Plasmodium falciparum malaria and HIV infections are common in southern Africa, where up to 66% of pregnant women have malaria infection  and 15-45% are HIV infected [2–7]. The vast majority of HIV-infected pregnant women do not have access to anti-retroviral treatment and 20–45% of them transmit the virus to their infants [8–11].
Maternal HIV viral load is the single most important risk factor for HIV mother-to-child transmission (MTCT). Various studies estimate that a 10-fold increase in maternal HIV viral load is associated with up to a 2.5-fold increase in HIV MTCT [9,10,12–14]. Malaria infection may increase HIV replication and viral load by activating the immune system by a mechanism similar to that found with vaccination and bacterial infections [15–19]. Previous studies found that non-pregnant HIV-infected adults with symptomatic malaria had sevenfold higher HIV-1 plasma concentrations than those uninfected with malaria , and pregnant women with peripheral malaria parasitemia had twofold higher HIV-1 plasma concentrations than those without malaria infection . The latter study did not examine the relationship between placental malaria and HIV-1 viral load, which is important since some pregnant women have no detectable peripheral parasitemia despite the presence of sequestered parasites in the placenta [22,23].
Placental malaria is associated with placental monocytic infiltration [24–26] and increased expression of CCR5 on placental macrophages . In HIV-infected pregnant women, this could increase the placental HIV reservoir. In vitro, malaria antigens stimulate HIV replication through the increased expression of tumor necrosis factor α (TNF-α)  and a similar process could occur in the placenta, where malaria causes a placental pro-inflammatory cytokine response [29–31]. These effects could increase placental and systemic HIV-1 viral load and subsequently increase HIV-1 MTCT.
The present study is a cross-sectional analysis of HIV-infected pregnant women at delivery to determine whether placental P. falciparum malaria infection is associated with increased peripheral and/or placental HIV-1 viral concentrations. The study group comprised a subset of women who participated in a prospective cohort study investigating the effect of malaria on HIV-1 MTCT.
The study was conducted between December 2000 and June 2002 at Queen Elizabeth Central Hospital (QECH) in Blantyre, Malawi. Blantyre district has an estimated population of 950 000, 40% of whom live in the urban and periurban areas. Malaria transmission is perennial but peaks during the rainy and post-rainy season (December to April), and P. falciparum causes over 98% of malaria infections. QECH is the main tertiary referral hospital in southern Malawi but also serves as a district hospital for Blantyre.
Pregnant women admitted in the antenatal ward were screened for eligibility to participate in the main cohort study after undergoing routine medical and obstetric assessment. Women were ineligible if they were in the active phase of labor, were participating in other research studies, lived outside Blantyre district, were less than 15 years of age or had hypertension, multiple gestations or altered consciousness. Informed consent was sought from eligible women; as part of the consenting process, women were told that participation in the study required that they be tested for HIV and be informed of the results, as only HIV-infected women would be enrolled in the cohort study. Consenting women received HIV pre-test counseling, and a blood sample was collected by venipuncture. A portion of the sample was used to screen for HIV and malaria and to measure hemoglobin concentrations. Subsequently, women were notified of their HIV and malaria status and received HIV post-test counseling. Malaria-infected women were treated with 1575 mg sulfadoxine–pyrimethamine (SP) or oral quinine sulfate 600 mg twice daily for 5 days.
HIV-infected women were enrolled in the cohort study. Blood collected at enrollment was used for T cell subset counts and peripheral HIV-1 RNA assays. Before the onset of active labor, demographic and medical information was obtained using a standardized questionnaire. Upon the onset of active labor, women received 200 mg oral nevirapine (Roxane Laboratories, Columbus, Ohio, USA). After delivery, an incision was made in the middle of the maternal surface of the placenta, 2 cm long and through half the thickness of the placental tissue. Placental blood was collected for placental HIV-1 RNA assays and thick placental blood films were prepared for malaria microscopy. A full-thickness biopsy was taken from the pericentric area of the placenta and fixed in 10% neutral buffered formalin for placental malaria histopathology.
The study was approved by the Malawi College of Medicine Research and Ethics Committee and by institutional review boards at the University of Michigan and the University of North Carolina at Chapel Hill.
