Placental Isolates Research Articles

Infection and Persistence of Coxiella burnetii Clinical Isolate in the Placental Environment.

Infection by Coxiella burnetii, the etiological agent of Q fever, poses the risk of causing severe obstetrical complications in pregnant women. C. burnetii is known for its placental tropism based on animal models of infection. The Nine Mile strain has been mostly used to study C. burnetii pathogenicity but the contribution of human isolates to C. burnetii pathogenicity is poorly understood. In this study, we compared five C. burnetii isolates from human placentas with C. burnetii strains including Nine Mile (NM) as reference. Comparative genomic analysis revealed that the Cb122 isolate was distinct from other placental isolates and the C. burnetii NM strain with a set of unique genes involved in energy generation and a type 1 secretion system. The infection of Balb/C mice with the Cb122 isolate showed higher virulence than that of NM or other placental isolates. We evaluated the pathogenicity of the Cb122 isolate by in vitro and ex vivo experiments. As C. burnetii is known to infect and survive within macrophages, we isolated monocytes and placental macrophages from healthy donors and infected them with the Cb122 isolate and the reference strain. We showed that bacteria from the Cb122 isolate were less internalized by monocyte-derived macrophages (MDM) than NM bacteria but the reference strain and the Cb122 isolate were similarly internalized by placental macrophages. The Cb122 isolate and the reference strain survived similarly in the two macrophage types. While the Cb122 isolate and the NM strain stimulated a poorly inflammatory program in MDM, they elicited an inflammatory program in placenta macrophages. We also reported that the Cb122 isolate and NM strain were internalized by trophoblastic cell lines and primary trophoblasts without specific replicative profiles. Placental explants were then infected with the Cb122 isolate and the NM strain. The bacteria from the Cb122 isolate were enriched in the chorionic villous foetal side. It is likely that the Cb122 isolate exhibited increased virulence in the multicellular environment provided by explants. Taken together, these results showed that the placental isolate of C. burnetii exhibits a specific infectious profile but its pathogenic role is not as high as the host immune response in pregnant women.

Antimicrobial Resistance Pattern in E. coli Isolated from Placental Tissues of Pregnant Women in Low-Socioeconomic Setting of Pakistan.

Infections during pregnancy can culminate in adverse pregnancy outcomes, including preterm birth (PTB). Pakistan is among the top ten nations with high PTB-associated neonatal mortality rates, where access to prenatal as well as neonatal care is only afforded by the privileged few. Societal stigma further discourages women seeking healthcare for minor infections. Microbial pathogens associated with genitourinary infections can lead to gestational complications culminating in earlier onset of labor. In this study, association of Escherichia coli (E. coli) with PTB in Pakistani women of low-socioeconomic status is examined. 57 paired vaginal swabs and placenta samples from mothers with full term and preterm deliveries were collected and processed for isolation and molecular characterization of extraintestinal pathogenic E. coli (ExPEC). ExPEC isolated from vaginal swabs and placenta showed phylotype B2 being most prevalent (Vagina n = 3 (9), 33%) (Placenta n = 4 (12), 33%) in preterm cases followed by phylotype B1 (Vagina n = 2 (9), 22%) (Placenta n = 3 (12), 25%) and untypeable strains. Antibiotic susceptibility profiling showed a large percentage of resistant isolates to multiple antibiotics, including carbapenem and included extended-spectrum beta-lactamase (ESBL) producers. Our study is the first to report different phylotypes of E. coli from placental tissues in preterm deliveries which may be a cause for concern for maternal and neonatal health. ExPEC from vaginal swabs and placental of females delivering preterm shows the pathogenic phylotype B2 dominance with a large percentage of isolates resistant to multiple antibiotics, including carbapenem and included ESBL producers. The placental isolates may indicate ascending infection from vagina or urinary tract which may lead to preterm birth.

A single full-length VAR2CSA ectodomain variant purifies broadly neutralizing antibodies against placental malaria isolates.

