MINIREVIEW

A Look Inside: Oral Sampling for Detection of Non-oral

Infectious Diseases
Ethan D. Valinetz,a Gerard A. Cangelosib

Department of Medicine, University of Washington, Seattle, Washington, USA

a

b Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, Washington, USA

ABSTRACT Efforts to control transmissible infectious diseases rely on the ability to

screen large populations, ideally in community settings. These efforts can be limited
by the requirement for invasive or logistically difficult collection of patient samples,
such as blood, urine, stool, sputum, and nasopharyngeal swabs. Oral sampling is an
appealing, noninvasive alternative that could greatly facilitate high-throughput sam-
pling in community settings. Oral sampling has been described for the detection of
dozens of human pathogens, including pathogens whose primary sites of infection
are outside of the oral cavity, such as the respiratory pathogens Mycobacterium tu-
berculosis and SARS-CoV-2. Oral sampling can demonstrate active infections as well
as resolving or previous infections, the latter through the detection of antibodies. Its
potential applications are diverse, including improved diagnosis in special popula-
tions (e.g., children), population surveillance, and infectious disease screening. In this
minireview, we address the use of oral samples for the detection of diseases that pri-
marily manifest outside the oral cavity. Focusing on well-supported examples, we
describe applications for such methods and highlight their potential advantages and
limitations in medicine, public health, and research.

KEYWORDS diagnosis, screening, saliva, swabs, respiratory diseases, pediatric infec-

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tious disease, COVID-19, SARS-CoV-2, tuberculosis, HIV, HCV, Ebola, malaria,
Pneumocystis, parvovirus, Mycobacterium tuberculosis, hepatitis C virus, human immuno-
deficiency virus, respiratory disease

ransmissible microbial pathogens range from primordial “companions” of human-

T kind such as Mycobacterium tuberculosis, the causative agent of tuberculosis (TB),
to newly arisen zoonotic infections such as SARS-CoV-2, the virus that causes COVID-
19. Efficient diagnostic methods are needed to rapidly identify infected people, not
only for patient care but also to reduce the spread of disease.
Infectious disease case-finding has been greatly facilitated by new molecular patho-
gen detection methods, some of which are fast and easy to use in the clinic, at point of
care (POC), and in community settings. Unfortunately, sample acquisition remains a Citation Valinetz ED, Cangelosi GA, 2021. A
look inside: oral sampling for detection of non-
critical bottleneck. Samples that require invasive procedures, such as blood, bron- oral infectious diseases. J Clin Microbiol 59:
chioalveolar lavage (BAL) fluid, or induced sputum, cannot be collected with high e02360-20. https://doi.org/10.1128/JCM.02360
-20.
throughput in settings such as schools and workplaces. Other samples such as urine or
Editor Alexander J. McAdam, Boston Children's
stool require privacy and sometimes-unreliable self-sampling protocols. Hospital
This article addresses oral sampling, an emerging noninvasive sampling strategy Copyright © 2021 American Society for
developed to address these challenges. As a portal between the interiors of our bodies Microbiology. All Rights Reserved.
and the external world, the mouth is an accessible and logical place to look for patho- Address correspondence to Gerard A.
Cangelosi, [email protected].
gens, especially pathogens of the airways. Noninvasive oral sampling refers to methods
Accepted manuscript posted online
of sample collection from the oral cavity that are simple, can be performed on almost 22 April 2021
all patients and in any setting, and produce little or no discomfort. These traits are de- Published 20 September 2021
sirable for screening large populations with high throughput, especially in community

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settings. Because most such methods do not require specialized training, many are
easily adapted for self-collection. This increases the capacity for screening of large pop-
ulations without overburdening public health and health care workers, and helps to
facilitate POC testing.
Noninvasive oral sampling can be versatile and is not confined to detection of oral
diseases. In many cases, a method used to detect one pathogen can also be applied to
others. For example, there is some evidence that a multiplex PCR test to detect several
respiratory viruses may be performed similarly using saliva samples compared to naso-
pharyngeal swabs (1). Because of the nature of this technology, it is also possible that
such multiplex PCRs could be used to detect biologically dissimilar pathogens that
cause overlapping clinical syndromes.
In this article we discuss non-oral pathogens that have been detected via oral sam-
pling, either in research or clinical context, with an emphasis on distinct applications of
noninvasive oral sampling.

NONINVASIVE ORAL SAMPLING TYPES

Examples of noninvasive oral samples include tongue and buccal swabs, saliva, and
oral rinses. Many such methods are routine for diagnosing and screening for oral infec-
tions; however, this article focuses on the use of oral sampling for detecting infectious
agents whose primary site of infection is outside the oral cavity. Exhaled breath (2) is
not addressed here because it is designed to be sample derived from the lungs, not
the mouth.
The use of tongue swabs has been described for the detection of a variety of patho-
gens. In addition to its distinctive native flora (3), microbes from other parts of our air-
ways can accumulate on the tongue dorsum due to its position in the mouth and its
coarse surface architecture. A typical sample collection process involves firmly scraping
the surface of the tongue with a swab for about 10 s followed by transferring the swab
to a tube with storage buffer (4, 5).
Different areas of the buccal mucosa have been explored for infectious disease sam-
pling, but most such studies have focused on the inner surface of the cheeks (5, 6).
Other studies collected material from the outer gums and mucosa between the gums

