Saliva: Newer avenues in the era of molecular biology, diagnostic and prognostic application

Abhishek Singh Nayyar; Mubeen Khan; Bharat Deosarkar; Soniya Bharat Deosarkar; KV Chalapathi; G Kartheek; B Kartheeki

doi:10.4103/jmedsci.jmedsci_88_16

REVIEW ARTICLE

Year : 2018 | Volume : 38 | Issue : 1 | Page : 7-15

Saliva: Newer avenues in the era of molecular biology, diagnostic and prognostic application

Abhishek Singh Nayyar¹, Mubeen Khan², Bharat Deosarkar³, Soniya Bharat Deosarkar⁴, KV Chalapathi⁵, G Kartheek⁶, B Kartheeki¹
¹ Department of Oral and Maxillo-facial Medicine and Radiology, Saraswati-Dhanwantari Dental College and Hospital and Post-Graduate Research Institute, Parbhani, Maharashtra, India
² Department of Oral and Maxillo-facial Medicine and Radiology, Government Dental College and Research Institute, Bangalore, Karnataka, India
³ Department of Conservative Dentistry and Endodontics, Saraswati-Dhanwantari Dental College and Hospital and Post-Graduate Research Institute, Parbhani, Maharashtra, India
⁴ Department of Prosthodontics and Crown and Bridge, Saraswati-Dhanwantari Dental College and Hospital and Post-Graduate Research Institute, Parbhani, Maharashtra, India
⁵ Department of Oral and Maxillo-facial Pathology and Microbiology, Care Dental College and Hospital, Guntur, India
⁶ Department of Oral and Maxillo-facial Pathology and Microbiology, KIMS Dental College and Hospital, Amalapuram, Andhra Pradesh, India

Date of Submission	27-Aug-2016
Date of Decision	27-Aug-2016
Date of Acceptance	16-Nov-2017
Date of Web Publication	14-Feb-2018

Correspondence Address:
Dr. Abhishek Singh Nayyar
Reader Cum Associate Professor, Department of Oral Medicine and Radiology, Parbhani, Maharashtra
India

Source of Support: None, Conflict of Interest: None

Check

DOI: 10.4103/jmedsci.jmedsci_88_16

Abstract

The salivary fluid has an old history of study, but its physiological importance has only been recognized recently. In the past 50 years, the pace of salivary research has accelerated with the advent of new techniques that illuminated the biochemical and physicochemical properties of saliva. The interest in saliva increased, further, with the finding that saliva is filled with hundreds of components that might serve to detect systemic diseases and/or act as an evidence of exposure to various harmful substances as well as provide biomarkers of health and disease. The role of saliva in the diagnosis as well as monitoring of glycemic control has, also, been attracting attention of clinical researchers in recent times although results have been conflicting. To conclude, saliva is a whole, diverse fluid that serves various purposes discussed in detail in the literature. The recent introduction of molecular biology opens up, once again, new vistas and a new search of the role of salivary fluid as a potential diagnostic tool which has an added advantage of being noninvasive. The present review presents such insight into the possible use of salivary fluid as a potential diagnostic and prognostic tool for the search of numerous diseases as well as for monitoring the treatment outcomes and assesses prognosis in such varied states of derangements of metabolic functions.

Keywords: Saliva, diagnostics, systemic diseases, forensic applications

How to cite this article:
Nayyar AS, Khan M, Deosarkar B, Deosarkar SB, Chalapathi K V, Kartheek G, Kartheeki B. Saliva: Newer avenues in the era of molecular biology, diagnostic and prognostic application. J Med Sci 2018;38:7-15

How to cite this URL:
Nayyar AS, Khan M, Deosarkar B, Deosarkar SB, Chalapathi K V, Kartheek G, Kartheeki B. Saliva: Newer avenues in the era of molecular biology, diagnostic and prognostic application. J Med Sci [serial online] 2018 [cited 2023 Mar 26];38:7-15. Available from: https://www.jmedscindmc.com/text.asp?2018/38/1/7/225574

