Multiple origins and regional dispersal of resistant dhps in African Plasmodium falciparum malaria.

BACKGROUND: Although the molecular basis of resistance to a number of common antimalarial drugs is well known, a geographic description of the emergence and dispersal of resistance mutations across Africa has not been attempted. To that end we have characterised the evolutionary origins of antifolate resistance mutations in the dihydropteroate synthase (dhps) gene and mapped their contemporary distribution. METHODS AND FINDINGS: We used microsatellite polymorphism flanking the dhps gene to determine which resistance alleles shared common ancestry and found five major lineages each of which had a unique geographical distribution. The extent to which allelic lineages were shared among 20 African Plasmodium falciparum populations revealed five major geographical groupings. Resistance lineages were common to all sites within these regions. The most marked differentiation was between east and west African P. falciparum, in which resistance alleles were not only of different ancestry but also carried different resistance mutations. CONCLUSIONS: Resistant dhps has emerged independently in multiple sites in Africa during the past 10-20 years. Our data show the molecular basis of resistance differs between east and west Africa, which is likely to translate into differing antifolate sensitivity. We have also demonstrated that the dispersal patterns of resistance lineages give unique insights into recent parasite migration patterns.

The implication of dihydrofolate reductase and dihydropteroate synthetase gene mutations in modification of Plasmodium falciparum characteristics.

BACKGROUND: The Plasmodium falciparum dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) are enzymes of central importance in parasite metabolism. The dhfr and dhps gene mutations are known to be associated with sulphadoxine/pyrimethamine (SP) resistance. OBJECTIVE: To investigate the effects of dhfr/dhps mutations on parasite characteristics other than SP resistance. METHOD: Parasite infections obtained from 153 Sudanese patients with uncomplicated falciparum malaria treated with SP or SP + chloroquine, were successfully genotyped at nine codons in the dhfr/dhps genes by PCR-ELISA. RESULTS & CONCLUSION: Mutations were detected in dhfr at N51I, S108N and C59R, and in at dhps at A/S436F, A437G, K540E and A581G, the maximum number of mutations per infection were five. Based on number of mutant codons per infection (multiplicity of mutation, MOM), the infections were organized into six grades: wild-types (grade 0; frequency, 0.03) and infections with MOM grades of 1 to 5, with the following cumulative frequency; 0.97, 0.931, 0.866, 0.719, 0.121, respectively. There was no significant association between the MOM and SP response. Importantly, immunity, using age as a surrogate marker, contributed significantly to the clearance of parasites with multiple dhfr/dhps mutations. However, these mutations have a survival advantage as they were associated with increased gametocytogenesis. The above implications of dhfr/dhps mutations were associated with MOM 2 to 5, regardless of the gene/codon locus.

Allelic polymorphism of MSP2 gene in severe P. falciparum malaria in an area of low and seasonal transmission.

The severe malaria (SM) and uncomplicated malaria (UM) infections are expected to have different genetic makeup. In this study, blood samples were obtained from 325 donors with SM and UM and malaria-free donors (including asymptomatic submicroscopic malaria--ASUM), from Eastern Sudan. The SM group included patients with cerebral malaria (CM), severe malarial anemia (SMA), and other complications. The MSP2 locus was exploited for parasite genotyping. We found that the genetic diversity of the parasite population was marked (51 genotypes). The overall multiplicity of infection (MOI) was 1.5, and it was comparable between SM and UM. However, the MOI in ASUM (1.0) and fatal CM (1.14) was comparable and significantly lower than in UM (1.53), SMA (1.52), and nonfatal CM (1.7). The ratio of the IC1 to FC27 allele families was comparable between SM and UM, and the distribution of the allele sizes was correlated (correlation coefficient = 0.59 and 0.718; P < 0.001). It is interesting to note that the FC27 genotype was overrepresented in ASUM (68.2%) and was not recognized in fatal CM, while in mixed-clone infections, the clearance of IC1 after quinine treatment was faster than FC27 clearance. Finally, the composition of the multiclone infections (IC1 and FC27) was suggesting a stronger cross-immunity within rather than between MSP2 gene families.

Drug resistance-virulence relationship in Plasmodium falciparum causing severe malaria in an area of seasonal and unstable transmission.

