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Previous Article | Next Article 
Journal of Clinical Microbiology, December 1998, p. 3737-3739, Vol. 36, No. 12
PanBio Pty Ltd.,
Received 18 June 1998/Returned for modification 8 July
1998/Accepted 1 September 1998
Saliva was collected prospectively from patients presenting with
suspected dengue infection 4 to 8 days after the onset of symptoms and
assayed by a commercial dengue immunoglobulin M (IgM) and IgG capture
enzyme-linked immunosorbent assay (ELISA) (PanBio Dengue Duo ELISA).
Laboratory diagnosis was based on virus isolation and on
hemagglutination inhibition (HAI) assay and an in-house IgM and IgG
capture ELISA. With a positive result defined as either salivary IgM or
IgG levels above the cutoff value, an overall sensitivity of 92% was
obtained for both primary- and secondary-dengue patients (22 of 24),
while no patients with non-flavivirus infections (n = 11) and no healthy laboratory donors (n = 17) showed
elevation of salivary antidengue antibody (100% specificity). Salivary
IgG levels correlated well with serum HAI titer (r = 0.78), and salivary IgG levels could be used to distinguish between
primary- and secondary-dengue virus infections.
In terms of morbidity, mortality,
and economic costs, dengue is the most important mosquito-borne disease
in the world, with an estimated 100 million cases annually
(13). Initial infection with one of the four serotypes of
dengue virus (primary-dengue virus infection) may lead to dengue fever,
which is a self-limiting, febrile disease with a low mortality
rate, while reinfection with a different dengue serotype
(anamnestic or secondary-dengue virus infection) may lead to
more-serious forms of the disease (e.g., dengue hemorrhagic fever
or dengue shock syndrome) (1, 9, 14). Recently, commercial
tests have been described for the detection of anti-dengue
immunoglobulin M (IgM) and IgG antibodies in serum (2, 11, 12, 21,
23). Potential problems with the use of serum include the
requirement of consent and cooperation of the patient, which is often
unavailable due to social or religious reasons, the need for a trained
venipuncturist and the need to separate serum before testing, and the
difficulty and added risk of venipuncture in children, the group most
commonly affected by dengue in areas where infection is endemic.
Most body fluids contain antibodies, although at much lower levels than
those in blood. Thus, these sources of antibody are unsuitable as
diagnostic specimens, in spite of the obvious advantages and
convenience of samples such as saliva. Salivary antibodies have been
reported to be useful for the diagnosis of a number of infections,
including AIDS, leptospirosis, measles, mumps, hepatitis A and B, and
rubella (3-6, 15-17). In this study we examined the
ability of the PanBio Dengue Duo enzyme-linked immunosorbent assay
(ELISA) to detect both IgM and IgG antibodies to dengue with saliva samples.
Sera and saliva samples were collected prospectively from patients
presenting at the Kamphaeng Phet Provincial Hospital in northern
Thailand. Saliva was collected by using a commercially available
collection device (Omni-Sal; Salivary Diagnostic Systems, Singapore).
This device dilutes saliva twofold in the buffer provided. After
collection, saliva was stored at The Dengue Duo ELISA has been shown to be useful in the diagnosis of
dengue infection with sera (2, 12). It detects IgM and IgG
separately by a capture assay format and was performed by the procedure
recommended by the manufacturer (2), except that saliva was
diluted 1:2 in the assay diluent provided before the addition of 100 µl to each well of the assay plate (final dilution, 1:4). Positive,
negative, and calibrator control sera used in the kit were also run
alongside the saliva samples, though these were diluted 1:100 in the
diluent provided. Results were expressed as the ratio of the absorbance
in test samples divided by the absorbance of the calibrator sera. A
ratio of 0.6 was found to give the best distinction between dengue
infection and other conditions. A positive sample was defined as having
a sample/calibrator absorbance ratio of High sensitivity and specificity were obtained when saliva was used for
the detection of anti-dengue virus antibodies, with 22 of 24 (92%) of
dengue virus infections showing elevation of either IgM or IgG (Table
1). Of the patients with dengue virus infection, 8 showed elevation of both salivary IgM and IgG (all secondary infections); 3 showed elevation of salivary IgM only (two
primary infections and one secondary infection); 11 showed elevation of
salivary IgG only (all secondary infections); and 2 with secondary
infections were negative for both IgM and IgG. The date of the onset of
symptoms was also available for 24 patients. Salivary antibodies were
elevated in 2 of 2 patients by day 4, in 4 of 6 patients at day 5, and
in all 16 patients tested between days 6 and 8. None of the 11 patients
with clinically suspected dengue and no laboratory evidence of
infection produced a positive result in the saliva assay (100%
specificity). Furthermore, none of the saliva samples from 17 healthy
laboratory donors showed elevation of anti-dengue IgM or IgG by this
assay (Table 1). Specificity needs to be evaluated among patients with
non-dengue flavivirus infections such as Japanese encephalitis, yellow
fever, West Nile fever, and tick-borne encephalitis. Some
false-positive results should be expected due to antibody
cross-reactivity. However, these diseases can often be distinguished on
the basis of clinical and epidemiological information.
Salivary IgM levels in primary-dengue virus infections were higher than
those found in secondary-dengue virus infection, while salivary IgG
levels were generally higher in secondary infections (Fig.
1). Similar results have been reported
with sera from dengue patients (2, 8, 11, 18, 20).
