Genomic surveillance of malaria parasites in an indigenous community in the Peruvian Amazon.

Hard-to-reach communities represent Peru's main challenge for malaria elimination, but information about transmission in these areas is scarce. Here, we assessed Plasmodium vivax (Pv) and P. falciparum (Pf) transmission dynamics, resistance markers, and Pf hrp2/3 deletions in Nueva Jerusalén (NJ), a remote, indigenous community in the Peruvian Amazon with high population mobility. We collected samples from November 2019 to May 2020 by active (ACD) and passive case detection (PCD) in NJ. Parasites were identified with microscopy and PCR. Then, we analyzed a representative set of positive-PCR samples (Pv = 68, Pf = 58) using highly-multiplexed deep sequencing assays (AmpliSeq) and compared NJ parasites with ones from other remote Peruvian areas using population genetics indexes. The ACD intervention did not reduce malaria cases in the short term, and persistent malaria transmission was observed (at least one Pv infection was detected in 96% of the study days). In Nueva Jerusalen, the Pv population had modest genetic diversity (He = 0.27). Pf population had lower diversity (He = 0.08) and presented temporal clustering, one of these clusters linked to an outbreak in February 2020. Moreover, Pv and Pf parasites from NJ exhibited variable levels of differentiation (Pv Fst = 0.07-0.52 and Pf Fst = 0.11-0.58) with parasites from other remote areas. No artemisin resistance mutations but chloroquine (57%) and sulfadoxine-pyrimethamine (35-67%) were detected in NJ's Pf parasites. Moreover, pfhrp2/3 gene deletions were common (32-50% of parasites with one or both genes deleted). The persistent Pv transmission and the detection of a Pf outbreak with parasites genetically distinct from the local ones highlight the need for tailored interventions focusing on mobility patterns and imported infections in remote areas to eliminate malaria in the Peruvian Amazon.

Plasmodium vivax genomic surveillance in the Peruvian Amazon with Pv AmpliSeq assay.

BACKGROUND: Plasmodium vivax is the most predominant malaria species in Latin America, constituting 71.5% of malaria cases in 2021. With several countries aiming for malaria elimination, it is crucial to prioritize effectiveness of national control programs by optimizing the utilization of available resources and strategically implementing necessary changes. To support this, there is a need for innovative approaches such as genomic surveillance tools that can investigate changes in transmission intensity, imported cases and sources of reintroduction, and can detect molecular markers associated with drug resistance. METHODOLOGY/PRINCIPAL FINDINGS: Here, we apply a modified highly-multiplexed deep sequencing assay: Pv AmpliSeq v2 Peru. The tool targets a newly developed 41-SNP Peru barcode for parasite population analysis within Peru, the 33-SNP vivaxGEN-geo panel for country-level classification, and 11 putative drug resistance genes. It was applied to 230 samples from the Peruvian Amazon (2007-2020), generating baseline surveillance data. We observed a heterogenous P. vivax population with high diversity and gene flow in peri-urban areas of Maynas province (Loreto region) with a temporal drift using all SNPs detected by the assay (nSNP = 2909). In comparison, in an indigenous isolated area, the parasite population was genetically differentiated (FST = 0.07-0.09) with moderate diversity and high relatedness between isolates in the community. In a remote border community, a clonal P. vivax cluster was identified, with distinct haplotypes in drug resistant genes and ama1, more similar to Brazilian isolates, likely representing an introduction of P. vivax from Brazil at that time. To test its applicability for Latin America, we evaluated the SNP Peru barcode in P. vivax genomes from the region and demonstrated the capacity to capture local population clustering at within-country level. CONCLUSIONS/SIGNIFICANCE: Together this data shows that P. vivax transmission is heterogeneous in different settings within the Peruvian Amazon. Genetic analysis is a key component for regional malaria control, offering valuable insights that should be incorporated into routine surveillance.

Effect of spatiotemporal variables on abundance, biting activity and parity of Nyssorhynchus darlingi (Diptera: Culicidae) in peri-Iquitos, Peru.

