Original Article

Influence of Saliva Collection Method on the Detection of Severe

Acute Respiratory Syndrome Coronavirus 2 (SARS‑CoV‑2) and
Immunoglobulin G (IgG) Antibodies in the Saliva: A Cross‑Sectional
Study
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Supriya Kheur, Avinash Sanap1, Chandrashekhar Raut2, Madhura Shekatkar, Avinash Kharat1, Madhusudan Barthwal3,
Jitendra Bhawalkar4, Mohit Kheur5, Ramesh Bhonde1

Department of Oral Background: Although the nasopharyngeal swabs (NPS) are considered as the gold
nYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC1y0abggQZXdgGj2MwlZLeI= on 03/06/2024

Abstract
Pathology and Microbiology,
Dr. D. Y. Patil Dental College
standard specimen for the clinical diagnosis of severe acute respiratory syndrome
and Hospital, Dr. D. Y. Patil coronavirus 2 (SARS‑CoV‑2) virus in the coronavirus disease 2019 (COVID‑19),
Vidyapeeth, 1Regenerative they pose several limitations such as the high risk of exposure, discomfort to
Medicine Laboratory, the patients, and requirement of trained healthcare professionals. Aim: This
Dr. D. Y. Patil Vidyapeeth, study aimed to investigate “saliva” as an alternate source and the influence
2
Central Research Facility, of the method of saliva collection on the sensitivity of SARS‑CoV‑2 detection.
Dr. D. Y. Patil Medical
College, Hospital and
Materials and Methods: In this cross‑sectional study, patients were screened for
Research Centre, the COVID‑19 infection with NPS. Saliva was collected from the same patients by
Dr. D. Y. Patil Vidyapeeth, four different methods (expectoration, drooling, gargling, and using salivary swabs)
3
Department of Respiratory and stored at 80°C. Saliva samples of the patients who were detected positive
Medicine, Dr. D. Y. Patil for SARS‑CoV‑2 were analyzed for viral load by RT‑qPCR and immunoglobulin
Medical College, Hospital G (IgG) levels by ELISA. Results: Out of 350 patients screened, 43 patients
and Research Centre,
4
Department of Community
were included in the study, which were found to be positive for COVID‑19 as
Medicine, Dr. D. Y. Patil evidenced by RT‑PCR in the NPS (positivity rate‑12.2%). Expectorated saliva
Medical College, Hospital exhibited 78.5% sensitivity and drooling method showed 22.2% sensitivity,
and Research Centre, whereas the salivary swab and gargling method yielded 21.42% and 16.66%
Dr. D. Y. Patil Vidyapeeth, sensitivity, respectively. Furthermore, the sensitivity of SARS‑CoV‑2 detection was
Pimpri, 5Department of reduced to 18.1% and 0.0% in the saliva collected by salivary swab and gargling
Maxillofacial Prosthodontics,
M. A. Rangoonwala College
method above the cycle threshold value 25.0 (NPS). Conclusion: Interestingly,
of Dental Sciences, Pune, salivary IgG showed better concordance with the viral load as compared to the
India serum IgG (R20.23 vs 0.04, P = 0.044). Expectorated saliva is a better specimen
as compared to the drooling, gargling, and salivary swabs for SARS‑CoV‑2 viral
detection for the clinical diagnosis of COVID‑19.
Submission: 24‑01‑2023,
Decision: 26‑03‑2023, Keywords: Diagnosis, IgG antibodies, nasopharyngeal swab, saliva,
Acceptance: 06-04-2023,
Web Publication: 19-02-2024 SARS‑CoV‑2

