Salivary biomarkers in COVID-19 patients

ABSTRACT

The use of salivary biomarkers in diagnosis, treatment, and overall prognosis of coronavirus disease 2019 (COVID-19) has been developed recently. Salivary biomarkers are extremely promising as they are fast to obtain and involve noninvasive collection of specimens. Monitoring patients in real time is necessary in this pandemic. Saliva is another biofluid with major advantages at the molecular level. Methods that detect viral presence in the host secretions measure the current infection by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), whereas the detection of human antibodies against SARS-CoV-2 evaluates the past exposure to the virus. There is an urgent need to increase the active research for the detection of SARS-CoV-2 in saliva because diagnostics may provide a reliable and cost-effective method and is suitable for the fast and early detection of COVID-19 infection. Salivary biomarkers have a potential to be a vital guide in determining coronavirus disease. Many people still do not get results of COVID-19 tests due to imbalance between supply and demand at large testing centers. The use of saliva has various advantages compared to collection of nasopharyngeal swabs. New techniques should be developed for detecting salivary biomarkers that help in diagnosis of COVID-19.

Introduction

The coronavirus disease pandemic has defined the 21^st century. In late 2019, the respiratory tract infection coronavirus 2 appeared in the Chinese city of Wuhan.[1] Coronavirus disease is a new viral pneumonia that is extremely transmissible all over the world. The severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) RNA is single stranded. The sickness is caused by a chiropteran-derived enveloped coronavirus with a high mutation rate.[2] Spread through air, surface contact, and even fecal–oral transfer from one person to another are responsible for the spike in infected cases and the widespread occurrence of the corona disease. Assays of viral-laden reverse transcription polymerase chain reaction (PCR) sputum and swabs from the oropharyngeal and nasal cavities are used to determine coronavirus disease 2019 (COVID-19) case positivity.[3] Since swab collection techniques are intrusive and linked with a higher risk of cross infection, researchers are currently focusing on the utilization of biofluids in the diagnosis of COVID-19.

Saliva, the primary head and neck biofluid, has been the preferred medium and is emerging as a clinically useful tool due to its advantages such as easy collection and management of specimens for noninvasive detection of salivary biomarkers, which do not require a specialist training. Salivary immunoglobulin A (IgA) represents an important immune biomarker secreted by healthy people and patients in different clinical conditions, or in response to acute stress and exercise. In the oral cavity, it has a key role among mucosal defense proteins, playing a central role in the first line of defense against the adhesion of pathogens and their penetration into the tissues.

Biomarkers are biological indicators of health. They are objectively tested and analyzed as an indicator of ordinary biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention.[4] Saliva is a kind of biofluid comprising factors such as biomarkers that include cytokines, DNA and RNA molecules, circulating and tissue-derived cells, and extracellular vesicles (EVs). Their study may provide important information for early diagnosis.

Salivary levels of tumor necrosis factor-α (TNF-?), interleukin?1?(IL?1), interleukin?4?(IL?4), interleukin?6?(IL?6), and interleukin?8?(IL?8) have been identified as significant indicatorsα), interleukin-1 (IL-1), interleukin-4 (IL-4), interleukin-6 (IL-6), and interleukin-8 (IL-8) have been identified as significant indicators.[5]

Saliva, a Diagnostic Tool

Saliva of humans is transparent with a mild acidity (pH = 6.0–7.0). It contains a combination of secretions from several salivary glands, which include the parotid, submandibular, and sublingual glands and other minor glands underneath the mucosa of the mouth and the gingival crevicular fluid (GCF). Salivary gland examination has evolved into a sophisticated science and is performed as a subset of the vast field of molecular examination. Saliva is used in screening and diagnosis, as it is a conveniently available specimen that may be obtained using noninvasive techniques and includes various hormones, medicines, and antibodies. Molecular examination, a wide range of fields, including drug development, plays a key role in the identification of salivary biomarkers for the diagnosis of oral and systemic disorders.[6]

