Article Type : Review Article
Authors : Burgess J
Keywords : Covid-19; DNA; Oral cancer
Recent scientific advancements have been made in
the use of saliva to identify several systemic diseases, oral cancer,
periodontal disease, and viral infections - including Covid-19. Saliva contains
a wealth of constituents that can serve as potential biomarkers of disease
including DNA and RNA fragments, proteins, proteomes, hormones, and antigens.
The future of saliva disease assessment (salivary diagnostics) will depend,
ultimately, on the development of ‘point-of-care’ devices that can provide
immediate feedback in the clinical, research, or public health setting. This
represents the future of salivary diagnostics and in some cases that future is
now.
The potential for saliva as a diagnostic aide was
recognized in 2002 by the US based National Institute of Dental and
Cranio-facial Research (the NIDCR). In light of the current pandemic, research
groups throughout the US and other countries have initiated studies that seek
to elucidate the diagnostic possibilities of using saliva to detect the
Sars-CoV-2 virus (the virus that causes Covid-19) but it should be appreciated
that prior to this current interest there has been much research assessing
saliva as a potential diagnostic aide for a number of diseases [1]. This review article briefly outlines some of the more interesting
innovations in salivary biomarker research and comments on the future of
salivary diagnostics.
Salivary
molecular biomarkers that have the potential to be indicators of systemic
conditions such as cancer (including oral cancer), periodontal disease,
autoimmune disease, viral and bacterial disease, and cardiovascular disease
include: DNA, ctDNA, RNA, meta-RNA, RNA fragments, minor proteins, nucleic
acids, various peptides, proteomes (the entire protein complement expressed by
a cell), hormones, antigens, antibodies, and drugs. What makes saliva such a
valuable target for molecular diagnostic research is that it is easy to
retrieve and collection does not involve discomfort.
Some
of the more recent studies underscoring the promise of saliva in the diagnoses
of disease include those in the fields of brain research, systemic diseases
(including Sjogren’s syndrome), oral cancer and other cancers, periodontal
disease and infection.
As reported
May 6-9, 2017, in an abstract presented at a Pediatric Academic Societies
Annual Meeting in San Francisco, and subsequently in the Journal of Neurotrauma
[2], micro-RNA fragments found in saliva were found to be
predictive of pediatric brain concussion in children with traumatic brain
injury. Micro-RNA fragments (mRNA) are short (length: 19-24 nt) non-coding molecules that are
important in regulating numerous cellular processes including those involving
the brain [3]. This preliminary
research suggests that salivary mRNA fragments easily acquired and measured in
the clinical setting, may be useful for identifying pediatric traumatic brain
injury (TBI). As the authors indicate, concentrations
of mRNA-320c (one of many fragments) were directly correlated with child and
parent reports of attention difficulty. The test was shown to be 90 percent
accurate. This contrasts with a concussion survey now commonly used that has
less than 70 percent accuracy. However,
additional studies assessing the influence of orthopedic injury and exercise on
peripheral mRNA patterns are needed in adults and children to further
understand the potential utility of this strategy for diagnosing concussion.
Researchers
have identified potential salivary transcriptomic profiles for other diseases
such as acute myocardial infarction, diabetes mellitus, Sjogren’s syndrome,
cystic fibrosis, and Parkinson’s disease in addition to breast, pancreatic,
ovarian, and gastric and lung cancer as well as melanoma [4].
A
good salivary assay for identifying systemic disease should have high sensitivity, specificity, and functionality. The literature
reflects optimism that these fundamental research parameters, although in some
cases not currently at acceptable levels, will be improved in the future.
Consequently, much work remains to be done before measurement of salivary
proteins and nucleic acids yields meaningful diagnostic capability.
Nonetheless, research to date has revealed some interesting results with
respect to salivary proteomes and some systemic diseases.
