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 Table of Contents  
REVIEW ARTICLE
Year : 2020  |  Volume : 32  |  Issue : 1  |  Page : 41-45

Hidden wonders in a spit: Novel technologies for salivary diagnostics


Department of Oral Medicine and Radiology, Babu Banarasi Das College of Dental Sciences, Lucknow, Uttar Pradesh, India

Date of Submission22-Nov-2019
Date of Decision11-Mar-2020
Date of Acceptance12-Mar-2020
Date of Web Publication17-Apr-2020

Correspondence Address:
Dr. Areeba Shahid
Post Graduate Student, Department of Oral Medicine and Radiology, Babu Banarasi Das College of Dental Sciences, Lucknow - 226 010, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jiaomr.jiaomr_192_19

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   Abstract 


Oral cancer is a major contributor to death worldwide. This life-threatening disease still lacks inexpensive screening. Novel strategies for early detection and therapy response are required. Saliva, as “mirror of the body,” reflects body's physiological and pathological state. Saliva is the epitome of a noninvasive, readily available, and easy to collect, transport, and store “biofluid.” Since the emphasis is switching more toward high impact personalized medicine, pioneering “Point-of-care salivary technologies” are being developed. These facilitate biomarker identification, as an alternative to invasive tissue biopsy. The concept that a patient can take a single drop of their own saliva and test it using portable wireless devices like biosensors, actuators, microelectronics, etc., like Oral Fluid NanoSensor Test (OFNASET) for oral cancer detection is being introduced. An upcoming contemporary tool that integrates both therapeutics and diagnostics is “Liquid biopsy.” It is a PCR-based sequencing technique that detects circulating tumor cells (CTCs) and fragments of tumor DNA shed into the bloodstream by cells undergoing apoptosis or necrosis. This review focuses on promising futuristic salivary technologies, which shall bring breakthroughs in terms of precision and efficiency. Oral physicians and oncologists will have ease in detecting and shaping diagnosis of oral cancer in near decade and beyond.

Keywords: Liquid biopsy, point-of-care technology, salivary diagnostics


How to cite this article:
Shahid A, Channaiah SG, Misra N, Narayanappa SM. Hidden wonders in a spit: Novel technologies for salivary diagnostics. J Indian Acad Oral Med Radiol 2020;32:41-5

How to cite this URL:
Shahid A, Channaiah SG, Misra N, Narayanappa SM. Hidden wonders in a spit: Novel technologies for salivary diagnostics. J Indian Acad Oral Med Radiol [serial online] 2020 [cited 2020 Jun 4];32:41-5. Available from: http://www.jiaomr.in/text.asp?2020/32/1/41/282612




   Introduction Top


Oral cancer is a commonly prevailing type of cancers worldwide, showing associate incidence and death rate double as high in men (2.3% and 1.7%, respectively) compared to women (1.2% and 0.8%, respectively). It has a multifaceted etiology associated with several risk factors. Generally, according to late-stage diagnosis, fewer than 50% of patients with oral and pharyngeal cancers survive over 5 years. Lack of awareness among the general public regarding the signs, symptoms, and risk factors for oral cancer, accompanied by a disappointing absence of early detection, are believed to be responsible for the diagnostic delay. In 2005, researchers documented that periodic examination of the oral cavity can result in a declination of mortality rate in high-risk individuals.[1],[2] Tissue biopsy is the gold standard in the diagnosis of oral cancer, but it is invasive, costly, time-consuming, and potentially harmful. This life-threatening disease lacks accurate, efficient, and inexpensive screening tests and requires minimally invasive novel strategies for the early detection.[3]

Point-of-care diagnostics is an evolving paradigm exploring technologies that allow patients and healthcare providers to gain medical information rapidly and conveniently by use of portable devices. It seeks to achieve ”bed-side” diagnosis, removing the time delay that is caused by the conventional workflow of collecting samples and transporting them to a central lab for testing.[4]

