Post-treatment haemolysis in African children with hyperparasitaemic falciparum malaria; a randomized comparison of artesunate and quinine.

BACKGROUND: Parenteral artesunate is the treatment of choice for severe malaria. Recently, haemolytic anaemia occurring 1 to 3 weeks after artesunate treatment of falciparum malaria has been reported in returning travellers in temperate countries. METHODS: To assess these potential safety concerns in African children, in whom most deaths from malaria occur, an open-labelled, randomized controlled trial was conducted in Kinshasa, Democratic Republic of Congo. 217 children aged between 6 months and 14 years with acute uncomplicated falciparum malaria and parasite densities over 100,000/µL were randomly allocated to intravenous artesunate or quinine, hospitalized for 3 days and then followed for 42 days. RESULTS: The immediate reduction in haemoglobin was less with artesunate than with quinine: median (IQR) fall at 72 h 1.4 g/dL (0.90-1.95) vs. 1.7 g/dL (1.10-2.40) (p = 0.009). This was explained by greater pitting then recirculation of once infected erythrocytes. Only 5% of patients (in both groups) had a ≥ 10% reduction in haemoglobin after day 7 (p = 0.1). One artesunate treated patient with suspected concomitant sepsis had a protracted clinical course and required a blood transfusion on day 14. CONCLUSIONS: Clinically significant delayed haemolysis following parenteral artesunate is uncommon in African children hospitalised with acute falciparum malaria and high parasitaemias. TRIAL REGISTRATION: ClinicalTrials.gov ; Identifier: NCT02092766 (18/03/2014).

A Toll-like receptor-1 variant and its characteristic cellular phenotype is associated with severe malaria in Papua New Guinean children.

Genetic factors are likely to contribute to low severe malaria case fatality rates in Melanesian populations, but association studies can be underpowered and may not provide plausible mechanistic explanations if significant associations are detected. In preparation for a genome-wide association study, 29 candidate single-nucleotide polymorphisms (SNPs) with minor allele frequencies >5% were examined in a case-control study of 504 Papua New Guinean children with severe malaria. In parallel, an immunological substudy was performed on convalescent peripheral blood mononuclear cells (PBMCs) from cases and controls. Following stimulation with a Toll-like receptor (TLR) 1/2 agonist, effector cytokines and chemokines were assayed. The only significant genetic association observed involved a nonsynonymous SNP (TLR1rs4833095) in the TLR1 gene. A recessive (TT) genotype was associated with reduced odds of severe malaria of 0.52 (95% confidence interval (0.29-0.90), P=0.006). Concentrations of pro-inflammatory cytokines interleukin-1ß and tumour necrosis factor α were significantly higher in severe malaria cases compared with healthy controls, but lower in children with the protective recessive (TT) genotype. A genetic variant in TLR1 may contribute to the low severe malaria case fatality rates in this region through a reduced pro-inflammatory cellular phenotype.

Fc gamma receptor IIa-H131R polymorphism and malaria susceptibility in sympatric ethnic groups, Fulani and Dogon of Mali.

It has been previously shown that there are some interethnic differences in susceptibility to malaria between two sympatric ethnic groups of Mali, the Fulani and the Dogon. The lower susceptibility to Plasmodium falciparum malaria seen in the Fulani has not been fully explained by genetic polymorphisms previously known to be associated with malaria resistance, including haemoglobin S (HbS), haemoglobin C (HbC), alpha-thalassaemia and glucose-6-phosphate dehydrogenase (G6PD) deficiency. Given the observed differences in the distribution of FcγRIIa allotypes among different ethnic groups and with malaria susceptibility that have been reported, we analysed the rs1801274-R131H polymorphism in the FcγRIIa gene in a study of Dogon and Fulani in Mali (n = 939). We confirm that the Fulani have less parasite densities, less parasite prevalence, more spleen enlargement and higher levels of total IgG antibodies (anti-CSP, anti-AMA1, anti-MSP1 and anti-MSP2) and more total IgE (P < 0.05) compared with the Dogon ethnic group. Furthermore, the Fulani exhibit higher frequencies of the blood group O (56.5%) compared with the Dogon (43.5%) (P < 0.001). With regard to the FcγRIIa polymorphism and allele frequency, the Fulani group have a higher frequency of the H allele (Fulani 0.474, Dogon 0.341, P < 0.0001), which was associated with greater total IgE production (P = 0.004). Our findings show that the FcγRIIa polymorphism might have an implication in the relative protection seen in the Fulani tribe, with confirmatory studies required in other malaria endemic settings.