Introduction Address for correspondence: Dr. Supriya Kheur,

R apid and accurate diagnosis of the novel coronavirus

disease 2019 (COVID‑19) is of paramount
importance as a central controlling measure of the
Department of Oral Pathology and Microbiology, In
Charge – Regenerative Medicine Laboratory, Dr. D. Y. Patil
Dental College and Hospital, Dr. D. Y. Patil Vidyapeeth Pimpri,
Pune ‑ 411 044, Maharashtra, India.
outbreak in healthcare centers and the community.[1] E‑mail: [email protected]
Nasopharyngeal (NP), as well as oropharyngeal (OP),
This is an open access journal, and articles are distributed under the terms of the Creative
swabs are preferred for the clinical diagnosis of Commons Attribution‑NonCommercial‑ShareAlike 4.0 License, which allows others to
remix, tweak, and build upon the work non‑commercially, as long as appropriate credit is
given and the new creations are licensed under the identical terms.
Access this article online
Quick Response Code: For reprints contact: [email protected]
Website:
https://journals.lww.com/mjdy
How to cite this article: Kheur S, Sanap A, Raut C, Shekatkar M, Kharat A,
Barthwal M, et al. Influence of saliva collection method on the detection
DOI: of severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) and
10.4103/mjdrdypu.mjdrdypu_87_23 immunoglobulin G (IgG) antibodies in the saliva: A cross‑sectional study.
Med J DY Patil Vidyapeeth 2024;17:52-60.

52 © 2024 Medical Journal of Dr. D.Y. Patil Vidyapeeth | Published by Wolters Kluwer - Medknow
 Kheur, et al.: Saliva collection methods for detection of SARS‑CoV‑2 virus in saliva

severe acute respiratory syndrome coronavirus were advised SARS‑CoV‑2 testing before the surgeries
2 (SARS‑CoV‑2). However, there are several as by the surgeons. Only SARS‑CoV‑2 positive patients
inconveniences associated with NP swabs, one of which diagnosed with reverse transcription-polymerase chain
is the close contact of healthcare workers with patients, reaction (RT-PCR) testing by NP swabs as per the
posing a high risk of viral transmission. Further, OP or standard criteria were included in the study.
NP swabs are uncomfortable for patients and frequently
Sample collection procedures
cause pain and discomfort and, in some cases, might
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Nasopharyngeal swab (NPS): NPS was collected as per

cause bleeding.[2] Additional complications such as a
the guidelines provided by the center for disease control
retailed swab, epistaxis, cerebrospinal fluid leakage as
and prevention (CDC).[22] Sterile synthetic fiber swabs
well as sneezing reaction while collecting the samples
with a flexible shaft were used by trained healthcare
are reported which compromise the safety of patients
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personnel for NPS collection. The swab was inserted

and healthcare workers.[3]
into the nasopharynx through the nostrils until resistance
Recently, several studies have explored biological was encountered. The swab was kept as it is for a few
fluids such as saliva as a source for the detection of seconds and gently rubbed and rolled for several seconds
SARS‑CoV‑2.[4-14] It has been postulated by various until it absorbed nasal secretion. The same procedure
authors that the use of saliva may prove more accurate was applied to the other nostril. The swab was then
in the detection of SARS‑CoV‑2 and might prove immediately kept in a viral transport medium (VTM).
