Interferon-γ (IFNG) microsatellite repeat and single nucleotide polymorphism haplotypes of IFN-α receptor (IFNAR1) associated with enhanced malaria susceptibility in Indian populations.

Pro-inflammatory cytokines IFNγ and IFNα function through their cellular receptors IFNγR1 and IFNαR1, respectively to mediate immune processes during malaria infection. A total of 21 SNPs, 2 ins/del polymorphisms and a microsatellite repeat, selected on the basis of their reported association with infectious diseases including malaria in world populations, were analysed for association with Plasmodium falciparum malaria susceptibility in a case-control study with adult patients and ethnically-matched controls drawn from a disease meso- to hyperendemic and a nonendemic region of India. Among the five IFNG SNPs tested, an intron 3 and a 3'UTR SNP associated with disease in the endemic region. In addition, large (CA)n repeats of IFNG intron 1 associated with protection from severe malaria in the endemic region (severe vs. control, odds ratio=0.21, 95% CI=0.08-0.52, P=1.3 × 10(-4)). The TA11CAG haplotype (rs2069705 T/C, rs2430561 A/T, rs3138557 (CA)n, rs2069718 T/C, rs2069727 A/G, rs2069728 G/A) carrying a short CA11 repeat also exhibited very strong association with severe malaria, particularly in the endemic region (severe vs. control, OR=14.56, 95% CI=3.39-85.81, P=3 × 10(-5)). One SNP each from the IFNA8 and IFNA17 of IFNA gene cluster had a protective effect in the non-endemic region but not in the endemic region. A promoter and an intron 2 SNP of IFNAR1 were risk factors for disease and the IFNAR1 haplotype GCCAGG (rs2843710 C/G, rs2850015 C/T, +6993 C/T, rs2243594 A/G, rs1012335 G/C, rs2257167 G/C) carrying both the risk alleles strikingly associated with disease manifestation in the endemic region (severe vs. control, OR=27.14, 95% CI=3.12-1254, P=2 × 10(-5); non-severe vs. control, OR=61.87, 95% CI=10.08-2521, P=1 × 10(-8)). The data indicates dissimilar contribution of cytokine and cytokine receptor variants to disease in populations residing in areas of differential malaria endemicity.

MBL2 variations and malaria susceptibility in Indian populations.

Human mannose-binding lectin (MBL) encoded by the MBL2 gene is a pattern recognition protein and has been associated with many infectious diseases, including malaria. We sought to investigate the contribution of functional MBL2 gene variations to Plasmodium falciparum malaria in well-defined cases and in matched controls. We resequenced the 8.7 kb of the entire MBL2 gene in 434 individuals clinically classified with malaria from regions of India where malaria is endemic. The study cohort included 176 patients with severe malaria, 101 patients with mild malaria, and 157 ethnically matched asymptomatic individuals. In addition, 830 individuals from 32 socially, linguistically, and geographically diverse endogamous populations of India were investigated for the distribution of functional MBL2 variants. The MBL2 -221C (X) allelic variant is associated with increased risk of malaria (mild malaria odds ratio [OR] = 1.9, corrected P value [P(Corr)] = 0.0036; severe malaria OR = 1.6, P(Corr) = 0.02). The exon1 variants MBL2*B (severe malaria OR = 2.1, P(Corr) = 0.036; mild versus severe malaria OR = 2.5, P(Corr) = 0.039) and MBL2*C (mild versus severe malaria OR = 5.4, P(Corr) = 0.045) increased the odds of having malaria. The exon1 MBL2*D/*B/*C variant increased the risk for severe malaria (OR = 3.4, P(Corr) = 0.000045). The frequencies of low MBL haplotypes were significantly higher in severe malaria (14.2%) compared to mild malaria (7.9%) and asymptomatic (3.8%). The MBL2*LYPA haplotypes confer protection, whereas MBL2*LXPA increases the malaria risk. Our findings in Indian populations demonstrate that MBL2 functional variants are strongly associated with malaria and infection severity.

Variations in ncRNA gene LOC284889 and MIF-794CATT repeats are associated with malaria susceptibility in Indian populations.

