli and colleagues (2020) noted that tissue biopsy with histopathological examination is still considered the gold standard to diagnose OSCC.  In a systematic review, these investigators examined the diagnostic test accuracy of 2 salivary biomarkers in adults suspected of OSCC.  The Cochrane Library, Medline, and Embase databases were searched for clinical studies examining the diagnostic accuracy of salivary biomarkers in detecting OSCC.  Studies were eligible for inclusion if only singular salivary biomarkers were assessed in 3 or more studies.  Studies examining combined salivary biomarkers or evaluating patients with oral potentially malignant disorders only were excluded.  The reporting of the review followed the PRISMA check-list.  A total of 6 studies, recruiting 775 subjects, were included in this review for only 2 salivary biomarkers, cytokeratin 19 fragment (CYFRA 21-1) and matrix metalloproteinase 9 (MMP-9).  The sensitivity and specificity (with 95 % CIs) for CYFRA 21-1 studies ranged from 0.84 (0.75 to 0.91) to 0.94 (0.83 to 0.99) and from 0.84 (0.71 to 0.93) to 0.96 (0.80 to 1.00), respectively.  In MMP-9 studies, sensitivity (with 95 % CIs) ranged from 0.76 (0.67 to 0.83) to 1.00 (0.78 to 1.00) and specificity from 0.27 (0.12 to 0.46) to 1.00 (0.78 to 1.00).  The overall quality of the included studies was poor.  The authors concluded that due to a lack of strong and high-quality evidence, considerable uncertainty remained surrounding the use of singular salivary biomarkers for the detection of OSCC.

Salivary Testing of Pepsin for the Diagnosis of Gastro-Esophageal Reflux Disease / Laryngo-Pharyngeal Reflux

Dy and colleagues (2016) examined the sensitivity of salivary pepsin compared with multi-channel intraluminal impedance with pH testing (pH-MII), endoscopy, and gastro-esophageal reflux disease (GERD) questionnaires.  These investigators prospectively recruited 50 children from Boston Children's Hospital who were undergoing pH-MII to evaluate for GERD.  Subjects completed 24-hour pH-MII testing, completed symptom and quality of life (QOL) questionnaires, and provided a saliva specimen that was analyzed using the PepTest lateral flow test.  A subset of patients also underwent bronchoscopy and esophago-gastro-duodenoscopy (EGD); receiver operating characteristic curve (ROC) analyses were performed to determine the sensitivity of salivary pepsin compared with each reference standard; 21 of the 50 patients (42 %) were salivary pepsin-positive, with a median salivary pepsin concentration of 10 ng/ml (IQR, 10 to 55 ng/ml).  There was no significant difference in the distributions of acid, non-acid, total reflux episodes, full column reflux, or any other reflux variable in patients who were pepsin-positive compared with those who were pepsin-negative (p > 0.50).  There was no significant correlation between the number of reflux episodes and pepsin concentration (p > 0.10).  There was no positive relationship between salivary pepsin positivity, any extra-esophageal symptoms or QOL scores, or inflammation on bronchoscopy or EGD (p > 0.30).  The authors concluded that salivary pepsin measurement had a low sensitivity for predicting pathological GERD in children.

Bozzani and colleagues (2020) noted that GERD is a digestive disorder characterized by nausea, regurgitation, and heartburn.  Gastro-esophageal reflux is the primary cause of laryngeal symptoms, especially chronic posterior laryngitis.  The best diagnostic test for this disease is esophageal impedance-pH monitoring; however, it is poorly utilized due to its high cost and invasiveness.  Salivary pepsin measured using a lateral flow device (Pep-test) has been suggested as an indirect marker of laryngo-pharyngeal reflux (LPR).  In a multi-center, non-interventional, pilot study, these researchers tested the reliability of Pep-test in diagnosing LPR in un-investigated primary care attenders presenting with chronic laryngeal symptoms, and examined the raw pepsin concentration in patients with LPR.  This trial was carried out on 86 suspected patients with LPR and 59 asymptomatic subjects as controls in 3 Italian primary care settings.  A reflux symptom index questionnaire was used to differentiate patients with LPR (score of greater than 13) from controls (score of less than 5).  Two saliva samples were collected, and comparisons between the groups were performed using 2-sided statistical tests, according to variable distributions.  There was no statistical difference in the salivary pepsin positivity between LPR patients and controls, whereas the pepsin intensity value was higher in controls than in LPR patients.  The authors concluded that a high prevalence of pepsin positivity was observed in asymptomatic controls.  These investigators stated that pepsin measurement should not be considered as a diagnostic test for LPR in the primary care setting.

In a systemic review and meta-analysis, Guo et al (2022) examined the use of Pep-test for diagnosis of LPR and GERD.  PubMed, Embase, and the Cochran Library (from January 1980 to January 26, 2020) were searched for pepsin in saliva for LPR/GERD diagnosis.  Sensitivity, specificity, PLR, NLR, DOR, and AUC data were summarized to examine the accuracy.  A total of 16 articles that included 2,401 patients and 897 controls were analyzed.  The pooled sensitivity and specificity for the diagnosis of GERD/LPR with Pep-test were 62 % (95 % CI: 49 % to 73 %) and 74 % (95 % CI: 50 % to 90 %), respectively.  The summarized DOR and AUC were 5.0 (95 % CI: 2 to 19) and 0.70 (95 % CI: 0.66 to 0.74), respectively.  The authors concluded that Pep-test showed moderate diagnostic value for LPR and GERD.  These researchers stated that further large-scale studies with standard protocols should be carried out to verify its usefulness.

The authors stated that this study had several drawbacks.  First, although 16 studies were enrolled, the sample size was relatively small, with 2,401 patients and 897 controls included.  Furthermore, there were no standard protocols for Pep-test, such as the best time and frequency of saliva sampling, which might result in inconsistence of diagnostic value.  The different protocols, study design, patient cohort, and cut-off value among studies might lead to heterogeneity.  Finally, the summarized data in some articles constrained these researchers from conducting a more detailed analysis.

Salivary Testing of Telomere Length for the Diagnosis of Depression

Whisman and Richardson (2017) examined the association between depressive symptoms and salivary telomere length in a probability sample of middle-aged and older adults, and evaluated age and sex as potential moderators of this association and examined if this association was incremental to potential confounds.  Subjects were 3,609 individuals from the 2008 wave of the Health and Retirement Study; telomere length assays were performed using quantitative real-time polymerase chain reaction (qRT-PCR) on DNA extracted from saliva samples.  Depressive symptoms were assessed via interview, and health and lifestyle factors, traumatic life events, and neuroticism were assessed via self-report.  Regression analyses were conducted to examine the associations between predictor variables and salivary telomere length.  After adjusting for demographics, depressive symptoms were negatively associated with salivary telomere length (b = -0.003; p = 0.014).  Furthermore, this association was moderated by sex (b = 0.005; p = 0.011), such that depressive symptoms were significantly and negatively associated with salivary telomere length for men (b = - 0.006; p < 0.001); but not for women (b = - 0.001; p = 0.644).  The negative association between depressive symptoms and salivary telomere length in men remained statistically significant after additionally adjusting for cigarette smoking, body mass index (BMI), chronic health conditions, childhood and lifetime exposure to traumatic life events, and neuroticism.  The authors concluded that higher levels of depressive symptoms were associated with shorter salivary telomeres in men, and this association was incremental to several potential confounds.  They stated that shortened telomeres may help account for the association between depression and poor physical health and mortality.

Cordant Health Solutions Comprehensive Oral Fluid Rx Evaluation (CORE)

Cordant Health Solutions’ Comprehensive Oral fluid Rx Evaluation (CORE), a new drug testing technology, is designed to correlate drug concentrations in oral fluid with drug levels in blood plasma; it is used for individualized drug testing for pain management.

Borg and colleagues (2017) noted that synthetic cannabinoids are a group of psycho-active compounds that mimic the effects of Δ9-tetrahydrocannabinol, the primary psycho-active constituent of marijuana.  The Drug Enforcement Administration (DEA) has classified many of the most common cannabinoids as Schedule 1 controlled substances.  As a result, several novel synthetic cannabinoid series have emerged in the illicit drug market, including PINACA, FUBINACA, PB-22, AKB-48 and multiple derivatives of these compounds.  The authors’ laboratory developed and validated an analytical method for the analysis 32 synthetic cannabinoid metabolites in urine samples.  Included in this method are metabolites that are constituents of the new generation of synthetic cannabinoids.  Following enzymatic hydrolysis, target analytes were recovered by liquid-liquid extraction utilizing 1-chlorobutane:isopropyl alcohol (70:30) as the organic ratio.  Chromatographic separation and detection was achieved using an Agilent Technologies 1290 liquid chromatograph (LC) coupled to a 6460-triple quadrupole mass spectrometer (MS) with a Jetstream electrospray source.  Linearity for all analytes was established along the range of 0.5 to 200 ng/ml.  Both intra-day and inter-day accuracy as well as precision data were all within acceptable limits, ± 20 % error and ± 15 % relative standard deviation (SD), respectively.  Recovery ranged from 48 % to 104 %.  This method has shown to be selective and specific, providing no evidence of interference or carry-over concerns.  Finally, 11 distinct synthetic cannabinoids were detected in 23 of 25 donor samples analyzed with the method.  The authors concluded that these data represented a validated LC tandem-MS method to accurately identify and quantitate synthetic cannabinoid metabolites in urine samples, incorporating new generation derivatives.

Shaparin and associates (2017) stated that interpretation limitations of urine drug testing and the invasiveness of blood toxicology have motivated the desire for the development of simpler methods to evaluate biologically active drug levels on an individualized patient basis.  Oral fluid is a matrix well-suited for the challenge because collections are based on simple non-invasive procedures and drug concentrations better correlate to blood drug levels as oral fluid is a filtrate of the blood.  Well-established pharmacokinetic models were utilized to generate oral fluid steady state concentration ranges to assess the interpretive value of the alternative matrix to monitor steady state plasma oxycodone levels.  Paired oral fluid and plasma samples were collected from patients chronically prescribed oxycodone and quantitatively analyzed by LC tandem-MS.  Steady state plasma concentration ranges were calculated for each donor and converted to an equivalent range in oral fluid.  Measured plasma and oral fluid oxycodone concentrations were compared with respective matrix-matched steady state ranges, using each plasma steady state classification as the control.  A high degree of correlation was observed between matrices when classifying donors according to expected steady state oxycodone concentration.  Agreement between plasma and oral fluid steady state classifications was observed in 75.6 % of paired samples.  The authors concluded that the findings of this study supported novel application of basic pharmacokinetic knowledge to the pain management industry, simplifying and improving individualized drug monitoring and risk assessment through the use of oral fluid drug testing.  They stated that many benefits of established therapeutic drug monitoring in plasma can be realized in oral fluid for patients chronically prescribed oxycodone at steady state.

