Journal of the International Clinical Dental Research Organization

REVIEW ARTICLE
Year
: 2021  |  Volume : 13  |  Issue : 2  |  Page : 101--108

Saliva in coronavirus disease-2019: A reliable diagnostic tool and imperative transmitter: A review


Jayati Pandey, Darshan R Prasad Hiremutt, Amit Mhapuskar 
 Department of Oral Medicine and Radiology, Bharati Vidyapeeth (Deemed to be University) Dental College and Hospital, Pune, Maharashtra, India

Correspondence Address:
Dr. Darshan R Prasad Hiremutt
Department of Oral Medicine and Radiology, Bharati Vidyapeeth Dental College and Hospital, Katraj - Dhankawadi, Pune - 411 043, Maharashtra
India

Abstract

Coronavirus disease-2019 (COVID-19) caused by zoonotic virus severe acute respiratory syndrome coronavirus-2 was first reported in Wuhan, China, in December 2019, in 41 patients with a perplexing pneumonia. Ever since, it has wreaked havoc in the entire world and was declared a pandemic by the World Health Organization (WHO) on March 11, 2020. According to the WHO, 2019-nCoV principally spreads through respiratory droplets and saliva, thus making dental care and other aerosol-generating practices precarious in nature. Saliva is a bio mixture secreted from major and minor salivary glands which plays a vital role in prompt diagnosis and close contact transmission of the disease. In this article, we discuss the role of saliva in the diagnosis and as a potent transmitter of COVID-19 infection.



How to cite this article:
Pandey J, Prasad Hiremutt DR, Mhapuskar A. Saliva in coronavirus disease-2019: A reliable diagnostic tool and imperative transmitter: A review.J Int Clin Dent Res Organ 2021;13:101-108


How to cite this URL:
Pandey J, Prasad Hiremutt DR, Mhapuskar A. Saliva in coronavirus disease-2019: A reliable diagnostic tool and imperative transmitter: A review. J Int Clin Dent Res Organ [serial online] 2021 [cited 2022 Jul 3 ];13:101-108
Available from: https://www.jicdro.org/text.asp?2021/13/2/101/335878


Full Text



 Introduction and Background



Coronavirus disease-2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was first reported in Hubei, Wuhan, China, in December 2019 diagnosed in 41 patients with perplexing pneumonia. Since then, it has wreaked havoc in the entire world and was declared a pandemic by the World Health Organization (WHO) on March 11, 2020.[1]

Saliva is a clear, slightly acidic muco-serous exocrine secretion. Whole saliva is a complex bio mixture of fluids from major, minor salivary glands and gingival crevicular fluid which can play a vital role in prompt diagnosis and close contact transmission of the disease.[2] SARS-CoV-2 affects the upper and lower respiratory tract and subsequently enters the gingival crevicular fluid through circulating blood. It also attaches to the salivary glands through the angiotension-converting enzyme II (ACE-2) receptors; hence, saliva shows positivity toward this virus. Hence, saliva shows positivity toward this virus.[3]

Although the nucleotide sequence of SARS-CoV (China, 2002) and SARS-CoV-2 shows a similarity of 79%,[4],[5] SARS-CoV has a higher mortality rate, whereas the novel coronavirus has established rapid human-to-human transmission such that every infected person further infects at least three individuals or more.[6] With a similar external subdomain of receptor-binding domain (RBD), 2019-nCoV spike shares the same host-cell receptor – ACE2 – with SARS-CoV spike, but in a higher affinity than SARS-CoV spike. ACE2 receptors are present abundantly on the lungs and salivary glands, thus making them probable sites for viral attachment.[2]

 Transmission



The mode of transmission of COVID-19 is primarily via respiratory droplets, saliva, and discharge from the nose, but the virus is also found in sputum and feces. Droplet particles more than 5-10 μm in diameter are referred to as respiratory droplets and less than 5 μm are referred to as droplet nuclei.[7]