BACKGROUND: There are increasing evidences on the role of non-coding RNA (ncRNA) as key regulator of cellular homeostasis. LOC284889 is an uncharacterized ncRNA gene on reverse strand to MIF mapped to 22q11.23. MIF, a lymphokine, regulates innate immune response by up-regulating the expression of TLR4, suppressing the p53 activity and has been shown to be involved in malaria pathogenesis. METHODS: In this study, the possible effect of MIF variations on malaria susceptibility was investigated by re-sequencing the complete MIF gene along with 1 kb each of 5' and 3' region in 425 individuals from malaria endemic regions of the Orissa and Chhattisgarh states of India. The subjects comprised of 160 cases of severe malaria, 101 of mild malaria and 164 ethnically matched asymptomatic controls. Data were statistically compared between cases and controls for their possible association with Plasmodium falciparum malarial outcome. RESULTS: It is the first study, which shows that the allele A (rs34383331T > A) in ncRNA is significantly associated with increased risk to P. falciparum malaria [severe: OR = 2.08, p = 0.002 and mild: OR = 2.09, P = 0.005]. In addition, it has been observed that the higher MIF-794CATT repeats (>5) increases malaria risk (OR = 1.61, p = 0.01). Further, diplotype (MIF-794CATT and rs34383331T > A) 5 T confers protection to severe malaria (OR = 0.55, p = 0.002) while 6A (OR = 3.07, p = 0.001) increases malaria risk. CONCLUSIONS: These findings support the involvement of ncRNA in malarial pathogenesis and further emphasize the complex genetic regulation of malaria outcome. In addition, the study shows that the higher MIF-794CATT repeats (>5) is a risk factor for severe malaria. The study would help in identifying people who are at higher risk to malaria and adapt strategies for prevention and treatment.

A rare non-synonymous c.102C>G SNP in the IFNB1 gene might be a risk factor for cerebral malaria in Indian populations.

Interferon beta1 (IFNB1) is a type I interferon that is mainly known for its antiviral activity, but it also regulates a number of anti-inflammatory and immunomodulatory functions. Studies on mouse models of cerebral malaria have established that IFNB1 regulates severe malaria pathogenesis and increases overall survival against malaria. It down-regulates pro-inflammatory cytokines: TNF, IFNG and ICAM-1, resulting in decreased adherence of Plasmodium falciparum parasitized RBC to capillary wall, entry into the brain and delayed onset of death. Therefore, we hypothesized that variations in IFNB1 gene could regulate malarial pathogenesis. We re-sequenced the complete IFNB1 gene along with 900bp of 5' up-stream and 500bp of 3'-UTR in 437 individuals from malaria endemic regions of the Orissa and Chhattisgarh states of India. The subjects comprised of 173 cases of severe malaria, 101 of mild malaria, and 156 ethnically matched asymptomatic controls. Data were statistically compared between cases and controls for their possible association with P. falciparum malarial outcome. Two single nucleotide polymorphisms (SNPs): a synonymous c.153C>T (rs1051922) and a non-synonymous substitution c.102C>G (rs139262191, p.Ser34Arg) were identified. The genotype and allele distribution of c.153C>T did not differ significantly between the study groups [mild, χ(2)2=4.10, p-value<0.13 and severe χ(2)2=0.06, p-value<0.97]. Interestingly, the rare non-synonymous SNP (rs139262191) was observed only in malaria patients. The differences between all cases and controls did not reach statistical significance, however, a statistically significant difference was observed between the asymptomatic control group and the cerebral malaria group [OR=20.32, 95% CI=1.08-382.63, p-value=0.044]. Moreover, the genotypes between cerebral malaria positive and negative groups were not significantly different [OR=5.58, 95% CI=0.61-50.97, p-value=0.123]. Our findings suggest that the IFNB1 variant, p.Ser34Arg, might be a risk factor for cerebral malaria in Indian populations.

IL-4 haplotype -590T, -34T and intron-3 VNTR R2 is associated with reduced malaria risk among ancestral indian tribal populations.

