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African Journal of Biomedical Research
Ibadan Biomedical Communications Group
ISSN: 1119-5096
Vol. 6, Num. 2, 2003, pp. 73-77
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African Journal of Biomedical Research, Vol. 6, No. 2, May, 2003, pp. 73-77
TETANUS TOXOID ANTIBODY LEVEL IN ASYMPTOMATIC PLASMODIUM
FALCIPARUM MALARIA PARASITEAMIC PREGNANT WOMEN.
1 *ONYENEKWE
C.C, 1 MELUDU S.C, 2 ARINOLA O.G., 2 SALIMONU L.S.
1Departments of Chemical and immunology,
College of Health Sciences, Nnamdi Azikiwe University, Nnewi Campus, PMB
5001 Nnewi, Anambra State . Nigeria .
2Immunology
Unit, Chemical Pathology Department, College of Medicine, University College
Hospital, PMB 5116 Ibadan, Oyo State. Nigeria .
*Author for correspondence
Code Number: md03014
ABSTRACT
The present study was designed to investigate if the presence of asymptomatic
malaria parasiteamia in pregnant women will compromise their ability to respond
to full dose of tetanus toxoid immunization during their antenatal clinic
visits. Hence, 90 apparently healthy pregnant women who had completed the
tetanus toxoid immunization during the current pregnancy were recruited at
the antenatal clinic and were divided into two groups based on the antenatal
record of malaria paras during the immunization period. Sixty (66.7%) of
the pregnant women were seroreactive for Plasmodium falciparum histidine
rich protein- (HRP)-2 while 30 (33.3%) were seronegative for Plasmodium falciparum
HRP-2. The malaria parasite density range for the seroreactive group was
between 322 and 1045 parasites per ml of blood. The blood concentration of
Tetanus toxoid antibody response in both groups of seroreactive and seronegative
HRP-2 pregnant women did not show any significant difference in tetanus toxoid
antibody response (p>O.2). This result showed that the presence of asymptomatic
IPlasmodium falciparum malaria parasiteamia in the pregnant women during
the immunization schedule did not compromise the ability to respond to tetanus
toxoid immunization. Hence asymptomatic malaria may not contribute to the
prevalence of neonatal tetanus in Nigeria , however, there
is need to treat these pregnant women for asymptomatic malaria when detected
in order to reduce the burden of malaria on them.
Key words: Tetanus toxoid, asymptomatic malaria, specific antibody, pregnant
women.
INTRODUCTION
Tetanus is considered a major health problem in the developing countries (
Campbell , 2000). However, the numerous financial and human resources that
have been invested into the eradication of neonatal tetanus have not yielded
the desired goal in the developing countries. Since the effectiveness of tetanus
toxoid immunization has been convincingly demonstrated in many field and hospital
based studies (Newell et al 1971, Black et a! 1980, Cliff et al 1985, EPI 1988).
It is necessary for extensive research to be conducted in developing countries
experiencing high neonatal tetanus rates in order to discover the actual reason
behind the observed results. Immunity against tetanus toxoid is antibody mediated
and belongs to the IgG class of immunoglobulin (WHO/EPI). This could suggest
that adequate immunization of the pregnant women may lead to effective protection
of their neonates (WHO/EPI), through placental transfer of the antibody to
the neonates. However, in Nigeria , pregnant women are not routinely screened
for the presence of asymptomatic malaria parasiteamia and hence such pregnant
women are not treated for malaria during pregnancy. However the recent report
of high incidence rate of asymptomatic malaria parasireamia amongst pregnant
women in Nigeria (Onyenekwe, 2002), led to the design of this study in order
to know the possible impact of this disease on one of the most prevalent immunization
programmes in Nigeria. Therefore, the present study was designed to check the
tetanus specific antibody levels in immunized pregnant women with antenatal
record of asymptomatic malaria parasiteamia during the tetanus toxoid inununization
schedules.
MATERIALS AND METHODS
Subjects:
90 apparently healthy pregnant women aged 19-4Oyears attending the antenatal
clinic of Obstetric and Gynaecology Department were studied, These pregnant
women had received fill dose of tetanus toxoid immunization in the course of
their ahtenatal visits. After the immunization of the pregnant women was completed,
they were divided into 2 groups based on the antenatal record of asymptomatic
malaria parasiteamia during the immunization schedules. Sixty of the pregnant
women had asymptomatic malaria parasiteamia and were reactive to Plasmodium
falciparum }IRP-2 during the period of immunization. These were called
positive subjects. The remaining 30 pregnant women had no asymptomatic malaria
parasiteamia and were not reactive to Plasmodium falciparum HRP-2 during
the period of immunization. These were called negative subjects. Malaria parasiteamia
was used as the bases of dividing the pregnant women into groups because of
the high prevalence of asymptomatic malaria parasiteaniia amongst pregnant
women in Nigeria . Informed consent was obtained from the pregnant women and
the University of Ibadan/University College Hospital, Board of Ethical Committee,
Post-Graduate Institute for Medical Research and Training; Ibadan approved
the design of this study.
