Curdlan sulphate in human severe/cerebral Plasmodium falciparum malaria

Preclinical studies have shown that curdlan sulphate (CRDS), a sulphated 1-->3-beta-D glucan, inhibits Plasmodium falciparum in vitro and down-modulates the immune response. A direct, non-specific effect on cytoadherence and rosetting may be predicted, as has been described with other sulphated polysaccharides, e.g. heparin. The anticoagulant effect of CRDS is 10-fold lower than heparin. Curdlan sulphate has, therefore, emerged as a candidate for adjunct medication in the treatment of severe/cerebral malaria. Two clinical studies were conducted using CRDS as adjunct medication to conventional therapy (artesunate) in patients with severe and severe/cerebral malaria. Both studies were double-blind and placebo-controlled to evaluate the efficacy and safety of the combination. Curdlan sulphate appeared to reduce the severity of the disease process, e.g. fever clearance time was shortened. Due to the small number of patients, there was no difference in mortality. The two treatment arms in both studies showed similar results for all laboratory parameters. The only adverse event recorded during CRDS treatment was an increase in activated partial thromboplastin time. This can be monitored easily. It seems that the patients who may benefit most are severe/cerebral cases with no organ damage on admission.     

Cytokine gene haplotypes with a potential effect on susceptibility to malaria in sympatric ethnic groups in Mali

Cytokines are important players in the immune responses, and an unbalance in pro- and anti-inflammatory cytokine responses may affect parasitemia and pathology in a Plasmodium falciparum infection. Polymorphisms in cytokine genes may affect not only the levels of the protein, but many down-stream functions, such as production of C-reactive protein and immunoglobulin isotype switching. Susceptibility to malaria has been shown to differ between individuals with different genetic backgrounds, as indicated by studies in Fulani and non-Fulani ethnic groups. The aim of this study was to investigate possible interethnic differences in totally twelve single nucleotide polymorphisms (SNPs) in the genes encoding the cytokines IL-1β, IL-6, IL-10 and TNF. These SNPs are present in the promoter region of the genes, and have previously been associated with cytokine expression and with disease outcome in malaria. The results from the present study suggest that the Fulani ethnic group has a more pro-inflammatory response, due to high frequencies of high-producing alleles of IL1β and low-producing alleles of IL10. IL-6 could potentially also contribute to the relatively lower susceptibility to malaria in the Fulani ethnic group, whereas the TNF polymorphisms analysed in this study rather seem to associate with the severity of the infection and not the susceptibility for the infection itself. We therefore suggest that the polymorphisms analysed in this study all show a potential to influence the relatively lower susceptibility to malaria seen in the Fulani ethnic group as compared to the other sympatric ethnic groups.     

Pfcrt K76T mutation and its associations in imported Plasmodium falciparum malaria cases

Over 3 years (1999-2002), 305 cases of falciparum malaria were diagnosed in Toulouse, France. After retrospective analysis, only 131 patients entered the study. The diagnosis of malaria was ensured by optical methods (QBC then thin smear examination), the results of which were checked from 1999 to mid-2001 by a conventional PCR method, replaced at that time by a real-time PCR using LightCycler. To detect Pfcrt K(lysine)76T(threonine) mutation, a real-time PCR assay was developed, the sensitivity of which was one mutated parasite per microliter, or 2% mutant asexual forms in a mixed population. Eighty-one patients harbored only mutant parasites, and 11 had a K76K/T76-mixed infection. The distribution of K76T mutation was significantly affected by the use of a chloroquine + proguanil (CQ + P) prophylaxis (P = 0.00037). Among 96 subjects who had no exposure to chloroquine or any history of CQ + P prophylaxis, the mean parasitemia was higher in K76-infected patients (P = 0.038), which suggested a lack of virulence in the falciparum mutant population.     

