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Future Virology

Review

Saliva specimen sampling:

anoninvasive method for
diagnosis and basic investigation
of viral hepatitisA, Band C
Luciane Almeida Amado Leon
Laboratory of Technological Development in Virology, Institute Oswaldo Cruz Fiocruz, Av. Brasil 4365, Rio de
Janeiro, 21045-900, Brazil n l_amado@ioc.fiocruz.br

Saliva is a biological fluid that is easy to collect and manipulate. Collection of

saliva samples is less expensive, noninvasive and painless compared with blood
collection. Due to these advantages, saliva has been investigated as an
alternative fluid to serum for diagnostic and epidemiological purposes. The aim
of this article was to the review research on salivary biomarkers of viral hepatitisA,
B and C, highlighting their current use, collection devices, and potential
applications for diagnosis and epidemiological studies. This paper also explores
recent findings of saliva as a possible source of viral hepatitis transmission.

Saliva is an oral fluid that plays a key physiologic

role in preliminary digestion (amylase is involved
in digestion of starch), taste perception (carbonic
anhydrase), homeostasis and active protection
and maintenance the oral health. The defense
compounds of saliva can be related to a specific
immune response such as immunoglobulins or
to a nonspecific oral defense such as lysozymes,
peroxidases, mucins, lactoferrins, and histatins
and defensins, which possess bacteria-killing
properties.
Saliva originates mainly from salivary glands:
the parotid, sublingual and submandibular. Once
saliva enters the oral cavity, it mixes with fluid
originated within oropharyngeal mucosae (blood
cells, bacteria, fungi and viruses), oral epithelial
cells, food debris and blood-derived compounds,
such as plasmatic proteins, erythrocytes and leucocytes. Saliva also contains crevicular fluid (CF),
a plasma transudate, which constantly flows from
the crevice between the gum margin and the teeth
[1]. CF is the component in saliva that contains the
highest concentrations of immunoglobulins IgA,
IgG and IgM. IgA is the main salivary immunoglobulin (>85%) and is produced directly by
the B lymphocytes present near the glands. IgM
and IgG are derived from CF or from plasma and
constitute the remaining 515% of the salivary
immunoglobulins.
The variety of components of the saliva not only
protect the integrity of oral tissues, but also provide
salivary biomarkers that are being explored as a
means of monitoring health [2] and diseases [3]. The
importance of saliva as a diagnostic fluid has been
recognized since 1992 by the New York Academy
of Sciences, who raised awareness of the potential
10.2217/FVL.13.41 2013 Future Medicine Ltd

of saliva-based diagnostics in a major conference

on the subject [4]. Continuing research has led to
the development of more sensitive salivary assays,
which have advanced the diagnosis and research
of several infectious and systemic diseases [2].
Nowadays, saliva is increasingly being used in the
diagnosis of several viral diseases, such as HIV [5,6],
hepatitisA [7,8], hepatitisB [9], hepatitisC [10,11] and
herpes simplex [12], and as an investigational tool.
Salivary analysis has a great potential diagnostic
role, since many salivary compounds can derive
from plasma. Therefore, several biochemical and
immunological parameters, as immunoglobulins,
can be measured in both blood and saliva. The
ability to analyze saliva to monitor health and
disease is a highly desirable goal for oral health
promotion and research. In the laboratory, saliva
is relatively easy to collect in sufficient quantities
for analysis, and the costs of collection tend to be
lower than those for serum. For healthcare professionals, saliva tests are safer than blood tests,
which are more likely to result in exposure to
blood-borne diseases, such as hepatitis and HIV.
For the patient, the noninvasive collection techniques for saliva can reduce discomfort, mainly of
concern in children, thereby simplifying collection
of serial samples for monitoring disease states over
time. These advantages have led to a proliferation
of noninvasive home testing kits for saliva.
The aim of this article was to review current
studies regarding the use of saliva, collection
devices and its potential applications for diagnosis
and epidemiological studies of viral hepatitis A, B
and C. This review also explores recent findings
regarding the role of the saliva in viral hepatitis
transmission.
Future Virol. (2013) 8(6), 575588

Keywords
collection n diagnosis
surveillance
ntransmission n viral hepatitis
n

nsaliva n

part of

ISSN 1746-0794

575

Review

Amado Leon

Saliva collection

Successful detection and measurement of salivary immunoglobulins and other viral markers
requires optimal collection, processing and storage. Several factors may influence the rate of salivary flow and its composition, and these factors
should be taken into account when saliva is used
as a diagnostic fluid [13]. Since the component in
saliva that contains the highest concentrations of
immunoglobulins is the CF, the conditions under
which saliva is collected can have an influence on
antibody detection.
Whole saliva, glandular duct saliva, CF and
mucosal transudate are different types of saliva
specimens with regards to the different compounds, including ions, drugs, hormones and
immunoglobulins, that can be collected by specific methods [3]. These methods of sampling
require a high degree of training for the specialist collecting the specimen. For this reason these
sampling methods are not commonly used [14].
Early studies on the detection of virus antibodies in saliva used whole saliva that was collected
by being dribbled into a sterile, wide-mouthed
container [15,16]. However, dribbling was considered distasteful for the patients, and also samples
with insufficient volume were frequently received.
Also, pipetting untreated saliva was difficult.
This led to the development of devices designed
to collect oral fluid. Today several methods and
devices are available (Table1). The devices are simple, safe and convenient to use, and provide an
adequate homogeneous specimen with low viscosity. Nevertheless, it is essential that devices used
to collect oral fluid are tested to ensure reasonable sensitivity and specificity for the detection
of viral antibodies. A transport buffer provided
with the Omni-SAL (Saliva Diagnostic Systems)
and OraSure (OraSure Technologies) devices
contains antimicrobial agents and protein stabilizers. Specimens may be stored at temperatures
of 437C for 21 days or at -20C for longer
periods[17].
Saliva as a diagnostic & basic
investigation tool of viral hepatitisA, B&C

Saliva has many advantages as a sample for antibody and genome detection, compared with
blood. It is simple, safe, painless and cheap to
collect compared with venipuncture. However,
although saliva antibody profiles indicate those in
blood, they are present at lower concentrations [15].
HepatitisA

There are few studies investigating HAV diagnostic capability through oral fluids. The collection
576

Future Virol. (2013) 8(6)

of saliva as an alternative to blood collection

offers potential advantages for research and epidemiological studies of outbreaks of hepatitisA.
It allows a noninvasive investigation of all individuals, mainly children, identifies subclinical
cases, which occur frequently among children,
and facilitates tracking of vaccination candidates.
A review of the available scientific literature in
the databases PubMed, MEDLINE, LILACS
and SciELO shows that many studies have been
conducted using saliva to detect specific antibodies against hepatitisA, usually with promising
results (Table2).
Since 1989, the use of salivary samples to
diagnose acute cases of hepatitisA outbreaks has
being investigated by Parry [15] and Bull [18], and
has been demonstrated to be a convenient and
satisfactory alternative to serum for the diagnosis
ofhepatitisAinfection.
A follow-up study conducted in 29 acute-phase
patients indicated that IgM anti-HAV persisted
at moderate levels for 24months and were not
usually detectable thereafter [19]. A study conducted by OFarrell to evaluate the reliability
ofsalivaas a sample for the detection ofhepatitisAimmunoglobulins under various sampling
conditions revealed that sampling time and tooth
brushing had a significant effect on the total antiHAV titer, while eating had no significant effect
[20]. Although tooth brushing can cause variation in anti-HAV titres, this variation is not sufficient to generate a false-negative result. These
results demonstrate the usefulness ofsalivafor the
detection ofhepatitisAantibodies, regardless of
sampling times, eating or tooth brushing.
Oba [21] and Amado [8] investigated the usefulness of saliva in HAV antibody detection in
Brazilian populations. These studies examined
the possibility of using commercially available
HAV serum ELISA kits with a few modificationsto detect IgM and total anti-HAV in oral
fluid and compared outcomes with the results of
serum ELISA test. The tests achieved a specificity
of 100% and the sensitivity was approximately
80% for total anti-HAV. However, for IgM antiHAV, Oba achieved a sensitivity of 100% [21],
while Amado achieved a sensitivity of 79% [8],
using saliva samples from a population (n=19)
involved in an outbreak of hepatitisA, collected
by Omni-SAL device. These discrepancies could
be related to the collection device used, the larger
samples tested by Amado [8], and were probably
mainly due to the different populations studied
(involved in an outbreak vs clinically suspected
cases). Amado used saliva samples collected by the
OraSure device for diagnosis of patients involved
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Saliva specimen sampling: noninvasive method for diagnosis & investigation of viral hepatitisA, B& C

Review

Table 1. Characteristics of commercial devices for the collection of oral fluid specimens.
Collection Description
device

Transport

Characteristics

Manufacturer

OraSure

An absorbent pad designed to collect

up to 2 ml of saliva, a centrifuge tube
containing preservative and a cap. The
user places the device between the
lower cheek and gum for 25min