BACKGROUND: In malaria endemic regions of the Peruvian Amazon, rainfall together with river level and breeding site availability drive fluctuating vector mosquito abundance and human malaria cases, leading to temporal heterogeneity. The main variables influencing spatial transmission include location of communities, mosquito behaviour, land use/land cover, and human ecology/behaviour. The main objective was to evaluate seasonal and microgeographic biting behaviour of the malaria vector Nyssorhynchus (or Anopheles) darlingi in Amazonian Peru and to investigate effects of seasonality on malaria transmission. METHODS: We captured mosquitoes from 18:00 to 06:00 h using Human Landing Catch in two riverine (Lupuna, Santa Emilia) and two highway (El Triunfo, Nuevo Horizonte) communities indoors and outdoors from 8 houses per community, during the dry and rainy seasons from February 2016 to January 2017. We then estimated parity rate, daily survival and age of a portion of each collection of Ny. darlingi. All collected specimens of Ny. darlingi were tested for the presence of Plasmodium vivax or Plasmodium falciparum sporozoites using real-time PCR targeting the small subunit of the 18S rRNA. RESULTS: Abundance of Ny. darlingi varied across village, season, and biting behaviour (indoor vs outdoor), and was highly significant between rainy and dry seasons (p < 0.0001). Biting patterns differed, although not significantly, and persisted regardless of season, with peaks in highway communities at ~ 20:00 h in contrast to biting throughout the night (i.e., 18:00-06:00) in riverine communities. Of 3721 Ny. darlingi tested for Plasmodium, 23 (0.62%) were infected. We detected Plasmodium-infected Ny. darlingi in both community types and most (20/23) were captured outdoors during the rainy season; 17/23 before midnight. Seventeen Ny. darlingi were infected with P. vivax, and 6 with P. falciparum. No infected Ny. darlingi were captured during the dry season. Significantly higher rates of parity were detected in Ny. darlingi during the rainy season (average 64.69%) versus the dry season (average 36.91%) and by community, Lupuna, a riverine village, had the highest proportion of parous to nulliparous females during the rainy season. CONCLUSIONS: These data add a seasonal dimension to malaria transmission in peri-Iquitos, providing more evidence that, at least locally, the greatest risk of malaria transmission is outdoors during the rainy season mainly before midnight, irrespective of whether the community was located adjacent to the highway or along the river.

Population genomic evidence of structured and connected Plasmodium vivax populations under host selection in Latin America.

Pathogen genomic epidemiology has the potential to provide a deep understanding of population dynamics, facilitating strategic planning of interventions, monitoring their impact, and enabling timely responses, and thereby supporting control and elimination efforts of parasitic tropical diseases. Plasmodium vivax, responsible for most malaria cases outside Africa, shows high genetic diversity at the population level, driven by factors like sub-patent infections, a hidden reservoir of hypnozoites, and early transmission to mosquitoes. While Latin America has made significant progress in controlling Plasmodium falciparum, it faces challenges with residual P. vivax. To characterize genetic diversity and population structure and dynamics, we have analyzed the largest collection of P. vivax genomes to date, including 1474 high-quality genomes from 31 countries across Asia, Africa, Oceania, and America. While P. vivax shows high genetic diversity globally, Latin American isolates form a distinctive population, which is further divided into sub-populations and occasional clonal pockets. Genetic diversity within the continent was associated with the intensity of transmission. Population differentiation exists between Central America and the North Coast of South America, vs. the Amazon Basin, with significant gene flow within the Amazon Basin, but limited connectivity between the Northwest Coast and the Amazon Basin. Shared genomic regions in these parasite populations indicate adaptive evolution, particularly in genes related to DNA replication, RNA processing, invasion, and motility - crucial for the parasite's survival in diverse environments. Understanding these population-level adaptations is crucial for effective control efforts, offering insights into potential mechanisms behind drug resistance, immune evasion, and transmission dynamics.

Genomic surveillance of malaria parasites in an indigenous community in the Peruvian Amazon.

Hard-to-reach communities represent Peru's main challenge for malaria elimination, but information about transmission in these areas is scarce. Here, we assessed Plasmodium vivax (Pv) and P. falciparum (Pf) transmission dynamics, resistance markers, and Pf hrp2/3 deletions in Nueva Jerusalén (NJ), a remote, indigenous community in the Peruvian Amazon with high population mobility. We collected samples from November 2019 to May 2020 by active (ACD) and passive case detection (PCD) in NJ. Parasites were identified with microscopy and PCR. Then, we analyzed a representative set of positive-PCR samples (Pv = 68, Pf = 58) using highly-multiplexed deep sequencing assays (AmpliSeq) and compared NJ parasites with ones from other remote Peruvian areas using population genetics indexes. The ACD intervention did not reduce malaria cases in the short term, and persistent malaria transmission was observed (at least one Pv infection was detected in 96% of the study days). In Nueva Jerusalen, the Pv population had modest genetic diversity (He = 0.27). Pf population had lower diversity (He = 0.08) and presented temporal clustering, one of these clusters linked to an outbreak in February 2020. Moreover, Pv and Pf parasites from NJ exhibited variable levels of differentiation (Pv Fst = -0.52 & Pf Fst = 0.11-0.58) with parasites from other remote areas. No artemisin resistance mutations but chloroquine (57%) and sulfadoxine-pyrimethamine (35-67%) were detected in NJ's Pf parasites. Moreover, pfhrp2/3 gene deletions were common (32-50% of parasites with one or both genes deleted). The persistent Pv transmission and the detection of a Pf outbreak with parasites genetically distinct from the local ones highlight the need for tailored interventions focusing on mobility patterns and imported infections in remote areas to eliminate malaria in the Peruvian Amazon.

