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Journal of Clinical Microbiology, May 1998, p. 1189-1192, Vol. 36, No. 5
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Immunoglobulin A-Specific Capture Enzyme-Linked Immunosorbent
Assay for Diagnosis of Dengue Fever
Antoine
Talarmin,*
Bhety
Labeau,
Josiane
Lelarge, and
Jean-Louis
Sarthou
Centre National de Référence pour
la Surveillance de la Dengue et de la Fièvre Jaune, Institut
Pasteur de la Guyane, Cayenne, French Guiana
Received 4 November 1997/Returned for modification 16 December
1997/Accepted 26 January 1998
 |
ABSTRACT |
Dengue fever (DF) is usually diagnosed by testing for dengue virus
immunoglobulin M (IgM) by a capture enzyme-linked immunosorbent assay
(ELISA) (MAC-ELISA). However, IgM can last for months, and its presence
might reflect a previous infection. We have tested the use of
anti-dengue virus IgA capture ELISA (AAC-ELISA) for the diagnosis of DF
by comparing the results of MAC-ELISAs and AAC-ELISAs for 178 serum
samples taken from patients with confirmed cases of DF. IgM appears
more rapidly (mean delay of positivity, 3.8 days after the onset of DF)
than IgA (4.6 days) but lasts longer; the peak IgA titer is obtained on
day 8. The specificity and the positive predictive value of AAC-ELISA
are 100%; its sensitivity and negative predictive value (NPV) are also
100% between days 6 and 25 after the onset of DF, but they decrease
drastically when data for tests conducted with specimens from the first
days of infection are included, because the IgA titers, like the IgM titers, have not yet risen. AAC-ELISA is a simple method that can be
performed together with MAC-ELISA and that can help in interprating DF
serology.
| |
INTRODUCTION |
Dengue fever (DF) is a tropical
mosquito-borne viral infection caused by four serotypes, dengue virus
type 1 (DEN-1), DEN-2, DEN-3, and DEN-4. DF is a major public
health problem that is responsible for millions of cases of
illness and thousands of deaths in tropical countries every year
(8). The increasing importance of DF and dengue hemorragic
fever in Asia, South America, and the Caribbean (8)
underlines the importance of early detection in controlling the spread
of the disease. The hemagglutination inhibition assay (HI) has been the
reference test for a long time (6), but the simple
immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay
(ELISA) (MAC-ELISA) that is now available allows poorly equipped
laboratories to perform confirmatory laboratory diagnoses
(2).
The presence of specific IgM to dengue viruses in patients with acute
DF is useful for detecting the numbers of recently infected individuals
during an epidemic (1). However, IgM can persist for more
than 8 months (4, 5). Therefore, in countries where DF is
endemic and where many serotypes of dengue viruses are cocirculating, as in southeast Asia and French Guiana, it is sometimes very difficult to interpret a positive result for patients presenting with febrile illness, since the presence of IgM might reflect infection up to 8 months previously.
It was reported that IgA increases at the same time as IgM in patients
with DF but persists for a shorter period of time (7). In
order to test the usefulness of specific IgA for diagnosing dengue
virus infection, we compared the results of MAC-ELISA and IgA capture
ELISA (AAC-ELISA) with reference sera from patients confirmed to have
DF, sera from patients with febrile illnesses due to other causes,
sequential sera from patients confirmed to have DF, and sera sent to
our laboratory for the diagnosis of dengue virus infection.
| |
MATERIALS AND METHODS |
Sera. (i) Reference sera.
Sera from the collection of the
Centre National de Référence pour la Surveillance de la
Dengue et de la Fièvre Jaune (CNR), Institut Pasteur, Cayenne,
French Guiana, were used as reference sera when seroconversion or a
fourfold rise in the HI titers was observed between sera from the acute
phase and sera from the recovery phase and when dengue virus was
detected in an acute-phase sample either by isolation on Aedes
pseudoscutellaris cell culture (AP 61) or by reverse
transcription-PCR by previously described methods routinely used in our
laboratory (11, 12). We thus tested 178 serum samples from
80 patients, with 45 serum samples being from patients infected with
DEN-1, 123 being from patients infected with DEN-2, and 10 being from
patients infected with DEN-4. Thirty-seven patients had primary
infections, 25 patients had secondary infections, and the infections of
the other patients were unknown on the basis of the criteria
established by the World Health Organization (14).