BACKGROUND: Interleukin 4 (IL-4) is an anti-inflammatory cytokine, which regulates balance between T(H)1 and T(H)2 immune response, immunoglobulin class switching and humoral immunity. Polymorphisms in this gene have been reported to affect the risk of infectious and autoimmune diseases. METHODS: We have analyzed three regulatory IL-4 polymorphisms; -590C>T, -34C>T and 70 bp intron-3 VNTR, in 4216 individuals; including: (1) 430 ethnically matched case-control groups (173 severe malaria, 101 mild malaria and 156 asymptomatic); (2) 3452 individuals from 76 linguistically and geographically distinct endogamous populations of India, and (3) 334 individuals with different ancestry from outside India (84 Brazilian, 104 Syrian, and 146 Vietnamese). RESULTS: The -590T, -34T and intron-3 VNTR R2 alleles were found to be associated with reduced malaria risk (P<0.001 for -590C>T and -34C>T, and P = 0.003 for VNTR). These three alleles were in strong LD (r²>0.75) and the TTR2 (-590T, -34T and intron-3 VNTR R2) haplotype appeared to be a susceptibility factor for malaria (P = 0.009, OR = 0.552, 95% CI = 0.356 -0.854). Allele and genotype frequencies differ significantly between caste, nomadic, tribe and ancestral tribal populations (ATP). The distribution of protective haplotype TTR2 was found to be significant (χ²3 =  182.95, p-value <0.001), which is highest in ATP (40.5%); intermediate in tribes (33%); and lowest in caste (17.8%) and nomadic (21.6%). CONCLUSIONS: Our study suggests that the IL-4 polymorphisms regulate host susceptibility to malaria and disease progression. TTR2 haplotype, which gives protection against malaria, is high among ATPs. Since they inhabited in isolation and mainly practice hunter-gatherer lifestyles and exposed to various parasites, IL-4 TTR2 haplotype might be under positive selection.

Deletion of the APOBEC3B gene strongly impacts susceptibility to falciparum malaria.

APOBEC3B, a gene involved in innate response, exhibits insertion-deletion polymorphism across world populations. We observed the insertion allele to be nearly fixed in malaria endemic regions of sub-Saharan Africa as well as populations with high malaria incidence in the past. This prompted us to investigate the possible association of the polymorphism with falciparum malaria. We studied the distribution of APOBEC3B, in 25 diverse Indian populations comprising of 500 samples and 176 severe or non-severe Plasmodium falciparum patients and 174 ethnically-matched uninfected individuals from a P. falciparum endemic and a non-endemic region of India. The deletion frequencies ranged from 0% to 43% in the Indian populations. The frequency of the insertion allele strikingly correlated with the endemicity map of P. falciparum malaria in India. A strong association of the deletion allele with susceptibility to falciparum malaria in the endemic region (non-severe vs. control, Odds ratio=4.96, P value=9.5E(-06); severe vs. control, OR=4.36, P value=5.76E(-05)) was observed. Although the frequency of deletion allele was higher in the non-endemic region, there was a significant association of the homozygous deletion genotype with malaria (OR=3.17, 95% CI=1.10-10.32, P value=0.0177). Our study also presents a case for malaria as a positive selection force for the APOBEC3B insertion and suggests a major role for this gene in innate immunity against malaria.

Distinct cytokine profiles define clinical immune response to falciparum malaria in regions of high or low disease transmission.

The immune effector response to Plasmodium falciparum infection involves a finely-tuned interplay between different cell types and cytokines. However, the processes by which they mediate the development of clinical immunity, in areas of different endemicity, are poorly understood. We analyzed circulating levels of pro-inflammatory (TNF, IFN-γ, IL-12, IL-16) and anti-inflammatory (IL-4, IL-10, IL-13) cytokines in control and patient groups drawn from a P. falciparum-endemic and a non-endemic region of India. The endemic region control population exhibited a lower pro- to anti-inflammatory cytokine ratio, indicating a shift towards a high basal Th2 response. Levels of IL-10 contributed most towards the region-specific difference in basal cytokine response. IL-10 was also the strongest predictor of disease in the endemic region, while IL-12, along with IL-10 and IL-6, contributed most to disease outcome in the non-endemic region. A low, mean IFN-γ/IL-10 ratio was associated with disease severity in the endemic region (p < 0.0001). In contrast, a low mean IL-12/IL-10 ratio correlated with disease outcome in the non-endemic region (p < 0.0001). In the endemic region, IL-13 correlated negatively with IFN-γ in severe patients (Spearman's ρ: -0.49; p : 0.013), while in the non-endemic region, IL-13 correlated negatively with IL-6 in severe malaria patients (Spearman's ρ: -0.485; p : 0.001). In conclusion, levels of pro- and anti-inflammatory cytokines and the relative balance between the Th1 and Th2 response, illustrates how populations residing in areas of varying disease endemicity may respond to P. falciparum-induced immune challenge.

