The relationship between reported fever and Plasmodium falciparum infection in African children

Background

The genetic diversity of Plasmodium falciparum has been extensively studied in various parts of the world. However, limited data are available from Pakistan. This study aimed to establish molecular characterization of P. falciparum field isolates in Pakistan measured with two highly polymorphic genetic markers, i.e. the merozoite surface protein 1 (msp-1)and 2 (msp-2).

Methods

Between October 2005 and October 2007, 244 blood samples from patients with symptomatic blood-slide confirmed P. falciparum mono-infections attending the Aga Khan University Hospital, Karachi, or its collection units located in Sindh and Baluchistan provinces, Pakistan were collected. The genetic diversity of P. falciparum was analysed by length polymorphism following gel electrophoresis of DNA products from nested polymerase chain reactions (PCR) targeting block 2 of msp-1 and block 3 of msp-2, including their respective allelic families KI, MAD 20, RO33, and FC27, 3D7/IC.

Results

A total of 238/244 (98%) patients had a positive PCR outcome in at least one genetic marker; the remaining six were excluded from analysis. A majority of patients had monoclonal infections. Only 56/231 (24%) and 51/236 (22%) carried multiple P. falciparum genotypes in msp-1 and msp-2, respectively. The estimated total number of genotypes was 25 msp-1 (12 KI; 8 MAD20; 5 RO33) and 33 msp-2 (14 FC27; 19 3D7/IC).

Conclusions

This is the first report on molecular characterization of P. falciparum field isolates in Pakistan with regards to multiplicity of infection. The genetic diversity and allelic distribution found in this study is similar to previous reports from India and Southeast Asian countries with low malaria endemicity.     

High levels of circulating cardiac proteins indicate cardiac impairment in African children with severe Plasmodium falciparum malaria

A role of the heart in the pathophysiology of severe Plasmodium falciparum malaria has recently been suggested. The objective of the present study was to substantiate this finding in a large group of African children and to correlate results with metabolic conditions in these children. Furthermore, the impact of a potential cardiac impairment on outcome in severe cases was assessed. Results may have important implications on the currently ongoing debate on fluid management in severe malaria patients. Plasma levels of N-terminal pro-brain natriuretic peptide (NT-proBNP), heart-type fatty acid-binding protein (H-FABP), myoglobin and creatine kinase muscle-brain (CK-MB) were compared in 400 African children with severe and mild falciparum malaria. Plasma levels of these markers were correlated with lactate and glucose blood levels, indicators for hypovolemia, and with clinical outcome. Children suffering from severe malaria and children who died (n = 22) exhibited high to very high levels of cardiac markers, respectively. Cardiac factors themselves were not predictive of fatal outcome, while, in multivariate analysis, lactic acidosis was the most important biochemical predictor of death in the severe malaria group. Lactic acidosis and hypoglycemia, however, result in cardiac impairment as defined by elevated levels of circulating cardiac proteins. Our results point to hypovolemia as a major underlying cause of lactic acidosis and hypoglycemia in African children with severe falciparum malaria. These deleterious metabolic conditions contribute to myocardial affection which was evident but not predictive per se of fatal outcome.     

Low antibodies against Plasmodium falciparum and imbalanced pro-inflammatory cytokines are associated with severe malaria in Mozambican children: a case-control study

