Malaria in pregnant Cameroonian women: the effect of age and gravidity on submicroscopic and mixed-species infections and multiple parasite genotypes.
ABSTRACT Polymerase chain reaction (PCR)-based methods were used to investigate malaria in pregnant women residing in Yaounde, Cameroon. Microscopy and species-specific PCR-based diagnosis show that at delivery 82.4% of the women were infected with Plasmodium falciparum (27.5% blood-smear positive and 54.9% submicroscopic infections). The prevalence of P. malariae and P. ovale was 7.6% and 2.5%, respectively, with 9.4% infected with more than one species. Based on genotyping of the merozoite surface protein 1 (msp-1) and msp-2 alleles, the mean number of genetically different P. falciparum parasites in peripheral blood was 3.4 (range = 1-9) and 3.5 (range 1-8) in the placenta. Plasmodium falciparum detected by microscopy and PCR as well as mixed-species infections were significantly higher in women < or = 20 years old and paucigravidae, but maternal anemia was associated only with microscopic detection of parasites. Neither submicroscopic infections nor number of parasite genotypes decreased significantly with age or gravidity. Thus, pregnancy-associated immunity helps reduce malaria to submicroscopic levels, but does not reduce the number of circulating parasite genotypes.
- SourceAvailable from: Hélène M De Nys
Dataset: De Nys et al. 2014
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ABSTRACT: Background The diversity of malaria parasites ( Plasmodium sp.) infecting chimpanzees ( Pan troglodytes ) and their close relatedness with those infecting humans is well documented. However, their biology is still largely unexplored and there is a need for baseline epidemiological data. Here, the effect of pregnancy, a well-known risk factor for malaria in humans, on the susceptibility of female chimpanzees to malaria infection was investigated. Methods A series of 384 faecal samples collected during 40 pregnancies and 36 post-pregnancies from three habituated groups of wild chimpanzees in the Taï National Park, Côte d’Ivoire, were tested. Samples were tested for malaria parasites by polymerase chain reaction (PCR) and sequencing. Data were analysed using a generalized linear mixed model. Results Probability of malaria parasite detection significantly increased towards the end of pregnancy and decreased with the age of the mother. Conclusions This study provides evidence that susceptibility to malaria parasite infection increases during pregnancy, and, as shown before, in younger individuals, which points towards similar dynamics of malaria parasite infection in human and chimpanzee populations and raises questions about the effects of such infections on pregnancy outcome and offspring morbidity/mortality.Malaria Journal 10/2014; · 3.49 Impact Factor
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ABSTRACT: Malaria during pregnancy results in adverse outcomes for mothers and infants. Intermittent preventive treatment (IPT) with sulphadoxine-pyrimethamine (SP) is the primary intervention aimed at reducing malaria infection during pregnancy. Although submicroscopic infection is common during pregnancy and at delivery, its impact throughout pregnancy on the development of placental malaria and adverse pregnancy outcomes has not been clearly established.Malaria Journal 07/2014; 13(1):274. · 3.49 Impact Factor
MALARIA IN PREGNANT CAMEROONIAN WOMEN: THE EFFECT OF AGE AND
GRAVIDITY ON SUBMICROSCOPIC AND MIXED-SPECIES INFECTIONS AND
MULTIPLE PARASITE GENOTYPES
ANNIE WALKER-ABBEY, ROSINE R. T. DJOKAM, ANNA ENO, ROSE F. G. LEKE, VINCENT P. K. TITANJI,
JOSEPHINE FOGAKO, GRACE SAMA, LUCY H. THUITA, ELIZA BEARDSLEE, GEORGES SNOUNOU,
AINONG ZHOU, AND DIANE WALLACE TAYLOR
Department of Biology, Georgetown University, Washington, District of Columbia; Faculty of Medicine and Biomedical Sciences,
Biotechnology Center, University of Yaoundé, Yaoundé, Cameroon; Unité de Parasitologie Biomédicale, Institut Pasteur, Paris,
France; AZ DataClinic, Inc., Rockville, Maryland
residing in Yaoundé, Cameroon. Microscopy and species-specific PCR-based diagnosis show that at delivery 82.4% of
the women were infected with Plasmodium falciparum (27.5% blood-smear positive and 54.9% submicroscopic infec-
tions). The prevalence of P. malariae and P. ovale was 7.6% and 2.5%, respectively, with 9.4% infected with more than
one species. Based on genotyping of the merozoite surface protein 1 (msp-1) and msp-2 alleles, the mean number of
genetically different P. falciparum parasites in peripheral blood was 3.4 (range ? 1–9) and 3.5 (range 1–8) in the
placenta. Plasmodium falciparum detected by microscopy and PCR as well as mixed-species infections were significantly
higher in women ? 20 years old and paucigravidae, but maternal anemia was associated only with microscopic detection
of parasites. Neither submicroscopic infections nor number of parasite genotypes decreased significantly with age or
gravidity. Thus, pregnancy-associated immunity helps reduce malaria to submicroscopic levels, but does not reduce the
number of circulating parasite genotypes.
