RESUMO
BACKGROUND: With increasing interest in eliminating malaria from the Caribbean region, Haiti is one of the two countries on the island of Hispaniola with continued malaria transmission. While the Haitian population remains at risk for malaria, there are a limited number of cases annually, making conventional epidemiological measures such as case incidence and prevalence of potentially limited value for fine-scale resolution of transmission patterns and trends. In this context, genetic signatures may be useful for the identification and characterization of the Plasmodium falciparum parasite population in order to identify foci of transmission, detect outbreaks, and track parasite movement to potentially inform malaria control and elimination strategies. METHODS: This study evaluated the genetic signals based on analysis of 21 single-nucleotide polymorphisms (SNPs) from 462 monogenomic (single-genome) P. falciparum DNA samples extracted from dried blood spots collected from malaria-positive patients reporting to health facilities in three southwestern Haitian departments (Nippes, Grand'Anse, and Sud) in 2016. RESULTS: Assessment of the parasite genetic relatedness revealed evidence of clonal expansion within Nippes and the exchange of parasite lineages between Nippes, Sud, and Grand'Anse. Furthermore, 437 of the 462 samples shared high levels of genetic similarity-at least 20 of 21 SNPS-with at least one other sample in the dataset. CONCLUSIONS: These results revealed patterns of relatedness suggestive of the repeated recombination of a limited number of founding parasite types without significant outcrossing. These genetic signals offer clues to the underlying relatedness of parasite populations and may be useful for the identification of the foci of transmission and tracking of parasite movement in Haiti for malaria elimination.
Assuntos
DNA de Protozoário/análise , Plasmodium falciparum/genética , Polimorfismo de Nucleotídeo Único , HaitiRESUMO
In regions with high malaria endemicity, the withdrawal of chloroquine (CQ) as first-line treatment of Plasmodium falciparum infections has typically led to the restoration of CQ susceptibility through the reexpansion of the wild-type (WT) allele K76 of the chloroquine resistance transporter gene (pfcrt) at the expense of less fit mutant alleles carrying the CQ resistance (CQR) marker K76T. In low-transmission settings, such as South America, drug resistance mutations can attain 100% prevalence, thereby precluding the return of WT parasites after the complete removal of drug pressure. In French Guiana, despite the fixation of the K76T allele, the prevalence of CQR isolates progressively dropped from >90% to <30% during 17 y after CQ withdrawal in 1995. Using a genome-wide association study with CQ-sensitive (CQS) and CQR isolates, we have identified a single mutation in pfcrt encoding a C350R substitution that is associated with the restoration of CQ susceptibility. Genome editing of the CQR reference strain 7G8 to incorporate PfCRT C350R caused a complete loss of CQR. A retrospective molecular survey on 580 isolates collected from 1997 to 2012 identified all C350R mutant parasites as being CQS. This mutation emerged in 2002 and rapidly spread throughout the P. falciparum population. The C350R allele is also associated with a significant decrease in piperaquine susceptibility in vitro, suggesting that piperaquine pressure in addition to potential fitness costs associated with the 7G8-type CQR pfcrt allele may have selected for this mutation. These findings have important implications for understanding the evolutionary dynamics of antimalarial drug resistance.
Assuntos
Cloroquina/uso terapêutico , Resistência a Medicamentos/genética , Evolução Molecular , Proteínas de Membrana Transportadoras/genética , Mutação , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Alelos , Guiana Francesa , Marcadores Genéticos , Genoma , Genótipo , Haplótipos , Humanos , Concentração Inibidora 50 , Malária/tratamento farmacológico , Fenótipo , Plasmodium falciparum/efeitos dos fármacos , Prevalência , Análise de Componente Principal , Quinolinas/química , Estudos RetrospectivosRESUMO
Plasmodium vivax, one of the five species of Plasmodium parasites that cause human malaria, is responsible for 25-40% of malaria cases worldwide. Malaria global elimination efforts will benefit from accurate and effective genotyping tools that will provide insight into the population genetics and diversity of this parasite. The recent sequencing of P. vivax isolates from South America, Africa, and Asia presents a new opportunity by uncovering thousands of novel single nucleotide polymorphisms (SNPs). Genotyping a selection of these SNPs provides a robust, low-cost method of identifying parasite infections through their unique genetic signature or barcode. Based on our experience in generating a SNP barcode for P. falciparum using High Resolution Melting (HRM), we have developed a similar tool for P. vivax. We selected globally polymorphic SNPs from available P. vivax genome sequence data that were located in putatively selectively neutral sites (i.e., intergenic, intronic, or 4-fold degenerate coding). From these candidate SNPs we defined a barcode consisting of 42 SNPs. We analyzed the performance of the 42-SNP barcode on 87 P. vivax clinical samples from parasite populations in South America (Brazil, French Guiana), Africa (Ethiopia) and Asia (Sri Lanka). We found that the P. vivax barcode is robust, as it requires only a small quantity of DNA (limit of detection 0.3 ng/µl) to yield reproducible genotype calls, and detects polymorphic genotypes with high sensitivity. The markers are informative across all clinical samples evaluated (average minor allele frequency > 0.1). Population genetic and statistical analyses show the barcode captures high degrees of population diversity and differentiates geographically distinct populations. Our 42-SNP barcode provides a robust, informative, and standardized genetic marker set that accurately identifies a genomic signature for P. vivax infections.