ABSTRACT: BACKGROUND: The factors involved in the progression from Plasmodium falciparum infection to severe malaria (SM) are still incompletely understood. Altered antibody and cellular immunity against P. falciparum might contribute to increase the risk of developing SM. METHODS: To identify immune responses associated with SM, a sex- and age-matched case-control study was carried out in 134 Mozambican children with SM (cerebral malaria, severe anaemia, acidosis and/or respiratory distress, prostration, hypoglycaemia, multiple seizures) or uncomplicated malaria (UM). IgG and IgM against P. falciparum lysate, merozoite antigens (MSP-119, AMA-1 and EBA-175), a Duffy binding like (DBL)-alpha rosetting domain and antigens on the surface of infected erythrocytes were measured by ELISA or flow cytometry. Plasma concentrations of IL-12p70, IL-2, IFN-gamma, IL-4, IL-5, IL-10, IL-8, IL-6, IL- 1beta, TNF, TNF-beta and TGF-beta1 were measured using fluorescent bead immunoassays. Data was analysed using McNemar's and Signtest. RESULTS: Compared to UM, matched children with SM had reduced levels of IgG against DBLalpha (P < 0.001), IgM against MSP-119 (P = 0.050) and AMA-1 (P = 0.047), TGF-beta1 (P <0.001) and IL-12 (P = 0.039). In addition, levels of IgG against P. falciparum lysate and IL-6 concentrations were increased (P = 0.004 and P = 0.047, respectively). Anti-DBLalpha IgG was the only antibody response associated to reduced parasite densities in a multivariate regression model (P = 0.026). CONCLUSIONS: The lower levels of antibodies found in children with SM compared to children with UM were not attributable to lower exposure to P. falciparum in the SM group. IgM against P. falciparum and specific IgG against a rosetting PfEMP1 domain may play a role in the control of SM, whereas an imbalanced pro-inflammatory cytokine response may exacerbate the severity of infection. A high overlap in symptoms together with a limited sample size of different SM clinical groups reduced the power to identify immunological correlates for particular forms of SM.     

Stool microbiota composition is associated with the prospective risk of Plasmodium falciparum infection

Background

In humans it is unknown if the composition of the gut microbiota alters the risk of Plasmodium falciparum infection or the risk of developing febrile malaria once P. falciparum infection is established. Here we collected stool samples from a cohort composed of 195 Malian children and adults just prior to an intense P. falciparum transmission season. We assayed these samples using massively parallel sequencing of the 16S ribosomal RNA gene to identify the composition of the gut bacterial communities in these individuals. During the ensuing 6-month P. falciparum transmission season we examined the relationship between the stool microbiota composition of individuals in this cohort and their prospective risk of both P. falciparum infection and febrile malaria.

Results

Consistent with prior studies, stool microbial diversity in the present cohort increased with age, although the overall microbiota profile was distinct from cohorts in other regions of Africa, Asia and North America. Age-adjusted Cox regression analysis revealed a significant association between microbiota composition and the prospective risk of P. falciparum infection; however, no relationship was observed between microbiota composition and the risk of developing febrile malaria once P. falciparum infection was established.

Conclusions

These findings underscore the diversity of gut microbiota across geographic regions, and suggest that strategic modulation of gut microbiota composition could decrease the risk of P. falciparum infection in malaria-endemic areas, potentially as an adjunct to partially effective malaria vaccines.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1819-3) contains supplementary material, which is available to authorized users.     

Genetic diversity of expressed Plasmodium falciparum var genes from Tanzanian children with severe malaria

ABSTRACT: BACKGROUND: Severe malaria has been attributed to the expression of a restricted subset of the var multigene family, which encodes for Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1 mediates cytoadherence and sequestration of infected erythrocytes into the post-capillary venules of the vital organs such as the brain, lung or placenta. Var genes are highly diverse and can be classified in three major groups (ups A, B and C) and two intermediate groups (B/A and B/C) based on the genomic location, gene orientation and upstream sequences. The genetic diversity of expressed var genes in relation to severity of disease in Tanzanian children was analysed. METHODS: Children with defined severe (SM) and asymptomatic malaria (AM) were recruited. Fulllength var mRNA was isolated and reversed transcribed into var cDNA. Subsequently, the DBL and N-terminal domains, and up-stream sequences were PCR amplified, cloned and sequenced. Sequences derived from SM and AM isolates were compared and analysed. RESULTS: The analysis confirmed that the var family is highly diverse in natural Plasmodium falciparum populations. Sequence diversity of amplified var DBL-1alpha and upstream regions showed minimal overlap among isolates, implying that the var gene repertoire is vast and most probably indefinite in endemic areas. var DBL-1alpha sequences from AM isolates were more diverse with more singletons found (p<0.05) than those from SM infections. Furthermore, few var DBL-1alpha sequences from SM patients were rare and restricted suggesting that certain PfEMP1 variants might induce severe disease. CONCLUSIONS: The genetic sequence diversity of var genes of P. falciparum isolates from Tanzanian children is large and its relationship to disease severity has been studied. Observed differences suggest that different var genes might have fundamentally different roles in the host-parasite interaction. Further research is required to examine clear disease-associations of var gene subsets in different geographical settings. The importance of very strict clinical definitions and appropriate large control groups needs to be emphasized for future studies on disease associations of PfEMP1.     

