Severe malaria in Gambian children is not due to lack of previous exposure to malaria.

The reasons why only a small proportion of African children infected with Plasmodium falciparum develop severe or fatal malaria are not known. One possible reason is that children who develop severe disease have had less previous exposure to malaria infection, and hence have less acquired immunity, than children who develop a mild clinical attack. To investigate this possibility we have measured titres of a wide range of anti-P. falciparum antibodies in plasma samples obtained from children with severe malaria, children with mild malaria and from children with other illnesses. Mean antibody levels in patients with malaria were higher than those in patients with other conditions but, with only one exception, there were no significant differences in antibody titres between cases of severe or mild malaria. A parasitized-erythrocyte agglutination assay was used to estimate the diversity of parasite isolates to which children had been exposed; plasma samples obtained from children with cerebral malaria recognized as many isolates as did samples obtained from children with mild disease. Our findings do not provide any support for the view that the development of severe malaria in a small proportion of African children infected with P. falciparum is due to lack of previous exposure to the infection.     

Immunoglobulin E, a pathogenic factor in Plasmodium falciparum malaria.

Most children and adults living in areas where the endemicity of Plasmodium falciparum malaria is high have significantly elevated levels of both total immunoglobulin E (IgE) and IgE antimalarial antibodies in blood. This elevation is highest in patients with cerebral malaria, suggesting a pathogenic role for this immunoglobulin isotype. In this study, we show that IgE elevation may also be seen in severe malaria without cerebral involvement and parallels an elevation of tumor necrosis factor alpha (TNF). IgE-containing serum from malaria immune donors was added to tissue culture plates coated with rabbit anti-human IgE antibodies or with P. falciparum antigen. IgE-anti-IgE complexes as well as antigen-binding IgE antibodies induced TNF release from peripheral blood mononuclear cells (PBMC). Nonmalaria control sera with no IgE elevation induced significantly less of this cytokine, and the TNF-inducing capacity of malaria sera was also strongly reduced by passing them over anti-IgE Sepharose columns. The cells giving rise to TNF were adherent PBMC. The release of this cytokine probably reflects cross-linking of their low-affinity receptors for IgE (CD23) by IgE-containing immune complexes known to give rise to monocyte activation via the NO transduction pathway. In line with this, adherent monocytic cells exposed to IgE complexes displayed increased expression of CD23. As the malaria sera contained IgG anti-IgE antibodies, such complexes probably also play a role in the induction of TNF in vivo. Overproduction of TNF is considered a major pathogenic mechanism responsible for fever and tissue lesions in P. falciparum malaria. This overproduction is generally assumed to reflect a direct stimulation of effector cells by certain parasite-derived toxins. Our results suggest that IgE elevation constitutes yet another important mechanism involved in excessive TNF induction in this disease.     

Increased plasma levels of soluble IL-2R are associated with severe Plasmodium falciparum malaria.

Plasma samples from children with mild and severe Plasmodium falciparum malaria and from children with unrelated diseases were collected to investigate whether the clinical outcome of infection was associated with plasma factors which reflected the activity of different cells of the immune system. Children with severe P. falciparum malaria had significantly higher plasma levels of soluble IL-2R than children with mild malaria. Plasma levels of IL-2R and levels of parasitaemia were significantly correlated. Neither parasitaemia nor plasma levels of tumour necrosis factor-alpha (TNF-alpha), IL-6, lymphotoxin (LT), interferon-gamma (IFN-gamma), IL-4, soluble IL-4R or soluble CD8 differed significantly between the two groups of children with malaria. High plasma levels of soluble CD8 were associated with failure of lymphocytes to produce IFN-gamma in vitro following stimulation with P. falciparum antigen. We conclude that soluble IL-2R is a useful marker of disease severity independently of the association with levels of parasitaemia, and that functional regulation of different lymphocyte subsets occurs during acute malaria episodes.     