Placental malaria (PM) is a deadly syndrome most frequent and severe in first pregnancies. PM results from accumulation of Plasmodium falciparum-infected erythrocytes (IE) that express the surface antigen VAR2CSA and bind to chondroitin sulfate A (CSA) in the placenta. Women become PM-resistant over successive pregnancies as they develop anti-adhesion and anti-VAR2CSA antibodies, supporting VAR2CSA as the leading PM-vaccine candidate. However, the first VAR2CSA subunit vaccines failed to induce broadly neutralizing antibody and it is known that naturally acquired antibodies target both variant-specific and conserved epitopes. It is crucial to determine whether effective vaccines will require incorporation of many or only a single VAR2CSA variants. Here, IgG from multigravidae was sequentially purified on five full-length VAR2CSA ectodomain variants, thereby depleting IgG reactivity to each. The five VAR2CSA variants purified ~0.7% of total IgG and yielded both strain-transcending and strain-specific reactivity to VAR2CSA and IE-surface antigen. In two independent antibody purification/depletion experiments with permutated order of VAR2CSA variants, IgG purified on the first VAR2CSA antigen displayed broad cross-reactivity to both recombinant and native VAR2CSA variants, and inhibited binding of all isolates to CSA. IgG remaining after depletion on all variants showed significantly reduced binding-inhibition activity compared to initial total IgG. These findings demonstrate that a single VAR2CSA ectodomain variant displays conserved epitopes that are targeted by neutralizing (or binding-inhibitory) antibodies shared by multiple parasite strains, including maternal isolates. This suggests that a broadly effective PM-vaccine can be achieved with a limited number of VAR2CSA variants.

Identification of Microorganisms associated with intraamniotic infection among women with preterm birth at Ruhengeri Referral Hospital, Rwanda: A case control study

Background: Preterm birth is a global public health threat for maternal and child health. Each year, 15 million neonates are born preterm worldwide, with 40% resulting from intrauterine infections. Materials and Methods: This cross-sectional and case–control study was conducted from October to February 2019 at Ruhengeri Referral Hospital. A total of 120 swab samples were collected from 40 women, of which 20 were full-term delivery, while the other 20 were preterm delivery. The three samples, including the placental membranes, amniotic fluids, and fetal membranes, were collected immediately after birth. A sterile cotton swab was used to collect the samples and put into swab Stuart sterile plastic container to avoid sample contamination. Samples were transported in a tightly covered carrier to the clinical microbiology laboratory at INES Ruhengeri for microbiological investigation. Gram staining, culture, and biochemical tests were performed. The independent t-test was used to test for significant differences between the means of the two groups, while the Chi-square test (χ2) was used to test for significant association with microorganisms and intra-amniotic infections. Results: A half of the participants were in the age range of 24–29 years. Non-albicans candida (32.7%) and mold (27.9%) were the predominant microorganisms isolated. Non-albicans candida and mold were common to preterm and full-term samples. Staphylococcus species were observed in placental and fetal membrane samples. Escherichia coli, Klebsiella species, Streptococcus species, and Candida albicans were observed among women with preterm birth samples. There was a statistically significant difference between the two means in the amniotic fluid isolates (t = 4.023, P < 0.007), placental membrane isolates (t = 7.17, P < 0.0004), and fetal membrane isolates (t = 6.7, P < 0.0006). Association with microorganisms and intra-amniotic infection was statistically significant with E. coli (χ2 = 3.98, P < 0.05), Streptococcus species (χ2 = 5.53, P < 0.019), non-albicans candida (χ2 = 8.37, P < 0.004), and C. albicans (χ2 = 3.98, P < 0.05). Conclusions: Invasion of the amniotic fluid, placenta, and fetal membranes by pathogenic microorganisms may be associated with the incidence of preterm labor and birth. Early diagnosis is recommended to avoid both maternal and fetal complications.

Is There a Role for Placental Cultures in Cases of Clinical Chorioamnionitis Complicating Preterm Premature Rupture of Membranes?

Objective To assess the role of placental cultures in cases of preterm premature rupture of membranes (PPROM) complicated by chorioamnionitis and to determine the effect of positive cultures on short-term neonatal outcomes. Design A retrospective single-center study. The medical records of all women with PPROM between January 1, 2011, and December 31, 2015, were reviewed. Cases were divided into placental culture positive (group A) and placental culture negative (group B) groups. Maternal and pregnancy characteristics as well as short-term neonatal outcomes were compared between groups. Results During the 5-year study period, 61 cases of clinical chorioamnionitis complicating PPROM were diagnosed: 25 cases were culture positive (group A) and 36 were culture negative (group B). Neonatal outcome measures, including Apgar score at 5 minutes (p = 0.028; odds ratio [OR]: 5.27; confidence interval [CI]: 1.19-23.34), respiratory distress syndrome (p = 0.026; OR: 4.11; CI: 1.18-14.25), and neonatal infection (p < 0.0001; OR: 11.59; CI: 3.37-39.87) were significantly more common in group A newborns, regardless of gestational age at delivery as was the composite neonatal outcome (p = 0.017; OR: 7.35: CI: 1.42-37.79). Placental isolates were primarily Streptococci and Escherichia coli. Conclusion Placental cultures may be an essential predictor of neonatal morbidity in PPROM and may contribute to the modification of neonatal treatment.

