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Epidemiology and Social Science

Association of HIV and Malaria With Mother-to-Child Transmission, Birth Outcomes, and Child Mortality

Brahmbhatt, Heena MPH, PhD*; Sullivan, David MD; Kigozi, Godfrey MD; Askin, Fred MD§; Wabwire-Mangenm, Fred MD, PhD; Serwadda, David MD; Sewankambo, Nelson MD#; Wawer, Maria MD**; Gray, Ronald MD*

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JAIDS Journal of Acquired Immune Deficiency Syndromes: April 1, 2008 - Volume 47 - Issue 4 - p 472-476
doi: 10.1097/QAI.0b013e318162afe0
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Abstract

Malaria and HIV are among the most prevalent infectious diseases in sub-Saharan Africa, and coinfections are common. In malaria-endemic areas, frequency and severity of malaria is greater in pregnant women compared to nonpregnant women.1,2 Pregnant women infected with malaria have increased risk of maternal anemia, abortion, stillbirth, and premature delivery and/or low birth weight.3-5 Low birth weight, preterm delivery, and infant mortality are increased in children born to HIV-positive mothers compared to HIV-negative mothers.6-8 Women infected with HIV have a diminished immunity to malaria infection, and placental and clinical malaria are more common in HIV-positive women compared with HIV-negative women.4,9,10 Coinfection with malaria results in an increase in HIV viral load.11-13 Therefore, coinfection of malaria and HIV-1 during pregnancy may have adverse consequences for morbidity and mortality of infants, and prevention of these infections during pregnancy could be an important public health priority.

Studies assessing the impact of HIV and malaria coinfection on mother-to-child HIV transmission (MTCT) are contradictory. In Uganda, we found a significant increase in vertical HIV transmission associated with placental malaria (adjusted relative risk [RR] = 2.89, 95% confidence interval [CI]: 1.66 to 3.24),14 and a study in Cameroon15 found higher MTCT rates correlated with peak rainy seasons with higher malaria transmission, suggesting an association between MTCT and higher rates of malaria during pregnancy. However, a study in Mombasa, Kenya, found no impact of placental malaria on MTCT,16 whereas a study in Kisumu, Kenya, found an increased risk of MTCT only at higher maternal parasitic densities compared with low parasitic densities.17 The latter study used placental blood smear to diagnose placental malaria.

The difference in findings of the association between malaria and MTCT could be due to the epidemiology of malaria in different settings, which could affect maternal immunity, or differences in diagnosis of placental malaria. Placental histology is more sensitive for detection of placental malaria (91%) than is a placental blood film (63%) or peripheral blood film (47%).18 Histopathology to detect the histidine-rich protein 2 (HRP-II) has also been shown to have high sensitivity and specificity in detecting severe malaria.19,20 We used histopathology, with HRP-II immunohistochemistry staining, to detect malaria parasites in the placentas. In addition, we used a rapid immunochromatographic test (BinaxNOW ICT Malaria; Binax, Scarborough, ME) for detection of Plasmodium falciparum and Plasmodium vivax antigens in maternal serum (80% sensitivity compared to polymerase chain reaction [PCR] and 94% to 100% sensitivity compared to blood smear for detection of P. falciparum, and 89% sensitivity and 95% specificity compared to placental histology).20-23

We previously reported an association between MTCT and placental malaria diagnosed with H&E stain, which is suboptimal for detection of placental malaria.14 We now report on this association using the HRP-II stain, which has higher sensitivity and specificity for placental malaria detection. Our study also assesses the impact of coinfection with HIV and malaria on adverse birth outcomes and child mortality. The majority of these mothers' placentas (60%) were not analyzed in the original study; hence, the association of placental malaria and MTCT is assessed among a new cohort of mothers using more sensitive methods to detect placental malaria.

