The aim of this study was to investigate the allelic dimorphism of the EBA-175 in P. falciparum isolates from Brazilian malaria endemic area, Rondonia State, in three different time points (time point-1: 1993/94; time point-2: 2001/2002; time point-3: 2007/2008 in order to evaluate whether the allelic dimorphism of the EBA-175 in Brazil was present and if this dimorphism is temporally stable in the same area. Two fragments, C-fragment (714 bp) and F-fragment (795 bp), were identified in the studied area. Overall, C-fragment (83.2%) was observed in a higher frequency than F-fragment (16.8%). This data differ from results reported by three independent research groups in high endemicity malaria areas of Ghana, Gabon and Burkina Faso where F-fragment was observed in a higher frequency than C-fragment [11, 13, 19]. Interesting, the results reported here are in agreement with the results observed in low endemicity malaria area of Sudan where C-fragment has been shown to be the most common . The lower frequency of F-fragment in the studied area can be explained by: immunological selection of these Plasmodium populations in infected studied individuals and/or genetic background of the human studied population [21, 22]. Also it can be speculated that a random selection may be the reason for this distribution of the EBA-175 allelic frequencies.
The differences observed in the distribution of C- and F-fragments among geographical regions could be explained by random shifts in parasite allele frequencies due to genetic drift in genetically isolated populations . In this case, allele frequencies might be predicted to change over time. Another hypothesis would be that differences in the genetic background among study population may select for different EBA-175 alleles. In this case, allele frequencies might be predicted to be stable over time . One study performed by Soulama and co-workers analysed the seasonal distribution of the EBA-175 allelic forms in four villages in Burkina Faso, where malaria transmission in markedly seasonal, and showed that the distribution of EBA-175 alleles were relatively stable over 8 months . In the present study, the distribution of C- and F-fragments were analysed in three time points over 15 years. Similar allelic frequencies suggests that the distribution of these two EBA-175 allelic forms is stable over time, corroborating the hypothesis that host genetic background can influence the distribution of the EBA-175 allelic forms.
In areas of high P. falciparum genetic diversity, the individuals have a higher probability of being infected simultaneously with more than two clones of the parasite, which has been called the multiclonal [24, 25] or mixed infection. Considering that the complexity of infections, reflected by the number of mixed infections, can increase the likelihood of cross-fertilization of Plasmodium spp. in the mosquito and thus increase the genetic diversity [26, 27], the frequency of single and mixed infections in P. falciparum isolates from studied population was evaluated. In the present work, a low frequency of mixed infection (3/181, 1.7%) was found. These results are similar to a study with the P126 protein that reveals low frequency of mixed infection in Brazilian isolates from the same area . The three mixed infections reported in the present work were observed only in the time point-1. Probably these three mixed infections were found due the high endemicity in the studied area in the first year of sample set (API = 549) when compared with the second (API = 173) and the third (API = 85) sample sets, since several studies indicated that there is a positive relationship between transmission intensity and complexity of infection [29, 30]. Alternatively, the lower sample size in time points-2 and -3 may account for underestimation of mixed infection.