The pathogenesis of severe Plasmodium falciparum malaria is still obscure, but is believed to be multi-factorial, and among the important factors are intrinsic parasite-properties. Here we investigated the association between clinical manifestation of P. falciparum malaria (an indicator of virulence) and two parasite properties--drug resistance and gametocyte production. Among 996 P. falciparum infections detected in the out-patient clinic of Gedarif Hospital in eastern Sudan, there was no significant association between the incidence of severe versus mild disease and the presence of resistant alleles at the chloroquine-resistance transporter locus (pfcrt-T76) and the multi-drug-resistance locus (pfmdr1-Y86). However, among severe cases, there was a significantly lower prevalence of parasites carrying resistant alleles among patients that died versus survived. There was a trend towards a higher gametocyte rate among severe malaria patients compared with uncomplicated malaria cases. These results are discussed in relation to the fitness of drug resistant parasites.

Cerebral malaria is frequently associated with latent parasitemia among the semi-immune population of eastern Sudan.

The accurate diagnosis of malaria starts with clinical suspicion, confirmed by reliable laboratory results. A hospital-based study, described here, was carried out in a malaria mesoendemic area in eastern Sudan, where the inhabitants are semi-immune to malaria, and the fever threshold of parasitemia is not above the detection level of microscopy. Thus, we hypothesized that patients with symptoms highly suggestive of cerebral malaria (CM), but aparasitemic by microscopy, may have submicroscopic parasitemia. Patients in our malaria clinic were screened by microscopy, and 120 individuals were selected for the study, including febrile patients with and without microscopically detectable parasitemia, and apparently healthy individuals. In the two former groups there were patients with severe anemia and deep coma. Polymerase chain reaction (PCR) for parasite detection and ELISA tests for measuring serum antibody levels were carried out on all blood samples. A majority of the febrile patients who were parasite negative by microscopy showed the presence of a Plasmodium falciparum infection by PCR. The occurrence of P. falciparum infection with parasitemia below the detection level of microscopy was recognized more often in patients with CM symptoms than in those with severe malarial anemia (SMA), and in older rather than younger patients. Patients clinically suspected (CS) of having CM ((CS)CM) mostly were infected with a single clone, and a large proportion of them acquired antibodies (Abs) against merozoite surface protein (MSP) antigens (Ags). The therapeutic response to quinine treatment was comparable between patients with (CS)CM and CM. In conclusion, uniquely in this setting, CM can be associated with sub-patent parasitemia; thus, a diagnostic tool more sensitive than microscopy is needed.

Raised plasma insulin level and homeostasis model assessment (HOMA) score in cerebral malaria: evidence for insulin resistance and marker of virulence.

OBJECTIVE: To study the glycaemic profile of patients with severe malaria (SM). METHODS: For this purpose, 110 SM patients were recruited. Pre-treatment random blood glucose and plasma insulin were measured in a subset of donors. An ex-vivo experiment was developed for estimation of glucose consumption by parasitized erythrocytes. RESULTS: Hyperglycaemia was frequent in SM but more commonly associated with cerebral malaria (CM), while hyperinsulinaemia was recognized in severe-malarial-hypotension (median, 25 %-75 %, 188.2, 93.8-336.8 pmol/L). The plasma insulin level was positively correlated with age (CC = 0.457, p < 0.001) and negatively with parasitaemia (CC = -0.368, p = 0.045). Importantly, fatal-CM was associated with hyperglycaemia (12.22, 6.5-14.6 mmol/L), hyperinsulinaemia (141.0, 54.0-186.8 pmol/L) and elevated homeostasis model assessment (HOMA) values. However, there was a trend of higher glucose consumption by parasites in CM compared with that in uncomplicated malaria (UM). CONCLUSION: Hyperglycaemia, hyperinsulinaemia and elevated HOMA are evidence for insulin resistance and possibly pancreatic B-cell dysfunction in fatal-CM.

Bimodal transmission of cerebral malaria and severe malarial anemia and reciprocal co-existence of sexual and asexual parasitemia in an area of seasonal malaria transmission.