Complications and mortality patterns due to Plasmodium falciparum malaria in hospitalized adults and children,Rourkela, Orissa,India

Of 1857 Plasmodium falciparum malaria patients hospitalized from 1995 to 1998, 608 had severe malaria and 83 died. Acute renal failure, jaundice and respiratory distress were common in adults whereas children frequently had severe anaemia. Cerebral malaria occurred equally in adults and children but recovery from coma was quicker in children. Multiple complications caused high mortality in adults.     

Deletion of the APOBEC3B gene strongly impacts susceptibility to falciparum malaria

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

Temporal and spatial variation in MSP1 clonal composition of Plasmodium falciparum in districts of Assam, Northeast India

Polymorphism in MSP1 gene generated by insertion/deletion of repeats causing repeat length polymorphisms is widely used as a marker for parasite genotyping. Elucidating Plasmodium falciparum clonal composition in relation to transmission intensity and other epidemiological factors in endemic areas is crucial to understanding the dynamics of host–parasite relationship and the development of immunity in malaria. We have examined here the allelic diversity of P. falciparum and attempted to understand the polymorphism and distribution of alleles of MSP1 with transition in transmission season and with differences in malaria epidemiology between sites. MSP1 diversity expressed as mean number of distinct alleles per isolate was 0.68 at Dimakusi and was much higher (p = 0.007) than seen at Guabari (0.336) and Kondoli (0.45) as was multiplicity of infection at 4.12, indicating the highest diversity at this site. Size polymorphism of the allelic families at Guabari was distinctly different from Kondoli but shared similarity with Dimakusi. Infections in high transmission summer season tended to be more complex with higher number of alleles. The frequency of alleles of RO33 and MAD20 allelic families at Guabari was found to be different between the two transmission periods. A 380 base pair allele of RO33 was over represented in high transmission summer season and seen frequently in isolates with high parasitaemia. At Kondoli allele distribution of only MAD20 was found to be different in each study year. Study site and ethnicity but not age of the study population were identified as risk factors in infection complexity. The present study demonstrates that allelic composition of P. falciparum varied with study site and between periods of high and low transmission as well as in different years of study.     

Clinical pattern of severe Plasmodium falciparum malaria in Sudan in an area characterized by seasonal and unstable malaria transmission

A hospital-based study was carried out in Gedarif town, eastern Sudan, an area of markedly unstable malaria transmission. Among the 2488 diagnosed malaria patients, 4.4% fulfilled the WHO criteria for severe malaria, and seven died of cerebral malaria. The predominant complication was severe malarial anemia (45.4%), followed by convulsions (21%), cerebral malaria (16. 4%) and hypotension (11.8%). Severe malaria was recognized in all age groups, but 44.5% of patients were aged 2 to 4 years. The mean ages of patients with severe anemia (5.6 years) and convulsions (5.9 years) were significantly lower than the mean ages of patients with cerebral malaria (14.1 years) or hypotension (35.2 years). Patients with convulsions and cerebral malaria had significantly higher mean parasite count (69972 and 56110 parasites/microL, respectively) than patients with severe anemia (24637 parasites/microL) or hypotension (13667 parasites/microL). The mean blood glucose level was higher in patients with cerebral malaria than in patients with anemia, and higher in patients who died than in patients who survived. In this setting, the clinico-epidemiological pattern of severe malaria varies considerably from that of hyperendemic regions in sub-Saharan Africa, and there is considerable variation between the individual complications of severe malaria.     

Increased frequency of malaria attacks in subjects co-infected by intestinal worms and Plasmodium falciparum malaria

The influence of intestinal worm infections on malaria was studied in individuals from Dielmo, Senegal in 1998. Results suggest that, compared with those infected, individuals free of helminths had the same degree of protection against malaria as that provided by sickle-cell trait, the most potent factor of resistance to malaria identified to date.     

