 |
|
|
July 1997 |
Inhibition ELISA :
An Alternative in the Serological Study of Cases of Dengue
In recent years, dengue and dengue hemorrhagic fever (DHF) have emerged
as a health problem in the tropics and subtropics and the disease is now considered the
most important arbovirus disease in terms of morbidity and mortality.
The publication Dengue and Dengue Hemorrhagic Fever in the Americas: Guidelines
for Prevention and Control (Scientific Publication No. 548, PAHO, 1994) summarizes the
experiences of a group of experts in the subject and provide guidelines for better control
of this disease. The Guidelines suggest the need to strengthen and develop active
surveillance systems with a significant laboratory component. In addition to the
IgM-capture ELISA for detection of dengue-specific IgM antibodies, which has been
recommended as a very useful tool in serological surveillance, the
hemoagglutination-inhibition (HI) technique, which detects total immunoglobulin and
utilizes paired serum samples, continues to be applicable as a serological technique
capable of confirming the presence of dengue infection or to classify a case suspected to
be dengue from a clinical standpoint as probable when high titers of antibodies are
detected in a single serum sample. HI also makes it possible to determine the presence of
a primary or secondary infection.
At the International Dengue Seminar, 1st. Rio Dengue session which took
place from 6 to 9 October, 1996 in Rio de Janeiro, there was discussion of the need to
utilize easily attainable immuno-enzymatic methods in diagnostic laboratories in order to
be able to replace HI and provide similar results.
The Virology Department of the Pedro Kouri Institute of Tropical
Medicine in Havana developed an ELISA Inhibition Method (EIM) which adds the dengue
antigen (sucrose acetone) at a dilution of 1:40 to polystyrene plates previously
sensitized for 18 hours at 4° C with anti-dengue human
immunoglobulin at a concentration of 10ug/ml and then blocked with bovine albumin at 1%.
The next step is to add the sera to be tested in double dilutions from 1:20, incubating
for 1 hour and finally adding the peridoxase antidengue conjugate, developing the reaction
with orthophenylenediamine (OPD) and hydrogen peroxide. Between each step the
corresponding washings are carried out utilizing PBS-Tween20 and incubations are done at
37° C. The antibody titer for each serum is considered to
be that at which a % of inhibition >=50% in comparison with the average DO value of the
negative control sera is observed. A test is considered valid if the -/+ ratio of the
controls is greater than or equal to 5.1 The EIM showed a sensitivity,
specificity, and coincidence of 100%, 83%, and 93% respectively when compared to HI in a
study with single serum samples.
This system has been used satisfactorily since 1987 in the study of
sera received through Dengue Surveillance which has been carried out in Cuba since the
epidemic of 1981; a total of 2878 serum pairs have been processed as of 1995. It has also
been used in the sero-epidemiological surveys carried out in Ecuador (1988 )2 and Panama (1994) in order to determine the prevalence of
antibodies to this agent, using blood samples taken on filter paper.3
Finally, in a comparative study with HI using 182 sera, it showed a
satisfactory correlation (r=.93 p;0.001 and one r2=0.86) and made it possible
through simple linear regression to determine the expected values for EIM that correspond
to different values of HI. This latter analysis made it possible to know that sera with HI
antibody titers of 1\1280 (classified as probable dengue cases using HI) correspond to EIM
titers of 1\5120 and that sera with HI antibody titers of 1\2560 (classified as cases of
secondary infection) correspond to EIM titers of 1\10240.4
The results obtained for several years in the application of this
system to the serological study of dengue allow us to recommend it as an easily used
diagnostic alternative in the region’s laboratories, which would make it possible to
conduct sero-epidemiological studies to define the prevalence of dengue antibodies within
an area or country. If applied in the study of samples received for sero-epidemiological
monitoring in each country, it would, based on the antibody titers observed, make it
possible to classify cases as primary or secondary dengue infection as well as identify
probable cases of infection.
Source: Virology Department of the Pedro Kouri Institute of
Tropical Medicine, Collaborating Center of PAHO/WHO for the Study of Viral Diseases,
Havana, Cuba.
References:
1. Vazquez et al. Rev. Med. Trop. 41(1): 18, 1989; Fernandez et al. Mem. Oswaldo Cruz,
vol. 85(3), 3/47, 1990).
2. Guzman et al. Arthropode-Borne Virus Information Exchange, June 1991.
3. Vazquez et al. Inst. Med. Trop. Sao Paulo, vol.33(4), 309, 1991.
4. Vazquez et al. Rev. Cub. Med. Trop., in press.
Return to issue index
Index of Online Bulletins