Polymerase chain reaction (PCR)–based methods were used to investigate malaria in pregnant women
During pregnancy, Plasmodium falciparum infections can
have an adverse effect on both the mother and the developing
fetus.1–3In malaria-endemic areas, the prevalence of clinical
and asymptomatic malaria is highest in young women and
those in their first and second pregnancies.1,3With successive
pregnancies, women acquire a gravidity-dependent form of
immunity, resulting in a decrease in both prevalence and se-
verity of infection.1–3Antibodies that block the binding of
parasites to chondrointin sulfate A in the placenta4,5and ma-
laria-specific memory lymphocytes with homing receptors for
the placenta6are thought to help mediate pregnancy-asso-
In the last decade, the development of molecular ap-
proaches has shed light on the prevalence and number of
genetically-different parasites, i.e., multiplicity of infection
(MoI) circulating in the blood of individuals living in endemic
areas. These techniques are beginning to increase our under-
standing of the level of immunity acquired by pregnant
women. Polymerase chain reaction (PCR)–based detection
methods show that at least twice as many pregnant women
are infected with malaria as indicated by microscopy.7,8For
example, in a cross-sectional study, 32% of pregnant Ghana-
ian women were peripheral blood smear positive, but 63%
were positive by PCR.7Likewise, 29% of samples from preg-
nant Senegalese women were slide positive, but 85% were
positive by PCR for the P. falciparum merozoite surface pro-
tein 1 (msp-1) or msp-2 genes.8Thus, a large number of preg-
nant women harbor submicroscopic infections, i.e., parasite
DNA is detected in their blood, but parasites are not seen on
blood smears. Although species-specific primers have been
used to determine the prevalence of P. malariae, P. ovale, and
P. vivax in population-based studies,9the prevalence of
mixed-species infections in pregnant women has not been re-
The relationship between acquisition of pregnancy-
associated immunity and MoI remains unclear, since the MoI
was reported to decrease with increasing gravidity in one
study,10but not in another.11High MoI as well as submicro-
scopic infections have also been associated with mild mater-
nal anemia,7,10,12but this is not a universal finding.11Thus,
additional studies using PCR-based approaches are needed to
increase our understanding of the extent to which gravidity-
dependent immunity controls/eliminates parasites and influ-
ences maternal anemia.
The current study was conducted in the city of Yaoundé,
Cameroon where malaria transmission is perennial. A previ-
ous study showed that the prevalence of asymptomatic P.
falciparum infections (i.e., detection of parasite in peripheral
blood smears) in Yaoundé is significantly higher in women
? 20 years of age and primigravidae.13However, after adjusting
the results for age, gravidity, chemoprophylaxis, and other
covariates, only young age remained statistically significant
(adjusted odds ratio [OR] ? 3.4, 95% confidence interval
[CI] ? 1.7–7.1) in this urban setting. Thus, we sought to ex-
plore the relationship between age and gravidity with respect
to the prevalence of submicroscopic and mixed-species (i.e.,
more than one species of malaria in the peripheral blood or
placenta) infections and number of different parasite geno-
types in the peripheral blood and placenta of women living in
Yaoundé, Cameroon. In addition, we sought to determine if
the level of submicroscopic and mixed-species infections and
MoI decreased with maternal age and gravidity and if they
were associated with maternal anemia at delivery.
