Keywords
COVID-19 - saliva - gingival crevicular fluid
Introduction
The global pandemic of coronavirus disease 2019 (COVID-19) has affected close to 1%
of the world’s population to date. Coronavirus patients, according to the World health
Organization, present with dry cough, tiredness, fever, diarrhea, sore throat, headache,
discoloration of fingers and toes, loss of taste or smell, aches and pains, rash on
the skin, and conjunctivitis. This infection spreads from human to human. The virus
transmission is either direct or indirect. Direct transmission is through droplet
infection, sneeze, or cough, or through contact, such as saliva, ocular contact, or
contact of mucous membranes of the nose and eyes.[1]
[2]
Many published articles and reviews have stated the mouth as the principal source
of infection and also the importance of saliva in diagnosis of the disease. Compared
with nasopharyngeal swabs, saliva is said to be more sensitive to coronavirus nucleic
acid detection.[3]
Oral cavity is intimately related to the pharynx. Hence respiratory infections can
harbor and multiply in the oral cavity and vice versa. Gingival sulcus has been said
to be a microbiological niche of various respiratory diseases.[4]
[5]
This article aims to present a literature review of the association of oral fluids
in diagnosis of coronavirus infection.
Role of Saliva
Coronavirus has been found in various human secreta such as saliva, feces, and urine.
Angiotensin-converting enzyme-2 (ACE-2) receptors, the principal receptors for coronavirus,
are found in high numbers in minor salivary glands of humans.[6] Hence in severe forms of infection due to high viral loads of the virus, it is detected
in saliva, and the destruction of salivary glands is seen in later stages of the disease.[7]
Hyposalivation is the causative factor of dry mouth.[8] Patients having COVID-19 infection present with hyposalivation and dry mouth.[7] Additionally, there is decrease in salivary flow rate with increase in age. Hyposalivation
leads to reduction of antiviral properties and proteins in saliva, which makes the
patient more susceptible to infections.[9] Hyposalivation can be due to medication (e.g., diabetic or hypertension medication)
or other systemic diseases, inflammatory processes, and infections. COVID-19 has also
been found to be more severe in patients above 50 years of age.[10]
[11]
[12] Hence, there might be a possible correlation between the two.
Antibody response to COVID-19 has been widely studied in blood samples of patients.
Total immunoglobulin G (IgG) levels, but not IgA or IgM levels, were found to be higher
in COVID-19 patients compared with controls. This study provides evidence that the
IgG response to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike
persists in the saliva and the serum, and that this response can be correlated between
the two biofluids, particularly for IgG.[13] Isho et al in their study stated that IgA and IgM degrade faster as compared with
IgG. They proved that the same level of IgG in saliva and serum was detected since
the onset of symptoms for a minimum of 3 months.[13]
Viral ribonucleic acid (RNA) could still be detected for 20 days or even longer in
deep throat saliva specimens of one-third of included patients, suggesting the viral
RNA could stay for a longer period of time instead of dying out after antibody application.[14]
Salivary Fluid in the Identification of Coronavirus
Saliva is a popular noninvasive diagnostic tool for various diseases.
The three approaches for collecting salivary sample that have been used to date are
swabs, sputum, and direct collection from the salivary duct.[15]
[16]
[17]
[18] Recommended tests for detection of COVID-19 is nasopharyngeal and oropharyngeal
swabbing; the major disadvantage of these techniques is that they are invasive and
there is poor patient cooperation. On the other hand, saliva collection is noninvasive,
hence has better patient cooperation, especially when multiple tests are required
for monitoring viral load. Due to the risk of bleeding during nasopharyngeal or oropharyngeal
swabbing, saliva collection can be done especially in patients with bleeding and clotting
disorders (e.g., thrombocytopenia). Saliva had 90% consistency rate comparable to
nasopharyngeal swabs in detection of respiratory infections.
To et al conducted a study that reported that deep throat saliva method of diagnosis
of COVID-19 is highly sensitive. They tested the S gene of COVID-19, in which 11 patients out of 12 tested positive when real-time reverse
transcription (RT) quantitative polymerase chain reaction (qPCR) was used.[15]
In another study conducted by To et al, they analyzed the temporal profile of the
virus load and tested for viral RNA. In this study they asked the patient to cough
out a sample early in the morning. This sample consisted of saliva along with nasopharyngeal
and bronchopharyngeal secretions. Total 20 out of 23 patients showed detectable viral
RNA in saliva. The temporal profile showed maximum increase of viral load in saliva
during the first week of symptomatic phase; thereafter decrease was seen.[16]
In the above study, To et al also analyzed viral RNA loads after termination of treatment.
Viral RNA was detected in deep throat saliva after antibody application in one-third
of the study sample for 20 days or more. This showed that viral RNA can persist for
a long time after antibody application instead of dying. A patient after resolution
of symptoms tested negative twice before testing positive again 2 days after the last
negative result. This showed that even after recovery of clinical symptoms, patients
may express viral RNA in saliva for a longer duration. However, they could not prove
whether the virus expressed in saliva after recovery of clinical symptoms was shedding
virus or of infectious nature.[16]
The most accurate test for COVID-19 in its acute stage is RT-PCR, as it is very sensitive
and specific for the virus.[19] However, there are numerous disadvantages of this technique, such as expensive instruments
and chemicals that are needed along with experienced technicians. The time required
to perform the procedure and obtain results is also more as compared with other diagnostic
tests.