Genetic diversity of Plasmodium vivax malaria in China and Myanmar

Genetic diversity and population structure of Plasmodium vivax parasites are valuable to the prediction of the origin and spread of novel variants within and between populations, and to the program evaluation of malaria control measures. Using two polymorphic genetic markers, the merozoite surface protein genes PvMSP-3α and PvMSP-3β, we investigated the genetic diversity of four Southeast Asian P. vivax populations, representing both subtropical and temperate strains with dramatically divergent relapse patterns. PCR amplification of PvMSP-3α and PvMSP-3β genes detected three and four major size polymorphisms among the 235 infections examined, respectively, while restriction analysis detected 15 and 19 alleles, respectively. Samples from different geographical areas differed dramatically in their PvMSP-3α and PvMSP-3β allele composition and frequency. Samples tended to cluster on the basis of their PCR-RFLP polymorphism. These results indicated that different parasite genotypes were circulating in each endemic area, and that geographic isolation may exist. Multiple infections were detected in all four parasite populations, ranging from 20.5% to 31.8%, strongly indicating that P. vivax populations were highly diverse and multiple clonal infections are common in these malaria-hypoendemic regions of Southeast Asia.     

Distinct haplotypes of dhfr and dhps among Plasmodium falciparum isolates in an area of high level of sulfadoxine–pyrimethamine (SP) resistance in eastern Sudan

Typing of polymorphic microsatellites that are linked to drug resistance genes has shed light on the origin and pattern of spread of some anti-malarial drugs. Recent surveys revealed spread of a high-level pyrimethemine resistant lineage of Plasmodium falciparum, of Asian origin, across Africa. Here, we examined mutations in dihydrofolate reductase, dhfr [chromsosome 4], the dihydropteroate synthase, dhps [chromosome 8] associated with resistance to sulfadoxine–pyrimethamine (SP), and neighboring microsatellites among P. falciparum isolates in Asar village, eastern Sudan. This area lies at the fringes of malaria endemicity, where the remote P. falciparum parasites have some distinct genetic characteristics. Overall, 89% (84/94) of the examined isolates carried double mutations at dhfr (N51I and S108N), but the 59R and I164L mutations were not seen. Similarly, the majority, 43% (35/81) of the isolates carried double mutations at dhps (437G, 540E). Analysis of neighboring microsatellites revealed one major dhfr haplotype with mutations (51I, 108N) and one dhps haplotype with mutations (436S, 437G, 540E). These haplotypes differ from the major ones thought to drive resistance to SP across Africa. The resistant haplotypes of dhfr and dhps, in Asar, share some microsatellites with the wild genotypes suggesting that they were generated locally. Among isolates successfully examined, 40% shared identical haplotypes of the 2 loci, comprising a dominant resistant lineage. Undoubtedly, this lineage plays an important role in clinical failure to SP in this area.     

The effects of untreated bednets on malaria infection and morbidity on the Kenyan coast

A study was conducted in order to determine whether children that slept under untreated bednets were protected against both malaria infection and clinical disease compared with children not sleeping under bednets. The study was conducted in Kilifi District, Kenya, during the malaria season (June-August, 2000) and involved 416 children aged < or = 10 years. Data collected from a cross-sectional survey showed evidence of protection against malaria infection among children sleeping under untreated bednets in good condition compared with those not using nets (adjusted odds ratio [AOR] = 0.4, 95% CI 0.22-0.72, P = 0.002). There was no evidence of a protective effect against infection when comparing those that used untreated bednets that were worn and those not using nets (AOR = 0.75, 95% CI 0.34-1.63, P = 0.47). When these same children were followed-up during the malaria season, there was evidence of a lower rate of clinical malaria among those that used untreated nets in good condition (adjusted incidence rate ratio = 0.65, 95% CI 0.45-0.94, P = 0.022), while the rate of clinical malaria among those that used untreated bednets that were worn was similar to that of those that did not use bednets. In the face of persistent failure of communities to take up net retreatment, there is hope that untreated nets will offer some protection against malaria infection and disease compared with not using nets at all.     

