Severe Malaria: Seeking Solutions to a Complex and Deadly Disease

Web Sites

Bernhard Nocht Institute for Tropical Medicine
For 100 years the Bernhard Nocht Institute for Tropical Medicine has dedicated itself to research regarding tropical illnesses, treatment of patients affected, and to the vocational training of doctors in the field of tropical medicine. In fact, vaccines and modern medicine have forced back several infectious diseases, particularly in the industrialized countries.

CDC — Malaria
A comprehensive Web site by the Centers for Disease Control and Prevention of the United States.

Karolinska Institute
The Karolinska Institute hosted the Severe Malaria conference and sponsors research into the disease.

Liverpool School of Tropical Medicine-AntiMal
Among its many projects, the Liverpool School of Tropical Medicine coordinates Anti-Mal, a project funded by the European Commission as part of its 6th Framework Programme and which is coordinated by Professor Steve Ward.  The project comprises leading groups of malaria researchers from a consortium of 31 institutions from ten European and two African countries.

Malaria Foundation International
The MFI is a nonprofit organization, dedicated to the fight against malaria since 1992. This website was created in 1995 to provide a central source of information about this devastating disease. The MFI works in partnership with many individuals and groups who have since joined this cause. The MFI's goals are to support awareness, education, training, research, and leadership programs for the immediate and long term development and application of tools to combat malaria.

National Institute of Allergy and Infectious Diseases (NIH)
NIAID sponsors research on malaria, provides resources for researchers, lists partners, and releases reports and summaries of their activities.

Nature Magazine — Malaria Focus
Nature published the complete genome sequence of Plasmodium falciparum, the main cause of human malaria and the complete sequence of Plasmodium yoelii, the infectious agent in rodent malaria. The malaria special issue also contains state-of-the-art global genomic analysis of the primary sequence.

Swedish Institute for Infectious Disease Control (SMI)
SMI is a government expert authority with a mission to monitor the epidemiology of infectious disease among Swedish citizens and promote control and prevention of these diseases. SMI gives expert advice and support to local, regional and central authorities with operative or political responsibilities for infectious disease control.

SMI also participates in several external expert committees and advisory groups, including the science council of the National Food Administration, the preparedness council of the National Board of Health and Welfare and the AIDS council of the National Institute of Public Health.

The Walter and Eliza Hall Institute of Medical Research
The Web site of the Infection and Immunity Division provides information from the labs of several conference participants, including Alan Cowman, James Beeson, Brendan Crabb, and Louis Schofield.

Wellcome Trust — Malaria
The Wellcome Trust is one of the world's leading supporters of research in tropical medicine. Over the last ten years it has funded £150 million of research on malaria, and has supported a number of initiatives that aim to tackle the huge burden of malaria in the developing world. The Wellcome Trust partners with the Kenya Medical Research Institute to conduct research on malaria and other important infectious diseases.

The Trust also produces a CD-ROM on malaria, which gives an up-to-date overview of the subject from basic science through to the latest treatment and control guidelines.

World Health Organization-Malaria
The World Health Organization's Web site on malaria contains basic information about malaria as well as information about worldwide efforts to eradicate the disease and guidelines for treatment (PDF, 1.85 MB).

Journal Articles

Severe Malaria: Complicated and Simple

Bejon P, Mwacharo J, Kai O, et al. 2007. The induction and persistence of T Cell IFN-γ responses after vaccination or natural exposure is suppressed by Plasmodium falciparum. J. Immunol. 179: 4193-4201.

Bejon P, Berkley JA, Mwangi T, et al. 2007. Defining childhood severe falciparum malaria for intervention studies. PloS Med. 4: e251. FULL TEXT

Dondorp AM, Silamut K, Charunwatthana P, et al. 2007. Levamisole inhibits sequestration of infected red blood cells in patients with falciparum malaria. J. Infect. Dis. 196: 460-466.

Dondorp AM, Day NP. 2007. The treatment of severe malaria. Trans. R. Soc. Trop. Med. Hyg. 101: 633-634.

Idro R, Ndiritu M, Ogutu B, et al. 2007. Burden, features, and outcome of neurological involvement in acute falciparum malaria in Kenyan children. JAMA 297: 2232-2240.

Medana IM, Idro R, Newton CR. 2007. Axonal and astrocyte injury markers in the cerebrospinal fluid of Kenyan children with severe malaria. J. Neurol. Sci. 258: 93-98.