Introduction

The salivary fluid is an exocrine secretion consisting of approximately 99% water with a variety of electrolytes including sodium, potassium, calcium, magnesium, chlorides, bicarbonates, phosphates, and proteins represented by enzymes, immunoglobulins and other antimicrobial factors, mucosal glycoproteins, and traces of albumin with glucose and nitrogenous products such as urea and ammonia secreted mainly by three pairs of major salivary glands, namely parotid, submandibular, and sublingual glands. A plethora of minor salivary glands distributed over the buccal mucosa, lips, and along the mucosa of the upper aerodigestive tract present from the nasal cavity to the larynx and pharynx, also, participate in this secretion. Together, they are responsible for the remaining 5% of saliva secreted in humans.^{[1],[2],[3],[4]} It is considered that humans secrete approximately 0.5 L of saliva per day in response to stimulation of the sympathetic and parasympathetic sections of the autonomic nervous system.^[3],[4],[5] Whole saliva is a multiglandular secretion complex consisting of gingival fluid, desquamated epithelial cells, microorganisms and products of their metabolism, food debris, leukocytes, and mucus from the nasal cavity and the larynx and pharynx. Saliva has varied functions from tissue repair to protection, digestion, taste, and antimicrobial action, in the maintenance of tooth integrity and antioxidant defense system.^[6],[7] The average daily volume of saliva production is 500–1000 ml with the submandibular gland producing around 70% of the total volume, parotid contributing for 25%, and the sublingual gland contributing to about 5% of the total salivary secretion. The contribution of minor salivary glands toward the total volume of saliva, although, has more or, less local effects.^[3],[4] The functions of saliva with the split-up of the various individual constituents are summarized in [Table 1] while the methods of collection of resting/unstimulated and stimulated saliva are summarized in [Table 2].

Table 1: Functions of saliva and salivary components^[7]

Click here to view

Table 2: Methods of collection of saliva^[6],[8]

Click here to view

Saliva as Diagnostic Fluid

The salivary fluid has an old history of study, but its physiological importance has only been recognized recently. In the past 50 years, the pace of salivary research has accelerated with the advent of newer techniques that have illuminated the biochemical and physicochemical properties of saliva. The interest in saliva increased, further, with the finding that saliva is filled with hundreds of components that might serve to detect systemic diseases and/or act as an evidence of exposure to various harmful substances and provide biomarkers of health and disease.^{[9],[10],[11],[12]} Many researchers have made use of sialometry and sialochemistry to diagnose systemic illnesses, monitoring general health, and as an indicator of risk for diseases creating a close relationship between oral and systemic health. However, since several factors can influence salivary secretion and composition, a strictly standardized collection must be made, so the above-mentioned examinations are able to reflect the real functioning of the salivary glands and serve as an efficient means for monitoring the systemic illnesses and health.^{[13],[14],[15],[16],[17],[18],[19],[20],[21],[22],[23],[24],[25]} The aim of the present literature review was to present such insight into the possible use of salivary fluid as a potential diagnostic and prognostic tool for the search of numerous diseases as well as for monitoring the treatment outcomes and to assess prognosis in such varied states of derangements of metabolic functions apart from investigating the composition and functions of saliva as well as to describe the factors that influence salivary flow and its biochemical composition.

Serum Versus Saliva

Currently, sera samples are used for the diagnosis and for monitoring the control of the disease processes and assessing the prognosis for most of the diseases. However, collection of sera samples has its own disadvantages including being an invasive procedure, being painful, and being associated with the risk of transmission of numerous infectious disease processes in cases where a strict asepsis is not followed. Thus, a simpler screening criterion which is noninvasive is an absolute necessity to make case finding easier for the clinicians and for the frequent monitoring of the disease processes. Furthermore, the ability to monitor health status, disease onset, progression, and treatment outcomes through noninvasive means is a highly desirable goal in health-care management.^[9] Like serum, saliva is a complex biological adjunct containing a variety of hormones, antibodies, enzymes, antimicrobial, and growth factors. Many of these enter saliva from the serum by passing through the spaces between the cells by transcellular (passive intracellular diffusion and/or active transport) or paracellular (extracellular ultrafiltration) routes. Therefore, most of the components found in the serum are, also, present in saliva, thus making saliva functionally equivalent to serum in reflecting the physiological status of the body, including the hormonal, nutritional, and various metabolic variations.^[13] The pace of research in relation to the salivary diagnostics and proteomics, however, could not reach the extent that was expected with the advent of newer techniques in the recent decades. The major problems in clinical salivary diagnostics are attributed mainly due to nonstandardized collection procedures and difficulty in interpretations caused due to the great diurnal variations of salivary secretion and the individual differences, in general. The major advantages of using saliva as a diagnostic fluid are its noninvasiveness, ease of collection, no requirement of special equipments and/or trained staff, and its usefulness in blood dyscrasias along with a likely better compliance with the children and geriatric patients.^{[9],[10],[11]}

Salivary Analysis as a Diagnostic Tool in Various Pathologic Conditions

Analysis of saliva is done commonly for the diagnosis of the following conditions:^{[13],[14],[15],[16],[17],[18],[19],[20],[21],[22],[23],[24],[25]}

Hereditary diseases

Cystic fibrosis ^[15],[16]
21-hydroxylase deficiency.

