Molecular Characterization of Severe Plasmodium falciparum Malaria Parasitemia and Antimalarial Drug-resistance in Eastern Sudan

Abstract

A hospital-based study was carried out in Gedarif town, an area of markedly unstable malaria transmission in eastern Sudan during the two malaria transmission seasons (2000-2002),. Among the 2488 diagnosed malaria patients, 110 fulfilled the WHO (2000) criteria for severe malaria (SM), and seven died of cerebral malaria (CM). The predominant complication was severe malarial anemia (SMA, 45.45%), followed by convulsions associated malaria (CAM, 20.9%), cerebral malaria (16.4%) and hypotension (HTN, 11.8%). DNA was extracted from 616 parasites obtained from 231 donors with asymptomatic submicroscopic malaria (ASUM), uncomplicated malaria (UM) and SM. The MSP2 locus was exploited for determination of parasite genotype. 51 parasite genotypes were detected. There was no correlation between the number of infectious clones and initial parasite count or patient age. In all infections, the mean clone number (MCN) was 1.5 clone/infection, and it was comparable between SM and UM. However detailed analysis revealed that; individuals with ASUM had significantly lower MCN compared with UM, SMA, and non-fatal CM. The frequency of IC1 and FC27 allele families was comparable between SM and UM and the distribution of the individual allele sizes was correlated. Further analysis showed that, 1. FC27 was associated with the mildest form of malaria and was not recognized in fatal CM, 2. Faster clearance of IC1 genotype in the multi-clone infections after quinine treatment 3. Multi-clone infections with different genotype families (IC1 and FC27) were far more frequent than that with the same genotype family, suggesting stronger cross-immunity within rather than between MSP2 gene families. The diversity of infection (DOI) was estimated by using different molecular markers (pfcrt76, pfmdr1 86, glurp and MSP2) separately and by considering them together to generate a multilocus genetic profile for each isolate. pfcrt and pfmdr1, are not usually used for parasite genotyping, here they revealed DOI of 0.043 and 0.064, respectively. However, when used in combination with the most polymorphic markers (MSP2 and GLURP) they disclosed a hidden diversity that would have not been detected. The DOI as estimated by MSP2 and GLURP was, 0.553 and 0.435, respectively. However, combining all 4 molecular markers (multi-locus genetic profile), had revealed a finger print pattern of diversity, DOI of 0.936, which almost indicated that there were one unique strain for each patient, and that was comparable between SM and UM (although for UM, GLURP was not analyzed). Not only, the genetic make up of parasites were different, but clinical data suggest that the virulence markers should also be extremely diverse. Another hospital-based study was carried out in New Halfa town, where 120 individuals were enrolled in the study, including febrile patients with and without microscopically detectable parasitemia, and apparently healthy individuals. 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 P. 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 SMA, and in older rather than younger patients. Patients clinically suspected to have cerebral malaria (CSCM) mostly had a single clone infection and a large proportion of them acquired antibodies against MSP antigens. The therapeutic response to quinine treatment was comparable between patients with CSCM and CM. In a 28-days in-vivo study of pyrimethamine/sulfadoxine (SP) and SP plus chloroquine (CQ) efficacy, 260 patients with uncomplicated P. falciparum malaria were enrolled in rural Eastern Sudan. The results revealed a comparable treatment failure (TF) for SP alone (32.99%) or with CQ (31.7%). Patients who achieved adequate clinical response (ACR), were significantly older than patients who had TF. Regarding gametocytogenesis; a. the microscopic gametocyte productivity was significantly higher in patients with TF when compared to ACR. Thus, gametocytemia was more associated with younger age b. gametocyte counts were comparable between TF and ACR groups of patients until Day7 of follow up, thereafter, at D14, D21 and D28, gametocytes count was significantly higher in patients with TF. However, the longevity of gametocytes in patients with TF and ACR were comparable. Parasite isolates from 168 patients (including all patients with TF) were genotyped at the dhfr, dhps and pfcrt loci. The molecular image of the in-vivo phenotype, revealed the following findings: the prevalence of dhfr mutations c51-I, c108-N, and double mutant c51I/108N, were estimated to be 91.9%, 92.3% and 79.7%, respectively, while only one isolate (0.6%) was found carrying c59R and no c164L mutation was recognized. The dhps mutations; c437G, c540E and double mutant c437/c540 had a prevalence rate of 90.2%, 79.3% and 58.5%, respectively, while those of c436F and c581G were, 0.6% and 13%, respectively. For pfcrt gene at c72-76, 92.26% were the mutant type CVIET, only 6.55% were the wild type CVMNK and 2 patients harbored mixed infection. Based on the multiplicity of mutation score (MOM) ranging from 1 to 5, the prevalence according to the MOM score were; 0.97, 0.931, 0.866, 0.719, 0.121, respectively.