High concordance of Pfdhfr and Pfdhps genotypes between matched peripheral and placental isolates of delivered women in Bobo-Dioulasso, Burkina Faso

Whether maternal peripheral parasites constitute a representative sample of the overall population infecting the individual, remains unknown in Burkina Faso. We therefore compared Pfdhfr and Pfdhps genotypes between matched peripheral and placental isolates. PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of polymorphic codons of the Pfdhfr gene (51, 59, 108 and 164) and the Pfdhps gene (437 and 540) was performed in 18 matched peripheral and placental dried blood spots of delivered women in Bobo-Dioulasso. Both Pfdhfr and Pfdhps genes were successfully genotyped in 94.4% (17/18) of the matched samples. Only 8.8% (3/34) of genotypes were of the wild type, while 20.6% (7/34), 20.6% (7/34), 23.5% (8/34) and 26.5% (9/34) comprised one, two, three and four mutations, respectively. None of the samples carried both Pfdhfr I164L and Pfdhps K540E mutations. A concordance of 82.4% was observed in matched samples for both the Pfdhfr and Pfdhps genes. Setting placental alleles as the reference, a concordance of 100% was obtained with Pfdhfr mutation S108N, Pfdhfr mutation C59R+S108N, and Pfdhfr mutation N51I+C59R +S108N, respectively. Likewise, a concordance of 85.7% was observed with the Pfdhps mutation A437G. For epidemiological purposes, peripheral blood Pfdhfr and Pfdhps genotyping is sufficient for monitoring SP resistant molecular markers in pregnant women.

Abstract 52: Isolation, Characterization and Partial Direct Differentiation of Patient Derived Human Primary Cardiac and Placental Fibroblasts From Surgical Discard Tissue into Induced Mesoderm Progenitor Cell

Background: Human primary cells more closely mimic the physiological state of cells in vivo and generate more relevant data representing living systems. Access to the surgical discard tissue enables the possibility of patient derived, disease specific, cellular bio-bank. In achieving this goal, target cellular lines must be isolated, characterized and fully expanded under specific Standard Operating Procedures(SOP). In this project, we aim in achieving and implementing such goal for Fibroblasts derived from two distinct patient tissues. In parallel, in light of cardiac regenerative therapy, with reference to the published work on Cardiomyocyte Direct Differentiation on mouse fibroblasts into induced cardiomyocyte-like cells (iCMs) by transduction with MicroRNAs (1,133a, 208a and 499) we sought to determine whether primary human cardiac and placental fibroblasts could have the potential in direct differentiation to induced cardiomyocyte-like cells (iCM) through sequential administration of small molecule (ITD-1) in combination with MicroRNAs. Methods and Results: Primary right atrial appendage cardiac and smooth chorion laeve placental fibroblast isolates were generated from surgical discard patient derived tissues subjected for identification of the most efficient isolation method through available enzymatic and mechanical techniques. We have shown through FACS analysis that both groups extracellular marker expression can be limited to CD90+CD31-CD44+FBSP+ which constitutes the majority of cultured primary fibroblasts. Respectively, intercellular expression of DDR2+ VIM+ COL1+CD90+ markers was validated through immunofluorescence microscopy. Transcriptional profiling of Noted lines showed expression of Fibroblast specific genes at higher rate.Furthermore, sorted placental fibroblast isolates were subjected to (ITD-1)treatment and mature MicroRNA mimic cocktail. Transcriptional profiling analysis through RT-PCR concluded down regulation of most distinct fibroblast markers and partial expression of MESP1.

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-016-1342-2) contains supplementary material, which is available to authorized users.