Infectious diseases: Saliva contains immunoglobulins (IgA, IgM, and IgG) that originate from two sources: the salivary glands and serum. Antibodies against viruses, bacteria, fungi, and parasite can be detected in saliva and can aid in the diagnosis of the following infections:

Helicobacter pylori^[17],[18]
Shigella
Pneumococcal pneumonia
Lyme disease
Taenia solium
Entamoeba histolytica
Mycobacterium tuberculosis^[19]
Human immunodeficiency virus ^[20],[21]
Hepatitis
Measles ^[22]
Mumps
Rubella
Rotavirus
Herpes simplex virus-1
Dengue

Monitoring of levels of hormones ^[23],[24]

Cortisol
Aldosterone
Testosterone
Dehydroepiandrosterone
Progesterone
Insulin

Hormones associated with bone turnover ^[25]

Salivary osteocalcin and pyridinoline in osteopenia and osteoporosis for bone mineral density (BMD)
BMD/t-scores

Autoimmune diseases

Sjogren's syndrome

Malignancies ^{[26],[27],[28],[29],[30],[31],[32],[33],[34],[35],[36],[37]}

p53 and CA 125 antibodies in oral squamous cell carcinoma (OSCC)^[27],[28]
Salivary defensin-1 in OSCC
Cancer antigen 15-3 (CA15-3) and c-erbB-2 (erb) and in breast carcinoma ^[33],[34]
CA 125 in epithelial ovarian carcinoma ^[35]
Prostate-specific antigen in prostatic adenocarcinoma ^[36]
High levels of nitrates in carcinoma of the digestive tract.

The oral manifestations of leukemias, also, occur early in the course of disease and these oral features can at times act as diagnostic indicators. A rise in salivary amylase levels in leukemic patients has, also, been reported.

Detection of Drugs in Saliva

Saliva can, also, be used as a medium to detect the presence and levels of the various drugs of therapeutic benefits and associated with drug abuse for which it plays a clinically significant role in the diagnostic and prognostic applications as is being reflected with the names of the drugs the presence of which can be and is usually detected in saliva in [Table 3].^{[38],[39],[40]}

Table 3: Detection of drugs in saliva^{[38],[39],[40]}

Click here to view

Forensic Evidence in Saliva

Saliva is deposited on the skin or object surface in enough amounts to allow typing of the deoxyribonucleic acid (DNA). Polymerase chain reaction (PCR) allows replication of thousands of copies of a specific DNA sequence in vitro enabling the study of small amounts of DNA.^{[41],[42],[43],[44]}

Detection of Oral Manifestations With Relevance to the Diagnosis of Systemic Diseases

Some systemic diseases affect salivary glands directly or indirectly and may influence the quantity and quality of saliva. These characteristic changes may contribute to the diagnosis and early detection of these diseases. Common examples of such diseases include ^{[13],[14],[15],[16],[17],[18],[19],[20],[21],[22],[23],[24],[25]}

Cystic fibrosis
HIV infection
Sjögren's syndrome
Rheumatoid diseases
Myasthenia gravis
Sarcoidosis
Hormonal disorders
Malnutrition
Dehydration
Vitamin deficiency
Hypertension
Cirrhosis
Parkinsonism ^{More Details}
Renal disease
Diabetes mellitus
Various malignancies.^{[26],[27],[28],[29],[30],[31],[32],[33],[34],[35],[36],[37]}