(ii) Sera from patients with other febrile illnesses.
Sera
taken during the recovery phase from 112 patients who showed no
increase in HI titers over that in acute-phase sera were tested. Ten
patients were known to be infected with Coxiella burnetii, six were known to be infected with Mayaro virus, one was known to have
yellow fever, and two were known to be infected with West Nile virus;
the causes of infection in the other patients were unknown.
(iii) Sequential sera.
Sixty-one sequential serum samples
from day 1 or 2 of infection to day 6 or more of infection from 14 patients infected with dengue virus confirmed by virus detection and
seroconversion were obtained from a previous study. They were used to
evaluate the delay of positivity of the AAC-ELISA.
(iv) Other sera.
A total of 442 patient serum samples sent
to CNR for the diagnosis of dengue virus infection were tested for the
presence of specific IgM and IgA. When paired samples were available,
an increase in HI titers was observed, but the virus could not be identified in the acute-phase sample. Four serum samples collected in
Tahiti during the DEN-3 epidemic in 1992 were included in the study.
MAC-ELISA and AAC-ELISA.
The best dilutions of the different
reagents for the MAC-ELISA and the ACC-ELISA were determined in
preliminary tests with positive and negative reference sera. The method
used was a modification of that described previously (10).
Briefly, each well of flat-bottom microplates was coated with 100 µl
of either goat anti-human IgA diluted 1:250 in phosphate-buffered
saline (PBS) or goat anti-human IgM diluted 1:500 in PBS (both
antibodies were from Sigma Laboratories, L'Isle d'Abeau Chesnes,
France). The microplates were incubated for 1 h at 37°C and
1 h at 25°C in a humidified box and were washed with PBS
containing 0.05% Tween 20 (PBS-T).
The sera were diluted at 1:100 in PBS containing 0.5% Tween 20 and 5%
nonfat dried milk (PBS-T-NDM) and dispensed at 100 µl per well in
duplicate. Six negative and two positive reference serum samples were
included on each plate as controls. The plates were then incubated for
1 h at 37°C, and after washing with PBS-T, 100 µl (16 hemagglutination units) of tetravalent dengue virus antigen or an
uninfected control sample diluted in PBS-T-NDM was dispensed into one
of a pair of wells. The antigens were prepared by extraction of the
antigens from the brains of suckling mice with sucrose-acetone. After
incubation at 4°C overnight, the plates were washed with PBS-T, and
bound antigens were detected with anti-dengue virus mouse ascitic fluid
prepared in our laboratory and diluted 1:10,000 in PBS-T-NDM, followed
by the addition of conjugated goat anti-mouse IgG peroxidase (Sigma
Laboratories) diluted 1:1,000 in PBS-T-NDM. Each of these two layers
was subjected to incubation for 1 h at 37°C followed by washing.
Tetramethylbenzidine was used as the substrate.
When positive reference sera appeared blue while the negative reference
sera were still clear, 50 µl of 0.5 N sulfuric acid
was added to each
well to block the reaction. The optical density
(OD) at 450 nm was read
in an ELISA reader (model LP 300; Sanofi
Diagnostics Pasteur, Marnes la
Coquette, France).
For the AAC-ELISA, the mean ± standard deviation OD values for
the negative controls were determined. A result was considered
negative
when the OD values were less than the mean value for
the negative
control plus 2 standard deviations, indeterminate
when the OD values
exceeded 2 standard deviations but scored less
than 3 standard
deviations, and positive when the OD values were
above 3 standard
deviations.
The results for the MAC-ELISA were calculated by dividing the
absorbance of the antigen-containing well by the absorbance
of the
uninfected control well for each specimen, as described
previously
(
3). A ratio of 3 or more was considered positive.
Statistical methods.