There is currently insufficient evidence to support the clinical value of Cordant Health Solutions’ Comprehensive Oral Fluid Rx Evaluation (CORE).

Measurement of Salivary Levels of Cortisol for Predicting the Efficacy of Sleep-Promoting Treatment  in Children with Postural Tachycardia Syndrome

In a prospective study, Lin and colleagues (2017) determined the value of salivary cortisol concentrations in predicting the efficacy of sleep-promoting treatment in children with postural tachycardia syndrome (POTS).  This trial involved 40 children with POTS and 20 healthy children (controls); POTS was diagnosed using the head-up or head-up tilt test.  Patients with POTS received a sleep-promoting treatment: greater than 8 hours of sleep every night and a mid-day nap in an appropriate environment; no drinking water or exercising before bedtime; and urination before bedtime.  The Pittsburgh Sleep Quality Index was used to evaluate sleep quality, and symptom scores were used to assess POTS severity.  Salivary samples were collected upon awakening, 30 minutes after awakening, at 12:00 p.m., 4:00 p.m., and 8:00 p.m., and at bed-time before treatment; ELISA was used to measure salivary cortisol concentrations.  Cortisol concentrations were significantly higher in patients with POTS than in the controls at all time-points (p < 0.05 for all); PSQI scores were significantly higher in patients with POTS (7.2 ± 3.0) than in the controls (1.35 ± 1.39; t = -10.370, p < 0.001).  Salivary cortisol concentrations at awakening were significantly higher in responders than in non-responders (4.83 ± 0.73 versus 4.05 ± 0.79 ng/ml, t = -3.197, p = 0.003).  The area under the receiver operating characteristic curve was 75.8 %, (95 % CI: 59.3 % to 92 %).  Cut-off at-awakening salivary cortisol concentrations of  greater than 4.1 ng/ml yielded 83.3 % sensitivity and 68.7 % specificity in predicting therapeutic efficacy.  The authors concluded that at-awakening salivary cortisol concentrations may predict the efficacy of sleep-promoting treatment in patients with POTS.

Measurement of Salivary Levels of Hemoglobin for Screening Periodontal Disease

Nomura and colleagues (2018) stated that periodontal disease is a common inflammatory disease.  It affects about 20 to 50 % of global population in both developed and developing countries.  Early detection of slight changes of periodontal tissue plays an important role in prevention of onset and progression of periodontal disease.  Thus, there is a need of a screening test to assess periodontal tissue for health check-ups.  Salivary levels hemoglobin (Hb) has been proposed to assess the conditions of the inflammation of gingiva.  In a systematic review, these investigators evaluated the evidences for Hb as periodontal screening test.  They carried out a literature search of report published using PubMed databases.  A total of 55 articles were retrieved and 16 were selected.  The review focused on correlation coefficient with periodontal clinical parameters or sensitivity and specificity.  As a result, 14 studies calculated sensitivity and specificity of Hb; 6 studies measured salivary levels hemoglobin at laboratory: 3 studies used polyclonal antibody reactions and other studies used colorimetric tests; 8 studies used paper strip method: 4 studies used monoclonal antibody reaction and 4 studies used colorimetric tests.  Youden's indexes by antibody reaction were better than those of colorimetric methods.  The authors concluded that further studies are needed to set the cut-off values stratified by gender, age and number of remaining teeth.

Measurement of Salivary Levels of Matrix Metalloproteinase-8 (MMP-8) for Diagnosis of Periodontal Disease

de Morais and associates (2018) noted that periodontal disease is characterized as a disorder of the oral microbiota resulting in an immune response which, in turn, leads to the destruction of periodontal tissue.  Matrix metalloproteinase-8 (MMP-8) has been reported as the major metalloproteinase involved in periodontal disease, being present at high levels in gingival crevicular fluid and salivary fluid (SF).  In a systematic review, these researchers examined the evidence regarding the expression of MMP-8 in gingival crevicular fluid and SF in patients with periodontal disease, analyzing its validity as a possible biomarker in the diagnosis of periodontal disease.  The literature review was carried out using the PubMed/Medline, CENTRAL and Science Direct databases.  Studies concerning the use of MMP-8 in the diagnosis of periodontal disease that evaluated its effectiveness as a biomarker for periodontal disease were selected.  The search strategy provided a total of 6,483 studies.  After selection, 6 articles met all the inclusion criteria and were included in the present systematic review.  The studies demonstrated significantly higher concentrations of MMP-8 in patients with periodontal disease compared with controls, as well as in patients presenting more advanced stages of periodontal disease.  The authors concluded that the findings on higher MMP-8 concentrations in patients with periodontal disease compared with controls imply the potential adjunctive use of MMP-8 in the diagnosis of periodontal disease.

Measurement of Salivary Levels of IgA, IgG, IgM, Interferon-Gamma, Interleukin-4, Interleukin-6, Interleukin-8 / Tumor Necrosis Factor-Alpha in Oral Lichen Planus

Mozaffari and colleagues (2017) stated that tumor necrosis factor-α (TNF-α) has a role in the progression of the oral lichen planus (OLP).  In a meta-analysis, these researchers evaluated the salivary and serum TNF-α levels in patients with OLP.  They searched in the databases of PubMed/Medline, Science direct, Scopus, Web of Science, and Cochrane Library for studies reported from 1983 to 2016.  All studies were checked for evaluation of salivary and serum levels of TNF-α in patients with OLP compared with healthy controls.  A total of 12 studies were included in the meta-analysis.  The mean difference of 7 studies reporting salivary TNF-α levels in patients with OLP versus healthy controls was 25.90 pg/ml (95 % CI: 15.31 to 36.49; p < 0.00001) and 7 studies reporting serum TNF-α levels was 1.65 pg/ml (95 % CI: -0.82 to 4.11; p = 0.19).  The authors concluded that in patients with OLP, the higher levels of TNF-α in saliva compared with serum suggested that measurement of this marker in saliva may be more useful than in serum for determining diagnostic and therapeutic aims.

Mozaffari and associates (2018a) noted that interleukin-6 (IL-6) is a cytokine that contributes to the pathogenesis of OLP.  In a meta-analysis, these investigators assessed IL-6 levels in the serum and saliva of patients with OLP compared with healthy controls.  They searched studies in 5 databases: PubMed/Medline, Scopus, ScienceDirect, Web of Science, and Cochrane Library, from 1983 to October 31, 2016.  A total of 11 studies were analyzed for the meta-analysis study.  The reviewers independently evaluated the quality of each included study using the Newcastle-Ottawa Quality Assessment Scale (NOS).  A random-effects meta-analysis, using Comprehensive Meta-Analysis software version 2.0, was used to reflect the variation in studies.  Heterogeneity between estimates was evaluated by the Q and I2 statistics and for the Q statistic; heterogeneity was considered for p < 0.1.  A total of 11 studies included 529 OLP patients and 333 healthy controls.  The review identified 2 different biomaterials used for IL-6 assays: saliva and serum.  The mean quality score of 11 studies was 7 (high quality).  Estimates pooled from 6 studies showed significant high saliva IL-6 levels in OLP patients compared with healthy controls (the standardized difference in means (SDM) = 4.534, 95 % CI: 1.915 to 7.153, p = 0.001).  Also, estimates pooled from 7 studies showed significantly high serum IL-6 levels in OLP patients compared with healthy controls (SDM = 1.482, 95 % CI:  0.524 to 2.439, p = 0.002).  The authors concluded that the higher levels of IL-6 in saliva compared with serum suggested that measurement of this marker in saliva may be more useful than serum for diagnostic and therapeutic aims.

The authors stated that this study had several drawbacks.  First, most studies for the evaluation of salivary IL-6 levels used small sample sizes.  Second, the kits used for measurement of IL-6 levels had different sensitivities and specificities.  Third, in some studies, OLP patients and healthy controls were not age- and sex-matched.  Fourth, the numbers of patients based on the type of OLP in meta-analyses were different.  These limitations may create a high heterogeneity in the results of meta-analysis, although there was no publication bias between the studies, and these were of high quality.

Mozaffari and colleagues (2019a) stated that cytokines have regulatory and leading roles in the immunopathogenesis of oral lichen planus (OLP).  These investigators reported the findings of a meta-analysis that examined serum and salivary interferon-gamma (IFN-γ) levels in patients with OLP compared with those in controls and the correlation of this cytokine with the progression of OLP.  Four databases -- PubMed, Web of Science, Scopus, and Cochrane Library -- were searched, from their start dates to November 2017, for reports in all languages on the effect of OLP on salivary and serum IFN-γ.  A total of 11 studies were included and analyzed in this meta-analysis.  The pooled mean difference (MD) values were estimated to be 3.60 pg/ml (p = 0.23) and -0.02 pg/ml (p = 1.00) for serum and salivary levels of IFN-γ, respectively, in the patients with OLP compared with controls.  The pooled MD values were -2.52 pg/ml (p = 0.03) and -2.01 pg/ml (p = 0.20) for serum and salivary IFN-γ levels in the erosive type, respectively, compared with the non-erosive type.  The authors concluded that according to the results of this meta-analysis, there was no statistically significant differences in IFN-γ levels between the OLP group and the control group both in serum and salivary levels and also between erosive and non-erosive types of OLP at the salivary level; so this cytokine is not considered to have an important role in the pathogenesis or severity of OLP.

