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    Comparison of the number of merozoite surface protein 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.

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    Changes in the multiplicity of infection (MoI) with increasing gravidity for merozoite surface protein 1 (msp-1) and msp-2 in the peripheral and placental blood of women residing in Yaoundé, Cameroon. Shaded bars = msp-1; dark bars = msp-2.

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MALARIA IN PREGNANT CAMEROONIAN WOMEN: THE EFFECT OF AGE AND GRAVIDITY ON SUBMICROSCOPIC AND MIXED-SPECIES INFECTIONS AND MULTIPLE PARASITE GENOTYPES

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  • 1 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

Polymerase chain reaction (PCR)–based methods were used to investigate malaria in pregnant women 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 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 ≤ 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.

INTRODUCTION

During pregnancy, Plasmodium falciparum infections can have an adverse effect on both the mother and the developing fetus.1–3 In malaria-endemic areas, the prevalence of clinical and asymptomatic malaria is highest in young women and those in their first and second pregnancies.1,3 With successive pregnancies, women acquire a gravidity-dependent form of immunity, resulting in a decrease in both prevalence and severity of infection.1–3 Antibodies that block the binding of parasites to chondrointin sulfate A in the placenta4,5 and malaria-specific memory lymphocytes with homing receptors for the placenta6 are thought to help mediate pregnancy-associated immunity.

In the last decade, the development of molecular approaches 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 understanding 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.7,8 For example, in a cross-sectional study, 32% of pregnant Ghanaian women were peripheral blood smear positive, but 63% were positive by PCR.7 Likewise, 29% of samples from pregnant Senegalese women were slide positive, but 85% were positive by PCR for the P. falciparum merozoite surface protein 1 (msp-1) or msp-2 genes.8 Thus, a large number of pregnant 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,9 the prevalence of mixed-species infections in pregnant women has not been reported.

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,10 but not in another.11 High MoI as well as submicroscopic infections have also been associated with mild maternal anemia,7,10,12 but this is not a universal finding.11 Thus, additional studies using PCR-based approaches are needed to increase our understanding of the extent to which gravidity-dependent immunity controls/eliminates parasites and influences maternal anemia.

The current study was conducted in the city of Yaoundé, Cameroon where malaria transmission is perennial. A previous 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.13 However, 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 explore 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 genotypes 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, Cameroon, 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. Consenting women provided information relative to their pregnancies, 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 maternal peripheral blood was collected. In addition, a sample of maternal placental blood was obtained using the pool-biopsy method.14 Briefly, 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 containing 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 infections.

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.

Isolation of DNA.

Extraction of DNA from blood samples was performed as described by Snounou and others.15 Cryopre-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 extraction 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 μL of DNA solution was equivalent to 5 μL 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 sequences 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 products were then analyzed electrophoretically by size fractionation on agarose gels (1.5% agarose; 0.5% NuSieve® agarose; BioWhittaker Molecular Applications, Rockland, ME) as described by Snounou and others9 After 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 families.17 For 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. Subsequently, separate nested reactions detecting three allelic families (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 corresponding to the Indochina (IC) and FC27 allelic families.

Separation of the secondary msp-1 PCR products was carried 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 analyzed by electrophoresis on 2% Metaphor agarose gels. Following electrophoresis, the gels were stained with ethidium bromide, visualized under UV trans-illumination, and photographed. When possible, products from the paired peripheral and placental samples were run side-by-side to allow for direct size comparison. For each sample, the MoI was determined using the genetic marker with the largest number of fragments.

Statistical analysis.

For unpaired between/multi-group comparisons, the univariate chi-square test or multivariate logistic regression model was used to evaluate binary variables, 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 paired t-test was used for comparing MoI and the McNemar’s test was used for comparing percentage of mixed infections.

RESULTS

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. falciparum 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 peripheral 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 infections.

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. However, 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 paucigravidae (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 microscopy 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 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 (Figure 1). Furthermore, 2.0 (range = 1–6) and 1.9 (range = 1–5) msp-2 genotypes were found in the peripheral and placental blood, respectively.

The MoI was significantly lower in women with submicroscopic infections compared with women whose infections were detected by microscopy. The mean number of genotypes in the peripheral blood of women with submicroscopic infections 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) compared 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 (multiple 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 peripheral 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.

Approximately 79.1% of the women reported taking antimalarial 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 microscopic, 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). Similarly, 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 gravidity.

Effect of microscopic, submicroscopic, and mixed infections 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 submicroscopic and mixed infections or the number of parasite genotypes in the placenta and maternal anemia was found (Table 5).

DISCUSSION

This is the first study to describe the prevalence of submicroscopic 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,18 27.5% 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 diagnosis 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 previously 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 chemoprophylaxis 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 prevalence of slide-positive, submicroscopic infections, or the number 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 primigravidae in Yaoundé are more susceptible to asymptomatic infections than multigravidae.13 This observation was confirmed 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 microscopically detected placental parasites compared with only 21.5% of women with ≥ 3 pregnancies (P = 0.026) (Table 3). Similar results were found using PCR-specific primers for P. falciparum (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,11 and the prevalence of submicroscopic infections was reported to actually increase with gravidity in Ghanaian women.7 Thus, the prevalence of submicroscopic infections does not appear to decrease in parallel 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,22 and Saute and others in Mozambique11 failed to find a decrease in MoI with increasing gravidity/parity. In the current study in Cameroon, a significant 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.10 Furthermore, the majority of women in the above studies were infected with more than one parasite genotype, e.g., 98% of peripheral blood samples from women in Dakar had multi-genotypes,21 84% in Gabon,22 83.5% in the current study in Cameroon, and 69% in Malawi.23 Thus, 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 infected.

