International Dental Journal (2007) 57, 295-299

Saliva as a diagnostic fluid*

Lakshman Samaranayake
Hong Kong SAR, China
The use of saliva as a diagnostic fluid for various human ailments is gaining popularity as it offers distinct advantages over serum. These include the non-invasive nature of saliva collection compared with phlebotomy, simplicity of collection even for individuals with a modest training and the cost-effective applicability for screening large populations. Whole saliva is most frequently used for diagnosis of systemic diseases since it is readily collected and contains serum constituents while gland-specific saliva is useful for investigating pathology of major salivary glands. Broadly, saliva analysis is currently used for the diagnosis of infectious and malignant diseases, hereditary disorders, autoimmune diseases, and endocrine disorders, as well as for the assessment of therapeutic drug levels, particularly in monitoring drug abuse. This review addresses the current status of salivary diagnostics and their future potential. Key words: Saliva, diagnosis, systemic, disease, drug, hormone

In ancient Greece, phlegm and saliva were regarded by Hippocrates as one of the four humours fundamental to disease and health. Saliva indeed is a mirror of our blood as these biouids and their molecular components share many similarities1. Realisation of this fact and the possible utility of saliva as a diagnostic bio-uid during the past couple of decades have led many researchers to develop saliva-based technology to detect the transition between health and disease. Furthermore, recent advances in technology including genomics, proteomics, transcriptomics and microuidics have led the way for using saliva beyond basic assessment of oral health characteristics to where it could be used to evaluate features of overall health including disease progression. It is generally thought that the quality of saliva is highly variable and difcult to handle in the laboratory due to its highly degradable nature and with millions of bacteria and other organic components. Further, compared with blood, other factors that affect salivary quality include the type of saliva, i.e. mixed vs pure (glandular), the specic collection methodology used and the physiological factors such as diurnal quality variations2. However, recent research has now established dened ways to work with saliva that are consistent, reproducible, and which keep these molecules in an un-degraded, stable state. Perhaps the widest current use of such technology is in the detection of viral infections, in particular human immunodeciency virus (HIV) disease. As will be seen later, the commercially available kits that are
2007 FDI/World Dental Press 0020-6539/07/05295-05

in current use have very high sensitivity with negligible false positivity rates (i.e., high specicity). Other recent studies from the US have yielded promising results on the feasibility of the use of saliva for the diagnosis of cancers including breast cancer, and oral carcinomas. The relative advantages of saliva as a diagnostic uid compared with blood are many and these include simplicity in collection essentially being non-invasive and pain free, relatively cheap technology compared to blood tests, and cost effective applicability for screening large populations. In order for a diagnostic method including salivary diagnostics to be widely applicable it should satisfy the following criteria: Analytic precision and accuracy Excellent sensitivity and specicity Good operational predictive value and efciency Value for money or cost/benet ratio in terms of rapid point of care diagnosis.

What follows is a highlight of the uses of saliva in assessing the health and disease of individuals, and related uses in diagnostic sciences for miscellaneous applications such as drug abuse. It must, however, be stated at the outset that salivary diagnostic science is yet to mature and its true potential not yet realised.

*This manuscript is an outcome of a project undertaken by the Science Committee of the FDI