In a meta-analysis, Mozaffari and colleagues (2019b) examined the serum and salivary levels of IL-4 in connection with several OLP variants.  The search was performed from 1995 in Cochrane Library and from 1983 in Scopus, PubMed, and Web of Science to September 2018.  The quality of the studies included in the meta-analysis was assessed using the Newcastle-Ottawa Scale (NOS).  The analyses were varied out by Review Manager 5.3 using MD and 95 % CIs.  Of the 108 studies retrieved in the databases, only 10 were included and analyzed in quantitative synthesis.  The pooled MD of the serum and salivary IL-4 levels in OLP patients compared with the controls was 6.36 (pg/ml) (95 % CI: 1.47 to 11.24; p = 0.01) and 2.67 pg/ml (95 % CI: 2.66 to 2.68; p < 0.00001), respectively.  In addition, the pooled MD of serum and salivary IL-4 level was 1.30 pg/ml (95 % CI: -0.35 to 2.95; p = 0.12) and 1.83 pg/ml (95 % CI: 0.26 to 3.40; p = 0.02), respectively, in patients with erosive, erythematous, bullous, and ulcerative variants of OLP compared with patients with reticular OLP.  The authors concluded that this meta-analysis found that OLP patients present elevated serum and salivary IL-4 levels, thus indicating that IL-4 may represent a potential salivary biomarker for the disease.  By contrast, clinicians must be aware that even other factors (e.g., secondary infection) may influence its concentration.

In a systematic review and meta-analysis, Mozaffari and colleagues (2018b) examined serum and salivary interleukin-8 (IL-8) levels of the OLP patients compared with the healthy controls.  Five databases including PubMed/Medline, Web of Science, Science Direct, Cochrane Library and Scopus were searched for the evaluation of serum and salivary IL-8 levels of the OLP patients compared with the healthy controls in the English abstract.  The NOS was used for checking the quality of the studies.  A random-effect model was used for calculating the MD and 95 % CIs.  A total of 9 studies were included in the meta-analysis.  The pooled estimate showed a significant difference between 2 groups that the salivary IL-8 level in the OLP patients was higher than the healthy controls (MD = 766.32 pg/ml, 95 % CI: 394.90 to 1137.75; p < 0.0001) and also the serum IL-8 level in the OLP patients was higher than the healthy controls (MD = 8.38 pg/ml, 95 % CI: 3.32 to 13.44; p = 0.001).  The authors concluded that the higher levels of IL-8 in saliva compared with serum suggested that measurement of this marker in saliva may be more useful than serum measurements for determining therapeutic and diagnostic aims.  Moreover, these researchers stated that other confounding factors such as the type of OLP and genetics must be considered when interpreting these findings.

The authors stated that this study had several drawbacks including variable timing of saliva collection, different kits and methods of IL-8 assays, variation in criteria for selection of the healthy controls, varying type and severity of OLP across studies, lack of uniform matching of age and sex between the OLP patients and the healthy controls, and the need to estimate a mean value and SD in several studies.

Mozaffari and colleagues (2018c) noted that immunoglobulins (IgA, IgG, and IgM) are significant anti-inflammatory factors.  In a meta-analysis, these researchers evaluated the serum and salivary levels of Igs as more important immunoglobulins in patients affected by OLP compared to the healthy controls.  Four databases, including PubMed/Medline, Scopus, Web of Science, and Cochrane Library as well as Iranian databases were checked up to January 2018 without language restriction.  The quality of each involved study was done using the NOS.  A random-effects model analysis was performed using RevMan 5.3 software and applying the MD plus 95 % CIs.  The CMA 2.0 software was applied to calculate the publication bias among the studies.  Out of 70 studies found in the databases, 8 studies were involved and analyzed in the meta-analysis.  The meta-analysis included 282 OLP patients and 221 healthy controls.  The pooled MDs of serum levels of IgA, IgG, and IgM were -0.13 g/L [95 % CI: -0.24 to -0.02; p = 0.02], 1.01 g/L [95 % CI: -0.91 to 2.93; p = 0.30], and -0.06 g/L [95 % CI: -0.25 to 0.14; p = 0.56], respectively; whereas, the salivary IgA and IgG levels were 71.54 mg/L [95 % CI: 12.01 to 131.07; p = 0.02] and 0.59 mg/L [95 % CI: -0.20 to 1.38; p = 0.14], respectively.  The authors concluded that considering the few studies performed on saliva, the results suggested that the salivary levels, especially IgA level had higher values than the serum levels.  Thus, the salivary immunoglobulins could play a significant function in the OLP pathogenesis.  Moreover, these researchers stated that clinicians / investigators should be aware of the effective factors involved in OLP in order to find better and more accurate results.

The authors stated that this study had several drawbacks.  First, there were only a few studies on saliva.  Second, there were different measurement methods of Ig.  Third, heterogeneity was among most sub-groups.

Examination of Salivary Microbiota Patterns for the Diagnosis of Minimal Hepatic Encephalopathy

Bajaj and colleagues (2019) stated that minimal hepatic encephalopathy (MHE) is epidemic in cirrhosis, but testing strategies often have poor concordance.  Altered gut/salivary microbiota occur in cirrhosis and could be related to MHE.  These researchers examined microbial signatures of individual cognitive tests and defined the role of microbiota in the diagnosis of MHE.  Out-patients with cirrhosis underwent stool collection and MHE testing with psychometric hepatic encephalopathy score (PHES), inhibitory control test, and EncephalApp Stroop; a subset provided saliva samples.  Minimal hepatic encephalopathy diagnosis/concordance between tests was compared.  Stool/salivary microbiota were analyzed using 16srRNA sequencing.  Microbial profiles were compared between patients with/without MHE on individual tests.  Logistic regression was used to evaluate clinical and microbial predictors of MHE diagnosis.  A total of 247 patients with cirrhosis (123 prior overt HE, MELD 13) underwent stool collection and PHES testing; 175 underwent inhibitory control test and 125 underwent Stroop testing; 112 patients also provided saliva samples.  Depending on the modality, 59 % to 82 % of patients had MHE.  Inter-test kappa for MHE was 0.15 to 0.35.  Stool and salivary microbiota profiles with MHE were different from those without MHE.  Individual microbiota signatures were associated with MHE in specific modalities.  However, the relative abundance of Lactobacillaceae in the stool and saliva samples was higher in MHE, regardless of the modality used, whereas autochthonous Lachnospiraceae were higher in those without MHE, especially on PHES.  On logistic regression, stool and salivary Lachnospiraceae genera (Ruminococcus and Clostridium XIVb) were associated with good cognition independent of clinical variables.  The authors concluded that specific stool and salivary microbial signatures existed for individual cognitive testing strategies in MHE.  These researchers stated that the presence of specific taxa associated with good cognitive function regardless of modality could potentially be used to circumvent MHE testing.

Salivary MicroRNA as a Biomarker of Traumatic Brain Injury / for Detection of Prolonged Concussion Symptoms

Johnson and colleagues (2018) noted that approximately 1/3 of children who experienced a concussion develop prolonged concussion symptoms.  At present, there are no objective or easily administered tests for predicting prolonged concussion symptoms.  Several studies have identified alterations in epigenetic molecules known as microRNAs (miRNAs) following traumatic brain injury (TBI).  No studies have examined if miRNA expression can detect prolonged concussion symptoms.  In a prospective cohort study, these researchers evaluated the efficacy of salivary miRNAs for identifying children with concussion who are at risk for prolonged symptoms.  This trial included 52 patients aged 7 to 21 years presenting for evaluation of concussion within 14 days of initial head injury, with follow-up at 4 and 8 weeks.  All subjects had a clinical diagnosis of concussion.  Salivary miRNA expression was measured at the time of initial clinical presentation in all subjects.  Patients with a Sport Concussion Assessment Tool (SCAT3) symptom score of 5 or greater on self-report or parent report 4 weeks after injury were designated as having prolonged symptoms.  Of the 52 included participants, 22 (42 %) were female, and the mean (SD) age was 14 (3) years.  Participants were split into the prolonged symptom group (n = 30) and acute symptom group (n = 22).  Concentrations of 15 salivary miRNAs spatially differentiated prolonged and acute symptom groups on partial least squares discriminant analysis and demonstrated functional relationships with neuronal regulatory pathways.  Levels of 5 miRNAs (miR-320c-1, miR-133a-5p, miR-769-5p, let-7a-3p, and miR-1307-3p) accurately identified patients with prolonged symptoms on logistic regression (area under the curve [AUC], 0.856; 95 % CI: 0.822 to 0.890).  This accuracy exceeded accuracy of symptom burden on child (AUC, 0.649; 95 % CI: 0.388 to 0.887) or parent (AUC, 0.562; 95 % CI: 0.219 to 0.734) SCAT3 score.  Levels of 3 miRNAs were associated with specific symptoms 4 weeks after injury; miR-320c-1 was associated with memory difficulty (R, 0.55; false detection rate, 0.02), miR-629 was associated with headaches (R, 0.47; false detection rate, 0.04), and let-7b-5p was associated with fatigue (R, 0.45; false detection rate, 0.04).  The authors concluded that salivary miRNA levels may identify the duration and character of concussion symptoms, which could reduce parental anxiety and improve care by providing a tool for concussion management.  These researchers stated that further validation of this approach is needed.

Hicks and associates (2018) examined the accuracy and physiological relevance of circulating miRNA as a biomarker of pediatric concussion.  These investigators compared changes in salivary miRNA and cerebrospinal fluid (CSF) miRNA concentrations following childhood TBI.  A case-cohort design was used to compare longitudinal miRNA concentrations in CSF of 7 children with severe TBI against 3 controls without TBI.  The miRNAs "altered" in CSF were interrogated in saliva of 60 children with mild TBI and compared with 18 age- and sex-matched controls.  The miRNAs with parallel changes (Wilcoxon rank sum test) in CSF and saliva were interrogated for predictive accuracy of TBI status using a multi-variate regression technique.  Spearman rank correlation identified relationships between miRNAs of interest and clinical features.  Functional analysis with DIANA mirPath identified related mRNA pathways.  There were 214 miRNAs detected in CSF, and 135 (63 %) were also present in saliva; 6 miRNAs had parallel changes in both CSF and saliva (miR-182-5p, miR-221-3p, mir-26b-5p, miR-320c, miR-29c-3p, miR-30e-5p).  These miRNAs demonstrated an AUC of 0.852 for identifying mild TBI status; 3 of the miRNAs exhibited longitudinal trends in CSF and/or saliva following TBI, and all 3 targeted mRNAs related to neuronal development.  Concentrations of miR-320c were directly correlated with child and parent reports of attention difficulty.  The authors concluded that salivary miRNA represented an easily measured, physiologically relevant, and accurate potential biomarker for TBI.  Moreover, they stated that further studies assessing the influence of orthopedic injury and exercise on peripheral miRNA patterns are needed.