Assuntos
Código de Barras de DNA Taxonômico/métodos , DNA de Protozoário/genética , Malária Vivax/parasitologia , Plasmodium vivax/isolamento & purificação , África/epidemiologia , Ásia/epidemiologia , Sequência de Bases , Mapeamento Cromossômico , Marcadores Genéticos/genética , Humanos , Malária Vivax/epidemiologia , Plasmodium falciparum/classificação , Plasmodium falciparum/genética , Plasmodium falciparum/isolamento & purificação , Plasmodium vivax/classificação , Plasmodium vivax/genética , Polimorfismo de Nucleotídeo Único , América do Sul/epidemiologiaRESUMO
Identifying the source of resurgent parasites is paramount to a strategic, successful intervention for malaria elimination. Although the malaria incidence in Panama is low, a recent outbreak resulted in a 6-fold increase in reported cases. We hypothesized that parasites sampled from this epidemic might be related and exhibit a clonal population structure. We tested the genetic relatedness of parasites, using informative single-nucleotide polymorphisms and drug resistance loci. We found that parasites were clustered into 3 clonal subpopulations and were related to parasites from Colombia. Two clusters of Panamanian parasites shared identical drug resistance haplotypes, and all clusters shared a chloroquine-resistance genotype matching the pfcrt haplotype of Colombian origin. Our findings suggest these resurgent parasite populations are highly clonal and that the high clonality likely resulted from epidemic expansion of imported or vestigial cases. Malaria outbreak investigations that use genetic tools can illuminate potential sources of epidemic malaria and guide strategies to prevent further resurgence in areas where malaria has been eliminated.
Assuntos
Antimaláricos/farmacologia , Cloroquina/farmacologia , Surtos de Doenças , Resistência a Medicamentos/genética , Malária Falciparum/epidemiologia , Plasmodium falciparum/isolamento & purificação , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Análise por Conglomerados , Colômbia , Código de Barras de DNA Taxonômico , Feminino , Loci Gênicos/genética , Haplótipos , Humanos , Malária Falciparum/parasitologia , Masculino , Pessoa de Meia-Idade , Panamá/epidemiologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Polimorfismo de Nucleotídeo Único , Proteínas de Protozoários/genética , Adulto JovemRESUMO
Plasmodium falciparum with reduced sensitivity to artemisinin derivatives has been observed in endemic areas, but the molecular mechanisms for this reduced sensitivity remain unclear. We evaluated the association between in vitro susceptibility of P. falciparum isolates obtained from southwest Nigeria and polymorphisms in selected putative transporter genes (PFE0775C, PF13_0271, pfmrp1, pfcrt, and pfmdr1). Modified schizont inhibition assay was used to determine the in vitro parasite susceptibility to artemether (ATH). Polymorphisms in selected genes were detected by polymerase chain reaction followed by direct DNA sequencing. The half-maximal inhibitory concentration (IC(50)) geometric mean (GM) for all P. falciparum isolates was 1.78 nM (range, 0.03-10.43 nM). Polymorphisms at codons 241, 86, and 76 of PFE0775C, pfmdr1, and pfcrt genes, respectively, were associated with reduced susceptibility to ATH. A new S263P single-nucleotide polymorphism on the PFE0775C gene was also detected in 27% of the isolates. Patient isolates harboring V241L or S263P polymorphisms on the PFE0775C gene showed increased IC(50) (GM: 3.08 nM and 1.79 nM, respectively). Plasmodium falciparum isolates harboring mutant Y86 pfmdr1 and P263 PFE0775C alleles showed a 2.5-5.5-fold increase in ATH IC(50.) This study shows that polymorphisms on the PFE0775C and pfmdr1 genes are associated with reduced sensitivity to ATH in fresh isolates of P. falciparum from Nigeria.