Consequently, the comparison of salivary IgM and salivary IgG levels
could be used to distinguish between primary- and secondary-dengue
infection, as observed in other studies with sera (2, 8, 11,
18). In this study, patients with primary-dengue infections had
elevated levels of IgM without detectable IgG, while the majority of
patients with secondary dengue (86%) showed elevated levels
of IgG with or without detectable IgM. This finding indicates that the
cutoff selected for IgG detects high levels of IgG characteristic of
secondary- but not of primary- or past dengue virus infections (7,
10, 22). This result is also reflected in the excellent
correlation between salivary IgG levels and HAI titer (Fig.
2).
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Detection of Specific Antibodies in Saliva during
Dengue Infection
ABSTRACT
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80°C until assayed blindly by the
Dengue Duo ELISA. Diagnosis was based on assay of blood or sera by
using in-house ELISA, hemagglutination inhibition assay (HAI), or viral
isolation performed at the Armed Forces Research Institute of Medical
Sciences (AFRIMS) in Bangkok, Thailand (8, 21). Of the 35 patients from Thailand enrolled in the study, 2 had primary dengue, 22 had secondary dengue, and 11 had no laboratory evidence of dengue
infection despite the presence of clinical symptoms compatible with
dengue fever. Saliva was also collected from 17 healthy Australian
laboratory staff members.
0.6, and a negative sample
was defined as having a sample/calibrator absorbance ratio of <0.6.
Dengue virus infection was characterized by the elevation of either IgM
or IgG, with a negative sample defined as having both IgM and IgG ratios of <0.6.
TABLE 1.
Elevation of salivary anit-dengue antibodies in dengue
and nondengue patients
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FIG. 1.
Comparison of anti-dengue IgG and IgM antibody levels in
saliva. Based on viral isolation, HAI, and an in-house ELISA, 2 patients were diagnosed with primary dengue (squares), 22 were
diagnosed with secondary dengue (triangles), and 11 had no evidence of
dengue infection (circles). Seventeen laboratory donors are also
represented (diamonds). Cutoff ELISA ratios of 0.6 for IgM and IgG are
shown by broken lines.
View larger version (13K):
[in a new window]
FIG. 2.
Correlation between salivary IgG levels determined by
the PanBio Dengue Duo ELISA and serum antibody levels determined by
HAI. Individual assay values are shown by open circles, while the mean
ELISA IgG ratio for different groupings is shown by a horizontal bar.
There was a significant association between mean ELISA IgG ratio and
HAI titer (analysis of variance, F = 14.2; P < 0.0001) as well as a significant correlation between individual
IgG levels and HAI titer (Pearson's r = 0.78, P < 0.0001).
Salivary IgG and IgM levels determined by the Dengue Duo ELISA were compared to serum levels of IgM and IgG found by the AFRIMS in-house ELISA (8) (Fig. 3). Serum and salivary IgG levels showed excellent correlation (Pearson's r = 0.93; P <0.0001), though the correlation between IgM levels in serum and saliva was not significant (Pearson's r = 0.25, P = 0.2014). The serum and salivary ELISAs gave the same result (positive or negative) for IgM in 20 of 30 serum samples, while the same result was obtained for IgG in 25 of 30 serum samples. The majority (8 of 10) of discrepant sera in the IgM assays were positive in the serum test and negative in the saliva test, while most (4 of 5) of the discrepant serum samples in the IgG assays were positive by the saliva test and negative by the serum test.
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Saliva has been reported to be an alternative to sera as a source of antibodies for the diagnosis of a number of diseases, including AIDS, leptospirosis, measles, and mumps, and for hepatitis A and B and rubella infections (3-6, 15-17). This study suggests that saliva is a useful alternative to sera for the diagnosis of dengue. The use of the antibody capture assay format has been shown to be superior to indirect ELISA for the detection of salivary antibodies, and this superiority is likely to be due to the removal of competing antibody in the capture step (15). Antibody concentrations in saliva are 19.9 mg/100 ml (IgA), 1.4 mg/100 ml (IgG), and 0.2 mg/100 ml (IgM), and these levels are approximately 1/10, 1/800, and 1/400 of those in serum (15). An IgA capture ELISA has been reported to have utility in dengue diagnosis, though IgA appears more slowly than IgM and is shorter-lived (19). In this study, investigation of IgA levels in saliva by an in-house antibody capture ELISA gave results similar to those of the IgM capture ELISA, with fewer than half the patients with secondary-dengue infection having elevated IgA (data not shown).
Saliva-based assays should be particularly useful for epidemiological studies (to document recent dengue virus infections in a healthy population or among those experiencing mild disease) and for the diagnosis of acute disease when blood collection is difficult due to cultural factors or difficult venous access, especially in very young children.
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ACKNOWLEDGMENTS |
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This work was supported by the U.S. Army Medical Research and Material Command, PanBio Pty, Ltd. (Brisbane, Australia), through a cooperative research and development agreement and by NIH (AI-34533). The Dengue Duo ELISA was developed by PanBio through an Australian Government-sponsored Co-operative Research Centre for Diagnostic Technologies.
We thank the physicians and nursing staff of the Kamphaeng Phet Provincial Hospital Pediatric Ward for excellent patient care and the Department of Virology, AFRIMS, for specimen collection, processing, and testing (enzyme immunoassay, HAI, and virus isolation) and database management.
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FOOTNOTES |
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* Corresponding author. Mailing address: PanBio Pty Ltd., 116 Lutwyche Rd., Windsor, Queensland 4030, Australia. Phone: 61-7-33571177. Fax: 61-7-33571222. E-mail: peter_devine{at}panbio.com.au.
Present address: Department of Virus Diseases, Walter Reed Army
Institute of Research, Washington, D.C.
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