Expression of Plasmodium falciparum erythrocyte membrane protein 1 in experimentally infected humans

BACKGROUND: Parasites causing severe malaria in non-immune patients express a restricted subset of variant surface antigens (VSA), which are better recognized by immune sera than VSA expressed during non-severe disease in semi-immune individuals. The most prominent VSA are the var gene-encoded Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family, which is expressed on the surface of infected erythrocytes where it mediates binding to endothelial receptors. Thus, severe malaria may be caused by parasites expressing PfEMP1 variants that afford parasites optimal sequestration in immunologically naïve individuals and high effective multiplication rates. METHODS: var gene transcription was analysed using real time PCR and PfEMP1 expression by western blots as well as immune plasma recognition of parasite cultures established from non-immune volunteers shortly after infection with NF54 sporozoites. RESULTS: In cultures representing the first generation of parasites after hepatic release, all var genes were transcribed, but GroupA var genes were transcribed at the lowest levels. In cultures established from second or third generation blood stage parasites of volunteers with high in vivo parasite multiplication rates, the var gene transcription pattern differed markedly from the transcription pattern of the cultures representing first generation parasites. This indicated that parasites expressing specific var genes, mainly belonging to group A and B, had expanded more effectively in vivo compared to parasites expressing other var genes. The differential expression of PfEMP1 was confirmed at the protein level by immunoblot analysis. In addition, serological typing showed that immune sera more often recognized second and third generation parasites than first generation parasites. CONCLUSION: In conclusion, the results presented here support the hypothesis that parasites causing severe malaria express a subset of PfEMP1, which bestows high parasite growth rates in individuals with limited pre-existing immunity

Development of a Fetal Weight Chart using Serial Trans-Abdominal Ultrasound in an East African Population: A Longitudinal Observational Study.

To produce a fetal weight chart representative of a Tanzanian population, and compare it to weight charts from Sub-Saharan Africa and the developed world. A longitudinal observational study in Northeastern Tanzania. Pregnant women were followed throughout pregnancy with serial trans-abdominal ultrasound. All pregnancies with pathology were excluded and a chart representing the optimal growth potential was developed using fetal weights and birth weights. The weight chart was compared to a chart from Congo, a chart representing a white population, and a chart representing a white population but adapted to the study population. The prevalence of SGA was assessed using all four charts. A total of 2193 weight measurements from 583 fetuses/newborns were included in the fetal weight chart. Our chart had lower percentiles than all the other charts. Most importantly, in the end of pregnancy, the 10(th) percentiles deviated substantially causing an overestimation of the true prevalence of SGA newborns if our chart had not been used. We developed a weight chart representative for a Tanzanian population and provide evidence for the necessity of developing regional specific weight charts for correct identification of SGA. Our weight chart is an important tool that can be used for clinical risk assessments of newborns and for evaluating the effect of intrauterine exposures on fetal and newborn weight

Plasmodium falciparum associated with severe childhood malaria preferentially expresses PfEMP1 encoded by group A var genes.

Parasite-encoded variant surface antigens (VSAs) like the var gene-encoded Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family are responsible for antigenic variation and infected red blood cell (RBC) cytoadhesion in P. falciparum malaria. Parasites causing severe malaria in nonimmune patients tend to express a restricted subset of VSA (VSA(SM)) that differs from VSA associated with uncomplicated malaria and asymptomatic infection (VSA(UM)). We compared var gene transcription in unselected P. falciparum clone 3D7 expressing VSA(UM) to in vitro-selected sublines expressing VSA(SM) to identify PfEMP1 responsible for the VSA(SM) phenotype. Expression of VSA(SM) was accompanied by up-regulation of Group A var genes. The most prominently up-regulated Group A gene (PFD1235w/MAL7P1.1) was translated into a protein expressed on the infected RBC surface. The proteins encoded by Group A var genes, such as PFD1235w/MAL7P1.1, appear to be involved in the pathogenesis of severe disease and are thus attractive candidates for a vaccine against life-threatening P. falciparum malaria

Accuracy of Malaria Rapid Diagnostic Tests in Community Studies and their Impact on Treatment of Malaria in an Area with Declining Malaria Burden in North-Eastern Tanzania.

Despite some problems related to accuracy and applicability of malaria rapid diagnostic tests (RDTs), they are currently the best option in areas with limited laboratory services for improving case management through parasitological diagnosis and reducing over-treatment. This study was conducted in areas with declining malaria burden to assess; 1) the accuracy of RDTs when used at different community settings, 2) the impact of using RDTs on anti-malarial dispensing by community-owned resource persons (CORPs) and 3) adherence of CORPs to treatment guidelines by providing treatment based on RDT results. Data were obtained from: 1) a longitudinal study of passive case detection of fevers using CORPs in six villages in Korogwe; and 2) cross-sectional surveys (CSS) in six villages of Korogwe and Muheza districts, north-eastern, Tanzania. Performance of RDTs was compared with microscopy as a gold standard, and factors affecting their accuracy were explored using a multivariate logistic regression model. Overall sensitivity and specificity of RDTs in the longitudinal study (of 23,793 febrile cases; 18,154 with microscopy and RDTs results) were 88.6% and 88.2%, respectively. In the CSS, the sensitivity was significantly lower (63.4%; χ2=367.7, p<0.001), while the specificity was significantly higher (94.3%; χ2=143.1, p<0.001) when compared to the longitudinal study. As determinants of sensitivity of RDTs in both studies, parasite density of<200 asexual parasites/μl was significantly associated with high risk of false negative RDTs (OR≥16.60, p<0.001), while the risk of false negative test was significantly lower among cases with fever (axillary temperature ≥37.5 °C) (OR≤0.63, p≤0.027). The risk of false positive RDT (as a determinant of specificity) was significantly higher in cases with fever compared to afebrile cases (OR≥2.40, p<0.001). Using RDTs reduced anti-malarials dispensing from 98.9% to 32.1% in cases aged ≥5 years. Although RDTs had low sensitivity and specificity, which varied widely depending on fever and parasite density, using RDTs reduced over-treatment with anti-malarials significantly. Thus, with declining malaria prevalence, RDTs will potentially identify majority of febrile cases with parasites and lead to improved management of malaria and non-malaria fevers