Mannose-binding lectin variant associated with severe malaria in young African children

Mannose-binding lectin (MBL) is a serum protein which initiates innate immune responses to microbial pathogens by binding to non-self surface oligosaccharides. MBL deficiency is the most common congenital immunodeficiency of human and has been shown to predispose to infections, particularly in children and immune compromised. In a matched case-control study among 870 Ghanaian children, we examined the influence of six polymorphisms of the MBL2 gene on Plasmodium falciparum infection and severe malaria. A missense mutation resulting in low MBL activity (MBL2*C) was found in 35% of healthy controls, but in 42% of asymptomatically infected children (P=0.01), and in 46% of patients with severe malaria (P=0.007). Heterozygosity for MBL2*C was associated with increased odds of infection (odds ratio (OR), 1.6; 95% confidence interval (CI), 1.1-2.1), severe malaria (OR, 1.7; 95% CI, 1.2-2.4), and of severe anemia in particular (OR, 2.3; 95% CI, 1.4-3.8). The population attributable fraction of severe malaria cases attributable to MBL2*C heterozygosity was 17%. Our results suggest that the MBL pathway of the complement system is a critical determinant of both, susceptibility to P. falciparum infection and manifestation of severe malaria, particularly in young children in whom specific immune responses are weak or absent.     

Literacy and recent history of diarrhoea are predictive of Plasmodium falciparum parasitaemia in Kenyan adults

Background

Evaluating copy numbers of given genes in Plasmodium falciparum parasites is of major importance for laboratory-based studies or epidemiological surveys. For instance, pfmdr1 gene amplification has been associated with resistance to quinine derivatives and several genes involved in anti-oxidant defence may play an important role in resistance to antimalarial drugs, although their potential involvement has been overlooked.

Methods

The^ΔΔCt method of relative quantification using real-time quantitative PCR with SYBR Green I detection was adapted and optimized to estimate copy numbers of three genes previously indicated as putative candidates of resistance to quinolines and artemisinin derivatives: pfmdr1, pfatp6 (SERCA) and pftctp, and in six further genes involved in oxidative stress responses.

Results

Using carefully designed specific RT-qPCR oligonucleotides, the methods were optimized for each gene and validated by the accurate measure of previously known number of copies of the pfmdr1 gene in the laboratory reference strains P. falciparum 3D7 and Dd2. Subsequently, Standard Operating Procedures (SOPs) were developed to the remaining genes under study and successfully applied to DNA obtained from dried filter blood spots of field isolates of P. falciparum collected in São Tomé & Principe, West Africa.

Conclusion

The SOPs reported here may be used as a high throughput tool to investigate the role of these drug resistance gene candidates in laboratory studies or large scale epidemiological surveys.     

Features and prognosis of severe malaria caused by Plasmodium falciparum, Plasmodium vivax and mixed Plasmodium species in Papua New Guinean children

Background

Mortality from severe pediatric falciparum malaria appears low in Oceania but Plasmodium vivax is increasingly recognized as a cause of complications and death. The features and prognosis of mixed Plasmodium species infections are poorly characterized. Detailed prospective studies that include accurate malaria diagnosis and detection of co-morbidities are lacking.

Methods and Findings

We followed 340 Papua New Guinean (PNG) children with PCR-confirmed severe malaria (77.1% P. falciparum, 7.9% P. vivax, 14.7% P. falciparum/vivax) hospitalized over a 3-year period. Bacterial cultures were performed to identify co-incident sepsis. Clinical management was under national guidelines. Of 262 children with severe falciparum malaria, 30.9%, 24.8% and 23.2% had impaired consciousness, severe anemia, and metabolic acidosis/hyperlactatemia, respectively. Two (0.8%) presented with hypoglycemia, seven (2.7%) were discharged with neurologic impairment, and one child died (0.4%). The 27 severe vivax malaria cases presented with similar phenotypic features to the falciparum malaria cases but respiratory distress was five times more common (P = 0.001); one child died (3.7%). The 50 children with P. falciparum/vivax infections shared phenotypic features of mono-species infections, but were more likely to present in deep coma and had the highest mortality (8.0%; P = 0.003 vs falciparum malaria). Overall, bacterial cultures were positive in only two non-fatal cases. 83.6% of the children had alpha-thalassemia trait and seven with coma/impaired consciousness had South Asian ovalocytosis (SAO).

