Identification of glycosaminoglycan binding regions in the Plasmodium falciparum encoded placental sequestration ligand, VAR2CSA

<p>Abstract</p> <p>Background</p> <p>Pregnancy malaria is caused by <it>Plasmodium falciparum</it>-infected erythrocytes binding the placental receptor chondroitin sulfate A (CSA). This results in accumulation of parasites in the placenta with severe clinical consequences for the mother and her unborn child. Women become resistant to placental malaria as antibodies are acquired which specifically target the surface of infected erythrocytes binding in the placenta. VAR2CSA is most likely the parasite-encoded protein which mediates binding to the placental receptor CSA. Several domains have been shown to bind CSA <it>in vitro</it>; and it is apparent that a VAR2CSA-based vaccine cannot accommodate all the CSA binding domains and serovariants. It is thus of high priority to define minimal ligand binding regions throughout the VAR2CSA molecule.</p> <p>Methods</p> <p>To define minimal CSA-binding regions/peptides of VAR2CSA, a phage display library based on the entire <it>var2csa </it>coding region was constructed. This library was screened on immobilized CSA and cells expressing CSA resulting in a limited number of CSA-binding phages. Antibodies against these peptides were affinity purified and tested for reactivity against CSA-binding infected erythrocytes.</p> <p>Results</p> <p>The most frequently identified phages expressed peptides residing in the parts of VAR2CSA previously defined as CSA binding. In addition, most of the binding regions mapped to surface-exposed parts of VAR2CSA. The binding of a DBL2X peptide to CSA was confirmed with a synthetic peptide. Antibodies against a CSA-binding DBL2X peptide reacted with the surface of infected erythrocytes indicating that this epitope is accessible for antibodies on native VAR2CSA on infected erythrocytes.</p> <p>Conclusion</p> <p>Short continuous regions of VAR2CSA with affinity for multiple types of CSA were defined. A number of these regions localize to CSA-binding domains and to surface-exposed regions within these domains and a synthetic peptide corresponding to a peptide sequence in DBL2 was shown to bind to CSA and not to CSC. It is likely that some of these epitopes are involved in native parasite CSA adhesion. However, antibodies directed against single epitopes did not inhibit parasite adhesion. This study supports phage display as a technique to identify CSA-binding regions of large proteins such as VAR2CSA.</p

Marked reduction in fertility among African women with urogenital infections:A prospective cohort study

<div><p>Background</p><p>There is paucity of data on risk factors for reduced fertility in low-income countries.</p><p>Objective</p><p>To investigate factors associated with fertility among women in rural north eastern Tanzania.</p><p>Subjects and methods</p><p>A cohort of 1248 non-pregnant women was followed with urine pregnancy testing every third month or more regularly if they reported a missed menstrual period. Pregnancy was confirmed with trans-abdominal ultrasound. Information regarding general health, socioeconomic status and obstetric-gynaecological history was collected. Factors associated with conceiving within 180 days were identified using multivariate logistic regression analyses.</p><p>Results</p><p>Among the 1248 women, 736 were followed for 180 days and 209 of these had an ultrasound confirmed pregnancy. During the follow-up period, 169/736 women were diagnosed with urogenital infections, including suspected sexually transmitted or reproductive tract infections, urinary tract infection, and vaginal candidiasis. Urogenital infections were significantly associated with reduced odds of conceiving within 180 days (adjusted OR (AOR) 0.21, 95% CI 0.11–0.36). Being above 30 years of age was also negatively associated with odds of conceiving (AOR 0.45, 95% CI 0.26–0.77). In contrast, women who recently stopped using hormonal contraceptives (AOR 2.86, 95% CI 1.45–5.70) and women with low socioeconomic status (AOR 1.56, 95% CI 1.04–2.33) were significantly more likely to become pregnant within 180 days.</p><p>Conclusion</p><p>Urogenital infection seems to be a major health factor associated with reduced chances of conceiving. Considering the availability of effective treatment options for these diseases, public health authorities should increase awareness of diagnostic tools in settings with limited resources in order to improve fertility.</p></div

Cellulose filtration of blood from malaria patients for improving <i>ex vivo</i> growth of <i>Plasmodium falciparum</i> parasites

BACKGROUND: Establishing in vitro Plasmodium falciparum culture lines from patient parasite isolates can offer deeper understanding of geographic variations of drug sensitivity and mechanisms of malaria pathogenesis and immunity. Cellulose column filtration of blood is an inexpensive, rapid and effective method for the removal of host factors, such as leucocytes and platelets, significantly improving the purification of parasite DNA in a blood sample. METHODS: In this study, the effect of cellulose column filtration of venous blood on the initial in vitro growth of P. falciparum parasite isolates from Tanzanian children admitted to hospital was tested. The parasites were allowed to expand in culture without subcultivation until 5 days after admission or the appearance of dead parasites and parasitaemia was determined daily. To investigate whether the filtration had an effect on clonality, P. falciparum merozoite surface protein 2 genotyping was performed using nested PCR on extracted genomic DNA, and the var gene transcript levels were investigated, using quantitative PCR on extracted RNA, at admission and 4 days of culture. RESULTS: The cellulose-filtered parasites grew to higher parasitaemia faster than non-filtered parasites seemingly due to a higher development ratio of ring stage parasites progressing into the late stages. Cellulose filtration had no apparent effect on clonality or var gene expression; however, evident differences were observed after only 4 days of culture in both the number of clones and transcript levels of var genes compared to the time of admission. CONCLUSIONS: Cellulose column filtration of parasitized blood is a cheap, applicable method for improving cultivation of P. falciparum field isolates for ex vivo based assays; however, when assessing phenotype and genotype of cultured parasites, in general, assumed to represent the in vivo infection, caution is advised. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12936-017-1714-2) contains supplementary material, which is available to authorized users

Haplotypes of the Endothelial Protein C Receptor (EPCR) Gene are Not Associated with Severe Malaria in Tanzania.