Respiratory droplets are an amalgamation of moisture and droplet nuclei of microorganisms which transmit when an infected person talks, sneezes, or coughs. They may enter into the body of a healthy individual via mouth, nose, eyes, or direct inhalation; the sizes of these droplets determine how far they travel by air. Large droplets (diameter larger than 60 μm) settle on objects and individuals in the near proximity of the source of infection, whereas smaller droplets (diameter lesser than or equal to 60 μm) undergo transmission up to a short distance. These small droplets dwindle into droplet nuclei (due to physical and biological decay) and are capable of traveling large distances[8] [Figure 1]. Emerging evidence on SARS-CoV-2 being airborne in a closed environment has also been found.[9] Airborne transmission may be possible in specific circumstances and settings in which procedures or treatments generate aerosols.[7]{Figure 1}

According to the New England Journal of Medicine, SARS-CoV-2 remains viable on plastic and stainless-steel surfaces for about 72 h, 4 h on copper surfaces, and a minimum 24 h on cardboard surfaces, which triggers a great concern regarding items delivered by mail or delivery services.[10] According to a scientific review by Kampf et al., viruses remain present for up to 4 days on glass and wood surfaces, while on other materials such as aluminum, it will survive for about 8 h only.[11] The incubation period of the virus ranges from 2 days to 14 days, 5 days on an average, and the infectious period starts 2 days before the onset of symptoms of the disease. Many infected cases do not show any symptoms of the disease and act as carriers or asymptomatic cases.[12]

 R0 Value (Reproductive Number)



R0 is the average number of secondary infections produced by an infectious case in a population where everyone is susceptible to the infection and it is used to measure the transmission potential of a communicable disease. When R0 is >1, it means that each individual affected by a transmittable disease is expected to infect a number of subjects that increase exponentially with the increase of the R0 value and the disease is expected to spread through the susceptible population. Conversely, when R0 is <1, each case transmits the disease to one or less than one individual, and the disease is expected to die out in the population. Although the concept of R0 is very intuitive, its calculation is based on complex models and may lead to misinterpretations.[6],[13]

Importantly, a disease's R0 value only applies when everyone in a population is completely vulnerable to the disease. This means:

No one has been vaccinatedNo one has had the disease beforeThere is no way to control the spread of the disease.

This combination of conditions is rare nowadays due to advancements in medicine. Many diseases that were deadly in the past can now be contained and sometimes cured.[14]

The basic reproductive number (R0) of COVID-19 has been initially estimated by the WHO to range between 1.4 and 2.5, as declared in the statement regarding the outbreak of SARS-CoV-2, dated January 23, 2020.[15]

According to a recent study published online in Emerging Infectious Diseases, the R0 for COVID-19 has now increased and is a median of 5.7, that's about double an earlier R0 estimate of 1.4–2.5. 5.7 means that one person with COVID-19 can potentially transmit the coronavirus to 5–6 people, rather than the two to three researchers originally thought. Researchers calculated the new number based on data from the original outbreak in Wuhan, China. They used parameters like the virus incubation period (4.2 days) – how much time elapsed from when people were exposed to the virus and when they started to show symptoms. The researchers estimated a doubling time of 2–3 days, which is much faster than earlier estimates of 6–7 days. The doubling time is how long it takes for the number of coronavirus cases, hospitalizations, and deaths to double. The shorter the time, the faster the disease is spreading. With an R0 of 5.7, at least 82% of the population need to be immune to COVID-19 to stop its transmission through vaccination and herd immunity.[16]

Ever since the first case of COVID-19, the D614G mutation in the SARS-CoV-2 spike protein has reduced S1 shedding and increased the infectivity of the virus. This is one of the reasons for the current and sudden increase in the R0 value.[17]

The study authors say active surveillance, contact tracing, quarantine, and strong physical distancing measures are needed to stop the coronavirus from being transmitted.[18] As of July 13, 2020, the R0 value of coronavirus in India had dipped to 1.11.[19]

 Viral Load of Saliva



There are mainly three different pathways by which SARS-CoV-2 can reach the oral cavity and saliva: primarily from the virus being present in the upper and lower respiratory tract from where it enters the oral cavity with the liquid droplets regularly exchanged by these organs.[20],[21] Second, it may reach the gingival crevicular fluid from the bloodstream[22] and finally by infecting the major and minor salivary glands by attaching to their ACE-2 receptors.

