Lasse S. Vestergaard

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 (VSASM) that differs from VSA associated with uncomplicated malaria and asymptomatic infection (VSAUM). We compared var gene transcription in unselected P. falciparum clone 3D7 expressing VSAUM to in vitro–selected sublines expressing VSASM to identify PfEMP1 responsible for the VSASM phenotype. Expression of VSASM 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.

A progressive declining in the burden of malaria in north-eastern Tanzania

BackgroundThe planning and assessment of malaria interventions is complicated due to fluctuations in the burden of malaria over time. Recently, it has been reported that the burden of malaria in some parts of Africa has declined. However, community-based longitudinal data are sparse and the reasons for the apparent decline are not well understood.MethodsMalaria prevalence and morbidity have been monitored in two villages in north-eastern Tanzania; a lowland village and a highland village from 2003 to 2008. Trained village health workers treated presumptive malaria with the Tanzanian first-line anti-malarial drug and collected blood smears that were examined later. The prevalence of malaria parasitaemia across years was monitored through cross-sectional surveys.ResultsThe prevalence of malaria parasitaemia in the lowland village decreased from 78.4% in 2003 to 13.0% in 2008, whereas in the highland village, the prevalence of parasitaemia dropped from 24.7% to 3.1% in the same period. Similarly, the incidence of febrile malaria episodes in the two villages dropped by almost 85% during the same period and there was a marked reduction in the number of young children who suffered from anaemia in the lowland village.ConclusionThere has been a marked decline in malaria in the study villages during the past few years. This decline is likely to be due to a combination of factors that include improved access to malaria treatment provided by the trained village helpers, protection from mosquitoes by increased availability of insecticide-impregnated bed nets and a reduced vector density. If this decline in malaria morbidity is sustained, it will have a marked effect on the disease burden in this part of Tanzania.

Sequential, Ordered Acquisition of Antibodies to Plasmodium falciparum Erythrocyte Membrane Protein 1 Domains

The binding of erythrocytes infected with mature blood stage parasites to the vascular bed is key to the pathogenesis of malignant malaria. The binding is mediated by members of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family. PfEMP1s can be divided into groups, and it has previously been suggested that parasites expressing group A or B/A PfEMP1s are most pathogenic. To test the hypothesis that the first malaria infections in infants and young children are dominated by parasites expressing A and B/A PfEMP1s, we measured the plasma Ab level against 48 recombinant PfEMP1 domains of different groupings in 1342 individuals living in five African villages characterized by markedly different malaria transmission. We show that children progressively acquire a broader repertoire of anti-PfEMP1 Abs, but that the rate of expansion is governed by transmission intensity. However, independently of transmission intensity, Abs are first acquired to particular duffy binding ligand-like domains belonging to group A or B/A PfEMP1s. The results support the view that anti-PfEMP1 Ab responses effectively structure the expenditure of the repertoire of PfEMP1 maintained by the parasite. Parasites expressing certain group A and B/A PfEMP1s are responded to first by individuals with limited previous exposure, and the resulting Abs reduce the fitness and pathogenicity of these parasites during subsequent infections. This allows parasites expressing less pathogenic PFEMP1s to dominate during later infections. The identification of PfEMP1 domains expressed by parasites causing disease in infants and young children is important for development of vaccines protecting against severe malaria.

Review of key knowledge gaps in glucose-6-phosphate dehydrogenase deficiency detection with regard to the safe clinical deployment of 8-aminoquinoline treatment regimens: a workshop report

The diagnosis and management of glucose-6-phosphate dehydrogenase (G6PD) deficiency is a crucial aspect in the current phases of malaria control and elimination, which will require the wider use of 8-aminoquinolines for both reducing Plasmodium falciparum transmission and achieving the radical cure of Plasmodium vivax. 8-aminoquinolines, such as primaquine, can induce severe haemolysis in G6PD-deficient individuals, potentially creating significant morbidity and undermining confidence in 8-aminoquinoline prescription. On the other hand, erring on the side of safety and excluding large numbers of people with unconfirmed G6PD deficiency from treatment with 8-aminoquinolines will diminish the impact of these drugs. Estimating the remaining G6PD enzyme activity is the most direct, accessible, and reliable assessment of the phenotype and remains the gold standard for the diagnosis of patients who could be harmed by the administration of primaquine. Genotyping seems an unambiguous technique, but its use is limited by cost and the large range of recognized G6PD genotypes. A number of enzyme activity assays diagnose G6PD deficiency, but they require a cold chain, specialized equipment, and laboratory skills. These assays are impractical for care delivery where most patients with malaria live. Improvements to the diagnosis of G6PD deficiency are required for the broader and safer use of 8-aminoquinolines to kill hypnozoites, while lower doses of primaquine may be safely used to kill gametocytes without testing. The discussions and conclusions of a workshop conducted in Incheon, Korea in May 2012 to review key knowledge gaps in G6PD deficiency are reported here.