The pad is transported to a

laboratory in a vial that contains a
preservative, which stabilizes the
sample for up to 21days at
3998F

The pad draws oral fluid,

which is rich in antibodies
and contains far fewer
contaminants than
typically found in saliva

OraSure
Technologies

ORACOL

An absorbent foam swab designed to

collect up to 1 ml of saliva, a
centrifuge tube and a cap. The user
moves pad in a similar manner to
tooth brushing for 1 min

The pad is transported to a

laboratory in a centrifuge tube

The extraction of saliva

from foam requires
centrifugation

Malvern Medical
Developments

OmniSAL

A compressed, absorbent cotton pad

attached to a plastic stem. The user
places the pad under the tongue and
absorbs fluid from the floor of the
mouth

The collection pad is inserted into a

stoppered transport tube
containing 1 ml of phosphatebuffered saline, pH 7.0, protease
inhibitors, surfactants, antimicrobial
agents and 0.2% sodium azide as a
preservative

The device incorporates an Saliva Diagnostic

indicator on the plastic
Systems
stem that turns from white
to blue when an adequate
amount of sample has
been collected

Salivette

Saliva is obtained by the user chewing The pad is transported to a

a compressed cylinder of cotton (saliva laboratory in a centrifuge tube
collector) for approximately 1 min

Orapette

A rayon ball that is placed in the

mouth for collecting saliva from
around the teeth and gum until the
rayon is completely saturated

in a hepatitisA outbreak (n=52) and showed a

higher sensitivity of 96%, when compared with
the previously described study. These discrepant results indicate that saliva samples are more
feasible for diagnosis of hepatitisA in outbreak
settings [7].
A similar study conducted in order to evaluate HAV vaccination status in Bangladesh found
a slightly lower sensitivity and specificity of the
saliva test compared with those two studies
(88.6and 90%, respectively) [22]. According to
Ahmed [22], this discrepancy may be due to the
use of a special device for the collection of saliva
used by those investigators.
Although oral fluid collection has many
advantages compared with venipuncture, some
problems may arise if the choice of assay is not
appropriate to detect antibodies in oral fluid
samples. The choice of one of four configurations
(immunoassay indirect, competitive, sandwich
or capture assay) depends on the concentration
of immunoglobulin. Owing to the lower concentration of saliva IgG and IgM, many studies
has shown the need for in-house development
of the immunoassay. In a study conducted by
Tourinho and colleagues to develop and optimize an in-house competitive ELISA for the
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In order to stimulate
Sarstedt
salivation, a version with a
citric acid preparation is
available

The fluid is collected in a stoppered A small rayon ball and a

tube suitable for transport and
snapped-cap plunger that
testing
screws into a receiving
container

Trinity Biotech

detection of anti-HAV in oral fluid, the importance of establishing a cutoff ratio for the detection of antibodies against HAV in oral fluid
specimens was demonstrated [23]. Comparison
of different oral fluid cutoff values showed that
a reduction of approximately 17% was essential
to increase test accuracy. At an optimal oral fluid
cutoff value set at 0.351, sensitivity and specificity reached 91.7 and 86.2%, respectively. The
results of this study were in accordance with
another study, which conducted an in-house
ELISA and radioimmunoa ssay to detect total
anti-HAV, both based on an antibody-capture
technique [24]. In this study, both assays were
validated using 120 paired serumoral fluid
samples, and the ELISA had a slightly superior
sensitivity to that of the oral fluid radioimmunoassay (92.5 vs 87%, respectively), while both had
100% specificity. Furthermore, owing to lower
anti-HAV IgG concentrations, the assay cutoff
value was reviewed within a group of vaccinated
individuals.
Oral fluid has also been used in several epidemiological studies. According to MorrisCunnington, the minimally invasive nature
of oral fluid sample collection makes the selfcollection of samples possible, reducing costs
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Randomly sampled from
general practices
countrywide
HepatitisA-acutely infected
patients

HepatitisA-acutely infected
patients

Persons aged less than

45years

Patients referred to the

National Reference Center
ofHAV

Children from a childcare

center

813year-old children

Morris-Cunnington
etal. (2004)

Mackiewicz etal.
(2004)

Poovorawan etal.
(2005)

Duval etal. (2005)

Future Virol. (2013) 8(6)

Randomly sampled
Asymptomatic and patients
clinically suspected of acute
viral hepatitis
HAV-vaccinated students

Quoilin etal. (2007) General population

Amado etal. (2008) Children from a daycare

center

Travelers to HAVendemic
areas

Volunteers and health

professionals

Matheson etal.
(2010)

Tourinho etal.
(2011)

Individuals living in
difficultto-access areas

Children from a daycare

center

RT-PCR: Reverse transcription PCR.

Tourinho etal.
(2012)

Amado etal. (2011)

Ahmed etal. (2009) Young adults and children

Randomly sampled

Ochnio etal. (2007) Patients aged 2039 years

HAV-immune and
-nonimmune individuals

Asymptomatic and patients

clinically suspected of acute
viral hepatitis

HAV-unvaccinated
individuals

HAV-immune and
-nonimmune individuals

Rio de Janeiro, Brazil

Patients clinically suspected

of acute viral hepatitis

Amado etal. (2006) Patients referred to the

Brazilian Reference Center
for Viral Hepatitis

ELISA

IgG anti-HAV

IgG anti-HAV

ELISA

ELISA

IgM andtotal anti-HAV ELISA

Anti-HAV IgG

Total and IgM antiHAV, ELISA, RT-PCR

and HAV RNA

Real-time PCR

ELISA

ELISA

Technique

Total anti-HAV

Assay optimization and

epidemiology study

Rio de Janeiro and

Pantanal
MatoGrosso, Brazil

Total anti-HAV

Total anti-HAV

IgG

To evaluate the reliability HAV RNA

of saliva for sequence
investigation in
outbreaks

To identify candidates
for HAV vaccination

Standardization of saliva
assay

Prevalence

ELISA

Real-time PCR,
RT-PCR

ELISA

ELISA

ELISA

Detection of HAV RNA in Total and IgM antiHAV, Real-time PCR,

and HAV RNA
RT-PCR, ELISA
saliva

Seroprevalence study

Population-based
surveys

Diagnosis

Prevalence estimation

Investigation of a
hepatitisA outbreak

To evaluate the excretion HAV RNA

of HAV in saliva and its
reliability for sequence
investigation

Rio de Janeiro, Brazil

Bangladesh

Rio de Janeiro, Brazil

Nova Scotia, Canada

Rio de Janeiro, Brazil

Flanders, Belgium

Vancouver, Canada

Qubec, Canada

Bangkok, Thailand

Villejuif, France

Total anti-HAV

Postal population-based
survey

England and Wales

HAV markers

Diagnosis and prevalence IgM and IgG anti-HAV

estimation

Objective

Bratislava, Slovakia

Study area

Randomly sampled

Randomly sampled

General

Madar etal. (2002)

Sampling

Study population

Author (year)

Table 2. Studies on oral fluids for HAV RNA and anti-HAV detection in the past 10years.

[99]

[29]

[22]

[23]

[98]

[27]

[33]

[97]

[96]

[95]

[94]

[28]

[24]

[93]

Ref.

Review
Amado Leon

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Saliva specimen sampling: noninvasive method for diagnosis & investigation of viral hepatitisA, B& C

and potentially increasing response rates in

epidemiological studies [25]. Apopulation-based
survey of immunity using oral fluid collected by
post showed the feasibility of this new methodological approach. This study described that there
was no requirement for specially trained personnel to be employed to collect serum samples and
allowed the collection of comprehensive riskfactor data. The sampling approach was most
successful in children and was less expensive
than venipuncture in terms of equipment, as
the syringe, rubber gloves and a spot plaster cost
approximately US$4.00 per person compared
with a US$1.14 oral fluid swab [25].
The introduction of PCR methods has led to
the use of oral fluids as a source for detecting
viruses. Molecular investigation of hepatitisA is
important to determine the sources of infection
and is also essential to identify links between
apparently sporadic cases or apparently distinct
outbreaks [26], thereby allowing the implementation of corrective measures. Since HAV RNA
can be present in serum samples during the
window period of infection, its detection is an
important tool for diagnosing asymptomatic
individuals, as well as acute hepatitis cases of
unknown etiology. Amado optimized molecular techniques, nested PCR and real-time PCR,
for the detection of HAV RNA in saliva [27].
In both tests evaluated, viral RNA was more
frequently detected in oral fluid samples than
in serum samples (50vs 42%, respectively for
nested PCR; and 60.8vs 51%, respectively, for
real-time PCR), indicating that analysis of oral
fluid could be useful for molecular investigation
during hepatitisA outbreaks. In this study, the
difference between saliva and serum viral load
was not significant, with a mean standard deviation of 1.73.24103 copies/ml. Mackiewicz
detected HAV RNA in saliva samples from five
of six patients and reported a 2-log difference
between serum and saliva HAV load [28]. The
authors concluded that molecular investigation
of saliva in HAV infection might be a promising
tool for obtaining a broader understanding of
HAV epidemiology.
The use of saliva samples was recently
described in a molecular epidemiological study
of HAV outbreaks that identified and characterized the HAV genome from saliva specimens [29].
Interestingly, comparison of the HAV genotypes
of matched serum and saliva specimens revealed
patients for whom the viral genotype in their
salivary specimen was different from the subgenotypes of their serum sample. Since mixed
HAV infections are uncommon [30], molecular
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Review

cloning was conducted to investigate this result,

and revealed that the distribution of genotypes in
saliva was different from that in serum, because
all the clones from saliva were classified as IB
whereas all those in serum were IA. This unexpected discrepancy between saliva and serum
provided some interesting data that challenge
the current understanding of the source of
salivary HAV RNA. Therefore, further studies
are necessary to investigate the biological and
clinical meaning of this uneven distribution of
HAV subgenotypes in serum and saliva from
patients with mixed infections [29]. This study
showed the important role that saliva can play
in the molecular epidemiological analysis of a
hepatitisA outbreak [29].
HepatitisB