296

Viral diseases

A number of viruses that cause disease are either shed in saliva or egress into saliva through crevicular or serum exudates of the healthy or diseased periodontium3. Of these, the most important saliva diagnostic test that has been developed to date is a kit for detecting the human immunodeciency virus (HIV) infection. Whole saliva can be used for detecting antibodies directed toward specic HIV viral protein epitopes and the test has very high sensitivity and specicity3,4. For instance, in one study using an enzyme-linked uorescence technique in combination with Western blot assays, saliva was superior to serum and urine with regard to both sensitivity and specicity5. Indeed the Federal Drug Administration (FDA) of United States of America approved saliva HIV testing kits for public use in as early as in 2003. Other developments in this regard include a commercially available, self-contained kit for use in community outreach and surveillance studies that does not require trained laboratory personnel. Further, saliva can be used to monitor the disease activity either in HIV-infection or other chronic inammatory diseases through assessment of beta 2 microglobulin and/or soluble tumour necrosis factor -receptor levels6,7. Saliva is also a useful alternative to serum for the diagnosis of variants of viral hepatitis. For instance, acute hepatitis A (HAV) and hepatitis B (HBV) can be diagnosed based on the presence of IgM antibodies in saliva. Comparison of serum and saliva levels of antibody to HAV revealed excellent agreement8. Recently, Hepatitis A Virus RNA detection in saliva has been suggested as a useful marker for tracing and monitoring infection in community settings9. Similarly, analysis of saliva is highly sensitive and specic for the diagnosis of viral hepatitis B as well as hepatitis C with a sensitivity and specicity approaching 100%3,10,11. Other uses of saliva for detecting viral diseases include determining immunisation efcacy for measles, mumps, and rubella (MMR) vaccine12, and to monitor the immune response to vaccination and infection with the rotavirus infection a common infant diarrhoeal disease in the developing world13. Whist the saliva diagnostics for the foregoing infections are far advanced there is a spectrum of other viral infections that await further technical renements. These, essentially based on plolymerase chain reaction (PCR) methodology or its advancements such as mutiplex PCR include tests for the evaluation of human herpes viruses. As most human viruses ranging from HHV1 to HHV8 are shed in saliva the latter would be an ideal source for testing and monitoring the dormancy or the sub-clinical activity of these viruses either in health or disease states. A number of investigators have measured the shedding of Epstein-Barr virus, cytomegalovirus and herpes viruses 6,7 and 8 in the saliva of HIV infected patients14,15. It is highly likely that further renements of molecular technology should lead to wide use
International Dental Journal (2007) Vol. 57/No.5

of saliva as an effective, simple and a valuable diagnostic tool for evaluation of human herpes virus carriage in saliva or indeed their clinical manifestations16.
Bacterial infections

The two most common plaque related oral bacterial infections are caries and gingivitis. Many investigators have studied the feasibility of salivary diagnostics as a predictor of caries susceptibility or for gingivitis /periodontitis17,18. With regard to caries, saliva samples can be used to establish the numbers of Streptococcus mutans and Lactobacillus species, the two major caries associated pathogens. In order to detect caries associated ora, parafn wax stimulated whole saliva samples are collected and a dip slide containing the selective growth medium (which permits the growth of only the specic organism) is dipped into the collected saliva. Afterwards the slide is incubated within the container at 370C for upto 24 hrs and the resultant colonies of the putative pathogen are assessed in a semi-quantitative manner. In general the approximate gures for the cariogenic ora are: High caries activity > 106 /mL S mutans and/or 105m/L lactobacillus spp Low caries activity : < 105 /mL S mutans and/or 104m/L lactobacillus spp. The presence of high salivary levels of S mutans or lactobacilli does not necessarily mean that the patient has an increased risk of developing dental caries, as it is a disease of multifactorial aetiology19. Other factors such as the diet, buffering capacity of saliva, uoride content of enamel and degree of oral hygiene should also be considered. Although this particular test is at best a generalised approximation of the caries risk it could be utilised to identify patients who have unusually high numbers of potential pathogens so that these data can be taken into account when integrating all the factors that may contribute to the carious process in an individual patient. In addition, the test could be utilised to monitor the efcacy of caries prevention techniques such as dietary and oral hygiene advice and the use of antimicrobial agents such as chlorhexidine20. Due to the foregoing reasons salivary-based tests for monitoring plaque-related infections cannot be construed as truly diagnostic, rather they are harbingers of a patients risk potential for disease and the consequent need for preventive measures. This approach has proven successful as a public health, preventive measure especially in Scandinavian countries for many decades21. With regard to periodontal disease and the detection of major periodontopathogenic bacteria, salivary diagnostics have proven less successful. Tests are, however, now available for chair-side assessment of Porphymonas gingivalis, a pathogen closely associated with periodontal