Relationship between the Pfcrt T76 and the Pfmdr-1 Y86 mutations in Plasmodium falciparum and in vitro/in vivo chloroquine resistance in Burkina Faso, West Africa

The relationship between Pfcrt T76 and Pfmdr-1 Y86 mutations in Plasmodium falciparum was explored in samples from patients with uncomplicated malaria and tested in vitro and in vivo with chloroquine (CQ) in Burkina Faso. The two mutations were strongly related. The Pfcrt T76 mutation was found in 82% of the samples having the Pfmdr-1 Y86 mutation too (odds ratio (OR)=4.8 [95% CI: 1.7-13.3]; P=0.002). However, only half (16/34) of samples with Pfcrt T76 mutation had also the Pfmdr-1 Y86 mutation. The latter was apparently associated with in vitro resistance (OR=4.8 [95% CI: 1.4-16.5]; P=0.01) but such association disappeared (P=0.77) after adjusting for the presence of the Pfcrt T76 mutation. This suggests that the occurrence of the Pfmdr-1 Y86 mutation is dependent on that of Pfcrt T76 mutation and could explain previous reports linking the Pfmdr-1 Y86 mutation with CQ resistance (CQR). The isolates carrying both the Pfcrt K76 and Pfmdr-1 N86 alleles (wild/wild (WW)) and the single mutant Pfmdr-1 Y86 (WM) had the lowest IC50 geometric mean (GMIC50) values, while those carrying both Pfcrt T76/Pfmdr-1 Y86 alleles (mutant/mutant (MM)), and the single mutant Pfcrt T76 (MW) had the highest. Among pre-treatment samples there was a strong linkage disequilibrium with an excess of MM and WW and a deficit of single mutants (MW and WM), suggesting that parasite fitness is higher for the former and lower for the latter.     

Genetic diversity of Plasmodium vivax Duffy Binding Protein II (PvDBPII) under unstable transmission and low intensity malaria in Sri Lanka

Elucidating the genetic diversity of the Duffy Binding Protein II (PvDBPII), a leading vaccine candidate for vivax malaria, in different geographical settings is vital. In Sri Lanka malaria transmission is unstable with low intensity. A relatively high level of allelic diversity, with 27 polymorphic nucleotides and 33 (aa) haplotypes was detected among the PvdbpII gene in 100 local Plasmodium vivax isolates collected from two hypoendemic areas, and from a non endemic area of the country. Mutations, recombination and balancing selection seem to maintain the observed local allelic diversity of PvdbpII. Lack of gene flow was evidenced by high Fst values between the two endemic study sites. Some of the aa polymorphisms may alter the binding and expression capacity of predicted T cell epitopes in PvDBPII. Of the 8 binding inhibitory linear B cell epitopes, 2 (H2 and M1) in the vicinity of the exact binding region of PvDBPII appeared to be highly conserved in Sri Lankan, Iran and Colombian isolates, while H3, M2, M3 and L3 neutralizing epitopes seem to be polymorphic globally, with H1 and L2 conserved in Colombian, South Korean and Iran isolates. In comparison to the reference Sal-1 strain, among 402 world-wide isolates (302 global and 100 local), 121 aa polymorphisms and 138 haplotypes were recorded of which 3 aa polymorphisms and 21 haplotypes seem to be unique to Sri Lanka. PvdbpII phylogeny suggests that local P. vivax parasites represent a sample of the global population. The ubiquitous presence of some PvDBPII aa haplotypes among both local and global P. vivax isolates may aid future vaccination strategies based on PvDBPII.     