Blood sample collection:
During the antenatal clinic visits by the pregnant women, asymptomatic Plasmodium
falciparum malaria parasite screening was performed using Plasmodium
falciparum HRP-2 seroreactivity and blood parasites detection and results
of the investigation were recorded during the tetanus toxoid immunization.
Two weeks after the full dose of tetanus toxoid immunization of the pregnant
women, blood samples were collected by vene-puncture and dispensed into sterile
containers. The separated sera were used for the determination of tetanus
toxoid antibody level in the pregnant women.
This was done by Plasmodium falciparum histidine rich protein 2 seroreactivity
based on indirect enzyme linked immunosorbent assay (Cellabs PTY Ltd Australia).
The procedure as described by the manufacturers is briefly reported. Microwells
were coated with monoclonal anti- Plasmodium falciparum histidine rich
protein (HRP)-2. 100 m l of test sample, reference positive control or reference
negative control was added respectively to the coated wells. The wells were
incubated at room temperature for 60 minutes to allow the fixing of the HIR
antigen to the specific antibody coated to the wells. Other blood components
were removed by washing each well with 0.01 M phosphate buffered saline (PBS)-Tween
solution. The conjugate of enzyme labelled anti-human globulin was added to
all the wells. This was allowed for incubation at room temperature for 60 minutes.
The conjugate antibody will bind any antigen fixed to the well. The wells were
washed as described above. The enzyme substrate solution was added and incubated
in the dark for 15 minutes. This allows for colour development that indicates
the presence of malaria antigen in the blood under test.
Interpretation of Result of histidine rich protein-2 showed that sixty pregnant
women were sero-reactive to Plasmodium falciparum HRP-2 while the remaining
30 were sero negative to Plasmodium falciparum HRP-2.
Detection of Plasmodium falciparum parasites:
Plasmodium falciparum Malaria parasites were detected by microscopic
examination of Giemsa stained thin and thick blood films and parasitaemia was
expressed as malaria parasites density per ml of blood as described by Rooth
et. Al (1991). The asymptomatic malaria parasites range detected in the Plasmodium
falciparum HRP-2 sero-reactive pregnant women was between 322 and 1045
malaria parasites per microlitre of blood.
Determination of tetanus toxoid antibody:
The serum tetanus toxoid antibody concentration was determined by indirect
enzyme linked immunosorbent assay. The procedure is briefly described. Microwells
were coated with tetavax adsorbed tetanus vaccine (Pasteur Meriux, France).
The serum samples for the determination of tetanus toxoid antibody were added
in 100j volumes to the respective coated wells. The wells were incubated for
60 minutes at 37°c to allow the specific antibody to fix to the antigen.
Other components not bound were washed off the wells with 0.OIM phosphate buffered
saline (PBS)-Tween solution. The conjugate enzyme labelled anti-human globulin
binds the tetanus antibody fixed to the well. Other conjugate antibody not
bound was washed-off. 100 m l of the enzyme substrate chromogen TMB (3,3,5,5-tetramethylbenzidine)
was added to each well and incubated in the dark at room temperature for 15
minutes. The reaction was stopped with 2.5m1 HC1 and the absorbance of the
colour developed was read on a dynatech MR 250 micro-plate reader (Guernsey
Channel, Islands ). Un-reacted well containing only the substrate solution
and stopping reagent was used to blank the micro-plate reader. The absorbance
was proportional to the concentration of the tetanus toxoid antibody in the
serum.
Statistical analysis: This was performed using stac-pac Gold
package. The mean (+lsd) was determined for the variables in each group while
the analysis of variance was used to determine the level of significance between
the variables. Level of significance was considered at p-value <0.05.
RESULTS
Table 1 shows the HRP-2 Seroreactivity rates and malaria parasites range (per
ml of blood) among the tetanus toxoid immunized pregnant women. Sixty (66.7%)
of the pregnant women were HRP-2 seroreactive while 30(33.3%) of the pregnant
women were FIRP-2 seronegative. Similarly, the malaria parasites density range
as observed in the HRP-2 seroreactive pregnant women was between 322 to 1045
parasites per ml of blood.
Table 1: Shows the HRP-2 Seroreactivity rates and malaria parasites density range (per
ml of blood) among the tetanus toxoid immunized pregnant women
Malaria parasite status
|
Asymptomatic (positive) Pregnant women |
Aparasiteamic (negative) Pregnant women |
HRP-2 Seroreactivity |
60 (66.7%)
positive |
30 (33.3%)
negative |
Malaria parasites density |
322 <1045/mi of blood
|
Nil
|
HRP-2= Plasmodium falciparum histidine rich protein-2
Table 2 shows the mean (± 1 Sd) serum tetanus toxoid antibody concentration
expressed as absorbance in the asymptomatic malaria parasiteamia positive and
aparasiteamic negative groups of pregnant women immunized with tetanus toxoid.
The mean serum tetanus toxoid antibody absorbance was 0.710 ± 0,230
and 0.790 ± 0.220 in asymptomatic malaria parasiteamia positive and
aparasiteamic negative groups respectively. There was no significant difference
in mean tetanus toxoid antibody absorbance between the two groups (p>0.2).