METHODS

Data for this study came from mothers and children who were enrolled in the Rakai Community Cohort Study (RCCS) between 1994 and 2000.24,25 Annual surveys including censuses, interviews, and collection of samples for HIV and sexually transmitted infections (STI) testing were conducted on all consenting adults aged 15 to 59 years resident in 56 communities.24,25 Pregnant women were identified either by interview or urinary hCG testing at varying durations of gestation and were seen postpartum. During the prepartum visit by the midwife, mothers were provided with a bucket containing 10% formol saline and were instructed to deposit the placenta into the bucket immediately after the delivery. Cotton wool was placed on top of the placenta to ensure that it was completely saturated with formalin. Approximately 70% of the mothers provided placentas, and for a majority of these women (70%), the placentas were collected within 3 days of birth. Pregnant women were given 1 dose of sulfadoxine-pyrimethamine during the first prepartum visit (on average, at 5 months gestation) and advised to go to a clinic for a second sulfadoxine-pyrimethamine dose.

Maternal serum, urine, and self-collected vaginal swabs were also obtained at the postpartum visit for repeat STI and HIV testing. For 70% of mothers, the postpartum visit was within 3 days of delivery. Women were tested for HIV during pregnancy and postpartum using 2 independent enzyme immunoassays (Vironostika HIV-1, Organon Teknika, Charlotte, NC; and Cambridge Biotech, Worcester, MA), with Western blot (Bio-Merieux-Vitek, St. Louis, MO) confirmation of discordant results or seroconverters. Children born to these mothers were assessed after birth to ascertain birth weight and chest, upper arm, and head circumferences. Preterm birth was determined using the New Ballard Score, which uses 7 physical and 6 neuromuscular signs of maturity,26 and last menstrual period was used to determine gestational age for children for whom the Ballard Score was missing (16% of infants). Because it was not possible to see all infants on the day of birth, given the prevalence of home deliveries in this rural setting, heel-stick filter paper samples or serum were obtained from infants seen within 3 days of birth and at 4 to 6 weeks of life. Infant HIV infection was detected by reverse transcriptase RNA-PCR (RT-PCR) using the Amplicor HIV-1 Monitor 1.5 Assay (Roche Molecular Systems, Branchburg, NJ). In-utero and intrapartum HIV transmission was defined as a positive HIV PCR test between birth and 6 weeks of life, and breast-feeding transmission was defined as a negative HIV PCR at 6 weeks followed by a positive HIV PCR at a later time.

Formalin-fixed, paraffin-embedded placental disc tissue, including membranes and umbilical cord tissue, were sectioned for hematoxylin and eosin staining. Giemsa staining and immunohistochemistry were also performed on the placental disc with a primary monoclonal antibody, 3A4,27 to P. falciparum histidine-rich-II and a secondary peroxidase antibody (Fig. 1).28 The microscopists read slides blinded to relational data. Parasite densities were calculated by assessing immunohistochemistry placental slides at 20× magnification. Five random fields, each of 1 square mm of the placental slides, were averaged to obtain density of parasites, expressed as number mm2. A high prevalence of formalin pigment precluded assessment of pigment that was due to chronic or past malaria infection. Maternal malaria was also diagnosed with 20 μL of serum using the BinaxNOW ICT (Binax, Portland, ME), which detects P. falciparum-specific histidine-rich protein and pan-Plasmodium aldolase antigens in the blood up to 2 weeks after clearance of infection.

F1-9
FIGURE 1:
Improved placental histologic diagnosis of malaria. A placenta stained by H&E (A, D), Giemsa (B, E), and immunohistochemistry for HRP-II (C, F). In C and F, Plasmodium-infected erythrocytes are highlighted by red staining. Tissues A-C are at 20× magnification, and D-F are at 60× magnification.

The study was approved by institutional review boards in Uganda and the United States.