To validate anti-dengue virus IgA
detection as a predictor of acute DF, the sensitivity, specificity,
positive and negative predictive values, and accuracy were calculated
for reference sera and were compared with the results of MAC-ELISA.
| |
RESULTS |
Anti-dengue virus IgA titers were always increased when DF had
been confirmed by virus detection and serology (Table
1). As seen with the sequential sera,
however, the positivity for anti-dengue virus IgA often occurs 1 day
later than that for IgM (Fig. 1). The
mean delay for positivity for IgM was 3.8 days after the onset of DF,
and the mean delay for positivity for IgA was 4.6 days after the onset
of DF. The peak IgA titer occurs on day 8 after the onset of DF, and
its decrease is more rapid than that of IgM; after 40 days, IgA is
usually not found (Fig. 2). No difference in IgA titers was found between patients with secondary and primary infections or between patients with DF due to DEN-1, DEN-2, or DEN-4.
The results of the AAC-ELISA were also positive for four DEN-3-positive
serum samples collected on days 7 to 13 after the onset of illness.
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TABLE 1.
Comparison of the results of AAC-ELISA and MAC-ELISA for
detection of dengue virus-positive reference sera
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FIG. 1.
Percentages of positive ( ), indeterminate ( ), and
negative ( ) results for AAC-ELISA (A) and MAC-ELISA (B) for 14 patients from whom sequential sera were available.
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FIG. 2.
Evolution of the mean ratios of the absorbance for the
well containing the serum specimen divided by the mean of the
absorbance values for the negative wells for AAC-ELISA ( ) and by the
absorbance for the uninfected control well for MAC-ELISA ( ) after
the onset of DF. The means were determined for 178 reference serum
samples and 61 sequential serum samples. Numbers in parentheses
represent the numbers of serum samples tested.
|
|
The agreement between tests for IgA and IgM was 100% for reference
sera taken before day 3 after the onset of the disease and for those
collected between days 6 and 25 after the onset of disease.
Discrepancies were seen between days 3 and 5 and after day 25 after the
onset of disease (Table 1). Sera from most of the patients with
infections other than DF and even those from patients recently infected
with West Nile and yellow fever viruses gave negative results for
dengue virus IgA (Table 2); two serum samples gave indeterminate results. In contrast, three patients with
low HI titers were found to be positive for IgM, and for these patients
there was no increase in the HI titer between the acute- and the
recovery-phase sera. The positive predictive value and the specificity
of the AAC-ELISA, determined with reference sera, were 100%, but its
sensitivity and its negative predictive value were high only between
days 6 and 25 after the onset of disease (Table 2). For sera taken
between days 1 and 29 after the onset of disease, the MAC-ELISA was
more sensitive and less specific than the AAC-ELISA, but the
differences were not significant (Table 2).
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TABLE 2.
Results of AAC-ELISA and MAC-ELISA with reference sera
and sera from patients with other febrile illnesses
|
|
For the sera sent to CNR for the diagnosis of DF, the discrepancies
between the results of tests for IgM and IgA were greater (Table
3), with many serum samples being dengue
virus IgM positive and dengue virus IgA negative. Most of these sera
were taken between days 3 and 5 and after day 25 after the onset of
fever; however, two serum samples collected between days 6 and 19 after
the onset of fever remained IgA negative, whereas a significant rise in the anti-dengue virus HI titers was observed.
| |
DISCUSSION |
In our study, the results of the AAC-ELISA were always positive
when DF was confirmed, but they were positive about 1 day later than
those of MAC-ELISA. The finding that IgA did not appear in two patients
presenting with seroconversion to flaviviruses but in whom no virus
could be isolated or detected by reverse transcription-PCR did not
appear to be due to IgA deficiency since the levels of IgA in serum
were normal. Therefore, either the seroconversion was due to another
flavivirus circulating in the Amazonian forest complex (9)
or dengue virus IgA is sometimes not present during DF. Since similar
levels of dengue virus IgA were detected in patients with DF due to
DEN-1, DEN-2, DEN-3, and DEN-4, the lack of increase in the IgA titer
does not seem to be the result of an infection with a particular
serotype of dengue virus. Further studies, especially in countries
where dengue viruses are the only flaviviruses present, are
necessary to answer to this question.