Lactic acid dehydrogenase (LDH) is usually liberated during tissue destruction and it can be co-related to the lung damage that takes place in COVID-19 patients.[23] The salivary viral load and LDH levels in the bloodstream were found to be directly proportionate. Thus, suggesting that saliva is not only a bio mixture that could be used for the qualitative detection of SARS-CoV-2, but it also represents a useful tool to follow the course of the illness together with other biological markers.[5]

 Invasion into Host Tissues



Phylogenetic analysis revealed that SARS-CoV-2 is closely related (88%–89% similarity) to two bat-derived SARS-like coronaviruses, namely bat-SL-CoVZC45 (GenBank accession no. MG772933.1) and bat-SL-CoVZXC21 (GenBank accession no. MG772934.1), but it is more distant from SARS-CoV (~79% similarity) and Middle East respiratory syndrome-CoV (~50% similarity).[5],[6]

Once the virus enters the human host, its first step is to attach to the surface and recognize the cell surface receptor of the host cell for invasion.[24] Studies indicate that ACE-2 plays an important role in cellular entry of the virus, thus ACE-2 expressing cells may act as target cells and are susceptible to COVID-19 infection.[21],[25] Besides, ACE-2 receptor was proven to be expressed in 72 human tissues and is abundantly present in the epithelium of the lung,[26] nasal, and oral mucosa. Previous research has proved the expression of ACE-2 in buccal and gingival tissue, epithelial cells of tongue, T-cells, B-cells, and fibroblasts of oral mucosa.[7],[27] Liu et al. analyzed rhesus macaques and found ACE-2 was also expressed in epithelial cells lining on minor salivary gland ducts.[28] The ACE-2 expression in minor salivary glands was greater than that in the lungs, indicating that a target for COVID-19 may possibly be salivary glands. Furthermore, SARS-CoV-2 RNA can be found in the saliva before lung lesions appear. This could account for asymptomatic infections.[29]

Li H. et al. revealed that a putative FURIN-cleavage site reported recently in the spike protein of 2019-nCov may facilitate the virus-cell fusion, which explains the transmission features of the virus.[30] In addition, Ma Y et al. found that the presence of FURIN-cleavage site, which was absent in SARS-CoV, caused distinct clinical symptoms of SARS-CoV-2.[31] Similar results were also reported in Li X.'s study from China.[32]

Immunohistochemistry experiments performed by Bing-peng Lin et al. indicated that the expression level of ACE-2 protein was significantly higher in the lip, tongue, and buccal mucosa, especially the epithelial cells in the basal layer, although the mRNA expression level is not as high. The percentage of FURIN-positive cells in lip, tongue, and gingiva was higher than that of buccal and palatal mucosa[33] [Figure 2].{Figure 2}

 Tests for Severe Acute Respiratory Syndrome Coronavirus-2



Studies on previously confirmed COVID-19 patients show 91% sensitivity for saliva tests and 98% sensitivity for nasopharyngeal (NP) tests.[34]

Real-time reverse transcription–polymerase chain reaction (rRT-PCR) on NP and oropharyngeal (OP) specimens represents the gold standard for the qualitative detection of SARS-CoV-2 infection. The Centers for Disease Control and Prevention (CDC) is now recommending the collection of only NP swabs, although OP swabs remain an acceptable specimen type.[35]

The collection of NP or OP samples requires a close contact between the health-care worker and the infected patient, thus leading to a high risk of cross-infection. During collection of the NP sample, the swab goes almost 7 cm or 2.7 inches inside the nose to reach the nasopharynx,[36] this causes discomfort to the patient and may induce bleeding in the thrombocytopenic patients.[37]

