_{What your saliva tells about your health…}

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WHY DROOL IS COOL

One of the most innocuous body secretions that becomes conspicuous only by its absence (as in dry mouth conditions), saliva serves more than being just a lubricant. Apart from its functions as a digestive fluid, buffer- maintaining oral homeostasis and antimicrobial actions, it’s use as a diagnostic fluid has been explored for decades in diagnosing both oral and systemic conditions.

During the pandemic the role of salivary diagnostics was reprised for detecting SARS CoV 2, which could be detected in saliva at high titres. The ease of handling this accessible and abundant secretion makes it an ideal sample for large scale disease screening processes in point of care settings. It also shows promise for prognostic evaluation and monitoring of chronic conditions, explaining the robust research currently dedicated to the development of countless salivary assays.

Saliva- The Ideal screening fluid: A lot of factors suggest that in case of diagnostics, drool is cool…

• Availability: A normal person produces 600 mL of saliva per day allowing sufficient sample collection.
• Ease of acquiring: Saliva collection, being a non-invasive and relatively straightforward process, does away with requirement of skilled technicians or major equipment.
• Easier to store and transport: Unlike blood saliva is relatively easy to store for longer durations and proves to be more cost effective in terms of logistics.
• Safe: Being noninvasive the collection poses minimal risk of cross infections
• Potential to diagnose both systemic and oral disorders: A composite collection of salivary pharyngeal, and gingival crevicular secretions, saliva reflects presence of both oral and systemic disease markers.

Limitations:

There are, however, certain challenges to overcome while using saliva as a diagnostic sample

• Complex composition: Saliva is a composite of diverse organic and inorganic constituents diluted in 99% water. This indicates presence of minute amounts (measurable in picograms, or nanograms) of a hoard of possible markers, that need to be picked up by sensitive analyze
• Variations: Salivary composition undergoes a lot of change in the course of the day depending on whether it is stimulated or unstimulated saliva.
• Contamination: Exposed to the oral microflora, saliva is prone to contamination and salivary proteins are prone to degradation by host derived or bacterial enzymes.

Sample Collection:

There are different methods of collecting saliva depending upon specific sites or type of secretion requiring assessment.

Commercially available collection and storage kits:

• Oragene the most commonly used sophisticated technique uses preservation buffers to protect the integrity of the sample until processing.
• Saligene, an alternative technique utilizes a collection tube into which saliva is expectorated to a predetermined volume following which a plunger is used to cap the tube and simultaneously release the buffer into the specimen which can then be sent for further processing.
• Oracol involves saliva collection through an absorbent foam swab which picks up 1 mL of saliva. Oracol is used in salivary diagnosis of measles, human immunodeficiency virus, hepatitis A and B, mumps and rubella.
• Verofy is a unique method which utilizes high-quality immunochromatographic strips for delivery of immediate results.
• Other commercially available collection kits include Salimetrics, DNA·SAL (Oasis Diagnostics), ORAcollect · (DNA Genotek), Oracol (Malvern Medical Developments).

Scope of Salivary diagnostics:

Where are we? Current status of Salivary Diagnostics

Historically, the use of saliva as a screening tool for cystic fibrosis was identified in the early 1960s, but its full diagnostic potential was discovered three decades later, when studies revealed distinct advantages of saliva over serum.

                Viral Infections

HIV detection saw the earliest use of saliva as a diagnostic fluid. An FDA-approved, commercially available HIV test, OraSure®, detects salivary antibodies against the p24 antigen of HIV. It is reported to have a sensitivity of 99.3%, specificity of 99.8%.

Other viruses which could be detected in saliva by rapid tests include hepatitis A, hepatitis B, dengue, Ebola, Zika, measles, mumps, rubella and most recent- SARS CoV2 RNA

Oral Microbiome

Phylochip™ and Bactochip™, employ rapid, high-output NGS and 16S rRNA microarrays that enable to better define the composition of the human oral microflora. Apart from well-established role in diagnosing oral conditions like caries activity, periodontitis and oral cancer, deviations in the oral microbiome help indicate risk for systemic conditions like Crohn’s, pancreatic cancer and even obesity.

Malignancies

A prototype device called electric field-induced release and measurement (EFIRM) has been developed by David Wong of UCLA and his colleagues, that can accurately detect mutational biomarkers using a drop of saliva for a malignancy called non-small cell lung cancer. Wong calls this concept of cancer detection: LIQUID BIOPSIES. His team are working on broadening the scope of this technique to include diagnosis of other oral and systemic cancers. According to Wong, “If such mutations can be picked up through screening of saliva, he said, it could offer physicians and patients an opportunity for early intervention and treatment of cancers that have a five-year survival rate of about 60%.”

Drug abuse and therapeutics:

Gas chromatography or salivary analysis by or the ELISA technique can be used for cigarette consumption detection, organic substances detection, heavy metal intoxication detection, and hormone detection.

Atomic absorption spectroscopy or mass spectroscopy techniques can be used to detect salivary metals like Cadmium, Lead, Mercury, Nickel, Zinc, etc.