The 5q31 region in two African populations as a facet of natural selection by infectious diseases.

Cases of extreme natural selection could lead either to rapid fixation or extinction of alleles depending on the population structure and size. It may also manifest in excess of heterozygosity and the locus concerned will be displaying such drastic features of allele change. We suspect the 5q31 in chromosome 5 to mirror situation of such extreme natural selection particularly that the region encompasses genes of type 2 cytokine known to associate with a number of infectious and non-infectious diseases. We typed two sets of single nucleotide polymorphisms (SNPS) in two populations: an initial limited set of only 4 SNP within the genes of IL-4, IL-13, IL-5 and IL-9 in 108 unrelated individuals and a replicating set of 14 SN P in 924 individuals from the same populations with disregard to relatedness. The results suggest the 5q31 area to be under intense selective pressure as indicated by marked heterozygosity independent of Linkage Disequilibrium (LD); difference in heterozygosity, allele, and haplotype frequencies between generations and departure from Hardy-Weinberg expectations (DHWE). The study area is endemic for several infectious diseases including malaria and visceral leishmaniasis (VL). Malaria caused by Plasmodiumfalciparum, however, occurs mostly with mild clinical symptoms in all ages, which makes it unlikely to account for these indices. The strong selection signals seems to emanate from recent outbreaks of VL which affected both populations to varying extent.

Variation in human genes encoding adhesion and proinflammatory molecules are associated with severe malaria in the Vietnamese.

The genetic basis for susceptibility to malaria has been studied widely in African populations but less is known of the contribution of specific genetic variants in Asian populations. We genotyped 67 single-nucleotide polymorphisms (SNPs) in 1030 severe malaria cases and 2840 controls from Vietnam. After data quality control, genotyping data of 956 cases and 2350 controls were analysed for 65 SNPs (3 gender confirmation, 62 positioned in/near 42 malarial candidate genes). A total of 14 SNPs were monomorphic and 2 (rs8078340 and rs33950507) were not in Hardy-Weinberg equilibrium in controls (P<0.01). In all, 7/46 SNPs in 6 genes (ICAM1, IL1A, IL17RC, IL13, LTA and TNF) were associated with severe malaria, with 3/7 SNPs in the TNF/LTA region. Genotype-phenotype correlations between SNPs and clinical parameters revealed that genotypes of rs708567 (IL17RC) correlate with parasitemia (P=0.028, r(2)=0.0086), with GG homozygotes having the lowest parasite burden. Additionally, rs708567 GG homozygotes had a decreased risk of severe malaria (P=0.007, OR=0.78 (95% CI; 0.65-0.93)) and death (P=0.028, OR=0.58 (95% CI; 0.37-0.93)) than those with AA and AG genotypes. In summary, variants in six genes encoding adhesion and proinflammatory molecules are associated with severe malaria in the Vietnamese. Further replicative studies in independent populations will be necessary to confirm these findings.

The impact of malaria-protective red blood cell polymorphisms on parasite biomass in children with severe Plasmodium falciparum malaria.

Severe falciparum malaria is a major cause of preventable child mortality in sub-Saharan Africa. Plasma concentrations of P. falciparum Histidine-Rich Protein 2 (PfHRP2) have diagnostic and prognostic value in severe malaria. We investigate the potential use of plasma PfHRP2 and the sequestration index (the ratio of PfHRP2 to parasite density) as quantitative traits for case-only genetic association studies of severe malaria. Data from 2198 Kenyan children diagnosed with severe malaria, genotyped for 14 major candidate genes, show that polymorphisms in four major red cell genes that lead to hemoglobin S, O blood group, α-thalassemia, and the Dantu blood group, are associated with substantially lower admission plasma PfHRP2 concentrations, consistent with protective effects against extensive parasitized erythrocyte sequestration. In contrast the known protective ATP2B4 polymorphism is associated with higher plasma PfHRP2 concentrations, lower parasite densities and a higher sequestration index. We provide testable hypotheses for the mechanism of protection of ATP2B4.

Variable major lipoprotein is a principal TNF-inducing factor of louse-borne relapsing fever.

Massive release of tumor necrosis factor is responsible for the potentially fatal larisch-Herxheimer reaction that follows antibiotic treatment of relapsing fever due to Borrelia recurrentis. We have undertaken the quantitative purification of the components of B. recurrentis that stimulate human monocytes to produce tumor necrosis factor. We show that the predominant factor inducing tumor necrosis factor is a variable lipoprotein homologous to the variable major protein of B. hermsii. We found antibodies to different forms of variable major protein in two patients with louse-borne relapsing fever. The three purified variable major proteins studied here differ in their ability to induce tumor necrosis factor production, which may partly explain the variable clinical severity of borrelial infection. These results may be of considerable relevance for the pathogenesis of Lyme disease and other forms of human borreliosis.