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and the inner lining of the lips. Technically, these swabs come into contact with buccal
mucosa as well, but are more often described as collecting oral fluid, which is likely a
combination of saliva and gingival crevicular fluid (7–9). Some studies refer to this sam-
ple type as oral mucosal transudate, despite nearly identical descriptions of sample col-
lection (10).
Collection of oral fluid with swabs can be contrasted with collection of saliva. For
SARS-CoV-2 alone, multiple collection methods have been described, including cough-
ing out, drooling out, spit saliva, collecting saliva with a pipette (for example from a
patient requiring endotracheal intubation), and directly sampling from the salivary
gland (11–15). The least invasive methods, such as the drooling technique, have been
approved as high-yield samples for COVID-19 testing (12).
Oral rinses, sometimes referred to as “gargle lavages,” have also been described for
the detection of infectious diseases. These typically involve gargling sterile saline solu-
tion before spitting out into a sterile container. This material is likely a mixture of sam-
ple types, including saliva and oropharyngeal secretions (16–18). However, distinctions
have been noted between oral rinses, defined as swishing sterile water without any
throat gargling, and samples collected by throat gargling (19).
Once noninvasive oral samples have been obtained, they can be tested in a variety
of ways depending on the pathogen and the clinical context. When there is concern
for an acute infection, in most cases the direct detection of pathogen cells, antigens, or
nucleic acid is most appropriate. Real-time PCR, also known as quantitative PCR (or
qPCR), is a laboratory technique that allows for the detection and quantification of spe-
cific nucleic acid sequences. The cycle threshold value (CT value) represents how many
cycles of PCR were required in order to detect the nucleic acid sequence; higher CT

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values indicate that more PCR cycles were needed before the sequence was detected
and therefore suggests a lower amount of the nucleic acid. There are also applications
for antibody testing of noninvasive oral samples, including screening for chronic infec-
tious diseases and population surveillance for immunity.
Oral sampling has been explored for diverse reasons, but there are specific situa-
tions that stand out as areas of particular need for noninvasive, high-throughput sam-
pling. The following sections explore several of these.

MINIMIZING HEALTH CARE WORKER EXPOSURE TO INFECTIOUS DISEASES

Many infectious diseases require samples obtained by health care workers for diag-
nosis. This can pose a significant hazard to health care workers, especially those who
are subject to repeated exposures that cumulatively increase the risk to the health care
worker over time.
Pulmonary tuberculosis. Oral sampling has been evaluated for the diagnosis of
pulmonary TB through the detection of TB DNA by PCR. Sputum production for pulmo-
nary TB diagnosis has several challenges. First, the quality of sputum generation has a
significant impact on test performance. Some people with TB infection are not able to
produce high quality sputum due to a weak cough or low disease burden with minimal
cough symptoms. For those who produce minimal to no sputum on their own, an
induced sputum can be obtained, which involves administering a hypertonic solution
via nebulizer to the patient. This requires medical equipment, specialized training, and
that the health care worker be present when the patient produces sputum, which pla-
ces the health care worker at increased risk. For these reasons, an alternative sampling
method would be useful.
Luabeya et al. (5) conducted a study in South Africa to analyze and describe the per-
formance of oral swab analysis to diagnose pulmonary TB via manual qPCR. One goal
was to compare sampling sites, including tongue, buccal, and gum swabs. They found
that tongue swabs obtained using Whatman OmniSwabs yielded stronger DNA signals
via Cq measurement compared to gum swabs and buccal swabs collected using the
same product. These swabs were obtained by firmly brushing the dorsum of the
tongue for approximately 10 s. The study also compared the performance of tongue

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swabs (tested by a manual IS6110-targeted qPCR) to sputum GeneXpert MTB/RIF and
found that the sensitivity of at least one of two tongue swabs collected on separate
days relative to sputum Xpert was 91.8%. Tongue swabs and sputum Xpert performed
identically (sensitivity of 83.1%) when compared to patients with confirmed TB via spu-
tum Xpert or culture. This is because some of the patients with sputum cultures posi-
tive for TB had negative Xpert testing from sputum but were positive from oral swab
testing, and vice versa (5).
The results of Luabeya et al. (5) offer an important reminder that the oral cavity is
internally heterogeneous, such that “oral swabs” collected from tongue dorsa may
yield different results from swabs collected from the buccal mucosa. In some cases,
this may have delayed the acceptance of oral sampling for infectious diseases. For
example, an early assessment of saliva as a specimen for detection of TB by PCR
showed very low sensitivity relative to sputum testing (38.5%) (20). The notion of oral
sampling for TB was not explored further until surface sampling (buccal mucosa,
tongue dorsa) was evaluated a few years later.
Independent studies of oral swabbing for TB have yielded a mixture of results. One
study evaluated the performance of buccal swabs obtained from 33 patients in Peru
with confirmed pulmonary TB by sputum culture. These swabs were analyzed using
the first-generation GeneXpert system rather than home-brew IS6110 PCR, and found
a sensitivity of 45% relative to positive culture (21). Relative to the South African study,
this one used different analytical methods and relied on buccal swabbing rather than
tongue swabbing. Another study conducted in Moldova found a sensitivity of only
36.3% relative to bacteriologically confirmed TB. This study used buccal, not tongue,

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swabbing and extended storage of samples in a proprietary storage formulation (22).