MATERIALS AND METHODS
Study design. The study was conducted in Yaoundé, Cam-
eroon, where malaria is perennial with periods of increased
transmission occurring during the two rainy seasons. Samples
were collected at Central Hospital (June 1995 to March 1996),
Biyem Assi Hospital (August 1996–November 1996), and
Central Hospital (December 1997 to December 1998). The
purpose of the study was explained to each woman. Consent-
ing women provided information relative to their pregnan-
Am. J. Trop. Med. Hyg., 72(3), 2005, pp. 229–235
Copyright © 2005 by The American Society of Tropical Medicine and Hygiene
cies, including age, number of previous pregnancies, and use
of antimalarial prophylaxis during pregnancy. Among the
women consecutively recruited, samples from 278 women
who had normal, vaginal singleton deliveries were evaluated
in this study. Because of their rarity, samples from women
with spontaneous abortions, stillbirths, and multiple births
were excluded from the analysis. Relevant information on the
women in the study is shown in Table 1. All malaria-positive
women had asymptomatic infections. Prior to initiating the
study, the project received approval from the Institutional
Review Board of Georgetown University and the Ethical
Committee of the Ministry of Health of Cameroon.
Sample collection. After delivery, an 8-mL sample of ma-
ternal peripheral blood was collected. In addition, a sample of
maternal placental blood was obtained using the pool-biopsy
method.14Briefly, a small piece of the placenta was removed
(5 cm × 5 cm × 5 cm) and intervillous blood was allowed to
pool into the site. The blood was collected in a tube contain-
ing EDTA and stored on ice until processed. A piece of the
placenta was also collected for parasitologic studies.
Analysis of blood samples and placental tissue. Thick and
thin blood films were made using maternal peripheral blood
and impression smears were prepared using placental tissue.
The slides were stained with Dif-Quick (Baxter Healthcare
Corp., Miami, FL). Two hundred microscopic fields of thick
films were examined for the presence of parasites. When
parasites were seen, the percent parasitemia was estimated by
determining the number of parasites per 2,000 erythrocytes.
In peripheral blood smears, the species of Plasmodium
present were recorded. In placental smears, speciation was
more difficult and slides were only recorded as having mixed
Heparinized, microhematocrit tubes were filled with a
sample of peripheral blood, centrifuged, and the packed cell
volume (PCV) was determined. Women with a PCV < 30%
were considered to have anemia. Samples of peripheral and
placental blood were centrifuged, plasma was removed, and
the erythrocyte pellet was washed twice with phosphate-
buffered saline and frozen until used.
Detection of Plasmodium species by PCR analysis. Isola-
tion of DNA. Extraction of DNA from blood samples was
performed as described by Snounou and others.15Cryopre-
served erythrocytes were defrosted and lysed using 0.05%
saponin. Pellets of parasites obtained after centrifugation
were further lysed using 2% sodium dodecyl sulfate to release
the DNA. The DNA was purified by phenol/chloroform ex-
traction according to the protocol of Sambrook and others,16
and precipitated with sodium acetate and ethanol. In some
cases, DNA was isolated using the Puregene DNA isolation
kit (Gentra Systems Inc., Minneapolis, MN). The DNA was
dried and then dissolved in TE buffer (10 mM Tris-HCl, 1
mM EDTA, pH 7.5) such that 1 ?L of DNA solution was
equivalent to 5 ?L of packed erythrocytes.
Amplification by PCR. Parasite DNA amplification by the
PCR was carried out on all DNA samples using a genus-
specific primer pair and four species-specific primer pairs in
nested PCRs following the method of Snounou and others.9
These primer pairs recognize and allow amplification of se-
quences of genes in the small subunit ribosomal RNA of P.
falciparum, P. malariae, P. ovale, and P. vivax. Although P.
vivax is absent in Africa, P. vivax-specific oligonucleotides
were used as an internal control. The amplified DNA prod-
ucts were then analyzed electrophoretically by size fraction-
ation on agarose gels (1.5% agarose; 0.5% NuSieve? agarose;
BioWhittaker Molecular Applications, Rockland, ME) as de-
scribed by Snounou and others9After electrophoresis, the
gels were stained with ethidium bromide, visualized under
ultraviolet (UV) trans-illumination, and photographed.