Role of Salivary Biomarkers in COVID-19 patients

Mucosal immunity, especially by secretory IgA, plays a key role in host defense in the fight against respiratory infections, and findings show that it may also play a role in COVID-19. Individually and collectively, salivary IgA, a readily accessible marker of mucosal immunity, may be a useful indicator of immune responses, illness gravity, clinical risk, and herd immunity. Saliva can be utilized as a biomarker for ongoing patient evaluation as well as surveillance of the public, due to its nonintrusive nature and convenience for grouping.[6]

Mechanism that is based on the system

Abundant serum peptides and proteins accompany the saliva that is secreted from the parotid gland, submandibular, and sublingual glands. Blood-borne chemicals are transported via the walls of salivary glands into the fluid between the interstitial spaces, from which secretion into the intraglandular ducts occurs. Moreover, depending on the physical characteristics of the individual, the mechanism of biomolecule secretion involving acinar or ductal cells of the salivary epithelium can involve intracellular or paracellular passive diffusion and active transport, as well as transcytosis.

Intracellular diffusion of COVID-19–induced biomarker begins at the ductal or acinar cells’ basal membrane, from where it undergoes cytoplasmic switch to the apical (luminal) cell membrane and is dumped into ducts immediately as part of the biofluid secretion. Paracellular diffusion of associated molecules involves ultrafiltration following insulin sensitive factor (ISF) emptying across the tense connections between the specialized salivary gland cells for subsequent release into ducts. Serum disease-preferential chemicals are transferred by EVs from the basal to apical cell membranes of the acinar cells for vesicular transfer into saliva. This secondary secretion of serum molecules occurs as a result of the discharge of GCF into saliva. Heightened hydrostatic pressure in the circulation of microorganisms causes dilation of the gingival arteriolar vasculature, loosening of endothelial cell connections, and a rush of blood-based illness outcome indicators into the GCF and, finally, into the saliva as a result. The leaking of serum biomarkers into saliva can also be caused by direct blood contamination of saliva.

Mechanisms with a local (intraoral) focus

Excess glandular protein output and an influx of proinflammatory cytokine and chemokine proteins into saliva ensue from COVID-19 fusion, entrance, and lysis of salivary gland cells that are specialized and oral epithelial cells, as well as local inflammation. Salivary gland acinar and ductal cells release microvesicular buddings and/or exosomal releases directly into biofluid harboring antiviral microRNAs (miRNAs) and proteins.[7]

Pathway for Secretion of Biomarker in Saliva of COVID-19 Patients

Figure 1 shows the pathway for secretion of COVID-19 biomarker in saliva.[7]

Figure 1.

Contributory factors for detection of salivary biomarkers in COVID 19 patients

Salivary Fluid as a Biomarker for COVID-19 Diagnosis and Detection

Utilization of saliva as a screening tool and as a quick diagnostic instrument allows for the identification of antibodies, cytokines, chemokines, and other bioanalytes, in addition to the detection of the virus. The role of salivary biomarkers at the point of care has highlighted the role of higher-level advancements, such as micro/nano electrosystems that are physical, paper-based techniques, fluorescent biosensors, photometric and electrochemical approaches, sequence analysis of RNA, liquid biopsy, and electrical field generation and measurement method, in featuring saliva as a medical examination fluid for the identification of coronavirus biomarkers.[8] Saliva can be tested for markers of inflammatory development, such as cytokines and chemokines. The results have suggested it to be helpful for diagnosis and treatment of both oral and systemic disorders. As a result, examining inflammation-related biomarkers in saliva can be used to develop an inflammatory outline of COVID-19. Some established indicators, such as C-reactive protein (CRP), malic acid, guanosine monophosphate, lactate dehydrogenase, and proteins linked to macrophage, platelet de-granulation which may be seen in saliva and are visible in this research. These findings substantiate the use of saliva-based determinants of metabolism of protein, and lipids as a noninvasive method for COVID-19 illness the sufferer classification.[9,10] Metabolomics is a technique for analyzing small molecules based on the cells, tissues, or liquids that have a metabolic profile. Biomarkers are molecules that are used in clinical practice to determine the stage of a disease in coronavirus patients. The concentration levels of IL-6 and IL-10 in the serum are used as determinants of disease progress. Biomarkers in saliva could be utilized to diagnose COVID-19 accurately and to identify disease prevalence, identify exposure, and forecast the severity of disease.[11]