Such variables may have reduced sensitivity of oral swabbing relative to earlier studies.
A more recent study conducted on a cohort of 103 Chinese patients with suspected
TB used tongue swabbing in combination with a loop-mediated isothermal amplifica-
tion (LAMP) molecular test for TB DNA. This study found low sensitivity (32% to 50%)
for single swab specimens relative to sputum bacteriology, but higher sensitivity
(82.6%) when three swab specimens were tested (23). As seen in the results of Luabeya
et al. (5), tongue swabs collected in the early morning appeared to yield better sensitiv-
ity. Importantly, this team also assessed sensitivity relative to an expanded bacteriolog-
ical definition of TB (either sputum or BAL fluid). LAMP analysis of oral swabs detected
three BAL-positive TB patients who were negative by sputum testing (23). Another
report, currently in preprint stage, describes a study conducted in Korea on a cohort of
272 patients, including 128 patients with confirmed TB (24). Oral swab specimens were
processed with a proprietary microfluidic device (“SLIM”) that concentrated bacilli in
the swab samples prior to qPCR analysis. Relative to clinically confirmed TB, the sensi-
tivity of the oral swab-based SLIM assay (65.6%) was higher than that of sputum testing
with the first-generation GeneXpert (43.4%) (24). These observations illustrate the
potential for oral swab-based tests to augment TB case finding relative to sputum test-
ing alone. It is possible that positive sputum production by some patients results in
deposition of bacilli on oral surfaces, such that the bacilli can be detected afterward
even if the patient does not produce positive sputa during their clinic visits.
SARS-CoV-2. The COVID-19 pandemic has led to significant health care worker ex-
posure that could potentially be mitigated by noninvasive sampling. Although the
gold standard for the detection of SARS-CoV-2 in patients is not known, many regard
the nasopharyngeal (NP) swab as a reference standard. NP swabbing requires special-
ized training, causes patients discomfort, and frequently leads to patients sneezing or
coughing, which may generate infectious aerosols. Noninvasive sampling reduces
these problems and, in many cases, enables self-sampling.
Several noninvasive oral sampling collection methods and samples have been
described for the detection of SARS-CoV-2 by PCR. The virus appears to be abundant
in the upper airways of symptomatic people, and the primary receptor molecule for

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the virus, ACE2, is expressed in oral mucosa and epithelia (25, 26). Most evaluations of
oral sampling have focused on saliva. Early in the pandemic, Azzi et al. (11) described
25 patients admitted to the hospital with COVID-19 infection based on positive NP
swabs. Saliva was collected via a drooling technique, unless the patient was critically ill
requiring mechanical ventilation, in which case a clinician obtained saliva intraorally
using a pipette. They detected SARS-CoV-2 RNA in 25/25 (100%) patients with COVID-
19 infection (10). Another study on 622 patients presenting to an outpatient COVID-19
screening clinic used a spitting out technique, which involved first pooling saliva in the
mouth for 1 to 2 min and then spitting into a container. All 622 patients also provided
an oropharyngeal (OP) swab, of which 39 were positive for SARS-CoV-2. Of those 39
patients, 33 (84.6%) also had detectable SARS-CoV-2 in their saliva. These researchers
also chose a subset of 50 patients with negative OP swabs and tested their saliva, and
1 of them had detectable SARS-CoV-2 in their saliva (27). Hanson et al. collected NP
swabs, anterior nasal swabs, and saliva collected by pooling in the mouth and then
spitting into a container. There was a very high concordance between NP swabs and
saliva (kappa = 0.912); SARS-CoV-2 was detected in 80/354 (22.5%) NP swabs and 81/
354 (22.9%) saliva samples (13). The performance of self-collected saliva for the detec-
tion of SARS-CoV-2 antigen has also been evaluated, with more limited success. In one
study the antigen was detected in only 11.7% of samples from 103 patients with con-
firmed COVID-19 infection (28), and another study demonstrated only 23.1% positive
percent agreement between antigen testing in saliva compared to NP samples (29).
Of note, some studies used saliva obtained through a coughing method. In some
cases it may be more appropriate to describe this as sputum. To et al. (15) described a
cohort of 23 patients who were asked to cough out saliva in the early morning. Twenty

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out of 23 (87%) patients with confirmed SARS-CoV-2 infection by NP swab were also
positive by collecting oropharyngeal saliva (15). Similarly, Procop et al. (14) described
obtaining “enhanced saliva,” which was self-collected by first sniffing strongly to
gather nasal secretions, followed by coughing to produce phlegm. NP swabs were col-
lected after this was done. Of 219 patients with both NP swabs and saliva samples, 39
had detectable SARS-CoV-2 by both NP swabs and saliva, yielding a 100% positive pre-
dictive agreement. They also reported a very high negative predictive agreement of
99.4% (14). This method appears to yield high-quality samples, but not all patients
might be able to produce similar quality samples if a strong cough is required.
Furthermore, it is unclear if it is superior to saliva collected simply by drooling or spit-
ting out. Additional studies with paired comparisons of these techniques would be
helpful.
Mittal et al. (18) described 50 symptomatic patients with confirmed COVID-19 infec-
tion by NP swab. Within 72 h of initial diagnosis, paired sampling was performed with
either NP swab or oropharyngeal swab paired with oral rinse obtained by gargling nor-
mal saline solution and then spitting into a sterile container. All of the oral rinse sam-
ples yielded detectable SARS-CoV-2 RNA. Patients were asked about discomfort with
each of the methods and, unsurprisingly, the oral rinse collection procedure was signif-
icantly better tolerated than NP or OP swab collection (18). Another study compared
oral rinses and saliva to NP or OP swabs in both symptomatic and asymptomatic health
care workers, with 285 health care workers participating in the study (224 were symp-
tomatic). Oral rinses were self-collected by swishing sterile water for 15 s, without any
throat gargling, and then spitting into a sterile container. Saliva samples were obtained
by bringing up saliva from the back of the throat and spitting into a sterile container.
Relative to NP swabs, oral rinses detected viral RNA in 64% of participants. Participants
who provided saliva samples had either matched OP swabs or NP swabs collected.
Relative to OP swabs, saliva had a sensitivity and specificity of 96.7% and 91.4%,
respectively. Using NP swabs as the reference, saliva had a sensitivity and specificity of
94.1% and 98.6%, respectively (19).
A meta-analysis published in August 2020 (12) compared SARS-CoV-2 detection in
saliva versus NP swabs, relative to confirmed COVID-19 disease. It reported 91% (confi-