Hypocalcemia in malaria. Study of correlations with other parameters

In a privileged observation of severe malaria contracted after laboratory contamination, we noticed a deep hypocalcemia 67 mg/l = 1.67 mmol/l. Since then we found again that hypocalcemia, connected with hypoalbuminemia, in ten other patients showing severe forms of malaria Plasmodium falciparum. The main biological datas of those four cases are summarized below. We made a systematic study of malaria cases, whatever may have been the intensity of parasitemia and clinical symptoms, and whatever the plasmodial species. For 26 non african paludisms , we found an average level of calcemia of 85 mg/l and an average hypoalbuminemia of 37 g/l = 536 mumol/l. For 30 african paludisms we found an average calcemia of 87.5 mg/l. That reduced value is statistically significant comparatively with the average value of 30 african people with no malaria 91.5 mg/l. The problem is to know if this hypocalcemia is connected only with hypoalbuminemia and if it may involve clinical or therapeutical effects.     

Blood Mononuclear Cell Nitric Oxide Production and Plasma Cytokine Levels in Healthy Gabonese Children with Prior Mild or Severe Malaria

Plasmodium falciparum malaria is an important cause of morbidity and mortality in children. Factors that determine the development of mild versus severe malaria are not fully understood. Since host-derived nitric oxide (NO) has antiplasmodial properties, we measured NO production and NO synthase (NOS) activity in peripheral blood mononuclear cells (PBMC) from healthy Gabonese children with a history of prior mild malaria (PMM) or prior severe malaria (PSM) caused by P. falciparum. The PMM group had significantly higher levels of NOS activity in freshly isolated PBMC and higher NO production and NOS activity in cultured PBMC. The investigation of NO-modulating cytokines (e.g., interleukin 12, gamma interferon, tumor necrosis factor alpha [TNF-alpha], and transforming growth factor beta1) as an explanation for differing levels of NOS activity revealed that plasma levels of TNF-alpha were significantly higher in the PSM group. Our results suggest that NOS/ NO and TNF-alpha are markers for prior disease severity and important determinants of resistance to malaria.     

Allelic polymorphisms in the repeat and promoter regions of the interleukin-4 gene and malaria severity in Ghanaian children

Immunoglobulin E has been associated with severe malaria suggesting a regulatory role for interleukin (IL)-4 and/or IgE in the pathogenesis of severe malaria. We have investigated possible associations between polymorphisms in the IL-4 repeat region (intron 3) and promoter regions (IL-4 +33CT and - 590CT) in Ghanaian children with severe malaria. There was a significantly higher frequency of IL-4 intron-3 B1B1 genotype in the cerebral malaria group [P < 0.0001, odds ratio (OR) = 8.7]. The genotype and allele frequencies of the IL-4 -590 and +33 polymorphisms did not differ between the four study groups. Carriers of IL-4 +33T/-590T with cerebral malaria had elevated total IgE compared to non-carriers (P = 0.03). Our data suggest that IL-4 and/or IgE play a regulatory role in the pathogenesis of severe or complicated malaria.     

Genetic diversity of P. falciparum and pathogenesis of the severe malarial anaemia in children under 5 years old in the province of Boulgou, Burkina Faso

The clinical presentation of malaria mainly the severe form may be related to Plasmodium falciparum msp-2 (merozoite surface protein 2) specific family To verify this hypothesis, during the high malaria transmission season in 2001; we analyzed the allelic polymorphism of the msp-2 gene of P. falciparum in children under 5 years old with different presentation of malaria in the regional Hospital and at community level in the Boulgou Province (Burkina Faso). A total of 405 children (107 severe malarial anaemia cases, 102 severe malaria cases without severe anaemia and 196 non severe malaria cases) were enrolled in the study. The frequencies of the FC27 were 89.2% in severe malarial anaemia children group, then 89.7% and 86.9% respectively in severe malaria non anaemic children cases and non severe malaria cases (P = 0.4). The frequencies of the 3D7 were 72.5%; 84.1% and 77% respectively severe malaria non anaemic children, severe malarial anaemia cases and non severe malaria cases (P = 0.7). The complexity of the FC27 genotypes was significantly higher in children with severe malaria (with and without severe anaemia) compared to the non severe malarial children (P < 0.001). No significant difference was pointed up in the complexity of the 3D7 genotypes.     