297

disease. Other salivary markers that have been studied as potential diagnostic tools for periodontal disease include proteins of host origin such as enzymes and immunoglobulins, host cells, hormones (cortisol), bacterial metabolites and volatile compounds. Of these, hostderived enzymes and other inammatory mediators originating from the gingival crevice appear to hold the greatest promise as salivary diagnostic tests for periodontal disease both on an individual basis or on a community wide assessment of risk for periodontal disease. The recent exciting developments on the potential role of periodontal disease as a risk factor for cardiovascular and cerebrovascular diseases22 and the incidence of preterm low-birth-weight babies23 bring new impetus to this aspect of salivary diagnostics. It is now well recognised that Helicobacter pylori are the causative agents of a signicant proportion of gastritis and duodenal ulcers (peptic ulcer disease) in humans and, it may also play a role in gastric cancer24. Studies have also shown that dental plaque biolms may act as a reservoir of H. pylori in some of these individuals. Hence attempts have been made to use saliva as a diagnostic aid for peptic ulcer disease. A nested PCR assay is now available to detect H. pylori DNA in saliva and conrm the presence of H. pylori infection in patients25. Other immunologic studies indicate that saliva may also be used for predicting risk for gastric adenocarcinoma26. Saliva from patients with a variety of other disorders including shigellosis, pigeon breeders disease and Lyme disease have been evaluated for the presence of specic antibodies, with mixed results27. But with the increased sensitivity of tests and advancing technology it is likely that saliva would be used as an important diagnostic uid for such disease entities.
Autoimmune disorders

Cardiovascular diseases

Cardiovascular disease is the commonest cause of death worldwide. Salivary markers such as salivary amylase have been used for postoperative follow up of patients undergoing cardiovascular surgery. The work of Adam et al.31 indicated that if salivary amylase levels were low in preoperative patients with ruptured aortic aneurysm, then this was associated with increased mortality. Others have found that salivary -amylase activity could be used as a good marker of catecholamine activity during evaluation of patients under various stressful situations32. Such investigations, while indicating the possible utility of saliva to assess the general health of the body, are as yet in the early stages of development and more work is required to conrm their general usefulness.
Endocrinology

When a bodys defences turn against itself, as seen in many autoimmune disorders, diseases such as Sjgrens syndrome may ensue. The latter is a chronic illness characterised by salivary and lacrimal gland dysfunction, serologic abnormalities, and multiple organ-system changes including rheumatoid arthritis28. Many have attempted to use saliva as well as other salivary parameters including sialography, salivary scintigraphy, and biopsies of minor salivary glands for the diagnosis of Sjgrens syndrome, with mixed results29. Of these, salivary ow rate determination or sialometry is the simplest whilst other tests need to be conducted in special laboratories or clinics. As these are invasive and expensive, a panel of simple chair-side tests that could be conducted that include ow rate, pH, buffer capacity, lactobacillus, and yeast concentration has been suggested as useful for the purpose30. These tests, performed on whole saliva, can provide persuasive evidence for the presence of Sjgrens syndrome that affects mainly the middle-aged, elderly population.

Measurements of salivary hormone levels are of clinical relevance if they accurately reect the serum hormone levels or if a constant correlation exists between salivary and serum hormone levels. Although there is wide disparity in these values for some hormones, salivary steroid levels are in general good indicators of their blood concentrations. Consequently, the use of saliva for monitoring of steroid hormone levels is now feasible and commercially available with dedicated websites on the internet (e.g. http://www.salivatest.com/journals/ saliva_ref.html ). At present, the following steroid levels can be assessed using mixed saliva: cortisol, estradiol, estriol, dehydroepiandrosterone, progesterone and testosterone. As opposed to these, serum levels of protein hormones such as prolactin and thyrotropin cannot be evaluated by salivary analyses as the latter molecules are too large to reach saliva through passive diffusion. The clinical utility of steroid hormone evaluation has been demonstrated in a wide variety of situations ranging from assessment of child health and development, mood and cognitive emotional behaviour, premenstrual depression, Cushings syndrome, ovarian function and monitoring full-term and preterm neonates.
Oncology

The use of saliva as a predictable and a sensitive marker for the detection of either oral or systemic cancers appears to be a practical reality. In a recent landmark study Wong et al.33, noticed seven messenger RNAs in particular that were present at a 3.5 fold higher level in patients with oral carcinomas (mRNA is the molecular intermediate between gene and protein, serving as a chemical record that an individual gene has been expressed). Wong and co-workers then reduced their list of signature mRNAs to four, based on statistical models that indicated the synchronised rise in expression of these four molecules increased the probability
Samaranayake: Saliva as a diagnostic fluid