Hormone Monitoring:

RIA is used to detect the presence and determine the quantity of testosterone, progesterone, estradiol cortisol, and cortisone in saliva. Of these, cortisol is the most studied and used test as several studies correlate salivary cortisol with pain or stress in both humans and animals

Concept of Salivaomics

Salivaomics involves genomics, transcriptomics (salivary RNA molecules and other discriminatory biomarkers), proteomics (salivary proteins), metabonomics and microRNA (miRNA) analysis of saliva. Professional salivaomics knowledge base (SKB) was set up by Wong. Precise molecular methods and nanotechnology are used to overcome the limitations concerning low concentration of analytes compared to blood. Current research focuses on establishing and correlating these biomarker levels with health and disease.

Recently, Bendifallah S et al conducted a prospective ENDO-miRNA study to analyze the human miRNome to differentiate patients with and without endometriosis and to define a saliva-based diagnostic miRNA signature for endometriosis with an internal cross validation. Their results supported the use of a saliva-based diagnostic miRNA signature for endometriosis in the diagnosis care pathways after an external validation to confirm these results.

Point of care diagnostics:

A sample as easily accessible as saliva can help bring the pathology lab right at doctors chairside or the patient’s bedside. This is the gist of point of care diagnostics, and the pandemic, with all its upheavals, brought such point of care diagnostic evaluations at the most primary levels of healthcare.

With the decided advantage of easy access in procuring salivary samples the researchers aim to extrapolate its uses for continuous monitoring of systemic status

Lab on a chip concept (LoC)

Put simply, LoC is a device which is capable of scaling the single or multiple laboratory functions down to chip-format integrating the principles of fluidics, electronics, optics and biosensors. The size of this chip may range from millimetres to a few square centimetres.

Welcome to the future:

Imagine a chip installed into your tooth, nestled as comfortably as any dental filling would be, constantly picking up molecular signals in your saliva and wirelessly giving digital readouts on your smart watch/phone.

Biosensors:

Biosensors are small analytical devices, that combine a biological element with a physicochemical component, generating a measurable signal aiming to detect a biological analyte.

Halima and colleagues developed an immunosensor for the detection of Cytokine Tumour necrosis Factor-α (TNF-α), an important inflammatory biomarker. The system involves an Ion Sensitive Field Effect Transistor (ISFET) with an anti-TNF-α antibody functionalized on the sensor’s surface. TNF-α was detected in saliva samples with a LOD of 1 pg/mL.  It could serve as a valuable tool to confirm risk for and monitor a host of inflammatory conditions.

To conclude:

We may appear to be tantalisingly close to the future that advanced salivary diagnostics promise to usher, but we have a long way to go before the tests fulfil all the criteria of an ideal diagnostic test summarised as ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free and Deliverable to end-users). Given the composite nature of saliva, and multifactorial aetiology of most diseases the translation of test result to accurate diagnosis needs a tremendous amount of research.

Like any diagnostic aid comes with the disclaimer that reminds clinicians to correlate the results clinically, the results of salivary diagnostics need to be treated with caution.

In the 5^th International Saliva Summit of India ‘SALSI 2023’ held in January this year, Dr Gargi Roy Goswami, Director, KROYNAS Pvt. Ltd., Chennai, India and Chairman, SALSI concluded her keynote address on a hopeful yet grounded note-

“Despite the challenges (in salivary diagnostics), research in this field holds great promise for advancing our understanding of oral health and developing new and more effective treatments. Interdisciplinary education and evidence-based research are crucial to achieve this goal.”

References:

1. Jayachandran, Sadaksharam. Review of salivary diagnostics – A current scenario. Journal of Indian Academy of Oral Medicine and Radiology 32(3): p 205-208, Jul–Sep 2020. | DOI: 10.4103/jiaomr.jiaomr_159_20
2. Yoshizawa t al Salivary Biomarkers: Toward Future Clinical and Diagnostic Utilities Clin Microbiol Rev.2013 Oct; 26(4): 781–791. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3811231/
3. https://www.thermofisher.com/blog/life-in-the-lab/5-benefits-of-saliva-testing-why-its-the-next-big-thing-for-in-vitro-diagnostics/?bid_pca_aup_ro1_co_1434_pjt8107_ext1905_0s0_blg_op_awa_kt_s00)_salivadiagnostic12
4. Shah S, Salivaomics: The current scenario J Oral Maxillofac Pathol.2018 Sep-Dec; 22(3): 375–381. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6306596/
5. 5th International Saliva Summit of India
6. Lino C et al Biosensors as diagnostic tools in clinical applications Volume 1877, Issue 3, May 2022, 188726
7. A guide to aid the selection of diagnostic tests Bull World Health Organ.2017 Sep 1; 95(9): 639–645.
8. Gupta t al Lab-on-chip Technology: A Review on Design Trends and Future Scope in Biomedical Applications, International Journal of Bio-Science and Bio-Technology Vol.8, No.5 (2016), pp. 311-322.

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