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dence interval [CI] 80 to 99%) sensitivity for saliva, comparable to but slightly lower
than the 98% (CI 89 to 100%) for NP swab. Additionally, these authors identified 18
registered, ongoing clinical trials of saliva-based tests for detection of the virus. As of
this writing, several commercial saliva tests have received FDA Emergency Use
Authorization.
The timing of sample collection relative to symptom onset may also be an impor-
tant factor when considering the use of noninvasive oral sampling. Nagura-Ikeda et al.
(28) described a cohort that included 88 symptomatic patients, 61 of whom presented
during the “early” phase of their infection, defined as within 9 days of symptom onset.
The remaining 17 patients had had symptoms for 10 days or more. Saliva samples were
self-collected by a spitting out method. They found that samples collected from indi-
viduals in this early phase of infection had a sensitivity (relative to NP swabs) of 65.6%
to 93.4%, depending on the specific PCR technique used. In contrast, samples from
those who presented after 10 days of symptom onset demonstrated a sensitivity of
22.3% to 66.7% (28). Another study prospectively demonstrated that the median viral
load from saliva was highest within a week of symptom onset, although a third of their
patients had detectable RNA for 20 days or longer (30). This suggests that the perform-
ance of some noninvasive samples may depend on when they are collected in relation
to symptom onset.
Despite the promise of saliva sampling for SARS-CoV-2, it has limitations, including
the potential for aerosol production by some methods and possible heterogeneity of
specimen quality. Oral swabbing may help to overcome some of these issues. Some
studies have specifically examined self-collected oral swab samples compared to
health care provider-obtained samples. Kojima et al. (31) compared “unsupervised”

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self-collected oral swabs to clinician-supervised self-collected oral swabs in 45 partici-

pants, 29 of whom had SARS-CoV-2 detected in at least one sample (oral, nasal, or NP
swab) (31). Patients were provided swabbing instructions, and in the clinician-super-
vised group, patients were corrected if they forgot a step or did not perform some-
thing correctly. In the unsupervised group, no corrections were made if a part of the
collection process was incorrect. In this study, participants were asked to first cough
deeply to collect secretions in their mouth, then rub a swab on the cheeks, gums, hard
palate, and tongue for 20 s. Clinician-observed self-collected oral fluid swabs detected
SARS-CoV-2 in 26/29 (90%) SARS-CoV-2-positive participants, compared to 19/29 (66%)
in unsupervised self-collected oral swabs, 23/27 (85%) in clinician-observed self-col-
lected nasal swabs, and 23/29 (79%) in clinician-collected NP swabs. As with some of
the saliva sampling studies, the preliminary cough may have produced sputum, such
that these samples may not have been purely oral. Nonetheless, the results suggested
that oral swabs self-collected under clinical supervision can detect SARS-CoV-2 well,
perhaps even better than NP swabs. However, the discrepancy between supervised
participants who received feedback versus those who did not suggests that optimiza-
tion of self-collection instructions is important. For example, the authors noted that
some of the unsupervised participants forgot to cough prior to collecting the sample
(31). Unsupervised samples can fall short in a variety of ways, and the use of sample ad-
equacy controls is critical (32).
Although ACE2, the receptor for SARS-CoV-2, is expressed in diverse oral surface tis-
sues, two studies reported especially high levels of expression in tongue dorsum cells
(25, 26). With its relative ease of access, the tongue dorsum may therefore be an inter-
esting site to explore for noninvasive SARS-CoV-2 sampling. Tu et al. compared self-col-
lected tongue, nasal, and midturbinate swabs to provider-collected NP swabs. In this
study conducted on 501 symptomatic patients, SARS-CoV-2 RNA was detected from
tongue swabs in 90% of patients with confirmed COVID-19 (4). This value was only
slightly lower than nasal swabbing (94%); however, signal strength measured by qPCR
was also lower in positive oral samples than in positive nasal samples. As a result of
this and other considerations, the FDA updated its guidance to recommend nasal
swabbing, but not oral swabbing, for noninvasive COVID-19 diagnosis. The study by Tu

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et al. (4) was conducted in the hectic early days of the COVID-19 pandemic in the
United States, and samples were stored in viral transport medium at refrigerator tem-
peratures for up to 4 days before testing. Given the abundance of bacteria in oral cav-
ities, it is possible that some oral swab samples degraded during this storage period, a
problem that can potentially be overcome by using alternative storage and transport
procedures, or POC testing.
Some recent studies have proposed strategies that combine oral (or oropharyngeal)
swabbing with more traditional noninvasive samples such as nasal swabbing for
COVID-19 diagnosis. The rationale is that the virus is not always evenly distributed
throughout the upper airways, and infected patients who are negative at one site may
sometimes be detected by sampling another site. A meta-analysis currently at preprint
stage found that while nasal swabbing, oropharyngeal swabbing, and saliva sampling
captured lower percent positives than traditional NP swabs (nasal swabs 0.82, 95% CI
0.73 to 0.90; oropharyngeal 0.84, 95% CI 0.57 to 1.0; saliva 0.88, 95% CI 0.81 to 0.93), a
combination of nasal and oropharyngeal swabs matched the performance of NP (0.97,
95% CI 0.90 to 1.0). A limitation of this idea is that oropharyngeal swabbing is nearly as
invasive as NP swabbing (33). However, another study found that a noninvasive proto-
col that combined noninvasive oral and nasal swabbing (in a sequential protocol using
a single swab) matched the sensitivity of NP swabbing (34).

DIAGNOSIS OF INFECTIOUS DISEASES IN CHILDREN

Diagnosing infectious diseases in children can be challenging if invasive sample col-
lection is required. This has led to the investigation of noninvasive sample collection,
including oral sampling.