Malaria morbidity and immunity among residents of villages with different Plasmodium falciparum transmission intensity in North-Eastern Tanzania

BackgroundThe relationship between the burden of uncomplicated malaria and transmission intensity is unclear and a better understanding of this relationship is important for the implementation of intervention programmes.MethodsA 6-month longitudinal study monitoring risk factors for anaemia and febrile malaria episodes was conducted among individuals aged below 20 years, residing in three villages of different altitude in areas of high, moderate and low malaria transmission intensity in North-Eastern Tanzania.ResultsThe burden of anaemia and malarial fever fell mainly on the youngest children and was highest in the village with high transmission intensity. Although a considerable percentage of individuals in all villages carried intestinal worms, logistic regression models indicated that Plasmodium falciparum was the only significant parasitic determinant of anaemia. Interestingly, children who carried low-density parasitaemia at the start of the study had a lower risk of contracting a febrile malaria episode but a higher risk of anaemia during the study period, than children who were slide negative at this point in time.ConclusionYoung children living in the high transmission village carried a very high anaemia burden, which could be attributed to malaria. The overall incidence of febrile malaria was also highest in the high transmission village particularly among those under five years of age. These data suggest that in rolling back malaria, available resources in prevention programmes should primarily be focussed on young children, particularly those residing in areas of high malaria transmission.

Occurrence of the Southeast Asian/South American SVMNT Haplotype of the Chloroquine-Resistance Transporter Gene in Plasmodium falciparum in Tanzania

Two main haplotypes, CVIET and SVMNT, of the Plasmodium falciparum chloroquine-resistance transporter gene (Pfcrt) are linked to 4-aminoquinoline resistance. The CVIET haplotype has been reported in most malaria-endemic regions, whereas the SVMNT haplotype has only been found outside Africa. We investigated Pfcrt haplotype frequencies in Korogwe District, Tanzania, in 2003 and 2004. The SVMNT haplotype was not detected in 2003 but was found in 19% of infected individuals in 2004. Amodiaquine use has increased in the region. The introduction and high prevalence of the SVMNT haplotype may reflect this and may raise concern regarding the use of amodiaquine in artemisinin-based combination therapies in Africa.

G6PD testing in support of treatment and elimination of malaria: recommendations for evaluation of G6PD tests

Malaria elimination will be possible only with serious attempts to address asymptomatic infection and chronic infection by both Plasmodium falciparum and Plasmodium vivax. Currently available drugs that can completely clear a human of P. vivax (known as “radical cure”), and that can reduce transmission of malaria parasites, are those in the 8-aminoquinoline drug family, such as primaquine. Unfortunately, people with glucose-6-phosphate dehydrogenase (G6PD) deficiency risk having severe adverse reactions if exposed to these drugs at certain doses. G6PD deficiency is the most common human enzyme defect, affecting approximately 400 million people worldwide.Scaling up radical cure regimens will require testing for G6PD deficiency, at two levels: 1) the individual level to ensure safe case management, and 2) the population level to understand the risk in the local population to guide Plasmodium vivax treatment policy. Several technical and operational knowledge gaps must be addressed to expand access to G6PD deficiency testing and to ensure that a patient’s G6PD status is known before deciding to administer an 8-aminoquinoline-based drug.In this report from a stakeholder meeting held in Thailand on October 4 and 5, 2012, G6PD testing in support of radical cure is discussed in detail. The focus is on challenges to the development and evaluation of G6PD diagnostic tests, and on challenges related to the operational aspects of implementing G6PD testing in support of radical cure. The report also describes recommendations for evaluation of diagnostic tests for G6PD deficiency in support of radical cure.