Pfemp1 and Var Genes

Bull PC, Kyes S, Buckee CO. 2007. An approach to classifying sequence tags sampled from Plasmodium falciparum var genes. Mol. Biochem. Parasitol. FULL TEXT

Duraisingh, MT, Voss TS, Marty AJ, et al. 2005. Perinuclear silencing and locus repositioning are linked to regulation of virulence genes in Plasmodium falciparum. Cell 121: 13-24. FULL TEXT

Freitas-Junior LH, Hernandez-Rivas R, Ralph SA, et al. 2005. Telomeric heterochromatin propagation and histone acetylation control mutually exclusive expression of antigenic variation genes in malaria parasites. Cell 121: 25-36. FULL TEXT

Kraemer SM, Kyes SA, Aggarwal G, et al. 2007. Patterns of gene recombination shape var gene repertoires in Plasmodium falciparum: comparisons of geographically diverse isolates. BMC Genomics 8: 45. FULL TEXT

Ralph SA, Scheidig-Benatar C, Scherf A. 2005. Antigenic variation in Plasmodium falciparum is associated with movement of var loci between subnuclear location. Proc. Nat. Acad. Sci. USA 102: 5414-5419. FULL TEXT

Voss TS, Tonkin CJ, Marty AJ, et al. 2007. Alterations in local chromatin environment are involved in silencing and activation of subtelomeric var genes in Plasmodium falciparum. Mol. Microbiol. Aug 28; [Epub ahead of print]

Pfemp1, Pathogenesis and Vaccine

Bir N, Yazdani SS, Avril M, et al. 2006. Immunogenicity of Duffy binding-like domains that bind chondroitin sulfate A and protection against pregnancy-associated malaria. Infect. Imm. 74: 5955-5963. FULL TEXT

Devi YS, Mukherjee P, Yazdani SS, et al. 2007. Immunogenicity of Plasmodium vivax combination subunit vaccine formulated with human compatible adjuvants in mice. Vaccine 25: 5166-5174.

Joergensen L, Vestergaard LS, Turner L, et al. 2007. 3D7-Derived Plasmodium falciparum erythrocyte membrane protein 1 is a frequent target of naturally acquired antibodies recognizing protein domains in a particular pattern independent of malaria transmission intensity. J. Immunol. 178: 428-435.

Normark J, Nilsson D, Ribacke U, et al. 2007. PfEMP1 motifs predict severity of Plasmodium falciparum malaria. Proc. Natl. Acad. Sci. USA, in press.

Rasti N, Namusoke F, Chene A, et al. 2006. Nonimmune immunoglobulin binding and multiple adhesion characterize Plasmodium falciparum-infected erythrocytes of placental origin. Proc. Natl. Acad. Sci. USA 103: 13795-13800. FULL TEXT

Saxena AK, Singh K, Su HP, et al. 2006. The essential mosquito-stage P25 and P28 proteins from Plasmodium form tile-like triangular prisms. Nat. Struct. Mol. Biol. 13: 90-91.

Saxena AK, Wu Y, Garboczi DN. 2007. Plasmodium p25 and p28 surface proteins: potential transmission-blocking vaccines. Eukaryot. Cell 6: 1260-1265.

Vogt AM, Pettersson F, Moll K, et al. 2006. Release of sequestered malaria parasites upon injection of a glycosaminoglycan. PLoS Pathog. 2: e100 FULL TEXT

Adhesion, Invasion, and More Vaccines

Harris PK, Yeoh S, Dluzewski AR, et al. 2005. Molecular identification of a malaria merozoite surface sheddase. PloS Pathog. 1: 241-251. FULL TEXT

Kubler-Kielb J, Majadly F, Wu Y, et al. 2007. Long-lasting and transmission-blocking activity of antibodies to Plasmodium falciparum elicited in mice by protein conjugates of Pfs25. Proc. Natl. Acad. Sci. USA 104: 293-298. FULL TEXT

Kumar KA, Garcia CR, Chandran VR, et al. 2007. Exposure of Plasmodium sporozoites to the intracellular concentration of potassium enhances infectivity and reduces cell passage activity. Mol. Biochem. Parasitol. 156: 32-40.

Kumar KA, Sano G, Boscardin S, et al. 2007. The circumsporozoite protein is an immunodominant protective antigen in irradiated sporozoites. Nature 444: 937-940.