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Tuberculosis. Pediatric pulmonary TB remains difficult to diagnose microbiologi-

cally. The gold standard relies on obtaining expectorated sputum of good quality.
Most children are unable to provide such a sample without induction, an invasive pro-
cedure. For this reason, noninvasive alternatives have been sought. One study enrolled
201 South African children with suspected pulmonary TB. Each child had two buccal
swabs and two induced sputum samples collected (35). DNA was extracted from each
swab in order to perform quantitative PCR specific for TB DNA and the sputum samples
were sent for Xpert MTB/RIF testing and culture. The sensitivity for either oral swab
being positive was low, at 43% among patients with TB confirmed by at least one
induced sputum culture. This was lower than the 64% sensitivity observed by Xpert
testing on one sputum sample. Interestingly, however, oral swabs were positive in 24%
of patients with unconfirmed TB (children with signs and symptoms of TB who were
started on TB treatment, but were negative by sputum testing) (35). The method cor-
rectly excluded 93% of children with no TB. Therefore, oral swabs have the potential to
augment pediatric TB screening and care, by detecting cases that were not detected
by sputum testing. As noted earlier, it is possible that positive sputum production by
some children results in deposition of bacilli on oral surfaces, such that the bacilli can
be detected afterward even if the child does not produce positive sputa during their
clinic visits. Differing PCR methods may have also contributed to the difference in sen-
sitivity between tongue swabs, which used manual PCR, and sputum, which uses
GeneXpert testing. The sensitivity of oral sampling may be further improved in children
by using tongue swabs instead of buccal swabs, as demonstrated in another study of
adult patients (5).
In contrast, another study conducted in Peru found that oral swabs had significantly
lower sensitivity for pediatric TB (6). That study used less sensitive qPCR methods than
the South African study (35). Specifically, DNA was not concentrated by ethanol precip-
itation. Such observations highlight the fact that oral swab samples may have relatively
low TB bacillary loads, especially in children.
Human immunodeficiency virus. Many infectious diseases require venipuncture
for gold standard testing. In small children, this can be challenging and may lead to
avoidance of testing. One example is blood testing for HIV infection. Dziva Chikwari et

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al. (10) compared blood-based HIV testing to testing using oral mucosal transudate
samples obtained with the OraQuick ADVANCE Rapid HIV-1/2 test kit, which detects
antibodies against HIV and displays the result on the swab device after 20 min (10).
The sample is collected by running the swab between the upper lip and gums, fol-
lowed by passing the swab between the lower lip and gums. This could be considered
a buccal mucosa swab or a swab that collects a mixture of gingival crevicular fluid and
saliva, but the manufacturer simply describes it as a device to collect oral fluid. In this
study, which looked at 1,776 treatment-naive children 18 months to 18 years of age in
Kenya and Zimbabwe, there were 71 children diagnosed with HIV infection by blood-
based testing (either by a 4th generation antigen/antibody combination test or a 3rd
generation antibody test). The test performed by oral sampling was positive in all 71 of
these children (100% sensitivity, 99.9% specificity). One limitation to this strategy,
which was pointed out by the authors, is that because it detects antibodies, it cannot
be used in newborn children due to circulating maternal antibodies (10).
Parvovirus B19 and SARS-CoV-2. Quantitative PCR testing for other pathogens
has been performed on oral samples from children in research contexts. For example,
Bodewes et al. (36) used oral sampling with the Oracol device, which uses a sponge to
collect oral fluid between the lip and gums, for the detection of parvovirus B19 DNA in
116 children with a compatible rash. This strategy exhibited 68% sensitivity relative to
serum IgM-EIA testing, and 61% sensitivity relative to serum DNA testing (36). While
oral sampling in this study did not perform as well as serum-based testing, it was capa-
ble of detecting IgM antibodies or DNA in a majority of children using a noninvasive
approach.

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Given the potential role of primary schools and child care in the spread of SARS-
CoV-2, there is considerable interest in identifying noninvasive methods for detecting
the virus in children, including asymptomatic children, in community settings. To date
there are only a couple of peer-reviewed studies that have investigated oral sampling
in children specifically (37, 38). Both were small studies (n = 11 to 12) with asymptom-
atic as well as symptomatic participants. The sensitivity of oral swabbing was modest
in these studies (73% to 82% relative to NP swabbing, with high CT values).
These observations established that parvovirus and SARS-CoV-2 can be detected in
the oral cavities of at least some children, but larger studies with improved methodolo-
gies are needed.

DIAGNOSIS OF INFECTIOUS DISEASES WHEN INVASIVE TESTING IS NOT POSSIBLE

OR PRACTICAL
Oral sampling may be especially helpful for patients who are unable to undergo
more invasive procedures. For example, the fungal pathogen Pneumocystis jirovecii can
cause a severe pneumonia, particularly in immunocompromised patients who are sus-
ceptible to a variety of infections that can cause a similar presentation. The typical
diagnostic modality includes a bronchoscopy with BAL for sample collection. This is
invasive, logistically challenging, and may not be safe for patients who are clinically
unstable. It is also expensive and it requires multiple staff members with special train-
ing. In search of a noninvasive alternative, investigators have evaluated the use of oral
washes or oral rinsing for the detection of P. jirovecii DNA by PCR, especially in patients
with HIV infection or other immunocompromising conditions. Larsen et al. (17)
described 108 patients living with HIV who presented with symptoms concerning for
Pneumocystis jirovecii pneumonia (PJP). Of these, 82% were confirmed to have PJP by
the gold standard direct microscopy from a BAL sample. Quantitative-touchdown PCR
was performed on oropharyngeal wash samples, which were obtained by the patient
gargling 10 ml of sterile saline solution for 60 s, and had a sensitivity of 88% and a
specificity of 85% relative to direct microscopy from BAL samples (17). Another study
examined 36 immunocompromised patients who presented clinically suspected PJP,
15 of whom were ultimately diagnosed with and treated for PJP. The diagnosis was
finalized by the agreement between infectious disease specialists and microbiologists