Development of WHO Recommendations for the Final Phase of Elimination and Prevention of Re-Establishment of Malaria.

The WHO recommends that all affected countries work toward the elimination of malaria, even those still experiencing a high burden of disease. However, malaria programs in the final phase of elimination or those working to prevent re-establishment of transmission after elimination could benefit from specific evidence-based recommendations for these settings as part of comprehensive and quality-controlled malaria guidelines. The WHO convened an external guideline development group to formulate recommendations for interventions to reduce or prevent malaria transmission in areas with very low- to low-transmission levels and those that have eliminated malaria. In addition, several interventions that could be deployed in higher burden areas to accelerate elimination, such as mass drug administration, were reviewed. Systematic reviews were conducted that synthesized and evaluated evidence for the benefits and harms of public health interventions and summarized critical contextual factors from a health systems perspective. A total of 12 recommendations were developed, with five related to mass interventions that could be deployed at higher transmission levels and seven that would be most appropriate for programs in areas close to elimination or those working to prevent re-establishment of transmission. Four chemoprevention, two active case detection, and one vector control interventions were positively recommended, whereas two chemoprevention and three active case detection interventions were not recommended by the WHO. None of the recommendations were classified as strong given the limited and low-quality evidence base. Approaches to conducting higher quality research in very low- to low-transmission settings to improve the strength of WHO recommendations are discussed.

Mutations Linked to Insecticide Resistance Not Detected in the Ace-1 or VGSC Genes in Nyssorhynchus darlingi from Multiple Localities in Amazonian Brazil and Peru.

Indoor residual spray (IRS), mainly employing pyrethroid insecticides, is the most common intervention for preventing malaria transmission in many regions of Latin America; the use of long-lasting insecticidal nets (LLINs) has been more limited. Knockdown resistance (kdr) is a well-characterized target-site resistance mechanism associated with pyrethroid and DDT resistance. Most mutations detected in acetylcholinesterase-1 (Ace-1) and voltage-gated sodium channel (VGSC) genes are non-synonymous, resulting in a change in amino acid, leading to the non-binding of the insecticide. In the present study, we analyzed target-site resistance in Nyssorhynchus darlingi, the primary malaria vector in the Amazon, in multiple malaria endemic localities. We screened 988 wild-caught specimens of Ny. darlingi from three localities in Amazonian Peru and four in Amazonian Brazil. Collections were conducted between 2014 and 2021. The criteria were Amazonian localities with a recent history as malaria hotspots, primary transmission by Ny. darlingi, and the use of both IRS and LLINs as interventions. Fragments of Ace-1 (456 bp) and VGSC (228 bp) were amplified, sequenced, and aligned with Ny. darlingi sequences available in GenBank. We detected only synonymous mutations in the frequently reported Ace-1 codon 280 known to confer resistance to organophosphates and carbamates, but detected three non-synonymous mutations in other regions of the gene. Similarly, no mutations linked to insecticide resistance were detected in the frequently reported codon (995) at the S6 segment of domain II of VGSC. The lack of genotypic detection of insecticide resistance mutations by sequencing the Ace-1 and VGSC genes from multiple Ny. darlingi populations in Brazil and Peru could be associated with low-intensity resistance, or possibly the main resistance mechanism is metabolic.

A new Plasmodium vivax reference genome for South American isolates.

