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Medical Research Unit, Albert Schweitzer Hospital, Lambaréné, Gabon
Institute for Tropical Medicine, Department of Parasitology, University of Tübingen, Tübingen, Germany
Department of Internal Medicine I, Division of Infectious Diseases, Medical University of Vienna, Vienna, Austria
Background.
Women with semi-immunity to malaria who live in regions where the disease is endemic are at increased risk for more frequent and severe episodes of malaria during pregnancy. Recent findings indicate that this increased risk might persist beyond delivery, but the underlying mechanisms for this change in risk are poorly understood.
Methods.
One hundred fifty women were included in a cohort study in Lambaréné, Gabon, and were actively followed up weekly for 10 weeks after delivery, as were nonpregnant control women who had been matched to them by location and age. Parasites in samples of placenta and blood were genotyped by use of polymerase chain reaction amplification of the merozoite surface antigen 2 gene and the subtelomeric variable open reading frame gene of Plasmodium falciparum.
Results.
Eleven puerperal women had cases of clinical malaria, compared with 1 control woman (rate ratio, 9.8; P = .006). Eighteen puerperal women had P. falciparum parasitemia, compared with 6 control women (rate ratio, 2.7; P = .03). Five of 16 puerperal women (31%) with parasitemia on follow-up had identical parasites in their placentas and blood, and 11 of these cases (69%) were the result of reinfection. Puerperal women remained at equal risk for the development of parasitemia throughout the first 10 weeks after delivery. Use of bed nets, use of chloroquine prophylaxis during pregnancy, presence of malaria episodes during pregnancy, gravidity, and age were not associated with the acquisition of parasitemia during follow-up.
Conclusions.
Compared with nonpregnant women, puerperal women have a considerably increased risk for the development of malaria and/or parasitemia. This increased risk is caused both by the recurrence of P. falciparum parasitemia and by the increased susceptibility to new infections, although the latter plays a more significant role.
Malaria remains one of the leading causes of morbidity and mortality in tropical regions. In areas of high endemicity, semi-immunity to Plasmodium falciparum is usually acquired early in childhood and, to a certain degree, protects adults from life-threatening consequences of this disease. However, during pregnancy, women with semi-immunity are at increased risk for more frequent and severe episodes of malaria [1, 2]. This phenomenon, most pronounced in primigravid women, is attributed, at least in part, to the adherence by a subtype of P. falciparum parasites to chondroitin sulfate A and hyaluronic acid in the placenta [3, 4].
Interestingly, women seem to be at an even higher risk for the development of malaria during the first 60 days after delivery than during pregnancy [5]. It is not known if parasitemia in puerperal women is caused by reinfection or by recurrence of infection by parasites that had been sequestered in the placenta during pregnancy. To date, the only study on malaria in puerperal women was performed in a rural area of Senegal, and it is not known to what extent the risk for the development of malaria would be altered in puerperal women living in regions that have other transmission characteristics.
The aim of this cohort study was, therefore, to determine the incidence of malaria in puerperal women living in a region in central Africa that has perennial transmission of the disease. Furthermore, we aimed to determine the origin of the parasites by genotyping and by comparing parasites in the placenta with those in the blood.
SUBJECTS, MATERIALS, AND METHODS
The present study was conducted in Lambaréné, a semiurban town surrounded by dense tropical rainforest and located in the central African country of Gabon. In this area, transmission of malaria is perennial and hyperendemic, with an entomological inoculation rate of 50 infectious mosquito bites per year, and P. falciparum isolates exhibit a high degree of resistance against chloroquine [6, 7, 8]. The major mother-child health care institutions in the region are located in 2 hospitals in Lambaréné.
During March 2003January 2004, women coming to either hospital to give birth were invited to participate in the study. Inclusion criteria were, on delivery, a thick blood smear result negative for P. falciparum parasites and permanent residence in Lambaréné or its vicinity. On admission to the hospital, demographic data (age, ethnicity, and parity) were collected. These women were questioned about their use of bed nets, use of chloroquine prophylaxis during pregnancy, and episodes of malaria during pregnancy. Information on episodes of malaria during pregnancy was taken from the mother-child health booklet kept by each puerperal woman. Only a thick blood smear result positive for P. falciparum parasites, documented in the health booklet, was regarded as an episode of malaria during pregnancy. A sample of the placenta was collected from each woman at delivery and was frozen at -80°C.
A week after recruitment of each puerperal woman into the study, a nonpregnant woman of the same age (±5 years) who had not given birth during the previous 6 months was invited to participate in the study as a control subject. Inclusion criteria for each member of the control group were a thick blood smear result negative for P. falciparum parasites and permanent residence within a 300-m radius of the matched puerperal woman's house. Information about the use of chloroquine prophylaxis and episodes of malaria was not available for control women. The study was approved by the ethics committees of the International Foundation for the Albert Schweitzer Hospital in Lambaréné. Written informed consent was obtained from all women who participated in the study.