Low interleukin-12 activity in severe Plasmodium falciparum malaria

We compared interleukin-12 (IL-12) and other cytokine activities during and after an acute clinical episode in a matched-pair case-control study of young African children who presented with either mild or severe Plasmodium falciparum malaria. The acute-phase, pretreatment plasma IL-12 and alpha interferon (IFN-alpha) levels, as well as the acute-phase mitogen-stimulated whole-blood production capacity of IL-12, were significantly lower in children with severe rather than mild malaria. IL-12 levels, in addition, showed strong inverse correlations both with parasitemia and with the numbers of circulating malaria pigment-containing neutrophils. Acute-phase plasma tumor necrosis factor (TNF) and IL-10 levels were significantly higher in those with severe malaria, and the concentrations of both of these cytokines were positively correlated both with parasitemia and with the numbers of pigment-containing phagocytes in the blood. Children with severe anemia had the highest levels of TNF in plasma. In all the children, the levels in plasma and production capacities of all cytokines normalized when they were healthy and parasite free. The results indicate that severe but not mild P. falciparum malaria in young, nonimmune African children is characterized by down-regulated IL-12 activity, contrasting markedly with the up-regulation of both TNF and IL-10 in the same children. A combination of disturbed phagocyte functions resulting from hemozoin consumption, along with reduced IFN-gamma responses, may contribute to these differential effects.     

IgE and tumor necrosis factor in malaria infection

IgE, the immunoglobulin instrumental in atopic diseases is also elevated in many infections. This paper reports on the occurrence and possible pathogenic role of IgE in human Plasmodium falciparum malaria, one of the most widely spread and severe infectious diseases world wide. Plasmodial infections induce IgE elevation in the blood of the majority of people living in malaria endemic areas and up to 5% of this IgE constitutes anti-malaria antibodies. Production of IgE is controlled by T cells and elevated IgE concentrations in the blood of malaria patients are the result of an increased ratio of T-helper 2 (Th2) over T-helper 1 (Th1) cells. The underlying Th1 to Th2 switch is controlled by a variety of environmental and genetic factors. The importance of the latter is demonstrated by the IgE levels occurring in monozygotic or dizygotic twins originating from malarious areas of Africa. While these levels were indistinguishable within monozygotic twin pairs, they were different within the dizygotic pairs. Comparison of the levels of total IgE or IgE anti-malaria antibodies in patients with uncomplicated malaria with those in patients with the severe form of the disease (cerebral malaria or severe malaria without cerebral involvement) indicate that these levels are significantly higher in the cases with severe disease. This is the reverse with IgG and suggests that IgE plays a role in malaria pathogenesis. An important pathogenic mediator causing malaria fever and tissue lesions is tumor necrosis factor (TNF), generally believed to be induced by toxins released from the parasite. However, sera from malaria patients can also cause TNF release from monocytes in a reaction dependent on the presence of IgE containing immune complexes or aggregates. This results in induction and cross-linking of Fcepsilon receptor II (CD23) and by binding to and activating these cells, IgE will contribute to a local over-production of TNF in capillaries and post-capillary venules where P. falciparum parasites or their products accumulate in the severe forms of this disease.     