LaRocca and co-workers (2019) noted that TBI is a major cause of death and disability worldwide, with mild TBI (mTBI) accounting for 85 % of cases.  mTBI is also implicated in serious long-term sequelae including second impact syndrome and chronic traumatic encephalopathy.  mTBI often goes un-diagnosed due to delayed symptom onset and limited sensitivity of conventional assessment measures compared with severe TBI.  Current efforts seek to identify accurate and reliable non-invasive biomarkers associated with functional measures relevant to long-term outcomes.  These investigators evaluated the utility of serum and salivary miRNAs to serve as sensitive and specific peripheral biomarkers of possible mTBI.  The primary objectives were to establish the relationship between peripheral measures of miRNA, objective quantification of head impacts, and sensitive indices of balance and cognitive function in healthy young adult athletes.  A secondary objective was to compare the sensitivity of miRNA versus commonly used blood-based protein biomarkers.  A total of 50 amateur mixed martial arts (MMA) fighters participated; 216 saliva and serum samples were collected at multiple time-points, both pre- and post-fight.  Levels of 10 serum proteins were compared in a subset of the fighters (n = 24).  Levels of miRNAs were obtained by next generation sequencing (NGS).  Functional outcomes were evaluated using a computerized assessment system that measured cognitive performance, body sway, and combined cognitive performance and body sway during dual task completion.  Data were analyzed using multi-variate logistic regression for predictive classification, analysis of variance, correlation analysis and principal component analysis.  These researchers identified a subset of salivary and serum miRNAs that showed robust utility at predicting TBI likelihood and demonstrated quantitative associations with head impacts as well as cognitive and balance measures.  In contrast, serum proteins demonstrated far less utility.  These investigators also found that the timing of the responses varied in saliva and serum, which was a critical observation for biomarker studies to consider. 

The authors concluded that this analysis has revealed a number of important findings for the mTBI biomarker field.  First, several salivary and serum miRNAs were robustly altered after a sport-related head impact.  Some of these miRNAs showed association with the quantity of head impacts, and affected processes involved in both adaptive and maladaptive responses.  Other miRNAs show changes that differed across time, which underscored the importance of designing studies to capture the time course of biomarker responses.  Both the hits to the head (HTH)-related and temporally-related sets of miRNAs were predicted to alter biological processes that were potentially highly-relevant for TBI studies.  Furthermore, the changes in some of these same miRNAs also showed associations with computerized functional outcome measures of both balance and cognitive function.  In contrast, this limited analysis of potential protein biomarkers did not yield strong associations with the number of head impacts, with the possible exception of UCHL1.  Clearly, much additional work needs to be done to determine the best molecular biomarkers of mTBI from peripheral biofluids and to relate these to the most sensitive functional measures.

The authors stated that this study had several limitations.  First, subjects were not systematically evaluated for general symptoms of concussion and mental responsiveness following the fight using a standardized assessment tool (such as the SCAT).  Second, these researchers were not able to use a full factorial model in their analysis to formally examine the effect of biofluid and time of sampling due to the use of slightly different sets of subjects across time.  Third, although these researchers quantified the HTH experienced during the MMA fight, there was considerable heterogeneity in the force of each head impact, depending on the fighters themselves and the trajectory and location of the blow to the head.  Finally, the data that were obtained may not be fully representative of what might occur in the general or young adult population because they were obtained from amateur mixed martial arts fighters.  Notably, however, the authors did point out that every subject in the study was employed in a full-time job or attended school, and only participated in MMA fighting as an athletic hobby.  Thus, this particular group of amateur MMA fighters may be fairly representative of young adult athletes who engage in contact sports.

Measurement of Salivary Levels of Creatinine and Urea for the Determination of Uremic State in Adults With Chronic Kidney Disease

In a systematic review and meta-analysis, Rodrigues and colleagues (2020) examined if salivary urea and creatinine levels accurately reflect their serum levels in blood samples of adults to detect chronic kidney disease. (CKD).  These researchers carried out a systematic review in 8 electronic data-bases.  The protocol was registered in PROSPERO; only diagnostic test studies were included.  The JBI critical appraisal tools assessed the risk of bias; a meta-analysis of proportions was conducted.  The GRADE tool assessed the quality of evidence and strength of recommendation across the studies included.  A total of 8 studies met the eligibility criteria and were included; 6 studies assessed salivary urea, and 6 studies assessed salivary creatinine.  All studies presented moderate risk of bias.  The meta-analysis depicted an overall sensitivity of 93.3 % (95 % CI: 88.6 to 97.9) for salivary creatinine levels and 87.5 % (95 % CI: 83.2 to 91.8) for salivary urea levels, while the overall specificity was 87.1 % (95 % CI: 82.8 to 91.3) and 83.2 % (95 % CI: 65.0 to 101.4) for salivary creatinine and urea levels, respectively.  The overall accuracy of salivary creatinine was 5.2 percentage points higher compared with salivary urea levels (90.8 % versus 85.6 %).  According to the GRADE tool, the analyzed outcomes were classified as having low-to-moderate level of certainty.  The authors concluded that compared with blood samples, salivary urea and creatinine levels presented high diagnostic values for CKD screening, but should not be considered equivalent to levels obtained from blood at stages 3, 4, or 5 of the disease.  These researchers stated that CKD patients could receive a clinically significant benefit from replacing blood with saliva for potentially monitoring renal function.

Furthermore, UpToDate reviews on “Overview of the management of chronic kidney disease in adults” (Rosenberg, 2020), and “Definition and staging of chronic kidney disease in adults” (Levey and Inker, 2020) do not mention measurements of salivary creatinine and urea as management options.

Measurement of Salivary Levels of Osteocalcin for the Determination of Periodontitis Severity

In a cross-sectional study, Shazam and colleagues (2020) examined osteocalcin levels in saliva of healthy and periodontitis patients and correlated these levels with periodontitis severity.  This trial was carried out in a hospital setting.  A total of 95 subjects enrolled in the study with 46 in group I (healthy individuals) and 49 in group II (mild, moderate, and severe chronic periodontitis patients).  A detailed assessment of clinical periodontal parameters and alveolar bone loss was made.  Un-stimulated saliva samples were collected from all study subjects and osteocalcin levels were quantitatively analyzed by sandwich enzyme-linked immunosorbent essay technique.  One-way analysis of variance, Spearman's correlation test, and Pearson's Chi-squared test were applied at a significance level of 95 %; p values of less than 0.05 were considered statistically significant.  The results showed a significant association of qualification with group II (p < 0.02).  Bone loss scores were also significantly associated with periodontitis severity (p < 0.01).  However, no statistically significant difference was observed between group I and group II in terms of mean salivary osteocalcin levels (p = 0.68).  Furthermore, an insignificant correlation was also observed between osteocalcin levels and periodontitis severity (p = 0.13).  The authors concluded that the overall study results showed that there was no significant difference between saliva osteocalcin levels of healthy and periodontitis patients.  In addition, there was a non-significant correlation between osteocalcin levels and periodontitis severity.  The findings of the present study supported the hypothesis that low osteocalcin levels in saliva might be considered as a poor indicator of periodontal disease progression and severity.

Salivary Hypermethylated DNA Biomarkers for Diagnosis of Oral Cancer

In a systematic review and meta-analysis, Adeoye and colleagues (2022) examined the DTA of hypermethylated DNA biomarkers in saliva and oral swabs for oral squamous cell carcinoma (OSCC) detection from the pre-validation studies available.  Electronic database searching of PubMed, Embase, Cochrane Library, Scopus, Web of Science, and LILACS was carried out to identify relevant articles that were published between January 1, 2000, and August 1, 2020.  Meta-analysis was performed based on 11 of 20 studies selected for review.  Included studies had high bias concerns on the QUADAS-2 study assessment tool.  These investigators found that salivary and oral swab hypermethylation markers had better specificity than sensitivity for oral cancer detection.  Summary sensitivity and specificity (95 % CI) of hypermethylation panels were 86.2 % (60 % to 96.2 %) and 90.6 % (85.9 % to 93.9 %) while for individual markers, summary sensitivity and specificity (95 % CI) were 70 % (56.9 % to 80.5 %) and 91.9 % (80.3 % to 96.9 %), respectively.  Respective positive and negative likelihood ratios for combined markers were 9.2 (5.89 to 14.36) and 0.15 (0.05 to 0.5), and 8.61 (3.39 to 21.87) and 0.33 (0.22 to 0.49) for single-application biomarkers.  The authors concluded that DNA hypermethylation biomarkers especially in combination have acceptable DTA that warrants further optimization with rigorous biomarker evaluation methods for conclusive determination of their efficacy.

Salivary Metabolites for Diagnosis of Cancer

In a systematic review, Assad and colleagues (2020) examined salivary metabolites and their diagnostic value in patients with cancer.  The systematic review was carried out in 2 phases and included studies that focused on the diagnostic value of salivary metabolites in humans with solid malignant neoplasms.  A total of 5 electronic databases were searched, and the risk of bias in individual studies was examined using the revised Quality Assessment of Diagnostic Accuracy Studies criteria (QUADAS-2).  All procedures were conducted according to the PRISMA guidelines.  Of the 1,151 studies retrieved, 25 were included; 13 studies used targeted and 12 untargeted metabolomics approaches.  Most studies included patients with breast and oral cancer.  Except for 1, all studies had case-control designs, and none fulfilled all quality assessments.  Overall, 140 salivary metabolites were described.  The most frequently reported metabolites were alanine, valine, and leucine.  Among the 11 studies that reported DTA values, proline, threonine, and histidine in combination and monoacylglycerol alone demonstrated the highest DTA for breast cancer.  Combined choline, betaine, pipecolinic acid, and L-carnitine showed better discriminatory performance for early oral cancer.  The authors concluded that this systematic review highlighted the current evidence on salivary metabolites that may be used as a future strategy to diagnose cancer.  Moreover, these researchers stated that further studies including larger sample sizes with confirmation of the results by untargeted analysis are needed.