Saliva is a conglomeration of a variety of enzymes, hormones, antibodies, antimicrobial constituents, and cytokines. Its composition mirrors that of blood and reflects virtually the entire spectrum of physiological and pathological states. Saliva collection is an easy, fast, cheap, safe, repeatable, inexpensive diagnostic source, does not require specialized equipment, and can be performed at home. There is an emerging need to move salivary diagnostics out of the lab into the clinical practice.[5],[6]

”Point of care” (POC) diagnostics that are relatively new to dentistry can offer quick, uncomplicated, value effective, and correct measurements directly from saliva specimen. For this, an integrated platform-based POC application is necessary, which includes sample processing, detection, and a user-friendly interface.[5]

”Liquid biopsy” is a noninvasive diagnostic tool, based on the detection of circulating tumor cells and components. Saliva can be a potent sample for liquid biopsy. One of the key advantages of studying liquid biopsies is that they provide a personalized snapshot of primary and metastatic tumors at successive time points, providing knowledge of the tumor burden to detect early evidence of recurrence. It provides a molecular profile for each patient and offers the great advantage of being less stressful for the patient.[1],[3]

This review discusses the far-reaching potential of saliva and a broad overview of new frontiers in salivary diagnostics with futuristic perspectives and anticipatory ease for dentists in a chairside screening of oral cancer.


   Saliva as Biomedia Top


Advantages of saliva as diagnostic medium:[5]

  • Inexhaustible biofluid.
  • Saliva collection is noninvasive and inexpensive.
  • Reduced anxiety and discomfort in patient.
  • Less time consuming.
  • Frequent, early-stage screening and risk assessments.
  • Procurement of repeated samples for longitudinal monitoring.
  • For professionals, saliva collection is safer than blood collection, which exposes healthcare providers to risks such as virus contraction.
  • Does not clot.
  • Easy storage and transport.


According to Wong [4,5] and few other authors, it was reported that a biological fluid such as saliva could be used as a diagnostic tool, if it meets the criteria of being easily and noninvasively collected and possesses validated and definitive biomarkers for a specific disease[7].


   Salivaomics Top


There are known five major diagnostic toolboxes of saliva:[6]

  • Proteomics - the study of proteins
  • Transcriptomics - the study of RNAs
  • Metabolomics - the study of metabolites
  • MicroRNA - the study of microRNAs
  • Microbiome - the study of microbiota.



   Modern Technologies in Salivary Diagnostics Top


A.) Liquid biopsy

IN CANCER RESEARCH

Liquid biopsies hold great promise as they enable multiple noninvasive global sampling. Allows a variety of clinical and investigational applications such as early detection, evaluation of early treatment response, monitoring of tumor dynamics, etc., Liquid biopsy has been described as an assessment of the genetic status of cancer tumors by analyzing cell materials.[6],[8] The ctDNA mutant fragments were observed in saliva samples of head and neck cancer patients.[4] If the links between distal cancers and the oral cavity can be thoroughly validated through investigation, it will open a new avenue of clinical utility to effectively and noninvasively diagnose cancers through saliva.

A new technique was developed at the University of California at Los Angeles (UCLA), School of Dentistry, called EFIRM. This process provides results in less than 30 min from a sample of saliva.[4] It enables disrupting and releasing the exosomal contents and on-site monitoring of the released biomarkers for specific cancer. The first step involves pipetting a sample of biofluid onto the surface of a biosensor and applying a pulsed electric field to release molecular content from the exosome. Biorecognition of those molecules is administered concurrently through a series of probes on the surface of the biosensor. This technique allows direct detection of specific mutations in saliva, thus eliminating the need for quantitative PCR. This promises a new launch for the noninvasive detection of tumor-specific proteins and gene mutations in saliva.[7]

Wei et al.[5] explored the detection of epidermal growth factor receptor (EGFR) mutation in saliva with non-small cell lung cancer patients, using EFIRM technology.

Circulating cell-free DNA (cfDNA)

Cell-free DNA can be detected in healthy individuals and malignancy. Under physiologic conditions, nonmutated cfDNA can be derived from nonmalignant cells undergoing apoptosis. It can also be found in saliva. HPV detection using cfDNA could also be of great value for OSCC.[1],[3]

Exosomes

Pan and Johnstone in 1983 explicated the use of “exosomes,” as an alternative in liquid biopsy. Exosomes can be abundantly released into numerous biological fluids including saliva, both in healthy and diseased conditions.

