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Population genomics and evidence of clonal replacement of Plasmodium falciparum in the Peruvian Amazon

ABSTRACT: Previous studies have shown that P. falciparum parasites in South America have undergone population bottlenecks resulting in clonal lineages that are differentially distributed and that have been responsible for several outbreaks different endemic regions. In this study, we explored the genomic profile of 18 P. falciparum samples collected in the Peruvian Amazon Basin (Loreto) and 6 from the Peruvian North Coast (Tumbes). Our results showed the presence of three subpopulations that matched previously typed lineages in Peru: Bv1 (n = 17), Clonet D (n = 4) and Acre-Loreto type (n = 3). Gene coverage analysis showed that none of the Bv1 samples presented coverage for pfhrp2 and pfhrp3. Genotyping of drug resistance markers showed a high prevalence of Chloroquine resistance mutations S1034C/N1042D/D1246Y in pfmdr1 (62.5%) and K45T in pfcrt (87.5%). Mutations associated with sulfadoxine and pyrimethamine treatment failure were found on 88.8% of the Bv1 samples which were triple mutants for pfdhfr (50R/51I/108N) and pfdhps (437G/540E/581G). Analysis of the pfS47 gene that allows P. falciparum to evade mosquito immune responses showed that the Bv1 lineage presented one pfS47 haplotype exclusive to Loreto and another haplotype that was present in both Loreto and Tumbes. Furthermore, a possible expansion of Bv1 was detected since 2011 in Loreto. This replacement could be a result of the high prevalence of CQ resistance polymorphisms in Bv1, which could have provided a selective advantage to the indirect selection pressures driven by the use of CQ for P. vivax treatment.

SUBMITTER: Villena F

PROVIDER: S-EPMC8551272 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundCharacterizing the parasite dynamics and population structure provides useful information to understand the dynamic of transmission and to better target control interventions. Despite considerable efforts for its control, vivax malaria remains a major health problem in Peru. In this study, we have explored the population genetics of Plasmodium vivax isolates from Iquitos, the main city in the Peruvian Amazon, and 25 neighbouring peri-urban as well as rural villages along the Iquitos-Nauta Road.Methodology/ resultsFrom April to December 2008, 292 P. vivax isolates were collected and successfully genotyped using 14 neutral microsatellites. Analysis of the molecular data revealed a similar proportion of monoclonal and polyclonal infections in urban areas, while in rural areas monoclonal infections were predominant (p = 0.002). Multiplicity of infection was higher in urban (MOI = 1.5-2) compared to rural areas (MOI = 1) (p = 0.003). The level of genetic diversity was similar in all areas (He = 0.66-0.76, p = 0.32) though genetic differentiation between areas was substantial (PHIPT = 0.17, p<0.0001). Principal coordinate analysis showed a marked differentiation between parasites from urban and rural areas. Linkage disequilibrium was detected in all the areas ([Formula: see text] = 0.08-0.49, for all p<0.0001). Gene flow among the areas was stablished through Bayesian analysis of migration models. Recent bottleneck events were detected in 4 areas and a recent parasite expansion in one of the isolated areas. In total, 87 unique haplotypes grouped in 2 or 3 genetic clusters described a sub-structured parasite population.Conclusion/significanceOur study shows a sub-structured parasite population with clonal propagation, with most of its components recently affected by bottleneck events. Iquitos city is the main source of parasite spreading for all the peripheral study areas. The routes of transmission and gene flow and the reduction of the parasite population described are important from the public health perspective as well for the formulation of future control policies.

Project description:BACKGROUND Nyssorhynchus dunhami, a member of the Nuneztovari Complex, has been collected in Brazil, Colombia, and Peru and described as zoophilic. Although to date Ny. dunhami has not been documented to be naturally infected by Plasmodium, it is frequently misidentified as other Oswaldoi subgroup species that are local or regional malaria vectors. OBJECTIVES The current study seeks to verify the morphological identification of Nuneztovari Complex species collected in the peri-Iquitos region of Amazonian Peru, to determine their Plasmodium infection status, and to describe ecological characteristics of their larval habitats. METHODS We collected Ny. nuneztovari s.l. adults in 2011-2012, and Ny. nuneztovari s.l. larvae and adults in 2016-2017. When possible, samples were identified molecularly using cytochrome c oxidase subunit I (COI) barcode sequencing. Adult Ny. nuneztovari s.l. from 2011-2012 were tested for Plasmodium using real-time PCR. Environmental characteristics associated with Ny. nuneztovari s.l. larvae-positive water bodies were evaluated. FINDINGS We collected 590 Ny. nuneztovari s.l. adults and 116 larvae from eight villages in peri-Iquitos. Of these, 191 adults and 111 larvae were identified by COI sequencing; all were Ny. dunhami. Three Ny. dunhami were infected with P. falciparum, and one with P. vivax, all collected from one village on one night. Ny. dunhami larvae were collected from natural and artificial water bodies, and their presence was positively associated with other Anophelinae larvae and amphibians, and negatively associated with people living within 250m. MAIN CONCLUSIONS Of Nuneztovari Complex species, we identified only Ny. dunhami across multiple years in eight peri-Iquitos localities. This study is, to our knowledge, the first report of natural infection of molecularly identified Ny. dunhami with Plasmodium. We advocate the use of molecular identification methods in this region to monitor Ny. dunhami and other putative secondary malaria vectors to more precisely evaluate their importance in malaria transmission.