Measurement of Salivary levels of IgG Antibodies to SARS-CoV-2 for Monitoring Seroprevalence and Vaccine Antibody Response

Heaney et al (2021) stated that saliva offers a non-invasive sampling method for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies; however, data comparing performance of salivary tests against commercially available serologic and neutralizing antibody (nAb) assays are lacking.  These researchers compared the performance of a laboratory-developed multiplex salivary SARS-CoV-2 IgG assay targeting antibodies to nucleocapsid (N), receptor binding domain (RBD) and spike (S) antigens to 3 commercially available SARS-CoV-2 serologic enzyme immunoassays (EIAs) (Ortho Vitros, Euroimmun, and BioRad) and nAb.  Paired salivary and plasma samples were collected from 101 eligible COVID-19 convalescent plasma (CCP) donors of greater than 14 days since PCR+ confirmed diagnosis.  Concordance was evaluated using positive and negative percent agreement (PPA and NPA), and Cohen's kappa coefficient.  The range between salivary and plasma EIAs for SARS-CoV-2-specific N was PPA: 54.4 % to 92.1 % and NPA: 69.2 % to 91.7 %, for RBD was PPA: 89.9 % to 100 % and NPA: 50.0 % to 84.6 %, and for S was PPA: 50.6 % to 96.6 % and NPA: 50.0 % to 100 %.  Compared to a plasma nAb assay, the multiplex salivary assay PPA ranged from 62.3 % (N) and 98.6 % (RBD) and NPA ranged from 18.8 % (RBD) to 96.9 % (S).  Combinations of N, RBD, and S and a summary algorithmic index of all 3 (N/RBD/S) in saliva produced ranges of PPA: 87.6 % to 98.9 % and NPA: 50 % to 91.7 % with the 3 EIAs and ranges of PPA: 88.4 % to 98.6 % and NPA: 21.9 % to 34.4 % with the nAb assay.  The authors concluded that a multiplex salivary SARS-CoV-2 IgG assay demonstrated variable, but comparable performance to 3 commercially available plasma EIAs and a nAb assay; and may be a viable alternative to assist in monitoring population-based seroprevalence and vaccine antibody response.

The authors stated that this study had several drawbacks.  First, the sample size was relatively small and included a limited number of samples from known-negative donors (as determined by standard plasma serological and nAb assays).  The cross-sectional design precluded evaluation of antibody dynamics over time, especially given the relatively short period during which sample collection was undertaken (i.e., relative to symptom resolution).  Nevertheless, this was consistent with most CCP collections and study populations, which provided insight into SARS-CoV-2 immunopathogenesis and screening options.  Second, the study population was primarily focused near Baltimore, MD and Washington DC; therefore, potentially limiting generalizability of these findings.  Third, the Ortho Vitros EIA was validated for serum rather than plasma; however, plasma samples have been previously shown to have reliable performance despite departure from the manufacturer's instructions.  Fourth, a different saliva collection device (OraSure, Bethlehem, PA) was used to establish the negative threshold for the multiplex SARS-CoV-2 IgG assay (Oracol+ S14, Malvern Medical Developments, Worcester, U.K.).  A larger study is recommended to test reproducibility of the findings following the manufacturer instructions for the assay kits and using the same saliva collection devices.

Alkharaan et al (2021) noted that declining humoral immunity in coronavirus disease 2019 (COVID-19) patients and possible re-infection have raised concern.  Mucosal immunity, especially salivary antibodies, may be short-lived although long-term studies are lacking.  Using a multiplex bead-based array platform, these investigators examined antibodies specific to SARS-CoV-2 proteins in 256 salivary samples from convalescent patients 1 to 9 months after symptomatic COVID-19 (n = 74, cohort 1), undiagnosed individuals with self-reported questionnaires (n = 147, cohort 2), and individuals sampled pre-pandemic (n = 35, cohort 3).  Salivary IgG antibody responses in cohort 1 (mainly mild COVID-19) were detectable up to 9 months post-recovery, with high correlations between S and N specificity.  At 9 months, IgG remained in blood and saliva in most patients.  Salivary IgA was rarely detected at this time-point.  In cohort 2, salivary IgG and IgA responses were significantly associated with recent history of COVID-19-like symptoms.  Salivary IgG tolerated temperature and detergent pre-treatments.  The authors concluded that unlike SARS-CoV-2 salivary IgA that appeared short-lived, specific salivary IgG appeared stable even after mild COVID-19, as for blood serology.  This non-invasive saliva-based SARS-CoV-2 antibody test with home self-collection may be a complementary alternative to conventional blood serology.

The authors concluded that one drawback of this study was the relatively small sample size and the predominantly male population.  Another drawback was that blood samples were not analyzed in the same way as saliva and, as several diagnostic assays were used, only binary data were provided.  Furthermore, because of the cross-sectional design, these investigators could not obtain baseline or longitudinal salivary samples.  Moreover, they could not examine individual possibilities of re-exposure or re-infection.  However, it was unlikely that humoral immunity was boosted because in Stockholm, where the study was carried out, the period of June to November 2020 (the 2nd wave) showed an increase in the daily incidence rate of COVID-19 from 30 to 400 cases/100,000 population.  These researchers stated that, despite waning immunity concerns, the present study showed that their multiplex bead-based immunoassays could detect antibodies against SARS-CoV-2 in late convalescence saliva up to 9 months after mild COVID-19.

Katz et al (2022) noted that SARS-CoV-2 circulating variants coupled with waning immunity pose a significant threat to the long-term care (LTC) population.  These investigators measured salivary IgG antibodies in residents and staff of an LTC facility to examine IgG response in saliva post-natural infection and vaccination and evaluate its feasibility to describe the seroprevalence over time.  They carried out salivary IgG sampling of all residents and staff who agreed to test in a 150-bed skilled nursing facility (SNF) during 3 seroprevalence surveys between October 2020 and February 2021.  The facility had SARS-CoV-2 outbreaks in May 2020 and November 2020, when 45 of 138 (32.6 %) and 37 of 125 (29.6 %) residents were infected, respectively.  These investigators offered 2 Federal vaccine clinics in January 2021.  They evaluated quantitative IgG in saliva to the N, S, and RBD antigens of SARS-CoV-2 over time post-infection and post-vaccination.  A total of 124 residents and 28 staff underwent saliva serologic testing on 1 or more survey visits.  Over 3 surveys, the SARS-CoV-2 seroprevalence at the facility was 49 %, 64 %, and 81 %, respectively.  IgG to S, RBD, and N antigens all increased post-infection.  Post-vaccination, the infection naive group did not have a detectable N IgG level, and N IgG levels for the previously infected did not increase post-vaccination (p < 0.001).  Fully vaccinated subjects with prior COVID-19 infection had significantly higher RBD and S IgG responses compared with those who were infection-naive before vaccination (p < 0.001 for both).  The authors concluded that positive SARS-COV-2 IgG in saliva was concordant with prior infection (anti N, S, RBD) and vaccination (anti S, RBD) and remained above positivity threshold for up to 9 months from infection.

The authors stated that this analysis had several drawbacks.  First, these researchers did not obtain serum samples to compare to saliva results of study participants, due to the invasive nature of phlebotomy; however, they had previously validated this by comparing saliva and serum responses from over 200 samples.  As part of this study, these investigators did not perform dedicated neutralization assays on saliva to ensure that saliva IgG antibody neutralized SARS-CoV-2; however, there is existing evidence of a clear correlation between IgG positivity and reduced risk for re-infection.  Second, these investigators were unable to examine IgG levels on some of the samples submitted to their laboratory, either due to insufficient saliva to saturate the swab (which could be a product of dry mouth or sampling technique), or because the IgG levels did not meet the established laboratory cut-offs, most likely if the sample was contaminated by recent intake of food/liquid.  This is a known limitation to salivary testing and could be overcome with the appropriate timing of sampling (e.g., early morning sampling, rubbing gums for more than 1 min).  Third, due to the transient presence of both staff and residents in the skilled nursing units in the facility, not all residents were tested on every survey.  The intent was to carry out a real-world analysis of overall immunity in a SNF at specific points in time; thus, these researchers included all individuals present in the facility who agreed to test at that survey, even if they were not at the facility during prior surveys.  This method may have introduced the potential for selection bias, as residents or staff who were tested on multiple occasions may have been more likely to remain in the facility because they had the milder disease (i.e., they were not hospitalized, or they died).  In addition, residents were more likely to be tested on multiple occasions if they resided on a long-term unit (versus a skilled unit).  Fourth, saliva serology samples were collected a minimum of 14 days from infection or vaccination; however, it is possible that these researchers captured some individuals during the period where their antibody response was still increasing, which may have decreased the slope of decay in the linear regression lines; however, a sub-analysis excluding individuals infected within 20 days of infection or vaccination did not yield any significant change in our results.  Fifth, this analysis was completed in February 2021, before the widespread circulation of the delta variant in the U.S., which may limit the applicability of these findings.  

Salivary Electrolytes as Biomarkers for Mouth Neoplasms and Oral Squamous Cell Carcinoma

In an observational study, Saavedra et al (2022) determined salivary electrolyte concentration of oral potentially malignant disorders (OPMD) and oral squamous cell carcinoma (OSCC) patients.  These investigators also carried out a related systematic review.  Unstimulated saliva from 18 patients with OSCC, 18 patients with OPMD, and 18 individuals without oral lesions was collected.  A biochemical analysis was carried out to evaluate salivary concentrations of potassium (K), phosphorus (P), sodium (Na), calcium (Ca), magnesium (Mg), zinc (Zn), copper (Cu), and iron (Fe).  Kruskal-Wallis test was performed, and p < 0.05 was interpreted as statistically significant.  The literature search for the systematic review retrieved 9 studies that associated salivary electrolyte levels with presence and progression of OSCC.  A highly significant increase was found in the salivary Mg levels in the OPMD group (5.41 µg/ml) in comparison with the OSCC (3.71 µg/ml) and control group (3.51 µg/ml) (p = 0.041).  No differences were observed in other salivary levels elements.  The results of the systematic review revealed that 1 study indicated a decrease, and 3 studies reported an increase in salivary Na levels in patients with OPMD and OSCC; 2 studies indicated a decrease in salivary K levels in OSCC, and the other 2 reported high Mg levels in OPMD and OSCC.  The authors concluded that high salivary Mg levels can be a potential biomarker indicating the presence of OPMD, however, the evidence is still contradictory; and more studies are needed.