Endothelial protein C receptor (EPCR) was recently identified as a key receptor for Plasmodium falciparum erythrocyte membrane protein 1 mediating sequestration of P. falciparum-infected erythrocytes in patients suffering from severe malaria. Soluble EPCR (sEPCR) inhibits binding of P. falciparum to EPCR in vitro and increased levels of sEPCR have been associated with the H3 haplotype of the EPCR encoding PROCR gene. It has been hypothesized that elevated sEPCR levels, possibly linked to the PROCR H3 genetic variant, may confer protection against severe forms of malaria. This study determined the frequencies of PROCR haplotypes H1-4 and plasma levels of sEPCR in a Tanzanian study population to investigate a possible association with severe malaria. Study participants were children under 5 years of age admitted at the Korogwe District Hospital (N = 143), and diagnosed as having severe malaria (N = 52; including cerebral malaria N = 17), uncomplicated malaria (N = 24), or an infection other than malaria (N = 67). In addition, blood samples from 71 children living in nearby villages were included. The SNPs defining the haplotypes of PROCR gene were determined by post-PCR ligation detection reaction-fluorescent microsphere assay. Individuals carrying at least one H3 allele had significantly higher levels of sEPCR than individuals with no H3 alleles (P < 0.001). No difference in the frequency of H3 was found between the non-malaria patients, malaria patients or the village population (P > 0.1). Plasma levels of sEPCR differed between these three groups, with higher sEPCR levels in the village population compared to the hospitalized patients (P < 0.001) and higher levels in malaria patients compared to non-malaria patients (P = 0.001). However, no differences were found in the distribution of H3 (P = 0.2) or levels of sEPCR (P = 0.8) between patients diagnosed with severe and uncomplicated malaria. Frequencies of SNPs determining PROCR haplotypes were in concordance with other African studies. The PROCR H3 allele was associated with higher levels of sEPCR, confirming earlier findings, however, in this Tanzanian population; neither PROCR haplotype nor level of sEPCR was associated with severe malaria, however, larger studies are needed to confirm these findings

Plasma Ang2 and ADAM17 levels are elevated during clinical malaria; Ang2 level correlates with severity and expression of EPCR-binding PfEMP1

The pathogenesis of Plasmodium falciparum malaria involves a complex interplay between parasite adhesion and inflammatory response that includes release of cytokines and activation of the endothelium with accompanying release of Angiopoitin 2 (Ang2) to the plasma. A-disintegrin and metalloproteinase 17 (ADAM17) is a protein responsible for releasing cytokines, including Tumor Necrosis Factor α (TNFα), and shedding of adhesion proteins. In this study, we show that plasma levels of ADAM17 are increased in Tanzanian children hospitalized with a malaria infection compared with asymptomatic children but similar to children hospitalized with other infectious diseases. The plasma levels of ADAM17 decreased during recovery after an acute malaria episode. Plasma levels of Ang2 were associated with markers of malaria severity and levels of var transcripts encoding P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) containing Cysteine Rich Inter Domain Region α1 (CIDRα1) domains predicted to bind Endothelial Protein C receptor (EPCR). ADAM17 levels were not associated with expression of var genes encoding different PfEMP1 types when controlling for age. These data are the first to report ADAM17 plasma levels in malaria-exposed individuals, and support the notion that parasite sequestration mediated by EPCR-binding PfEMP1 is associated with endothelial activation and pathology in severe paediatric malaria

Reliability of Rapid Diagnostic Tests in Diagnosing Pregnancy-Associated Malaria in North-Eastern Tanzania.

Accurate 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. A 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. From 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. Microscopy 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

Identification of a major rif transcript common to gametocytes and sporozoites of Plasmodium falciparum

Background: The Plasmodium falciparum parasite is transmitted in its sexual gametocyte stage from man to mosquito and as asexual sporozoites from mosquito to man. Developing gametocytes sequester preferentially in the bone marrow, but mature stage gametocytes are released to the bloodstream. Sexual stage parasite surface proteins are of interest as candidate target antigens for transmission blocking vaccines.Methods: In this study, the transcript profiles of rif and var genes, known to encode surface antigens in asexual blood stage parasites, were investigated at different stages of 3D7/NF54 gametocytogenesis and in sporozoites.Results: Gametocytes exhibited a rif transcript profile unlinked to the rif and var transcript profile of the asexual progenitors. At stage V, mature gametocytes produced high levels of a single rif gene, PF13_0006, which also dominated the rif transcript profile of sporozoites. All var genes appeared to be silenced in sporozoites.Conclusions: The most prominent variant surface antigen transcribed in both gametocytes and sporozoites of 3D7/NF54 is a single variant of the RIFIN protein family. This discovery may lead to the identification of the parasites binding ligands responsible for the adhesion during sexual stages and potentially to novel vaccine candidates