BACKGROUND: Plasmodium vivax is the second most important cause of human malaria worldwide, and accounts for the majority of malaria cases in South America. A high-quality reference genome exists for Papua Indonesia (PvP01) and Thailand (PvW1), but is lacking for South America. A reference genome specifically for South America would be beneficial though, as P. vivax is a genetically diverse parasite with geographical clustering. RESULTS: This study presents a new high-quality assembly of a South American P. vivax isolate, referred to as PvPAM (P. vivax Peruvian AMazon). The genome was obtained from a low input patient sample from the Peruvian Amazon and sequenced using PacBio technology, resulting in a highly complete assembly with 6497 functional genes. Telomeric ends were present in 17 out of 28 chromosomal ends, and additional (sub)telomeric regions are present in 12 unassigned contigs. A comparison of multigene families between PvPAM and the PvP01 genome revealed remarkable variation in vir genes, and the presence of merozoite surface proteins (MSP) 3.6 and 3.7. Three dhfr and dhps drug resistance associated mutations are present in PvPAM, similar to those found in other Peruvian isolates. Mapping of publicly available South American whole genome sequencing (WGS) data to PvPAM resulted in significantly fewer variants and truncated reads compared to the use of PvP01 or PvW1 as reference genomes. To minimize the number of core genome variants in non-South American samples, PvW1 is most suited for Southeast Asian isolates, both PvPAM and PvW1 are suited for South Asian isolates, and PvPAM is recommended for African isolates. Interestingly, non-South American samples still contained the least subtelomeric variants when mapped to PvPAM, indicating high quality of the PvPAM subtelomeric regions. CONCLUSIONS: Our findings show that the PvPAM reference genome more accurately represents South American P. vivax isolates in comparison to PvP01 and PvW1. In addition, PvPAM has a high level of completeness, and contains a similar number of annotated genes as PvP01 or PvW1. The PvPAM genome therefore will be a valuable resource to improve future genomic analyses on P. vivax isolates from the South American continent.

Natural Infection of Nyssorhynchus darlingi and Nyssorhynchus benarrochi B with Plasmodium during the Dry Season in the Understudied Low-Transmission Setting of Datem del Marañon Province, Amazonian Peru.

The persistence of malaria hotspots in Datem del Marañon Province, Peru, prompted vector control units at the Ministry of Health, Loreto Department, to collaborate with the Amazonian International Center of Excellence for Malaria Research to identify the main vectors in several riverine villages that had annual parasite indices > 15 in 2018-2019. Anophelinae were collected indoors and outdoors for two 12-hour nights/community during the dry season in 2019 using human landing catch. We identified four species: Nyssorhynchus benarrochi B, Nyssorhynchus darlingi, Nyssorhynchus triannulatus, and Anopheles mattogrossensis. The most abundant, Ny. benarrochi B, accounted for 96.3% of the total (7,550/7,844), of which 61.5% were captured outdoors (4,641/7,550). Six mosquitoes, one Ny. benarrochi B and five Ny. darlingi, were infected by Plasmodium falciparum or Plasmodium vivax. Human biting rates ranged from 0.5 to 592.8 bites per person per hour for Ny. benarrochi B and from 0.5 to 32.0 for Ny. darlingi, with entomological inoculation rates as high as 0.50 infective bites per night for Ny. darlingi and 0.25 for Ny. benarrochi B. These data demonstrate the risk of malaria transmission by both species even during the dry season in villages in multiple watersheds in Datem del Marañon province.

Molecular Surveillance of Malaria Using the PF AmpliSeq Custom Assay for Plasmodium falciparum Parasites from Dried Blood Spot DNA Isolates from Peru.

Malaria molecular surveillance has great potential to support national malaria control programs (NMCPs), informing policy for its control and elimination. Here, we present a new three-day workflow for targeted resequencing of markers in 13 resistance-associated genes, histidine rich protein 2 and 3 (hrp2&3), a country (Peru)-specific 28 SNP-barcode for population genetic analysis, and apical membrane antigen 1 (ama1), using Illumina short-read sequencing technology. The assay applies a multiplex PCR approach to amplify all genomic regions of interest in a rapid and easily standardizable procedure and allows simultaneous amplification of a high number of targets at once, therefore having great potential for implementation into routine surveillance practice by NMCPs. The assay can be performed on routinely collected filter paper blood spots and can be easily adapted to different regions to investigate either regional trends or in-country epidemiological changes.

Malaria Molecular Surveillance in the Peruvian Amazon with a Novel Highly Multiplexed Plasmodium falciparum AmpliSeq Assay.

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.

Effect of out-of-village working activities on recent malaria exposure in the Peruvian Amazon using parametric g-formula.

