Caroline Pehrson

Adhesion of Plasmodium falciparum infected erythrocytes in ex vivo perfused placental tissue: a novel model of placental malaria.

BackgroundPlacental malaria occurs when Plasmodium falciparum infected erythrocytes sequester in the placenta. Placental parasite isolates bind to chondroitin sulphate A (CSA) by expression of VAR2CSA on the surface of infected erythrocytes, but may sequester by other VAR2CSA mediated mechanisms, such as binding to immunoglobulins. Furthermore, other parasite antigens have been associated with placental malaria. These findings have important implications for placental malaria vaccine design. The objective of this study was to adapt and describe a biologically relevant model of parasite adhesion in intact placental tissue.ResultsThe ex vivo placental perfusion model was modified to study adhesion of infected erythrocytes binding to CSA, endothelial protein C receptor (EPCR) or a transgenic parasite where P. falciparum erythrocyte membrane protein 1 expression had been shut down. Infected erythrocytes expressing VAR2CSA accumulated in perfused placental tissue whereas the EPCR binding and the transgenic parasite did not. Soluble CSA and antibodies specific against VAR2CSA inhibited binding of infected erythrocytes.ConclusionThe ex vivo model provides a novel way of studying receptor-ligand interactions and antibody mediated inhibition of binding in placental malaria.

Plasmodium falciparum mutant haplotype infection during pregnancy associated with reduced birthweight, Tanzania.

Intermittent preventive treatment during pregnancy with sulfadoxine–pyrimethamine (IPTp-SP) is a key strategy in the control of pregnancy-associated malaria. However, this strategy is compromised by widespread drug resistance from single-nucleotide polymorphisms in the Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthetase genes. During September 2008–October 2010, we monitored a cohort of 924 pregnant women in an area of Tanzania with declining malaria transmission. P. falciparum parasites were genotyped, and the effect of infecting haplotypes on birthweight was assessed. Of the genotyped parasites, 9.3%, 46.3%, and 44.4% had quadruple or less, quintuple, and sextuple mutated haplotypes, respectively. Mutant haplotypes were unrelated to SP doses. Compared with infections with the less-mutated haplotypes, infections with the sextuple haplotype mutation were associated with lower (359 g) birthweights. Continued use of the suboptimal IPTp-SP regimen should be reevaluated, and alternative strategies (e.g., intermittent screening and treatment or intermittent treatment with safe and effective alternative drugs) should be evaluated.

Factors associated with and causes of perinatal mortality in northeastern Tanzania

Objective. To identify factors associated with perinatal mortality in northeastern Tanzania. Design. Prospective cohort study. Setting. Northeastern Tanzania. Population. 872 mothers and their newborns. Methods. Pregnant women were screened for factors possibly associated with perinatal mortality, including preeclampsia, small‐for‐gestational age, preterm delivery, anemia, and health‐seeking behavior. Fetal growth was monitored using ultrasound. Finally, the specific causes of the perinatal deaths were evaluated. Main outcome measure. Perinatal mortality. Results. Forty‐six deaths occurred. Key factors associated with perinatal mortality were preterm delivery (adjusted odds ratio (OR) 14.47, 95% confidence interval (CI) 3.23–64.86, p < 0.001), small‐for‐gestational age (adjusted OR 3.54, 95%CI 1.18–10.61, p = 0.02), and maternal anemia (adjusted OR 10.34, 95%CI 1.89–56.52, p = 0.007). Adherence to the antenatal care program (adjusted OR 0.027, 95%CI 0.003–0.26, p = 0.002) protected against perinatal mortality. The cause of death in 43% of cases was attributed to complications related to labor and specifically to intrapartum asphyxia (30%) and neonatal infection (13%). Among the remaining deaths, 27% (7/26) were attributed to preeclampsia and 23% (6/26) to small‐for‐gestational age. Of these, 54% (14/26) were preterm. Conclusions. Preeclampsia, small‐for‐gestational age and preterm delivery were key risk factors and causes of perinatal mortality in this area of Tanzania. Maternal anemia was also strongly associated with perinatal mortality. Furthermore, asphyxia accounted for a large proportion of the perinatal deaths. Interventions should target the prevention and handling of these conditions in order to reduce perinatal mortality.