Traditionally, hepatitisB is diagnosed through

HBV antigens (HBsAg and HBeAg) or antibody detection in serum or plasma, and hence
requires trained healthcare workers for collection
and laboratory facilities for testing. Over the past
few years, alternative fluids for viral diagnosis,
such as saliva, have been widely studied. Saliva
as an alternative fluid for HBV testing could be
a useful tool for epidemiological purposes, especially among children, intravenous drug users
or hemophiliacs, for whom blood collection is
difficult. It could also be useful for field collection of samples in remote areas or in nonclinical
settings by individuals with minimal training [9].
Several studies have evaluated the detection of
antibodies against hepatitisB in saliva. However,
the results can differ in terms of the sensitivity
and specificity of oral fluid for HBV antibody
detection according to the collection device,
HBV markers evaluated and the assay applied.
Hutse conducted a study to validate the
detection of HBsAg in oral fluid, collected by
the OR ACOL (Malvern Medical Developments) collection device, compared with serum
using the commercial ELISA [31]. In this study,
a very good concordance between serum and
oral fluid results using the modified ELISA was
demonstrated. The sensitivity and specificity of
salivary hepatitisB surface antigens were 90.7
and 100%, respectively. The authors concluded
that the modified ELISA applied to oral fluid
samples is applicable mainly for epidemiological
surveys, but that this test needs further improvement before introduction as a suitable screening
test alternative for serum [31]. In a Russian study,
the presence of HBV markers was investigated
in the saliva and serum of acute hepatitis B
patients in order to evaluate the diagnostic and
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Review

Amado Leon

epidemiological value of saliva samples [32]. The

frequency of HBsAg in saliva was found to be
correlated with the frequency of its detection in
serum. Interestingly, the frequency of HBeAg
in saliva was significantly higher than in serum
samples in the acute phase, and was persistent
in saliva more than 1month after its clearance
from serum. Furthermore, after beginning antiviral treatment, HBsAg disappeared faster from
saliva than serum in most of the patients. The
authors suggested that saliva could be a source
of HBV diagnosis and treatment monitoring.
Cruz evaluated the use of a modified ELISA
for HBsAg among whole-saliva and oral fluid
(Salivette [Sarstedt] collection device) samples in
a Brazilian population [9]. Furthermore, in order
to optimize the assay, they evaluated the transport buffer for the oral fluids, sample volume and
incubation period with conjugate as well as the
cutoff values. Using the optimized protocol, the
sensitivities of whole saliva and oral fluid were
93.6 and 85.1%, respectively, whereas specificities of whole saliva and oral fluid were 92.6and
94.1%, respectively. Altogether these data demonstrate the importance of standardization of the
assay and establishing which specimens are the
best choice according to the laboratory facilities
available in order to develop a feasible an accurate
test for HBV infection surveillance using saliva.
The prevalence of hepatitisBsurface antigen
antibodies was measured in oral fluid samples
collected by postal survey, using the collector OraSure, in the Belgian population [33].
This study performed among 150 chronically
infected HBV patients described a high correlation between HBV DNA in matched serum and
saliva samples, with a mean log HBV DNA in
serum of 5.8 copies/ml and in saliva of 3.2copies/ml [34]. Of the 147 patients positive for HBV
DNA in serum, 69 (47%) also had detectable
HBV DNA in saliva. None of the patients who
were negative for HBV DNA in serum were
positive for HBV DNA in saliva. In accordance
with these findings, another study conducted in
Sweden among chronic HBV carriers revealed
that, of 25 HBV DNA-positive patients, ten had
HBV DNA in their saliva samples [35]. van der
Eijk, reporting quantitative measurements of
HBV DNA levels in saliva and the relationship
with quantitative HBV DNA levels in serum
from 27 chronic HBsAg carriers, demonstrated
a high correlation (r=0.9) between HBV DNA
in serum and saliva that could be described
by the following equation: log HBV DNA in
saliva=1.01+(0.56log HBV DNA) [34]. In
addition, the authors described the detection of
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Future Virol. (2013) 8(6)

HBV DNA according to the HBeAg marker. The

authors showed that, among the HBeAg-positive
patients, HBV DNA detection in saliva was 80%
and in serum was 93%. Among the HBeAgnegative patients, HBV DNA was detected in
the saliva of 42% and in the serum of 75% of
cases. These findings indicate a higher frequency
of HBV DNA detection in saliva in patients with
high viremia. These findings indicate that the
frequency of detection and significant amounts
of HBV DNA found in saliva in chronic HBV
patients with high viremia in serum may have
implications for the understanding of hepatitisB
epidemiology.
HepatitisC

The diagnosis and management of HCV infection includes ELISAs for the detection of antiHCV antibodies, qualitative and quantitative
tests to detect HCV RNA, and methods to
determine the HCV genotype [36]. While the
detection of anti-HCV and HCV RNA in blood
is the traditional method of viral surveillance,
venipuncture is invasive, often painful and
expensive. The use of saliva samples to diagnose
HCV offers several potential advantages, such
as minimal training requirements for sample
collection. The use of a noninvasive collection
technique makes this method especially suitable
for seroprevalence studies when blood samples
are difficult to obtain (e.g., in intravenous drug
users, children and hemophiliacs).
Many studies have documented the suitability of saliva samples for epidemiological studies and the diagnosis of HCV infections using
ELISA and real-time PCR. However, several
discrepant results have been observed. Previous studies detecting anti-HCV antibodies in
oral fluid resulted in a sensitivity of 72% and a
specificity of 98% using the Salivette collection
device with the Ortho HCV 3.0 assay (Ortho
Diagnostic Systems, NJ, USA) [37]. In support
of these results, another article conducted among
105matched serum and saliva samples from Brazilian patients clinically suspected of acute viral
hepatitis demonstrated a sensitivity of 75% and
a specificity of 97.75% in anti-HCV detection
in oral fluid samples, using an OraSure collection device with UBI HCV EIA 4.0 (United
Biomedical Inc.). A sensitivity of 84 and 87%
and specificity 100% using the OraSure collection device with the Ortho HCV 3.0 assay
was reported previously by Allwright [38] and
Judd [39], respectively. De Cock, performing the
test on oral fluid collect using the ORACOL
device, with the modified protocol, detected 61
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Saliva specimen sampling: noninvasive method for diagnosis & investigation of viral hepatitisA, B& C

of 73anti-HCV-positive saliva samples, while

73 of 73 anti-HCV-negative samples were tested
negative, giving a sensitivity and specificity of
83.6and 100.0%, respectively [40].
A sensitivity of 94 and 98.2% and a specificity
of 99% were observed by Bello [41] and Sherman
[42], using the Abott assay. There are many factors
that might explain these different results, such
as: the collection devices employed; modified
antibody detection methods for salivary HCV
antibody testing; or the population studied.
All data presenting with higher sensitivities are
related to the use of the saliva antibody assay
in one specific HCV risk group such as HIV
carriers and prisoners that in general have high
antibody titers, probably in response to repeated
antigenic stimulation [38,41,42]. On the other
hand, the studies of McIntyre [37] and Amado [8]
demonstrated a lower sensitivity when the assay
was applied in suspected cases of hepatitis. These
findings suggest that low serum anti-HCV titers
could be associated with false-negative results in
saliva. This hypothesis is supported by a study
that demonstrated that subjects with low titers
of anti-HCV who are HCV RNA negative in
serum present negative results in oral fluid assays
[43]. A study carried out in Belgium to correlate
HCV RNA positivity in serum with salivary
HCV antibodies by testing 85paired serum and
saliva samples, observed that, in 70 cases of the
corresponding oral fluid samples, HCV antibodies were positive. The corresponding oral fluid
samples were positive for HCV RNA in 52 of
59 positive serum samples, while 18 of 26HCV
RNA negative serum samples were positive for
HCV in the corresponding oral fluid samples.
These results suggest that HCV antibody detection in oral fluid has a slightly higher sensitivity
when used to test patients whose serum samples
are positive for HCV RNA [44].
In recent years, oral swab rapid point-of-care
tests for detection of anti-HCV are commercially available. A review of the performance of
these tests showed that oral point-of-care tests
were highly sensitive (98%) with a specificity of
99% or higher compared with laboratory-based
tests [10]. With high accuracy, rapid turnaround
time, convenience and high patient acceptability, these rapid oral swab tests could help HCV
screening programs reach individuals unaware of
their status and expand testing into nonclinical
settings. Several studies published on the topic
of salivary HCV RNA prevalence show great
variability. Hermida detected a salivary prevalence of 52.4% among 74 patients with HCV
infection [45], similar to those reported by other
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researchers [4648], while a higher prevalence