In OSCC, exosomes have shown to be key components in the tumor microenvironment, increasing the transforming growth factor (TGF) signaling pathway, which contributes to the progression of OSCC. Few articles[1],[3] report a study conducted by Zlotogorski-Hurvitz et al., which mentions the morphological chracterisation of oral fluid-derived exosomes in oral cancer.

  • ADVANTAGES


    • Liquid biopsy is a minimally invasive technique. Arneth affirmed[8] liquid biopsy to be a less invasive technique that can be performed more frequently and with lower morbidity than conventional biopsy.
    • Important for taking temporal measurements of tumor burden and for analyzing the possibility of recurrence and malignancy.
    • Ilié and Hofman noted that liquid biopsy provides accurate genetic mutation information. They also noted that liquid biopsies are a source of fresh and reliable tumor derived cell components, uncontaminated by any preservative.
    • Sensitivity and high predictive power.


  • LIMITATIONS and CHALLENGES[8]


    • Aberrations in cfDNA levels among patients may compromise the genuineness and authenticity of the tests.
    • May only identify specific mutations within a limited number of loci, within a single gene.
    • It is an upscale routine laboratory technique. The current costs of liquid biopsies are substantially higher than the conventional biopsies.
    • Liquid biopsy has not yet reached the level of reliability required for widespread implementation in routine clinical diagnostics.
    • The post-processing lab work is much simpler, cheaper, and well-established for conventional biopsy.


  • FUTURE PERSPECTIVES OF LIQUID BIOPSY


  • Liquid biopsy for cancer is in its infancy. Research efforts are required to perform large, prospective multicenter studies. These will investigate the role of tumor cells in oral cancer, monitor tumor alteration and check for progression similar to classical salivary biomarkers, used currently.[1]


B.) Point-of-care platforms

POINT-OF-CARE TESTING

Point-of-care testing can be defined as testing performed close to the patient at the time that care is required.[5] Self-monitoring glucometer, coagulation (INR), and pregnancy testing kits are acclaimed samples of PoCT and became over-the-counter commodities to be sold within the market.[9] Regarding this, the World Health Organization (WHO) enlisted pointers which had the features for designing devices, known as the ASSURED criteria (Affordability, Sensitivity, Specific, User-friendly, Rapid and robust, Equipment, Delivered).[10]

PoCT replaces the specialist testing centers by using salivary samples other than blood and urine.[9] Currently, the amalgamation of emerging biotechnologies and salivary diagnostics has expanded the range of saliva-based diagnostics from the lab-based assay to clinics, as chairside tools for the oral diagnosticians.[5]

REQUIREMENTS FOR SALIVA POINT-OF-CARE PLATFORMS[5]

Accuracy

In saliva, the biomarkers are usually very dilute when compared with blood and urine. The low levels of biomarkers in saliva require the point-of-care tests which detect a small amount of a target molecule in the saliva matrix. Both high-sensitivity and high-specificity detections are highly desired in salivary diagnostics.

Multiplexibility



The use of biomarkers in combination rather than a single one has been shown to improve accuracy, indicating the importance of multiplexing assays.

Robustness



The robustness of the point-of-care platform is determined by its repeatability, intermediate precision, and reproducibility. Parameters for quality control include:

  1. Device performance to assess the instrument's durability, astuteness, and impartiality before, during, and after the analysis of a batch of saliva samples.
  2. Sample batch quality control to monitor assay performance, accuracy, and precision, and to estimate any errors associated with each sample batch.


Convenience and cost

The convenience or user-friendliness of the device is a key factor, especially for untrained operators and resource-limited areas. The saliva collection and processing procedures need to be small, simple, rapid, and robust, and the software should perform the measurements easily. Usually, the point-of care device for saliva must be operated within a humidity range of 10% to 90% and a temperature range of 4°C to 30°C. Most of the point-of-care devices contain a disposable part for an individual test, which determines the price of each test. The biosafety of the disposable part must be considered.