Genotyping of P. falciparum species. Determination of
the number of P. falciparum genotypes in blood samples was
performed by a nested PCR amplification of DNA fragments
that correspond to the three msp-1 and two msp-2 allelic fami-
lies.17For msp-1, the region of the gene amplified by the PCR
was polymorphic Block 2. In the initial PCR, oligonucleotide
primer pair M1-OF and M1-OR was used to amplify a region
of the gene spanning both Blocks 2 and Block 4. Subse-
quently, separate nested reactions detecting three allelic fami-
lies (K1, MAD20, and RO33) were carried out using three
family-specific primer pairs flanking the Block 2 region. For
msp-2, the central polymorphic region was analyzed by PCR
amplification. Oligonucleotide primer pair M2-OF and M2-
OR was used to amplify essentially the entire gene in the first
PCR. In the second PCRs, two family-specific primer pairs
were independently used that amplify DNA fragments corre-
sponding to the Indochina (IC) and FC27 allelic families.
Separation of the secondary msp-1 PCR products was car-
ried out by electrophoresis on 3% Metaphor? agarose gels
(Cambrex Bio Science, Rockland Inc., Rockland, ME). The
PCR fragments of the secondary msp-2 products were ana-
lyzed by electrophoresis on 2% Metaphor agarose gels. Fol-
lowing electrophoresis, the gels were stained with ethidium
bromide, visualized under UV trans-illumination, and photo-
graphed. When possible, products from the paired peripheral
and placental samples were run side-by-side to allow for di-
rect size comparison. For each sample, the MoI was deter-
mined using the genetic marker with the largest number of
Statistical analysis. For unpaired between/multi-group
comparisons, the univariate chi-square test or multivariate
logistic regression model was used to evaluate binary vari-
ables, including anemia and mixed infections. Differences in
MoI were evaluated using the likelihood ratio (LR) test based
on an appropriate Poisson model. The estimates of crude and
adjusted ORs were also reported for binary variables. For
comparisons between peripheral and placental samples, the
Description of the pregnant women in the study (n ? 278)
5 (grand multigravidae)
? 6 (grand multigravidae)
Women with anemia
Women taking chemoprophylaxis
WALKER-ABBEY AND OTHERS
paired t-test was used for comparing MoI and the McNemar’s
test was used for comparing percentage of mixed infections.
Description of study subjects. Characteristics of the
women enrolled in the study and their delivery outcomes are
shown in Table 1. Among the women, approximately half
were ? 25 years of age, 44.7% were in their first or second
pregnancies, and 31.9% had anemia. Overall, 79.1% reported
taking malarial chemoprophylaxis during pregnancy.
Prevalence of malaria. Microscopy showed that 22.6% of
the women were peripheral blood smear positive for P. falci-
parum and 26.8% of the women had placental parasites
(Table 2). The PCR analysis showed that 76.1% of the women
had circulating P. falciparum parasite DNA in their periph-
eral blood and 52.9% had parasite DNA in the placental
blood. Based on these results, a total of 82.4% of the women
were determined to be infected with P. falciparum by the
PCR (i.e., parasites were detected in either the peripheral or
placental blood) and 27.5% were diagnosed by microscopy.
Therefore, 54.9% of the women had submicroscopic infec-
Using species-specific primers, PCR analysis of peripheral
blood samples demonstrated that 9.4% of the women were
infected with more than one species of malaria parasite
(Table 2). An estimated 6.9% were infected with both P.
falciparum and P. malariae, 1.8% with P. falciparum and P.
ovale, and 0.7% were infected with all three species. How-
ever, only 4.0% of the women had more than one species of
malarial parasites detected in the placenta. Mixed infections
were therefore detected more frequently in peripheral blood
samples than in placental blood samples (P ? 0.0004, by
McNemar’s test). The overall prevalence of P. malariae and
P. ovale was 7.6% and 2.5%, respectively. Neither species was
detected in the absence of P. falciparum.
