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Orjuela-Sánchez P. Genetic Diversity of Plasmodium vivax populations: analysis of neutral genetic markers and genes potentially associated with drug resistance. [Ph.D. thesis]. São Paulo (Brasil): Instituto de Ciências Biomédicas da Universidade de São Paulo; 2010. In this work neutral markers (microsatellites and synonymous SNPs) and loci under selection (repetitive motifs of pvcsp and non-synonymous SNPs in pvcrt-o and pvmdr1) were used to study in detail the P. vivax genetic variation in the Brazilian Amazon region. Microsatellite typing revealed that Plasmodium vivax populations from the Brazilian Amazon region are more diverse than sympatric populations of P. falciparum, since they showed a higher number of multiple-clone infections, alleles per locus and virtual heterozygosity. However, both species showed significant linkage disequilibrium (LD). Contrasting the temporal stability of markers under selection, neutral polymorphisms from sympatric isolates of P. vivax and P. falciparum showed a high haplotype turnover rate, with few lineages persisting over time. Analysis of the central repeat domain of P. vivax circumsporozoite protein (CSP) in sympatric isolates of P. vivax from Brazil revealed nine csp haplotypes. Identical or nearly identical repeat sequences predominated in most arrays, suggesting the recent expansion of these domains. The analysis of 31 SNP sites across the chromosome 8 segment flanking the csp locus showed strong LD, suggesting that non-meiotic recombination has an important role in CSP diversity. Next, recurrent P. vivax infections were examined in two cohorts from rural Amazonia exposed to low levels of malaria transmission, one of them having high rates of P. vivax recurrence after chloroquine (CQ) primaquine treatment. Microsatellite analysis revealed that only two of 28 pairs of recurrence samples showed identical haplotypes and four pairs with genetically related haplotypes. Microsatellite typing of 99 isolates of P. vivax from one of these cohorts revealed an extraordinary diversity over time, with few isolates sharing the same haplotype. These results suggest that frequent recurrences did not favor the persistence or reappearance of clone lineages of parasites in this population. Since cases of P. vivax CQ resistance have been reported in the Brazilian Amazon region, we decided to analyze SNPs of coding and noncoding sequences of the pvmdr1 and pvcrt-o genes (orthologs of mdr1 and crt genes in P. falciparum and associated with drug resistance). These analyses revealed no associations between SNPs and sensitive or resistant phenotype to CQ among studied samples. Finally, 85 SNPs from chromosome 8 of P. vivax were genotyped among 234 parasites from different areas of malaria transmission in Brazil and Asia. Different levels of diversity and LD among populations were found, with the highest diversity and strongest LD in parasites from the area of lowest malaria transmission (Sri Lanka). Several clusters of contiguous markers with rare meiotic recombination and relatively conserved haplotype structure among populations were found, suggesting the existence of recombination hotspots in this chromosome region. Silent and nonsynonymous SNPs revealed substantial between-population divergence, being able to cluster parasites according to their continental and subcontinental origin. Marked geographic variation was also observed in allele frequencies of pvmdr1.

Diversidade genética em populações de Plasmodium vivax: análise de marcadores genéticos neutros e de genes potencialmente associados à resistência a drogas.