Conclusions

The low mortality from severe falciparum malaria in PNG children may reflect protective genetic factors other than alpha-thalassemia trait/SAO, good nutrition, and/or infrequent co-incident sepsis. Severe vivax malaria had similar features but severe P. falciparum/vivax infections were associated with the most severe phenotype and worst prognosis.     

Broadly reactive antibodies specific for Plasmodium falciparum MSP-119 are associated with the protection of naturally exposed children against infection

ABSTRACT: BACKGROUND: The 19 kDa C-terminal region of Plasmodium falciparum Merozoite Surface Protein-1 is a known target of naturally acquired humoral immunity and a malaria vaccine candidate. MSP- 119 has four predominant haplotypes resulting in amino acid changes labelled EKNG, QKNG, QTSR and ETSR. IgG antibodies directed against all four variants have been detected, but it is not known if these variant specific antibodies are associated with haplotype-specific protection from infection. METHODS: Blood samples from 201 healthy Kenyan adults and children who participated in a 12-week treatment time-to-infection study were evaluated. Venous blood drawn at baseline (week 0) was examined for functional and serologic antibodies to MSP-119 and MSP-142 variants. MSP-119 haplotypes were detected by a multiplex PCR assay at baseline and weekly throughout the study. Generalized linear models controlling for age, baseline MSP-119 haplotype and parasite density were used to determine the relationship between infecting P. falciparum MSP-119 haplotype and variant-specific antibodies. RESULTS: A total of 964 infections resulting in 1,533 MSP-119 haplotypes detected were examined. The most common haplotypes were EKNG and QKNG, followed by ETSR and QTSR. Children had higher parasite densities, greater complexity of infection (>1 haplotype), and more frequent changes in haplotypes over time compared to adults. Infecting MSP-119 haplotype at baseline (week 0) had no influence on haplotypes detected over the subsequent 11 weeks among children or adults. Children but not adults with MSP-119 and some MSP-142 variant antibodies detected by serology at baseline had delayed time-to-infection. There was no significant association of variant-specific serology or functional antibodies at baseline with infecting haplotype at baseline or during 11 weeks of follow up among children or adults. CONCLUSIONS: Variant transcending IgG antibodies to MSP-119 are associated with protection from infection in children, but not adults. These data suggest that inclusion of more than one MSP-119 variant may not be required in a malaria blood stage vaccine.     

Plasmodium falciparum-specific interleukin-2 and tumor necrosis factor-alpha expressing-T cells are associated with resistance to reinfection and severe malaria in healthy African children

The frequency of P. falciparum-specific interleukin (IL)-2-, interferon (IFN)-gamma-, tumor necrosis factor (TNF)-alpha- and IL-10-expressing CD3+ cells was studied in healthy Gabonese children segregated according to their clinical presentation at admission to a longitudinal study of severe and mild malaria. The percentage of IL-2- and TNF-alpha- expressing P. falciparum-specific CD3+ cells was significantly higher in the children with prior mild malaria and less frequent reinfections compared to the children with prior severe malaria and more frequent reinfections. No differences were shown for P. falciparum-specific IFN-gamma and IL-10 expression within CD3+ cells and parasite-non-specific expression of IL-2, IL-4, IL-6, IL-10, IL-13, TNF-alpha, and IFN-gamma within the CD4+, CD8+, TCRgamma\delta+ CD3+ and CD94+ CD3- cell populations, indicating that immunological determinants regulating the susceptibility to malaria in age-matched children are parasite-specific. The ability of P. falciparum-specific T cells to mount a rapid IL-2 and TNF-alpha response might be of significance in preventing severe disease and reinfection.     

