Parasite multiplication potential and the severity of Falciparum malaria

The multiplication rates and invasiveness of Plasmodium falciparum parasites isolated from adult Thai patients hospitalized with uncomplicated malaria (n=34) were compared with those from persons with severe malaria (n=42). To simulate severe malaria and control for host effects, the in vitro cultures were adjusted to 1% parasitemia and used the same red blood cell donor. P. falciparum isolates from persons with severe malaria had initial cycle multiplication rates in vitro that were 3-fold higher than those from uncomplicated malaria (median [95% confidence interval], 8.3 [7. 1-10.5] vs. 2.8 [1.7-3.9]; P=.001). Parasites causing severe malaria exhibited unrestricted red blood cell invasion, whereas those from uncomplicated malaria were restricted to a geometric mean of 40 (31%-53%) of red blood cells. P. falciparum parasites causing severe malaria were less selective and multiplied more at high parasitemias than those causing uncomplicated malaria.     

Platelet-mediated clumping of Plasmodium falciparum infected erythrocytes is associated with high parasitemia but not severe clinical manifestations of malaria in African children

Platelet-mediated clumping of Plasmodium falciparum-infected erythrocytes is an adhesive phenotype commonly found in field isolates that has previously been associated with severe malaria. Here, clumping was assessed in 131 isolates from Malian children. The clumping phenotype was seen in 6% (N = 51) of uncomplicated malaria, 24% (N = 51) of severe malaria, and 45% (N = 29) of high parasitemia non-severe malaria isolates. Multivariate analysis indicated that clumping was strongly positively associated with parasitemia (F(1,122) = 24.1, P < 0.001) but not with disease category (F(2,122) = 1.8, P = 0.17). Therefore platelet-mediated clumping in Malian P. falciparum isolates is primarily associated with high parasitemia and not with severe clinical manifestations of malaria.     

Platelet-induced autoagglutination of Plasmodium falciparum-infected red blood cells and disease severity in Thailand

The relationship of the platelet-mediated autoagglutination of Plasmodium falciparum-infected red blood cells (IRBCs) to disease severity was investigated in 182 Thai patients with falciparum malaria; it was evident in 43% of uncomplicated malaria (n=63), 41% of severe malaria (n=104), and 100% of cerebral malaria (n=15; P=.001) isolates. The median (range) number of IRBCs in agglutinates per 1000 IRBCs was significantly higher in cerebral malaria (6 [3-42]) than in severe (0 [0-52]) and uncomplicated (0 [0-24]) malaria (P=.01). In multivariate analyses, high parasitemia and cerebral malaria were associated independently with parasite agglutination.     

Blood group O protects against severe Plasmodium falciparum malaria through the mechanism of reduced rosetting

Malaria has been a major selective force on the human population, and several erythrocyte polymorphisms have evolved that confer resistance to severe malaria. Plasmodium falciparum rosetting, a parasite virulence phenotype associated with severe malaria, is reduced in blood group O erythrocytes compared with groups A, B, and AB, but the contribution of the ABO blood group system to protection against severe malaria has received little attention. We hypothesized that blood group O may confer resistance to severe falciparum malaria through the mechanism of reduced rosetting. In a matched case-control study of 567 Malian children, we found that group O was present in only 21% of severe malaria cases compared with 44-45% of uncomplicated malaria controls and healthy controls. Group O was associated with a 66% reduction in the odds of developing severe malaria compared with the non-O blood groups (odds ratio 0.34, 95% confidence interval 0.19-0.61, P < 0.0005, severe cases versus uncomplicated malaria controls). In the same sample set, P. falciparum rosetting was reduced in parasite isolates from group O children compared with isolates from the non-O blood groups (P = 0.003, Kruskal-Wallis test). Statistical analysis indicated a significant interaction between host ABO blood group and parasite rosette frequency that supports the hypothesis that the protective effect of group O operates through the mechanism of reduced P. falciparum rosetting. This work provides insights into malaria pathogenesis and suggests that the selective pressure imposed by malaria may contribute to the variable global distribution of ABO blood groups in the human population.     

Cytoadherence characteristics of Plasmodium falciparum-infected erythrocytes from Malawian children with severe and uncomplicated malaria.