(100%) of HCV RNA in saliva was showed by
Lins [49]. The authors detected HCV RNA in all
of the saliva samples from the 50 anti-HCV-positive patients. Many studies have demonstrated
that the HCV viral load is higher in serum than
in saliva of patients who are infected with HCV
[11,50,51] and that there is a significant relationship between plasma and saliva HCV RNA viral
load [45]. Comparison of the HCV viral loads in
the serum and saliva of infected patients showed
that the median viral RNA levels were 3.32 log10
copies in the saliva and 5.78 log10 copies in the
serum, indicating that the viral loads in saliva are
100-times lower than those in serum [11]. This
previous study demonstrated that HCV is present in a high quantity in the oral fluid of chronic
HCV patients. A study conducted in order to
quantify HCV loads within the saliva and CF
of anti-HCV-positive patients using real-time
reverse transcription PCR showed that HCV is
more common and almost twofold higher in the
CF than the saliva of HCV-seropositive patients.
In addition, the authors further suggest a trend
that patients with HCV RNA-positive saliva
showed higher viral loads in sera than patients
with HCV RNA-negative saliva. These findings
suggest that CF might be one of the sources of
HCV RNA within the saliva [51].
It has been suggested that the presence of
HCV RNA in saliva correlates with HCV viremia [48,5255]. Wang, in a longitudinal study of 23
subjects with chronic and acute HCV infection,
detected HCV RNA in 72% of the saliva samples
[43]. It was also observed that serum HCV RNA
load predicted the detection of HCV RNA in
saliva. Thus, these data demonstrate that salivary HCV RNA detection was associated with
serum HCV RNA load in individuals who were
chronically or acutely infected with HCV [43].
Fabris, evaluating the prevalence of HCV RNA
in different fractions of saliva taken from patients
with chronic hepatitisC to establish whether
HCV RNA positivity in serum, genotype, viral
load or disease severity exert any influence on
the detection of HCV RNA in saliva, demonstrated that HCV RNA positivity was unrelated
to genotype, duration of disease, HepatitisActivity Index scores or transaminase levels [48]. Furthermore, HCV RNA was detectable in the cell
fraction of saliva in a high proportion of highly
viremic patients with chronic hepatitisC. Hence,
these data confirmed that a relationship between
serum viral load and the presence of HCV in
saliva exists [48], with plasma viral load being the
only known predictable factor [45].
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Although detection of HCV in oral fluid

is easy and noninvasive compared with using
blood, saliva is still not recommended to be used
for diagnostic purposes as a substitute for blood
samples. However, the saliva of serum-positive
patients should be tested for HCV RNA; positive cases should take precautionary measures
regarding contact with healthy members of their
household to avoid infecting them [56].
Saliva as an infectious fluid
HAV

HAV is mainly transmitted through the fecaloral

route, by person-to-person contact and/or contaminated food and drinking water [57]. Although
few studies have demonstrated HAV RNA detection in saliva [2729], there are still questions about
the origin of salivary HAV and the infectious
potential of HAV-containing saliva.
Animal experiments have further shown the
presence of HAV in saliva [58,59] and that infectious virus is shed in saliva during the incubation period and in the early acute phase [60]. Pinto
demonstrated that HAV RNA may be detected
in saliva from 6 h postinoculation up to several
weeks after hepatitis onset [58]. In acutely infected
patients, HAV load in the saliva ranged from
102to 104 copies/ml, suggesting that saliva may
be infectious [27,61].
An experimental infection study in cynomolgus
monkeys demonstrated the viral antigen [62] and
an active replication of HAV in salivary glands
of these animals, through detection of the HAV
replicative intermediate (minus-strand RNA).
In this previous study, a specific real-time reverse
transcription PCR assay was used to quantify
the level of viral strand-specific RNA, especially
of the HAV minus-stranded RNA, in salivary
glands and liver. The viral titers obtained from
salivary glands were approximately 2.3104copies/mg, while the RNA replicative intermediate
detection was more intense in salivary glands
(103copies/mg) at the beginning of the infection
(15dpi) than in the liver (102copies/mg). A positive correlation was observed between the salivary
viral load and the level of replicative intermediate
in salivary glands, suggesting that the HAV viral
load in saliva samples may reflect a local site of
HAV replication. These results established one
of the mechanisms causing the presence of HAV
in saliva and also suggested that the saliva is a
potential source of HAV transmission [63].
Corroborating these findings, a previous study
demonstrated a higher frequency of HAV in saliva
than in corresponding serum sample of children
enrolled in a hepatitisA outbreaks and the HAV
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Future Virol. (2013) 8(6)

viral load in saliva was similar of matched serum

[7]. The HAV sequence analysis conducted to
investigate the source of HAV transmission during this outbreak showed that HAV isolated
from the drinking water shared a close identity
(97.599.3%) with HAV isolated from the saliva
of children enrolled in the study. These findings
suggest that the drinking fountain could have
been contaminated by the childrens hands or
HAV-containing saliva [7]. From an epidemiological point of view, saliva could have been one of the
sources of HAV transmission. Molecular epidemiological investigation of such an outbreak revealed
cases where viral genotypes in saliva specimens
were found to be different from the genotypes
detected in the corresponding serum specimens
[29]. This distribution of different genotypes of
HAV in blood and saliva also reinforces the occurrence of extrahepatic independent replication.
These results suggest that HAV replication in the
oral cavity and its consequent release into saliva
may play an infective role in saliva as a potential
source of person-to-person transmission [7].
HBV

Transmission of HBV is mainly blood-borne,

although the route of infection during horizontal transmission in childhood is unclear [35] and
following contact tracing 20% of acute HBV
infections remain unexplained [34].Therefore,
salivamay be an unexpected vehicle of HBV
DNA transmission. To further explore this
hypothesis, several studies have evaluated the
quantitative levels of HBV DNA insalivaand
compared these with the HBV DNA levels
measured in serum.
Several studies have reported that HBV DNA
in body fluids such as saliva, semen, urine, sweat
and tears can be detected by PCR [34,35,64]. Heiberg found high levels of HBV DNA in the
saliva of HBeAg-positive children, suggestingsaliva as a vehicle for horizontal transmission
of HBV among children [64]. Kidd-Ljunggren
observed that highly viremic HBeAg-positive
carriers had high copy numbers detected insaliva
fluid [35]. In accordance with this, van der Eijk
[34] demonstrated that, among 27chronic HBsAg
carriers, two patients HBeAg-negative/antiHBe-positive had a higher concentration of HBV
DNA in their saliva samples than in their serum
samples. One of the possible explanations of this
result could be a role of salivary glands as a site of
HBV replication. This hypothesis might explain
the higher level reached in saliva. According to
the authors, these findings suggest that saliva
can be a source of considerable amounts of HBV
future science group

Saliva specimen sampling: noninvasive method for diagnosis & investigation of viral hepatitisA, B& C

DNA. In patients with high viremia this finding

may have implications for the infectivity of saliva
and give insight in the possible routes of transmission of HBV. This has particular importance
for infection control programs and regulations,
underlining the importance of aiming towards
regular HBV DNA testing and thus infectivity
assessment of chronic carriers in order to prevent
transmission [35].
Approximately 30 years ago, the infectivity of saliva from HBV carriers was proven by
experimental transmission, using gibbons [65,66].
Recently, chimeric mice with severe combined
immunodeficiency and with transplanted
human hepatocytes have been used as an appropriate animal model for studying whether body
fluids such as tears and saliva from HBV-carrier
children were infectious for hepatitisB [67]. This
study demonstrated that saliva, as well as tears,
contained large amounts of HBV DNA in the
experimentally infected chimeric mice and that
the levels of HBV DNA in saliva and tear specimens from young chronic HBV carrier children
were extremely high, suggesting that saliva has
the potential to transmit HBV.
Epidemiological studies and genome sequencing also suggest salivary transmission in an
intrafamilial cluster of hepatitisB [68] and by
human bite [69]. Hui reported a case of acute
hepatitisB that developed after a patient was bitten by a chronic carrier of HBV who contained
the virus in his saliva [69]. The HBV in both
men had an identical genotype and sequence.
This finding prompted some studies about the
infection and location of HBV in extrahepatic
tissues. However, it still remains controversial
whether HBV can replicate outside the liver
after acute infection. The origin of the HBV in
saliva has been investigated in a previous study
through evaluation of the HBV markers and
DNA in parotid tissues from patients with positive serumHBVmarkers [70]. According to the
authors, the detection of HBsAg, HBcAg and
HBV DNA in parotid cells suggests that HBVin
saliva might originate from the infected salivary glands, and the infectious saliva could
transmitHBV.
Previous studies have reported that 10% of
HBV particles are infectious [69]. Therefore, all
body fluids from HBV carriers should be considered to be infectious. Hence, strict precautions
should be taken against contact with body fluids
from HBV carriers with high-level viremia, especially in countries implementing an immunizing
program focused on individuals at risk for HBV
infection.
future science group