HIV screening and measurement
HIV status was determined simultaneously with the Determine HIV-1/2 Rapid Test (Abbott Laboratories, Illinois, USA) and the SeroCard HIV-1/2 Rapid Test (Trinity Biotech, Co Wicklow, Eire), according to the manufacturers’ instructions. If these tests produced discordant results, the HIVSPOT HIV-1/2 Rapid Test (Genelabs Diagnostics, Singapore) was used to resolve the discrepancy.
HIV-1 RNA was assayed in peripheral and placental blood samples. The samples were centrifuged and the plasma fractions were stored at −80°C. HIV-1 RNA levels were measured using Amplicor HIV-1 Monitor version 1.5 (Roche Diagnostics, Branchburg, New Jersey, USA), which reliably quantifies HIV-1 subtypes A, C and D , the predominant subtypes in eastern and southern Africa. The assay has a threshold sensitivity of 400 copies/ml with a linear range of quantification of 400–750 000 copies/ml. Therefore, plasma HIV-1 RNA concentrations < 400 and > 750 000 copies/ml were assigned values of 400 and 750 000 copies/ml, respectively.
Peripheral and placental thick blood films were air dried and field stained. Two trained microscopists examined the films for asexual forms of P. falciparum parasites and classified a blood film negative if no parasites were detected after counting 200 leukocytes. Parasite densities were determined as described in . Fixed placental biopsies were embedded in wax and sectioned. Histological slides were made and stained with Gurr's modified Giemsa and/or haematoxylin and eosin. The slides were examined for the presence of malaria parasites, pigment and leukocytes, as described by Rogerson et al. .
Definition of malaria infection
Pregnant women may have placental malaria without detectable peripheral malaria parasites [22,23]. Therefore, in the main analysis, malaria infection was defined as the presence of P. falciparum parasites on placental histopathology, while samples with only malaria pigment or without parasites were classified as uninfected. The effects of peripheral and placental blood film parasitemia on HIV-1 viral load were also examined.
Hemoglobin concentrations were measured using a Hemocue hemoglobinometer (HemoCue AB, Ängelholm Sweden). T cell subset counts were performed using fluorescence activated cell sorting (FACScan, Becton Dickinson, San Jose, California, USA).
Data were entered into Microsoft Access and analyzed using SAS version 8.2 (SAS Institute, Cary, North Carolina, USA). Student's t-test was used to compare means of normally distributed continuous variables; the Wilcoxon rank sum test was used to compare continuous variables with non-normal distribution, and the chi-square test was used for comparison of categorical variables.
Before performing statistical analyses, crude plasma HIV-1 RNA concentrations and malaria parasite densities were log10 transformed to approximate normal distributions. The relationship between potential confounding variables and HIV-1 RNA concentrations was assessed using the Student's t-tests for dichotomous variables and analysis of variance (ANOVA) for variables with more than two categories. Relationships between continuous predictor variables and HIV-1 RNA concentrations were assessed using Pearson's correlations. The paired t-test was used to compare peripheral and placental HIV-1 RNA concentrations in the same women. To control for confounding, variables associated with either malaria or HIV-1 RNA concentrations at P values < 0.10 in univariate analyses were entered into multivariate models, which included the malaria status variables. In the final multivariate models, malaria status variables and any variable with a P value < 0.05 were retained. HIV-1 viral load was reported as geometric mean HIV-1 RNA concentration.
Of the 4923 women admitted in the antenatal ward at QECH between December 2000 and June 2002, 3148 (63.9%) were evaluated for eligibility to participate in the study and 2761 (87.7%) were eligible. In this group, 397 eligible women were discharged and consent was obtained from 1662 (70.3%) of the 2364 women who remained in the hospital. Women who agreed to participate were similar to those who were admitted in the antenatal ward with respect to age and gravidity (data not shown).
Of the 1662 women screened, 480 (28.9%) tested positive for HIV and were enrolled in the study. Only two of the HIV-infected women had prior knowledge of their HIV status, but neither was on antiretroviral treatment. At the time of enrollment, women had a mean gestation age of 37.0 weeks (SD, 4.4). Peripheral malaria parasitemia was detected in 12.7% (61/480) of the women, with a median parasite density of 6615 × 106/l [interquartile range (IQR), 1140–27 780]. A total of 387 women (80.6%) delivered at QECH and the median time from enrollment to delivery was 4.5 days (IQR, 1–17). Twelve women did not receive nevirapine since they were readmitted in the second stage of labor.