Cytoadherence of Plasmodium falciparum-infected erythrocytes to the microvascular endothelium is believed to be a key factor in the development of cerebral malaria. Erythrocyte rosette formation has been correlated with malaria severity in studies from east and west Africa. We cultured fresh isolates from Malawian children with severe (n = 76) or uncomplicated (n = 79) malaria to pigmented trophozoite stage and examined rosette formation and adherence to CD36, intercellular adhesion molecule-1 (ICAM-1), chondroitin sulfate A (CSA), and thrombomodulin (TM). Most (126 of 148) isolates bound to CD36, and 76 of 136 bound to ICAM-1. Fewer bound to CSA (40 of 148) or TM (23 of 148). After controlling for parasitemia, there was an inverse association between binding to CD36 (P = 0.004) or ICAM-1 (P = 0.001) and disease severity. Parasites from children with severe malaria anemia bound least to CD36, whereas ICAM-1 binding was lowest in children with cerebral malaria. There was no difference in rosette formation between any of the groups. In Malawian children, there was no evidence of a positive association between adherence to any of the receptors examined and disease severity. The negative association found raises the possibility that adherence to certain receptors could instead be an indicator of a less pathogenic infection.     

Analysis of circulating populations of Plasmodium falciparum in mild and severe malaria in two different epidemiological patterns in Madagascar

Objective To investigate whether the severity of Plasmodium falciparum attack in endemic areas was associated with the multiplicity of infection (MOI) and/or with a particular genotype(s). Method In two areas of different malaria transmission pattern in Madagascar (Sainte‐Marie – mesoendemic and Tsiroanomandidy – hypoendemic) the number and the proportions of msp‐2 genotypes within isolates were determined for each patient using a capillary electrophoresis genotyping method. DNA sequencing was performed to identify the msp‐2 allelic family of dominant clones. Results Eighty six uncomplicated and 33 severe cases were included in Sainte‐Marie and 48 uncomplicated and 69 severe cases were included in Tsiroanomandidy. We found no association between the MOI and severity of malaria as the same mean number of msp‐2 genotypes was found in isolates from uncomplicated and from severe malaria cases (3.72 and 3.73, respectively, P>0.05). The study of the association of dominant clones with clinical status showed no particular genotype or allelic family associated with malaria severity. Conclusions Severity of malaria was not associated with higher MOI in our study. Severity did not appear restricted to some particular genotypes either. On the contrary, severe malaria appeared to be caused by very common genotypes in the studied areas. More comprehensive explorations including immunity and genetic factors of the host are needed to acquire new information about this complex condition.     

Association of intraleukocytic Plasmodium falciparum malaria pigment with disease severity, clinical manifestations, and prognosis in severe malaria

Peripheral parasite density of Plasmodium falciparum is used as an indicator of malaria disease severity, but does not quantify central sequestration, which is important in the pathogenesis of severe disease. Malaria pigment, recognizable within the cytoplasm of phagocytic cells by light microscopy may represent a peripheral marker for parasite biomass. One hundred seventy-two index cases of severe malaria and 172 healthy age-, residence-, and ethnicity-matched controls with uncomplicated malaria in Bandiagara, Mali were analyzed prospectively for presence of malaria pigment. The presence of polymorphonuclear cell (PMN) and monocyte pigment was strongly associated with severe disease compared with uncomplicated malaria. Total PMN pigment burden in children with severe malaria was higher in those with cerebral manifestations and with combined cerebral manifestations and severe anemia (hemoglobin < or = 5 g/dL) but was not associated with hyperparasitemia (> 500,000 asexual forms/mm3). Additionally, pigmented PMNs/mm3 was associated with a fatal outcome in patients with severe malaria. This study validates the presence of malaria pigment in monocytes and neutrophils as a marker for disease severity, and demonstrates that pigmented neutrophils are associated with cerebral malaria and with death in children with severe malaria.     

The PfCRT (K76T) point mutation favours clone multiplicity and disease severity in Plasmodium falciparum infection

In Orissa, a malaria-hyperendemic area of India, we assessed the relationship between the PfCRT (K76T) point mutation of Plasmodium falciparum and the clinical severity of malaria. Forty uncomplicated and 36 severe malaria cases were selected, and parasite species, density and schizontaemia determined by examination of Giemsa-stained thick or thin blood films. The PfCRT point mutation was analysed by PCR-RFLP and genotypes of the parasite isolates investigated by nested PCR using the polymorphic region of the merozoite surface protein-2. We found that (i) the prevalence of the PfCRT point mutation was significantly higher (P < 0.01) in severe malaria cases and that (ii) heavy parasitaemia along with clone multiplicity was statistically more common (P < 0.01) in severe cases. These associations may be due to progression of uncomplicated to severe disease after chloroquine treatment failure and/or increased virulence of chloroquine-resistant parasites. The implications for antimalarial treatment policy are discussed.     