Review

HCV

HCV is transmitted mainly through the parenteral route. Sexual and vertical transmissions are
considered to be rare. However, epidemiologi
cal studies demonstrate that the transmission
routes of approximately 10 [71] to 40% [72] of
HCV infections are unknown. This situation
suggests that unknown routes of transmission,
distinct from the percutaneous route and from
at-risk sexual contact, exist. The potential role
of human saliva as a source of HCV infection
has been the subject of debate for more than
10years. Seroepidemiological surveys indicate
that living with HCV-infected patients could
constitute a risk factor for acquiring the disease
[73,74]. Ackerman found evidence that both sexual and nonsexual intrafamiliar transmission of
HCV does occur [75]. Through epidemiological
evidence, these studies suggest the possibility
of infection by contact with body fluids other
than blood.
Since 1990, there have been numerous studies on the presence of HCV RNA in saliva [76].
Although these studies are heterogeneous, it
has been demonstrated that HCV RNA can be
detected in up to 100% of the saliva samples
from theHCV-positive group [49]. An important point regarding HCV detection in saliva is
the possibility of HCV transmission by saliva.
These observations have led to several studies
focused on describing evidence about the origin
and infectivity of HCV RNA in saliva.
One of the first studies conducted to describe
the direct transmission of HCV by saliva demonstrated that chimpanzees that were inoculated with HCV-containing saliva developed an
infection [77], and two reports have suggested
that HCV is transmissible to humans via bite
injuries [77,78]. However, these studies did not
confirm the transmission of HCV by a human
bite through sequence analysis of the virus.
There are several reasons why HCV RNA
may be present in the saliva of patients with
detectable virus in the plasma. A transfer of
viral particles from the circulation to the saliva
via a concentration gradient could contribute,
since a direct relationship between the presence
of the virus in the saliva and the viral load in
blood has been demonstrated in previous studies [45,48,52,54,79]. However, some authors point
out that HCV RNA detection in saliva was
found not to be dependent on the viral load in
the serum [11,49] and that viral RNA detection
is not dependent upon the presence of occult
blood in the samples [45]. These findings indicate
that the detection of HCV RNA in saliva is not
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Amado Leon

necessarily a consequence of blood contamination secondary to mucosal lesions [45,48,80]. HCV

could also enter the saliva via the gingival sulcus
[81], but this is clearly not the sole route, since
HCV RNA has been found in saliva samples
from edentate patients [46]. Another possible
source could be peripheral blood mononuclear
cells for transferring HCV to saliva [48,82], but
the presence of HCV RNA in peripheral blood
mononuclear cells and in saliva are not closely
correlated [83].
Previous studies observed viral genotypes in
oral fluid specimens different from the genotypes
detected in the corresponding serum specimens
[84,85]. This observation could be explained by an
alternative mechanism for viral entry into saliva
through active HCV replication in the salivary
glands [86]. HCV RNA has been found in salivary glands obtained from chronically infected
patients [87]. However, these reports are derived
from case reports and have not been confirmed.
Toussirot detected HCV RNA in the serum and
saliva of five patients with primarySjgrens syndrome and chronically infected by HCV, and
RNA extracted from salivary gland specimens
tested positive in three cases [88]. In addition,
it was found that in the two patients with the
lower serum HCV RNA titers, HCV RNA was
not detected in their salivary glands, contrasting
with the others, who had the highest viremia.
These findings suggest that the HCV patients
with high circulating levels of HCV had a higher
probability of infecting their salivary glands,
which indicates that HCV may propagate and
reside within salivary gland tissue [87]. Arrieta,
using insitu hybridization and immunohisto
chemistry, detected HCV RNA as well as an
HCV core antigen in the epithelial cells of the
salivary gland biopsies from anti-HCV-positive
patients [86]. Although these findings suggest that
HCV replication can occur in salivary glands,
the hallmark of HCV replication in a tissue is
the presence of the HCV RNA negative strand
(the replicative intermediate). Laskus, employing assays optimized for identifying replicative
intermediates of HCV, found HCV quasispecies in different tissue samples, compatible with
independent replication at extrahepatic sites [89].
Altogether, these findings indicate that saliva may
play a significant role in the nonparenteral transmission of HCV [49].
Conclusion

The collection of saliva is simple, noninvasive,

requires no notably specialized equipment and is
generally acceptable to patients and clinicians[90].
584

Future Virol. (2013) 8(6)

It is recognized that there are several reports of

antibody detection and of frequency of oral carriage and load of HAV, HBV and HCV in saliva.
However, many of these results are discrepant,
since the reports have included different groups
of studied populations and used different methods and tools. These variations make it difficult
to define a conclusion about the feasibility and
applicability of the saliva samples on the diagnosis
and investigations of hepatitisA, B and C.
The general acceptance of collecting saliva
samples facilitates investigation via large epidemiological studies [33]. These advantages in combination with the great accuracy of the assays
make saliva a promising substitute for serum for
epidemiological surveillance purposes. However,
the data described in this present review demonstrate that to achieve an accurate and reliable
saliva test it is extremely important to standardize the immunoassay and to establish the most
suitable collection device. Development of an
assay for detection of antibodies in saliva requires
careful optimization of numerous parameters to
maximize sensitivity and specificity. The various approaches include: decreasing the dilution
effect of the sample buffer [9,40,91];exclusion of
the dilution step entirely [8];increasing the sample input [39];increasing the sample incubation
time [39]; andincreasing the time for conjugate
incubation [9,85]. Another approach to assay optimization was the use of an altered cutoff value
for saliva [23].
As a diagnosis tool for hepatitisA infection,
although some reports showed a good sensitivity and specificity of IgM anti-HAV tests using
saliva [7,21], these results are most relevant for
a specific population involved in outbreaks of
hepatitisA, which indicates that saliva could be
a feasible alternative for diagnosis of hepatitisA
in outbreaks. In relation to hepatitisB diagnosis, the results of the studies on the sensitivity
and specificity of oral fluid for anti-HBcAg and
-HBsAg detection mostly agree that oral fluid
can be used for HBV diagnosis. However, most
of the studies reported a sensitivity of approximately 80% for anti-HBcAg and approximately
90% for HBsAg. These findings indicate that
significant promise exists for the use of saliva
together with the modified commercial ELISA
for HBV infection diagnosis; however, further
studies should be carried out to establish the
use of saliva as a substitute for serum in HBV
diagnosis. The usual method for detection of
HCV infection is based on testing for HCV
serum antibodies. Several studies suggest that
salivary HCV antibodies may have the potential
future science group

Saliva specimen sampling: noninvasive method for diagnosis & investigation of viral hepatitisA, B& C

to substitute for HCV serum antibodies in

immunodiagnostic analysis. Nevertheless, one
significant issue before suggesting the clinical
use of salivary anti-HCV testing is the positive
and negative predictive value of the assay [91].
Therefore, the feasibility of the screening test
is mainly dependent on the prevalence of the
disease in the population tested, as well as on the
sensitivity and specificity of the test.
In general, many reports support that oral fluid
collection is a good tool for hepatitis epidemiological surveillance purposes. The convenience,
reliability and noninvasive nature of this method
make it an attractive tool for the selection of candidates for vaccination against hepatitisA and B
and in monitoring vaccine response.
In addition, the presence of viral nucleic acids
suggests that oral fluids are potentially infectious. However, this remains to be confirmed
by epidemiological and experimental studies.
Future perspective

With the growing number of new saliva collection devices, accurate assays and new techniques as point of care tests, there is a possibility

Review

to increase the use of oral specimens for broadbased diagnosis of viral hepatitis and potentially
for screening and epidemiological applications.
The recent US FDA approval of an oral HIV
rapid test (OraQuick HIV rapid test, OraSure
Technologies) for home use is a great illustration
of the value of saliva for diagnosis [92]. This offers
a great possbility that in the future the saliva
may be the next in-house self-test for screening
of viral hepatitis and/or as a preliminary test.
However, before the widespread implementation
of saliva tests, a concerted effort must be made
to overcome their lower accuracy compared with
serum-based assays.
Financial & competing interests disclosure

The author has no relevant affiliations or financial

involvement with any organization or entity with a
financial interest in or financial conflict with the subject matter or materials discussed in the manuscript.
This includes employment, consultancies, honoraria,
stock ownership or options, expert testimony, grants or
patents received or pending, or royalties.
No writing assistance was utilized in the production
of this manuscript.