Prevalence of malaria by age and gravidity. Microscopy
showed that the prevalence of P. falciparum malaria was
higher in women ? 20 years old (P ? 0.003) and pauci-
gravidae (i.e., women in their first and second pregnancies) (P
? 0.012) than in older mothers and multigravidae (Table 3).
Plasmodium falciparum parasites were also detected more
frequently in the placenta of these women by both micros-
copy and PCR. The prevalence of mixed-species infections
was also significantly higher in women ? 20 years old and
paucigavidae (P ? 0.039 and P ? 0.014, respectively), but
interestingly, the difference was not significant in the placenta
(P ? 0.077 and P ? 0.24, respectively). The prevalence of
submicroscopic infections was not statistically different
among women in the different age and gravidity groups.
Multiplicity of infection in peripheral and placental
blood. No single polymorphic genotype predominated since
each msp-1 KI and MAD20 and msp-2 IC and FC27 variant
was detected in less than 20% of the samples. A comparison
Percentage of pregnant women infected with malaria (n ? 278)*
Peripheral blood Placental bloodTotal†
MicroscopyPCR MicroscopyPCRMicroscopy PCR
P. falciparum + P. malariae
P. falciparum + P. ovale
All 3 species
Total mixed species
* PCR ? polymerase chain reaction.
† Parasites were detected in either the peripheral blood, placental blood, or both.
‡ Based on n ? 178.
§ Not determined (see Materials and Methods).
Effect of age and gravidity on prevalence of malaria*
Percentage of malaria-positive women in each group
Maternal age (years) Number of pregnancies
? 20 > 20
* PCR ? polymerase chain reaction.
† By chi-square test. Statistically significant P values are in bold.
‡ Parasites detected in the peripheral blood, placental blood, or both.
§ Percentage of women (no. of positive women/total no. in the group).
PCR-BASED ANALYSIS OF MALARIA IN PREGNANT WOMEN
of the distribution of allelic genotypes in the periphery and
placenta is shown in Figure 1. In pregnant women, an average
of 3.5 (range ? 1–9) msp-1 genotypes were detected in the
peripheral blood and 3.7 (range ? 1–8) in the placenta (Fig-
ure 1). Furthermore, 2.0 (range ? 1–6) and 1.9 (range ? 1–5)
msp-2 genotypes were found in the peripheral and placental
The MoI was significantly lower in women with submicro-
scopic infections compared with women whose infections
were detected by microscopy. The mean number of genotypes
in the peripheral blood of women with submicroscopic infec-
tions averaged 2.7 (range ? 1–9) compared with 4.1 (range ?
1–8) genotypes in slide-positive women (P < 0.001, by Poisson
regression LR test). For the placenta, 2.7 (range ? 2–7) com-
pared with 4.0 (range ? 1–8) genotypes were detected in
samples collected from women with submicroscopic and slide-
positive cases, respectively (P ? 0.047). A direct correlation
between the percent parasitemia and MoI in the peripheral
blood (Spearman r ? 0.40, P < 0.0001) and placenta (r ?
0.34, P ? 0.0001) was found. In addition, multiple genotypes
were more common in women who were slide positive (mul-
tiple genotypes were detected in 98.1% and 92.1% of the
peripheral and placental blood samples, respectively) than in
women with submicroscopic infections (72.2% and 73.2%
multiple genotypes, respectively) (P < 0.0001 and P ? 0.008
for peripheral and placental blood, by chi-square test).
The repertoire of genotypes differed between the periph-
eral and placental blood. An average of 1.6 (range ? 0–4)
msp-1 and 1.4 (range ? 0–4) msp-2 genotypes were detected
in both sites. Conversely, an average of 6.0 (range ? 0–12)
msp-1 and 5.8 (range ? 2–12) msp-2 genotypes were detected
in either the peripheral or placental blood, but not both.
When all genetic markers were considered (i.e., msp-1 K1,
MAD20, and RO33 and msp-2 FC27 and IC), none of the
women had identical genotypic patterns in their peripheral
and placental blood.