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based on review of the patients’ symptoms, radiography, and other laboratory findings.
Nine out of 15 patients (60%) with PJP infection had malignancies as the cause of their
immunosuppression; 3 out of 15 (20%) were people living with HIV. Oropharyngeal
wash samples were obtained in a similar method as above (gargling for only 30 s) and
PCR was performed on two targets (the mtSSU gene and the DHPS gene), which found a
100% sensitivity and 85.7% specificity for each target, relative to the patients who were
diagnosed by consensus between specialists (16). Although this was a small study, their
results are encouraging for the use of oral samples to diagnose PJP in situations where
BAL samples are not readily obtainable. A caveat to using any PCR-based test for the di-
agnosis of PJP is that detection of Pneumocystis jirovecii DNA may simply represent colo-
nization. However, in one study, only 4% of 100 asymptomatic patients with HIV infec-
tion had a positive PCR for Pneumocystis jirovecii (39). Clinical judgment is necessary to
interpret results in these cases.

NONINVASIVE ORAL SAMPLING FOR TROPICAL INFECTIOUS DISEASES

Noninvasive oral sampling has been described for a variety of tropical infectious dis-
eases. It could be useful in settings in which other sample types are not practical to
collect.
Malaria. Plasmodium spp. were estimated to infect 228 million people in 2018, with
405,000 deaths. Malaria diagnosis commonly involves light microscopy to examine pe-
ripheral blood smears with Giemsa staining and blood-based tests that detect malaria
antigens. The performance and ease of use of these methods vary, but the requirement
for blood sampling is a limitation in all cases.

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Saliva-based testing for malaria antigens has been described. Fung et al. (40)
obtained blood and saliva samples concurrently from 8 patients in the Philippines with
confirmed malaria infections by smear microscopy. Parasite density ranged from 800/
m l to 32,000/m l, in addition to one sample with a “packed field” that was estimated to
represent a density of .50,000/m l. Saliva was obtained by rinsing their mouths with
water and expectorating into a sterile tube. An antigen called PfHRP2 was detected by
enzyme-linked immunosorbent assay (ELISA) in saliva from all 8 patients. It was not
detected in any of 16 negative-control patients, indicative of 100% specificity (40).
Although this was a small study, the results suggest that saliva could be further
explored in malaria diagnostics. Saliva has also been studied in the context of subclini-
cal malaria in children. As with most of the studies reviewed here, it is unknown
whether there are temporal differences between the detection of these antigens in sa-
liva specimens versus blood-based testing, because the samples were obtained at the
same time. There may be differences in when tests from different sampling sites
become positive and this should be investigated further, in malaria as well as in other
infectious diseases. Subclinical infections may comprise as much as 80% of malaria’s
reservoir, making the detection of these infections important to reduce the burden of
malaria disease. Tao et al. (41) obtained saliva samples via the drooling technique from
364 children with known subclinical malaria infection in Cameroon and Zambia.
Subclinical malaria infection was diagnosed either by light microscopy or rapid antigen
detection from a blood sample. The children, some as young as 5 years old, collected
the samples themselves and the procedure was acceptable to them. The saliva was an-
alyzed for the presence of several malaria antigens to determine which would perform
the best. They identified one antigen, PSSP17, and developed a lateral flow immunoas-
say rapid test to detect it. The sensitivity of this assay ranged from 91 to 100%, depend-
ing on whether light microscopy or molecular testing was used as a reference (41). A
saliva-based test such as this could be scaled up more easily than blood-based testing.
This could significantly improve case detection and reduce the malaria reservoir.
Ebola virus. Ebola can cause hemorrhagic fever and is frequently fatal. The diagno-
sis is typically made via reverse transcriptase PCR (RT-PCR) testing for viral RNA in
blood. A noninvasive alternative may be helpful for several reasons, including protec-

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tion of health care workers and greater acceptability in some cultures. Delays in diag-
nosis associated with the reliance on blood samples have been reported (42).
There were two outbreaks of Ebola viral hemorrhagic fever in the Republic of the
Congo in 2003. Formenty et al. (42) describe 24 patients with suspected Ebola associ-
ated with those outbreaks. Serum and oral fluid samples, obtained by the Orasure de-
vice, were collected from the patients and tested for IgG antibodies, antigen, and RNA.
Antibodies were not detected in the oral fluid of seropositive patients; however, test-
ing for viral RNA in oral fluid yielded a sensitivity and specificity of 100%, relative to se-
rum viral RNA testing (42). However, a larger studied conducted in Sierra Leone by
Erickson et al. found low sensitivity for oral swabs relative to blood testing.
Nonetheless, those authors found the method to be highly sensitive (equivalent to
blood) for testing corpses, presumably because of the relatively high viral load in
patients who progress (43). Given the risks associated with venipuncture in such set-
tings, oral swabbing may be the preferred sampling method for very sick or deceased
patients.

ORAL SAMPLING FOR POPULATION SURVEILLANCE AND SCREENING OF INFECTIOUS

DISEASES
Infectious disease screening has many benefits, including earlier diagnosis leading
to decreased morbidity and mortality and prevention of spread of communicable dis-
eases. Mass screening can be useful to determine the overall prevalence of disease,
and to identify asymptomatic carriers. Oral sampling could simplify infectious disease
screening, especially in situations involving large numbers of people, given its noninva-
sive nature and the ability for patients to collect their own samples.