Methods Determining Salivary Biomarker

Enzyme-linked immunosorbent assay

Antibodies found in animals in response to specific biomarkers are commonly utilized in enzyme-linked immunosorbent assay (ELISA). A particular epitope of antigens binds to the polyclonal antibodies (pABs) immobilized on the surface. The enzyme-linked soluble antibody (sAB) binds to the other epitopes to form a sandwich-like complex. The antigen–pAB binding is identified by measuring the activity of the enzyme that converts the substrate into a colored product. Biomarker proteins in serum and body fluids can be detected and quantified using ELISA.[5]

Two-dimensional gel electrophoresis

The isolation of proteins depending on their molecular weights is commonly accomplished using gel electrophoresis. Depending on the isoelectric point mass and the molar mass of the various proteins, 1D or 2D gel electrophoresis may be used to separate the proteins from the material. The drawbacks of 2D gel electrophoresis include the need for a high number of samples and the technique being a time-consuming multistep technique.

Surface-enhanced Raman spectroscopy

Surface-enhanced Raman spectroscopy (SERS) is a spectroscopic vibration method that may be utilized to identify biomarker proteins on or near plasmonic nanostructures’ surfaces. The dispersed cross sections for molecules immobilized on the metallic structure are critical in SERS for biochemical experiments, clinical diagnosis, and environmental monitoring to be successful. It is important to address the shortcomings of SERS, such as the requirement for expensive apparatus and a lack of a better knowledge of the mechanics and fundamental principles of SERS, in order for it to be used directly in clinical settings.

Fluorescence-based detection

Fluorescence detection is the most extensively utilized optical detection technology in biological applications. The fluorescence-based detection of biomarker proteins employs a straightforward quantification procedure, large-scale sample detection, and very simple biomolecule labeling with fluorescent tags.

Mass spectrometry

Recent advancements in mass spectrometric technology for biomarker detection might have a significant influence on illness detection and treatment. We may investigate a salivary proteome in minute detail using mass spectrometry (MS). Modern MS can identify the presence or absence, degree of expression, and posttranslational changes of many biomarkers in a salivary proteome that has been changed by illnesses or therapies.

Applications

Saliva in diagnosis of disease

The notion of saliva omics was introduced in light of the significant progress achieved in salivary investigations. Genomic, transcriptomic, proteomic, metabonomic, and miRNA analysis are all part of saliva omics. With the creation of exact molecular techniques and nanoscience, the difficulties of using saliva for the findings due to its low analyte amount relative to blood have been resolved.

• 1) Lung cancer: Alterations in the epidermal growth factor receptor (EGFR) serve as a tumor-specific biomarker for non-small cell lung tumor. A novel core technology involving electric field–triggered release and measurement that relies on an electrochemical sensor with multiplexing to find out EGFR alteration in physiological fluids has been proven to be successful for detecting EGFR alteration in the saliva of a patient. This suggests that proteome biomarkers may hold the key to early lung cancer detection and prognosis.[12]

• 2) Cardiovascular diseases: Cardiovascular diseases (CVD) refer to a set of heart and blood vessel problems that encompass conditions including atherosclerosis, myocardial infarction, and coronary heart disease. CRP, myoglobin (MYO), creatinine kinase myocardial band (CKMB), cardiac troponins (cTn), and myeloperoxidase (MYO) are all salivary biomarkers of inflammation in the body.[13]

• 3) Diabetes: Diabetes is a metabolic condition that affects the body’s capacity to handle blood glucose due to low insulin production or insulin dysfunction. A positive link was found between α2-macroglobulin and HbA1c, which indicated that the levels of α2-macroglobulin in the saliva could reflect insulin sensitivity in patients with type 2 diabetes mellitus.[14]

• 4) Viral infection: Currently, salivary indicators such antigens and antibodies, as well as viral DNA and RNA are used to diagnose viral infections.