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

Plasmepsin II–III copy number accounts for bimodal piperaquine resistance among Cambodian Plasmodium falciparum

Multidrug resistant Plasmodium falciparum in Southeast Asia endangers regional malaria elimination and threatens to spread to other malaria endemic areas. Understanding mechanisms of piperaquine (PPQ) resistance is crucial for tracking its emergence and spread, and to develop effective strategies for overcoming it. Here we analyze a mechanism of PPQ resistance in Cambodian parasites. Isolates exhibit a bimodal dose–response curve when exposed to PPQ, with the area under the curve quantifying their survival in vitro. Increased copy number for plasmepsin II and plasmepsin III appears to explain enhanced survival when exposed to PPQ in most, but not all cases. A panel of isogenic subclones reinforces the importance of plasmepsin II–III copy number to enhanced PPQ survival. We conjecture that factors producing increased parasite survival under PPQ exposure in vitro may drive clinical PPQ failures in the field

Multiple var2csa-Type PfEMP1 Genes Located at Different Chromosomal Loci Occur in Many Plasmodium falciparum Isolates

BACKGROUND:The var2csa gene encodes a Plasmodium falciparum adhesion receptor which binds chondroitin sulfate A (CSA). This var gene is more conserved than other PfEMP1/var genes and is found in all P. falciparum isolates. In isolates 3D7, FCR3/It4 and HB3, var2csa is transcribed from a sub-telomeric position on the left arm of chromosome 12, but it is not known if this location is conserved in all parasites. Genome sequencing indicates that the var2csa gene is duplicated in HB3, but whether this is true in natural populations is uncertain. METHODOLOGY/PRINCIPAL FINDINGS:To assess global variation in the VAR2CSA protein, sequence variation in the DBL2X region of var2csa genes in 54 P.falciparum samples was analyzed. Chromosome mapping of var2csa loci was carried out and a quantitative PCR assay was developed to estimate the number of var2csa genes in P.falciparum isolates from the placenta of pregnant women and from the peripheral circulation of other malaria patients. Sequence analysis, gene mapping and copy number quantitation in P.falciparum isolates indicate that there are at least two loci and that both var2csa-like genes can be transcribed. All VAR2CSA DBL2X domains fall into one of two distinct phylogenetic groups possessing one or the other variant of a large (approximately 26 amino acid) dimorphic motif, but whether either motif variant is linked to a specific locus is not known. CONCLUSIONS/SIGNIFICANCE:Two or more related but distinct var2csa-type PfEMP1/var genes exist in many P. falciparum isolates. One gene is on chromosome 12 but additional var2csa-type genes are on different chromosomes in different isolates. Multiplicity of var2csa genes appears more common in infected placentae than in samples from non-pregnant donors indicating a possible advantage of this genotype in pregnancy associated malaria

CD36 selection of 3D7 Plasmodium falciparum associated with severe childhood malaria results in reduced VAR4 expression

<p>Abstract</p> <p>Background</p> <p>A subset of the <it>Plasmodium falciparum </it>erythrocyte membrane protein 1 (PfEMP1_SM) is involved in the cytoadherence of <it>P. falciparum</it>-infected red blood cells (iRBC) contributing to the pathogenesis of severe disease among young children in malaria endemic areas. The PfEMP1_SMare encoded by group A <it>var </it>genes that are composed of a more constrained range of amino acid sequences than groups B and C <it>var </it>genes encoding PfEMP1_UMassociated with uncomplicated malaria. Also, unlike <it>var </it>genes from groups B and C, those from group A do not have sequences consistent with CD36 binding – a major cytoadhesion phenotype of <it>P. falciparum </it>isolates.</p> <p>Methods</p> <p>A 3D7 PfEMP1_SMsub-line (3D7_SM) expressing VAR4 (PFD1235w/MAL8P1.207) was selected for binding to CD36. The protein expression of this parasite line was monitored by surface staining of iRBC using VAR4-specific antibodies. The serological phenotype of the 3D7_SMparasites was determined by flow cytometry using malaria semi-immune and immune plasma and transcription of the 59 <it>var </it>genes in 3D7 were analysed by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) using <it>var</it>-specific primers.</p> <p>Results</p> <p>A selection-induced increased adhesion of 3D7_SMiRBC to CD36 resulted in a reduced <it>var4 </it>transcription and VAR4 surface expression.</p> <p>Conclusion</p> <p>VAR4 is not involved in CD36 adhesion. The current findings are consistent with the notion that CD36 adhesion is not associated with particular virulent parasite phenotypes, such as those believed to be exhibited by VAR4 expressing parasites.</p