There are several advantages in using saliva specimens for the diagnosis of 2019-nCoV. First, saliva specimens can be provided by the patient easily without any invasive procedures. Therefore, the use of saliva specimens could reduce the risk of nosocomial 2019-nCoV transmission.[36] Cases of 2019-nCoV infection among health-care workers in India were found to be more than 15,200 as of July 18, 2020.[38] Second, the use of saliva will allow specimen collection outside the hospitals where airborne-infection isolation rooms are not available, such as in outpatient clinics or in the community. In the setting where a large number of individuals require screening, saliva would represent a practical noninvasive specimen type. Third, since health-care workers are not required to collect saliva specimens, the use of saliva specimens will eliminate the waiting time for specimen collection, and hence, the results would be available much sooner. This is especially important in busy clinical settings where the number of available staff is limited.[39] Thus, salivary specimens would reduce discomfort to the patient and health hazards to health-care workers during repeated sampling.

SARS in 2002 showed that viral load often peaked at day ten after symptom onset. Thus, early detection and isolation of cases was strategic for infection control and provides the window of opportunity for antiviral therapy to decrease the peak viral load.[39]

 Advantages and Disadvantages of Saliva Sampling [Table 1][40]

{Table 1}

Diagnostic value of saliva

Saliva shows the presence of malic acid, C-reactive protein, lactate dehydrogenase, guanosine monophosphate, and proteins associated with macrophage, platelet degranulation, and complement system pathways. These findings support and promote the usage of saliva biomarkers as a noninvasive approach for patient stratification in COVID-19 disease.[40]

Till now, three approaches have been reported to collect saliva–coughing out deep throat saliva, saliva swabs, and directly from salivary gland duct.[3]

Surprisingly, in two patients at the Circolo Hospital in Italy, the salivary samples proved positive for SARS-CoV-2, while their respiratory swabs showed negative results on the same days, indicating that salivary tests were more sensitive to the presence of the virus.[5]

Coughing out deep throat saliva

A study by To et al. showed that deep throat saliva has a high diagnostic rate for 2019-nCoV. Eleven samples from 12 confirmed COVID-19 patients were collected by coughing up saliva a few days after hospitalization and tested for the S gene of 2019-nCoV using rRT-PCR. 11 saliva specimens were positive for 2019-nCoV out of the 12 patients (91.67%). Six patients offered serial saliva, and five out of them showed a declining trend of virus with prolonged hospitalization.[39]

Another study by the same group used self-collected saliva from COVID-19 patients, tested 2019-nCoV RNA, and analyzed the temporal profile of 2019-nCoV load. For this study, saliva mixed with NP and bronchopulmonary secretions from deep throat was collected by coughing out in the morning. It was found that salivary viral load was highest during the 1st week after symptomatic onset and subsequently declined with time. In one patient, viral RNA was detected 25 days after symptomatic onset.[41]

To et al. also detected 2019-nCoV RNA in saliva after treatment, indicating that viral RNA could still be detected for 20 days or even longer in deep throat saliva specimens of one-third of included patients, suggesting that viral RNA stays a long period of time instead of dying out after antibody application. One patient with completely resolved symptoms was found 2019-nCoV RNA positive again after 2 days of negative results, suggesting that low levels of2019-nCoV RNA could still be excreted in saliva even after clinical recovery.[39]

Saliva in oral cavity

By harvesting oral swabs and testing RNA among 15 COVID-19 patients, Zhang et al. found that 50% were 2019-nCoV RNA positive in oral swabs, 26.7% had positive anal swabs, 40% had a positive blood test, and 20% were serum positive. Among all the positive swab tests put together, most of the positive results were from oral swabs at the early stage, while more positives came from anal swabs at late stage of COVID-19, suggesting that oral swabs may indicate early infection of 2019-nCoV but cannot be used as discharge criteria.[3],[42]