more beneficial over NPS.[15-19] Furthermore, salivary
immunoglobulin G (IgG) levels have been investigated Saliva: All the patients were instructed not to eat/drink
as a surrogate measure of systemic immunity for at least 2 h before the sample collection. Saliva was
against SARS‑CoV‑2.[10] Several investigations have collected by four different methods on the basis of origin,
demonstrated that saliva can identify SARS‑CoV‑2 with biological location, and exposure to the respiratory
a sensitivity of more than 80%.[4,5,9] Some studies, on secretions from the lungs. Salivary samples were collected
the other hand, have indicated lesser sensitivity (ranging between 10.30 and 11.30 am. Patients were supervised
from 25% to 70%).[20,21] Furthermore, research on the and assisted by the virologists and laboratory personnel
relative viral load present in saliva compared to the NPS from Dr. D. Y. Patil Dental College and Hospital,
has yielded conflicting mixed results.[6] Pune (India) for appropriate sample collection. The
auxiliary staff were from the COVID center who were
Therefore, to understand these variations in the given the approval for handling the coronavirus‑infected
outcomes of saliva‑based diagnosis of COVID‑19, the samples as per the national regulatory body.
current study hypothesized that the method of saliva
specimen collection might influence the sensitivity and Unstimulated Saliva: Passive unstimulated whole
concomitant SARS‑CoV‑2 viral load. In this study, the saliva (1–2 ml) was collected in a sterile and leak‑proof
sensitivity and relative viral load between the NPS and container by the drooling method by the patient himself/
saliva collected by drooling, expectoration, gargling, and herself under instructions from the healthcare workers.
salivary swabs are compared. In addition, the relative Gargled saliva: Patients were instructed to take 3 ml
efficacy of salivary and blood IgG as a determinant of phosphate buffer saline (PBS) (sterile) in the oral cavity
systemic immunity against SARS‑CoV‑2 is investigated. and gargle it for 10 seconds. Gargled saliva was then
collected in the sterile and leak‑proof container.
Materials and Methods
Salivary Swabs: Sterile swabs (salivary swabs) were
Patient enrollment
used to collect saliva samples. This method employed
This study was conducted at Dr. D. Y. Patil Medical
trained professionals. Patients were asked to open the
College and Hospital, Pune (India) from Nov 2020 to
mouth, and the swabs were gently rubbed on the parotid
Feb 2021. The study was approved by the Institutional
gland duct (Stenson’s duct) until it became wet. Swabs
Ethics Committee of Dr. D. Y. Patil Medical College and
were immediately put into the VTM inside a sterile
Hospital, Pune (India) (DYPV/EC/599/2020). Informed
container.
consent was obtained from the study participants before
the sample collection. Medical history, co‑morbidities, Expectorated saliva: Patients were instructed to
ongoing symptoms, and anthropometric data were expectorate into the 15 ml sterile falcon tubes containing
collected at the time of enrollment. The present study VTM. Self‑collection was supervised by the trained
included (1) the patients who reported to the fever clinic personnel to avoid entry of the sputum into the saliva
for the purpose of SARS‑CoV‑2 testing, (2) admitted while expectoration. The patient was allowed to
patients for COVID‑19 treatment, and (3) patients who expectorate saliva 2–3 times for 25 seconds.