Elevated secretion of reactive nitrogen and oxygen intermediates by inflammatory leukocytes in hyperacute experimental autoimmune encephalomyelitis: enhancement by the soluble products of encephalitogenic T cells.

Perivascular lesions within the central nervous system (CNS) of rats with hyperacute experimental autoimmune encephalomyelitis (HEAE) contained large numbers of peripheral blood mononuclear cells (PBMC) and polymorphonuclear leukocytes (PMN), cells enzymatically capable of producing reactive nitrogen and oxygen intermediates (RNI and ROI), which, in excess, are mediators of tissue damage. PBMC and PMN isolated from the CNS and periphery of HEAE-affected rats secreted significantly (p less than 0.01-0.0001) elevated levels of ROI and RNI compared with that of similar cell populations from pertussis- and saline-treated control animals. Coincubation of systemically derived PBMC and PMN with antigen-stimulated myelin basic protein-specific T cell lines led to further increases in ROI and RNI output of between 15.3 and 83.1%, an effect that could be largely attributed to heat-labile, soluble products released by these T cell lines. Our studies suggest a putative neuropathological role for ROI and RNI in HEAE, which may be mediated via cytokines emanating from autoreactive T lymphocytes.

Genetic association study for RSV bronchiolitis in infancy at the 5q31 cytokine cluster.

BACKGROUND: The pathophysiological basis of severe respiratory syncytial virus (RSV) bronchiolitis in infancy is poorly understood and has hindered vaccine development. Studies implicate the cell-mediated immune response in the pathogenesis of the disease. A recent twin study estimated a heritable contribution of 22% to RSV bronchiolitis. Genetic epidemiology provides a new approach to identifying important immune determinants of disease severity. METHODS: A comprehensive high-density gene-region association study for severe RSV bronchiolitis in infancy at 5q31 across 11 genes including the Th2-cytokine cluster was performed. A haplotype tagging approach was used to analyse genetic variation at 113 single nucleotide polymorphisms (SNPs) in 780 independent cases and 1045 controls. The study had sufficient power to detect small effects, perform extensive haplotype analysis and analyse both a principal phenotype and a refined age-limited phenotype enriched for first-exposure RSV infection. RESULTS: SNP associations were found at IL4 and a highly significant risk haplotype was identified across IL13 CNS-1 and IL4 (odds ratio 1.69, p<0.0001), present in both case-control and family-based analyses. All associations were strongest for a phenotype limited to <6 months of age, implicating this locus in primary RSV disease. The same risk haplotype has previously been shown to be associated with increased IL13 expression. CONCLUSIONS: A haplotype at IL13-1L4, which is associated with increased IL13 production, confers an increased risk of severe primary RSV bronchiolitis in early infancy. This study, together with previous studies implicating the same locus in atopic sensitisation, suggests that primary RSV bronchiolitis and atopy share a genetic contribution at the IL13-IL4 locus.

Interferon regulatory factor-1 polymorphisms are associated with the control of Plasmodium falciparum infection.

We describe the haplotypic structure of the interferon regulatory factor-1 (IRF-1) locus in two West African ethnic groups, Fulani and Mossi, that differ in their susceptibility and immune response to Plasmodium falciparum malaria. Both populations showed significant associations between IRF-1 polymorphisms and carriage of P. falciparum infection, with different patterns of association that may reflect their different haplotypic architecture. Genetic variation at this locus does not therefore account for the Fulani-specific resistance to malaria while it could contribute to parasite clearance's ability in populations living in endemic areas. We then conducted a case-control study of three haplotype-tagging single nucleotide polymorphisms (htSNPs) in 370 hospitalised malaria patients (160 severe and 210 uncomplicated) and 410 healthy population controls, all from the Mossi ethnic group. All three htSNPs showed correlation with blood infection levels in malaria patients, and the rs10065633 polymorphism was associated with severe disease (P=0.02). These findings provide the first evidence of the involvement in malaria susceptibility of a specific locus within the 5q31 region, previously shown to be linked with P. falciparum infection levels.

Haplotypic diversity in human CEACAM genes: effects on susceptibility to meningococcal disease.