Saliva in Oral Health and Disease, Disease Diagnostics, and Monitoring

Saliva contains a spectrum of immunologic and nonimmunologic proteins with antibacterial properties. In addition, some proteins are necessary for inhibiting the spontaneous precipitation of calcium and phosphate ions in the salivary glands and in their secretions. Both the acquired film and the biofilm have proteins derived from saliva. High numbers of Streptococcus mutans and Lactobacillus indicate a shift in oral microflora from healthy to more cariogenic environment. Secretory IgA is the largest immunologic component of saliva. It can neutralize viruses, bacteria, and enzyme toxins. It serves as an antibody for bacterial antigens and is able to aggregate bacteria inhibiting their adherence to oral tissues.^[45] Other immunologic components such as IgG and IgM occur in lesser quantity and probably originate from gingival fluid.^[3] Among the nonimmunologic salivary protein components, there are enzymes (lysozyme, lactoferrin, and peroxidase), mucin glycoproteins, agglutinins, histatins, proline-rich proteins, statherins, and cystatins.^[45] Lysozyme can hydrolyze the cellular wall of some bacteria, and because it is strongly cationic, it can activate the bacterial “autolisines” which are able to destroy bacterial cell wall components. Gram-negative bacteria are more resistant to this enzyme due to the protective function of their external lipopolysaccharide layer. Other antibacterial mechanisms have, also, been proposed for this enzyme including inhibiting bacterial aggregation and adherence.^{[46],[47],[48]} Lactoferrin links to free iron in the saliva causing bactericidal or bacteriostatic effects on various microorganisms requiring iron for their survival such as the S. mutans group. Lactoferrin, also, provides fungicidal, antiviral, anti-inflammatory, and immunomodulatory functions.^{[46],[47],[48],[49]} Peroxidase or sialoperoxidase offers antimicrobial activity because it serves as a catalyst for the oxidation of the salivary thiocyanate ion by hydrogen peroxide into hypothiocyanate, a potent antibacterial substance. As a result of its consumption, proteins and cells are protected from the toxic and oxidant effects of hydrogen peroxide.^[50] The proline-rich proteins, salivary mucins, and statherins inhibit the spontaneous precipitation of calcium phosphate salts and the growth of hydroxyapatite crystals on the tooth surface preventing the formation of salivary and dental calculus. They favor oral structure lubrication and it is probable that both are important in the formation of acquired film. Another function proposed for the proline-rich proteins is the capacity to selectively mediate bacterial adhesion to tooth surfaces.^[50],[51] The cystatins are also related to acquired film formation and to hydroxyapatite crystal equilibrium. Due to its proteinase inhibiting properties, it is surmised that they act in controlling proteolytic activity.^{[50],[51],[52],[53]} The histatins, a family of histidine-rich peptides, have antimicrobial activity against some strains of S. mutans and inhibit hemagglutination of the periopathogen Porphyromonas gingivalis.^[54],[55] They neutralize the lipopolysaccharides of the external membranes of Gram-negative bacteria and are potent inhibitors of Candida albicans growth and development through the union of positively loaded histatins with the biological membranes resulting in the destruction of their architecture and altering their permeability.^[56],[57] Other functions attributed to these peptides include participation in acquired film formation and inhibition of histamine release by the mast cells suggesting a role in the inflammatory process.^[45] Salivary agglutinin, a highly glycosylated protein frequently associated with other salivary proteins and with secretory IgA, is one of the main salivary components responsible for bacteria agglutination.^[50] In a healthy situation, there is no correlation between saliva secretion rate and dental caries. When the salivary secretion rate drops below a certain minimum, the predisposition for dental caries increases dramatically.^{[46],[47],[48]} Saliva secretion rate and buffering capacity have proven to be sensitive parameters in caries prediction models.^[58] Diagnostic kits for S. mutans and Lactobacillus counts are widely used in dental practice and can be conducted without laboratory facilities. Commercial kits are also available for determination of the salivary buffering capacity. Different saliva-based caries activity tests include:^[59],[60]

Lactobacillus colony count test
Snyder test
Reductase test
Buffer capacity test
Fosdick calcium dissolution test
S. mutans adherence test
S. mutans dip-slide test.

Similarly, during active periods of the periodontal disease, increased levels of inflammatory markers such as C-reactive protein (CRP), various interleukins, and matrix metalloproteinases (MMPs) can be demonstrated in the saliva.^{[61],[62],[63]} The traditional methods to diagnose periodontal disease rely on measuring the periodontal pocket depth and clinical attachment loss and evaluating the radiographs for assessing the bone loss. These assessments do not predict periodontal disease in their earliest state.^[64] Since periodontal disease is irreversible disease process, an early diagnosis is imperative. Researchers have been investigating ways to detect periodontal disease in their preclinical phases using genetic, microbial, and protein biomarkers.^[65] Since the early 1990s, much research was generated to learn about the biomarkers of periodontal disease. Gingival crevicular fluid (GCF) became an early medium to examine for such biomarkers due to its location within the sulcus and easy accessibility. The major advantages of using GCF for diagnostic purpose include a site-specific approach for the detection of the presence or absence of specific periodontal pathogens, gingival and periodontal inflammation, the host inflammatory-immune response to certain pathogenic species, and the host tissue destruction. However, relatively expensive, technique sensitive, requirement of multiple samples of individual tooth sites, and laboratory processing make GCF less preferred when compared to saliva.^[66],[67] Saliva is readily available and easier to collect than is GCF. Saliva, also, contains a plethora of biomarkers for periodontal disease activity including GCF and has emerged as the medium of choice to detect markers for periodontal disease.^[68] Significant advances are in development for the screening of periodontal disease. Researchers have reported that high levels of the inflammatory biomarkers such as CRP, various interleukins, and MMPs including MMP-8 have been associated with chronic and aggressive periodontal disease.^{[61],[62],[63]} MMP-8 has been identified as a major tissue destructive enzyme in periodontal disease. Consequently, MMP-8 is a promising candidate for diagnosing and, possibly more importantly, assessing the progression of periodontal disease.^[69] Similarly, serum and salivary aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase are considered to be the possible disease markers in periodontal disease.^[70] It is clear that individual susceptibility along with a variety of local and systemic conditions can influence the initiation and progression of periodontal disease. Therefore, it is important that advances in diagnostic testing are made to help identify early periodontal risk. The use of saliva-based diagnostics appears promising for future application to diagnose periodontal disease and to predict the various possible treatment outcomes.^[68],[69] Furthermore, several bacteria have been associated with periodontal disease which are susceptible to different antibiotics.^{[53],[54],[55]} Therefore, before antibiotic treatment, pathogens are determined by culturing or PCR techniques. In this, saliva plays a pivotal role apart from the GCF for which methylcellulose paper strips are used to collect fluid from the gingival crevices around the teeth. Urea, a buffer present in salivary fluid, is a product of amino acid and protein catabolism that causes a rapid increase in biofilm pH by releasing ammonia and carbon dioxide when hydrolyzed by bacterial ureases. Ammonia, a product of urea and another significant catabolic end product of amino acid metabolism, is potentially cytotoxic to gingival tissues. It is an important factor in the initiation of gingivitis and, further, periodontal disease because it increases the permeability of the sulcular epithelium to a plethora of toxic or antigenic substances in addition to the formation of low pH biofilms and plaque deposits detrimental to periodontal health.^[71] Furthermore, it has been shown that untreated periodontal disease can lead to systemic disorders such as cardiovascular disease and diabetes. Nevertheless, the recent focus on the potential role of periodontal disease as a risk factor for cardio- and cerebrovascular disease and diabetes brings new importance to this aspect of salivary analysis.^{[72],[73],[74]}