Table 2: Mean (±1sd) serum concentrations of tetanus toxoid
specific antibody expressed in absorbance in asymptomatic malaria parasiteamia
positive and negative pregnant women after full tetanus toxoid immunization.
Parameter
|
asymptomatic (Positive) pregnant women
|
aparasiteamic (Negative) pregnant women.
|
p-value
|
Tetanus toxoid antibody levels
|
0.710 ± 0.230
(n=60) |
0.790 ± 0.220
(n=30) |
p>0.2
|
sd = standard deviation
DISCUSSION
The present study observed the presence of asymptomatic Plasmodium falciparum positive
pregnant women, thus confirming that pregnant women may also present with asymptomatic
malaria. One of the possible reason for this phenomenon may be that the immune
systems of the pregnant women was quite able to contain the attained blood
malaria parasites density. Hence this study calls on concerned authority to
include asymptomatic malaria parasites screening as part of normal antenatal
routine checks in Nigeria and possible in other malaria endemic regions of
the world.
An epidemiological implication of not treating asymptomatic malaria parasiteamia
is that the infected pregnant women may serve as reservoirs for the spread
of malaria to the community and probable to their feotus in utero. In addition,
in the cases of stress or any condition that may suppress their inunune system,
clinical presentation of malaria may occur easily due to the presence of several
predisposing factors. Therefore the authors of the present study are of the
opinion that asymptomatic malaria parasiteamia be treated during antenatal
clinic visits as this will help to reduce the burden of malaria and possible
chances of precipitation of clinical malaria during pregnancy.
Asymptomatic malaria parasiteamia is a type of malaria parasiteanua that is
different from clinical malaria in presentation but in the presence of predisposing
factors may precipitate clinical malaria in the individual. The individuals
are known to present apparently healthy without any sign and symptoms of malaria.
Characteristic of this pattern of parasiteamia is the maintenance of parasites
threshold usually different from that observed in cases of acute or clinical
malaria infections. It has been shown that single infection can persist for
as long as 18 months in the absence of re-infection (Frank et al 2001, Krajden
et al 1991). This could suggest the impact of such asymptomatic existence of
infections on human and resources, The present study also observed that the
asymptomatic Plasmodium falciparum malaria parasiteamia positive pregnant
women on full tetanus toxoid immunization had similar tetanus toxoid specific
antibody response as observed in the aparasitèamic negative pregnant
women.
This result indicates that asymptomatic Plasmodium falciparum parasiteamia
did not pose any immediate threat to achieving the desired target for tetanus
toxoid immunization. Thus, there seem not to be any immunosuppressive effect
of the presence of asymptomatic malaria parasiteamia on the specific response
of infected pregnant women to tetanus toxoid immunization. In a study elsewhere,
the antibody responses of malaria infected pregnant women to tetanus toxoid
immunization was reported to be similar to that of non-pregnant healthy adults
(Brabin et al 1984).
Increase in the prevalence of neonatal tetanus has been reported in Nigeria
(Anti-Obong et al 1993, Owa and Makinde 1992). However, healthy Nigerian women
and pregnant women on malaria chemoprophylaxis have been shown to respond adequately
to tetanus toxoid immunization (Gilles 1983, Gini and Okafor 1992). This shows
that generation of tetanus specific antibody is not impaired in Nigeria women
and could be assumed that their neonates would be protected. However, certain
disease conditions, if present in a host, has been shown to induce immunosuppressive
effect on the ability of the individual to respond to other infections. Possibly
the incidence of any such disease may disrupt the ability of these pregnant
women to generate adequate tetanus specific antibody and transfer of such antibody
during pregnancy to their neonates (Gilles 1983, Gini and Okafor 1992).
Therefore the increase in prevalence of asymptomatic malaria in Nigerian pregnant
women may have nothing to do with the increase cases of neonatal tetanus in
Nigeria . The present study therefore suggests that the presence of asymptomatc Plasmodium
falciparum malaria parasiteamia during tetanus toxoid immunization may
not compromise the ability of the pregnant women to respond to tetanus toxoid
immunization. However it also suggests that in order to limit the burden of
malaria morbidity and mortality on pregnant women, it is necessary that treatment
be administered in cases of asymptomatic malaria infection to the affected
subjects in order to reduce the epidemiological impact.
ACKNOWLEDGEMENT:
We are gratethi to Prof. IF Adewole and Prof. KA Obisesan, both of Obstetric
and Gynaecology Department, University College Hospital , Ibadan for
allowiiig us assess to the patients. We are also grateful to Prof. Claude
Muller of immunology Department, Laboreitoire de Sante , Luxembourg ,
for providing the conjugate antibody. We are also grateful to Mrs. Omoyeme
Okiwelu and Mr Ekanem Inyang for their assistance in the laboratory and to
the staff members of both immunology unit and antenatal clinic. This study
was partly sponsored by Stephen Oluwole Awokoya foundation for Science Education
awarded to Onyenekwe Charles.
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