Rhabdomyolysis in falciparum malaria--a series of twelve cases (five children and seven adults).

There are very few reports in the literature of rhabdomyolysis in falciparum malaria and they mostly consist of single case studies. A report from Sri Lanka described a single patient with myoglobinuria and skeletal muscle necrosis. Taylor and Prosser reported a single case of rhabdomyolysis with renal failure. We report on 12 patients with evidence of skeletal muscle injury in severe malaria from Rourkela, Orissa State, India.

Can urine dipstick tests detect renal impairment in Plasmodium falciparum malaria in a rural setup?

Renal impairment in falciparum malaria leads to poor prognosis. Serum creatinine is the mainstay of diagnosis. However, the serum creatinine concentration is only observed when the glomerular filtration rate falls below 50%. We evaluated the use of the urine dipstick method to predict renal impairment in 77 patients. Twenty-three (29.8%) had haematuria and 52 (67.5%) had urinary protein > or = 300 mg/L. Renal impairment (plasma creatinine > or = 1.2 mg/dL) was observed in 17 patients. The sensitivity and specificity of haematuria in the detection of renal impairment was 94.1% and 90.8%, but for proteinuria it was 88.2% and 62.7%, respectively. There was a positive correlation of plasma urea and creatinine with haematuria (r = 0.56, P < 0.001; r = 0.46, P < 0.01) but not with proteinuria. The detection of haematuria using a dipstick seems to be a highly specific and sensitive method of observing renal impairment in malaria. This is probably the first study which utilizes a commonly available tool that can be easily adopted for early recognition in rural areas.

CR1 levels and gene polymorphisms exhibit differential association with falciparum malaria in regions of varying disease endemicity.

Complement receptor 1 (CR1/CD35) levels on erythrocytes and related CR1 polymorphisms have been associated with response to falciparum malaria in populations inhabiting malaria-endemic regions. Differences in disease association profiles of its low expression alleles have been observed in populations from different regions of the world. We analyzed the influence of CR1 levels and associated SNPs on susceptibility/resistance to falciparum malaria in Indian populations. Two CR1 SNPs [exon 22 (A/G) and intron 27 (A/T)] define the low expression (L) CR1 allele in populations inhabiting a Plasmodium falciparum-endemic and a nonendemic region of India. Populations of the endemic region have very low red blood cell surface CR1 levels and higher frequencies of the exon 22 and intron 27 mutant L alleles. Whereas low CR1 levels correlated with susceptibility to severe malaria in the nonendemic region, high CR1 levels were associated with manifestation of disease in the endemic region. In addition, the exon 22 L allele was a risk factor for severe malaria in the nonendemic region. Absence of correlation between levels of tumor necrosis factor-alpha, interferon-gamma, and interleukin-6 with CR1 levels in patients with severe disease indicated that RBC CR1 levels in individuals are not the major determinants of pro-inflammatory cytokine release during infection. Our results are interpreted in the context of differences in the pathogenesis of severe malaria in the malaria-endemic and nonendemic region.

Functional and immunological characterization of a Duffy binding-like alpha domain from Plasmodium falciparum erythrocyte membrane protein 1 that mediates rosetting.

The Duffy binding-like (DBL) domains are common adhesion modules present in Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) variants, which are responsible for immune evasion and cytoadherence. Knowledge about how immune responses are acquired against polymorphic DBL domains of PfEMP1 can aid in the development of vaccines for malaria. A recombinant DBLalpha domain, encoded by R29 var1, which binds complement receptor 1 to mediate rosetting by the P. falciparum laboratory strain R29, was expressed in Escherichia coli, renatured by oxidative refolding to its native form, and purified to homogeneity. Antibody levels in 704 plasmas obtained from residents of areas of different levels of malaria endemicity in Orissa (India) and Manhiça (Mozambique) were assessed by enzyme-linked immunosorbent assay. The refolded DBLalpha domain was pure, homogeneous, and functional in that it bound human erythrocytes with specificity and was capable of inhibiting rosetting. The proportion of individuals who had measurable anti-DBLalpha immunoglobulin G responses was low in areas of low malaria endemicity in Orissa (6.7%) but high in areas of high endemicity in Orissa (87.5%) and Manhiça (74.5%). Seroprevalence and antibody levels against the recombinant protein increased with the age of inhabitants from areas with high transmission rates (P < 0.001). Half of the children in these areas had seroconverted by the age of 5 years. These findings suggest that in spite of the extreme polymorphism of PfEMP1 DBLalpha domains, the acquisition of specific antibodies is rapid and age related and reflects the reduced risk of malaria in areas with high transmission rates. Further studies are required to elucidate the role of these antibodies in protection from malaria.