Influence of reported use of antimalarial prophylaxis. Ap-
proximately 79.1% of the women reported taking antima-
larial chemoprophylaxis during pregnancy, with 38.5% taking
chloroquine, 28.4% pyrimethamine, 2.5% amodiaquine, 1.8%
proguanil, and 7.9% other drugs. However, no effect of use of
chemoprophylaxis was found on the prevalence of micro-
scopic, submicroscopic, or mixed species infections (P ?
0.29–0.75). The mean MoI in the peripheral blood of women
who took antimalarials and those who did not was 3.3 and 3.0,
respectively (P ? 0.397, by Poisson regression LR test). Simi-
larly, the mean MoI in the placenta was 3.5 and 3.0 in women
who reported taking antimalarial compared with those who
did not (P ? 0.162, by Poisson regression LR test).
Association of MoI with age and gravidity. The MoI in the
peripheral blood and placenta did not differ significantly in
women ? 20 years old compared with those > 20 years old
(P ? 0.062 and P ? 0.092, respectively) (Table 4). Although
the number of circulating parasite genotypes in the peripheral
blood decreased from 3.6 in primigravidae to 3.1 in women
with ? 6 pregnancies, the decrease was not significant (P ?
0.774, by Poisson regression LR test). The number of msp-1
alleles decreased with increasing gravidity, but an off-setting
increase in msp-2 alleles occurred (Figure 2). Similarly, the
number of genotypes in the placenta decreased from 3.3 in
primigraviade to 3.0 in grand multiparous women, but the
trend was also not significant (Table 4 and Figure 2). Thus, no
significant decrease in MoI was seen with either age or gra-
Effect of age and gravidity on parasite diversity (MoI) and multiple genotypes*
Peripheral bloodPlacental blood
Number of pregnancies
* MoI ? multiplicity of infection; min ? minimum; max ? maximum.
† By chi-square test.
‡ Likelihood ratio test based on a univariate Poisson regression model of co-infections with Plasmodium malariae and P. ovale.
tein 1 (msp1) and msp2 alleles present in the peripheral blood and
placentas of the study group. The multiplicity of infection (MoI) is
based on the marker with the largest number of fragments.
Comparison of the number of merozoite surface pro-
WALKER-ABBEY AND OTHERS
Effect of microscopic, submicroscopic, and mixed infec-
tions and MoI on maternal anemia. After adjusting for age
and gravidity, the presence of microscopically detected
placental malaria was found to be a risk factor for maternal
anemia (adjusted OR ? ? 2.1, 95% CI ? 1.0–4.6) (Table 5).
However, no associations between presence of submi-
croscopic and mixed infections or the number of parasite
genotypes in the placenta and maternal anemia was found
This is the first study to describe the prevalence of submi-
croscopic and mixed-species infections and the number of
genetically different P. falciparum parasites in pregnant
women living in Yaoundé, Cameroon. In this urban setting,
where transmission rates are estimated to be 13 infectious
bites per year,1827.5% of the women were slide positive for
P. falciparum (Table 2) and had an average of 3.4 (range ?
1–9) different parasite genotypes in their peripheral blood
and 3.5 (range ? 1–8) in the placenta (Figure 1). Among the
women, 54.9% had submicroscopic P. falciparum infections,
with an average of 2.7 P falciparum genotypes in both the
peripheral and placental blood. Overall, 82.4% of the women
had asymptomatic P. falciparum infections (Table 2), with
7.6% being infected with P. malariae and 2.5% with P. ovale
in addition to P. falciparum (Table 2). Since accurate diag-
nosis of P. malariae and P. ovale by microscopy is difficult, the
prevalence of P. malariae and P. ovale in Yaoundé has been
considered to be low. Thus, a substantially higher number of
pregnant women are infected with these species than previ-
ously recognized. Based on these results, only 17.6% of the
women appear to be free of malarial parasites at delivery.
Women in Cameroon are encouraged to take chemopro-
phylaxis during pregnancy and 79.1% reported taking anti-
malarial prophylaxis during pregnancy (Table 1). However,
the use of antimalarials did not have an effect on the preva-
lence of slide-positive, submicroscopic infections, or the num-
ber of parasite genotypes in the peripheral blood or placenta.