Decreased hemoglobin concentrations,hyperparasitemia, and severe malaria are associated with increased Plasmodium falciparum gametocyte carriage

To determine factors influencing gametocyte carriage, a cross-sectional study was conducted among 512 patients admitted for Plasmodium falciparum malaria. After adjustments for potential confounders, hemoglobin concentrations were lower in gametocyte carriers 10.5 (+/-2.5) than in patients without gametocytes 12.5 (+/-2.3) (P < 0.0001). Hemoglobin concentrations were negatively correlated with peak gametocyte counts (Spearman's p = -0.37, P < 0.0001) and gametocyte carriage durations (Spearman's p = -(0.30, P < 0.0001). Adjustments for the duration of the malaria episode and other potential confounders did not alter the association (P < 0.0001). After adjustment for potential confounders, the median asexual parasitemia was higher in patients with gametocytes than in patients without gametocytes (P = 0.003). Severe malaria cases were more likely to have gametocytes (65%) than malaria with hyperparasitemia (38%) or mild malaria (31%) (P = 0.0001). These findings suggest that events surrounding anemia and tissue hypoxia stimulate Plasmodium falciparum gametocytogenesis.     

Bone marrow transplantation in children with neuroblastoma

Neuroblastoma is the third most frequent malignant tumor in childhood. One-third of the patients over one year of age at the time of diagnosis suffer from the disseminated form (stage IV). Despite highly aggressive chemotherapy survival rates are poor. One hundred and eighty-seven patients with neuroblastoma stage IV have been treated according to the German protocol NB 85. The probability of disease free survival is only 15% after 70 months. Treatment strategy in our protocol includes autologous and allogeneic bone marrow transplantation (BMT) for patients with stage IV (and greater than 1 year old). Twenty-two patients were grafted (7 allogeneic and 15 autologous). The conditioning regimen consisted mainly of high-dose melphalan (180 mg/m2) and total body irradiation (TBI) (3.4 Gy). Survival rates are discussed in the context of the chemotherapy protocol. Our own experience with autologous BMT is poor, despite of different purging methods. For this reason we decided to focus on allogeneic BMT. We have grafted five patients within the last 3 years. Three of them are alive and well, on died from veno-occlusive disease 70 days after BMT, and the remaining patient, grafted from a syngeneic donor, died from relapsing tumor. The main problem in neuroblastoma stage IV is resistance to chemotherapy. Intensification of the conditioning regimen or double autografting leads to a rate of toxic deaths close to 20% (Zucker, EBMT 1987) which is not tolerable. New improvements in the conditioning regimen have to be found to increase the effect of BMT.     

Plasma Plasmodium falciparum histidine-rich protein-2 concentrations are associated with malaria severity and mortality in Tanzanian children

Plasma Plasmodium falciparum histidine-rich protein-2 (PfHRP-2) concentrations, a measure of parasite biomass, have been correlated with malaria severity in adults, but not yet in children. We measured plasma PfHRP-2 in Tanzanian children with uncomplicated (n = 61) and cerebral malaria (n = 45; 7 deaths). Median plasma PfHRP-2 concentrations were higher in cerebral malaria (1008 [IQR 342-2572] ng/mL) than in uncomplicated malaria (465 [IQR 36-1426] ng/mL; p = 0.017). In cerebral malaria, natural log plasma PfHRP-2 was associated with coma depth (r = -0.42; p = 0.006) and mortality (OR: 3.0 [95% CI 1.03-8.76]; p = 0.04). In this relatively small cohort study in a mesoendemic transmission area of Africa, plasma PfHRP-2 was associated with pediatric malaria severity and mortality. Further studies among children in areas of Africa with higher malaria transmission and among children with different clinical manifestations of severe malaria will help determine the wider utility of quantitative PfHRP-2 as a measure of parasite biomass and prognosis in sub-Saharan Africa.     

Plasmodium falciparum infection and exoerythrocytic development in mice with chimeric human livers

The exoerythrocytic stage of Plasmodium falciparum has remained a difficult phase of the parasite life-cycle to study. The host and tissue specificity of the parasite requires the experimental infection of humans or non-human primates and subsequent surgical recovery of parasite-infected liver tissue to analyze this stage of the parasites development. This type of study is impossible in humans due to obvious ethical considerations and the cost and complexity in working with primate models has precluded their use for extensive studies of the exoerythrocytic stage. In this study we assessed, for the first time, the use of transgenic, chimeric mice containing functioning human hepatocytes as an alternative for modeling the in vivo interaction of P. falciparum parasites and human hepatocytes. Infection of these mice with P. falciparum sporozoites produced morphologically and antigenically mature liver stage schizonts containing merozoites capable of invading human red blood cells. Additionally, using microdissection, highly enriched P. falciparum liver stage parasites essentially free of hepatocyte contamination, were recovered for molecular studies. Our results establish a stable murine model for P. falciparum that will have a wide utility for assessing the biology of the parasite, potential anti-malarial chemotherapeutic agents and vaccine design.     