Malkin E, Long CA, Stowers AW, et al. 2007. Phase 1 study of two merozoite surface protein 1 (MSP1(42)) vaccines for Plasmodium falciparum malaria. PLoS Clin Trials 2: e12 FULL TEXT

Meissner M, Breinich MS, Gilson PR, Crabb BS. 2007. Molecular genetic tools in Toxoplasma and Plasmodium: achievements and future needs. Curr. Opin. Microbiol. [Epub ahead of print]

Nasr A, Iriemenam NC, Troye-Blomberg M, et al. 2007. Fc γ receptor IIa (CD32) polymorphism and antibody responses to asexual blood-stage antigens of Plasmodium falciparum malaria in Sudanese patients. Scand. J. Immunol. 66: 87-96.

O'Donnell RA, Hackett F, Howell SA, et al. 2006. Intramembrane proteolysis mediates shedding of a key adhesin during erythrocyte invasion by the malaria parasite. J. Cell Biol. 174: 1023-1033. FULL TEXT

Sanders PR, Cantin GT, Greenbaum DC, et al. 2007. Identification of protein complexes in detergent-resistant membranes of Plasmodium falciparum schizonts. Mol. Biochem. Parasitol. 154: 148-157.

Inflammation and Severe Malaria

Beare NA, Taylor TE, Harding SP, et al. 2006. Malarial retinopathy: a newly established diagnostic sign in severe malaria. Am. J. Trop. Med. Hyg. 75: 790-797.

Hansen DS, D'Ombrain MC, Schofield L. 2007. The role of leukocytes bearing Natural Killer Complex receptors and Killer Immunoglobulin-like Receptors in the immunology of malaria. Curr. Opin. Immunol. 19: 416-423.

Montgomery J, Mphande FA, Berriman M, et al. 2007. Differential var gene expression in the organs of patients dying of falciparum malaria. Mol. Microbiol. 65: 959-967.

Nie CQ, Bernard NJ, Schofield L, Hansen DS. 2007. CD4⁺ CD25⁺ regulatory T cells suppress CD4⁺ T-cell function and inhibit the development of Plasmodium berghei-specific TH1 responses involved in cerebral malaria pathogenesis. Infect. Immun. 75: 2275-2282. FULL TEXT

Ramjanee S, Robertson JS, Franke-Fayard B, et al. 2007. The use of transgenic Plasmodium berghei expressing the Plasmodium vivax antigen P25 to determine the transmission-blocking activity of sera from malaria vaccine trials. Vaccine 25: 886-894.

Tripathi AK, Sullivan DJ, Stins MF. 2007. Plasmodium falciparum-infected erythrocytes decrease the integrity of human blood-brain barrier endothelial cell monolayers. J. Infect. Dis. 195: 942-950.

van der Heyde HC, Nolan J, Combes V, et al. A unified hypothesis for the genesis of cerebral malaria: sequestration, inflammation and hemostasis leading to microcirculatory dysfunction. Trends Parasitol. 22: 503-508.

Pregnancy Associated with Malaria

Dahlback M, Lavstsen T, Salanti A, et al. 2007. Changes in var gene mRNA levels during erythrocytic development in two phenotypically distinct Plasmodium falciparum parasites. Malar. J. 6: 78. FULL TEXT

Dahlback M, Rask TS, Andersen PH, et al. 2006. Epitope mapping and topographic analysis of VAR2CSA DBL3X involved in P. falciparum placental sequestration. PloS Pathog. 2: e124. FULL TEXT

Francis SE, Malkov VA, Oleinikov AV, et al. 2007. Six genes are preferentially transcribed by the circulating and sequestered forms of Plasmodium falciparum parasites that infect pregnant women. Infect. Immun. Aug 13; [Epub ahead of print]

Fried M, Hixson KK, Anderson L, et al. 2007. The distinct proteome of placental malaria parasites. Mol. Biochem. Parasitol. 155: 57-65.

Gamain B, Smith JD, Viebig NK, et al. 2007. Pregnancy-associated malaria: parasite binding, natural immunity and vaccine development. Int. J. Parasitol. 37: 273-283.

Nielsen MA, Resende M, Alifrangis M, et al. 2007. Plasmodium falciparum: VAR2CSA expressed during pregnancy-associated malaria is partially resistant to proteolytic cleavage by trypsin. Exp. Parasitol. 117: 1-8.

Ward Sa, Sevene EJ, Hastings IM, et al. 2007. Antimalarial drugs and pregnancy: safety, pharmacokinetics, and pharmacovigilance. Lancet Infect. Dis. 7: 136-144.