Project description:Molecular surveillance for malaria has great potential to support national malaria control programs (NMCPs). To bridge the gap between research and implementation, several applications (use cases) have been identified to align research, technology development, and public health efforts. For implementation at NMCPs, there is an urgent need for feasible and cost-effective tools. We designed a new highly multiplexed deep sequencing assay (Pf AmpliSeq), which is compatible with benchtop sequencers, that allows high-accuracy sequencing with higher coverage and lower cost than whole-genome sequencing (WGS), targeting genomic regions of interest. The novelty of the assay is its high number of targets multiplexed into one easy workflow, combining population genetic markers with 13 nearly full-length resistance genes, which is applicable for many different use cases. We provide the first proof of principle for hrp2 and hrp3 deletion detection using amplicon sequencing. Initial sequence data processing can be performed automatically, and subsequent variant analysis requires minimal bioinformatic skills using any tabulated data analysis program. The assay was validated using a retrospective sample collection (n = 254) from the Peruvian Amazon between 2003 and 2018. By combining phenotypic markers and a within-country 28-single-nucleotide-polymorphism (SNP) barcode, we were able to distinguish different lineages with multiple resistance haplotypes (in dhfr, dhps, crt and mdr1) and hrp2 and hrp3 deletions, which have been increasing in recent years. We found no evidence to suggest the emergence of artemisinin (ART) resistance in Peru. These findings indicate a parasite population that is under drug pressure but is susceptible to current antimalarials and demonstrate the added value of a highly multiplexed molecular tool to inform malaria strategies and surveillance systems. IMPORTANCE While the power of next-generation sequencing technologies to inform and guide malaria control programs has become broadly recognized, the integration of genomic data for operational incorporation into malaria surveillance remains a challenge in most countries where malaria is endemic. The main obstacles include limited infrastructure, limited access to high-throughput sequencing facilities, and the need for local capacity to run an in-country analysis of genomes at a large-enough scale to be informative for surveillance. In addition, there is a lack of standardized laboratory protocols and automated analysis pipelines to generate reproducible and timely results useful for relevant stakeholders. With our standardized laboratory and bioinformatic workflow, malaria genetic surveillance data can be readily generated by surveillance researchers and malaria control programs in countries of endemicity, increasing ownership and ensuring timely results for informed decision- and policy-making.

Project description:BackgroundDifferent antigens are needed to characterize Plasmodium falciparum infection in terms of seroreactivity and targets for invasion inhibition, in order to guide and identify the proper use of such proteins as tools for the development of serological markers and/or as vaccine candidates.MethodsIgG responses in 84 serum samples from individuals with P. falciparum infection [classified as symptomatic (Sym) or asymptomatic (Asym)], or acute Plasmodium vivax infection, from the Peruvian Amazon region, were evaluated by enzyme-linked immunosorbent assays specific for a baculovirus-produced recombinant protein P. falciparum Merozoite Surface Protein 10 (rMSP10) and for non-EGF region selected peptides of PfMSP10 selected by a bioinformatics tool (PfMSP10-1, PfMSP10-2 and PfMSP10-3). Monoclonal antibodies against the selected peptides were evaluated by western blotting, confocal microscopy and inhibition invasion assays.ResultsSeroreactivity analysis of the P. falciparum Sym- and Asym-infected individuals against rMSP10 showed a higher response as compared to the individuals with P. vivax acute infection. IgG responses against peptide PfMSP10-1 were weak. Interestingly high IgG response was found against peptide PfMSP10-2 and the combination of peptides PfMSP10-1 + PfMSP10-2. Monoclonal antibodies were capable of detecting native PfMSP10 on purified schizonts by western blot and confocal microscopy. A low percentage of inhibition of merozoite invasion of erythrocytes in vitro was observed when the monoclonal antibodies were compared with the control antibody against AMA-1 antigen. Further studies are needed to evaluate the role of PfMSP10 in the merozoite invasion.ConclusionsThe rMSP10 and the PfMSP10-2 peptide synthesized for this study may be useful antigens for evaluation of P. falciparum malaria exposure in Sym and Asym individuals from the Peruvian Amazon region. Moreover, these antigens can be used for further investigation of the role of this protein in other malaria-endemic areas.

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Population genomics and evidence of clonal replacement of Plasmodium falciparum in the Peruvian Amazon

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