Executive summary
Saliva collection
The component in saliva that contains the highest concentrations of immunoglobulins is the crevicular fluid; therefore, the conditions
under which saliva is collected can have an influence on antibody detection.
Saliva sampling as a diagnostic & basic investigation method for viral hepatitis
Development of an assay for detection of antibodies in saliva requires careful optimization of numerous parameters to maximize
sensitivity and specificity.
Many reports provide evidence that oral fluid collection is a good tool for hepatitis epidemiological surveillance purposes. The
convenience, reliability and noninvasive nature of this method makes it an attractive tool for epidemiological studies, for selection of
nonimmune candidates for vaccination against hepatitisA and B, and in monitoring vaccine response.
A significant promise exists for the use of saliva together with the modified commercial ELISA for viral hepatitis diagnosis; however,
further studies should be carried out to establish the use of saliva as a substitute for serum in HBV diagnosis.
Saliva as an infectious fluid
The presence of viral nucleic acids in saliva suggests that oral fluids could be potentially infectious.

References
Papers of special note have been highlighted as:
n of interest
1.

2.

3.

Atkinson JC. The role of salivary

measurements in the diagnosis of salivary
autoimmune diseases. Ann. NY Acad. Sci.
694, 238251 (1993).
Lawrence HP. Salivary markers of systemic
disease: noninvasive diagnosis of disease and
monitoring of general health. J. Can. Dent.
Assoc. 68(3), 170174 (2002).
Chiappin S, Antonelli G, Gatti R, De Palo EF.
Saliva specimen: a new laboratory tool for
diagnostic and basic investigation. Clin. Chim.
Acta 383(12), 3040 (2007).

future science group

4.

Mandel ID. Salivary diagnosis: promises,

promises. Ann. NY Acad. Sci. 694, 110
(1993).

5.

Belza MJ, Rosales-Statkus ME, Hoyos J

etal. Supervised blood-based self-sample
collection and rapid test performance: a
valuable alternative to the use of saliva by
HIV testing programmes with no medical or
nursing staff. Sex. Transm. Infect. 88(3),
218221 (2012).

6.

Donnell-Fink LA, Arbelaez C, Collins JE

etal. Acceptability of fingerstick versus oral
fluid rapid HIV testing: results from the
universal screening for HIV infection in the
emergency room (usher Phase II)
randomized controlled trial. J. Acquir.
www.futuremedicine.com

Immune Defic. Syndr. 61(5), 588592

(2012).
7.

8.

Amado L, Villar L, de Paula V, Gaspar A.

Comparison between serum and saliva for the
detection of hepatitisA virus RNA. J. Virol.
Methods 148(12), 7480 (2008).
First report of HAV RNA detection and
quantification in saliva from subjects
enrolled in an outbreak study.
Amado LA, Villar LM, de Paula VS,
deAlmeida AJ, Gaspar AM. Detection of
hepatitisA, B, and C virus-specific antibodies
using oral fluid for epidemiological studies.
Mem. Inst. Oswaldo Cruz 101(2), 149155
(2006).

585

Review
9.

Amado Leon

Cruz HM, da Silva EF, Villela-Nogueira CA

etal. Evaluation of saliva specimens as an
alternative sampling method to detect
hepatitisB surface antigen. J. Clin. Lab. Anal.
25(2), 134141 (2011).

22. Ahmed M, Munshi SU, Nessa A, Ullah MS,

Tabassum S, Islam MN. High prevalence of

hepatitisA virus antibody among Bangladeshi
children and young adults warrants preimmunization screening of antibody in HAV
vaccination strategy. Indian J. Med. Microbiol.
27(1), 4850 (2009).

10. Drobnik A, Judd C, Banach D, Egger J, Konty

K, Rude E. Public health implications of rapid

hepatitisC screening with an oral swab for
community-based organizations serving highrisk populations. Am. J. Public Health 101(11),
21512155 (2011).

Importance of the cutoff ratio for detecting

antibodies against hepatitisA virus in oral
fluids by enzyme immunoassay. J. Virol.
Methods 173(2), 169174 (2011).
24. Morris-Cunnington MC, Edmunds WJ,

Miller E, Brown DW. A novel method of oral

fluid collection to monitor immunity to
common viral infections. Epidemiol. Infect.
132(1), 3542 (2004).

12. Lackner A, Kessler HH, Walch C, Quasthoff

S, Raggam RB. Early and reliable detection of

herpes simplex virus type 1 and varicella zoster
virus DNAs in oral fluid of patients with
idiopathic peripheral facial nerve palsy:
decision support regarding antiviral treatment?
J. Med. Virol. 82(9), 15821585 (2010).
13. Dawes C. Considerations in the development

of diagnostic tests on saliva. Ann. NY Acad.

Sci. 694, 265269 (1993).
14. Sagan C, Salvador A, Dubreuil D, Poulet PP,

Duffaut D, Brumpt I. Simultaneous

determination of metronidazole and
spiramycin I in human plasma, saliva and
gingival crevicular fluid by LC-MS/MS.
J.Pharm. Biomed. Anal. 38(2), 298306
(2005).
15. Parry JV. Detection of viral antibodies in

saliva specimens as an alternative to serum.

J.Clin. Chem. Clin. Biochem. 27(4), 245246
(1989).
16. Johnson AM, Parry JV, Best SJ, Smith AM, de

Silva M, Mortimer PP. HIV surveillance by

testing saliva. AIDS 2(5), 369371 (1988).
17. Gaudette D, North L, Hindahl M, Griffin K,

Klimkow N, Thieme T. Stability of clinically

significant antibodies in saliva and oral fluid.
J. Clin. Immun. 17, 171175 (1994).
18. Bull AR, Kimmance KJ, Parry JV, Perry KR.

Investigation of an outbreak of hepatitisA

simplified by salivary antibody testing.
Epidemiol. Infect. 103(2), 371376 (1989).
19. Parry JV, Perry KR, Panday S, Mortimer PP.

Diagnosis of hepatitisA and B by testing

saliva. J. Med. Virol. 28(4), 255260 (1989).
20. OFarrell BJ, Rajan E, Albloushi SS, Courtney

MG, Fielding J, Shattock AG. The reliability

of saliva as a sample for the detection of
hepatitisA immunoglobulins under various
sampling conditions. Clin. Diagn. Virol. 7(3),
153157 (1997).
21. Oba IT, Spina AM, Saraceni CP etal.

Detection of hepatitisA antibodies by ELISA

using saliva as clinical samples. Rev. Inst. Med.
Trop. Sao Paulo 42(4), 197200 (2000).

586

Apopulation-based prevalence study of

hepatitisA, B and C virus using oral fluid in
Flanders, Belgium. Eur. J. Epidemiol. 22(3),
195202 (2007).
34. van der Eijk AA, Niesters HG, Gtz HM

etal. Paired measurements of quantitative

hepatitisB virus DNA in saliva and serum of
chronic hepatitisB patients: implications for
saliva as infectious agent. J. Clin. Virol.
29(2), 9294 (2004).

23. Tourinho RS, Amado LA, Villar LM etal.

11. Menezes GB, Pereira FA, Duarte CA etal.

HepatitisC virus quantification in serum and

saliva of HCV-infected patients. Mem. Inst.
Oswaldo Cruz 107(5), 680683 (2012).

33. Quoilin S, Hutse V, Vandenberghe H etal.

Epidemiological study in which saliva

samples and questionnaires were collected
by patients and sent to the laboratory
bypost.