Novel Strategies

Barnes KI, Lindegardh N, Ogundahunsi O, et al. 2007. World Antimalarial Resistance Network (WARN) IV: Clinical pharmacology. Malar J. 6: 122. FULL TEXT

Medana IM, Turner GD. 2006. Human cerebral malaria and the blood-brain barrier. Int. J. Parasitol. 36: 555-568.

Price RN, Dorsey G, Ashley EA, et al. 2007. World Antimalarial Resistance Network (WARN) I: Clinical efficacy of antimalarial therapy. Malar J. 6: 119. FULL TEXT

Wong D, Prameya R, Dorovini-Zis K. 2007. Adhesion and migration of polymorphonuclear leukocytes across human brain microvessel endothelial cells are differentially regulated by endothelial cell adhesion molecules and modulate monolayer permeability. J. Neuroimmunol. 184: 136-148.

Genetic Resistance

Fairhurst RM, Baruch DI, Brittain NJ, et al. 2005. Abnormal display of PfEMP-1 on erythrocytes carrying haemoglobin C may protect against malaria. Nature 435: 1117-1121.

Gunasekera AM, Wickramachchi T, Neafsey DE, et al. 2007. Genetic diversity and selection at the Plasmodium vivax apical membrane antigen-1 (PvAMA-1) locus in a Sri Lankan population. Mol. Biol. Evol. 24: 939-947.

Khor CC, Vannberg FO, Chapman SJ, et al. 2007. Positive replication and linkage disequilibrium mapping of the chromosome 21q22.1 malaria susceptibility locus. Genes Immun. Aug 16; [Epub ahead of print]

Kwiatkowski DP, Luoni G. 2006. Host genetic factors in resistance and susceptibility to malaria. 2005. Parassitologia 48: 450-467.

Su X, Hayton K, Wellems TE. Genetic linkage and association analyses for trait mapping in Plasmodium falciparum. Nat. Rev. Genet. 8: 497-506.

Volkman SK, Sabeti PC, DeCaprio D, et al. 2007. A genome-wide map of diversity in Plasmodium falciparum. Nat. Genet. 39: 113-119.

Speakers:
Thomas Wellems, National Institute of Allergy and Infectious Diseases
Rolf Horstmann, Bernhard Nocht Institute for Tropical Medicine
Daniel Neafsey, The Broad Institute

A tale of two polymorphisms

Two brothers are infected with malaria. One experiences fever and malaise while the other ends up in a coma, with severe anemia and respiratory distress. Why the difference? Genetic polymorphisms—specifically, those that affect hemoglobin—are responsible, according to Thomas Wellems of the National Institute of Allergy and Infectious Diseases.

"Many polymorphisms that protect against severe P. falciparum malaria affect the hemoglobin of erythrocytes, reflecting the importance of these cells as hosts of the parasites during infection and disease," Wellems explained. "The most prominent polymorphisms are the point mutations responsible for hemoglobin C (HbC) and sickle cell hemoglobin (HbS)." His group's work has helped elucidate the mechanisms by which these mutations exert their influence to confer protection.

How might HbC protect against severe malaria? Some researchers suggest the gene influences the extent of parasite invasion of erythrocytes. "But no, the rates of P. falciparum invasion are the same for hemoglobin AA, AC, and CC cells," said Wellems. Does HbC influence parasite growth or multiplication within erythrocytes? "Unlikely," said Wellems, since the rates are the same in AA and AC cells. Moreover, "these two hypotheses do not explain the relatively greater protection conferred against severe versus uncomplicated malaria."

HbC causes an abnormal distribution and reduced display of PfEMP1 in erythrocytes.

Could HbC be conferring protection by affecting the interactions of parasitized erythrocytes with host tissues? Indeed, recent work by Wellems' group demonstrated that this is the case. As shown in previous presentations, in red blood cells containing normal hemoglobin A, malaria parasites generate knobby protrusions with the adhesion protein PfEMP1, enabling infected cells to attach to blood vessel walls and avoid destruction by the spleen. HbC impairs this attachment by causing abnormal distributions of knobs and reduced display of PfEMP1, Wellems explained.

"It's like bathmats with suction cups," he said. More expensive bathmats have many suction cups very close to each other—the most effective way of getting the mat to adhere to the bottom of the tub. By contrast, less expensive bathmats have large suction cups that are few and far between. The latter is what happens when HbC interacts with knobby red cells and the endothelium—altered display of the adhesion protein and reduced affinity for attachment.

