P.falciparum Immune Evasion Mechanisms: Possible Roles For Allelic Polymorphism and Antigenic Variation In Sudanese Isolates.

Abstract

Abstract Genetic diversity within P. falciparum and the continuous variability of the parasite antigens, makes malaria an important global threat and it the reason behind failure to produce an effective vaccine against malaria. Objectives: This study aimed to determine and characterize Plasmodium falciparum genotypic variations and to detect probable genetic diversity of genotypically identical Pfalciparum isolates in Sudan and to determine whether there is any association between the presence of specific MSP-1 antibodies and the carriage of multiple P. falcinarum infections. Study design: It was cross-sectional, hospital-based and experimental study that was carried out in four localities, Kasab Rural hospital, Eddouiem, Gazira and Khartoum. One hundred and eighty six P. fulciparum infected patients were enrolled in the study. Methods: Nested polymerase chain reaction was carried out to genotyped study isolates using P. falciparum merozoite surface protein (PIMSP-1) gene specific primers. Genetic diversity of all isolates was characterized by random amplified polymorphic DNA (RAPD) analysis using thirteen arbitrary oligonucleotide primers. RAPD was also used to study the polymorphism of different isolates before and after in vitra cultures. ELISA test was performed to determine the prevalence of lgG antibodies against four rMSP-1 fragments (C-terminus, WELL, K1 DI & K1 Dll). Results: The |gG seroprevalence was 85.2, 23.8, 23.9 81 35.2 % to MSP-1,5, WELL, K1 DI & K1 Dll respectively. There was a gradual increase in overall antibody prevalence reaching a peak in adolescence and decrease thereafter. The difference was only age»specific for the C-terminal fragment. The three reported families of msp-Ialleles (K1, MADZO and R033) were observed among the studied isolates. The predominant allele was of Klvariant while R033 was less frequent. The majority of the patients had multiple parasite clones and multiplicity of infection (MOI) was found to be 1.82, this MOI was found to be higher in Kasab, Eddouiem and Khartoum and was significantly associated with age and having malaria symptoms, but not with parasitemia and fiequency of infections. Patients with multiple infections had higher prevalence of antibodies to all antigen fragments studied, compared to those with single infection, but this was only significant for MSP-119. RAPD primers recognized marked polymorphism and difierent profiles among isolates with a .laccard’s Similarity Coefficient ra.nged from 0.25 to 0.80. Different MSP-1 alleles showed different RAPD banding patterns, no certain genotype was found to be unique a certain banding pattem. Forty nine isolates were successfully in vitra cultured. RAPD analysis revealed that only 29 isolates showed different banding patterns before and after in vitro culture Conclusion: P. falciparum isolates fi'om different parts of Sudan were genetically diverse and most of the infections were mixed with a high level of multiplicity of infection (MOI). This MOI extend the duration of the infection and delay the acquisition of protective immunity, making it difiicult to produce effective vaccines against these parasites and this is in turn increase the burden of the disease and poverty. Further longitudinal large size studies, including protein analysis and mono-clonal antibodies to identify functional antibodies are needed to evaluate the impact of this polymorphism on the immune response and in tum help in developing effective vaccine against malaria