Medical Journal of Dr. D.Y. Patil Vidyapeeth ¦ Volume 17 ¦ Issue 1 ¦ January-February 2024 53
 Kheur, et al.: Saliva collection methods for detection of SARS‑CoV‑2 virus in saliva

RT‑PCR analysis Statistical analysis

All the collected samples were stored at −80°C Statistical Package for Social Sciences version 17 (IBM)
and processed for RT‑PCR analysis within 24 h. software was used for statistical analysis. Categorical
RT‑PCR analysis was performed in house as per the variables were expressed as a percentage. Continuous
manufacturer’s instruction (TRUPCR® SARS‑CoV‑2 variables were compared with the Mann–Whitney U
Kit). The kit employed simultaneous detection of test. P value was considered significant when <0.05.
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the gene for the detection of the sarbecovirus (genus

B‑betacoronavirus (B‑βCoV)) and RdRP gene for the Results
detection of SARS‑CoV‑2. RNaseP was used as an The primary objective of this cross‑sectional study
endogenous internal control to check the extracted RNA was to identify the ideal sample collection method and
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quality, amplification procedure, and possible presence to validate the saliva for the SARS‑CoV‑2 detection.
of inhibitors to avoid false‑negative results. Results were Therefore, by and large, we enrolled patients who
expressed as cycle threshold (CT) values. reported for COVID‑19 testing (n = 43, male‑27,
female‑16). The median age of the patients was
IgG antibody analysis 49 years (range 16–76 years). Twenty‑eight patients
Detection of IgG antibodies in the saliva was presented with symptoms such as fever, dry cough,
performed by using ErbaLisa COVID‑19 IgG kit as difficulty in breathing, chest pain, sore throat, and
per the manufacturer’s instructions. Saliva samples of weakness while 15 patients were asymptomatic.
COVID‑19 positive patients confirmed by RT‑PCR The incidences of chronic co‑morbidities were
were subjected to IgG antibody estimation. Briefly, diabetes (16.2%), hypertension (13.9%), hypertension
diluted saliva samples (1:21) were added to the 96‑well along with diabetes (6.9%), chronic renal
plate coated with recombinant spike protein (S) and disease (2.3%), and an immune‑compromised condition
incubated for 20 mins at room temperature. Negative along with chronic renal disease (4.6%). Twenty‑four
control, positive control, and calibrator were added patients (55.8%) had no co‑morbidity or any other
without any dilution. Further, samples were removed, chronic medical illness. Twenty‑eight patients were
and the wells were washed with 1×washing buffer. hospitalized, out of which six were intubated. Saliva
of the incubated patients was collected with the help of
Horseradish peroxidase (HRP) anti‑human IgG enzyme
hospital staff. The characteristics of patients have been
conjugate was added to the wells and incubated for
summarized in Table 1.
20 mins. Wells were again washed with washing buffer,
and 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate was Out of 350 individuals examined, 43 were determined to
added. After 10 mins, stop solution was added, and be positive by RT‑PCR analysis of nasopharyngeal swabs
absorbance was measured at 450 nm using an enzyme- at the commencement. Saliva obtained by four different
linked immunosorbent assay (ELISA) reader. The methods from the COVID‑19‑positive patients (n = 43)
antibody index was calculated by dividing the mean was subjected to the RT‑PCR analysis simultaneously.
values of each sample by the cut‑off value (provided by We obtained the highest (78.5%, 11 out of 14 patients)
the manufacturer). detection sensitivity in the expectorated saliva whereas
the drooling method exhibited 22.2% sensitivity
For comparative analysis of the IgG in serum and (2 out of 9 patients). Saliva obtained by salivary swabs
saliva, blood samples were further collected from showed 21.42% sensitivity (3 out of 14 patients) and the
COVID‑19‑positive patients from the study group and gargling method exhibited 16.66% sensitivity (1 out of
allowed to clot at room temperature. IgG levels in 6 patients) [Figure 1].
serum were determined by nephelometry on Atellica®
Furthermore, to determine the influence of the
NEPH 630 automated system. Serum was separated collection method on the salivary SARS‑CoV‑2 load,
by centrifugation and stored at −80°C until further we measured the presence of e gene and RdRp gene
use. Before the IgG assay, serum samples were diluted as a measure of SARS‑CoV‑2 loading the saliva.
in a ratio of 1:400 using assay diluents. Further, The median CT values (e and RdRp gene) for saliva
diluted samples were incubated with antiserum to collected by expectoration were 27.0 ± 6.03 and
Human IgG (γ chain) for 4 h. Multipoint calibration 28.0 ± 6.48, respectively [Figure 2]. In contrast, NPS
was performed with the serially diluted N protein exhibited median CT values of 29.5.0 ± 6.18 and
standards simultaneously. IgG levels were expressed 31.0 ± 7.23, respectively. We found relatively higher
as IgG antibody index (mg/dL) upon correlation with viral load (lower CT values) in the expectorated saliva
calibrators. as compared to the NPS, although the difference was

54 Medical Journal of Dr. D.Y. Patil Vidyapeeth ¦ Volume 17 ¦ Issue 1 ¦ January-February 2024
 Kheur, et al.: Saliva collection methods for detection of SARS‑CoV‑2 virus in saliva

Table 1: Characteristics of the patients

Parameters Asymptomatic (n=15) Symptomatic (n=28) Total (n=43)
Age (n=43) 42.0 (30–65) 52.0 (16–76) 49 (16–76)
Sex
Male (n) 5 22 27 (62.8%)
Female (n) 10 6 16 (37.2%)
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Chronic co‑morbidities
Diabetes (n) 1 6 7 (16.2%)
Hypertension (n) 0 6 6 (13.9%)
Diabetes + Hypertension (n) 0 3 3 (6.9%)
Chronic renal disease (n) 1 0 1 (2.3%)
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Chronic renal disease+immunocompromised condition (n) 0 2 2 (4.6%)