Saliva constituents appear to be altered in Sjogren’s
syndrome (SS), an autoimmune disorder that causes, among other symptoms, dry
mouth, and dry eyes. In one preliminary study, twenty five proteins in whole
saliva, including ?-enolase, carbonic anhydrase I and II, and
salivary ?-amylase fragments in patients with SS were found to be up-regulated and sixteen proteins expressed by the acinar cells of the glands,
including lysozyme C, polymeric immunoglobulin receptor (pIgR), and calgranulin
A were down-regulated as compared with the salivary proteomes of individuals
without disease [5]. In another study 15
proteins in whole saliva differentiated non-SS subjects from those with SS [6]. There is also
evidence that the microRNA profiles of minor labial salivary glands differ
between normal subjects and those with SS [7]. These accumulated
studies suggest that specific proteomic salivary biomarkers may prove useful in
the development of a diagnostic panel that can be used on site to identify
patients with Sjogren’s syndrome.
In recent
years there has been a focus on micro RNA fragments (mRNAs) in saliva as
biomarkers of oral cancer because these cellular constituents are known to have
an effect on cell growth, proliferation, and apoptosis and also appear to serve
as oncogenes within different cancer types [8,9]. Multiple studies suggest that
mRNAs may prove useful in early detection of oral cancer and could potentially
lead to changes in how oral cancer is treated [10]. Yoshizawa and Wong provide a
good overview of oral cancer detection and salivary mRNAs [11].
In a study funded, by the ‘Early
Disease Research Network’ a working group within the National Cancer Institute,
salivary biomarkers were assessed for their utility in discriminating patients
with oral cancer from healthy subjects [12].
In this case-controlled study utilizing two independent laboratories,
395 subjects from five independent cohorts were assessed. The result was that
seven mRNAs and three proteins were found to be increased in oral squamous
carcinoma cancers versus controls in all the cohorts. Specifically, the
increase in two markers: IL-8 and SAT demonstrated good sensitivity and
specificity in predicting disease. These biomarkers were found to effectively
discriminate patients with oral OSCC from healthy controls.
Other studies have identified additional proteomic salivary biomarkers
potentially useful in detecting OSCC [13]. In a comparison of sera with saliva
in patients with OSCC, three tumor markers: specifically Cyfra 21-1, tissue
polypeptide antigen (TPA), and a cancer antigen CA125 were found to be
significantly more elevated in the saliva of diseased subjects [14]. And results of an additional study suggest that
five salivary proteins (M2BP, MRP14, profilin, CD59, and catalase) can
discriminate oral cancer with 90% accuracy [15].
Additional
research will help to fully elucidate candidate proteins that can be used to
indicate the presence of OSCC [16,17]. Thus
far, the research on salivary biomarkers is unclear regarding their
use in defining oral cancer disease severity and progression, but the discovery
of specific biomarkers that can be used to diagnose cancer presents a big step
forward in salivary diagnostics. Arellano, et al. [18] and Sannam, et al.
[19] provide excellent reviews covering the advancements in the
identification of OSCC via salivary proteomics.
A
few cancers, including those involving the ovaries, endometrial tissues,
fallopian tubes, the pancreas, stomach, esophagus, colon, liver, and breast
demonstrate an elevation of the cancer antigen 125 (CA 125) in blood. This
protein biomarker has also been found in at least one study to be elevated in
the saliva of individuals with malignant ovarian tumors. Saliva CA 125 levels were correlated with serum
levels in subjects with ovarian cancer in terms of sensitivity and specificity
(81.3 and 93.8 respectively). And in patients with endometriomas and pelvic
tuberculosis the false positive rate was significantly lower for saliva CA 125
than serum CA 125 (13.6, 10% versus 72.7, 80%) [20]. suggesting that
saliva CA 125 may have better diagnostic value for these conditions than CA 125
found in serum. However, subsequent research results are in conflict with the
above findings and suggest a lack of relationship between CA 125 levels and
epithelial ovarian cancer and benign gynecologic conditions so the issue of its
potential diagnostic use remains cloudy [21].
More recent research suggests that better specificity and sensitivity of CA125
as a tumor biomarker may occur when the assay is combined with another
biomarker HE4 [22] and the
presence of mRNAs may also help in determining therapeutic response [23]. The introduction of FDA-approved
algorithms is reported to have improved the ability to assess risk of ovarian
cancer from sera of patients with a pelvic mass. The extent to which this
applies to the same markers found in saliva remains unclear.
With
advancement of methodological techniques able to identify viral DNA, RNA, mRNA
proteins or salivary antibodies, a few viruses can now be identified in saliva.