After exposure of an individual to the virus, the antibodies can be diagnosed using
various test like enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay.[20]
The first test for COVID-19 was invented in March 2020. Following this, many advances
were seen in this field. By July 2020, U.S. Food and Drug Administration (FDA) approved
around 11 home kits for the diagnosis of COVID-19. Most of these kits work on the
principals of RT-PCR; other technologies used are loop-mediated isothermal amplification,
etc. Rapid antigen home kits are also in the process of being developed.
A decrease in the risk of hospital acquired infections and cross-infection for the
patient as well as medical professional is the major advantage of salivary tests,
as the patients can obtain their own samples for testing. Virus load in sputum and
salivary samples is said to be similar. However, the testing using nasopharyngeal
swab detects the virus longer than that in a salivary sample. The viral load in the
saliva decreases over the duration of the treatment.
The disadvantages of nasopharyngeal swabbing for RT-PCR are the possibility of obtaining
false positive results and the need for repeated testing.
In a systematic review and meta-analysis done by Butler-Laporte et al, they found
that the diagnostic sensitivity for saliva nucleic acid amplification testing (NAAT)
is ~83.2%, which is comparable to that reported for nasopharyngeal swab NAAT. As saliva
collection can be done without specialized professionals, saliva is a very good alternative
diagnostic tool for COVID-19.[21] A systematic review by Bastos et al concluded that nasopharyngeal swabbing remains
the gold standard for COVID-19 testing; however, salivary tests have shown to have
equal sensitivity with the added advantage of low cost.[22] A systematic review done by Czumbel et al found that the test sensitivities for
SARS-CoV-2 were 91% (confidence interval [CI]: 80–99%) and 98% (CI: 89–100%) for saliva
and nasopharyngeal swab samples, respectively. They concluded that nasopharyngeal
swabs have slightly higher sensitivity; however, the difference is not significant.[23]
Another mode of testing of COVID-19 is the use of serum samples. Studies have demonstrated
the presence of COVID-19 RNA in body fluids such as plasma, using diagnostic tests
like RT-qPCR[24]
[25]
[26] or droplet digital PCR.[27]
[28] In all of these studies the presence of viral RNA in blood has been associated with
increased disease severity, as it has mostly been found in patients admitted in the
intensive care unit.[24]
[25]
[26] Therefore, detection of COVID-19 RNA plasma can be considered to be an important
diagnostic tool for critically ill patients, especially in the intensive care unit,
to formulate a better treatment plan for the patient.
Various new salivary diagnostic tests for COVID-19 are currently under trial and being
developed by Nitte University Centre for Science Education and Research, Mangalore,
and Medanta Institute of Education and Research in Gurgaon.
A rapid salivary test for COVID-19 that can provide results in less than a second
is under trial at Sheba Medical Center, Israel. An at-home COVID-19 test, designed
by Stanford researchers to be easy to use and provide results within 30 minutes, will
be the focus of a study funded by the Stanford Medicine Catalyst Program. An at-home
COVID-19 diagnostic test invented by Manu Prakash, PhD, Associate Professor at Stanford
Medicine, is under trial and is expected to give results in 30 minutes and is easy
to use. Huergo et al are developing a diagnostic tool for COVID-19 using magnetic
bead-based immunoassay, which may be a rapid and low-cost alternative to ELISA in
the future.[29]
Rodriguez-Manzano et al have developed a hand-held device for rapid detection of COVID-19,
within 20 minutes. The device can be connected to mobiles for epidemiological survaillence.[30]
There are multiple FDA-approved diagnostic tests for COVID-19 available in the market.
Few of them are, namely, Curative-Korva SARS-CoV-2 Assay, Ubi SARS-CoV-2 ELISA, EliA
SARS-CoV-2-Sp1 IgG Test, RightSign COVID-19 IgG/IgM Rapid Test Cassette.
Role of Gingival Sulcus as a Niche
Oral colonization of respiratory infections has been seen in immunocompromised patients.
The gingival sulcus of the oral cavity harbors numerous bacteria as well as virus.
The gingival sulcus releases various enzymes such as sialidase, hexosaminidase, fucosidase,
and mannosidase, which play a role in modulating the respiratory surfaces and promoting
colonization of respiratory microbes.[19] As coronavirus has showed high concentrations in saliva, there might be a strong
correlation between the oral cavity and colonization of coronavirus in the gingival
sulcus. Furthermore, ACE-2 receptors present in gingiva and salivary glands are the
main receptors to which SARS-CoV-2 binds.
These evidences support the use of gingival crevicular fluid for the identification
of coronavirus ([Fig. 1]).
Fig. 1 Transmission of infection
Conclusion
Oral fluids such as saliva as well as gingival crevicular fluid could be used for
the detection of coronavirus. Both these methods are not invasive, hence can be used
especially in severe cases where multiple tests are done to monitor viral loads. COVID-19
binds to ACE-2 receptors in the body; these receptors are present in the salivary
glands, the oral mucosa, and the tongue in the oral cavity. Hence the patient might
present with symptoms such as ageusia, hyposalivation, and dry mouth. In severe cases
due to increased viral loads in later stages, destruction of the salivary glands may
be seen.
Gingival sulcus is a niche for colonization of various oral as well as respiratory
bacteria. ACE-2 receptors important in COVID-19 infection are found in the gingiva.
It is speculated that as viral loads can be detected in saliva, there is a strong
correlation between viral loads in saliva and colonization in the gingival sulcus.
Hence, oral fluid may be used as a diagnostic tool; however, further studies are required
to prove this correlation.