Limitations of the model for using saliva in diagnostics

Xerostomia accounts for one of the major limitations for using saliva in diagnostics. Many classes of drugs, particularly those that have anticholinergic action (antidepressants, anxiolytics, antipsychotics, antihistaminics, and antihypertensives), may cause reduction in salivary flow and alter composition of saliva leading to xerostomia of varying grades and associated adverse effects.^[75],[76] Numerous diseases, too, have an impact on salivary flow rates and composition, one among them being diabetes mellitus, apart from a plethora of other conditions including numerous autoimmune and/or inflammatory conditions such as Sjogren's syndrome and primary biliary cirrhosis, graft versus host disease, IG-G4-related sclerosing disease, degenerative diseases such as amyloidosis, granulomatous conditions including sarcoidosis, infections including HIV/AIDS, hepatitis C and malignancies such as lymphomas, and salivary gland agenesis or, aplasia. In addition, patients with salivary gland changes after exposure to radiation in the head and neck area for treatment of malignancies, also, pose such challenges.^[77],[78] Apart from the above-mentioned limitations, age-related degenerative changes seen in salivary glands, also, add to a significant fraction of geriatric population to suffer from xerostomia seen to varying grades.^{[79],[80],[81],[82],[83]}

Conclusion

Saliva is a whole, diverse fluid that serves various purposes discussed in detail in the literature. Furthermore, there has been sufficient literature that assays the role of saliva as a potential diagnostic fluid although with conflicting reports and results from the various studies conducted to prove the reliability of saliva as a potential diagnostic fluid. Although the recent introduction of molecular biology opens up new vistas and a new search for the role of salivary fluid as a potential diagnostic fluid, further studies and an active search are, still, warranted to establish the role of saliva in the diagnosis of various conditions as well as its suitability and sound usage for prognostic and forensic purposes.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1.	Berkovitz BK, Holland GR, Moxham BJ. Oral Anatomy, Histology and Embryology. 3^rd ed. New York: Mosby; 2002.
2.	Ferraris ME, Munõz AC. Histologia e Embriologia Bucodental. 2^nd ed. Rio de Janeiro: Guanabara Koogan; 2006.
3.	Edgar WM. Saliva: Its secretion, composition and functions. Br Dent J 1992;172:305-12.
4.	Humphrey SP, Williamson RT. A review of saliva: Normal composition, flow, and function. J Prosthet Dent 2001;85:162-9.
5.	Douglas CR. Tratado de Fisiologia Aplicada à Saúde. 5^th ed. São Paulo: Robe Editorial; 2002.
6.	Falcão DP, da Mota LM, Pires AL, Bezerra AC. Sialometry: Aspects of clinical interest. Rev Bras Reumatol 2013;53:525-31.
7.	Pink R, Simek J, Vondrakova J, Faber E, Michl P, Pazdera J, et al. Saliva as a diagnostic medium. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2009;153:103-10.
8.	Gutiérrez AM, Martínez-Subiela S, Eckersall PD, Cerón JJ. Saliva collection and handling advice: Salimetrics. J Clin Endocrinol Metabol 2009;36:1230-6.
9.	Castagnola M, Picciotti PM, Messana I, Fanali C, Fiorita A, Cabras T, et al. Potential applications of human saliva as diagnostic fluid. Acta Otorhinolaryngol Ital 2011;31:347-57.
10.	Sandhu SV, Bhandari R, Gupta S, Puri A. Salivary diagnostics: An insight. Indian J Dent Sci 2011;3:19-23.
11.	Mittal S, Bansal V, Garg S, Atreja G, Bansal S. The diagnostic role of saliva: A review. J Clin Exp Dent 2011;3:e314-20.
12.	Malamud D. Salivary diagnostics: The future is now. J Am Dent Assoc 2006;137:284, 286.