These include norovirus, rabies, human papillomavirus (HPV), Epstein-Barr
virus, herpes simplex viruses, hepatitis C virus, cytomegalovirus
(CMV), HIV, and now Covid-19 [24].
In April, the FDA authorized, by prescription only,
the use of a saliva test for diagnosing Covid-19. Multiple institutions including
Yale University have assessed saliva testing capability. In a letter to the
editor of the NEJM, August of 2020, it is reported that a Covid-19 saliva test
was used on 70 patients who had already tested positive for Covid-19 (confirmed
by positive nasal swab). The author, Anne Wyllie, notes that a higher
percentage of saliva samples were positive for SARS-CoV-2 at 10
days than the nasopharyngeal swab samples. She reports that at 1 to 5 days
after diagnosis, 81% (95% CI, 71-96) of the saliva samples were positive, as
compared with 71% (95% CI, 67-94) of the nasal specimens, suggesting that both
tests were relatively equivalent in terms of sensitivity (detection of the
disease). What remains a concern for both nasal swab and saliva tests, however,
is their generally low sensitivity (detection of actual disease) and their
degree of specificity (their ability to detect whose individuals who do not
have the disease) Nonetheless, the initial reported results related to use of
saliva are quite promising for its use in detecting Covid-19 disease [25].
With the Morbillivirus virus that causes measles
infection, the presence of salivary antibodies demonstrates 97% sensitivity and
100% specificity [26], for Paramyxoviridae virus that causes mumps 94%
sensitivity and 94% specificity [27], and for the Togaviridiae virus that causes
rubella, 98% sensitivity and 98% specificity [28].
Antibodies used to diagnose HIV infection are also found in saliva and
salivary assays have been shown to be as accurate as those associated with
serum, particularly when plasma virus exceeds 50
copies/mL (when there is active disease) [29]. A commercial product called
OraQuick has been FDA approved and is available for assessing HIV antibodies in
saliva [30], The testing kit contains a collection
stick, test tube, and testing information/directions. It is reported to be able
to detect antibodies to HIV-1 and HIV-2 within 20 minutes [31].
In
a move towards establishing more precise diagnosis of periodontal disease a
number of salivary constituents have been considered as potential biomarkers
including DNA from specific bacteria, inflammatory cytokines that are
host-derived, cell death host-derived proteins, and enzyme, protein, or calcium
derived factors from bone destruction [32]. The many
biomarkers associated with periodontal disease. The validated biomarkers
include: bacteria-derived DNA
salivary (porphyromonas gingivalis, prevotella intermedia, and tannerella
forsythia) host-derived in?ammatory mediators (in?ammatory cytokines (IL-1? and
MIP-1?), host-derived markers associated with soft tissue destruction (MMP-8,
MMP-9, HGF, lactate dehydrogenase, aspartate aminotransferase, and TIMP-2), and
host-derived markers associated with bone destruction
(alkaline phosphatase, osteonectin, RANKL, and calcium). Among the various salivary biomarkers
listed, P. gingivalis has been shown to satisfy all the requirements for an
ideal biomarker of periodontitis.
Presently periodontal disease remains
a clinical diagnosis established through visual examination, periodontal
probing of the gingival sulcus, and evaluation of radiographic imaging to
detect bone loss. For public health and research purposes, the Community
Periodontal Index (CPI) that includes a periodontal ‘probe’ and rating system
defining pocket depth was developed and adopted for use by the World Health
Organization [33]. Elements of the rating instrument, including a
version of the probe, currently represent the standard of care in clinical
practice in establishing a diagnosis of periodontal disease and its severity.
Tracking the specific cellular processes associated with periodontal disease
progression continues to be a problem in the context of clinical care.
However, recent advances in salivary
research suggest that periodontal disease progression may be effectively
tracked via integration of biologic measures (e.g. the presence of the specific
biomarkers in saliva mentioned above) coupled with standard clinical and
radiologic measures. Ebersole, et al., [34]
in
a case controlled study of 209 subjects, evaluate a specific kit (the Milliplex
Map Kit – EMD Millipore, Billerica, MA, USA) to detect saliva analytes related to the biological processes of
periodontitis. IL-1? and IL-6 (both cytokine inflammatory signals), MMP-8 (a
primary collagenase), and MIP-1? (also known as CCL3 -a chemokine macrophage
inflammatory protein) isolated alone or in combination, were found to
distinguish healthy subjects from those with gingivitis and periodontitis.