Salivary Cytokines in the Diagnosis and Prognosis of Oral Squamous Cell Carcinoma

Benito-Ramal et al (2023) stated that OSCC is gradually increasing its incidence; unfortunately, this entity is diagnosed at an advanced stage in most patients, a fact that implies greater difficulty in its treatment and a worse prognosis.  In a systematic review, these researchers examined if cytokines IL-6, IL-8 and TNF-α are potential salivary biomarkers that allow early diagnosis of cancer.  They carried out an electronic search in 3 databases (PubMed, Scopus and Web of Science).  These investigators used the following keywords: "salivary cytokines", "saliva cytokines", "salivary interleukins", "biomarkers", "oral squamous cell carcinoma" and "diagnosis", combined with the Boolean operators "AND" and "OR".  A total of 128 studies were identified and 23 were included in the final review and 15 in the meta-analysis.  It has been observed that the majority of OSCC patients expressed higher salivary concentrations of IL-6, IL-8 and TNF-α compared to the control (CL) and pre-malignant lesion (OPML) groups.  It has also been observed that the different pre-malignant lesions did not have statistically significant differences in the salivary concentration of the cytokines, and on the other hand, differences have been observed between the different TNM stages.  The meta-analysis has shown that the difference in concentration of IL-6, IL-8 and TNF-α was statistically significant between the CL group and the OSCC, and also between the CL group and OPML.  The authors concluded that this review highlighted the importance of early detection in order to improve the prognosis of OSCC, and the 3 salivary cytokines (IL-6, IL-8 and TNF-α) have been shown to be potentially useful in the early diagnosis and prognosis of OSCC.  Moreover, these researchers stated that future studies are needed to establish greater reliability of these biomarkers and thus be able to develop a valid diagnostic test.

Th authors stated that despite the extensive literature on this subject, future investigations with an adequate methodology are needed to conduct meta-analysis studies that determine reliable intervals for the salivary concentration of each biomarker.  Regarding the drawbacks of this review, these investigators found that several studies lacked the necessary data to perform their quantitative analysis.  Furthermore, among the studies that had been quantitatively analyzed, great variability was found in the mean value of the concentrations of pro-inflammatory cytokines.

Salivary Lactate Dehydrogenase as a Biomarker in Oral Potentially Malignant Disorders and Head and Neck Cancer

In a systematic review and meta-analysis, Iglesias-Velazquez et al (2022) examined if salivary lactate dehydrogenase (SaLDH) levels are increased in patients with oral cancer (OC) or OPMD when compared to a healthy control group (CG).  These investigators carried out a comprehensive search of specialized databases (PubMed/Medline, the Cochrane Library, Web of Science, Scopus, and OpenGrey), including observational analytical studies examining the SaLDH levels (in UI/L or μ/L) in OC or OPMD patients and compared them with a CG.  A total of 13 case-control studies were included.  A total of 755 patients were evaluated, including 303 OC cases, 149 OPMD cases, and 303 controls.  The meta-analysis showed that SaLDH levels were higher within the OC group than the CG (SMD 9.49; 95 % CI: 6.97 to 12; p = 0.00001).  Patients with oral leucoplakia (SMD 11.67; 95 % CI: 1.01 to 22.33; p = 0.03) and oral submucous fibrosis (SMD 25.83; 95 % CI: 1.74 to 53.40; p = 0.07) also presented higher levels than the CG.  Furthermore, OC patients had higher SaLDH levels than oral leucoplakia patients (SMD 5.62; 95 % CI: 2.14 to 9.11; p = 0.002).  Heterogeneity was high across all the evaluated studies.  The authors concluded that determination of SaLDH may be a useful method for screening and tracking OC and OPMD; however, new protocolized studies are needed to establish precise cut-off values to determine the levels that indicate the presence of OC or, if possible, of OPMD.

Lokesh Kumar et al (2023) noted that the prognosis of head and neck cancer (HNC) depends on its early detection, diagnosis, and treatment, which has advocated a search for a simple, reliable, non-invasive, cost-effective tool to aid in the same.  SaLDH has gained interest in recent years, meeting the above requisite.  In a systematic review and meta-analysis, these investigators examined the levels of SaLDH in patients with OPMD, HNC, and in the healthy CG; to find the correlation, grade-wise and gender-wise differences between them; and to examine if it can be used as a potent biomarker in OPMD and HNC.  They carried out a comprehensive search of the specialized 14 databases and 4 institutional repositories to identify studies evaluating SaLDH in OPMD and HNC patients either comparing or not comparing to the healthy control group in the systematic review process.  The meta-analysis was carried out with the eligible study data with the STATA version 16, 2019 software with 95 % CI and p ≤ 0.05 in the random-effects model.  A total of 28 studies of case-control, interventional, or uncontrolled non-randomized design evaluating SaLDH were included.  A total of 2,074 subjects were included, involving HNC, OPMD, and CG.  The SaLDH levels were significantly higher in HNC than in CG and oral leukoplakia (OL) (p = 0.00); in OL and oral submucous fibrosis (OSMF) than CG (p = 0.00); and higher in HNC than OSMF, however not significant (p = 0.49).  Furthermore, the SaLDH levels had no significant difference between males and females in CG, HNC, OL, and OSMF groups(p > 0.05).  It is evident that the epithelial transformations in the various OPMD and HNC, and the proceeding necrosis in the case of HNC, raised the LDH levels.  It was also worth noting that when degenerative alterations continued, the SaLDH levels rose correspondingly, which were higher in HNC than in OPMD.  Thus, it was essential to determine the cut-off values for SaLDH for establishing that the patient may have HNC or OPMD.  It would be easy to follow-up frequently and perform examinations such as biopsy for the cases with high SaLDH levels, thereby aiding in the early detection and improving the prognosis of HNC.  Moreover, the increased SaLDH levels were indicative of a lower degree of differentiation and an advanced disease leading to a poor prognosis.  Salivary sample collection is less invasive, simple, and more acceptable to the patient; however, saliva collection is a time-consuming procedure as it is mostly collected by the passive spit method.  Furthermore, it is more feasible to repeat the SaLDH analysis during the follow-up, but the method has recently gained interest for the past 10 years.  The authors concluded that SaLDH can be a potential biomarker for the screening, early detection, and follow-up of OPMD or HNC being simple, non-invasive, cost-effective, and readily acceptable modality.  However, these researchers stated that more studies with new standardized protocols are needed to determine the precise cut-off levels for HNC and OPMD.

Araujo et al (2023) examined the evidence on salivary biomarkers for HNC diagnosis.  The acronym PICOS was employed to develop the eligibility criteria and the focused review question: are liquid biopsies (salivary biomarkers) reliable for cancer detection in HNC patients?  Electronic database search encompassed PubMed, Embase, Scopus, Cochrane Library, Web of Science, and LILACS.  Risk of Bias (RoB) was evaluated via AMSTAR 2.  A total of 20 systematic reviews (SRs) were included.  Only 7 SRs were able to reach more solid conclusions regarding the retrieved findings by calculating the pooled sensitivity, specificity, and the overall area under the curve (AUC).  The authors concluded that despite the limitations, significant RoB, and lack of test metrics in primary studies, all SRs recognized and encouraged the potential role of saliva in the early diagnosis of oral cancer.

Viome CancerDetect-Oral & Throat Test

Viome’s CancerDetect Test is designed as a home test (takes only a few mins to collect saliva sample) for early detection oral cancer and throat cancer.  This test detects molecular features associated with OSCC, and/or throat cancer (OSCC or oropharyngeal squamous cell carcinoma [OPSCC]) in saliva samples.  The CancerDetect-Oral & Throat Test is recommended for use in adults with an elevated risk for oral and throat cancer (e.g., those aged 50 years or older or individuals who have a history of smoking, chewing tobacco, vaping, or using other forms of tobacco, heavy alcohol drinkers, or individuals with a high risk of HPV).  However, the Viome’s CancerDetect Test is not an FDA-approved or cleared test.

Banavar et al (2021) stated that despite advances in cancer treatment, the 5-year mortality rate for OC is 40 %, mainly due to the lack of early diagnostics.  To advance early diagnostics for high-risk and average-risk populations, these researchers developed and examined machine-learning (ML) classifiers using meta-transcriptomic data from salivary samples (n = 433) collected from OPMD, OC patients (n = 71) and normal controls (n = 171).  Their diagnostic classifiers yielded a receiver operating characteristics (ROC) area under the curve (AUC) up to 0.9, sensitivity up to 83 % (92.3 % for stage 1 cancer) and specificity up to 97.9 %.  The authors concluded that their meta-transcriptomic signature incorporates both taxonomic and functional microbiome features, and revealed a number of taxa and functional pathways associated with OC.  These researchers demonstrated the potential clinical use of an artificial intelligence (AI)/ML model for diagnosing OC early, opening a new era of non-invasive diagnostics, enabling early intervention and improved patient outcomes.  Moreover, these researchers stated that once an early diagnostic test is available at scale, they can routinely improve the accuracy of their test as they collect more “real-world evidence” to further train their machine-learning models.  This research was sponsored by Viome.