In the Amazon Region of Peru, occupational activities are important drivers of human mobility and may increase the individual risk of being infected while contributing to increasing malaria community-level transmission. Even though out-of-village working activities and other mobility patterns have been identified as determinants of malaria transmission, no studies have quantified the effect of out-of-village working activities on recent malaria exposure and proposed plausible intervention scenarios. Using two population-based cross-sectional studies in the Loreto Department in Peru, and the parametric g-formula method, we simulated various hypothetical scenarios intervening in out-of-village working activities to reflect their potential health benefits. This study estimated that the standardized mean outcome (malaria seroprevalence) in the unexposed population (no out-of-village workers) was 44.6% (95% CI: 41.7%-47.5%) and 66.7% (95% CI: 61.6%-71.8%) in the exposed population resulting in a risk difference of 22.1% (95% CI: 16.3%-27.9%). However, heterogeneous patterns in the effects of interest were observed between peri-urban and rural areas (Cochran's Q test = 15.5, p < 0.001). Heterogeneous patterns were also observed in scenarios of increased prevalence of out-of-village working activities and restriction scenarios by gender (male vs. female) and age (18 and under vs. 19 and older) that inform possible occupational interventions targetting population subgroups. The findings of this study support the hypothesis that targeting out-of-village workers will considerably benefit current malaria elimination strategies in the Amazon Region. Particularly, males and adult populations that carried out out-of-village working activities in rural areas contribute the most to the malaria seropositivity (recent exposure to the parasite) in the Peruvian Amazon.

Geographical distribution and genetic characterization of pfhrp2 negative Plasmodium falciparum parasites in the Peruvian Amazon.

Malaria rapid diagnostic tests (RDTs) have been evaluated in the Peruvian Amazon region and their performance has been variable. This region is known for being the first with documented evidence of wild Plasmodium falciparum parasites lacking pfhrp2 and pfhrp3 genes, leading to false-positive results with HRP2-based RDTs. In our attempt to further characterize the deletion pattern of these genes and their evolutionary relationship, 93 P. falciparum samples, collected in different communities from the Peruvian Amazon region between 2009 and 2010, were analyzed in this study. Genomic DNA was used to amplify 18S rRNA, pfmsp2 and pfglurp to confirm the diagnosis and DNA quality, respectively; pfhrp2, pfhrp3, and their flanking genes were amplified by PCR to assess the pattern of the gene deletions. In addition, microsatellite analysis were performed using seven neutral microsatellites (MS) and five microsatellite loci flanking pfhrp2. The data showed the absence of pfhrp3 gene in 53.76% (50/93) of the samples, reflecting a higher frequency than the proportion of pfhrp2 gene deletions (33.33%; 31/93). Among the flanking genes, the highest frequency of deletion was observed in the PF3D7_0831900 gene (78.49%; 73/93) for pfhrp2. MS marker analysis showed the presence of 8 P. falciparum lineages. The lineage Bv1 was the most prevalent among parasites lacking pfhrp2 and pfhrp3 genes. Additionally, using MS flanking pfhrp2 gene, the haplotypes α and δ were found to be the most abundant in this region. This study confirms the presence in this area of field isolates with deletions in either pfhrp2, pfhrp3, or both genes, along with their respective flanking regions. Our data suggest that some pfhrp2/pfhrp3 deletion haplotypes, in special the lineage Bv1, are widely dispersed within the Peruvian Amazon. The persistence of these haplotypes ensures a proportion of P.falciparum parasites lacking the pfhrp2/pfhrp3 genes in this area, which ultimately leads to false-negative results on PfHRP2-detecting malaria RDTs. However, additional studies are needed to not only confirm this hypothesis but also to further delineate the origin and genetic basis for the pfhrp2- and pfhrp3 gene deletions in wild P. falciparum parasites.

Band 3-mediated Plasmodium vivax invasion is associated with transcriptional variation in PvTRAg genes.