Changes in Plasmodium falciparum gametocytaemia in children with chloroquine-sensitive asexual infections

A non-compartmental pharmacokinetic model was used to describe the changes in gametocytaemia in nine children with chloroquine-sensitive Plasmodium falciparum malaria in whom asexual parasitaemia cleared within 72 h of chloroquine treatment. Peak gametocytaemia was 74 +/- 19.9 (se), range 24-198, geometric mean 58 sf (sexual forms)/microliter. Time to peak gamelocytaemia was 43.2 +/- 14.4, range 0-120 h. Following peak gametocytaemia, gametocytes persisted in blood for a period of 168-504 h. The decline from peak gametocytaemia was exponential with a half-life of gametocytaemia of 43.2 +/- 20.4, range 13.1-206 h. The mean pre-treatment sex ratio was male-biased and remained so till complete elimination of gametocytaemia. Peak microgametocytaemia, area under the curve of microgametocytaemia versus time, and the half-life of microgametocytaemia were significantly higher than those of macrogametocytaemia. The volume of blood completely cleared of macrogametocytaemia per unit time was significantly higher than that of microgametocytaemia. Macrogometocytes are cleared from the circulation faster than microgametocytes but chloroquine treatment of chloroquine-sensitive infections has little or no significant effect on gametocyte sex ratios in this group of children.     

Bone marrow hypoplasia associated with estrus in ferrets

Bone marrow hypoplasia was characterized in a group of female ferrets during prolonged estrus. All ferrets exhibited hematological changes characteristic of various degrees of bone marrow hypoplasia. Hematological findings included initial thrombocytosis and leukocytosis followed by thrombocytopenia, leukopenia and anemia. Platelet counts below 50,000/microliters were observed in 55% of the ferrets. Hemorrhagic anemia due to thrombocytopenia was the most common cause of death and the mortality rate was 40%. Histopathological findings included bone marrow hypoplasia affecting all cell lines and decreased splenic extramedullary hematopoiesis.     

Reduced T regulatory cell response during acute Plasmodium falciparum infection in Malian children co-infected with Schistosoma haematobium

Background

Regulatory T cells (Tregs) suppress host immune responses and participate in immune homeostasis. In co-infection, secondary parasite infections may disrupt the immunologic responses induced by a pre-existing parasitic infection. We previously demonstrated that schistosomiasis-positive (SP) Malian children, aged 4–8 years, are protected against the acquisition of malaria compared to matched schistosomiasis-negative (SN) children.

Methods and Findings

To determine if Tregs contribute to this protection, we performed immunologic and Treg depletion in vitro studies using PBMC acquired from children with and without S. haematobium infection followed longitudinally for the acquisition of malaria. Levels of Tregs were lower in children with dual infections compared to children with malaria alone (0.49 versus 1.37%, respectively, P = 0.004) but were similar months later, during a period with negligible malaria transmission. The increased levels of Tregs in SN subjects were associated with suppressed serum Th1 cytokine levels, as well as elevated parasitemia compared to co-infected counterparts.

Conclusions

These results suggest that lower levels of Tregs in helminth-infected children correlate with altered circulating cytokine and parasitologic results which may play a partial role in mediating protection against falciparum malaria.     

Specific receptor usage in Plasmodium falciparum cytoadherence is associated with disease outcome

Our understanding of the basis of severe disease in malaria is incomplete. It is clear that pathology is in part related to the pro-inflammatory nature of the host response but a number of other factors are also thought to be involved, including the interaction between infected erythrocytes and endothelium. This is a complex system involving several host receptors and a major parasite-derived variant antigen (PfEMP1) expressed on the surface of the infected erythrocyte membrane. Previous studies have suggested a role for ICAM-1 in the pathology of cerebral malaria, although these have been inconclusive. In this study we have examined the cytoadherence patterns of 101 patient isolates from varying clinical syndromes to CD36 and ICAM-1, and have used variant ICAM-1 proteins to further characterise this adhesive phenotype. Our results show that increased binding to CD36 is associated with uncomplicated malaria while ICAM-1 adhesion is raised in parasites from cerebral malaria cases.