"Reduced adherence of parasitized HbC erythrocytes to the endothelium decreases the pathogenic effects of the cells' sequestration in the microvasculature, mitigating inflammation in critical tissues," Wellems hypothesized. A comparable mechanism of protection may also operate with HbS, he suggested.

Comparing HbS and HbC, Wellems noted that the incidence of the former is higher where the incidence of the is lower, and vice versa, depending on the region. "Both confer greater protection against severe rather than uncomplicated malaria. However, HbS protects against all kinds of severe malaria, whereas HbC seems to protect preferentially against cerebral malaria. Furthermore, some evidence suggests greater protection by HbC versus HbS, or vice versa, in different ethnic populations, he observed. Wellems' group is continuing to investigate the mechanisms of protection against severe malaria that could form the basis of novel treatment strategies.

A bit about thalassemia

In other work on genetic resistance in malaria, Rolf Horstmann of the Bernhard Nocht Institute for Tropical Medicine Diseases observed that "the high prevalences of HbS, HbC and α-thalassemia (-α) in malaria-endemic areas are considered to result from some mode of balancing selection with malaria."

In a case-control study of 4797 African children, Horstmann and colleagues found that, as Wellems suggested, the HbS carrier state (HbAS) was negatively associated with all major forms of severe malaria, whereas the HbC carrier state (HbAC) was negatively associated selectively with cerebral malaria. Moreover, the alpha-thalassemia carrier state (-α/αα) was negatively associated selectively with a severe and possibly complicated form of anemia.

The findings suggest that HbAC and -α/αα confer protection by interfering with different pathophysiological events, Horstmann said. Furthermore, the geographical distributions of HbC and -α may be influenced by differences in the regional predominance of cerebral malaria or severe anemia as malaria complications. Horstmann theorized that alpha-thalassemia may protect against severe anemia "simply by being a chronic hemolytic anemia"—that is, it causes "a constantly accelerated red blood cell production and turnover," thereby reducing the risk of falling hemoglobin counts.

New tool

Further insights into the role of genetic polymorphisms in malaria protection and susceptibility will be forthcoming as a result of a collaborative effort between members of the Broad Institute and the Harvard School of Public Health to map single nucleotide polymorphism (SNP) diversity in the genome of P. falciparum, according to Daniel Neafsey of the Broad Institute of MIT and Harvard University. The aim of the project, he said, "is to attempt to ameliorate some of the burden of this disease through increased understanding of the parasite's genomic diversity and evolution."

In the first stage of the project, which is still ongoing, the team has focused on SNP discovery through genome-wide scanning. To date, more than 96,000 SNPs from 12 geographically diverse parasites have been identified. Most SNPs were of low frequency, suggesting that "the parasites are very diverse and that additional sequencing will be helpful in finding SNPs that are more common in the population," Neafsey said.

To increase the power of these genome-wide scans for selection, the researchers are working with a prototype chip that includes 3000 SNPs and is expandable to 5000 SNPs. "Even though this is just a prototype, we were able to learn a few interesting things by using it to genotype population samples of parasite from Senegal and Thailand," Neafsey said.

Senegalese strains are about 50% more diverse in the assayed SNPs than the Thai strains.

In-depth analyses of the data gleaned from the samples suggest that the Senegalese strains are about 50% more diverse in the assayed SNPs than the Thai strains, which is likely a function of the higher endemic and infection rates in Africa, Neafsey explained. Investigations of the nature of the SNPs that differentiate the two populations revealed that nonsynonymous SNPs (SNPs that are exposed to selection pressure and therefore have an impact on phenotype) comprise a little less than half of all the SNPs assayed on the chip, with the other half being a mixture of noncoding and synonymous-coding SNPs ("silent" SNPs).

Looking specifically at the differentiated SNPs—that is, SNPs that show fixed differences or are highly divergent in frequency between the populations—"we see that nonsynonymous SNPs make up almost 75% of that group, a highly significant enrichment," Neafsey said. "This suggests that natural selection is playing an important role in the divergence of the two parasite populations. Thus, just as malaria has had a significant evolutionary impact on the human genome, we find that the human immune system has reciprocally exerted a broad evolutionary impact across many protein sequences in the malaria genome," he observed. A better understanding of this impact could reveal novel strategies for mitigating the parasites' pathogenic effects.