298

that the saliva belonged to a cancer patient. These four mRNAs were from the following genes: Interleukin 1-beta (IL1B), Ornithine decarboxylase antizyme 1 (OAZ1), spermidine/spermine N1-acetyl transferase (SAT), and interleukin 8 (IL-8). To put this idea to the test, Wong et al.33, screened the saliva again to see how often they could correctly identify the samples from the cancer patients. The group could identify the saliva from cancer patients in nine out of 10 samples. Although this is a primary, exploratory study the data is exciting and paves way to further research in the direction. Previoulsy, Boyle et al.34, have shown the possible value of p53 in saliva as a marker for squamous cell carcinoma. Interestingly, they detected and identied tumour-specic mutations in p53 in preoperative salivary samples of individuals suffering from head and neck squamous cell carcinoma. Apart from these more recent molecular studies many have investigated the utility of proteins in saliva for the detection of systemic, non-oral, malignancies. These results indicate, for instance, higher levels of salivary kallikrein in patients with malignant tumours as compared with those with benign tumours or from healthy controls. Others have also shown that saliva contains the cancer antigen CA 125, a glycoprotein complex that is an often-used marker for ovarian cancer. Indeed according to some studies salivary CA 125 assay had a better diagnostic value than the comparable serum assay35. Saliva has also been used for monitoring patient response to chemotherapy for breast cancer or surgical treatment of the disease. Thus Streckfus et al.36, studied the protein product of the oncogene c-erbB-2, also known as HER-2/neu, and noted that it is elevated in the saliva of women diagnosed with breast cancer. In earlier studies, it has also been shown that the epidermal growth factor (EGF) is higher in the saliva of women with primary breast cancer or a recurrence of breast cancer when compared with women without disease37. The foregoing clearly illustrates the potential of salivary diagnostics in the management of both oral and non-oral malignancies. In particular, the salivary proteomic and mRNA studies and those in current development38 including electrochemical enzyme immunoassay procedures39 have the greatest promise in this regard.
Hereditary disorders

the salivary drug concentration is inuenced by the molecular and physicochemical characteristics of the drug and its interaction with the salivary tissues, as well as the extravascular drug metabolism. Additionally, salivary factors including the presence of food debris, sloughed epithelial cells, and pH determine the drug availability40. More improved saliva collection methods and preservatives that maintain the integrity have largely overcome these problems however. Monitoring of illicit drug use by salivary detection methods appears to be the most promising in this context. Currently, saliva can be used to detect opioids, barbiturates, benzodiazepines, amphetamines, cannabinoids, cocaine, phencyclidine, and ethanol (for a review see Kaumman and Lamster 27; Table 1). Saliva can also be used to detect recent marijuana use by means of radiommunoassay. In psychiatry, saliva has been used with partial success, to monitor responses in the treatment of anxiety and post-traumatic stress disorder by measuring salivary levels of 3-methoxy-4-hydroxyphenylglycol (MHPG) 41.
Conclusions

Whilst saliva is an appropriate diagnostic tool for some diseases, more research is warranted to identify the ideal candidate markers for monitoring and diagnosis of others. Furthermore, advances in genomics, proteomics and nanotechnology will contribute much to the understanding of the role of saliva as a diagnostic biouid. The maturation of salivary diagnostics has much promise, and is bound to be widespread in the not too distant future.
Table 1 Drug detection in saliva: Currently available methodology detects minute quantities of the following drugs and chemicals in saliva Therapeutic drugs Antipyrene Cyclosporin Digoxin Mathadone Paracetamol Quinine Carbamazepine Diazepam Lithium Oxprenolol Phenytoin Tolbutamide

Recreational drugs and drugs of abuse Amphetamines Benzodiazepines Cocaine Nicotine Phencyclidine Barbiturates Ethanol Marijuana Opioids

Attempts have been made by several investigators to use saliva as a diagnostic uid for hereditary diseases such as cystic brosis (CF), celiac disease and 21-Hydroxylase deciency - an inherited disorder of steroidogenesis. However, these have been met with partial success.
Drug monitoring