25. Morris-Cunnington MC, Edmunds WJ,

Miller E, Brown DW. A population-based

seroprevalence study of hepatitisA virus using
oral fluid in England and Wales. Am. J.
Epidemiol. 159(8), 786794 (2004).
26. Stene-Johansen K, Jenum PA, Hoel T, Blystad

H, Sunde H, Skaug K. An outbreak of

hepatitisA among homosexuals linked to a
family outbreak. Epidemiol. Infect. 129(1),
113117 (2002).
27. Amado LA, Villar LM, De Paula VS, Gaspar

AM. Comparison between serum and saliva

for the detection of hepatitisA virus RNA.
J.Virol. Methods 148(12), 7480 (2008).
28. Mackiewicz V, Dussaix E, Le Petitcorps MF,

Roque-Afonso AM. Detection of hepatitisA

virus RNA in saliva. J. Clin. Microbiol. 42(9),
43294331 (2004).
29. Amado LA, Villar LM, de Paula VS, Pinto

MA, Gaspar AM. Exposure to multiple

subgenotypes of hepatitisA virus during an
outbreak using matched serum and saliva
specimens. J. Med. Virol. 83(5), 768775
(2011).
30. de Paula V, Saback F, Gaspar A, Niel C. Mixed

infection of a child care provider with

hepatitisA virus isolates from subgenotypes Ia
and Ib revealed by heteroduplex mobility assay.
J. Virol. Methods 107(2), 223228 (2003).
31. Hutse V, Verhaegen E, De Cock L etal. Oral

fluid as a medium for the detection of

hepatitisB surface antigen. J. Med. Virol.
77(1), 5356 (2005).
32. Zhevachevsky NG, Nomokonova NY,

Beklemishev AB, Belov GF. Dynamic study of

HBsAg and HBeAg in saliva samples from
patients with hepatitis B infection: diagnostic
and epidemiological significance. J. Med. Virol.
61, 433438 (2000).

Future Virol. (2013) 8(6)

Demonstrated significant amounts

ofHBVDNA in saliva from
chronicHBVpatients with high viremia,
which has implications for the potential
infectivity of this body fluid.

35. Kidd-Ljunggren K, Holmberg A, Blckberg

J, Lindqvist B. High levels of hepatitisB

virus DNA in body fluids from chronic
carriers. J.Hosp. Infect. 64(4), 352357
(2006).
36. Seeff LB, Hoofnagle JH. National Institutes

of Health Consensus Development

Conference: management of hepatitis C:
2002. Hepatology 36(5 Suppl.1), S1S2
(2002).
37. McIntyre PG, Laszlo J, Appleyard K, Ogden

GR. Modified enzyme immunoassay to

detect hepatitisC virus antibodies in oral
fluid. Eur. J.Clin. Microbiol. Infect. Dis.
15(11), 882884 (1996).
38. Allwright S, Bradley F, Long J, Barry J,

Thornton L, Parry JV. Prevalence of

antibodies to hepatitisB, hepatitisC, and
HIV and risk factors in irish prisoners:
results of a national cross sectional survey.
BMJ 321(7253), 7882 (2000).
39. Judd A, Parry J, Hickman M etal.

Evaluation of a modified commercial assay in

detecting antibody to hepatitisC virus in
oral fluids and dried blood spots. J. Med.
Virol. 71(1), 4955 (2003).
40. De Cock L, Hutse V, Verhaegen E, Quoilin

S, Vandenberghe H, Vranckx R. Detection

of HCV antibodies in oral fluid. J. Virol.
Methods 122(2), 179183 (2004).
41. Bello PY, Pasquier C, Gourney P, Puel J,

Izopet J. Assessment of a hepatitisC virus

antibody assay in saliva for epidemiological
studies. Eur. J. Clin. Microbiol. Infect Dis
17(8), 570572 (1998).
42. Sherman KE, Creager RL, OBrien J,

Sargent S, Piacentini S, Thieme T. The use

of oral fluid for hepatitisC antibody
screening. Am. J. Gastroenterol. 89(11),
20252027 (1994).
43. Wang CC, Morishima C, Chung M etal.

High serum hepatitisC virus (HCV) RNA

load predicts the presence of HCV RNA in
saliva from individuals with chronic and

future science group

Saliva specimen sampling: noninvasive method for diagnosis & investigation of viral hepatitisA, B& C

acute HCV infection. J. Infect. Dis. 193(5),

672676 (2006).
n

Describes the frequency and pattern of

HCV RNA detection in saliva. Performed a
longitudinal study that included daily
self-collection of saliva samples from
chronic and acute HCV patients.

44. Cock De L, Hutse V, Vranckx R. Correlation

54. Mariette X, Loiseau P, Morinet F.

HepatitisC virus in saliva. Ann. Intern. Med.

122(7), 556 (1995).

R, Castro A, Diz Dios P. Detection of HCV

RNA in saliva of patients with hepatitisC
virus infection by using a highly sensitive test.
J.Virol. Methods 101(12), 2935 (2002).
46. Roy KM, Bagg J, McCarron B, Good T,

GD. HepatitisC virus in saliva of

haemophiliac patients attending an oral
surgery unit. Br. J. Oral Maxillofac. Surg.
34(2), 162165 (1996).
48. Fabris P, Infantolino D, Biasin MR etal.

High prevalence of HCV-RNA in the saliva

cell fraction of patients with chronic
hepatitisC but no evidence of HCV
transmission among sexual partners. Infection
27(2), 8691 (1999).
49. Lins L, Almeida H, Vitvisk L, Carmo T,

Paran R, Reis MG. Detection of hepatitisC

virus RNA in saliva is not related to oral
health status or viral load. J. Med. Virol.
77(2), 216220 (2005).

JE, Maynard JE. HepatitisA in day-care

centers. A community-wide assessment.
N.Engl. J. Med. 302(22), 12221227 (1980).

52. Wang JT, Wang TH, Sheu JC, Lin JT, Chen

DS. HepatitisC virus RNA in saliva of

patients with posttransfusion hepatitis and
low efficiency of transmission among spouses.
J.Med. Virol. 36(1), 2831 (1992).
53. Numata N, Ohori H, Hayakawa Y, Saitoh Y,

Tsunoda A, Kanno A. Demonstration of

hepatitisC virus genome in saliva and urine
of patients with type C hepatitis: usefulness of
the single round polymerase chain reaction
method for detection of the HCV genome.
J.Med. Virol. 41(2), 120128 (1993).

future science group

Shimokawa R, Fujisawa T. Tears from

children with chronic hepatitisB virus (HBV)
infection are infectious vehicles of HBV
transmission: experimental transmission of
HBV by tears, using mice with chimeric
human livers. J. Infect. Dis. 206(4), 478485
(2012).
68. Marie-Cardine A, Mouterde O, Dubuisson S,

Buffet-Janvresse C, Mallet E. Salivary

transmission in an intrafamilial cluster of
hepatitisB. J. Pediatr. Gastroenterol. Nutr.
34(2), 227230 (2002).

58. Pinto MA, Marchevsky RS, Baptista ML

etal. Experimental hepatitisA virus (HAV)

infection in Callithrix jacchus: early detection
of HAV antigen and viral fate. Exp. Toxicol.
Pathol. 53(6), 413420 (2002).

69. Hui AY, Hung LC, Tse PC, Leung WK,

Chan PK, Chan HL. Transmission of

hepatitisB by human bite confirmation by
detection of virus in saliva and full genome
sequencing. J. Clin. Virol. 33(3), 254256
(2005).

59. Cohen JI, Feinstone S, Purcell RH.

HepatitisA virus infection in a chimpanzee:

duration of viremia and detection of virus in
saliva and throat swabs. J. Infect. Dis. 160(5),
887890 (1989).

70. Chen L, Liu F, Fan X etal. Detection of

hepatitisB surface antigen, hepatitisB core

antigen, and hepatitisB virus DNA in parotid
tissues. Int. J. Infect. Dis. 13(1), 2023 (2009).

60. Asher LV, Binn LN, Mensing TL,

Marchwicki RH, Vassell RA, Young GD.

Pathogenesis of hepatitisA in orally
inoculated owl monkeys (Aotus trivirgatus).
J.Med. Virol. 47(3), 260268 (1995).
61. Mackiewicz V, Dussaix E, Le Petitcorps M,

Roque-Afonso A. Detection of hepatitisA

virus RNA in saliva. J. Clin. Microbiol.
42(9), 43294331 (2004).
62. Gaspar A, Vitral C, Yoshida C, Schatzmayr

H. Fast growth of a Brazilian hepatitisA

virus (HAF-203) in a primate cell line. Braz.
J.Med. Biol. Res. 26(2), 203206 (1993).
63. Amado LA, Marchevsky RS, de Paula VS

etal. Experimental hepatitisA virus (HAV)

infection in cynomolgus monkeys (Macaca
fascicularis): evidence of active extrahepatic
site of HAV replication. Int. J. Exp. Pathol.
91(1), 8797 (2010).

51. Suzuki T, Omata K, Satoh T etal.

Quantitative detection of hepatitisC virus

(HCV) RNA in saliva and gingival crevicular
fluid of HCV-infected patients. J. Clin.
Microbiol. 43(9), 44134417 (2005).

67. Komatsu H, Inui A, Sogo T, Tateno A,

57. Hadler SC, Webster HM, Erben JJ, Swanson

50. Rey D, Fritsch S, Schmitt C, Meyer P, Lang

JM, Stoll-Keller F. Quantitation of hepatitisC

virus RNA in saliva and serum of patients
coinfected with HCV and human
immunodeficiency virus. J. Med. Virol. 63(2),
117119 (2001).

HJ, Tingpalapong M. Experimental

transmission of hepatitisB virus by semen and
saliva. J. Infect. Dis. 142(1), 6771 (1980).