No co‑morbidities (n) 13 11 24 (55.8%)
CT
Nasopharyngeal Swab
e gene 30.0±5.67 28.5±5.48 29.0±5.48
RdRP gene 33.0±6.90 30.0±5.76 30.0±6.11
CT
Saliva
e Gene 25.0±6.90 29.5±4.87 29.0±5.42
RdRP gene 25.0±7.36 30.0±5.30 30.0±5.98

a b
Figure 1: Influence of saliva collection method on the SARS‑CoV‑2 diagnosis outcome: (a) Saliva collected by expectoration method exhibited the
highest detection sensitivity (78.5%), where drooling, gargling, and salivary swabs method showed 22.5%, 16.66%, and 21.42% detection sensitivity,
respectively. Data shown are the percentage of positive patients detected by saliva and nasopharyngeal swabs. (b) Outcome of RT‑PCR‑based diagnosis
of the SARS‑CoV‑2 in the saliva collected by four different methods. Data shown are the number of patients diagnosed either positive or negative

statistically non‑significant (P = 0.807). Furthermore, drooling, gargling, and salivary swabs was lower than
saliva collected by salivary swabs yielded median CT the NPS (P = NS).
values of 29.0 ± 5.1 (e gene) and 30 ± 5.1 (RdRp),
To analyze our findings further, we plotted the difference
whereas detection using NPS exhibited median CT
between the median CT values obtained by saliva in
values of 29.0 ± 5.1 (e gene) and 30.0 ± 5.1 (RdRp)
gene (P = NS). The median CT value for the saliva comparison with NPS. Expectorated saliva showed a
collected by the drooling method was 30.5 ± 0.7 (e difference of −1.5 (e gene) and −3 (RdRp) gene, whereas
gene) and 34 ± 0.0 (RdRp gene), whereas NPS drooled saliva showed +0.5 (e gene) and +5.5 (RdRp)
showed a median CT value of 30.0 ± 1.5 (e gene) [Figure 3]. Gargled saliva showed the difference
and 28.5 ± 4.94 (RdRp gene). The gargling method of +10 (e gene) and +13 (RdRp gene). Interestingly,
detected only 1 patient out of 6 patients screened there was no change in the median CT values of the e
(e gene, 24 ± 7.5 vs 14.0) and (RdRp, 28.0 ± 8.09 vs and RdRp genes between saliva obtained by salivary
15.0). The relative viral load in the saliva collected by swabs and saliva collected by NPS.

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 Kheur, et al.: Saliva collection methods for detection of SARS‑CoV‑2 virus in saliva
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Figure 2: Box plot representing the CT values of the SARS‑CoV‑2 in the saliva and nasopharyngeal swabs as detected by the RT‑PCR for (i) e gene (ii)
RdRp gene

b
Figure 3: (a) Difference between the median CT values of the saliva collected by four different methods and the nasopharyngeal swabs as evidenced
by the RT‑PCR analysis (i) e gene (ii) RdRp gene; data shown are median of the CT values. (b) Difference between the absolute CT values of the saliva
collected by four different methods and nasopharyngeal swabs. Patients diagnosed negative in the saliva have assigned CT value of 35

Furthermore, we determined the sensitivity of the saliva We evaluated IgG levels in blood serum and saliva
collected by different methods in COVID‑19 patients collected by gargling, expectoration, and salivary
who had CT values above 25 as estimated by using swabs in COVID‑19 patients to investigate the immune
NPS. Out of 10 patients, 7 patients were diagnosed response and as a putative predictor of SARS‑CoV‑2
infection. The Salivary IgG index was found to be
positive for SARS‑CoV‑2 in the saliva collected by
relatively higher in the expectorated saliva [Figure 4].
expectoration (70% sensitivity) whereas drooling, However, the difference between the saliva collection
salivary swab, and gargling method evidenced 22.2% groups was not significant (P = 0.141). Interestingly,
(2 out of 9 patients), 18.1% (2 out of 9 patients), and saliva IgG antibody index showed better association with
0.0% (0 out 3 patients) detection sensitivity, respectively. the viral load (CT value) of the patients as compared

56 Medical Journal of Dr. D.Y. Patil Vidyapeeth ¦ Volume 17 ¦ Issue 1 ¦ January-February 2024
 Kheur, et al.: Saliva collection methods for detection of SARS‑CoV‑2 virus in saliva
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b
Figure 4: Association between the SARS‑CoV‑2 viral load and IgG levels in saliva and serum in COVID‑19‑positive patients: (a) Mean IgG antibody
index was not significantly different in the saliva collected by gargling, expectoration, and salivary swabs. Data shown are mean IgG antibody index
in the saliva (b) IgG index in the saliva exhibited better association with the viral load as compared to the IgG levels in the serum (e gene, R2 0.23 vs
0.04, vs, P = 0.044), (RdRp gene, R2 0.21 vs 0.024, P = 0.055)