Their findings suggest that the salivary level of MIP-1? could have clinical
utility as a screening tool for identifying moderate to severe periodontal
disease and that sensitivity, specificity, and accuracy may be improved by
exploring combinations of the identified biomarkers.
Other salivary constituents including the enzymes
aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase
(LD), and alkaline phosphatase have also been considered useful as biomarkers
for diagnosing and screening periodontal disease [35]. Given that lactate dehydrogenase is related
to epithelial cell breakdown, a ‘kit’ allowing quick assessment of this enzyme
was tested on 70 healthy volunteers against standard periodontal examination.
Salivary LD level was positively correlated with bleeding on probing and the
sensitivity and specificity of the kit was 0.89 and 0.98 respectively, at a
cut-off value of 8.0 for LD level. Although the above study was
limited because it was cross sectional, it was concluded that the evaluated
‘kit’ could have utility in the early detection of gingivitis [36].
Additional studies suggest that assessment of salivary
biomarkers may also help in determining treatment response in the management of
gingivitis, with some biomarkers more important than others in defining
efficacy. In a study by Syndergaard, et al., mean biomarker concentrations were
found to decrease in the gingivitis groups following dental prophylaxis.
However, certain markers, specifically MIP-1?
and PGE2, remained significantly higher in the healthy
group [37]. Based on these results, the authors conclude that relative
change in the assessed biomarkers could prove helpful in identifying diseased
patients who, despite prophylaxis, might be at risk for continued chronic
gingival inflammation and the development of more destructive periodontal
disease.
With respect
to disease progression, the accumulated research evidence suggests that a panel
of optimal biomarkers must be carefully selected based on the pathogenesis of
periodontitis. The biggest hurdle for the diagnosis and tracking of
periodontitis progression using saliva may be validating specific disease
related biomarkers as well as the efficacy of point-of-care devices within
large diverse patient populations.
There are
currently two point-of-care devices that have been developed for the salivary
diagnosis of periodontitis: one is called the Integrated Micro?uidic
Platform for Oral Diagnostics (IMPOD), the other is a lab-on-a-chip (LOC)
system developed by the University of Texas. The IMPOD measures MMP-8 (a neutrophil collagenase, also known as matrix
metalloproteinase-8), TNF-? (a tumor necrosis
factor), IL-6, and CRP (C-reactive protein) in saliva and the LOC measures CRP,
MMP-8, and IL-1?. The LOC has shown good comparative accuracy with the enzyme-linked
immunosorbent assay (ELISA). The LOC device is currently undergoing clinical trials:
(NCT02403297 at ClinicalTrials.gov) [38].
The emergence of Covid-19 and the worldwide pandemic underscores the need
for non-invasive tests offering immediate diagnostic results. And recent
developments affirm that saliva can be used to diagnose this disease as well as
many others. Developments in the field of salivary diagnostics should lead to
significant advances in point-of-care precision medicine and dentistry. The
benefits of using saliva as a biomarker for disease are multiple. Collection of
fluid is non-invasive, simple, and easily accomplished by the patient or in the
clinical setting. Relative to collection of serum it is inexpensive, and
clotting is not a problem as it is with serum. Saliva contains physiological
markers for many conditions, both systemic as well as those localized to the oral
environment [39]. Salivary diagnostic
assessment will also allow for the screening of patients outside the clinical
setting, for purposes of treatment monitoring, and for epidemiological research
or public health screening. Combining point-of-care devices such as those
currently available or those being developed for assessment and monitoring of
periodontal disease and oral cancer, as well as systemic diseases, coupled with
improved medical and dental electronic software communication could lead to more
accurate disease tracking of patients by providers, researchers, and community
health screeners via the internet and through digital charting. As pointed out
by Yager, et al., “underserved communities and resource-limited areas may be
accessed more efficiently than by current cumbersome and poorly utilized
screening programs” [40]. Further, the use of salivary
diagnostics may increase access to treatment for identified at-risk individuals
not aware of developing disease and help in containing community spread of
viral infections such as Covid-19. Kaczor-Urbanowicz and colleagues
provide a nice overview of salivary diagnostics, the current views, and
directions [41].