Prognostic significance of skin and subcutaneous fat sequestration of parasites in severe falciparum malaria

Intradermal blood smear, histopathologic and immunohistologic studies were performed in severe malaria (n=10) and uncomplicated malaria (n=10) patients during positive parasitemia and within 6 hours after negative parasitemia by finger prick smears. Intradermal blood smears showed asexual forms and intraleukocytic pigments when finger prick blood smears showed negative results; however intradermal blood smear did not indicate disease severity within 6 hours after negative parasitemia by finger prick. Histopathologic findings showed 15 fold higher parasitized red blood cells sequestered in vessels of subcutaneous fatty tissue in severe malaria than in uncomplicated malaria (p<0.001) and may indicate disease severity. A panel of polyclonal antibodies against cytokines applied to skin biopsies clearly detected a higher titer against tumor necrosis factor-alpha (TNFalpha) and interleukin-10 (IL-10) in dermal vessels and stratum granulosum respectively, in severe malaria compared with uncomplicated malaria. Results of the study suggest that histopathology and immunohistology of skin and subcutaneous fatty tissue may indicate prognostic severity of malaria and may be associated with focal accumulation of cytokines.     

Short report: Positive correlation between rosetting and parasitemia in Plasmodium falciparum clinical isolates

Plasmodium falciparum isolates that form rosettes with uninfected red cells are associated with severe malaria in African children, although the mechanism by which rosetting contributes to severe disease is unknown. Here we have analyzed the relationship between rosetting and parasitemia in two samples of clinical isolates from children with malaria in Kilifi, Kenya. A consistent positive correlation was found between rosetting and parasitemia (Spearman's rank correlation coefficent p = 0.467, P < .001, n = 154, for 1993 study; p = .407, P < .001, n = 74, for 2000 study). Rosetting may enhance parasite growth and survival by facilitating invasion or promoting immune evasion, thus allowing higher parasitemia to develop and increasing the likelihood of severe malaria.     

Measures of capillary permeability in acute falciparum malaria: relation to severity of infection and treatment.

Capillary permeability was investigated in 32 Thai patients aged 14-49 years who had acute falciparum malaria with use of three distinct techniques: quantitation of the urinary albumin/creatinine ratio (ACR), estimation of the transcapillary escape rate of radiolabeled albumin (TER), and retinal photography/fluorescein angiography. Fourteen patients had uncomplicated infections and 18 were severe cases. The severely ill patients had significantly higher ACRs (median, 4.8 mg/mmol; 95% confidence limits, 2.4-19.9 mg/mmol) and TERs (median, 8.3%/h; 95% confidence limits, 6.2-13.2%/h) than the uncomplicated cases (ACR: median, 2.1 mg/mmol; 95% confidence limits, 6.2-13.2%/h) than the uncomplicated cases (ACR: median, 2.1 mg/mmol; 95% confidence limits, 1.0-8.8 mg/mmol; TER: median, 5.9%/h; 95% confidence limits, 3.8-10.6%/h; P = .014 and .042). Both variables were significantly associated with biochemical indices of disease severity including total serum bilirubin levels (rs greater than or equal to 0.398, P less than .025 in each case), but there were no significant differences between ACRs and TERs among comatose and noncomatose patients with severe infections (P greater than or equal to .08). Retinopathy (hemorrhages, cotton-wool spots, capillary nonperfusion, and/or extravasation of fluorescein) was found in eight severely ill patients and in two uncomplicated cases. Fluorescein leakage was evident in six patients. Although fluorescein leakage had the strongest parametric correlation with the presence of coma relative to both ACR and TER in the full patient series (r = 0.58, P less than .01), multiple linear regression analysis indicated that concentrations of plasma lactate (t = 2.998, P = .006) and serum creatinine (t = 2.200, P = .036) were the factors responsible for this association. These data do not support a role for tissue edema in the pathogenesis of cerebral malaria but reveal an association between markers of disease severity and a generalized increase in systemic capillary permeability.     

Serum antibody levels to glycosylphosphatidylinositols in specimens derived from matched Malian children with severe or uncomplicated Plasmodium falciparum malaria and healthy controls

Neutralizing antibodies to glycosylphosphatidylinositols (GPIs), which are Plasmodium falciparum surface protein anchor molecules implicated in malaria pathogenesis, are thought to protect against symptomatic malaria. Index cases of severe malaria in Malian children 3 months to 14 years of age were matched by age and residence to uncomplicated malaria and healthy controls. Serum antibodies to GPI (IgM and IgG) were measured at the time of severe malaria and after the malaria transmission season. The mean optical density values for IgM and IgG antibodies were higher in children with severe or uncomplicated malaria compared with healthy controls. Similarly, higher percentages of children with IgM and IgG antibodies to GPI were observed in the severe malaria group compared with matched healthy controls. IgG antibody levels to GPI were highest among children with cerebral malaria and children who died. The IgG antibody levels to GPI peaked during periods of malaria transmission and decreased after malaria transmission ended. A direct correlation between age and parasitemia and IgG antibodies to GPI was observed. In summary, higher levels of IgM and IgG antibodies to GPI in young children were associated with disease severity and were short-lived.     