Coronavirus disease 2019

Coronavirus disease is a kind of viral pneumonia that has its origin in the Chinese city of Wuhan. Coronavirus 2 causes severe acute respiratory disease. COVID-19, like SARS-CoV-2, may efficiently spread between people through droplets and fomites when the infector and infected are in close unprotected contact. Fever, cough, breathlessness, pain in muscle, less leukocyte counts, and radiographic proof of pneumonia are all evidence of COVID-19 infection.

Some viral subtypes have been found in the saliva for up to 4 weeks after onset, implying that a technology that is noninvasive for immediately detecting indicators utilizing saliva might improve illness diagnosis. Only 28% of COVID-19 patients generated sputum from the lower respiratory tract, indicating a notable restriction in sample a vailability for diagnostic evaluation. Saliva has previously been shown to have a higher degree of accuracy (higher than 90%) in detecting respiratory viruses, including coronaviruses, when compared to nasopharyngeal specimens. Saliva is a feasible biosample for coronavirus evaluation, when compared to nasopharyngeal or oropharyngeal swabs. The presence of coronavirus in patients’ saliva indicates the possibility of a salivary gland disease. On the other hand, the salivary specimens contain secretions going down from the nasopharynx or rising up from the lungs, as well as saliva released by major and small salivary glands.

Saliva would be a viable nonintrusive sample type in a situation when a large number of people need to be screened. salivary sample will reduce the amount of period it takes for assessment of the outcome. Diagnostic capacity of saliva for CORONAVIRUS and for viral propagation is critical for developing speedy diagnostic tests and effective preventative tactics.[15]

Challenges in Using Salivary Biomarker for Diagnosis of COVID-19

However, our knowledge of the scope of salivary analysis of COVID-19 infection is inadequate at the moment. Simple experimental designs and modest sample numbers are expected in research examining ways of diagnosing COVID-19 with saliva. When evaluating biomarkers in the saliva associated with coronavirus infection, other factors such as collection of saliva and its laboratory processing should be carefully evaluated. Because studies have indicated the presence of viral RNA in the saliva of asymptomatic and presymptomatic patients, evaluating the salivary load in asymptomatic carriers to find the extent of infection and the future test threshold is crucial.

When saliva is utilized to diagnose COVID-19, a number of limitations and problems are encountered. Because posterior oropharyngeal saliva and coughed-up saliva can have secretions from the posterior nasopharynx and salivary glands, as well as respiratory secretions from the tracheobronchial tree, certain results should be interpreted with caution. For future investigations, an extra uniform specimen collection ought to be designed and carried out. Due to a paucity of data in the research, no conclusions can be drawn about the likely impacts of these confounding variables on the usage of saliva in COVID-19 diagnosis.[15]

Conclusion

In Wuhan, Hubei (China), a pneumonia outbreak with an unknown source had surfaced. Since then, the number of COVID-19–infected patients exploded, which led the World Health Organization to proclaim a public health emergency of worldwide concern. The presence of coronavirus in the saliva of COVID-19 patients has been verified. Salivary samples enable various diagnostics techniques, such as examination of biomarkers that could be employed in quick diagnostic devices, in addition to the precise discovery of the pathogen. Alternatively, valuable biomarkers could be produced naturally in the salivary glands and in the case of COVID-19 infection, there is a paucity of knowledge about the salivary biomarkers that could be exploited for diagnosis. Worldwide efforts should be made to develop COVID-19 detection and monitoring techniques based on salivary biomarkers.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.