STATISTICAL METHODS

Maternal demographic characteristics and infant outcomes were assessed in the overall population and then stratified by placental malaria and maternal HIV status. Rates of MTCT were stratified by maternal HIV viral loads below or above the median and by the presence or absence of placental malaria. The χ2 test for trend was used to assess the MTCT risk associated with placental malaria, stratified by the mother's median HIV viral load. Univariate and multivariate relative risks of MTCT were calculated using a generalized linear model with a logit link and binomial distribution. The data were analyzed using Intercooled Stata statistical software for Windows, version 9 (Stata, College Station, TX).11

RESULTS

Approximately 59% of the mothers in this population were HIV positive (170 of 286). The prevalence of placental malaria was 32.1% in the overall population, but higher among HIV-positive mothers compared with HIV-negative mothers (37.1% vs. 25%, respectively; P = 0.039) according to immunohistochemistry. Figure 1 shows the histopathology results comparing H&E, Giemsa, and immunohistochemistry. HRP-II-based immunohistochemistry enabled more rapid detection of lower parasitemias corroborated by Giemsa stains. Mild inflammation in placental villi was seen in all placentas with a monocyte/macrophage predominance. There was no difference in the prevalence of chorioaminonitis (25%) regardless of malaria or HIV status. Prevalence of maternal serologic malaria detected by the ICT test was 31.1%, but no significant difference was observed between HIV-positive mothers compared with HIV-negative mothers (30.3% vs. 32.3%, P = 0.72).

For the MTCT analyses, the sample size was 109 mother-infant pairs with placental histopathology and 107 mother-infant pairs for mothers with a serologic malaria test. Of the infants in the study, 19 of 109 (17.4%) were HIV positive by 6 weeks after birth.

Table 1 summarizes HIV-positive mothers' demographic characteristics and birth outcomes by overall malaria infection, stratified by placental and serologic malaria. HIV-positive mothers with malaria were younger and more likely to be primigravida than mothers without malaria. The prevalence of low birth weight was higher among infants of mothers with malaria; this was statistically significant for infants whose mothers had both placental and rapid test results that were positive for malaria and for infants of mothers with serologic malaria alone. There were no statistically significant differences in the frequency of preterm births or child mortality by maternal malaria infection. Of the 107 mothers who had been tested for both serologic and placental malaria, approximately 21% (23 of 107) were positive for both tests, 15.9% (17 of 107) had placental malaria but tested negative for serologic malaria, and 6.5% (7 of 107) were positive on the ICT sera test but were negative for placental malaria. The ICT correctly identified 23 of 40 women who had placental malaria (57.5% sensitivity) and had a much higher specificity compared with placental histology (89.5%).

T1-9
TABLE 1:
Characteristics of Mothers and Their Children, by Mother's HIV Status

Table 2 shows the univariate and adjusted risk factors for MTCT in HIV-positive women. Risks of vertical transmission of HIV were significantly higher if the mother had placental malaria (RR = 3.6, 95% CI: 1.3 to 10.1), if the mother had a positive ICT sera test (RR = 3.2, 95% CI: 1.14 to 9.2), and if the infant was low birth weight (RR = 6.3, 95% CI: 2.2 to 18.5). The highest rates of MTCT were among mothers who had both placental and serologic malaria (30.4%), followed by mothers who had a positive ICT sera test but had no placental parasites (28.6%), then mothers who only had placental malaria (23.5%); the lowest risk of MTCT was among mothers who had no malaria infection (8.3%, χ2 for trend, P = 0.01). The multivariate adjusted risk of MTCT associated with placental malaria, adjusted for maternal HIV viral load, was 7.9 (95% CI: 1.4 to 58.5) compared to HIV-positive mothers who did not have placental malaria infection (P = 0.025). The risk of MTCT associated with maternal serologic malaria was not significantly increased after controlling for maternal HIV viral load (RR = 3.9, 95% CI: 0.78 to 19.5, P = 0.09). Controlling for gravidity did not alter the multivariate results.

T2-9
TABLE 2:
Risk Factors for MTCT

Figure 2 shows MTCT rates by presence or absence of placental malaria infection, stratified by the median maternal HIV viral load (median log10 viral load = 4.2). There was a progressive trend of MTCT associated with viral load and placental malaria. In mothers with high HIV viral load and placental malaria, the MTCT rate was 35.3%; in mothers who had HIV viral loads below the median and placental malaria, the MTCT rate was 28.6%; in mothers who had high HIV viral loads but no placental malaria, MTCT rate was 11.1%; and no infant HIV infections were observed among mothers with low viral load and no placental malaria (χ2 for trend, P < 0.001).