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.12,21–23 In the current study, using msp-1 and msp-2, none of the women had identical 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 placenta, the site from which the placental blood was collected. Fried and Duffy reported that P. falciparum-infected erythrocytes in the placenta do not bind to CD36, whereas parasites 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.4 It is currently unclear if the differences in composition of parasite genotypes are due to sequestration or to the natural cytoadherence of different parasite genotypes in the deep vasculature 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 placental 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 infections,7,11 this 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.10 Although 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.

Table 1

Description of the pregnant women in the study (n = 278)

%
Age, years
    ≤ 2018.9
    21–2532.4
    26–3027.3
    > 3021.4
Gravidity
    1 (primigravidae)22.7
    2 (secundigravidae)22.0
    3 (multigravidae)16.3
    4 (multigravidae)14.4
    5 (grand multigravidae)8.7
    ≥ 6 (grand multigravidae)15.9
Women with anemia31.9
Women taking chemoprophylaxis79.1
Table 2

Percentage of pregnant women infected with malaria (n = 278)*

Peripheral bloodPlacental bloodTotal†
Plasmodial speciesMicroscopyPCRMicroscopyPCRMicroscopyPCR
* 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).
P. falciparum22.676.126.852.927.582.4
P. falciparum + P. malariae1.1‡6.9–§2.56.9
P. falciparum + P. ovale01.81.41.8
All 3 species00.700.7
Total mixed species9.44.09.4
Table 3

Effect of age and gravidity on prevalence of malaria*

Percentage of malaria-positive women in each group
Maternal age (years)Number of pregnancies
≤ 20> 20P1–2≥ 3P
* 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).
Malaria positive‡
    Plasmodium falciparum§
        Microscopy45.1§ (23/51)23.9 (53/222)0.00335.3 (43/122)21.6 (33/153)0.012
        PCR88.5 (46/52)81.2 (181/223)0.21387.9 (109/124)78.4 (120/153)0.039
        Submicroscopic44.2 (23/52)57.0 (127/223)0.09852.4 (65/124)56.9 (87/153)0.460
    Mixed species28.0 (7/25)12.2 (18/147)0.03923.2 (16/69)9.5 (10/105)0.014
Placental malaria
    P. falciparum
        Microscopy42.0 (21/50)23.6 (51/216)0.00933.6 (40/119)21.5 (32/149)0.026
        PCR73.1 (38/52)48.2 (107/222)0.00266.7 (82/123)42.5 (65/153)< 0.0001
        Submicroscopic30.8 (16/52)25.1 (56/223)0.40432.3 (40/124)22.2 (34/153)0.061
        Mixed species13.2 (5/38)4.7 (5/107)0.0779.8 (8/82)4.6 (3/65)0.240
Table 4

Effect of age and gravidity on parasite diversity (MoI) and multiple genotypes*

Peripheral bloodPlacental blood
No.MoI (min–max)PMultiple genotypes (%)PNo.MoI (min–max)PMultiple genotypes (%)P
* 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.
Age, years
    ≤ 20313.5 (1–8)0.36287.10.524333.2 (1–7)0.44075.80.092
    > 201073.2 (1–9)82.2923.5 (1–8)88.0
Number of pregnancies
    1303.6 (1–9)0.77486.70.657343.3 (1–8)0.24070.60.066
    2413.1 (1–8)78.0383.5 (1–7)84.2
    3163.1 (1–8)93.8154.3 (2–7)100
    4163.5 (1–9)81.3133.2 (1–5)84.6
    ≥ 5363.1 (1–6)83.8263.0 (1–7)92.3
Table 5

Relationship between placental malaria and maternal anemia

Maternal anemia
%*Adjusted odds ratio†
* 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.
Placental malaria
    No infection26.6 (25/94)Referent
    Microscopic41.3 (19/46)2.1 (1.0–4.6)
    Submicroscopic34.2 (13/38)1.7 (0.7–4.0)
    Mixed species25.0 (1/4)1.0 (0.1–10.0)
Number of placental genotypes
    029.3 (29/99)Referent
    122.2 (4/18)0.9 (0.3–3.4)
    2–438.9 (21/54)1.8 (0.9–3.7)
    > 527.3 (3/11)1.1 (0.3–4.9)
Figure 1.
Figure 1.

Comparison of the number of merozoite surface protein 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.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 3; 10.4269/ajtmh.2005.72.229

Figure 2.
Figure 2.

Changes in the multiplicity of infection (MoI) with increasing gravidity for merozoite surface protein 1 (msp-1) and msp-2 in the peripheral and placental blood of women residing in Yaoundé, Cameroon. Shaded bars = msp-1; dark bars = msp-2.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 3; 10.4269/ajtmh.2005.72.229

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: taylordw@georgetown.edu. 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: rl23@georgetown.edu. 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: snounou@pasteur.fr. Ainong Zhou, AZ DataClinic, Inc., Rockville, MD, 20850, Telephone: 240-476-2148. E-mail: zhoua1@georgetown.edu.

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 herein.

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 Research program UO1 AI-135839 and the Human Immune Resistance to Malaria in Endemic Areas Program UO1 AI-43888.

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