Modified from Kaufman and Lamster27

Acknowledgements

Various drugs taken internally either for medicinal or recreational purposes can appear in saliva. However,
International Dental Journal (2007) Vol. 57/No.5

I am grateful to Professor Stephen Moss (editor), Professor Emeritus, New York University, USA, for the

299

kind permission given to modify and update my original text that appeared as a chapter in the monograph The benets of chewing, published by Health Education Enterprises, USA.
References
1. Slavkin HC. Toward molecularly based diagnostics for the oral cavity. J Am Dent Assoc 1998 129: 1138-1143. 2. Dawes C. Considerations in the development of diagnostic tests on saliva. Ann N Y Acad Sci 1993 694: 265-269. 3. Scully C, Samaranayake LP. Clinical Virology in Oral medicine and Dentistry. Cambridge University Press, 1992. 4. Scully C. HIV topic update: salivary testing for antibodies. Oral Dis 1997 3: 212-215. 5. Martinez PM, Torres AR, Ortiz de Lejarazu R et al. Human immunodeciency virus antibody testing by enzyme-linked uorescent and western blot assays using serum, gingival-crevicular transudate, and urine samples. J Clin Microbiol 1999 37: 1100-1106. 6. Grant RM, Piwowar EM, Katongole-Mbidde E et al. Comparison of saliva and serum for human immunodeciency virus type 1 antibody testing in Uganda using a rapid recombinant assay. Clin Diagn Lab Immunol 1996 3: 640-644. 7. Nishanian P, Aziz N, Chung J et al. Oral uids as an alternative to serum for measurement of markers of immune activation. Clin Diagn Lab Immunol 1998 5: 507-512. 8. Stuart JM, Majeed FA, Cartwright KA et al. Salivary antibody testing in a school outbreak of hepatitis A. Epidemiol Infect 1992 109: 161-166. 9. Mackiewicz V, Dussaix E, Le Petitcorps MF et al. Detection of hepatitis A virus RNA in saliva. J Clin Microbiol 2004 42: 4329-4331. 10. El-Medany OM, El-Din Abdel Wahab KS, Abu Shady EA et al. Chronic liver disease and hepatitis C virus in Egyptian patients. Hepatogastroenterology 1999 46: 1895-1903. 11. Thieme T, Yoshihara P, Piacentini S et al. Clinical evaluation of oral uid samples for diagnosis of viral hepatitis. J Clin Microbiol 1992 30: 1076-1079. 12. Brown DW, Ramsay ME, Richards AF et al. Salivary diagnosis of measles: a study of notied cases in the United Kingdom, 1991-3. BMJ 1994 308: 1015-1017. 13. Jayashree S, Bhan MK, Kumar R et al. Serum and salivary antibodies as indicators of rotavirus infection in neonates. J Infect Dis 1988 158: 1117-1120. 14. LaDuca JR, Love JL, Abbott LZ et al. Detection of human herpesvirus 8 DNA sequences in tissues and bodily uids. J Infect Dis 1998 178: 1610-1615. 15. Lucht E, Brytting M, Bjerregaard L et al. Shedding of cytomegalovirus and herpesviruses 6, 7, and 8 in saliva of human immunodeciency virus type 1- infected patients and healthy controls. Clin Infect Dis 1998 27: 137-141. 16. Pozo F, Tenorio A. Detection and typing of lymphotropic herpesviruses by multiplex polymerase chain reaction. J Virol Methods 1999 79: 9-19. 17. Lendenmann U, Grogan J, Oppenheim FG. Saliva and dental pellicle-a review. Adv Dent Res 2000 14: 22-28. 18. Rudney JD. Saliva and dental plaque. Adv Dent Res 2000 14: 29-39. 19. Samaranayake LP, MacFarlane TW. Clinical Oral Microbiology. Bristol; Wright,1989. 20. Samaranayake LP. Essential Microbiology for Dentistry. 3rd ed, Edinburgh: Churchill Livingstone, 2002. 21. Birkhed D, Edwardsson S, Andersson H. Comparison among a dip-slide test (Dentocult), plate count, and Snyder test for estimating number of lactobacilli in human saliva. J Dent Res 1981 60: 1832-1841.