TNFalpha-308A associated with shorter intervals of Plasmodium falciparum reinfections

A mutation at position -308 of the tumor necrosis factor alpha (TNF-308A) gene promoter has previously been associated with particular manifestations of infectious and non-infectious diseases. In a longitudinal study on malariological parameters in Gabon, TNF promoter variants of 98 children initially presenting with severe Plasmodium falciparum malaria, followed by a total of 504 reinfection events within 52 months, and 100 children initially presenting with mild malaria followed by a total of 342 reinfections were analyzed. Symptomatic P. falciparum reinfections occurred more quickly (median 11 weeks) in carriers of the TNF-308A allele, with severe malaria at enrollment, compared to carriers of other TNF promoter variants (median 16 weeks). The results show that this particular TNF promoter allele increases the risk of reinfection with the malaria parasite P. falciparum.     

Pertinence of the 2000 WHO criteria in non-immune children with severe malaria in Dakar, Senegal

The relevance of World Health Organization (WHO) criteria for severe malaria has not been assessed in non-immune children. The objectives of this study were (i) to evaluate the significance of 1990 WHO definition reconsidered in 2000 on distribution and lethality of severe cases in children admitted with falciparum malaria, and (ii) to contribute to the study of relevance of the WHO severe criteria in Dakar, an hypoendemic area in Senegal. PATIENTS AND METHODS: The 1990 WHO criteria, respiratory distress and platelet counts were prospectively collected in 1997-99 from children admitted to H?pital Principal de Dakar, Senegal, with falciparum malaria diagnosed on a thick blood film. This method allowed also the definition of severe cases according to 2000 WHO criteria. RESULTS: Among 311 patients (median age: 8 years old), according to the 2000 WHO criteria, the frequency of severe malaria cases was increased by 23% (75% versus 52%) and case-fatality rates thereof were decreased by 5% (17% versus 12%) compared with 1990 WHO definition. One death occurred among cases defined as severe on admission only according to criteria modified by WHO in 2000. A multivariate logistic regression model identified several independent prognostic factors: cerebral malaria, hypoglycaemia, respiratory distress, renal failure, collapse, abnormal bleedings, pupillary abnormalities and thrombocytopaenia defined as a platelet count below 100,000/mm3. A significant association (p < 0.001) was observed between platelet count increase and consciousness level improvement, evaluated on day of first platelet count control (time from admission: 1-7 d). Among survivors, a lesser improvement in coma score was associated with a decrease in platelet counts (p < 0.04). CONCLUSIONS: The 1990 WHO criteria, which predicted death among malaria cases in children living under stable falciparum transmission, are relevant in this series of non-immune children living in a low and seasonal transmission. Nevertheless new WHO criteria showed poor prognostic significance. However, the 2000 WHO definition was highly sensitive to detect severe malaria cases. These findings should be considered for managing severe malaria in migrant children.     

Immunoglobulin G isotype responses to erythrocyte surface-expressed variant antigens of Plasmodium falciparum predict protection from malaria in African children

We assessed immunoglobulin G (IgG) isotype responses to variant surface antigens (VSA) expressed on parasite-infected erythrocytes of a panel of heterologous isolates during and after acute episodes in groups of Gabonese children presenting with either mild or severe Plasmodium falciparum malaria. In the acute and convalescent phases IgG3 and IgG1 anti-VSA antibodies, respectively, predominated. In the absence of infection, the levels of both cytophilic isotypes waned, while those of IgG4 increased, particularly in those admitted with severe malaria. Prospective analyses showed significantly longer delays between malaria attacks associated both (i) with increasing IgG1 responses with specificity for VSA of isolates from children with mild malaria and (ii) with increasing IgG4 responses with specificity for VSA of isolates from children with severe malaria. These findings imply that the predictive value of prospectively measured cross-reactive VSA-specific IgG antibodies with respect to protection against malaria in African children depends both on their isotype and on their fine specificity.     