13.	Lee YH, Wong DT. Saliva: An emerging bio-fluid for early detection of diseases. Am J Dent 2009;22:241-8.
14.	González LF, Sánches MC. La saliva: Revisión sobre composición, función y usos diagnósticos: Primera parte. Univ Odontol 2003;23:18-24.
15.	Gonçalves AC, Marson FA, Mendonça RM, Ribeiro JD, Ribeiro AF, Paschoal IA, et al. Saliva as a potential tool for cystic fibrosis diagnosis. Diagn Pathol 2013;8:46.
16.	Minarowska A, Minarowski L, Karwowska A, Sands D, Dabrowska E. The activity of cathepsin D in saliva of cystic fibrosis patients. Folia Histochem Cytobiol 2007;45:165-8.
17.	Kabir S. Detection of Helicobacter pylori DNA in feces and saliva by polymerase chain reaction: A review. Helicobacter 2004;9:115-23.
18.	Silva DG, Stevens RH, Macedo JM, Hirata R, Pinto AC, Alves LM, et al. Higher levels of salivary MUC5B and MUC7 in individuals with gastric diseases who harbor Helicobacter pylori. Arch Oral Biol 2009;54:86-90.
19.	Eguchi J, Ishihara K, Watanabe A, Fukumoto Y, Okuda K. PCR method is essential for detecting Mycobacterium tuberculosis in oral cavity samples. Oral Microbiol Immunol 2003;18:156-9.
20.	Corstjens PL, Abrams WR, Malamud D. Detecting viruses by using salivary diagnostics. J Am Dent Assoc 2012;143:12S-8S.
21.	Delaney KP, Branson BM, Uniyal A, Phillips S, Candal D, Owen SM, et al. Evaluation of the performance characteristics of 6 rapid HIV antibody tests. Clin Infect Dis 2011;52:257-63.
22.	Oliveira SA, Siqueira MM, Brown DW, Camacho LA, Faillace T, Cohen BJ, et al. Salivary diagnosis of measles for surveillance: A clinic-based study in Niterói, state of Rio de Janeiro, Brazil. Trans R Soc Trop Med Hyg 1998;92:636-8.
23.	Figueiredo VC, Szklo M, Szklo AS, Benowitz N, Lozana JA, Casado L, et al. Determinants of salivary cotinine level: A population-based study in Brazil. Rev Saude Publica 2007;41:954-62.
24.	Raff H. Utility of salivary cortisol measurements in Cushing's syndrome and adrenal insufficiency. J Clin Endocrinol Metabol 2009;94:3647-55.
25.	McGehee JW Jr., Johnson RB. Biomarkers of bone turnover can be assayed from human saliva. J Gerontol 2004;59:196-200.
26.	Liu J, Duan Y. Saliva: A potential media for disease diagnostics and monitoring. Oral Oncol 2012;48:569-77.
27.	Warnakulasuriya S, Soussi T, Maher R, Johnson N, Tavassoli M. Expression of p53 in oral squamous cell carcinoma is associated with the presence of IgG and IgA p53 autoantibodies in sera and saliva of the patients. J Pathol 2000;192:52-7.
28.	Balan JJ, Rao RS, Premalatha BR, Patil S. Analysis of tumor marker CA 125 in saliva of normal and oral squamous cell carcinoma patients: A comparative study. J Contemp Dent Pract 2012;13:671-5.
29.	Tang H, Wu Z, Zhang J, Su B. Salivary lncRNA as a potential marker for oral squamous cell carcinoma diagnosis. Mol Med Rep 2013;7:761-6.
30.	Yoshizawa JM, Wong DT. Salivary microRNAs and oral cancer detection. Methods Mol Biol 2013;936:313-24.
31.	Bernabe DG, Tamae AC, Miyahara GI, Sundefeld ML, Oliveira SP, Biasoli ER. Increased plasma and salivary cortisol levels in patients with oral cancer and their association with clinical stage. J Clin Pathol 2012;65:934-9.
32.	Rai B, Kaur J, Jacobs R, Anand SC. A denosine deaminase in saliva as a diagnostic marker of squamous cell carcinoma of tongue. Clin Oral Investig 2011;15:347-9.
33.	Agha-Hosseini F, Mirzaii-Dizgah I, Rahimi A. Correlation of serum and salivary CA15-3 levels in patients with breast cancer. Med Oral Patol Oral Cir Bucal 2009;14:e521-4.
34.	Streckfus C, Bigler L, Dellinger T, Dai X, Kingman A, Thigpen JT, et al. The presence of soluble c-erbB-2 in saliva and serum among women with breast carcinoma: A preliminary study. Clin Cancer Res 2000;6:2363-70.