The parasite dynamics in severe malaria (SM) varies with malaria endemicity. This study was conducted in eastern Sudan, an area of seasonal and unstable malaria transmission. From the beginning of October to the end of December (malaria season) in the years 2000, 2001, and 2003, 99 patients with severe malarial anemia (SMA) and 54 patients with cerebral malaria (CM) were identified. There was marked variation in the incidence of SMA and CM (up to six folds) and in the CM/SMA incidence ratio, over 3 years. In the heavy season of 2003, CM peaked at the beginning of the season and declined within a month at a time that the SMA reached the peak. At diagnosis, the rate of gametocytemia had inclined from approximately 10% to 100% from the beginning to the end of the season. During follow-up, gametocytemia was more associated with SMA than with CM. Paradoxically, the late occurring SMA was associated with early gametocytemia (day 7) and the opposite was true in CM. In conclusion, within the season the transmission of CM and SMA was bimodal, the prevalence of the asexual and sexual parasitemia was reciprocal, and the peaks of transmission and gametocytemia were paradoxical.

The profile of IgG-antibody response against merozoite surface proteins 1 and 2 in severe Plasmodium falciparum malaria in Eastern Sudan.

In this study, antibodies (Ab) directed against three MSP antigens; MSP1(19), MSP2(A), and MSP2(B) were analyzed in blood samples obtained from 223 Sudanese patients who presented with either severe malaria (SM) or uncomplicated malaria (UM) and from 117 malaria-free donors (MF). The results showed that the prevalence of MSP Abs was associated with the clinical outcome of malaria infection, and the Ab prevalence was age-dependent (P<0.0005). More importantly, the prevalence of MSP Abs against the test antigens was lower in SM compared to UM (P=0.001 to 0.020), suggesting a protective role for these Abs against SM. Furthermore, the Ab responses between individual complications of SM were significantly different.

Genetic fingerprints of parasites causing severe malaria in a setting of low transmission in Sudan.

In this study we intended to examine the extent of genetic diversity of Plasmodium falciparum parasites causing severe malaria (SM). For this purpose, 100 parasite isolates were obtained from patients with SM and uncomplicated malaria, from an area of low and unstable malaria transmission in Sudan. The diversity of infection (DOI) was estimated by relating the number of the different parasite genotypes that were detected to the total number of parasites that were genotyped (parasite population/subpopulation). We used different molecular markers individually (pfcrt-76, pfmr1-86, GLURP size and MSP2 family and size) and as a group to set a multilocus genetic profile for each parasite isolate. The DOI as estimated by MSP2 and GLURP was 0.553 and 0.435, respectively. However, combination of all four molecular markers (multilocus genetic profile) revealed a fingerprint pattern of genetic diversity with a DOI of 0.936, indicating that in SM infection, diversity is the rule and homogeny is the exception. Furthermore, our clinical data suggest that the virulence markers might also be more diverse than expected. In conclusion, the results are unexpected and overturn the assumption that parasites causing SM are a limited subpopulation of virulent parasites or of a clonal nature. However, it was more likely that there was a genetically unique parasite in each infection.

The efficacy of sulfadoxine-pyrimethamine alone and in combination with chloroquine for malaria treatment in rural Eastern Sudan: the interrelation between resistance, age and gametocytogenesis.

OBJECTIVE: To compare the efficacy of sulfadoxine-pyremethamine (SP)+chloroquine (CQ) combination treatment against falciparum malaria with SP treatment alone. METHOD: In-vivo study of 254 patients with uncomplicated Plasmodium falciparum malaria in rural eastern Sudan, where the population is semi-immune. RESULTS: Sulfadoxine-pyremethamine treatment alone cured 68.3% (41/60) and SP+CQ cured 63.4% (123/194). Early and late treatment failures occurred in both treatment groups. Host age (as a marker for immunity) and parasite gametocytogenesis (as a marker for transmissibility) were significantly associated with SP resistance. Patients who were cured were significantly older (median age 21 years) than patients whose treatment failed (median age 12 years). Gametocyte production was significantly higher in patients with treatment failure (0.72 vs 0.45) and associated with younger age. Gametocyte counts were comparable between both groups until day 7 of follow up; thereafter, they were significantly higher in patients with treatment failure. However, the longevity of gametocytes was comparable in both treatment groups. CONCLUSION: Chloroquine did not improve the parasite response to SP. Age was strongly associated with clearance of SP-resistant parasites. The fast rise of SP resistance may partially be due to selection of SP resistant parasites and expansion of the resistant population through the gametocytogenic effect of SP.