T, Ullah M, Qureshi JA. Investigating the

concurrent presence of HCV in serum, oral
fluid and urine samples from chronic HCV
patients in Faisalabad, Pakistan. Arch. Virol.
154(9), 15231527 (2009).

Cameron S, Pithie A. Predominance of HCV

type 2a in saliva from intravenous drug users.
J.Med. Virol. 54(4), 271275 (1998).
47. Roy KM, Bagg J, Follett EA, Brewer A, Lowe

66. Scott RM, Snitbhan R, Bancroft WH, Alter

56. Shafique M, Ahmad N, Awan FR, Mustafa

between detection of antibodies against

hepatitisC virus in oral fluid and hepatitisC
virus RNA in serum. Eur. J. Clin. Microbiol.
Infect. Dis. 24(8), 566568 (2005).
45. Hermida M, Ferreiro MC, Barral S, Laredo

hepatitisB surface antigen. J. Infect. Dis.

135(1), 7985 (1977).

55. Caldwell SH, Sue M, Bowden JH etal.

HepatitisC virus in body fluids after liver

transplantation. Liver Transpl. Surg. 2(2),
124129 (1996).

First discussion of HAV replication in the

salivary glands of nonhuman primates,
which may have implications for the
potential infectivity of this fluid.

64. Heiberg IL, Hoegh M, Ladelund S, Niesters

HG, Hogh B. HepatitisB virus DNA in

saliva from children with chronic hepatitisB
infection: implications for saliva as a
potential mode of horizontal transmission.
Pediatr. Infect. Dis. J. 29(5), 465467
(2010).
65. Bancroft WH, Snitbhan R, Scott RM etal.

Transmission of hepatitisB virus to gibbons

by exposure to human saliva containing

www.futuremedicine.com

Review

Demonstrates the presence of HBV DNA

in parotid tissues from patients infected
with HBV and suggests that HBV in saliva
might originate from infectedsalivary
glands and that the infectious saliva could
transmitHBV.

71. Murphy EL, Bryzman SM, Glynn SA etal.

Risk factors for hepatitisC virus infection in

United States blood donors. NHLBI
Retrovirus Epidemiology Donor Study
(REDS). Hepatology 31(3), 756762 (2000).
72. Januszkiewicz-Lewandowska D, Wysocki J,

Rembowska J etal. Transmission of HCV

infection among long-term hospitalized oncohaematological patients. J. Hosp. Infect. 53(2),
120123 (2003).
73. Saltoglu N, Taova Y, Burgut R, Dndar IH.

Sexual and non-sexual intrafamilial spread of

hepatitisC virus: intrafamilial transmission of
HCV. Eur. J. Epidemiol. 14(3), 225228
(1998).
74. Alvarez-Muoz MT, Vences-Aviles MA,

Damacio L etal. HepatitisC virus RNA

(HCV-RNA) in blood donors and family
members seropositive for anti-HCV
antibodies. Arch. Med. Res. 32(5), 442445
(2001).
75. Ackerman Z, Ackerman E, Paltiel O.

Intrafamilial transmission of hepatitisC virus:

a systematic review. J. Viral Hepat. 7(2),
93103 (2000).

587

Review

Amado Leon

76. Takamatsu K, Koyanagi Y, Okita K,

Yamamoto N. HepatitisC virus RNA in

saliva. Lancet 336(8729), 1515 (1990).
77. Abe K, Inchauspe G. Transmission of

hepatitisC by saliva. Lancet 337(8735), 248

(1991).
78. Dusheiko GM, Smith M, Scheuer PJ.

HepatitisC virus transmitted by human bite.

Lancet 336(8713), 503504 (1990).
79. Blec L, Legoff J, Si-Mohamed A etal.

Mucosal humoral immune response to

hepatitisC virus E1/E2 surface glycoproteins
and HCV shedding in saliva and
cervicovaginal fluids from chronically HCVinfected patients. J. Hepatol. 38(6), 833842
(2003).
80. Liou TC, Chang TT, Young KC, Lin XZ, Lin

CY, Wu HL. Detection of HCV RNA in

saliva, urine, seminal fluid, and ascites.
J.Med. Virol. 37(3), 197202 (1992).
81. Maticic M, Poljak M, Kramar B etal.

Detection of hepatitisC virus RNA from

gingival crevicular fluid and its relation to
virus presence in saliva. J. Periodontol. 72(1),
1116 (2001).
82. Roy KM, Bagg J, Bird GL etal. Serological

and salivary markers compared with

biochemical markers for monitoring
interferon treatment for hepatitisC virus
infection. J.Med. Virol. 47(4), 429434
(1995).
83. Young KC, Chang TT, Liou TC, Wu HL.

Detection of hepatitisC virus RNA in

peripheral blood mononuclear cells and in
saliva. J. Med. Virol. 41(1), 5560 (1993).
84. Roy KM, Bagg J, McCarron B. The effect of

saliva specimen collection, handling and

storage protocols on hepatitisC virus (HCV)
RNA detection by PCR. Oral Dis. 5(2),
123127 (1999).

588

85. van Doornum GJ, Lodder A, Buimer M,

vanAmeijden EJ, Bruisten S. Evaluation of

hepatitisC antibody testing in saliva specimens
collected by two different systems in
comparison with HCV antibody and HCV
RNA in serum. J. Med. Virol. 64(1), 1320
(2001).
86. Arrieta JJ, Rodrguez-Iigo E, Ortiz-Movilla N

etal. Insitu detection of hepatitisC virus RNA

in salivary glands. Am. J. Pathol. 158(1),
259264 (2001).
87. Takamatsu K, Okayasu I, Koyanagi Y,

Yamamoto N. HepatitisC virus propagates in

salivary glands. J. Infect. Dis. 165(5), 973974
(1992).
88. Toussirot E, Le Hud G, Mougin C, Balblanc

JC, Bettinger D, Wendling D. Presence of

hepatitisC virus RNA in the salivary glands of
patients with Sjgrens syndrome and
hepatitisC virus infection. J.Rheumatol.
29(11), 23822385 (2002).
89. Laskus T, Radkowski M, Wang LF, Nowicki

M, Rakela J. Uneven distribution of hepatitisC

virus quasispecies in tissues from subjects with
end-stage liver disease: confounding effect of
viral adsorption and mounting evidence for the
presence of low-level extrahepatic replication.
J.Virol. 74(2), 10141017 (2000).
90. Mahboobi N, Porter SR, Karayiannis P,

Alavian SM. Oral fluid and hepatitisA, B

andC: a literature review. J. Oral Pathol. Med.
41(7), 505516 (2012).
91. Elsana S, Sikuler E, Yaari A, Shemer-Avni Y,

Margalith M. Salivary HCV-antibodies;

afollow-up cohort study of liver disease
patients. Clin. Lab. 47(78), 335338 (2001).
92. CDC. Advancing HIV prevention: new

strategies for a changing epidemic United

States, 2003. MMWR Morb. Mortal. Wkly Rep.
52, 329332 (2003).

Future Virol. (2013) 8(6)

93. Madar R, Straka S, Baska T. Detection of

antibodies in saliva an effective auxiliary

method in surveillance of infectious diseases.
Bratisl. Lek. Listy 103(1), 3841 (2002).
94. Poovorawan Y, Theamboonlers A,

Chongsrisawat V, Jantaradsamee P,
Chutsirimongkol S, Tangkijvanich P. Clinical
features and molecular characterization of
hepatitis A virus outbreak in a child care
center in Thailand. J. Clin. Virol. 32(1),
2428 (2005).
95. Duval B, De Serres G, Ochnio J, Scheifele D,

Glca V. Nationwide canadian study of

hepatitis A antibody prevalence among
children eight to thirteen years old. Pediatr.
Infect. Dis. J. 24(6), 514519 (2005).
96. Amado LA, Villar LM, de Paula VS,

deAlmeida AJ, Gaspar AM. Detection of

hepatitis A, B, and C virus-specific antibodies
using oral fluid for epidemiological studies.
Mem. Inst. Oswaldo Cruz 101(2), 149155
(2006).
97. Ochnio JJ, Scheifele DW, Marion SA et al.

Participant-collected, mail-delivered oral fluid

specimens can replace traditional serosurveys:
a demonstration-of-feasibility survey of
hepatitis A virus-specific antibodies in adults.
Can. J. Public Health 98(1), 3740 (2007).
98. Matheson K, Halperin B, McNeil S, Langley

JM, Mackinnon-Cameron D, Halperin SA.

Hepatitis A and travel amongst Nova Scotia
postsecondary students: evidence for a
targeted vs. universal immunization strategy.
Vaccine 28(51), 81058111 (2010).
99. Tourinho RS, de Almeida AJ, Amado LA,

Villar LM, Castro AR, de Paula VS. Could

oral fluid be used to evaluate anti-hepatitis A
virus status in individuals living in difficultto-access areas? Vaccine 30(45), 64216426
(2012).

future science group

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