Levels of Plasma Immunoglobulin G with Specificity against the Cysteine-Rich Interdomain Regions of a Semiconserved Plasmodium falciparum Erythrocyte Membrane Protein 1, VAR4, Predict Protection against Malarial Anemia and Febrile Episodes

ABSTRACT Antibodies to variant surface antigen have been implicated as mediators of malaria immunity in studies measuring immunoglobulin G (IgG) binding to infected erythrocytes. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is an important target for these antibodies, but no study has directly linked the presence of PfEMP1 antibodies in children to protection. We measured plasma IgG levels to the cysteine-rich interdomain region 1α (CIDR1α) of VAR4 (VAR4-CIDR1α), a member of a semiconserved PfEMP1 subfamily, by enzyme-linked immunosorbent assay in 561 Tanzanian individuals, who were monitored clinically for 7 months. The participants resided in Mkokola (a high-transmission village where malaria is holoendemic) or Kwamasimba (a moderate-transmission village). For comparison, plasma IgG levels to two merozoite surface protein 1 (MSP1) constructs, MSP1-19 and MSP1 block 2, and a control CIDR1 domain were measured. VAR4-CIDR1α antibodies were acquired at an earlier age in Mkokola than in Kwamasimba, but after the age of 10 years the levels were comparable in the two villages. After controlling for age and other covariates, the risk of having anemia at enrollment was reduced in VAR4-CIDR1α responders for Mkokola (adjusted odds ratio [AOR], 0.49; 95% confidence interval [CI], 0.29 to 0.88; P = 0.016) and Kwamasimba (AOR, 0.33; 95% CI, 0.16 to 0.68; P = 0.003) villages. The risk of developing malaria fever was reduced among individuals with a measurable VAR4-CIDR1α response from Mkokola village (AOR, 0.51; 95% CI, 0.29 to 0.89; P = 0.018) but not in Kwamasimba. Antibody levels to the MSP1 constructs and the control CIDR1α domain were not associated with morbidity protection. These data strengthen the concept of developing vaccines based on PfEMP1.

In vitro selection of Plasmodium falciparum 3D7 for expression of variant surface antigens associated with severe malaria in African children

P. falciparum‐infected red blood cells (IRBC) can adhere to endothelial host receptors through parasite‐encoded, clonally variant surface antigens (VSA). The VSA‐mediated IRBC adhesion and the acquired VSA‐specific antibody response have both been linked to IRBC organ tropism and disease severity. Parasites isolated from young children with severe malaria (SM) tend to express a limited and conserved set of VSA (VSASM) that are both stronger and more commonly recognized by IgG in the plasma of malaria‐exposed individuals than VSA (VSAUM) expressed by parasites causing uncomplicated malaria (UM) in older semi‐immune children.

Geographical and Temporal Conservation of Antibody Recognition of Plasmodium falciparum Variant Surface Antigens

ABSTRACT The slow acquisition of protection against Plasmodium falciparum malaria probably reflects the extensive diversity of important antigens. The variant surface antigens (VSA) that mediate parasite adhesion to a range of host molecules are regarded as important targets of acquired protective immunity, but their diversity makes them questionable vaccine candidates. We determined levels of VSA-specific immunoglobulin G (IgG) in human plasma collected at four geographically distant and epidemiologically distinct localities with specificity for VSA expressed by P. falciparum isolates from three African countries. Plasma levels of VSA-specific IgG recognizing individual parasite isolates depended on the transmission intensity at the site of plasma collection but were largely independent of the geographical origin of the parasites. The total repertoire of immunologically distinct VSA thus appears to be finite and geographically conserved, most likely due to functional constraints. Furthermore, plasma samples frequently had high IgG reactivity to VSA expressed by parasites isolated more than 10 years later, showing that the repertoire is also temporally stable. Parasites from patients with severe malaria expressed VSA (VSASM) that were better recognized by plasma IgG than VSA expressed by other parasites, but importantly, VSASM-type antigens also appeared to show substantial antigenic homogeneity. Our finding that the repertoire of immunologically distinct VSA in general, and in particular that of VSASM, is geographically and temporally conserved raises hopes for the feasibility of developing VSA-based vaccines specifically designed to accelerate naturally acquired immunity, thereby enhancing protection against severe and life-threatening P. falciparum malaria.