Malaria and Fetal Growth Alterations in the 3rd Trimester of Pregnancy: A Longitudinal Ultrasound Study

Background Pregnancy associated malaria is associated with decreased birth weight, but in-utero evaluation of fetal growth alterations is rarely performed. The objective of this study was to investigate malaria induced changes in fetal growth during the 3rd trimester using trans-abdominal ultrasound. Methods An observational study of 876 pregnant women (398 primi- and secundigravidae and 478 multigravidae) was conducted in Tanzania. Fetal growth was monitored with ultrasound and screening for malaria was performed regularly. Birth weight and fetal weight were converted to z-scores, and fetal growth evaluated as fetal weight gain from the 26th week of pregnancy. Results Malaria infection only affected birth weight and fetal growth among primi- and secundigravid women. Forty-eight of the 398 primi- and secundigravid women had malaria during pregnancy causing a reduction in the newborns z-score of −0.50 (95% CI: −0.86, −0.13, P = 0.008, multiple linear regression). Fifty-eight percent (28/48) of the primi- and secundigravidae had malaria in the first half of pregnancy, but an effect on fetal growth was observed in the 3rd trimester with an OR of 4.89 for the fetal growth rate belonging to the lowest 25% in the population (95%CI: 2.03–11.79, P<0.001, multiple logistic regression). At an individual level, among the primi- and secundigravidae, 27% experienced alterations of fetal growth immediately after exposure but only for a short interval, 27% only late in pregnancy, 16.2% persistently from exposure until the end of pregnancy, and 29.7% had no alterations of fetal growth. Conclusions The effect of malaria infections was observed during the 3rd trimester, despite infections occurring much earlier in pregnancy, and different mechanisms might operate leading to different patterns of growth alterations. This study highlights the need for protection against malaria throughout pregnancy and the recognition that observed changes in fetal growth might be a consequence of an infection much earlier in pregnancy.

Reliability of rapid diagnostic tests in diagnosing pregnancy-associated malaria in north-eastern Tanzania

BackgroundAccurate diagnosis and prompt treatment of pregnancy-associated malaria (PAM) are key aspects in averting adverse pregnancy outcomes. Microscopy is the gold standard in malaria diagnosis, but it has limited detection and availability. When used appropriately, rapid diagnostic tests (RDTs) could be an ideal diagnostic complement to microscopy, due to their ease of use and adequate sensitivity in detecting even sub-microscopic infections. Polymerase chain reaction (PCR) is even more sensitive, but it is mainly used for research purposes. The accuracy and reliability of RDTs in diagnosing PAM was evaluated using microscopy and PCR.MethodsA cohort of pregnant women in north-eastern Tanzania was followed throughout pregnancy for detection of plasmodial infection using venous and placental blood samples evaluated by histidine rich protein 2 (HRP-2) and parasite lactate dehydrogenase (pLDH) based RDTs (Parascreen™) or HRP-2 only (Paracheck Pf® and ParaHIT®f), microscopy and nested Plasmodium species diagnostic PCR.ResultsFrom a cohort of 924 pregnant women who completed the follow up, complete RDT and microscopy data was available for 5,555 blood samples and of these 442 samples were analysed by PCR. Of the 5,555 blood samples, 49 ((proportion and 95% confidence interval) 0.9% [0.7 -1.1]) samples were positive by microscopy and 91 (1.6% [1.3-2.0]) by RDT. Forty-six (50.5% [40.5 - 60.6]) and 45 (49.5% [39.4 – 59.5]) of the RDT positive samples were positive and negative by microscopy, respectively, whereas nineteen (42.2% [29.0 - 56.7]) of the microscopy negative, but RDT positive, samples were positive by PCR. Three (0.05% [0.02 - 0.2]) samples were positive by microscopy but negative by RDT. 351 of the 5,461 samples negative by both RDT and microscopy were tested by PCR and found negative. There was no statistically significant difference between the performances of the different RDTs.ConclusionsMicroscopy underestimated the real burden of malaria during pregnancy and RDTs performed better than microscopy in diagnosing PAM. In areas where intermittent preventive treatment during pregnancy may be abandoned due to low and decreasing malaria risk and instead replaced with active case management, screening with RDT is likely to identify most infections in pregnant women and out-performs microscopy as a diagnostic tool.

Placental Sequestration of Plasmodium falciparum Malaria Parasites Is Mediated by the Interaction Between VAR2CSA and Chondroitin Sulfate A on Syndecan-1

During placental malaria, Plasmodium falciparum infected erythrocytes sequester in the placenta, causing health problems for both the mother and fetus. The specific adherence is mediated by the VAR2CSA protein, which binds to placental chondroitin sulfate (CS) on chondroitin sulfate proteoglycans (CSPGs) in the placental syncytium. However, the identity of the CSPG core protein and the cellular impact of the interaction have remain elusive. In this study we identified the specific CSPG core protein to which the CS is attached, and characterized its exact placental location. VAR2CSA pull-down experiments using placental extracts from whole placenta or syncytiotrophoblast microvillous cell membranes showed three distinct CSPGs available for VAR2CSA adherence. Further examination of these three CSPGs by immunofluorescence and proximity ligation assays showed that syndecan-1 is the main receptor for VAR2CSA mediated placental adherence. We further show that the commonly used placental choriocarcinoma cell line, BeWo, express a different set of proteoglycans than those present on placental syncytiotrophoblast and may not be the most biologically relevant model to study placental malaria. Syncytial fusion of the BeWo cells, triggered by forskolin treatment, caused an increased expression of placental CS-modified syndecan-1. In line with this, we show that rVAR2 binding to placental CS impairs syndecan-1-related Src signaling in forskolin treated BeWo cells, but not in untreated cells.