Of the 387 women who delivered, 342 (88.4%) had placental blood film microscopy and 304 (78.6%) had placental biopsies examined histopathologically. Malaria parasites were detected in 11.4% (39/342) of the placental blood films, with a median parasite density of 5400 × 106/l (IQR, 330–26 145). Of the women who had placental histopathology, 74 (24.3%) had detectable malaria parasites. Assuming that placental histopathology is the gold standard for detecting malaria infection in pregnancy, placental blood film microscopy had a sensitivity of 39.1% and a specificity of 98.2%, while peripheral blood film microscopy had a sensitivity of 27.0% and a specificity of 94.4%. In the main analyses, histopathology results were used to define malaria infection.
Table 1 compares characteristics of all enrolled women with women who had placental histopathology results and those who did not. Women with placental histopathology results made more antenatal visits than those who did not. However, the proportions receiving two or more doses of SP intermittent preventive treatment (SP-IPT) and other characteristics were similar between the two groups.
Table 2 compares the characteristics of women with and without placental malaria based on placental histopathology. Compared with their uninfected counterparts, women with placental malaria made fewer antenatal clinic visits and were more likely to have been treated with SP within 2 weeks prior to enrollment. However, the proportions of women who received two or more doses of routine SP-IPT were similar between the two groups. Women with placental malaria had lower CD4 cell counts and hemoglobin concentrations than those without placental malaria. There were no differences between the two groups in the prevalence of documented fever at enrollment and the distribution of age, gravidity and educational level.
Association between placental histopathologic malaria and HIV-1 viral loads
Peripheral plasma HIV-1 RNA concentrations were measured in 69 of 74 (93.2%) women with histopathologic malaria and a consecutive sample of 200 of 230 (87.0%) women without histopathologic malaria. In univariate analysis, the geometric mean peripheral HIV-1 RNA concentration was 2.5-fold higher in women who had placental malaria than in those who did not (Table 3). Women with documented fever at enrollment had a higher peripheral geometric mean HIV-1 RNA concentration than those without fever (70 836 versus 29 921 copies/ml; P = 0.03). Peripheral HIV-1 RNA concentrations were correlated with CD4 cell count (r = −0.46; P < 0.0001) and hemoglobin concentration (r = −0.21; P = 0.0003) but not with age, gravidity, educational level or antimalarial drug use (data not shown). In multivariate analysis, after adjusting for CD4 cell count and hemoglobin concentration, the geometric mean peripheral HIV-1 RNA concentration was 1.7 times higher in women with placental malaria infection than in their uninfected counterparts (Table 3).
Placental plasma HIV-1 RNA concentrations were measured in 66 of 74 (89.1%) women with histopathologic malaria, and in a consecutive sample of 196 of 230 (85.2%) women without histopathologic malaria. In univariate analysis, the geometric mean placental HIV-1 RNA concentration was 2.4 times higher in women with placental malaria than in those without placental malaria (Table 4). Placental HIV-1 RNA concentration was significantly associated with CD4 cell count (r = −0.30; P < 0.0001) but weakly correlated with hemoglobin concentration (r = −0.12; P = 0.052) and age (r = −0.18; P = 0.08). Placental HIV-1 RNA concentration was not associated with documented fever at enrollment, educational level, gravidity or antimalarial drug use (data not shown). In multivariate analysis, placental HIV-RNA concentration was significantly associated with placental malaria, age and CD4 cell count but not with hemoglobin concentration. After adjusting for age and CD4 cell count, the geometric mean placental HIV-1 RNA concentration was 2.0 times higher in women with placental malaria than those without placental malaria (Table 4).
Associations between peripheral/placental blood film parasitemia and HIV-1 viral loads
Although placental histopathology was used to define malaria infection, the relationship between parasitemia determined from blood films and HIV-1 RNA concentrations was also examined. As summarized in Table 3, peripheral HIV-1 RNA concentration was not associated with either peripheral or placental blood film parasitemia. There was no correlation between peripheral HIV-1 RNA concentration and peripheral parasite density among women with peripheral parasitemia (r = 0.14; P = 0.30), but a positive correlation was observed between peripheral HIV-1 RNA concentration and placental parasite density (r = 0.37; P = 0.02) among women with placental blood parasitemia.