Identification of a gene in Mycoplasma hominis associated with preterm birth and microbial burden in intraamniotic infection

Microbial invasion of the amniotic cavity is associated with spontaneous preterm labor and adverse pregnancy outcome, and Mycoplasma hominis often is present. However, the pathogenic process by which M hominis invades the amniotic cavity and gestational tissues, often resulting in chorioamnionitis and preterm birth, remains unknown. We hypothesized that strains of M hominis vary genetically with regards to their potential to invade and colonize the amniotic cavity and placenta. We sequenced the entire genomes of 2 amniotic fluid isolates and a placental isolate of M hominis from pregnancies that resulted in preterm births and compared them with the previously sequenced genome of the type strain PG21. We identified genes that were specific to the amniotic fluid/placental isolates. We then determined the microbial burden and the presence of these genes in another set of subjects from whom samples of amniotic fluid had been collected and were positive for M hominis. We identified 2 genes that encode surface-located membrane proteins (Lmp1 and Lmp-like) in the sequenced amniotic fluid/placental isolates that were truncated severely in PG21. We also identified, for the first time, a microbial gene of unknown function that is referred to in this study as gene of interest C that was associated significantly with bacterial burden in amniotic fluid and the risk of preterm delivery in patients with preterm labor. A gene in M hominis was identified that is associated significantly with colonization and/or infection of the upper reproductive tract during pregnancy and with preterm birth.

VAR2CSA Signatures of High Plasmodium falciparum Parasitemia in the Placenta

Plasmodium falciparum infected erythrocytes (IE) accumulate in the placenta through the interaction between Duffy-binding like (DBL) domains of parasite-encoded ligand VAR2CSA and chondroitin sulphate-A (CSA) receptor. Polymorphisms in these domains, including DBL2X and DBL3X, may affect their antigenicity or CSA-binding affinity, eventually increasing parasitemia and its adverse effects on pregnancy outcomes. A total of 373 DBL2X and 328 DBL3X sequences were obtained from transcripts of 20 placental isolates infecting Mozambican women, resulting in 176 DBL2X and 191 DBL3X unique sequences at the protein level. Sequence alignments were divided in segments containing combinations of correlated polymorphisms and the association of segment sequences with placental parasite density was tested using Bonferroni corrected regression models, taking into consideration the weight of each sequence in the infection. Three DBL2X and three DBL3X segments contained signatures of high parasite density (P<0.003) that were highly prevalent in the parasite population (49–91%). Identified regions included a flexible loop that contributes to DBL3X-CSA interaction and two DBL3X motifs with evidence of positive natural selection. Limited antibody responses against signatures of high parasite density among malaria-exposed pregnant women could not explain the increased placental parasitemia. These results suggest that a higher binding efficiency to CSA rather than reduced antigenicity might provide a biological advantage to parasites with high parasite density signatures in VAR2CSA. Sequences contributing to high parasitemia may be critical for the functional characterization of VAR2CSA and the development of tools against placental malaria.

Improved Pregnancy Outcomes in Women Exposed to Malaria With High Antibody Levels Against Plasmodium falciparum

Antibodies against VAR2CSA, the Plasmodium falciparum variant surface antigen that binds placental chondroitin sulfate A, have been suggested to mediate protection against malaria in pregnancy but also to be markers of infection. Here, we aimed to identify clinically relevant antibody responses, taking into consideration variations in parasite exposure and human immunodeficiency virus type 1 (HIV) infection status. Levels of immunoglobulin G (IgG) against placental and pediatric isolates, VAR2CSA (DBL2X, DBL3X, DBL5ε, and DBL6ε domains), and other blood-stage antigens (DBLγ, DBLα, MSP119, AMA1, and EBA175) were measured in plasma specimens from 293 pregnant Mozambican women at delivery. Associations between antibody responses, factors influencing malaria exposure, HIV infection status, and pregnancy outcomes were assessed. Maternal antibodies were affected by placental infection, parity, season, and neighborhood of residence. HIV infection modified these associations and attenuated the parity-dependent increase in IgG level. High levels of antibody against AMA1, DBL3X, DBL6ε, placental isolates, and pediatric isolates were associated with increased weight and gestational age of newborns (P ≤ .036) among women with malaria episodes during pregnancy. Antiparasite IgGs in women at delivery are affected by HIV infection, as well as by variations in the exposure to P. falciparum. Heterogeneity of malaria transmission needs to be considered to identify IgGs against VAR2CSA and other parasite antigens associated with improved pregnancy outcomes.