Although some of the women may have misreported anti-
malarial usage or failed to take the drug consistently, the lack
of effectiveness is likely due to the high prevalence of multi-
drug-resistant of P. falciparum parasites in Cameroon.19,20
Results suggest that immunologic responses, rather than
chemoprophylaxis, are responsible for controlling the level of
parasitemias in pregnant women.
As in other African countries, young women and primi-
gravidae in Yaoundé are more susceptible to asymptomatic
infections than multigravidae.13This observation was con-
firmed in the current study because 42.0% of women ? 20
years were slide positive for placental malaria compared with
23.6% of women > 20 years (P ? 0.009), and 33.6% of
women in their first and second pregnancies had microscopi-
cally detected placental parasites compared with only 21.5%
of women with ? 3 pregnancies (P ? 0.026) (Table 3). Simi-
lar results were found using PCR-specific primers for P. fal-
ciparum (Table 3). Although a decrease in microscopic slide
positivity with age and gravidity is well established, only two
studies have evaluated the effect of age and gravidity on
prevalence of submicroscopic infections. In the current study,
no age or gravidity effect was found on the prevalence of
submicroscopic infections in Cameroonian women (Table 3).
Similarly, no decrease in submicroscopic infections was found
in pregnant women in Mozambique,11and the prevalence of
submicroscopic infections was reported to actually increase
with gravidity in Ghanaian women.7Thus, the prevalence of
peripheral and placental blood of women residing in Yaoundé, Cameroon. Shaded bars ? msp-1; dark bars ? msp-2.
Changes in the multiplicity of infection (MoI) with increasing gravidity for merozoite surface protein 1 (msp-1) and msp-2 in the
Relationship between placental malaria and maternal anemia
%*Adjusted odds ratio†
Number of placental genotypes
* Percentage of women who were anemic (no. of women who were anemic/total number
of women in the group).
† Adjusted for age and gravidity in a multivariate logistic regression model. Values in
parentheses are 95% confidence intervals.
PCR-BASED ANALYSIS OF MALARIA IN PREGNANT WOMEN
submicroscopic infections does not appear to decrease in par-
allel with that observed at the microscopic level.
Several studies have looked at the effect of gravidity on
MoI. Studies by Schleiermacher and others in Senegal,21
Kassberger and others in Gabon,22and Saute and others in
Mozambique11failed to find a decrease in MoI with increas-
ing gravidity/parity. In the current study in Cameroon, a sig-
nificant decreased in MoI with gravidity was also not seen
(Table 4). Beck and others reported a significant decrease in
MoI from 3.5 in Ghanaian primigravidae to 2.2 in women with
? 5 pregnancies, but the decrease was not observed until after
four pregnancies.10Furthermore, the majority of women in
the above studies were infected with more than one para-
site genotype, e.g., 98% of peripheral blood samples from
women in Dakar had multi-genotypes,2184% in Gabon,22
83.5% in the current study in Cameroon, and 69% in
Malawi.23Thus, results from molecular epidemiologic studies
come to similar biologic conclusions, namely, a substantial
number of pregnant women with asymptomatic infections
have submicroscopic malaria and are infected with multiple
parasite genotypes even after five pregnancies. Thus, the
acquisition of pregnancy-associated immunity does not result
in the elimination of the infection, nor does it decrease the
range of genetically distinct parasites with which a woman is
Four previous studies have compared the composition of P.
falciparum genotypes in peripheral and placenta blood, and
they found the genetic composition of parasites in the two
compartments to be quite different.12,21–23In the current
study, using msp-1 and msp-2, none of the women had iden-
tical parasite genotypes in their peripheral and placental
blood. The reason for this is unclear since maternal arterial
blood constantly flows into the intervillous space of the pla-
centa, the site from which the placental blood was collected.
Fried and Duffy reported that P. falciparum-infected eryth-
rocytes in the placenta do not bind to CD36, whereas para-
sites in the peripheral blood may bind to CD36, chondroitin
sulfate A, or both, thereby suggesting that the population of
parasites in the two compartments are different.4It is cur-
rently unclear if the differences in composition of parasite
genotypes are due to sequestration or to the natural cytoad-
herence of different parasite genotypes in the deep vascula-
ture during the 48-hour erythrocytic cycle.