F2-9
FIGURE 2:
MTCT rates by maternal HIV viral load, segregated above or below median, and placental malaria.

The median placental parasite density in mothers with placental malaria was 66 parasites/mm2. There was no significant difference in the median malaria parasite density in HIV-positive mothers versus HIV-negative mothers (HIV-positive mothers: 66 parasites/mm2, HIV-negative mothers: 42 parasites/mm2, ranksum P = 0.89), nor any difference in median HIV viral loads among mothers who had parasite densities higher versus lower than median (high parasite density, median log10 viral load = 4.43 cps/mL, vs. low parasite density, median log10 viral load = 4.49 cps/mL, ranksum P = 0.83).

DISCUSSION

We found that placental malaria is more prevalent in HIV-positive mothers compared with HIV-negative mothers, as has been reported previously,29 and children born to HIV-positive mothers coinfected with malaria were significantly more likely to be born low birth weight. Mother-to-child HIV transmission was significantly increased among mothers coinfected with placental malaria and among those whose infants were of low birth weight. MTCT was highest among children born to mothers with high HIV viral loads and concurrent placental malaria, and placental malaria was associated with increased MTCT, even at low maternal viral loads (Fig. 2). High maternal HIV viral load is known to be predictive of MTCT, but in this study, the risk of MTCT associated with placental malaria was present irrespective of maternal viral load.30 This suggests that malaria prevention during pregnancy could be important for prevention of MTCT.

It has been suggested that placental malaria might increase in MTCT by upregulating maternal HIV viral load. However, unlike other studies,12,16 in our study, there seems to be an independent mechanism by which placental malaria increases the risk of MTCT, because even after adjusting for maternal HIV viral load in the multivariate analyses, the risk of MTCT associated with placental malaria remained significantly higher. Another hypothesized mechanism is that higher maternal HIV viral load increases malaria parasite density in the placenta. However, unlike other studies,29,31 we did not find a higher malaria parasite density in HIV-positive mothers compared with HIV-negative mothers, nor did we find an increase in maternal HIV viral load among mothers with high malaria parasite densities.

It's important to highlight the importance of the method used to detect placental malaria. In our original study, placental histopathology used H&E stain alone, and the placental malaria rate was approximately 9%.14 When these placentas were rediagnosed using the HRP-II immunohistochemistry, the placental malaria rate was increased to 32%, indicating a marked increase in sensitivity. The method of detecting placental malaria may in part explain the differences between study findings, because use of low-sensitivity malaria diagnoses will reduce the power to detect an association between placental malaria and MTCT. For example, the study by Inion et al16 in Mombasa that found no association between placental malaria and MTCT had a placental malaria rate of 7% based on placental blood smear. Placental histology (using Giemsa and/or hematoxylin-eosin stain) has been shown to be a more sensitive method for detection of placental malaria compared to a peripheral blood film or placental blood film.18 In addition, this was a rural population-based study, whereas most other studies were conducted in hospital populations in urban locations. Thus, differences in the epidemiology of malaria in these settings could affect results.

Some of the strengths of our study are the use of highly sensitive methods for diagnosis of serologic and placental malaria and the use of PCR to diagnose HIV in infants in a rural, community-based cohort. Some of the limitations are that we only had maternal rapid test diagnosis at the postpartum visit, and hence we could not detect malaria episodes resolved during pregnancy or determine the effects of timing of infection during gestation.

CONCLUSIONS

Coinfection with HIV and peripheral malaria are risk factors for low birth weight and MTCT. This suggests that public health programs should emphasize an integrated approach focused on both MTCT and malaria prophylaxis during pregnancy to help reduce the burden of HIV and adverse birth outcomes in children in rural Africa.

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Keywords:

MTCT; vertical transmission; malaria; placental malaria; peripheral malaria

© 2008 Lippincott Williams & Wilkins, Inc.