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Minireview Journal of Clinical Microbiology

SARS-CoV-2. Oral sampling for screening purposes has been evaluated in the
COVID-19 pandemic, both to detect SARS-CoV-2 RNA directly and to detect SARS-CoV-
2 antibodies indicating prior infection. Yokota et al. screened 1,924 asymptomatic indi-
viduals with self-collected saliva and provider-collected NP swabs; RT-PCR and/or RT-
LAMP were used to detect SARS-CoV-2 RNA. These individuals were either part of a
contract tracing initiative, which included people who were exposed to someone with
confirmed SARS-CoV-2 infection, or an airport quarantine cohort of asymptomatic trav-
elers arriving in Japan. The sensitivity of saliva samples was estimated to be 92%, com-
pared to an estimated sensitivity of 86% for NP swabs; the specificity for both sample
types was .99.9%. These values were obtained by using a Bayesian latent class model,
given the lack of a gold standard for COVID-19 diagnosis (44). Another study included
401 people presenting with symptoms of COVID-19, contact with someone with con-
firmed COVID-19 infection, or general concern for COVID-19 infection. Each person had
an NP swab obtained by a health care worker as well as a saliva sample collected by
pooling saliva in the mouth for 1 to 2 min followed by spitting into a sterile container.
No transport medium was used for the saliva samples. Thirty-five out of 401 partici-
pants had detectable SARS-CoV-2 RNA from either the NP swab, the saliva, or both.
Most of these individuals (20/35, 57%) were asymptomatic. They found that relative to
the NP swabs, saliva had a sensitivity of 73.1%, a specificity of 97.6%, and an accuracy
of 96.0% (45). Although the reported sensitivity of saliva was lower in this study, it was
calculated using different methods than the study by Yokota et al. and still was able to
detect SARS-CoV-2 RNA in a significant proportion of participants. If the sensitivity of
saliva-based testing for COVID-19 diagnosis in this patient population is truly as low as
this study suggests, it is possible that more frequent testing could be a strategy to
improve the overall sensitivity of this test.
Oral sampling has also been used to detect antibodies against SARS-CoV-2, indica-
tive of current or prior infection. Pisanic and Randad et al. (46) described a cohort for
which saliva and serum samples were compared to detect IgG antibodies against
SARS-CoV-2. They obtained oral samples by using the Oracol collection device, which
collects a mixture of saliva and gingival crevicular fluid by brushing the gum line.
Thirty-three out of 128 patients (24%) who submitted saliva samples had previously

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had an illness confirmed to be consistent with COVID-19 infection through detection
of SARS-CoV-2 RNA. Of those 33 patients, 28 had samples obtained 10 days after onset
of illness, and all 28 had detectable IgG antibodies from oral samples. They also
obtained paired serum and saliva samples from a subset of patients, and showed that
the detection of IgG was highly correlated between the two sample types (46). These
results suggest that oral sampling is a viable option for monitoring evidence of prior
COVID-19 infection on a large scale given its high concordance with serologic testing
and its ability to be self-collected.
Tuberculosis. Oral sampling has been described in the context of active TB case-
finding. Lima et al. (47) describe the use of tongue swabs in 128 individuals who were
confirmed to have TB infection by sputum testing, conducted as part of a mass screen-
ing study in Brazilian prisons. Two tongue swabs were obtained from each of these
individuals; 128 negative controls with sputum negative by GeneXpert testing also had
tongue swabs collected. Investigators stratified by level of bacillary load in the sputum
and found that 32 out of 39 (82.1%) individuals with medium or high bacillary loads
were positive via Xpert testing on at least 1 out of 2 oral swabs. However, the sensitiv-
ity of oral swabs was much lower for those with low or very low bacillary loads, with
just 34/89 (38.2%) having a positive Xpert test from at least 1 out of 2 oral swabs col-
lected (47). The relatively poor sensitivity observed in this study compared to other
studies of oral swabbing for TB (5, 35) may be at least partly explained by the use of
GeneXpert to test swab samples. This commercial TB testing system is designed for
testing sputum but not swab samples. The use of testing methods specifically
designed for oral swabs may make it more feasible to exploit the high throughput of
this sampling method in screening programs. It is likely that analytical sensitivity would

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need to be increased, given the expectation of low pathogen burden in people with in-
cipient disease. Another possibility to increase the sensitivity of oral swabbing for TB
diagnosis in this setting would be to increase the number of swabs collected. Because
oral sampling is noninvasive, it would be relatively simple to collect more swabs,
although, when scaled up to large populations, this could place an additional burden
on the laboratories processing these samples. Further study of the potential increased
sensitivity from collecting additional samples would be valuable.
Hepatitis C. Mass screening for hepatitis C (HCV) could significantly improve diag-
nosis. Because there are several effective treatments available, many long-term compli-
cations of the infection could thereby be avoided. Currently, screening is performed by
testing for serum HCV antibodies. Oral sampling can be used to increase the capacity
for screening and has been demonstrated to be very sensitive.
Tang et al. (9) conducted a meta-analysis and literature review of diagnostic HCV
testing, which included several studies focused specifically on oral sampling. The
pooled sensitivity and specificity for HCV antibody testing on oral samples were 94%
and 100%, respectively. These studies involved various sample collection techniques,
but they demonstrated that the pooled sensitivity was highest among studies that
used the OraQuick ADVANCE collection kit, which involves swabbing the outer gums
to obtain oral fluid, a mixture of gingival crevicular fluid and saliva (9). The perform-
ance of a new POC test for the detection of anti-HCV antibodies known as the Well
Oral Anti-HCV test was recently described. This test involves a similar procedure as the
OraQuick and yields results in 15 to 20 min. Liu et al. (48) enrolled 1,179 patients, 486
of whom were known to have chronic HCV infection, and tested them using the Well
Oral test collected by a health care provider. The sensitivity and specificity of this POC
test relative to serum testing for HCV antibodies were 92% and 98%, respectively.
Some individuals also self-collected and the results of self-collected tests differed from
the provider-collected test in only 2 out of 199 participants (48). The use of a point-of-
care, self-collected HCV test could substantially improve screening.
HIV. Oral sampling to test for HIV has been studied extensively. A meta-analysis
that included studies using the OraQuick point-of-care test demonstrated a pooled
sensitivity of 98% and specificity of 99% for the detection of HIV-1/2 antibodies. In