The presence of placental malaria diagnosed by microscopy
was a significant risk factor for anemia (adjusted OR ? 2.1,
95% CI ? 1.0–4.6), but the presence of submicroscopic pla-
cental infections, mixed-species infections, or the number of
parasite genotypes did not significantly increase the risk of
anemia (Table 5). Since two previous studies reported no
association between anemia and submicroscopic infec-
tions,7,11this result was not surprising. The effect of mixed-
species infections on anemia in African women has not been
reported; thus, comparisons with previous studies cannot be
made. The presence of high peripheral and placental MoI was
also not a significant risk factor for anemia in the current
study (Table 5). Beck and others reported that Ghanian
women who had ? 3 pregnancies and harbored ? 4 parasite
genotypes had a 2.3 times greater chance of anemia.10Al-
though not statistically significant, Cameroonian women with
2–5 placental genotypes also tended to have an elevated
prevalence of anemia (adjusted OR ? 1.8, 95% CI ? 0.9–3.7)
(Table 5). However, since women with slide-positive cases of
malaria have a higher MoI than women with submicroscopic
infections (peripheral blood P < 0.001, placental blood P ?
0.047), the association between a high MoI and anemia may
be related to the level of parasitemia rather than the number
of genetically-different parasites per se.
In summary, in Yaoundé 82.4% of pregnant women had P.
falciparum malaria and 9.4% were infected with more than
one plasmodial species at delivery. Although, 3.4 different
parasite genotypes were detected in their peripheral blood
and 3.5 genotypes in the placenta, they had asymptomatic
infections. With increasing age and gravidity, the prevalence
of slide and PCR positivity for P. falciparum as well as co-
infections with P. malariae and P. ovale decreased, but the
prevalence of submicroscopic P. falciparum infections and
number of genetically different genotypes did not. Thus, the
acquisition of pregnancy-associated immunity does not result
in either the elimination of parasites or a decrease in the
number of infecting strains, but appears to aid in reducing
parasites to submicroscopic levels.
Received April 17, 2004. Accepted for publication August 11, 2004.
Acknowledgments: We express our gratitude to the Cameroonian
women who participated in this study. We are also indebted to all
members of the Malaria Research Team at the Biotechnology Center
in Yaoundé for conducting the field and microscopic studies reported
Financial support: This project was funded by the National Institute
of Allergy and Infectious Diseases, National Institutes of Health
through the International Collaborations in Infectious Disease Re-
search program UO1 AI-135839 and the Human Immune Resistance
to Malaria in Endemic Areas Program UO1 AI-43888.
Authors’ addresses: Annie Walker-Abbey, Lucy H. Thuita, Eliza
Beardslee, and Diane Wallace Taylor, Department of Biology, Room
406, Reiss Science Center, Georgetown University, 37th and O
Streets, NW, Washington, DC, 20057, Telephone: 202-687-5972,
E-mail: email@example.com. Rosine R. T. Djokam, Anna Eno,
Rose. F. G. Leke, Vincent P. K. Titanji, Josephine Fogako, and Grace
Sama, Faculty of Medicine and Biomedical Sciences, Biotechnology
Center, University of Yaoundé 1, Yaoundé, Cameroon, Telephone:
237-223-7479, E-mail: firstname.lastname@example.org. Georges Snounou,
Unité de Parasitologie Biomédicale, Centre National de la Recherche
Scientifique, Unité de Recherche Associée 2581, Institut Pasteur, 25
Rue du Dr. Roux, 75724 Paris Cedex 15, France, Telephone: 33-1-
46-01-37-35, Fax: 33-1-45-68-860, E-mail: email@example.com. Ain-
ong Zhou, AZ DataClinic, Inc., Rockville, MD, 20850, Telephone:
240-476-2148. E-mail: firstname.lastname@example.org.
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PCR-BASED ANALYSIS OF MALARIA IN PREGNANT WOMEN