Unlike peripheral HIV-1 RNA concentrations, placental HIV-1 RNA concentration was associated with peripheral and placental blood parasitemia in univariate analyses (Table 4). After adjusting for age and CD4 cell count in multivariate analyses, peripheral and placental blood parasitemia remained significantly associated with higher placental HIV-1 RNA concentration (Table 4). Among women with peripheral parasitemia, there was a borderline correlation between peripheral parasite density and placental HIV-1 RNA concentration (r = 0.31; P = 0.06). Among women with placental blood film parasitemia, a significant correlation was observed between placental blood parasite density and placental HIV-1 RNA concentration (r = 0.41; P = 0.01).
In this study of HIV-infected pregnant women, placental histopathologic malaria was associated with higher concentrations of both peripheral and placental HIV-1 RNA. After adjusting for confounders, women with placental histopathologic malaria had a 1.7-fold higher peripheral HIV-1 viral load and a 2.0-fold higher placental HIV-1 viral load than those without placental malaria. Although peripheral and placental blood film microscopy had a low sensitivity for detecting malaria infection, peripheral and placental parasitemias were also associated with higher placental HIV-1 viral load. Among women with placental parasitemia, placental parasite density was positively correlated with both placental and peripheral HIV-1 viral load.
In a previous Tanzanian study, peripheral malaria parasitemia in the second trimester of pregnancy was associated with a twofold higher peripheral HIV-1 viral load . The lack of significant association between peripheral parasitemia and peripheral HIV-1 viral load in our study may have been because of the low sensitivity of peripheral blood film microscopy, leading to the misclassification of some malaria-infected women. Peripheral parasitemia is a poor indicator of malaria infection in pregnancy because of the sequestration of malaria parasites in the placenta [22,23]. When we used placental histopathology to define malaria infection, there was a significant association between peripheral HIV-1 viral load and malaria infection, and the magnitude of increase in peripheral HIV-1 viral load associated with malaria was similar to that found in the Tanzanian study. Tanzanian women were enrolled around mid-pregnancy, when parasite prevalence and density are highest, while both decline towards term . This might explain the difference found in the two studies in the relationship between peripheral parasitemia and peripheral HIV-1 viral load.
The increase in peripheral HIV-1 viral load associated with placental histopathologic malaria was lower than that reported for non-pregnant adults, in whom symptomatic malaria was associated with a sevenfold higher peripheral HIV-1 viral load . Parasitemic subjects in that study were febrile, whereas only 9.5% of our malaria-infected women were (Table 2). Pro-inflammatory cytokines, such as TNF-α, interleukin-1β and interleukin-6, which are thought to cause malaria symptoms [36–38], are also associated with increased HIV-1 viral replication [39,40]. Therefore, parasitemic subjects in the previous study might have had a stronger systemic pro-inflammatory cytokine response and hence a higher peripheral HIV-1 viral load than the women we studied.
Malaria-infected placentas produce high levels of pro-inflammatory cytokines such as TNF-α [29,30,41] and contain increased numbers of monocytes [24–26]. P. falciparum malaria may increase HIV replication in the placenta, as it does in vitro, by enhancing production of TNF-α by mononuclear cells . However, in our study, placental HIV-1 RNA concentration was significantly lower than peripheral HIV-1 RNA concentration in women with placental malaria [mean difference 0.70 log10 copies/ml (5.0-fold); P < 0.0001] and in those without placental malaria [mean difference 0.74 log10 copies/ml (5.5-fold); P < 0.0001]. Several reasons might account for this observation. First, nevirapine, which women took at the onset of active labor (after peripheral blood samples had already been collected), might have substantially reduced placental HIV-viral load. However, among 12 women who did not receive nevirapine, placental HIV-1 RNA concentrations were still lower than peripheral HIV-1 RNA concentrations [mean difference 0.88 log10 copies/ml (7.5-fold); P = 0.001], suggesting that the use of nevirapine could not fully account for the lower placental HIV-1 viral load. Second, since placental blood samples were obtained by making an incision through placental tissues, there may have been some dilution of maternal intervillous blood with fetal (cord) blood. Therefore, placental HIV-1 viral load may have been higher if we had corrected for the dilutional effect. Third, it is possible that, despite the sequestration of malaria parasites in the placental compartment, malaria-induced immune activation is more generalized and high HIV-1 viral replication resulting from malaria infection may occur predominantly in other extraplacental sites, such as the lymphoreticular system.