Banavar et al (2022) noted that OSCC and OPSCC are the 2 major subtypes of HNC that can go undetected resulting in late detection and poor outcomes.  These researchers described the development and validation of a convenient and easy-to-use test, called CancerDetect for Oral & Throat cancer (CDOT), to detect markers of OSCC and/or OPSCC within a high-risk population using salivary meta-transcriptomics.  These researchers collected salivary samples from 1,175 unique individuals who were 50 years or older, or adults who had a history of tobacco use.  All salivary samples were processed via a meta-transcriptomic method to isolate microbial organisms and functions, as well as human transcripts.  Of the 1,175 samples, 945 were used to train a classifier using ML methods, resulting in a salivary RNA meta-transcriptomic signature.  The classifier was then independently validated on the 230 remaining samples unseen by the classifier, consisting of 20 OSCC (all stages), 76 OPSCC (all stages), and 134 negatives (including 14 pre-malignant).  On the validation cohort, the specificity of the CDOT test was 94 %, sensitivity was 90 % for participants with a histopathological diagnosis of OSCC, and 84.2 % for participants with a diagnosis of OPSCC.  Similar classification results were observed among patients in early stage (stages I and II) versus late stage (stages III and IV) of OSCC and OPSCC.  The authors concluded that while future studies with a larger number of patients with pre malignancies are needed, the current method is a practically useful, non-invasive method that can be easily incorporated in dentist offices, primary care centers and specialized cancer clinics for early detection of oral and throat cancers.

The authors stated that this study had several drawbacks.  While the model performed well in patients with OSCC and OPSCC and cancer-free patients, the number of participants with pre-malignant diseases is currently too low to discriminate between positives and negatives. The model was validated in former and current smokers as well as young and older participants; however, other populations at risk such as heavy drinkers and patients with HPV-OPSCC were not evaluated and deserve further investigation.  Finally, while the study participants were recruited from across the U.S. and Australia, the positive cases were recruited from a single center in Australia.  These investigators stated that a larger, multi-center validation including centers in the U.S. is forthcoming; and is expected to address most of these drawbacks.


The above policy is based on the following references:

  1. AACE Thyroid Task Force. American Association of Clinical Endocrinologists medical guidelines for clinical practice for the evaluation and treatment of hyperthyroidism and hypothyroidism. Endocr Pract. 2002;8(6):457-469..
  2. Adeoye J, Alade AA, Zhu W-Y, et al. Efficacy of hypermethylated DNA biomarkers in saliva and oral swabs for oral cancer diagnosis: Systematic review and meta-analysis. Oral Dis. 2022;28(3):541-558.
  3. AlAli AM, Walsh T, Maranzano M. CYFRA 21-1 and MMP-9 as salivary biomarkers for the detection of oral squamous cell carcinoma: A systematic review of diagnostic test accuracy. Int J Oral Maxillofac Surg. 2020;49(8):973-983.
  4. Alexandraki KI, Grossman AB. Novel insights in the diagnosis of Cushing's syndrome. Neuroendocrinology. 2010;92 Suppl 1:35-43.
  5. Alkharaan H, Bayati S, Hellstrom C, et al. Persisting salivary IgG against SARS-CoV-2 at 9 months after mild COVID-19: A complementary approach to population surveys. J Infect Dis. 2021;224(3):407-414.
  6. American Association of Clinical Endocrinologists (AACE). Medical guidelines for clinical practice for management of menopause. Endocrine Pract. 1999;5:355-366. 
  7. American Association of Clinical Endocrinologists (AACE). American Association of Clinical Endocrinologists (AACE) Reproductive Medicine Committee position statement on bioidentical hormones. Jacksonville, FL: AACE; updated July 15, 2007. Available at: Accessed November 7, 2013.
  8. American College of Obstetricians and Gynecologists (ACOG) Committee on Gynecologic Practice. ACOG Committee Opinion #322: Compounded bioidentical hormones. Obstet Gynecol. 2005;106(5 Pt 1):1139-1140.
  9. Araujo ALD, Santos-Silva AR, Kowalski LP. Diagnostic accuracy of liquid biopsy for oral potentially malignant disorders and head and neck cancer: An overview of systematic reviews. Curr Oncol Rep. 2023;25(4):279-292.
  10. Assad DX, Mascarenhas ECP, de Lima CL, et al. Salivary metabolites to detect patients with cancer: A systematic review. Int J Clin Oncol. 2020;25(6):1016-1036.
  11. Bajaj JS, Fagan A, White MB, et al. Specific gut and salivary microbiota patterns are linked with different cognitive testing strategies in minimal hepatic encephalopathy. Am J Gastroenterol. 2019;114(7):1080-1090.
  12. Banavar G, Ogundijo O, Toma R, et al. The salivary metatranscriptome as an accurate diagnostic indicator of oral cancer. NPJ Genom Med. 2021;6(1):105.
  13. Banavar G, Ogundijo O, Julian C, et al. Detecting salivary host and microbiome RNA signature for aiding diagnosis of oral and throat cancer. Oral Oncol. 2023;145:106480.
  14. Benito-Ramal E, Egido-Moreno S, González-Navarro B, et al. Role of selected salivary inflammatory cytokines in the diagnosis and prognosis of oral squamous cell carcinoma. A systematic review and meta-analysis. Med Oral Patol Oral Cir Bucal. 2023 Apr 26 [Online ahead of print].
  15. Bonamico M, Nenna R, Montuori M, et al. First salivary screening of celiac disease by detection of anti-transglutaminase autoantibody radioimmunoassay in 5000 Italian primary schoolchildren. J Pediatr Gastroenterol Nutr. 2011;52(1):17-20.
  16. Borg D, Tverdovsky A, Stripp R. A fast and comprehensive analysis of 32 synthetic cannabinoids using agilent triple quadrupole LC-MS-MS. J Anal Toxicol. 2017;41(1):6-16.
  17. Bozzani A, Grattagliano I, Pellegatta G, et al. Usefulness of Pep-Test for laryngo-pharyngeal reflux: A pilot study in primary care. Korean J Fam Med. 2020;41(4):250-255.
  18. Carroll T, Raff H, Findling JW. Late-night salivary cortisol for the diagnosis of Cushing syndrome: A meta-analysis. Endocr Pract. 2009;15(4):335-342.
  19. Carroll T, Raff H, Findling JW. Late-night salivary cortisol measurement in the diagnosis of Cushing's syndrome. Nat Clin Pract Endocrinol Metab. 2008;4(6):344-350.
  20. Cheng YS, Jordan L, Rees T, et al. Levels of potential oral cancer salivary mRNA biomarkers in oral cancer patients in remission and oral lichen planus patients. Clin Oral Investig. 2014;18(3):985-993.
  21. Committee on Gynecologic Practice and the American Society for Reproductive Medicine Practice Committee. Committee opinion no. 532: Compounded bioidentical menopausal hormone therapy. Obstet Gynecol. 2012;120(2 Pt 1):411-415.
  22. Contreras LN, Arregger AL, Persi GG, et al. A new less-invasive and more informative low-dose ACTH test: Salivary steroids in response to intramuscular corticotrophin. Clin Endocrinol (Oxf). 2004;61(6):675-682.
  23. de Morais EF, Pinheiro JC, Leite RB, et al. Matrix metalloproteinase-8 levels in periodontal disease patients: A systematic review. J Periodontal Res. 2018;53(2):156-163.
  24. DeSimone JA, Karia PS, Schmults, CD. Recognition and management of high-risk (aggressive) cutaneous squamous cell carcinoma. UpToDate [online serial]. Waltham, MA: UpToDate; reviewed April 2014.
  25. Doi M, Sekizawa N, Tani Y, et al. Late-night salivary cortisol as a screening test for the diagnosis of Cushing's syndrome in Japan. Endocr J. 2008;55(1):121-126.
  26. Dy F, Amirault J, Mitchell PD, Rosen R. Salivary pepsin lacks sensitivity as a diagnostic tool to evaluate extraesophageal reflux disease. J Pediatr. 2016;177:53-58.
  27. Elamin MB, Murad MH, Mullan R, et al. Accuracy of diagnostic tests for Cushing's syndrome: A systematic review and metaanalyses. J Clin Endocrinol Metab. 2008;93(5):1553-1562.
  28. Flyckt RL, Liu J, Frasure H, Wekselman K, et al. Comparison of salivary versus serum testosterone levels in postmenopausal women receiving transdermal testosterone supplementation versus placebo. Menopause. 2009;16(4):680-688.
  29. Grimley Evans J, Malouf R, Huppert F, van Niekerk JK. Dehydroepiandrosterone (DHEA) supplementation for cognitive function in healthy elderly people. Cochrane Database Syst Rev. 2006;(4):CD006221.
  30. Groschl M. Current status of salivary hormone analysis. Clin Chem. 2008;54(11):1759-1769.
  31. Gualtero DF, Suarez Castillo A. Biomarkers in saliva for the detection of oral squamous cell carcinoma and their potential use for early diagnosis: A systematic review. Acta Odontol Scand. 2016;74(3):170-177.
  32. Guardino CM, Schetter CD, Saxbe DE, et al. Diurnal salivary cortisol patterns prior to pregnancy predict infant birth weight. Health Psychol. 2016;35(6):625-633.
  33. Guerra EN, Acevedo AC, Leite AF, et al. Diagnostic capability of salivary biomarkers in the assessment of head and neck cancer: A systematic review and meta-analysis. Oral Oncol. 2015;51(9):805-818.
  34. Guo Z, Jiang J, Wu H, et al. Salivary peptest for laryngopharyngeal reflux and gastroesophageal reflux disease: A systemic review and meta-analysis. Medicine (Baltimore). 2021;100(32):e26756.
  35. Heaney CD, Pisanic N, Randad PR, et al. Comparative performance of multiplex salivary and commercially available serologic assays to detect SARS-CoV-2 IgG and neutralization titers. J Clin Virol. 2021;145:104997.
  36. Herbert V, Kava R. The miracle of melatonin? Priorities (American Council on Science and Health). 1995;7(4). Available at: Accessed February 15, 2002.
  37. Hicks SD, Johnson J, Carney MC, et al. Overlapping microRNA expression in saliva and cerebrospinal fluid accurately identifies pediatric traumatic brain injury. J Neurotrauma. 2018;35(1):64-72.
  38. Hodgson SF, Watts NB, Bilezikian JP, et al. .American Association of Clinical Endocrinologists medical guidelines for clinical practice for the prevention and treatment of postmenopausal osteoporosis: 2001 edition, with selected updates for 2003. Endocr Pract. 2003;9(6):544-564.
  39. Huppert FA, Van Niekerk JK. Dehydroepiandrosterone (DHEA) supplementation for cognitive function. Cochrane Database Syst Rev. 2006:(2):CD000304.
  40. Iglesias-Velazquez O, Lopez-Pintor RM, Gonzalez-Serrano J, et al. Salivary LDH in oral cancer and potentially malignant disorders: A systematic review and meta-analysis. Oral Dis. 2022;28(1):44-56.
  41. Institute for Clinical Systems Improvement (ICSI). Health care guideline: Menopause and hormone therapy (HT): Collaborative decision-making and management. Bloomington, MN: ICSI; October 2008.
  42. Institute for Clinical Systems Improvement (ICSI). Menopause and hormone therapy (HT): Collaborative decision-making and management. Bloomington, MN: ICSI; October 2006.
  43. Johnson JJ, Loeffert AC, Stokes J, et al. Association of salivary microRNA changes with prolonged concussion symptoms. JAMA Pediatr. 2018;172(1):65-73.
  44. Kamali M, Saunders EF, Prossin AR, et al. Associations between suicide attempts and elevated bedtime salivary cortisol levels in bipolar disorder. J Affect Disord. 2012;136(3):350-358.
  45. Katz MJ, Heaney CD, Pisanic N, et al. Evaluating immunity to SARS-CoV-2 in nursing home residents using saliva IgG. J Am Geriatr Soc. 2022;70(3):659-668.
  46. Klebanoff MA, Meis PJ, Dombrowski MP, et al; National Institute of Child Health and Human Development Maternal-Fetal Medicine Units Network. Salivary progesterone and estriol among pregnant women treated with 17-alpha-hydroxyprogesterone caproate or placebo. Am J Obstet Gynecol. 2008;199(5):506.e1-e7.
  47. Knorr U, Vinberg M, Kessing LV, Wetterslev J. Salivary cortisol in depressed patients versus control persons: A systematic review and meta-analysis. Psychoneuroendocrinology. 2010;35(9):1275-1286.
  48. Koopaie M, Kolahdooz S, Fatahzadeh M, Manifar S. Salivary biomarkers in breast cancer diagnosis: A systematic review and diagnostic meta-analysis. Cancer Med. 2022;11(13):2644-2661.
  49. LaRocca D, Barns S, Hicks SD, et al. Comparison of serum and saliva miRNAs for identification and characterization of mTBI in adult mixed martial arts fighters. PLoS One. 2019;14(1):e0207785.
  50. Levey AS, Inker LA. Definition and staging of chronic kidney disease in adults. UpToDate [online serial]. Waltham, MA: UpToDate; reviewed April 2020.
  51. Lin J, Zhao H, Shen J, Jiao F. Salivary cortisol levels predict therapeutic response to a sleep-promoting method in children with postural tachycardia syndrome. J Pediatr. 2017;191:91-95.
  52. Lokesh Kumar S, Naik Z, Lagali-Jirge V, et al. Salivary lactate dehydrogenase as a potential biomarker in oral potentially malignant disorders and head & neck cancer -- A systematic review and meta-analysis. Gulf J Oncolog. 2023;1(41):78-99.
  53. Maas C, Ringwald C, Weber K, et al. Relationship of salivary and plasma cortisol levels in preterm infants: Results of a prospective observational study and systematic review of the literature. Neonatology. 2014;105(4):312-318.
  54. Macey R, Walsh T, Brocklehurst P, et al. Diagnostic tests for oral cancer and potentially malignant disorders in patients presenting with clinically evident lesions. Cochrane Database Syst Rev. 2015;5:CD010276.
  55. Monteleone P, Scognamiglio P, Canestrelli B, et al. Asymmetry of salivary cortisol and α-amylase responses to psychosocial stress in anorexia nervosa but not in bulimia nervosa. Psychol Med. 2011;41(9):1963-1969.
  56. Mozaffari HR, Ramezani M, Mahmoudiahmadabadi M, et al. Salivary and serum levels of tumor necrosis factor-alpha in oral lichen planus: A systematic review and meta-analysis study. Oral Surg Oral Med Oral Pathol Oral Radiol. 2017;124(3):e183-e189.
  57. Mozaffari HR, Sharifi R, Hayati M, et al. Evaluation of serum and salivary interferon-γ levels in patients with oral lichen planus: A systematic review and meta-analysis of case-control studies. Oral Surg Oral Med Oral Pathol Oral Radiol. 2019a;127(3):210-217.
  58. Mozaffari HR, Sharifi R, Mirbahari S, et al. A systematic review and meta-analysis study of salivary and serum interleukin-8 levels in oral lichen planus. Postepy Dermatol Alergol. 2018b;35(6):599-604.
  59. Mozaffari HR, Sharifi R, Sadeghi M. Interleukin-6 levels in the serum and saliva of patients with oral lichen planus compared with healthy controls: A meta-analysis study. Cent Eur J Immunol. 2018a;43(1):103-108.
  60. Mozaffari HR, Zavattaro E, Abdolahnejad A, et al. Serum and salivary IgA, IgG, and IgM levels in oral lichen planus: A systematic review and meta-analysis of case-control studies. Medicina (Kaunas). 2018c;54(6).
  61. Mozaffari HR, Zavattaro E, Saeedi M, et al. Serum and salivary interleukin-4 levels in patients with oral lichen planus: A systematic review and meta-analysis. Oral Surg Oral Med Oral Pathol Oral Radiol. 2019b;128(2):123-131.
  62. National Comprehensive Cancer Network (NCCN). Breast cancer. NCCN Clinical Practice Guidelines in Oncology, Version 2.2017. Fort Washington, PA: NCCN; 2017.
  63. National Comprehensive Cancer Network (NCCN). Head and neck cancers. NCCN Clinical Practice Guidelines in Oncology, Version 2.2017. Fort Washington, PA: NCCN; 2017.
  64. National Institutes of Health (NIH). NIH State-of-the-Science Conference Statement on Management of Menopause-Related Symptoms. NIH Consensus and State-of-the-Science Statements. Bethesda, MD: NIH: March 21-23; 22(1). 
  65. Nieman LK, Biller BM, Findling JW, et al. The diagnosis of Cushing's syndrome: An Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab. 2008;93(5):1526-1540.
  66. Nieman LK, Biller BM, Findling JW, et al. Treatment of Cushing's syndrome: An Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 2015;100(8):2807-2831.
  67. No authors listed. Chronic hypoadrenalism. GPNotebook. General Practitioner Notebook. Warwickshire, UK: Oxbridge Solutions, Ltd.; 2005. Available at: Accessed September 16, 2005.
  68. No authors listed. Melatonin: Interesting, but not miraculous. Prescrire Int. 1998;7(38):180-187.
  69. Nomura Y, Okada A, Tamaki Y, Miura H. Salivary levels of hemoglobin for screening periodontal disease: A systematic review. Int J Dent. 2018;2018:2541204.
  70. North American Menopause Society (NAMS). The 2012 hormone therapy position statement of the North American Menopause Society. Cleveland, OH: NAMS; 2012. Available at: Accessed November 7, 2013.
  71. Novy BB. Saliva and biofilm-based diagnostics: A critical review of the literature concerning sialochemistry. J Evid Based Dent Pract. 2014;14 Suppl:27-32.
  72. Odeke S, Nagelberg SB. Addison disease. eMedicine Endocrinology Topic 42. Omaha, NE:; updated November 25, 2003. Available at: Accessed September 16, 2005.
  73. Porto-Mascarenhas EC, Assad DX, Chardin H, et al. Salivary biomarkers in the diagnosis of breast cancer: A review. Crit Rev Oncol Hematol. 2017;110:62-73.
  74. Punyani SR, Sathawane RS. Salivary level of interleukin-8 in oral precancer and oral squamous cell carcinoma. Clin Oral Investig. 2013;17(2):517-524.
  75. Raff H. Utility of salivary cortisol measurements in Cushing's syndrome and adrenal insufficiency. J Clin Endocrinol Metab. 2009;94(10):3647-3655.
  76. Rajkumar K, Ramya R, Nandhini G, et al. Salivary and serum level of CYFRA 21-1 in oral precancer and oral squamous cell carcinoma. Oral Dis. 2015;21(1):90-96.
  77. Rodrigues RPCB, de Andrade Vieira W, Siqueira WL, et al. Saliva as an alternative to blood in the determination of uremic state in adult patients with chronic kidney disease: A systematic review and meta-analysis. Clin Oral Investig. 2020;24(7):2203-2217.
  78. Rosenberg M. Overview of the management of chronic kidney disease in adults. UpToDate [online serial]. Waltham, MA: UpToDate; reviewed April 2020.
  79. Rubin GJ, Hotopf M, Papadopoulos A, Cleare A. Salivary cortisol as a predictor of postoperative fatigue. Psychosom Med. 2005;67(3):441-447.
  80. Rubio-Tapia A, Hill ID, Kelly CP, Calderwood AH, Murray JA. ACG clinical guidelines: Diagnosis and management of celiac disease. Am J Gastroenterol. 2013;108(5):656-676.
  81. Saavedra JA, La Rosa Novo D, Mesko MF, et al. Comparison of salivary electrolytes profile in oral potentially malignant disorders and oral squamous cell carcinoma. Asian Pac J Cancer Prev. 2022;23(3):1031-1039.
  82. Senneby A, Mejare I, Sahlin NE, et al. Diagnostic accuracy of different caries risk assessment methods. A systematic review. J Dent. 2015;43(12):1385-1393.
  83. Sereg M, Toke J, Patócs A, et al. Diagnostic performance of salivary cortisol and serum osteocalcin measurements in patients with overt and subclinical Cushing's syndrome. Steroids. 2011;76(1-2):38-42.
  84. Shaparin N, Mehta N, Kunkel F, et al. A novel chronic opioid monitoring tool to assess prescription drug steady state levels in oral fluid. Pain Med. 2017;18(11):2162-2169.
  85. Shazam H, Shaikh F, Hussain Z, et al. Evaluation of osteocalcin levels in saliva of periodontitis patients and their correlation with the disease severity: A cross-sectional study. Eur J Dent. 2020;14(3):352-359.
  86. The North American Menopause Society. The role of testosterone therapy in postmenopausal women: Position statement of The North American Menopause Society. Menopause. 2005;12(5):497-511.
  87. Whisman MA, Richardson ED. Depressive symptoms and salivary telomere length in a probability sample of middle-aged and older adults. Psychosom Med. 2017;79(2):234-242.