Modelling of human transplacental transport as performed in Copenhagen, Denmark

Placenta perfusion models are very effective when studying the placental mechanisms in order to extrapolate to real-life situations. The models are most often used to investigate the transport of substances between mother and foetus, including the potential metabolism of these. We have studied the relationships between maternal and foetal exposures to various compounds including pollutants such as polychlorinated biphenyls, polybrominated flame retardants, nanoparticles as well as recombinant human antibodies. The compounds have been studied in the human placenta perfusion model and to some extent in vitro with an established human monolayer trophoblast cell culture model. Results from our studies distinguish placental transport of substances by physicochemical properties, adsorption to placental tissue, binding to transport and receptor proteins and metabolism. We have collected data from different classes of chemicals and nanoparticles for comparisons across chemical structures as well as different test systems. Our test systems are based on human material to bypass the extrapolation from animal data. By combining data from our two test systems, we are able to rank and compare the transport of different classes of substances according to their transport ability. Ultimately, human data including measurements in cord blood contribute to the study of placental transport.

Pre-clinical and clinical development of the first placental malaria vaccine

ABSTRACT Introduction: Malaria during pregnancy is a massive health problem in endemic areas. Placental malaria infections caused by Plasmodium falciparum are responsible for up to one million babies being born with a low birth weight every year. Significant efforts have been invested into preventing the condition. Areas covered: Pub Med was searched using the broad terms ‘malaria parasite placenta’ to identify studies of interactions between parasite and host, ‘prevention of placental malaria’ to identify current strategies to prevent placental malaria, and ‘placental malaria vaccine’ to identify pre-clinical vaccine development. However, all papers from these searches were not systematically included. Expert commentary: The first phase I clinical trials of vaccines are well underway. Trials testing efficacy are more complicated to carry out as only women that are exposed to parasites during pregnancy will contribute to endpoint measurements, further it may require extensive follow-up to establish protection. Future second generation vaccines may overcome the inherent challenges in making an effective placental malaria vaccine.

Burkitt lymphoma expresses oncofetal chondroitin sulfate without being a reservoir for placental malaria sequestration

Burkitt lymphoma (BL) is a malignant disease, which is frequently found in areas with holoendemic Plasmodium falciparum malaria. We have previously found that the VAR2CSA protein is present on malaria‐infected erythrocytes and facilitates a highly specific binding to the placenta. ofCS is absent in other non‐malignant tissues and thus VAR2CSA generally facilitates parasite sequestration and accumulation in pregnant women. In this study, we show that the specific receptor for VAR2CSA, the oncofetal chondroitin sulfate (ofCS), is likewise present in BL tissue and cell lines. We therefore explored whether ofCS in BL could act as anchor site for VAR2CSA‐expressing infected erythrocytes. In contrast to the placenta, we found no evidence of in vivo sequestering of infected erythrocytes in the BL tissue. Furthermore, we found VAR2CSA‐specific antibody titers in children with endemic BL to be lower than in control children from the same malaria endemic region. The abundant presence of ofCS in BL tissue and the absence of ofCS in non‐malignant tissue encouraged us to examine whether recombinant VAR2CSA could be used to target BL. We confirmed the binding of VAR2CSA to BL‐derived cells and showed that a VAR2CSA drug conjugate efficiently killed the BL‐derived cell lines in vitro. These results identify ofCS as a novel therapeutic BL target and highlight how VAR2CSA could be used as a tool for the discovery of novel approaches for directing BL therapy.

Plasmodium falciparum Infection Early in Pregnancy has Profound Consequences for Fetal Growth

Malaria during pregnancy constitutes a large health problem in areas of endemicity. The World Health Organization recommends that interventions are initiated at the first antenatal visit, and these improve pregnancy outcomes. This study evaluated fetal growth by ultrasonography and birth outcomes in women who were infected prior to the first antenatal visit (gestational age, <120 days) and not later in pregnancy. Compared with uninfected controls, women with early Plasmodium falciparum exposure had retarded intrauterine growth between gestational ages of 212 and 253 days (difference between means, 107 g [95% confidence interval {CI}, 26-188]; P = .0099) and a shorter pregnancy duration (difference between means, 6.6 days [95% CI, 1.0-112.5]; P = .0087). The birth weight (difference between means, 221 g [95% CI, 6-436]; P = .044) and the placental weight (difference between means, 84 g [95% CI, 18-150]; P = .013) at term were also reduced. The study suggests that early exposure to P. falciparum, which is not targeted for prevention by current control strategies, has a profound impact on fetal growth, pregnancy duration, and placental weight at term.