Antibodies to Escherichia coli-Expressed C-Terminal Domains of Plasmodium falciparum Variant Surface Antigen 2-Chondroitin Sulfate A (VAR2CSA) Inhibit Binding of CSA-Adherent Parasites to Placental Tissue

Placental malaria (PM) is characterized by infected erythrocytes (IEs) that selectively bind to chondroitin sulfate A (CSA) and sequester in placental tissue. Variant surface antigen 2-CSA (VAR2CSA), a Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) protein family member, is expressed on the surface of placental IEs and mediates adherence to CSA on the surface of syncytiotrophoblasts. This transmembrane protein contains 6 Duffy binding-like (DBL) domains which might contribute to the specific adhesive properties of IEs. Here, we use laboratory isolate 3D7 VAR2CSA DBL domains expressed in Escherichia coli to generate antibodies specific for this protein. Flow cytometry results showed that antibodies generated against DBL4ε, DBL5ε, DBL6ε, and tandem double domains of DBL4-DBL5 and DBL5-DBL6 all bind to placental parasite isolates and to lab strains selected for CSA binding but do not bind to children's parasites. Antisera to DBL4ε and to DBL5ε inhibit maternal IE binding to placental tissue in a manner comparable to that for plasma collected from multigravid women. These antibodies also inhibit binding to CSA of several field isolates derived from pregnant women, while antibodies to double domains do not enhance the functional immune response. These data support DBL4ε and DBL5ε as vaccine candidates for pregnancy malaria and demonstrate that E. coli is a feasible tool for the large-scale manufacture of a vaccine based on these VAR2CSA domains.

Transcription of var Genes Other Than var2csa in Plasmodium falciparum Parasites Infecting Mozambican Pregnant Women

Increased susceptibility to Plasmodium falciparum infection during pregnancy has been attributed to the accumulation of infected erythrocytes in the placenta. This phenomenon is mediated by a var gene coding for VAR2CSA, which adheres to chondroitin sulphate A. However, the contribution of parasites transcribing other var genes to maternal infections has not been well characterized. Transcription of var2csa and var groups A, B, and C was measured by real-time polymerase chain reaction in 30 placental and 21 peripheral P. falciparum isolates from pregnant women and in 42 isolates from nonpregnant adults and children. Associations of infections with non-var2csa isolates with maternal parasitemia and immune responses were assessed. Placental parasites showed the highest levels of var2csa. ABC var genes were transcribed by 20 (67%) of 30 placental isolates and were associated with higher parasitemia compared with infections by parasites only transcribing var2csa (P = .004). Peripheral isolates from pregnant women transcribed ABC var genes at levels similar to those of parasites infecting nonpregnant adults with clinical malaria (P[varA] = .420, P[varB] = .808, and P[varC] = .619). Transcripts of var2csa are abundant in pregnancy-associated P. falciparum infections; however, ABC var types are also common, especially in peripheral blood, with transcription levels similar to those of infections out of pregnancy. These findings are of interest for the design of malaria vaccines for pregnant women.

Insight into Antigenic Diversity of VAR2CSA-DBL5ε Domain from Multiple Plasmodium falciparum Placental Isolates

BackgroundProtection against pregnancy associated malaria (PAM) is associated with high levels of anti-VAR2CSA antibodies. This protection is obtained by the parity dependent acquisition of anti-VAR2CSA antibodies. Distinct parity-associated molecular signatures have been identified in VAR2CSA domains. These two observations combined point to the importance of identifying VAR2CSA sequence variation, which facilitate parasitic evasion or subversion of host immune response. Highly conserved domains of VAR2CSA such as DBL5ε are likely to contain conserved epitopes, and therefore do constitute attractive targets for vaccine development.Methodology/Principal FindingsVAR2CSA DBL5ε-domain sequences obtained from cDNA of 40 placental isolates were analysed by a combination of experimental and in silico methods. Competition ELISA assays on two DBL5ε variants, using plasma samples from women from two different areas and specific mice hyperimmune plasma, indicated that DBL5ε possess conserved and cross-reactive B cell epitopes. Peptide ELISA identified conserved areas that are recognised by naturally acquired antibodies. Specific antibodies against these peptides labelled the native proteins on the surface of placental parasites. Despite high DBL5ε sequence homology among parasite isolates, sequence analyses identified motifs in DBL5ε that discriminate parasites according to donor's parity. Moreover, recombinant proteins of two VAR2CSA DBL5ε variants displayed diverse recognition patterns by plasma from malaria-exposed women, and diverse proteoglycan binding abilities.Conclusions/SignificanceThis study provides insights into conserved and exposed B cell epitopes in DBL5ε that might be a focus for cross reactivity. The importance of sequence variation in VAR2CSA as a critical challenge for vaccine development is highlighted. VAR2CSA conformation seems to be essential to its functionality. Therefore, identification of sequence variation sites in distinct locations within VAR2CSA, affecting antigenicity and/or binding properties, is critical to the effort of developing an efficient VAR2CSA-based vaccine. Motifs associated with parasite segregation according to parity constitute one such site.