35.	Lee YH, Kim JH, Zhou H, Kim BW, Wong DT. Salivary transcriptomic biomarkers for detection of ovarian cancer: For serous papillary adenocarcinoma. J Mol Med (Berl) 2012;90:427-34.
36.	Shiiki N, Tokuyama S, Sato C, Kondo Y, Saruta J, Mori Y, et al. Association between saliva PSA and serum PSA in conditions with prostate adenocarcinoma. Biomarkers 2011;16:498-503.
37.	Zhang L, Xiao H, Zhou H, Santiago S, Lee JM, Garon EB, et al. Development of transcriptomic biomarker signature in human saliva to detect lung cancer. Cell Mol Life Sci 2012;69:3341-50.
38.	Drummer OH. Drug testing in oral fluid. Clin Biochem Rev 2006;27:147-59.
39.	Cone EJ, Huestis MA. Interpretation of oral fluid tests for drugs of abuse. Ann N Y Acad Sci 2007;1098:51-103.
40.	Guinan T, Ronci M, Kobus H, Voelcker NH. Rapid detection of illicit drugs in neat saliva using desorption/ionization on porous silicon. Talanta 2012;99:791-8.
41.	Pfaffe T, Cooper-White J, Beyerlein P, Kostner K, Punyadeera C. Diagnostic potential of saliva: Current state and future applications. Clin Chem 2011;57:675-87.
42.	Miller CS, Foley JD, Bailey AL, Campell CL, Humphries RL, Christodoulides N, et al. Current developments in salivary diagnostics. Biomark Med 2010;4:171-89.
43.	Sweet D, Hildebrand D. Saliva from cheese bite yields DNA profile of burglar: A case report. Int J Legal Med 1999;112:201-3.
44.	Kamodyová N, Durdiaková J, Celec P, Sedláčková T, Repiská G, Sviežená B, et al. Prevalence and persistence of male DNA identified in mixed saliva samples after intense kissing. Forensic Sci Int Genet 2013;7:124-8.
45.	Schenkels LC, Veerman EC, Nieuw Amerongen AV. Biochemical composition of human saliva in relation to other mucosal fluids. Crit Rev Oral Biol Med 1995;6:161-75.
46.	Axelsson P. Diagnosis and Risk Prediction of Dental Caries. Vol. 2. Illinois: Quintessence Books; 2000.
47.	Tenovuo J, Lagerlöf F. Saliva. In: Thylstrup A, Fejerskov O, editors. Textbook of Clinical Cariology. 2^nd ed. Copenhagen: Munksgaard; 1994.
48.	Larsen MJ, Bruun C. A química da cárie dentária e o flúor: Mecanismos de ação. In: Thylstrup A, Fejerskov O, editors. Cariologia Clínica. 3^rd ed. São Paulo: Livraria Editora Santos; 2001.
49.	Nikawa H, Samaranayake LP, Tenovuo J, Pang KM, Hamada T. The fungicidal effect of human lactoferrin on Candida albicans and Candida krusei. Arch Oral Biol 1993;38:1057-63.
50.	Amerongen AV, Veerman EC. Saliva – The defender of the oral cavity. Oral Dis 2002;8:12-22.
51.	Edgar M, Dawes C, O'Mullane D. Saliva and Oral Health. 3^rd ed. London: BDJ Books; 2004.
52.	Tabak LA, Levine MJ, Mandel ID, Ellison SA. Role of salivary mucins in the protection of the oral cavity. J Oral Pathol 1982;11:1-7.
53.	Blankenvoorde MF, Henskens YM, van't Hof W, Veerman EC, Nieuw Amerongen AV. Inhibition of the growth and cysteine proteinase activity of Porphyromonas gingivalis by human salivary cystatin S and chicken cystatin. Biol Chem 1996;377:847-50.
54.	MacKay BJ, Denepitiya L, Iacono VJ, Krost SB, Pollock JJ. Growth-inhibitory and bactericidal effects of human parotid salivary histidine-rich polypeptides on Streptococcus mutans. Infect Immun 1984;44:695-701.
55.	Murakami Y, Tamagawa H, Shizukuishi S, Tsunemitsu A, Aimoto S. Biological role of an arginine residue present in a histidine-rich peptide which inhibits hemagglutination of Porphyromonas gingivalis. FEMS Microbiol Lett 1992;77:201-4.
56.	Sugiyama K. Anti-lipopolysaccharide activity of histatins, peptides from human saliva. Experientia 1993;49:1095-7.
57.	Xu T, Levitz SM, Diamond RD, Oppenheim FG. Anticandidal activity of major human salivary histatins. Infect Immun 1991;59:2549-54.
58.	Dawes C. Physiological factors affecting salivary flow rate, oral sugar clearance and the sensation of dry mouth in man. J Dent Res 1987;66:648-53.