to IgG levels in the serum (e gene, R20.23 vs 0.04, vs, wide range of sensitivity. It is apparent that these studies
P = 0.044), (RdRp gene, R2 0.21 vs 0.024, P = 0.055), have employed different methods of saliva collection.[18]
irrespective of the method of collection. Therefore, investigation on the comparative efficiency
of the various methods of saliva specimen collection is
Discussion important, as the detection rate in saliva specimens is
Saliva possesses several advantages over NPS as a modulated by the collection method.[2]
clinical specimen for diagnosis of COVID‑19 disease Our study revealed that expectorated saliva yielded
such as low exposure to the health care workers, better sensitivity (78.5%) among the four methods
being relatively comfortable for patients, and further investigated for saliva specimen collection. Also, the
eliminating the requirement of trained professionals median viral load was relatively higher in the saliva,
for specimen collection. The sensitivity and relative although the difference was not statistically significant.
SARS‑CoV‑2 viral load in saliva and NPS can be Our findings are in agreement with previously obtained
influenced by the method of saliva specimen collection results, where expectorated saliva has been shown
as justified in many studies with varied results showing a to produce 74% (160 out of 217) sensitivity whereas

Medical Journal of Dr. D.Y. Patil Vidyapeeth ¦ Volume 17 ¦ Issue 1 ¦ January-February 2024 57
 Kheur, et al.: Saliva collection methods for detection of SARS‑CoV‑2 virus in saliva

91.7% (11 out of 12) sensitivity could be observed in 0.0% sensitivity, whereas expectorated saliva showed
NPSofCOVID‑19 positive samples.[13,14] It has been 70% sensitivity. These findings suggest drooling,
previously reported that the release of SARS‑CoV‑2 salivary swabs, and gargling may not be reliable
virus particles from the lower and upper respiratory saliva collection methods in patients. Moreover,
tract into the oral cavity can be a predominant source of the viral load in the saliva can be dynamic and
saliva for clinical diagnosis.[23] Our study confirmed that may shed 10–15 days post‑infection. Interestingly,
expectorated saliva exhibits better detection sensitivity,
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a study done by Nagura‑Ikeda et al.[30] reported

which can be attributed to the sufficient viral entry from variations in the sensitivity of the saliva samples
the lower and upper respiratory tract. collected at different intervals after the onset of the
The whole saliva collected from the mouth is a mixture symptoms (0–9 days 65.6 to 93.4%, after 10 days
22.2 to 66.7%). The very fact that saliva is a carrier
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of glandular secretions, gingival crevicular fluid, serum,