Plasmodium falciparum population dynamics in a cohort of pregnant women in Senegal

BackgroundPregnant women acquire protective antibodies that cross-react with geographically diverse placental Plasmodium falciparum isolates, suggesting that surface molecules expressed on infected erythrocytes by pregnancy-associated malaria (PAM) parasites have conserved epitopes and, that designing a PAM vaccine may be envisaged. VAR2CSA is the main candidate for a pregnancy malaria vaccine, but vaccine development may be complicated by its sequence polymorphism.MethodsThe dynamics of P. falciparum genotypes during pregnancy in 32 women in relation to VAR2CSA polymorphism and immunity was determined. The polymorphism of the msp2 gene and five microsatellites was analysed in consecutive parasite isolates, and the DBL5ε + Interdomain 5 (Id5) part of the var2csa gene of the corresponding samples was cloned and sequenced to measure variation.ResultsIn primigravidae, the multiplicity of infection in the placenta was associated with occurrence of low birth weight babies. Some parasite genotypes were able to persist over several weeks and, still be present in the placenta at delivery particularly when the host anti-VAR2CSA antibody level was low. Comparison of diversity among genotyping markers confirmed that some PAM parasites may harbour more than one var2csa gene copy in their genome.ConclusionsHost immunity to VAR2CSA influences the parasite dynamics during pregnancy, suggesting that the acquisition of protective immunity requires pre-exposure to a limited number of parasite variants. Presence of highly conserved residues in surface-exposed areas of the VAR2CSA immunodominant DBL5ε domain, suggest its potential in inducing antibodies with broad reactivity.

Functional and immunological characterization of the var2CSA-DBL5ɛ domain of a placental Plasmodium falciparum isolate

Pregnancy-associated malaria (PAM) arises from sequestration of Plasmodium falciparum-parasitized erythrocytes (PE) in the placenta, leading to chronic symptoms in the expectant mother and serious consequences for fetal development. Placental sequestration has been linked to binding of chondroitin sulphate A (CSA) by the var2CSA variant of PfEMP1 expressed on the PE surface, and a substantial body of evidence shows that the immune response to var2CSA gives an effective protection against PAM. We have expressed the var2CSA-DBL5ɛ domain, derived from a placental isolate from Senegal, as soluble product in Escherichia coli and have shown using different criteria that the recombinant protein is obtained with the native conformation. Using surface plasmon resonance techniques, we have examined binding of DBL5ɛ to placental chondroitin sulphate proteoglyan and CSA; however, the recombinant protein also binds to other sulphated oligosaccharides, with higher affinity in some cases, indicating that the single domain lacks the specificity for CSA shown by the complete extra-cellular region of var2CSA and placental parasites. Recombinant DBL5ɛ was specifically recognized by sera from malaria-exposed Senegalese women in a parity-dependent manner but by sera not from children or males from the same endemic region. Conversely, DBL5ɛ induced antibodies in mice that recognized placental isolates from Benin but not isolates from children. The presence of universal epitopes thus supports DBL5ɛ as an interesting component of var2CSA to be considered for vaccine development.

Maternal and neonatal sepsis caused by Haemophilus influenzae type d

A 29-year-old pregnant woman was admitted to hospital with signs of sepsis and threatened pre-term labour. The premature neonate also showed signs of sepsis. Haemophilus influenzae biotype III was cultured from a midstream urine sample taken from the mother, maternal placental swabs and neonatal blood cultures. The placental and neonatal isolates were both found to be serotype d by PCR, and were indistinguishable by PFGE.