22. Joshipura KJ, Douglass CW, Willett WC. Possible explanations for the tooth loss and cardiovascular disease relationship. Ann Periodontol 1998 3: 175-183. 23. Offenbacher S, Jared HL, OReilly PG et al. Potential pathogenic mechanisms of periodontitis associated pregnancy complications. Ann Periodontol 1998 3: 233-250. 24. Kountouras J. Detecting Helicobater pylori. Diagnostic tests for Helicobacter pylori. Gut 1998 42: 900-901. 25. Jiang C, Li C, Ha T et al. Identication of H. pylori in saliva by a nested PCR assay derived from a newly cloned DNA probe. Dig Dis Sci 1998 43: 1211-1218. 26. Vaira D, Holton J, Menegatti M et al. New immunological assays for the diagnosis of Helicobacter pylori infection. Gut 1999 45: 123-127. 27. Kaufman E, Lamster IB. The diagnostic applications of saliva-a review. Crit Rev Oral Biol Med 2002 13: 197-212. 28. Daniels TE. Sjogrens syndrome: clinical spectrum and current diagnostic controversies. Adv Dent Res 1996 10: 3-8. 29. Streckfus C, Bigler L, Navazesh M et al. Cytokine concentrations in stimulated whole saliva among patients with primary Sjogrens syndrome, secondary Sjogrens syndrome, and patients with primary Sjogrens syndrome receiving varying doses of interferon for symptomatic treatment of the condition: a preliminary study. Clin Oral Investig 2001 5: 133-135. 30. Sreebny L, Zhu WX. Whole saliva and the diagnosis of Sjogrens syndrome: an evaluation of patients who complain of dry mouth and dry eyes. Part 1: Screening tests. Gerodontology 1996 13: 35-43. 31. Adam DJ, Milne AA, Evans SM et al. Serum amylase isoenzymes in patients undergoing operation for ruptured and non-ruptured abdominal aortic aneurysm. J Vasc Surg 1999 30: 229-235. 32. Chatterton RT, Vogelsong KM, Lu YC et al. Salivary alpha-amylase as a measure of endogenous adrenergic activity. Clin Physiol 1996 16: 433-448. 33. Li Y, St John MA, Zhou X et al. Salivary transcriptome diagnostics for oral cancer detection. Clin Cancer Res 2004 10: 8442-8450. 34. Boyle JO, Mao L, Brennan JA et al. Gene mutations in saliva as molecular markers for head and neck squamous cell carcinomas. Am J Surg 1994 168: 429-432. 35. Chen DX, Schwartz PE, Li FQ. Saliva and serum CA 125 assays for detecting malignant ovarian tumors. Obstet Gynecol 1990 75: 701-704. 36. Streckfus C, Bigler L, Tucci M et al. A preliminary study of CA153, c-erbB-2, epidermal growth factor receptor, cathepsin-D, and p53 in saliva among women with breast carcinoma. Cancer Invest 2000 18: 101-109. 37. Navarro MA, Mesia R, Diez-Gibert O et al. Epidermal growth factor in plasma and saliva of patients with active breast cancer and breast cancer patients in follow-up compared with healthy women. Breast Cancer Res Treat 1997 42: 83-86. 38. Vitorino R, Lobo MJ, Ferrer-Correira AJ et al. Identication of human whole saliva protein components using proteomics. Proteomics 2004 4: 1109-1115. 39. Ivnitski D SR, Ivnitski N. Hand-held amperometric sensor for saliva and other oral uid-based diagnostics. Analytica Chimica Acta 2004 504: 265-269. 40. Siegel IA. The role of saliva in drug monitoring. Ann N Y Acad Sci 1993 694: 86-90. 41. Aurer A, Aurer-Kozelj J, Stavljenic-Rukavina A et al. Inammatory mediators in saliva of patients with rapidly progressive periodontitis during war stress induced incidence increase. Coll Antropol 1999 23: 117-124.
Correspondence to: Professor Lakshman Samaranayake, Dean and Chair of Oral Microbiology, Faculty of Dentistry, The University of Hong Kong, 34 Hospital Road, Sai Ying Pun, Hong Kong SAR, China. Email: lakshman@hku.hk

Samaranayake: Saliva as a diagnostic fluid