Variations in host genes encoding adhesion molecules and susceptibility to falciparum malaria in India.

BACKGROUND: Host adhesion molecules play a significant role in the pathogenesis of Plasmodium falciparum malaria and changes in their structure or levels in individuals can influence the outcome of infection. The aim of this study was to investigate the association of SNPs of three adhesion molecule genes, ICAM1, PECAM1 and CD36, with severity of falciparum malaria in a malaria-endemic and a non-endemic region of India. METHODS: The frequency distribution of seven selected SNPs of ICAM1, PECAM1 and CD36 was determined in 552 individuals drawn from 24 populations across India. SNP-disease association was analysed in a case-control study format. Genotyping of the population panel was performed by Sequenom mass spectroscopy and patient/control samples were genotyped by SNaPshot method. Haplotypes and linkage disequilibrium (LD) plots were generated using PHASE and Haploview, respectively. Odds-ratio (OR) for risk assessment was estimated using EpiInfotrade mark version 3.4. RESULTS: Association of the ICAM1 rs5498 (exon 6) G allele and the CD36 exon 1a A allele with increased risk of severe malaria was observed (severe versus control, OR = 1.91 and 2.66, P = 0.02 and 0.0012, respectively). The CD36 rs1334512 (-53) T allele as well as the TT genotype associated with protection from severe disease (severe versus control, TT versus GG, OR = 0.37, P = 0.004). Interestingly, a SNP of the PECAM1 gene (rs668, exon 3, C/G) with low minor allele frequency in populations of the endemic region compared to the non-endemic region exhibited differential association with disease in these regions; the G allele was a risk factor for malaria in the endemic region, but exhibited significant association with protection from disease in the non-endemic region. CONCLUSION: The data highlights the significance of variations in the ICAM1, PECAM1 and CD36 genes in the manifestation of falciparum malaria in India. The PECAM1 exon 3 SNP exhibits altered association with disease in the endemic and non-endemic region.

Polymorphisms of TNF-enhancer and gene for FcgammaRIIa correlate with the severity of falciparum malaria in the ethnically diverse Indian population.

BACKGROUND: Susceptibility/resistance to Plasmodium falciparum malaria has been correlated with polymorphisms in more than 30 human genes with most association analyses having been carried out on patients from Africa and south-east Asia. The aim of this study was to examine the possible contribution of genetic variants in the TNF and FCGR2A genes in determining severity/resistance to P. falciparum malaria in Indian subjects. METHODS: Allelic frequency distribution in populations across India was first determined by typing genetic variants of the TNF enhancer and the FCGR2A G/A SNP in 1871 individuals from 55 populations. Genotyping was carried out by DNA sequencing, single base extension (SNaPshot), and DNA mass array (Sequenom). Plasma TNF was determined by ELISA. Comparison of datasets was carried out by Kruskal-Wallis and Mann-Whitney tests. Haplotypes and LD plots were generated by PHASE and Haploview, respectively. Odds ratio (OR) for risk assessment was calculated using EpiInfotrade mark version 3.4. RESULTS: A novel single nucleotide polymorphism (SNP) at position -76 was identified in the TNF enhancer along with other reported variants. Five TNF enhancer SNPs and the FCGR2A R131H (G/A) SNP were analyzed for association with severity of P. falciparum malaria in a malaria-endemic and a non-endemic region of India in a case-control study with ethnically-matched controls enrolled from both regions. TNF -1031C and -863A alleles as well as homozygotes for the TNF enhancer haplotype CACGG (-1031T>C, -863C>A, -857C>T, -308G>A, -238G>A) correlated with enhanced plasma TNF levels in both patients and controls. Significantly higher TNF levels were observed in patients with severe malaria. Minor alleles of -1031 and -863 SNPs were associated with increased susceptibility to severe malaria. The high-affinity IgG2 binding FcgammaRIIa AA (131H) genotype was significantly associated with protection from disease manifestation, with stronger association observed in the malaria non-endemic region. These results represent the first genetic analysis of the two immune regulatory molecules in the context of P. falciparum severity/resistance in the Indian population. CONCLUSION: Association of specific TNF and FCGR2A SNPs with cytokine levels and disease severity/resistance was indicated in patients from areas with differential disease endemicity. The data emphasizes the need for addressing the contribution of human genetic factors in malaria in the context of disease epidemiology and population genetic substructure within India.