Hypertriglyceridemia as an indicator of the severity of<Emphasis Type="Italic"> falciparum</Emphasis> malaria in returned travelers: a clinical retrospective study

To test the hypothesis that the magnitude of plasma triglyceride changes could be related to the severity of falciparum malaria, we performed a retrospective case-control study from January 1999 to December 2000 among hospitalized patients with fever who were returning to France from the tropics. Plasma triglycerides were measured in patients with severe falciparum malaria (n=13), mild falciparum malaria (n=169), non-falciparum malaria (n=20) and controls (n=55). Triglyceride level was significantly higher in the malaria group than in controls [mean values were 2.17±1.43 mmol/l versus 1.30±0.70 mmol/l, respectively (P<0.0001)]. Triglyceride level was also significantly higher in severe than in mild malaria [4.78±1.93 mmol/l versus 1.94±1.11, respectively (P<0.00001)]. Hypertriglyceridemia (>1.80 mmol/l) was noted in all the patients with severe malaria, compared to 37% of patients with mild disease (P<0.001). Although further studies are needed, these results define the relevance of hypertriglyceridemia as an indicator of the severity of falciparum malaria.     

Increased plasma concentrations of sICAM-1, sVCAM-1 and sELAM-1 in patients with Plasmodium falciparum or P. vivax malaria and association with disease severity.

Increased serum concentrations of soluble intercellular adhesion molecule-1 (sICAM-1), soluble endothelial leucocyte adhesion molecule-1 (sELAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1) were detected in Danish malaria patients infected with sequestering Plasmodium falciparum or non-sequestering P. vivax parasites, as well as in patients with sepsis or meningitis. Levels of soluble adhesion molecules remained elevated in the P. falciparum patients for several weeks after initiation of treatment. Plasma concentrations of sICAM-1, sVCAM-1 and sELAM-1 were higher in Gambian children with severe P. falciparum malaria than in children with mild malaria. Plasma levels of sVCAM-1 and sELAM-1 were significantly correlated. Plasma levels of sELAM-1 and sVCAM-1 may reflect endothelial inflammatory reactions and these reactions may be harmful for humans infected with malaria parasites.     

Anti-phospholipid antibodies in patients with Plasmodium falciparum malaria.

Plasma levels of antibodies against phosphatidylinositol (PI), phosphatidylcholine (PC) and cardiolipin (CL) were measured by enzyme-linked immunosorbent assay (ELISA) in patients from malaria endemic area of Sudan and The Gambia. Some Sudanese adults produced IgM antibodies against all three types of phospholipids (PL) during an acute Plasmodium falciparum infection. The anti-PL antibody titre returned to preinfection levels in most of the donors 30 days after the disease episode. IgG titres against PI, PC and CL were low. In Gambian children with malaria, IgM antibody titres against PI and PC were significantly higher in those with severe malaria than in those with mild malaria. These results show that a proportion of malaria patients produce anti-PL antibodies during infection and that titres of these antibodies are associated with the severity of disease.     

Antibody responses to a C-terminal fragment of the Plasmodium falciparum blood-stage antigen Pf332 in Senegalese individuals naturally primed to the parasite

Previous studies have shown that antibodies from humans exposed continuously to malaria recognize the Plasmodium falciparum asexual blood-stage antigen Pf332. Here we analysed the antibody responses to a C-terminal fragment of Pf332, designated C231, in individuals from Senegal, by measuring the serum levels of immunoglobulin M (IgM), IgG class and subclass and IgE antibodies. IgG antibody reactivity with crude P. falciparum antigen was detected in all the donors, while many of the children lacked or had low levels of such antibodies against C231. The antibody levels increased significantly with age for both crude P. falciparum antigen and C231, and in the older age groups most of the donors displayed antibodies to C231. This was also true for IgM, IgE and IgG subclass reactivity against C231. Moreover, the ratio of IgG1/IgG2 was considerably lower for C231 than for crude P. falciparum antigen, and in age groups 10-14 and 15-19 years the levels of IgG2 against C231 even exceeded that of IgG1. The IgG2/IgG3 ratios suggest that C231 gives similar levels of IgG2 and IgG3, except for children aged 4-9 years, where IgG3 was higher. Raw IgM, IgG class and subclass and IgE antibody levels to C231 tended to be higher in those who did not experience a malaria attack, but following linear multivariate analysis the trends were not significant.     