expectorated airway surface liquid and mucus, epithelial of the virus further emphasizes its role in disease
and immune cells from the oral mucosa and upper transmission and underpinning the use of a mask for
airways, oral microbes, and viruses.[18,24] Drooled saliva preventing the spread of the virus. These varying
is a convenient and relatively safe method of whole degrees of sensitivities may be due to the status of
saliva specimen collection for diagnosis. However, COVID‑19 patients (symptomatic/asymptomatic), viral
in our study, drooled saliva method exhibited 22.5% clearance due to the generation of antibodies defense,
sensitivity. Previous studies by Chong et al. and Sutjipto and possible contamination of viral particles from
et al. have reported sensitivity of 52.95% and 38–52%, upper and lower respiratory tract sample collection.[13]
respectively, in drooled saliva, whereas in contrast, Furthermore, the build‑up of systemic immunity against
Vaz et al., Williams et al., Yokota et al., Landry et al., the SARS‑CoV‑2 virus may affect the temporal viral
and Iwasaki et al. have reported sensitivity of 94.4%, load in the saliva. Taken together, these factors may
84.6%, 85.7%, 92%, and 88.8%, respectively.[8-9,13,21,25-27] limit the efficacy of saliva as a specimen for accurate
diagnosis of COVID‑19 infection by drooling, salivary
Furthermore, the collection of saliva by using swab, and gargling method of saliva collection.
salivary swabs showed sensitivity in the range of
53–85%.[6,8,13,16,20,25,28] It has been reported that salivary Apart from RT‑PCR‑based SARS‑CoV‑2 detection,
glands express ACE2, the major surface receptor for salivary IgG has been reported as an antibody‑based
SARS‑CoV‑2 virus entry inside the cell, and may be one diagnosis marker for COVID‑19 disease.[10] Our data
of the sources of SARS‑CoV‑2 in the saliva.[23] Although unequivocally demonstrate a better association between
we did not find a difference in the median viral load, our the salivary IgG and SARS‑CoV‑2 viral load compared
result showed 21.42% sensitivity in the saliva collected to the blood serum IgG. Salivary levels of IgG can
by salivary swabs. A previous study conducted by remain stable up to 105 days post‑onset of symptoms,
Sutjipto et al. showed 60%, 50%, and 36.8% sensitivity which suggests that salivary IgG can be reliably used
in between 0–7, 8–14, and >15 days post‑onset for COVID‑19 detection, disease progression, and
symptoms, which suggests that saliva collected by throat surveillance instead of serum IgG.[10] Our data clearly
swabs are comparable to the salivary swabs and may demonstrate the inverse proportion of viral load with
gradually lose their sensitivity over the time period.[13] salivary IgG levels advocating checking salivary IgG
The detection levels of the SARS‑CoV‑2 virus were rather than serum IgG for surveillance studies.
shown to be the least in the gargling technique. Out of 6 On the basis of this study, we hypothesize that the
NPS‑positive cases, only one case was detected positive oral cavity may not be a primary reservoir for virus
in saliva, exhibiting only 16.66% sensitivity. This is proliferation. It is essentially the viral particle shedding
in contrast to the 100% positivity reported by Mittal into the oral cavity via respiratory pathways. Thus, the
et al.[29] in the paired saliva (gargled) and NPS. As per collection method for saliva for SARS‑CoV‑2 diagnosis
our statistical evaluation of the median CT values, this is very imperative to get accurate results. The method
can be explained based on the fact that the difference in of expectorating saliva would be preferred as it also
median values was higher in the gargled saliva. So, if receives the respiratory secretions and hence has more
the viral load is higher, salivary detection of the virus is chances of harboring the viral particles, irrespective of
more predictable despite the method used. the temporal status of the patient. Thus expectorated
Interestingly, we determined the detection sensitivity salivary samples become an easy and efficient way for
of COVID‑19 patients in the saliva that had CT monitoring the SARS‑CoV‑2 patients. However, further
values above 25. Saliva collected by drooling, salivary studies are required to confirm these observations in
swabs, and gargling reflected 22.2%, 18.1%, and larger clinical settings.

58 Medical Journal of Dr. D.Y. Patil Vidyapeeth ¦ Volume 17 ¦ Issue 1 ¦ January-February 2024
 Kheur, et al.: Saliva collection methods for detection of SARS‑CoV‑2 virus in saliva

Limitations of the study of nasopharyngeal swabs and safe procedures for covid‑19
testing based on anatomical knowledge. J Korean Med Sci
Although we could enroll overall 43 patients in the 2022;37:1-10.
study, the number of patients in the individual method 4. To KKW, Tsang OTY, Yip CCY, Chan KH, Wu TC, Chan JMC,
of the collection still was low. Also, we could not take et al. Consistent detection of 2019 novel coronavirus in saliva.
longitudinal samples of the same patients as most of Clin Infect Dis 2020;71:841-3.
the patients were unlikely to visit the hospital once 5. Altawalah H, AlHuraish F, Alkandari WA, Ezzikouri S. Saliva
specimens for detection of severe acute respiratory syndrome
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discharged or were asymptomatic. In our observation,

coronavirus 2 in Kuwait: A cross‑sectional study. J Clin Virol
most of the similar studies have reported this problem.[14] 2020;132:104652. doi: 10.1016/j.jcv.2020.104652.
We strictly adhered to the protocol while collecting the 6. Pasomsub E, Watcharananan SP, Boonyawat K, Janchompoo P,
saliva by drooling, gargling, and using salivary swabs Wongtabtim G, Suksuwan W, et al. Saliva sample as a
so that the saliva would not get mixed with secretions non‑invasive specimen for the diagnosis of coronavirus disease
nYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC1y0abggQZXdgGj2MwlZLeI= on 03/06/2024