Cytoadherence characteristics of Plasmodium falciparum isolates in Thailand using an in vitro human lung endothelial cells model

Using an in vitro model of human lung endothelial cells, we studied different characteristics of Plasmodium falciparum isolates as potential factors for malaria severity in 2 Thai patient groups: 27 with complicated malaria and 42 with uncomplicated malaria. In regard to binding properties, no association existed between cytoadherence and rosette phenotypes (P = 0.1) and hypothrombocytemia increased the cytoadherence level (P = 0.007). Cytoadherence was significantly associated with malaria severity (P = 0.05) in contrast to rosette formation (P = 0.9). Intercellular adhesion molecule-1 and chondroitin-4-sulfate were major receptors of cytoadherence in those with complicated malaria compared with those with uncomplicated malaria (P < 10(-4)). Chondroitin-4-sulfate could act as a putative receptor for malaria complications in non-pregnant women. CD36 was the main receptor in patients with uncomplicated malaria (P < 10(-3)). Vascular cell adhesion molecule-1 and E-selectin played a minor role in 2 groups (P = 0.6). Qinghaosu derivatives were more efficient than other antimalarial drugs, but a positive correlation was observed between the 50% inhibitory concentrations of halofantrine and quinine and the number of adhesive parasitized red blood cells, suggesting their influence on cytoadherence.     

Thiamine deficiency and malaria in adults from southeast Asia

Beriberi or thiamine deficiency is common in countries where malaria is endemic. The hypotheses that subclinical thiamine deficiency complicates malaria and may be associated with lactic acidosis and coma were investigated in a prospective study conducted in Kanchanaburi, Thailand. 77 consecutive patients who presented to Paholpolpayuhasena Hospital between May and July 1992 with malaria or other febrile illnesses and 50 healthy relatives and volunteers were enrolled. The activation coefficient for transketolase activity in erythrocytes was used to measure thiamine deficiency. The mean Box-Cox transformed coefficients were 0.166 among cases with severe malaria (n = 23), 0.145 in cases with uncomplicated malaria (n = 54), 0.138 in healthy volunteers (n = 27), 0.137 in febrile controls (n = 10), and 0.122 in healthy relatives of patients (n = 13). 12 patients (52%) with severe malaria and 10 (19%) of those with uncomplicated malaria had coefficients above the normal range. Thiamine deficiency occurred in significantly more patients with cerebral malaria than in those with uncomplicated malaria (odds ratio, 4.8; 95% confidence interval, 1.68-13.8). The 12 patients who died from severe malaria had higher coefficient values than the 115 patients and controls who survived. Finally, the 15 patients with lactic acidosis had significantly higher coefficient values than those without this complication. A study of thiamine administration to patients with cerebral malaria is recommended to allow distinctions between the findings recorded in this study and the possibility of a causal link.     

Hemoglobin E: a balanced polymorphism protective against high parasitemias and thus severe P falciparum malaria

Hemoglobin E is very common in parts of Southeast Asia. The possible malaria protective effects of this and other inherited hemoglobin abnormalities prevalent in Thailand were assessed in a mixed erythrocyte invasion assay. In vitro, starting at 1% parasitemia, Plasmodium falciparum preferentially invaded normal (HbAA) compared to abnormal hemoglobin (HbH, AE, EE, HCS, beta-thalassemia E) red cells (HRBCs). The median (range) ratio of parasitization of HRBCs (n = 109) compared to the controls of different major blood groups was 0.40 (0.08, 0.98), less than half that of the normal red cells (NRBCs) compared to their controls 0.88 (0.53, 1.4; P =.001). The median (range) parasitemia in the HRBCs was 2% (0.1%-9%) compared to 5.2% (1.2%-16.3%) in the NRBCs (P =.001). The proportion of the RBC population that is susceptible to malaria parasite invasion can be described by a selectivity index (SI; observed number of multiply invaded RBCs/number predicted). The heterozygote AE cells differed markedly from all the other cells tested with invasion restricted to approximately 25% of the RBCs; the median (range) SI was 3.8 (1-15) compared with 0.75 (0.1-0.9) for EE RBCs (P <.01). Despite their microcytosis, AE cells are functionally relatively normal in contrast to the RBCs from the other hemoglobinopathies studied. These findings suggest that HbAE erythrocytes have an unidentified membrane abnormality that renders the majority of the RBC population relatively resistant to invasion by P falciparum. This would not protect from uncomplicated malaria infections but would prevent the development of heavy parasite burdens and is consistent with the "Haldane" hypothesis of heterozygote protection against severe malaria for hemoglobin E.     