Diversity of the sarco/endoplasmic reticulum Ca-ATPase orthologue of Plasmodium falciparum (PfATP6)

The sarco/endoplasmic reticulum Ca(2+)-ATPase orthologue of Plasmodium falciparum (PfATP6) has been suggested to be involved in the mechanism of action and resistance to artemisinins, the main constituent of artemisinin-based combination therapy (ACT). In previous studies only six single-nucleotide polymorphisms (SNPs) have been described in clinical samples and field isolates. Our aim was to sequence a large number of clinical samples with different geographical origins to further explore the natural diversity of PfATP6. We sequenced three genetic regions of PfATP6 in 388 samples from 17 countries, mainly Zanzibar and Tanzania, and identified 33 SNPs, of which 29 were non-synonymous and 4 synonymous. To our knowledge 29 of these SNPs have not been described previously. Three mutations were found in high frequency in Zanzibar and Tanzania; E431K, N569K and A630S were present in respectively 31% (95% CI, 26-37%), 36% (95% CI, 30-42%), and 2% (95% CI, 1-5%) of Zanzibar samples and in 39% (95% CI, 29-51%), 29% (95% CI, 16-45%) and 7% (95% CI, 1-22%) of the Tanzania Mainland samples. No variation was found in position 263, suggested to be involved in artemisinin binding to PfATP6, or in position 769, proposed to be related to decreased sensitivity to artemether in vitro. A considerable difference in diversity was observed between the three genetic regions. In conclusion our findings show that PfATP6 is a more diverse gene than previously demonstrated. This natural variation may constitute a starting ground for artemisinin-driven progressive selection of resistant parasites.     

Travel as a risk factor for uncomplicated Plasmodium falciparum malaria in the highlands of western Kenya

In the 1980s, highland malaria returned to the tea estates of western Kenya after an absence of nearly a generation. In order to determine the importance of travel for the spread of malaria in this region, we prospectively collected blood films and travel, demographic and geographic information on well persons and outpatients on tea estates near the western rim of the Rift Valley. Risk factors for malaria asexual parasitaemia included: tribal/ethnic group, home province and home district malaria endemicity. Travel away from the Kericho tea estates within the previous two months showed an odds ratio (OR) for parasitaemia of 1.59 for well persons and 2.38 for outpatients. Sexual stages of malaria parasites (gametocytes) had an OR of 3.14 (well persons) and 2.22 (outpatients) for those who had travelled. Increased risk of malaria parasitaemia with travel was concentrated in children aged <5 years. An increase in population gametocytaemia is possibly due to increased chloroquine resistance and suppressed infections contracted outside of the tea estates.     

Differences in automated depolarization patterns of Plasmodium faiciparum and P. vivax malaria infections defined by the Cell-Dyn® CD4000 haematology analyser

Of 1014 samples submitted for full blood count analysis and malaria screening, 854 were designated malaria-negative by blood film microscopy, 79 were unequivocally identified as Plasmodium vivax and 81 as P. falciparum. All samples were additionally analysed with the Abbott Cell-Dyn® CD4000 haematology instrument, and leucocyte differential plots of 90° polarized vs. 90° depolarized (NEU-EOS plot) and 90° depolarized vs. 0° light (EOS I plot) scatter were specifically examined for abnormal depolarization patterns. Depolarization pattern types were correlated with microscopy (species) results, and these correlations were consolidated by polymerase chain reaction analysis. All 854 microscopically-designated malaria-negative samples showed a type 1 (normal) CD4000 depolarization pattern. Abnormal pattern types 2, 3a and 3b were entirely restricted to one of the two malaria categories. Plasmodium falciparum malaria showed two CD4000 pattern types only; a ‘normal’ type 1 pattern was seen in 36/75 (48%) cases and the remaining 39 cases were all abnormal pattern type 3a. In contrast, most (79/85) P. vivax malaria cases showed a distinctive clustered EOS I population (types 2 and 3b patterns) that was not seen with P. falciparum. Automated depolarization analysis provides an effective means of detecting malaria-associated haemozoin, and the patterns of intracellular haemozoin further appear to provide species differentiation between P. falciparum and P. vivax.