Adhesion between the opacity-associated adhesin (Opa) proteins of Neisseria meningitidis and human carcino-embryonic antigen cell adhesion molecule (CEACAM) proteins is an important stage in the pathogenesis of meningococcal disease, a globally important bacterial infection. Most disease is caused by a small number of meningococcal genotypes known as hyperinvasive lineages. As these are also carried asymptomatically, acquisition of them alone cannot explain why only some hosts develop meningococcal disease. Our aim was to determine whether genetic diversity in CEACAM is associated with susceptibility to meningococcal disease. Frequency distributions of alleles, genotypes and haplotypes were compared in four CEACAM genes in 384 case samples and 190 controls. Linkage disequilibrium among polymorphic sites, haplotype structures and relationships were also analysed. A number of polymorphisms were observed in CEACAM genes but the diversity of CEACAM1, to which most Opa proteins bind, was lower, and a small number of high-frequency haplotypes were detected. Dose-dependent associations of three CEACAM haplotypes with meningococcal disease were observed, with the effect of carrying these haplotypes amplified in homozygous individuals. Two haplotypes were protective while one haplotype in CEACAM6 was associated with a twofold increase in disease susceptibility. These data imply that human CEACAM may be one determinant of human susceptibility to meningococcal disease.

Variation in the ICAM1 gene is not associated with severe malaria phenotypes.

Evidence from autopsy and in vitro binding studies suggests that adhesion of erythrocytes infected with Plasmodium falciparum to the human host intercellular adhesion molecule (ICAM)-1 receptor is important in the pathogenesis of severe malaria. Previous association studies between polymorphisms in the ICAM1 gene and susceptibility to severe malarial phenotypes have been inconclusive and often contradictory. We performed genetic association studies with 15 single nucleotide polymorphisms (SNPs) around the ICAM1 locus. All SNPs were screened in a family study of 1071 trios from The Gambia, Malawi and Kenya. Two key non-synonymous SNPs with previously reported associations, rs5491 (K56M or 'ICAM-1(Kilifi)') and rs5498 (K469E), were tested in an additional 708 Gambian trios and a case-control study of 4058 individuals. None of the polymorphisms were associated with severe malaria phenotypes. Pooled results across our studies for ICAM-1(Kilifi) were, in severe malaria, odds ratio (OR) 1.02, 95% confidence interval (CI) 0.96-1.09, P=0.54, and cerebral malaria OR 1.07, CI 0.97-1.17, P=0.17. We assess the available epidemiological, population genetic and functional evidence that links ICAM-1(Kilifi) to severe malaria susceptibility.

Inhibition of intraerythrocytic development of Plasmodium falciparum by proteinase inhibitors.

A group of inactivators of cysteinyl proteinases which function by covalent bond formation have been examined for their ability to inhibit the development of Plasmodium falciparum within red blood cells. The most effective of these caused inactivation of the parasite near 10(-8) M concentration. The range of inhibitory action varied with peptide structure in a manner characteristic of affinity labels for proteinases suggesting that the target of inhibition was an unidentified proteinase, probably of the cysteinyl type, but different from cathepsins B and L.

Multiple sclerosis: increased expression of interleukin-2 receptors on lymphocytes.

The percentage of interleukin-2-receptor-positive peripheral blood lymphocytes in MS patients was significantly higher in acute relapse than in remission or in controls. After stimulation by phytohemagglutinin, the expression of interleukin-2 receptor on peripheral blood lymphocytes of MS patients was within the range of healthy controls, implying no general impairment of receptor expression. These results confirm other evidence that there is a small population of activated T lymphocytes in acute relapse of MS.

Astrocytic hypertrophy: an important pathological feature of chronic experimental autoimmune encephalitis in aged rats.

Experimental autoimmune encephalomyelitis (EAE) was induced in young (2-3 month old), middle-aged (12-13 month old) and geriatric (24-26 month old) Lewis (JC) rats by active immunisation with myelin basic protein (MBP) in complete Freund's adjuvant (CFA). It was found that aged Lewis (JC) rats developed a more chronic form of EAE than younger rats of the same strain, a phenomenon observed in both male and female rats despite males developing more severe disease than females at all ages. Middle-aged recipients also developed more severe disease than young recipients when EAE was induced by the adoptive transfer of lymphocytes from actively immunised young donors, suggesting that disease chronicity in middle-aged animals is a property of the central nervous system (CNS) milieu. Histological studies demonstrated that disease chronicity did not correlate with the number of inflammatory lesions in the CNS, young animals containing substantial numbers of CNS lesions following recovery and lesions being largely absent from middle-aged animals which still exhibited signs of disease. No significant differences were found in the degree of fibrin deposition or demyelination between young and middle-aged or symptomatic and asymptomatic animals. However, astrocytic hypertrophy was found to correlate with manifestation of disease in both young and middle-aged animals and in particular with disease chronicity in middle-aged animals. In parallel studies, no significant differences were found in the levels of the inflammatory mediators tumor necrosis factor (TNF)-alpha, prostaglandin E (PGE)2, reactive nitrogen intermediates (RNI) and corticosterone in young and middle-aged animals. However, markedly elevated corticosterone levels were found in both young and middle-aged animals with the development of clinical signs which returned to baseline levels with the resolution of clinical signs. Elevated levels of RNI were evident in animals immediately prior to and during the early stages of symptomatic EAE. Although these results suggest that nitric oxide may play a role in the pathogenesis of disease, whereas corticosterone may play a role in the immunoregulation of the disease, these factors cannot explain differences in disease chronicity evident in middle-aged animals.(ABSTRACT TRUNCATED AT 400 WORDS)