The primary endpoints of the study were numbers of episodes of malaria and episodes of P. falciparum parasitemia per person-year of follow-up. An episode of malaria was defined as asexual P. falciparum parasitemia with >100 parasites/l of blood, fever (tympanic temperature >37.5°C), history of fever within the previous 24 h, or presence of other symptoms associated with malaria (e.g., headache, chills, or vomiting). Puerperal women were monitored weekly for 10 weeks, starting at delivery, and were encouraged to visit the health care center in case of illness. Control women were monitored weekly for 10 weeks and were given pregnancy tests at the time of recruitment into the study and at the final follow-up visit. An assessment of health status, tympanic temperature, and a blood sample collected on filter paper (Schleicher & Schuell) were obtained from each woman at each visit. Thick blood smear analyses were performed at these visits, as described elsewhere [9]. Asymptomatic subjects with parasitemia were closely monitored until resolution of parasitemia or commencement of clinical signs of malaria. In the case of the development of malaria caused by P. falciparum, women were treated with a 7-day oral regimen of 600 mg of quinine sulfate taken 3 times per day. In the case of the development of malaria caused by P. malariae or P. ovale, women were treated with chloroquine.
DNA was extracted from parasites in placentas and blood samples by use of a commercially available kit (QIAamp Blood Minikit; Qiagen), according to the manufacturer's instructions. The multicopy subtelomeric variable open reading frame (stevor) gene of P. falciparum was amplified, by polymerase chain reaction (PCR), from parasites in the placentas of women presenting with a positive thick blood smear result during follow-up [10]. In addition, parasites from a random selection of placentas from 18 (of 130) women who did not present with a positive thick blood smear result during follow-up were genotyped. To compare placental parasites with parasites that appeared in the blood during follow-up, merozoite surface antigen 2 (msa-2) gene-length polymorphisms were amplified by use of a nested PCR protocol, as described elsewhere, and were analyzed [11]. If the msa-2 gene could not be amplified despite detection of the stevor gene, the sample was treated with GenomiPhi-polymerase (Amersham Biosciences), to increase the amount of available DNA. To detect msa-2, primers specific for the 3D7 and FC27 strains of P. falciparum were used for the nested PCR. After separation by agarose-gel electrophoresis, populations of parasites in the blood and in the placenta of the same woman were compared according to the size and number of DNA fragments.
If a woman missed a follow-up visit, any period of elapsed time >14 days between her visits was excluded from the person-time calculation. Women with asymptomatic parasitemia were monitored until symptoms occurred. Two women had self-limiting episodes of asymptomatic P. falciparum parasitemia and also had recurrences of parasitemia that caused symptoms either 34 or 38 days after the first episode. The genotypes of the parasites involved in the 2 episodes in each of these women could not be compared, however, because the msa-2 gene could not be amplified from the parasites taken during 1 episode in each woman. For the present analysis, only the symptomatic episode was counted, because a recurrence seemed somewhat more likely than a reinfection. However, no significant differences in interpretation resulted when these episodes were evaluated as 2 distinct episodes of parasitemia in each woman.
Fisher's exact test and McNemar's test were used for 2 × 2 tables. Differences between groups were analyzed by use of the t test and the Wilcoxon rank sum test. Ninety-five percent confidence intervals (CIs) were calculated for proportions and rate ratios. All tests were 2-tailed, and a significance level of P = .05 was used.
RESULTS
One hundred fifty women in the puerperium were recruited for this prospective study. All were matched by age and location to nonpregnant control subjects, except for 1 for whom a matching partner was not found. Puerperal women had a higher median number of children than did control women (2 children vs. 1 child, respectively; P < .001). The use of bed nets in the 2 groups was comparable (67% for puerperal women vs. 68% for control women; P = .9). Similarly, no significant differences in educational level and ethnicity were found (data not shown). Of puerperal women, 74% had multiple pregnancies, 19% had an episode of malaria during pregnancy, and 77% used chloroquine prophylaxis, although none continued prophylaxis after delivery. Follow-up was terminated prematurely for 2 puerperal women and for 3 control women because they withdrew consent. Another 10 control women had positive pregnancy test results during follow-up and were excluded from the analysis. The incidence of P. falciparum parasitemia and episodes of malaria was calculated for the 284 women remaining in the study group.
Eleven cases (7%) of malaria were observed in puerperal women, versus 1 case (1%) in control women (P = .006; table 1). Puerperal women had 0.4 more episodes of malaria per person-year (95% CI, 0.120.67) than did control women and also had a 10-fold increased risk for the development of malaria (table 1) compared with control women. The incidence of all P. falciparum infections (including both malaria and asymptomatic parasitemia) was 0.46 more episodes per person-year (95% CI, 0.060.87) for puerperal women than for control women, with a 2.7-fold increased risk for the development of malaria in puerperal women (table 1). Of women with positive thick blood smear results, puerperal women had an increased risk for the development of malaria, compared with that in control women, that approached statistical significance (relative risk, 3.7 [95% CI, 0.622.8]; P = .059).