The rapid decrease in the dengue virus IgA titer (about 40 days) is
very interesting for countries where many serotypes of dengue viruses
cocirculate. In French Guiana in 1996 and 1997, dengue viruses were
very active, with an epidemic due to DEN-1 and DEN-2 occurring from
September 1996 to April 1997 (13). Individuals could therefore be
infected with both viruses within a short period of time. All DF-like
syndromes are not necessarily due to dengue viruses, however, and the
presence of dengue virus IgM after a febrile syndrome is not always
proof of recent infection but may be due to DF that occurred some
months previously. For example, two of the three patients positive for
dengue virus IgM but for whom a diagnosis of acute DF could be
eliminated were known to have had DF five and seven months earlier,
respectively. These patients illustrate the limitation of MAC-ELISA
alone for diagnosing DF in countries where it is endemic. An indicator
of a very recent infection, such as the presence of IgA, is therefore
useful. The limit of positivity was chosen in order to eliminate false
positivity. By using this cutoff value, the positive predictive value
of the AAC-ELISA is excellent, and positivity by this test is proof of recent DF. Although the sensitivity of AAC-ELISA is not very good, it
is almost the same as that of MAC-ELISA. The absence of IgA coupled
with the presence of IgM is usually due to a delay after onset of DF
that is too short. Indeed, in our study IgM antibodies were detected
about 1 day sooner than IgA antibodies when sequential sera were
tested, even though many trials were performed to improve the
sensitivity of the AAC-ELISA. However, a positive MAC-ELISA result
in association with a negative AAC-ELISA result at the beginning of a
febrile illness can also be due to an older infection. This has been
confirmed at least three times with sera from patients with other
febrile illnesses. It was also probably the case for sera sent to CNR
for the diagnosis of DF, but this is difficult to prove since a
recovery-phase sample was not available. Knowledge of the date of the
disease's onset and the availability of a recovery-phase serum sample
are important in drawing a conclusion. However, when the dengue virus
IgA is absent from a blood sample obtained between days 6 and 25 after the onset of the disease, a diagnosis of DF is highly improbable,
since the negative predictive value is 100% during this period (Table
2). The presence of IgM coupled with the absence of dengue virus IgA
during this period probably indicates an old infection. Indeterminate
AAC-ELISA results in association with positive MAC-ELISA results
usually reflect DF at a very early or late phase (Fig. 1; Table 1).
Assays of sequential sera from 14 patients indicated that at the
beginning of DF, when the results of MAC-ELISA are positive, the
results of AAC-ELISA are usually positive or indeterminate (Fig. 1).
In conclusion, the presence of dengue virus IgA is a better indicator
of dengue virus infection than the presence of IgM on days 6 to 25 after the onset of infection. Since the date of onset is not always
known, however, MAC-ELISA remains the best tool for the serological
diagnosis of DF. Use of the two assays in conjunction provides useful
information for interpreting the results of dengue virus serology. The
experimental procedures for AAC-ELISA and MAC-ELISA are quite similar,
and the only limiting factor is the doubling of the cost of dengue
virus serology.
IgA detection may also be useful for determining the rates of infection
during a DF epidemic. Since the IgA titer decreases rapidly, it gives
an indication only of the individuals infected during the past month;
thus, successive testing can provide an indication of the progression
of the epidemic. Dengue virus IgA could be determined in saliva since
this immunoglobulin is excreted in large quantities. If reliable,
testing for IgA in saliva would be useful in large-scale
epidemiological studies and for the diagnosis of DF in babies and
children, eliminating the need for venipuncture.
| |
ACKNOWLEDGMENTS |
We are grateful to B. Le Guenno, Institut Pasteur, Paris, France,
for providing sera from yellow fever and West Nile fever patients and
to E. Chungue, Institut Malardé, Papeete, Tahiti, for
providing sera from patients infected with DEN-3.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institut Pasteur
de la Guyane, BP 6010, 97306 Cayenne cedex, French Guiana. Phone: (594)
29.26.09. Fax: (594) 30.94.16.
| |
REFERENCES |
| 1.
|
Boisier, P.,
J. M. Morvan,
S. Laventure,
N. Charrier,
E. Martin,
A. Ouledi, and J. Roux.
1994.