Salivary gland

To rule out contamination of respiratory secretion, Chen et al. collected saliva directly from the opening of salivary gland and found 2019-nCoV nucleic acid, suggesting that salivary glands were 2019-nCoV infected. Out of 31 COVID-19 patients, 13 were nucleic acid positive by OP swab and four of them were positive in saliva. Three out of these four cases were critically ill patients in need of ventilator support, suggesting 2019-nCoV nucleic acid positive in salivary-gland-originated saliva as an indicator of the severity of COVID-19.[3],[43]

Many companies like Ventnostics, USA, are now looking at saliva for rapid, at home testing. Their device, “SalivaSpot,” is currently in development and will act as a rapid COVID-19 testing unit by giving results in 10 min using the patients' saliva sample.

 Clinical Manifestations



Most patients present with fever and dry cough, while some experience headache, sore throat, anosmia, fatigue, shortness of breath, and other atypical symptoms (muscle pain, confusion, diarrhea, and vomiting). Chest computed tomography of patients revealed bilateral pneumonia, with ground-glass opacity and patchy shadows. The undiagnosed cases may be more in number as most patients present with mild symptoms that closely resemble seasonal allergies and common flu.[44]

 Oral Manifestations of Coronavirus Disease-2019



According to a study by Chen et al., the two major oral-related symptoms, dry mouth and amblygeustia, were manifested by a relatively high proportion of COVID-19 patients, suggesting that oral symptoms can also be considered as initial symptoms of COVID-19 infection.[43]

The CDC included recent loss of taste sensation (ageusia/dysgeusia) as an early symptom of COVID-19.[34] Other oral manifestations reported so far include oral unspecific ulcerations (affecting both keratinized and nonkeratinized epithelium), xerostomia, opportunistic fungal infections, recurrent oral herpes simplex virus-1 infection, fixed drug eruptions, and gingivitis.[45] There is no conclusive evidence whether oral lesions associated with COVID-19 are typical of direct viral invasion or occurring as a result of systemic deterioration or following adverse drug reactions. However, the most likely reason is that oral tissues (salivary glands and tongue) show high degree of ACE2 expression and the presence of FURIN (an enzyme that facilitates cellular entry of SARS-CoV-2).[3],[4],[46]

Patients suffering from mild-to-moderate forms of COVID-19 showed a light red tongue with a white coating, whereas those who suffered from severe forms had a purple tongue with a yellow coating. Greasy coating was a significant characteristic of patients with COVID-19. These features of the tongue are known as “covid-tongue.”[47]

Protective measures to minimize saliva-mediated spread of diseases in dental clinics:

Patient screening is most important to avoid COVID-19 transmission in dental clinics. Dental health-care personnel should be familiar with COVID-19 symptoms and able to identify a suspected COVID-19 patient. Future development of saliva-based rapid tests on either SARS-CoV-2 nucleic acid or antibodies has the potential to identify suspected cases in the dental clinic during the pandemic periodPersonal protective equipment is important for infection control in the dental clinic, particularly considering that splatter/droplets contain potential saliva-borne pathogens. Protective goggles or face shields, masks, gloves, and caps should be regularly worn by the practitioner (Meng et al., 2020; Samaranayake and Peiris, 2004) [Figure 3]aA recent meta-analysis has shown that a preprocedural mouth rinse (0.2% povidone-iodine or 1% hydrogen peroxide) can significantly reduce microbial load in dental aerosols, this would be most useful in cases when rubber dam cannot be used. However, clinical studies are still needed to evaluate their capability for reducing the salivary load of SARS-CoV-2 and saliva-contaminated droplets/aerosols during dental proceduresWhen performing intraoral examinations, the use of three-way syringe should be avoided to minimize splatter/droplets during the pandemic period of COVID-19. Intraoral X-ray examination will stimulate saliva secretion and coughing, and thus, extraoral radiographies, such as panoramic radiography and cone-beam CT, could be used as alternatives (Meng et al., 2020)The use of rubber dams can significantly minimize the production of saliva-contaminated splatters, droplets, and aerosols, particularly when high-speed dental handpieces and ultrasonic devices were used. The application of a rubber dam can significantly reduce airborne particles in an approximately 3-foot diameter of the operational field by 70% (Samaranayake, Reid, and Evans, 1989). When a rubber dam is applied, extra high-volume evacuation should be used along with a regular saliva ejector to reduce splatters and droplets (Samaranayake and Peiris, 2004), and four-hand operation is necessary [Figure 3]b. If rubber dam isolation is not possible, manual devices such as Carisolv and hand scalers are recommended for caries removal and periodontal scaling, to minimize saliva contaminationHigh-speed dental handpieces without anti-retraction valves may aspirate and expel debris and fluids during dental procedures. When a rubber dam is not applied, saliva can be aspirated into the handpiece, and saliva-borne microbes may further contaminate the dental unit waterlines, thus causing cross-infection. A study has shown that the anti-retraction high-speed dental handpiece can significantly reduce the backflow of oral microorganisms into the tubes of handpiece and dental unit (Hu, Li, Zuo, and Zhou, 2007). Therefore, the use of dental handpieces without anti-retraction function should be prohibited during the epidemic period of COVID-19 and the use of electrical handpieces is suggested.{Figure 3}

Usually, splatters and droplets can contaminate a 3-ft diameter range, while aerosols produced can spread further and cause long-lasting contamination and potential transmission (Zemouri, de Soet, Crielaard, and Laheij, 2017). A recent study published in the New England Journal of Medicine demonstrated that aerosol and fomite transmission of SARS-CoV-2 is plausible, since coronavirus remains viable and infectious in aerosols for hours, and on inanimate surfaces up to days (van Doremalen et al., 2020). Aerosol-generating dental procedures could spread saliva-contaminated droplets/aerosols to various surfaces and equipment in the dental office, which requires specific disinfection tactics. Good ventilation is critical for the reduction of aerosols in clinical settings. Strict and regular surface disinfection with alcohol or chlorine disinfectant is also important during the outbreak of COVID-19. Saliva-containing waste generated by patients with suspected or confirmed contagious diseases is regarded as infectious medical waste and should be properly disposed of accordingly.[48]

Antibody titer test

COVID-19 serology testing relies on targeted antibodies binding to SARS-CoV-2-specific antigens. If the patient has developed antibodies in their blood against SARS-CoV-2, the corresponding antibodies will recognize and bind to the antigens, indicating past exposure to SARS-CoV-2. The platforms for COVID-19 serology tests on the market today include lateral flow assays, enzyme-linked immunosorbent assays, and chemiluminescent immunoassays. Accurate interpretation of serology tests depends on antigen specificity and on the type of antibody being detected. Viral antigens used to detect antibodies for SARS-CoV-2 are spike protein, nucleocapsid, and RBD. IgM, IgG, IgA, and total antibody count are the primary targets of COVID-19 serology tests.[49]

Saliva-based tests

FDA approved SalivaDirect in August 2020 for emergency use only to Yale School of Public Health, USA. The test for rapid detection of SARS-CoV-2 will further reduce the demand for scarce testing resources.[50]

SalivaSpot by Ventnostics is a rapid saliva-based test which gives results in 10 min. It is still in the development phase and will be able to give three types of results, that is, show if the patient has been infected with COVID-19 previously if currently infected or safe and not infected.[51]

 Conclusion



At the beginning of this pandemic, more weightage was given to breaking the chain and reducing the spread of the virus. But as the virus spreads at an uncontainable rate all over the globe, we now have to focus on safer, effective, and efficient testing and development of a vaccine for the same. Thus, further research is needed to study the role of saliva as an unrealized diagnostic tool as salivary diagnostics may impart a fitting and economic point-of-care platform for nCoV infections.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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