Immunization with VAR2CSA-DBL5 Recombinant Protein Elicits Broadly Cross-Reactive Antibodies to Placental Plasmodium falciparum -Infected Erythrocytes

Pregnancy-associated malaria is a severe clinical syndrome associated with the sequestration of Plasmodium falciparum-infected erythrocytes in the placenta. Placental binding is mediated by VAR2CSA, a member of the large and diverse P. falciparum erythrocyte membrane 1 (PfEMP1) protein family. To better understand if conserved regions in VAR2CSA can be targeted by antibodies, we immunized rabbits with VAR2CSA-DBL1 and -DBL5 recombinant proteins produced in Pichia pastoris and developed a panel of seven chondroitin sulfate A (CSA)-binding parasites from diverse geographic origins. Overall, no two parasites in the panel expressed the same VAR2CSA sequence. The DBL1 domains averaged 80% amino acid identity (range, 72 to 89%), and the DBL5 domains averaged 86% amino acid identity (range, 83 to 99%), similar to a broader sampling of VAR2CSA sequences from around the world. Whereas antibodies generated against the VAR2CSA-DBL1 recombinant protein had only limited breadth and reacted with three or four parasites in the panel, immunization with DBL5 recombinant proteins elicited broadly cross-reactive antibodies against all or most parasites in the panel, as well as to fresh clinical isolates from pregnant women. These findings demonstrate that the major PfEMP1 variant expressed by placental isolates exposes strain-transcendent epitopes that can be targeted by vaccination and may have application for pregnancy malaria vaccine development.

High prevalence of sulfadoxine/pyrimethamine-resistant alleles of Plasmodium falciparum isolates in pregnant women at the time of introduction of intermittent preventive treatment with sulfadoxine/pyrimethamine in Gabon

The frequency of dhfr and dhps point mutations was assessed in Plasmodium falciparum isolates from pregnant women in Libreville. PCR-restriction fragment length polymorphism of polymorphic codons of the dhfr gene (51, 59 and 108) and the dhps gene (436, 437 and 540) was performed in matched peripheral and placental blood samples. The proportion of multiple mutations was high (98%), and was not different between women with and without a history of intermittent preventive treatment with sulfadoxine/pyrimethamine (IPTp/SP). The prevalence of triple dhfr mutation was 80%, and that of quadruple and quintuple mutations was 53% and 22%, respectively. The Glu540 mutation was present in two isolates. The concordance of resistant alleles in matched peripheral and placental isolates was >90% for both genes. These findings underline the need for a regular assessment of the relationship between the presence of resistant isolates and in vitro/in vivo IPTp/SP efficacy, and evaluation of an alternative drug.

Impact of Human Immunodeficiency Virus Infection in Pregnant Women on Variant-Specific Immunity to Malaria

Human immunodeficiency virus (HIV) increases susceptibility to Plasmodium falciparum infection, and this has most clearly been demonstrated in pregnant women. Variant surface antigens on the surfaces of erythrocytes infected with P. falciparum are major targets of protective immunity. We studied the impact of HIV infection on pregnant women's humoral immunity to variant surface antigens expressed by placental and pediatric isolates of P. falciparum. By flow cytometry, sera from HIV-infected women more frequently lacked antibodies to these antigens than sera from HIV-uninfected women. This difference was similar in magnitude for pediatric isolates (unadjusted odds ratio [OR] = 6.36; 95% confidence interval [CI] = 1.14, 35.32; P < 0.05) and placental isolates (unadjusted OR = 6.47; 95% CI = 0.75, 55.64; P < 0.10). We divided women into high and low responders on the basis of their antibody levels. After adjustment for CD4 count, maternal age, and gravidity, we found that HIV-infected women more frequently had low responses to both pediatric isolates (OR = 5.34; 95% CI = 1.23, 23.16; P = 0.025) and placental isolates (OR = 4.14; 95% CI = 1.71, 10.02; P = 0.002). The relative quantity of antibodies to both pediatric isolates (P = 0.035) and placental isolates (P = 0.005) was lower in HIV-infected women than in HIV-uninfected women. HIV infection has a broad impact on variant-specific immunity, which may explain the susceptibility of infected individuals to clinical malaria episodes.