Epidémie de dengue 1 sur l'île de la Grande Comore (République Fédérale Islamique des Comores). Mars-mai 1993.
Ann. Soc. Belge Med. Trop.
74:217-229[Medline].
|
| 2.
|
Bundo, K., and A. Igarashi.
1985.
Antibody-capture ELISA for detection of immunoglobulin M in sera from Japanese encephalitis and dengue hemorragic fever patients.
J. Virol. Methods
11:15-22[Medline].
|
| 3.
|
Cardosa, M. J., and I. Zuraini.
1991.
Comparison of an IgM capture ELISA with a dot enzyme immunoassay for laboratory diagnosis of dengue virus infections.
Southeast Asian J. Trop. Med. Public Health
22:337-340[Medline].
|
| 4.
|
Chen, W. J.,
K. P. Hwang, and A. H. Fang.
1991.
Detection of IgM antibodies from cerebrospinal fluid and sera of dengue fever patients.
Southeast Asian J. Trop. Med. Public Health
22:659-663[Medline].
|
| 5.
|
Chow, L., and S. T. Hsu.
1989.
MAC-ELISA for the detection of IgM antibodies to dengue type 1 virus.
Chin. J. Microbiol. Immunol. (Taiwan)
22:278-285.
|
| 6.
|
Clarke, D. H., and J. Casals.
1958.
Techniques for hemagglutination and hemagglutination-inhibition with arthropod-borne viruses.
Am. J. Trop. Med. Hyg.
7:561-573.
|
| 7.
|
Groen, J.,
E. Balentien,
V. Deubel,
V. Vorndam, and A. D. M. E. Osterhaus.
1997.
Kinetics of IgA serum antibodies in monkeys immunized with live attenuated dengue virus type 2 vaccine and in patients with a dengue virus infection, p. 24.
In
Program and abstracts of the 4th International Symposium on Dengue Fever.
|
| 8.
|
Gubler, D. J.
1988.
Dengue, p. 223-260.
In
J. P. Monath (ed.), The arboviruses: epidemiology and ecology. CRC Press, Inc., Boca Raton, Fla.
|
| 9.
|
Karabatsos, N. (ed.).
1985.
International catalogue of arboviruses.
The American Society of Tropical Medicine and Hygiene, San Antonio, Tex.
|
| 10.
|
Kuno, G.,
I. Gomez, and D. J. Gubler.
1987.
Detecting artificial anti-dengue IgM immune complexes using an enzyme-linked immunosorbent assay.
Am. J. Trop. Med. Hyg.
36:153-159.
|
| 11.
|
Lanciotti, R. S.,
C. H. Calisher,
D. J. Gubler,
G.-J. Chang, and A. V. Vorndam.
1992.
Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction.
J. Clin. Microbiol.
30:545-551[Abstract/Free Full Text].
|
| 12.
|
Reynes, J. M.,
A. Laurent,
V. Deubel,
E. Telliam, and J. P. Moreau.
1994.
The first epidemic of dengue hemorrhagic fever in French Guiana.
Am. J. Trop. Med. Hyg.
51:545-553.
|
| 13.
|
Talarmin, A.,
M. Senes,
P. Maurer,
F. Fouque,
B. Labeau,
G. du Fou, and J. L. Sarthou.
1997.
Dengue 1 outbreak in Kourou, French Guiana, p. 18.
In
Program and abstracts of the 4th International Symposium on Dengue Fever.
|
| 14.
|
World Health Organization.
1986.
Dengue haemorragic fever: diagnosis, treatment and control.
World Health Organization, Geneva, Switzerland.
|
Journal of Clinical Microbiology, May 1998, p. 1189-1192, Vol. 36, No. 5
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Top
Abstract
Introduction