Fc gamma receptor IIa (CD32) polymorphism and antibody responses to asexual blood-stage antigens of Plasmodium falciparum malaria in Sudanese patients

In a prospective clinical study in New Halfa Teaching Hospital, the possible association between FcgammaRIIa-R/H131 polymorphism and anti-malarial antibody responses with clinical outcome of Plasmodium falciparum malaria among Sudanese patients was investigated. A total of 256 individuals were consecutively enrolled, comprising 115 patients with severe malaria, 85 with mild malaria and 56 malaria-free controls. Genotyping of FcgammaRIIa-R/H131 dimorphism was performed using gene-specific polymerase chain reaction (PCR) amplification with allele-specific restriction enzyme digestion of the PCR product. The antibody responses to asexual blood-stage antigens were assessed by an enzyme-linked immunosorbent assay. The frequency of the FcgammaRIIa-R/R131 genotype was significantly higher in those with severe malaria when compared with patients with mild malaria, while the FcgammaRIIa-H/H131 genotype showed a significant association with mild malaria. A reduced risk of severe malaria with IgG3 antibodies in combination with the H/H131 genotype was observed. Furthermore, low levels of IgG2 antibodies reactive with the Pf332-C231 antigen were also associated with lower risk of severe malaria in individuals carrying the H131 allele. The levels of IgG1 and IgG3 antibodies were statistically significantly higher in the mild malaria patients when compared with the severe malaria patients. Taken together, our study revealed that the FcgammaRIIa-R/R131 genotype is associated with the development of severe malaria, while the H/H131 genotype is more likely to be associated with mild malaria. Our results also revealed that the natural acquisition of immunity against clinical malaria appeared to be more associated with IgG1 and IgG3 antibodies, signifying their roles in parasite-neutralizing immune mechanisms.     

Levels and interactions of plasma xanthine oxidase, catalase and liver function parameters in Nigerian children with Plasmodium falciparum infection

Elevated plasma levels of xanthine oxidase and liver function parameters have been associated with inflammatory events in several human diseases. While xanthine oxidase provides in vitro protection against malaria, its pathophysiological functions in vivo and interactions with liver function parameters remain unclear. This study examined the interactions and plasma levels of xanthine oxidase (XO) and uric acid (UA), catalase (CAT) and liver function parameters GOT, GPT and bilirubin in asymptomatic (n=20), uncomplicated (n=32), and severe (n=18) falciparum malaria children aged 3-13 years. Compared to age-matched control (n=16), significant (p<0.05) elevation in xanthine oxidase by 100-550%, uric acid by 15.4-153.8%, GOT and GPT by 22.1-102.2%, and total bilirubin by 2.3-86% according to parasitaemia (geometric mean parasite density (GMPD)=850-87100 parasites/microL) was observed in the malarial children. Further comparison with control revealed higher CAT level (16.2+/-0.5 vs 14.6+/-0.4 U/L; p<0.05) lacking significant (p>0.05) correlation with XO, but lower CAT level (13.4-5.4 U/L) with improved correlations (r=-0.53 to -0.91; p<0.05) with XO among the asymptomatic and symptomatic malaria children studied. 75% of control, 45% of asymptomatic, 21.9% of uncomplicated, and none of severe malaria children had Hb level>11.0 g/dL. Multivariate analyses further revealed significant (p<0.05) correlations between liver function parameters and xanthine oxidase (r=0.57-0.64) only in the severe malaria group. We conclude that elevated levels of XO and liver enzymes are biochemical features of Plasmodium falciparum parasitaemia in Nigerian children, with both parameters interacting differently to modulate the catalase response in asymptomatic and symptomatic falciparum malaria.