No obvious pattern was observed for the recurrence of P. falciparum parasitemia (figure 1). Three cases of P. malariae parasitemia (2 in control women and 1 in puerperal women) and 2 cases of P. ovale parasitemia (both in puerperal women) were detected during follow-up.
The effects of additional risk factors for parasitemia are shown in table 2. None of these variables, including use of chloroquine prophylaxis, was significantly associated with the development of P. falciparum parasitemia during follow-up. Use of bed nets was associated with an increased risk for the development of P. falciparum parasitemia, although this association was not statistically significant.
Genotyping of P. falciparum parasites.
Parasites taken from placentas from 16 of 18 puerperal women could be genotyped; no placenta sample was available in 2 cases. In 8 of these 16 cases (50%), the placenta was positive for P. falciparum parasites, and in 8 of these 16 cases (50%) the placenta was negative for P. falciparum parasites. Eleven of 18 women (61%) whose thick blood smear results were negative for P. falciparum parasites during follow-up had placental parasites. Matched-pair analysis showed no relationship between the presence of placental parasites and the development of P. falciparum parasitemia during the puerperium (P = .64).
Parasites from the 8 P. falciparumpositive placenta-blood pairs were genotyped and compared. Five pairs had identical parasites, and 3 pairs had parasites of different genotypes, representing new infections that occurred during the puerperium. In total, therefore, 5 of 16 puerperal women (31% [95% CI, 11%59%]) had a recurrence of infection with placental parasites, and 11 of 16 puerperal women (69% [95% CI, 41%89%]) had an infection with parasites of a different genotype.
DISCUSSION
Our findings indicate that puerperal women have a 10-fold increased risk for the development of malaria, compared with control women, confirming results from a study conducted by Diagne et al. in Senegal [5]. In Gabon, the risk was greater than that in Senegal (10-fold vs. 4-fold, respectively), although the present study had wide 95% CIs. This difference in risk may be the result of different definitions of malaria or of the higher endemicity of P. falciparum in Gabon than in Senegal. Also, Diagne et al. used self-controls to compare the incidences of P. falciparum parasitemia before pregnancy, during pregnancy, and after delivery for every single woman, rather than recruiting a control group. In the present study, the observed increase in the number of cases of malaria was similarly reflected in a higher incidence of P. falciparum parasitemia, which included asymptomatic infections. Puerperal women experienced more cases of parasitemia and had malaria disproportionately more often than did control women. They seemed to remain equally at risk for the development of malaria throughout the first 10 weeks after delivery.
Although parity is generally considered to be a major risk factor for malaria during pregnancy [1], we found no association between parity and the risk of malaria in the postpartum period in our study population. Interestingly, use of bed nets was associated with the incidence of P. falciparum parasitemia. That insecticide-treated bed nets are virtually nonexistent in the study area and that the use of bed nets might be associated with a high prevalence of mosquitoes in a given microenviroment might explain this association.
Genotyping suggested that the main mechanism responsible for the majority of puerperal malaria cases was new infections being acquired during the puerperium rather than parasites that were trapped in the placenta resurfacing in the blood, although obviously both mechanisms play a role. However, reactivation of P. falciparum strains infecting the placenta at levels below the limit of detection by PCR cannot be ruled out. These findings also explain why women who have parasites in their placentas are not at an increased risk for the development of malaria, compared with women who do not have parasites in their placentas.
Several mechanisms may be responsible for the increased risk for the development of malaria in puerperal women, including parasite factors, host factors, and entomological factors. During the puerperium, IgG antibodies against pregnancy-associated malaria antigens increase at the expense of antibodies to variant surface antigens that are common in parasite strains infecting other populations [12, 13, 14]. After the removal of the placenta at delivery, puerperal women might therefore become more vulnerable to parasitemia caused by parasites undergoing an antigenic shift in their binding preference from chondroitin sulfate A to common variant surface antigens. This molecular mechanism might explain the recurrence of placental parasites in the blood after delivery. It is also known that pregnant women have a different immunological response to antigenic stimulation than nonpregnant women do, possibly because of changes in hormone levels and adaptations of the immune system to accommodate the fetal allograft [14, 15]. As was shown recently, the percentage of CD4+ CD25+ regulatory T cells that modulate immune responses increases during the second and third trimesters of pregnancy and gradually returns to normal levels during the puerperium, reflecting the pattern of increased incidence of malaria during and after pregnancy [16]. Because specific T-1type immune responses, which are pivotal for successful immunity to malaria, are altered during pregnancy, the ability to control new infections and asymptomatic parasitemia might diminish, thus putting women at increased risk for the development of malaria [17]. Finally, puerperal women might also exhibit an increased attractivity to Anopheles species similar to that shown during pregnancy, and general behavioral changes may play an additional role [18]. Most likely, all these factors and other undefined mechanisms contribute to the overall finding of an increased incidence of malaria during the puerperium.
Acknowledgments
We are grateful for the fruitful discussions with the participating women and for their sustained enthusiasm for this study. The cooperation of the staff of the mother-child health care institutions at the Albert Schweitzer Hospital and the General Hospital of Lambaréné is thankfully acknowledged.
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