Assuntos
Antimaláricos/farmacologia , Artemisininas/farmacologia , Proteínas de Transporte/genética , Resistência a Medicamentos/genética , Plasmodium falciparum/efeitos dos fármacos , Polimorfismo Genético , Artemeter , Proteínas de Transporte/metabolismo , Criança , DNA de Protozoário/genética , DNA de Protozoário/isolamento & purificação , Regulação da Expressão Gênica , Humanos , Concentração Inibidora 50 , Testes de Sensibilidade Parasitária/métodos , Reação em Cadeia da Polimerase/métodos , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
BACKGROUND: The ideal malaria parasite populations for initial mapping of genomic regions contributing to phenotypes such as drug resistance and virulence, through genome-wide association studies, are those with high genetic diversity, allowing for numerous informative markers, and rare meiotic recombination, allowing for strong linkage disequilibrium (LD) between markers and phenotype-determining loci. However, levels of genetic diversity and LD in field populations of the major human malaria parasite P. vivax remain little characterized. RESULTS: We examined single-nucleotide polymorphisms (SNPs) and LD patterns across a 100-kb chromosome segment of P. vivax in 238 field isolates from areas of low to moderate malaria endemicity in South America and Asia, where LD tends to be more extensive than in holoendemic populations, and in two monkey-adapted strains (Salvador-I, from El Salvador, and Belem, from Brazil). We found varying levels of SNP diversity and LD across populations, with the highest diversity and strongest LD in the area of lowest malaria transmission. We found several clusters of contiguous markers with rare meiotic recombination and characterized a relatively conserved haplotype structure among populations, suggesting the existence of recombination hotspots in the genome region analyzed. Both silent and nonsynonymous SNPs revealed substantial between-population differentiation, which accounted for ~40% of the overall genetic diversity observed. Although parasites clustered according to their continental origin, we found evidence for substructure within the Brazilian population of P. vivax. We also explored between-population differentiation patterns revealed by loci putatively affected by natural selection and found marked geographic variation in frequencies of nucleotide substitutions at the pvmdr-1 locus, putatively associated with drug resistance. CONCLUSION: These findings support the feasibility of genome-wide association studies in carefully selected populations of P. vivax, using relatively low densities of markers, but underscore the risk of false positives caused by population structure at both local and regional levels.
Assuntos
Estudo de Associação Genômica Ampla , Desequilíbrio de Ligação , Plasmodium vivax/genética , Polimorfismo de Nucleotídeo Único , Animais , Genética Populacional , Seleção GenéticaRESUMO
Understanding the genetic structure of malaria parasites is essential to predict how fast some phenotypes of interest originate and spread in populations. In the present study, we used highly polymorphic microsatellite markers to analyze 74 Plasmodium vivax isolates, which we collected in cross-sectional and longitudinal surveys performed in an area of low malaria endemicity in Brazilian Amazonia, and to explore the transmission dynamics of genetically diverse haplotypes or strains. P. vivax populations are more diverse and more frequently comprise multiple-clone infections than do sympatric Plasmodium falciparum isolates, but these features paradoxically coexist with high levels of inbreeding, leading to significant multilocus linkage disequilibrium. Moreover, the high rates of microsatellite haplotype replacement that we found during 15 months of follow-up most likely do not result from strong diversifying selection. We conclude that the small-area genetic diversity in P. vivax populations under low-level transmission is not severely constrained by the low rates of effective meiotic recombination, with clear public health implications.
Assuntos
Variação Genética , Malária Vivax/transmissão , Repetições de Microssatélites/genética , Plasmodium vivax/genética , Animais , Brasil/epidemiologia , Estudos Transversais , Haplótipos , Humanos , Desequilíbrio de Ligação/genética , Estudos Longitudinais , Malária Vivax/epidemiologia , Plasmodium vivax/classificação , Plasmodium vivax/patogenicidade , Polimorfismo Genético , População Rural , Fatores de TempoRESUMO
Genetic variation allows the malaria parasite Plasmodium falciparum to overcome chemotherapeutic agents, vaccines and vector control strategies and remain a leading cause of global morbidity and mortality. Here we describe an initial survey of genetic variation across the P. falciparum genome. We performed extensive sequencing of 16 geographically diverse parasites and identified 46,937 SNPs, demonstrating rich diversity among P. falciparum parasites (pi = 1.16 x 10(-3)) and strong correlation with gene function. We identified multiple regions with signatures of selective sweeps in drug-resistant parasites, including a previously unidentified 160-kb region with extremely low polymorphism in pyrimethamine-resistant parasites. We further characterized 54 worldwide isolates by genotyping SNPs across 20 genomic regions. These data begin to define population structure among African, Asian and American groups and illustrate the degree of linkage disequilibrium, which extends over relatively short distances in African parasites but over longer distances in Asian parasites. We provide an initial map of genetic diversity in P. falciparum and demonstrate its potential utility in identifying genes subject to recent natural selection and in understanding the population genetics of this parasite.