from the upper and lower respiratory tract. However, the 2019: A cross‑sectional study. Clin Microbiol Infect 2021;27:285.
e1‑4.
possibility of prior contamination cannot be overruled.
7. Azzi L, Carcano G, Gianfagna F, Grossi P, Gasperina DD,
Genoni A, et al. Saliva is a reliable tool to detect SARS‑CoV‑2.
Conclusion J Infect 2020;81:e45-50.
Though saliva has been argued to be a good substitute 8. Vaz SN, Santana DS de, Netto EM, Pedroso C, Wang WK,
for NPS, a lot of research is still required in optimizing Santos FDA, et al. Saliva is a reliable, non‑invasive
specimen for SARS‑CoV‑2 detection. Brazilian J Infect Dis
the use of saliva specimens for community diagnosis 2020;24:422-7.
and monitoring the patients. The key constituent is the 9. Iwasaki S, Fujisawa S, Nakakubo S, Kamada K, Yamashita Y,
method of saliva collection. In this study, we aimed Fukumoto T, et al. Comparison of SARS‑CoV‑2 detection in
to iron out the practical difficulties and challenges nasopharyngeal swab and saliva. J Infect 2020;81:e145-7.
associated with the influence of the collection method on 10. Isho B, Abe KT, Zuo M, Jamal AJ, Rathod B, Wang JH,
et al. Persistence of serum and saliva antibody responses to
the outcome. Here, we report that expectorated salivary SARS‑CoV‑2 spike antigens in COVID‑19 patients. Sci Immunol
collection method yields better sensitivity as compared to 2020;5:1-21.
other collection methods. Our study further showed that 11. Wyllie AL, Fournier J, Casanovas‑Massana A, Campbell M,
salivary IgG could be a better biomarker as compared to Tokuyama M, Vijayakumar P, et al. Saliva is more sensitive
serum IgG for correlating with the viral load. for SARS‑CoV‑2 detection in COVID‑19 patients than
nasopharyngeal swabs TT – Published article: Saliva or
Acknowledgment nasopharyngeal swab specimens for detection of SARS‑CoV‑2.
Thanks are due to DR. D. Y. Patil Medical College, MedRxiv 2020. doi: 10.1101/2020.04.16.20067835.
12. Nagura‑ikeda M, Imai K, Tabata S, Miyoshi K, Murahara N,
Hospital and Research Centre, Pimpri, Pune, India
Mizuno T. Crossm clinical evaluation of self‑collected saliva by
and Dr. D. Y. Patil Dental College and Hospital, quantitative. J Clin Microbiol 2020;58:1-9.
Dr. D. Y. Patil Vidyapeeth, Pimpri, Pune, India for 13. Sutjipto S, Lee PH, Tay JY, Mendis SM, Abdad MY,
providing infrastructural support for the smooth conduct Marimuthu K, et al. The effect of sample site, illness duration,
of the project. The authors would like to thank Mr. Amit, and the presence of pneumonia on the detection of SARS‑CoV‑2
by real‑time reverse transcription PCR. Open Forum Infect Dis
Mr. Purushottam, and late Mr. Umesh for their assistance
2020;7:1-9. doi: 10.1093/ofid/ofaa335.
in saliva sample collection. 14. Rao M, Rashid FA, Sabri FSAH, Jamil NN, Zain R, Hashim R,
Financial support and sponsorship et al. Comparing nasopharyngeal swab and early morning saliva
for the identification of severe acute respiratory syndrome
In house funding was given by Dr. D.Y. Patil Vidyapeeth, coronavirus 2 (SARS‑CoV‑2). Clin Infect Dis 2021;72:E352-6.
Pimpri, Pune (India) to carry out the study. 15. Fogarty A, Joseph A, Shaw D. Pooled saliva samples for
COVID‑19 surveillance programme. Lancet Respir Med
Conflicts of interest
2020;8:1078-80.
There are no conflicts of interest. 16. Hwang S, Tan S, Tan P, Siau C. Self‑swab and saliva collection
for the diagnosis of covid‑19. What do patients feel about them?
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