59.	D'Amario M, Barone A, Marzo G, Giannoni M. Caries-risk assessment: The role of salivary tests. Minerva Stomatol 2006;55:449-63.
60.	Larmas M. Saliva and dental caries: Diagnostics tests for normal dental practices. Int Dent J 1992;42:199-208.
61.	Malamud D, Rodriguez-Chavez IR. Saliva as a diagnostic fluid. Dent Clin North Am 2011;55:159-78.
62.	Priyanka N, Kalra N, Shanbhag N, Kumar K, Brijet BS, Uma SR, et al. Recent approaches in saliva as a credible periodontal diagnostic and prognostic marker. Arch Oral Sci Res 2012;2:40-6.
63.	Patil PB, Patil BR. Saliva: A diagnostic biomarker of periodontal diseases. J Indian Soc Periodontol 2011;15:310-7. [PUBMED] [Full text]
64.	Zia A, Khan S, Bey A, Gupta ND, Mukhtar-Un-Nisar S. Oral biomarkers in the diagnosis and progression of periodontal disease. Biol Med 2011;3:45-52.
65.	Scannapieco FA, Ng P, Hovey K, Hausmann E, Hutson A, Wactawski-Wende J. Salivary biomarkers associated with alveolar bone loss. Ann N York Acad Sci 2007;1098:496-7.
66.	Giannobile WV. Salivary diagnostics for periodontal diseases. J Am Dent Assoc 2012;143:6S-11S.
67.	Kim JJ, Kim CJ, Camargo PM. Salivary biomarkers in the diagnosis of periodontal diseases. J Calif Dent Assoc 2013;41:119-24.
68.	Chapple IL. Periodontal diagnosis and treatment – Where does the future lie? Periodontol 2000 2009;51:9-24.
69.	Herr AE, Hatch AV, Throckmorton DJ, Tran HM, Brennan JS, Giannobile WV, et al. Microfluidic immunoassays as rapid saliva-based clinical diagnostics. Proc Natl Acad Sci U S A 2007;104:5268-73.
70.	Totan A, Greabu M, Totan C, Spinu T. Salivary aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase: Possible markers in periodontal diseases? Clin Chem Lab Med 2006;44:612-5.
71.	Macpherson LM, Dawes C. Urea concentration in minor mucous gland secretions and the effect of salivary film velocity on urea metabolism by Streptococcus vestibularis in an artificial plaque. J Periodontal Res 1991;26:395-401.
72.	Spielmann N, Wong DT. Saliva: Diagnostics and therapeutic perspectives. Oral Dis 2011;17:345-54.
73.	Ligtenberg AJ, de Soet JJ, Veerman EC, Amerongen AV. Oral diseases: From detection to diagnostics. Ann N Y Acad Sci 2007;1098:200-3.
74.	Wong DT. Salivaomics. J Am Dent Assoc 2012;143:19S-24S.
75.	Stack KM, Papas AS. Xerostomia: Etiology and clinical management. Nutr Clin Care 2001;4:15-21.
76.	Thomson WM, Chalmers JM, Spencer AJ, Slade GD. Medication and dry mouth: Findings from a cohort study of older people. J Public Health Dent 2000;60:12-20.
77.	Guggenheimer J, Moore PA. Xerostomia: Etiology, recognition and treatment. J Am Dent Assoc 2003;134:61-9.
78.	Atkinson JC, Baum BJ. Salivary enhancement: Current status and future therapies. J Dent Educ 2001;65:1096-101.
79.	Nagler RM. Salivary glands and the aging process: Mechanistic aspects, health-status and medicinal-efficacy monitoring. Biogerontology 2004;5:223-33.
80.	Percival RS, Challacombe SJ, Marsh PD. Flow rates of resting whole and stimulated parotid saliva in relation to age and gender. J Dent Res 1994;73:1416-20.
81.	Azevedo LR, Damante JH, Lara VS, Lauris JR. Age-related changes in human sublingual glands: A post mortem study. Arch Oral Biol 2005;50:565-74.
82.	Moreira CR, Azevedo LR, Lauris JR, Taga R, Damante JH. Quantitative age-related differences in human sublingual gland. Arch Oral Biol 2006;51:960-6.
83.	Shern RJ, Fox PC, Li SH. Influence of age on the secretory rates of the human minor salivary glands and whole saliva. Arch Oral Biol 1993;38:755-61.

Tables

[Table 1], [Table 2], [Table 3]

This article has been cited by
1	Acute phase proteins, saliva and education in laboratory science: an update and some reflections
	José J. Cerón
	BMC Veterinary Research. 2019; 15(1)
	[Pubmed] \| [DOI]