Pfcrt haplotypes and in-vivo chloroquine response in Sundergarh district, Orissa, India.

The Plasmodium falciparum chloroquine resistance transporter (Pfcrt) K76T mutation and haplotype (amino acids 72-76) were analyzed as markers of chloroquine (CQ) resistance in the blood samples of patients from two sites of different intensities of malaria transmission (high, n=70; low, n=68) in Sundergarh district of Orissa, India and correlated with the in-vivo response. Early treatment failure (ETF) was significantly more frequent in the high endemic area (32.9 vs. 7.4%, P<0.001), with children below 5 years suffering more. A high frequency of pfcrt K76T mutation was observed in both the areas (87.1 vs. 79.4%, P=0.22). Patients carrying pfcrt 76T were the most likely to develop ETF (odds ratio 36; 95% CI 3.35-1653.3; P<0.001). The ratio of 76T:K76 was 22:9 and 11:14, respectively, in high and low endemic areas (odds ratio 3.1; 95% CI 0.9-11.03; P=0.04), which may be used as a measure of drug pressure. Sequences of pfcrt codons 72-76 showed 16 of the CQ-resistant haplotypes to be SVMNT, 5 CVMNT and 12 CVIET. The CQ-sensitive haplotypes were mostly CVMNK in 10 samples; CVIEK in 2 samples. Both Southeast Asian and South American haplotypes were present, with the latter predominating.

Epidemiology of malaria transmission and development of natural immunity in a malaria-endemic village, San Dulakudar, in Orissa state, India.

We describe the epidemiology of malaria in San Dulakudar, a village in Sundargarh District in the state of Orissa in eastern India. Malaria transmission is perennial with Plasmodium falciparum, accounting for greater than 80% of malaria cases. Transmission intensity varies with season with high transmission after the monsoon rains in autumn and winter, low transmission in summer, and intermediate transmission in spring. The anthropophagic mosquito Anopheles fluviatilis was identified as the main vector for malaria transmission. Based on observations of spleen rates and supported by data on malaria parasite prevalence and malaria incidence, San Dulakudar can be classified as a hyperendemic area for P. falciparum malaria. Parasite prevalence and malaria incidence rates decrease with age, suggesting that residents of San Dulakudar develop immunity to malaria. The study demonstrates the presence of regions in the Indian subcontinent such as Sundargarh District where P. falciparum is the primary cause of malaria and where malaria transmission rates are comparable to those found in many parts of Africa.

Plasmodium falciparum infection elicits both variant-specific and cross-reactive antibodies against variant surface antigens.

Naturally acquired antibodies to Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP-1), the variant surface antigens expressed on the surface of infected erythrocytes, are thought to play a role in protection against P. falciparum malaria. Here, we have studied the development of antibodies to PfEMP-1 in adult malaria patients living in Rourkela, India, an area with a low malaria transmission rate, and prevalence of antibodies to PfEMP-1 in residents of San Dulakudar, India, a village in which P. falciparum malaria is hyperendemic. Convalescent-phase sera from adult malaria patients from Rourkela agglutinate homologous P. falciparum isolates as well as some heterologous isolates, suggesting that they develop partially cross-reactive antibodies to PfEMP-1 following infection. Adult sera from San Dulakudar agglutinate diverse P. falciparum isolates, suggesting that they have antibodies with wide recognition of diverse PfEMP-1. Mixed-agglutination assays using pairs of P. falciparum isolates confirm the presence of both variant-specific and partially cross-reactive antibodies in convalescent-phase sera from Rourkela and adult sera from San Dulakudar. Analysis of PfEMP-1 sequences suggests a molecular basis for the observed cross-reactivity.