INTEGRATED SYSTEM FOR POINT-OF-CARE TESTING[5]

Saliva collection

Conventionally, there are three methods for collecting saliva which include the following:[11]

  1. salivary rinse
  2. whole unstimulated saliva
  3. whole stimulated saliva.


One of the main advantages for using a salivary rinse is that the specimen is not restricted to the anterior mouth and allows a more complete sampling of the entire oropharynx. The saliva point-of-care platform must provide a collector to collect and process saliva samples in an easy and repeatable fashion.

Several different types of collector have been developed for specific applications, such as the OraQuick® (OraSure, USA) for salivary proteins, Oracollect® (DNAgenotek, USA), and SalivaGene® (Stratec, Germany)[5]

Detection

Current biological sensing techniques require specialized instrumentation and trained personnel. Sample processing in the point-of-care device requires effective, simple, and reliable fluid control.

User interface and data presentation

For the point-of-care application, a user-friendly interface is important to allow untrained personnel to carry out measurements.

INTEGRATED POINT-OF-CARE MULTIPLEXING PLATFORMS FOR ORAL CANCER DETECTION IN SALIVA

A fully functional point-of care platform has been developed in salivary diagnostics (SDx) at UCLA by Dr. David Wong's laboratory, California. This device is a robust electrochemical platform, integrating sample collection and processing, and is capable of performing sensitive and specific multiplexing assays. It can detect both salivary proteins and nucleic acids, and can measure up to eight different biomarkers in a single test in less than 15 min. All the tests are consistently performed under ambient conditions. No sample extraction or amplification is needed. There is minimal manual operation; the operator only needs to insert the sensor and cartridge and push the menu button. Readable diagnostic results are displayed once the testing is complete.[5]

The SDx point-of-care platform has been tested in an Indian cohort study,[5] and the results correlated well with results obtained using tradition ELISA and quantitative PCR assays for protein and RNA biomarkers, respectively. The clinical study assessed two salivary biomarkers for oral cancer: IL-8 mRNA and IL-8 protein. A multiplex assay of these two biomarkers directly from 28 cancer and 28 matched control saliva samples showed significant clinical discrimination. The electrochemical sensor yielded 90% sensitivity and 90% specificity for both IL-8 mRNA and IL-8 protein. With the SDx point-of-care platform, the salivary test will screen and assess the risk of oral cancer, with only test-positive patients being directed to biopsy.

EMERGING POC TECHNOLOGIES WITH SCOPE IN CANCER DETECTION[9]

Besides biomarkers, systems like biochip and biosensor also make up distinct diagnostic modes.

Biosensors

The biosensor may be a bioanalytical device which has the power to mimic any biological material. The first biosensor to be fabricated was an enzyme-based glucose sensor.[12]

Saliva-based biosensors

The application of saliva-based biosensors for exfoliated cells in the oral cavity allows screening and assimilation of potential biomarkers for oral cancer. Weigum et al.[13] constructed a new nano-biochip cellular analysis technique for characterizing oral malignant and premalignant lesions.

Fluorescent Biosensors

Can be used for cancer detection by the use of fluorescent probes in gene expression.

Biological Micro-Electro-Mechanical Systems (BioMEMS)

PoC lab-on-chip systems use simple miniature (BioMEMS) devices for the detection of cells, proteins, microbes, viruses, and DNA in biological specimens. They are based on micro/nanoscale fabrication systems which help in increasing the sensitivity of sensors, increased reliability, increased performance, reduced detection time, and cost-effectiveness.

Smartphone-Based Biosensors

They imitate the same designing of miniature computers, acting as reasonable, handy analytical laboratory devices. Recent advancements in smartphones electronics have made its use as a smart detector and powerful tool in mutation detection.

Microfluids

The field of microfluidics comprises the microscale usage of fluids through small channels. It possesses high potential and is often used for specialized manipulation of samples to concentrate or extract the precise salivary components.

Accurate PoC diagnostics require no prepreparation and screening for biomarker recognition. It is a noninvasive testing, as seen in already-patented devices, like Oral Fluid Nano Sensor Test (OFNASET) for the detection of oral cancer, OraRisk for HPV test, etc.[9]

FUTURE OF POINT-OF-CARE TECHNOLOGIES IN SALIVARY DIAGNOSTICS[5]

Advances in three areas are likely to make the concept of sophisticated point-of-care diagnostic devices practical in the near term.