Complexity of the msp2 locus and the severity of childhood malaria, in south-western Nigeria

As the genetic diversity of Plasmodium falciparum infections in humans is implicated in the pathogenesis of malaria, the association between P. falciparum diversity at the merozoite surface protein-2 (msp2) locus and the severity of childhood malaria was investigated in Ibadan, in south-western Nigeria. The 400 children enrolled had acute uncomplicated malaria (144), cerebral malaria (64), severe malarial anaemia (67) or asymptomatic infections with P. falciparum (125). Nested PCR was used to investigate the msp2 genotype(s) of the parasites infecting each child. In terms of the complexity of infection and frequency of polyinfection, the children with asymptomatic infection were significantly different from those with uncomplicated malaria or severe malaria. The median number of FC27 alleles detected was higher in the asymptomatic children than in the symptomatic. After controlling for age and level of parasitaemia (with 'asymptomatic infection' as the reference category), a child in whom no FC27 alleles were detected was found to be at five-fold greater risk of uncomplicated malaria, and a child without polyinfection was found to have a three-fold increased risk of severe malarial anaemia and a six-fold increased risk of cerebral malaria. It therefore appears that msp2 genotypes are associated with asymptomatic carriage and that children with mono-infections are more likely to develop severe malaria than children with polyinfection.     

Comparing the understanding of subjects receiving a candidate malaria vaccine in the United States and Mali

Initial responses to questionnaires used to assess participants' understanding of informed consent for malaria vaccine trials conducted in the United States and Mali were tallied. Total scores were analyzed by age, sex, literacy (if known), and location. Ninety-two percent (92%) of answers by United States participants and 85% of answers by Malian participants were correct. Questions more likely to be answered incorrectly in Mali related to risk, and to the type of vaccine. For adult participants, independent predictors of higher scores were younger age and female sex in the United States, and male sex in Mali. Scores in the United States were higher than in Mali (P = 0.005). Despite this difference participants at both sites were well informed overall. Although interpretation must be qualified because questionnaires were not intended as research tools and were not standardized among sites, these results do not support concerns about systematic low understanding among research participants in developing versus developed countries.     

High frequency of PfCRT 76T in two Malian villages and its prevalence in severe relative to non-severe malaria

We investigated PfCRT 76T mutation in severe and non-severe malaria in Southern Mali. One hundred and ninety three severe malaria cases were each matched against two non-severe malaria cases. Patients with G6PD deficiency and any known hemoglobin abnormality were excluded. PfCRT 76T was present in 60.8% (n = 386) non-severe malaria cases and in 77.2% (n = 193) severe malaria cases (p < 0.0001). In children 5 years or younger, these proportions were 62.9% (n = 294) vs. 73.5% (n = 147), respectively (p < 0.01). PfCRT 76T was therefore associated with malaria severity in this setting of Mali.     

Association of Schistosoma haematobium infection with protection against acute Plasmodium falciparum malaria in Malian children

Plasmodium falciparum and Schistosoma haematobium are co-endemic parasitic diseases with worldwide distribution. Evidence suggests interactions occur between helminthic and malaria infections, although it is unclear whether this effect is beneficial or harmful to the host. Malian children 4-14 years of age with asymptomatic S. haematobium infection (SP) (n = 338) were prospectively matched by age, sex, and residence to children without schistosomiasis (SN) (n = 338) who were cleared of occult intestinal parasites, and followed-up for one malaria transmission season (25 weeks). The time to the first clinical malaria infection, incidence of malaria episodes, and parasitemia were recorded. Age associated protection from malaria in children with schistosomiasis was observed. SP children (4-8 years of age) compared with SN children demonstrated delayed time to first clinical malaria infection (74 versus 59 days; P = 0.04), fewer numbers of malaria episodes (1.55 versus 1.81 infections; P = 0.03) and lower geometric mean parasite densities (6,359 versus 9,874 asexual forms/mm(3); P = 0.07) at first infection. No association between schistosomiasis and P. falciparum malaria was observed in children 9-14 years of age. We conclude that underlying schistosomiasis is associated with protection against clinical falciparum malaria in an age-dependent manner.