Tumor necrosis factor and interleukin-1 synergy in the context of malaria pathology.

Reports linking human malarial illness and pathology with serum tumor necrosis factor (TNF) levels are now common, although the association is not always precise. Possible reasons for this discrepancy include the reported variation in levels of interleukin-1 (IL-1), a cytokine known to synergize with TNF. We have examined the extent of synergy between recombinant human TNF and either recombinant human IL-1 alpha or recombinant human IL-1 beta in producing hypoglycemia and increasing plasma levels of nitric oxide in malaria (Plasmodium vinckei)-infected CBA mice. Very low concentrations of either IL-1 alpha or IL-1 beta, with negligible effects on their own, greatly enhanced the effectiveness of TNF in bringing about these changes. In particular, synergy in generating nitric oxide, a mediator argued to induce cerebral malaria, was profound. Thus, variation in generation of IL-1 during infection provides one explanation for the poor correlation sometimes encountered between serum TNF levels and clinical condition.

Increased lymphotoxin in human malarial serum, and the ability of this cytokine to increase plasma interleukin-6 and cause hypoglycaemia in mice: implications for malarial pathology.

The origin of illness and pathology in malaria is now largely attributed to high levels of circulating tumour necrosis factor (TNF), released from cells of macrophage lineage after triggering by the products of malarial schizogony. The role of lymphocytes and their products in malarial pathology is not yet known. This paper reports the presence of a related cytokine, lymphotoxin, which is produced only by lymphocytes, in the serum of malarial patients. This is the first report of raised serum levels of lymphotoxin in a systemic disease state. When injected into mice, recombinant human lymphotoxin induced hypoglycaemia and increased serum levels of interleukin-6. These changes, which are seen in severe experimental and human malaria, were also provoked by TNF. Both of these cytokines acted synergistically with interleukin-1, which has also been reported to be raised in malaria, to produce these alterations. These observations imply that lymphotoxin, as well as TNF, may contribute to the hypoglycaemia and raised serum interleukin-6 observed in malaria. This reduces the likelihood of effectively blocking the pathology of this disease by the use of neutralizing antibody directed against just one member of this family of functionally overlapping mediators.

Association between serum levels of reactive nitrogen intermediates and coma in children with cerebral malaria in Papua New Guinea.

Serum levels of reactive nitrogen intermediates (RNI; nitrate plus nitrite) were measured in 92 patients with cerebral malaria in the Madang Province of Papua New Guinea. RNI levels were compared to disease severity and clinical outcome, and correlated with both the depth of coma on admission and its duration. Median levels were higher among children with deeper coma than among those with lighter coma (35.6 microM vs. 16.7 microM; P = 0.008) and also among children with longer duration of coma (72 h; 59.3 microM vs. 19.3 microM; P = 0.004). RNI levels also correlated with clinical outcome, fatal cases having significantly higher RNI levels than survivors (41.2 microM vs. 18.5 microM; P = 0.014). Thus, high RNI levels are associated with indices of disease severity and may predict outcome in children with cerebral malaria. These data are consistent with the hypothesis that nitric oxide is involved in the pathogenesis of coma in human cerebral malaria.

In vitro induction of nitric oxide by an extract of Plasmodium falciparum.

Malarial illness and pathology is generally accepted to be caused by material released when the infected red cells burst at schizogony. The released material has been partially purified and shown to stimulate macrophages to make TNF. We have extended this work to show that these same preparations, isolated from parasitized erythrocytes, induce the mouse macrophage cell line RAW 264.7 to produce inducible nitric oxide synthase and release nitric oxide. By using cytokine-specific antisera we have found that this induction is independent of TNF and IL-1 alpha and partly independent of IL-1 beta.