•First, thedevelopment of microfluidic devices and methodologies, which drastically reduce the fluid transfer errors.

• Second, the advent of many new transduction mechanisms allowing the design of very small and mechanically robust devices.

• Third, improvements in nanoscience. Nanomaterials have shown emerging potential in salivary diagnostics with improved biocompatibility and additional binding sites.

The future application of the integrated spittle collection system embedded in the point-of- care platform will further improve the performance of the currently used ones.


   Conclusion Top


Noninvasiveness is the prime quality required for early detection of diseases. Salivary PoCT diagnostics is replacing the laboratory work and offers competent, quick, and easy automation. The upcoming decade envisions breakthroughs in terms of precision, dynamics, and chairside investigations. Personalized medicine, with the help of battery-powered microfluidic devices, will bring revolution. Moreover, it allows clinicians to be accurate, be more consistent, capture clinical data quickly, provides patient satisfaction, and maintains streamline workflow. Salivary diagnostics' impact on the healthcare system is gigantic, being noninvasive, suitable, and well-credentialed, while introducing bioinformatics will highly improve its standards and performance.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Lousada-Fernandez F, Rapado-Gonzalez O, Lopez-Cedrun JL, Lopez-Lopez R, Muinelo-Romay L, Suarez-Cunqueiro MM. Liquid biopsy in oral cancer. Int J Mol Sci 2018;19:1-15.  Back to cited text no. 1
    
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Fedele S. Diagnostic aids in the screening of oral cancer. Head Neck Oncol 2009;1:1-5.  Back to cited text no. 2
    
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Kahlert C. Liquid biopsy: Is there an advantage to analyzing circulating exosomal DNA compared to cfDNA or are they the same? Cancer Res 2019;79:1-4  Back to cited text no. 3
    
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Aro K, Wei F, Wong DT, Tu M. Saliva liquid biopsy for point-of-care applications. Front Public Health 2017;77:1-7.  Back to cited text no. 4
    
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Wei F, Wong TWD. Point-of-care platforms for salivary diagnostics. Chin J Dent Res 2012;15:1-15.  Back to cited text no. 5
    
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Kaczor-Urbanowicz KE. Salivary Diagnostics. Salivary Glands-New Approaches in Diagnostics and Treatment. Chapter 4 InTechOpen; 2018. p. 51-65.  Back to cited text no. 6
    
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Kaczor-Urbanowicz KE, Carreras-Presas CM, Aro K, Tu M, Garcia-Godoy F, Wong TWD. Saliva diagnostics – Current views and directions. Exp Biol Med 2016;0:1-14.  Back to cited text no. 7
    
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Arneth B. Update on the types and usage of liquid biopsies in the clinical setting: a systematic review. BMC Cancer 2018;527:1-12.  Back to cited text no. 8
    
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Khan RS, Khurshid Z, Asiri FYI. Advancing Point-of-Care (PoC) Testing Using Human Saliva as Liquid Biopsy. Diagnostics 2017;7:1-12.  Back to cited text no. 9
    
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Khurshid Z. Salivary point-of-care technology. Eur J Dent 2018;12:1-2.  Back to cited text no. 10
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Franzmann EJ, Donovan MJ. Effective early detection of oral cancer using a simple and inexpensive point of care device in oral rinses. Expert Rev Mol Diagn 2018;18:837-44.  Back to cited text no. 11
    
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Lakshmi KR, Nelakurthi H, Kumar AS, Rudraraju A. Oral fluid-based biosensors: A novel method for rapid and noninvasive diagnosis. Indian J Dent Sci 2017;9:60-6.  Back to cited text no. 12
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Weigum SE, Floriano PN, Redding SW, Yeh CK, Westbrook SD, McGuff HS, et al. Nano-bio-chip sensor platform for examination of oral exfoliative cytology. Cancer Prev Res (Phila) 2010;3:518-28.  Back to cited text no. 13
    




 

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