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comparison, the sensitivity and specificity of blood-based testing were both .99%.
The most significant difference between oral sampling and blood-based testing was
seen when the data were stratified by settings with high or low HIV prevalence, and
pooled positive predictive value (PPV) was calculated. In high-prevalence settings, the
PPV was about the same when using oral and blood-based testing (98.65% in oral sam-
pling, 98.50% in blood-based testing). However, in low-prevalence settings, the PPV
was significantly lower using OraQuick (88.55% in oral sampling, 97.65% in blood-
based testing) (8). A subsequent study compared several different oral sampling kits.
The DPP HIV 1/2 Assay, which involves swabbing the outer gums for 15 to 30 s to col-
lect oral fluid, was shown to be 100% sensitive and specific when tested in 507 patients
(7). Further study using this assay is warranted to confirm these results.

SUMMARY
The oral cavity is a complex compartment with diverse microenvironments, some of
which are highly aerated and continuously washed with saliva, while others are sheltered
and anaerobic. It harbors one of the most complex microbial communities in the human
body. As a major portal between our bodies and the outside world, it is a useful and very
accessible place to look for pathogen biomarkers of infectious disease. As illustrated by the
examples in this article (summarized in Table 1), the list includes diseases that are not nor-
mally associated with the oral cavity. In some cases, identical methods can potentially be
used for multiplex screening for multiple pathogens. With rapid improvements in molecu-
lar and point-of-care detection technologies, other examples of oral sampling may be
worth visiting or revisiting. By design, oral sampling is an easy idea to evaluate.

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TABLE 1 Summary of oral samples and techniques

Reference Sample type Sample collection notes Analyte used
Tuberculosis
Luabeya et al. (5) Tongue swab, buccal swab, gum swab OmniSwab, PurFlock DNA
Nicol et al. (35)a Buccal swab OmniSwab, PurFlock DNA
Flores et al. (6)a Buccal swab OmniSwab DNA
Mesman et al. (21) Buccal swab OmniSwab DNA
Lima et al. (47)b Tongue swab DNA
Shenai et al. (20) Saliva, exhaled breath condensate Salivette saliva collector DNA
Molina-Moya et al. (22) Buccal swab PrimeSwab DNA
Song et al. (23) Tongue swab DNA
Kang et al. (24) Oral mucosal transudated OMNIgene ORAL OMR-110 DNA
SARS-CoV-2
Han et al. (37)a Saliva RNA
Kam et al. (38)a Buccal swab Mini UTM Kit with flocked swabs RNA
Azzi et al. (11) Saliva Drooling technique RNA
Williams et al. (27) Saliva Spitting out technique RNA
Hanson et al. (13) Saliva Pooling in mouth then spitting RNA
To et al. (30) Oropharyngeal saliva Coughing out early morning saliva RNA, viral culture
Procop et al. (14)c Enhanced saliva Sniffing strongly, coughing out RNA
Mittal et al. (18)c Oral rinse RNA
Babady et al. (19) Oral rinse and saliva Spitting out technique for saliva RNA
Czumbel et al. (12) Saliva RNA
Kojima et al. (31)c Tongue, buccal, gum, hard palate swab Copan flocked swab RNA
Tu et al. (4)c Tongue swab Copan flocked swab RNA
Yokota et al. (44)c Saliva RNA
Senok et al. (45)c Saliva Drooling technique RNA
Pisanic et al. (46)c Oral mucosal transudated Oracol device Antibodies
Nagura-Ikeda et al. (28)c Saliva Spitting out technique RNA and antigen
Agulló et al. (29) Saliva Antigen
Kandel et al. (34)c Oral rinse, tongue and buccal swab RNA
HIV
Dziva Chikwari et al. (10)a Oral mucosal transudated OraQuick ADVANCE Antibodies
Pant Pai et al. (8) Oral mucosal transudated OraQuick ADVANCE Antibodies
Beelaert et al. (7) Oral mucosal transudated DPP HIV 1/2 Assay Antibodies

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Parvovirus B19
Bodewes et al. (36)a Oral mucosal transudated Oracol device DNA and antibodies
Pneumocystis jirovecii
Larsen et al. (17)d Oral rinse DNA
Goterris et al. (16) Oral rinse DNA
Fraczek et al. (39) Oral rinse DNA

Malaria
Fung et al. (40) Saliva Rinse mouth then expectorate Antigen
Tao et al. (41)a Saliva Drooling technique Antigen

Ebola
Formenty et al. (42) Oral mucosal transudated Orasure device RNA, antigen, and antibodies
Erickson et al. (43) Oral swab RNA

Hepatitis C
Tang et al. (9) Oral mucosal transudated OraQuick ADVANCE Antibodies
Liu et al. (48)c Oral mucosal transudated Well Oral Anti-HCV test, OraQuick Antibodies
aStudy involving children.
bActive TB case finding.
cSelf-collected samples.

dAlso referred to as gingival crevicular fluid.

ACKNOWLEDGMENTS
The writing of this article was supported by University of Washington Host Defense
Research Training Grant (T32AI007044, to E.D.V.), and by grants from the National Institutes
of Allergy and Infectious Diseases and the Bill & Melinda Gates Foundation.

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Minireview Journal of Clinical Microbiology

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