Women with malaria had significantly lower CD4 cell counts than malaria-uninfected women. Therefore, the association between placental malaria and HIV-1 viral load might have been a result of an increased risk of malaria in subjects with advanced immunosuppression. We cannot exclude this possibility, but placental malaria was still associated with peripheral and placental HIV-1 viral load after adjusting for CD4 cell count and other covariates. This suggests that placental malaria might independently increase HIV-1 viral load, consistent with findings of a previous study of HIV-infected non-pregnant adults, which showed that treatment of acute malaria resulted in a decline in HIV-1 viral load .
The government of Malawi recommends the use of two doses of SP-IPT to prevent the adverse effects malaria infection in pregnancy. In this study, the prevalence of placental malaria was lower in women who took the recommended doses of SP-IPT than in those who did not, but the difference was not statistically significant (21.2 and 28.5%, respectively; P = 0.15). Previous studies in Kenya and Malawi have shown poor effectiveness of the two dose SP-IPT regimen against malaria infection in the third trimester among HIV-infected women [42,43], suggesting defects in host parasite clearance secondary to HIV infection. These findings might explain the lack of association between the use of SP-IPT and HIV-1 viral load in our study. Further studies are needed to identify antimalarial drug regimens that are effective at reducing placental malaria infection in HIV-infected women, which may also help to reduce HIV-1 viral load.
A possible source of selection bias in our study was the exclusion from analysis of 36.7% (176/480) of enrolled women for whom we lacked placental histopathology results. These excluded women made fewer antenatal visits than those included in the analysis, suggesting that they were generally poor utilizers of health services. However, they were similar to those included in the study with respect to CD4 cell counts, hemoglobin concentrations, peripheral malaria prevalence and the use of SP-IPT. Therefore, it is highly unlikely that selection bias could have affected the validity of our findings.
Several studies have estimated that a 10-fold (unit log10) increase in HIV-1 viral load is associated with approximately a 2.5-fold increase in the odds of HIV MTCT [9,10,12–14]. Assuming a linear relationship between HIV-1 viral load and HIV MTCT, the 1.7-fold (0.25 log10) increase in peripheral HIV-1 viral load associated with placental malaria in our study may result in a 25% increase in the odds of HIV MTCT and a concomitant increase in infant mortality. This would be consistent with the findings of a previous study, which showed that infants born to women coinfected with placental malaria and HIV had 2.7–7.7 times higher postneonatal mortality than those born to women infected with HIV alone .
In sub-Saharan Africa, the vast majority of HIV-infected pregnant women are not treated with antiretroviral medications because of high drug costs and the lack of resources to run effective antiretroviral treatment programs. Similarly, the performance of elective cesarean sections as a measure of preventing HIV MTCT is impractical because of the limited capacity of health-care systems to meet the potential demand for this intervention. Consequently, there is an urgent need to find feasible and low-cost interventions to reduce the rates of HIV MTCT. If malaria increases HIV-1 MTCT by increasing HIV-1 viral load, this would provide an additional rationale for intensifying malaria control and treatment programs in sub-Saharan Africa. Such programs might not only reduce HIV viral load but also the prevalence of low birthweight and maternal anemia, which are important risk factors for infant and maternal mortality, respectively.
We would like to thank Sioban Harlow, Terrie Taylor, Rosemary Rochford, Daniel Remick and Frank Anderson for their contributions in writing this paper. We also thank Mrs Njiragoma, Mrs Munthali, Mrs Mnapo, Mrs Jere, Mrs Katimba Banda and Mrs Kumwenda for collecting research data, Patrick Mkundika and Maxwell Kanjala for analyzing laboratory specimens and Kelvin Gwadani, Sarah White, Pelani Malange and Dongling Zeng for data management and analysis.
Sponsorship: This work was supported by NIH grant AI 49084, the NIH-FIC grant 5 D43 TW00908 and the Center for AIDS Research at the University of North Carolina. SJR is a Wellcome Trust Senior Overseas Fellow in Biomedical Sciences. Nevirapine was provided by Global Strategies for HIV Prevention.
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