The Plasmodium vivax reticulocyte invasion process is still poorly understood, with only a few receptor-ligand interactions identified to date. Individuals with the Southeast Asian ovalocytosis (SAO) phenotype have a deletion in the band 3 protein on the surface of erythrocytes, and are reported to have a lower incidence of clinical P. vivax malaria. Based on this observation, band 3 has been put forward as a receptor for P. vivax invasion, although direct proof is still lacking. In this study, we combined functional ex vivo invasion assays and transcriptome sequencing to uncover a band 3-mediated invasion pathway in P. vivax and potential band 3 ligands. Invasion by P. vivax field isolates was 67%-71% lower in SAO reticulocytes compared with non-SAO reticulocytes. Reticulocyte invasion was decreased by 40% and 27%-31% when blocking with an anti-band 3 polyclonal antibody and a PvTRAg38 peptide, respectively. To identify new band 3 receptor candidates, we mRNA-sequenced schizont-stage isolates used in the invasion assays, and observed high transcriptional variability in multigene and invasion-related families. Transcriptomes of isolates with low or high dependency on band 3 for invasion were compared by differential expression analysis, which produced a list of band 3 ligand candidates with high representation of PvTRAg genes. Our ex vivo invasion assays have demonstrated that band 3 is a P. vivax invasion receptor and confirm previous in vitro studies showing binding between PvTRAg38 and band 3, although the lower and variable inhibition levels observed suggest the involvement of other ligands. By coupling transcriptomes and invasion phenotypes from the same isolates, we identified a list of band 3 ligand candidates, of which the overrepresented PvTRAg genes are the most promising for future research.

Evidence-Based Malaria Control and Elimination in the Amazon: Input from the International Center of Excellence in Malaria Research Network in Peru and Brazil.

Malaria remains endemic in 17 countries in the Americas, where 723,000 cases were reported in 2019. The majority (> 90%) of the regional malaria burden is found within the Amazon Basin, which includes nine countries and territories in South America. Locally generated evidence is critical to provide information to public health decision makers upon which the design of efficient and regionally directed malaria control and elimination programs can be built. Plasmodium vivax is the predominant malaria parasite in the Amazon Basin. This parasite species appears to be more resilient to malaria control strategies worldwide. Asymptomatic Plasmodium infections constitute a potentially infectious reservoir that is typically missed by routine microscopy-based surveillance and often remains untreated. The primary Amazonian malaria vector, Nyssorhynchus (formerly Anopheles) darlingi, has changed its behavior to feed and rest predominantly outdoors, reducing the efficiency of core vector control measures such as indoor residual spraying and distribution of long-lasting insecticide-treated bed nets. We review public health implications of recent field-based research carried out by the Amazonia International Center of Excellence in Malaria Research in Peru and Brazil. We discuss the relative role of traditional and novel tools and strategies for better malaria control and elimination across the Amazon, including improved diagnostic methods, new anti-relapse medicines, and biological larvicides, and emphasize the need to integrate research and public health policymaking.

Malaria Resilience in South America: Epidemiology, Vector Biology, and Immunology Insights from the Amazonian International Center of Excellence in Malaria Research Network in Peru and Brazil.

The 1990s saw the rapid reemergence of malaria in Amazonia, where it remains an important public health priority in South America. The Amazonian International Center of Excellence in Malaria Research (ICEMR) was designed to take a multidisciplinary approach toward identifying novel malaria control and elimination strategies. Based on geographically and epidemiologically distinct sites in the Northeastern Peruvian and Western Brazilian Amazon regions, synergistic projects integrate malaria epidemiology, vector biology, and immunology. The Amazonian ICEMR's overarching goal is to understand how human behavior and other sociodemographic features of human reservoirs of transmission-predominantly asymptomatically parasitemic people-interact with the major Amazonian malaria vector, Nyssorhynchus (formerly Anopheles) darlingi, and with human immune responses to maintain malaria resilience and continued endemicity in a hypoendemic setting. Here, we will review Amazonian ICEMR's achievements on the synergies among malaria epidemiology, Plasmodium-vector interactions, and immune response, and how those provide a roadmap for further research, and, most importantly, point toward how to achieve malaria control and elimination in the Americas.

Drug resistance and population structure of Plasmodium falciparum and Plasmodium vivax in the Peruvian Amazon.

Malaria is a major health problem in Peru despite substantial progress achieved by the ongoing malaria elimination program. This study explored the population genetics of 63 Plasmodium falciparum and 170 P. vivax cases collected in the Peruvian Amazon Basin between 2015 and 2019. Microscopy and PCR were used for malaria detection and positive samples were genotyped at neutral and drug resistance-associated regions. The P. falciparum population exhibited a low nucleotide diversity (π = 0.02) whereas the P. vivax population presented a higher genetic diversity (π = 0.34). All P. falciparum samples (n = 63) carried chloroquine (CQ) resistant mutations on Pfcrt. Most P. falciparum samples (53 out of 54) carried sulfadoxine (SD) resistant mutations on Pfdhfr and Pfdhps. No evidence was found of artemisinin resistance mutations on kelch13. Population structure showed that a single cluster accounted for 93.4% of the P. falciparum samples whereas three clusters were found for P. vivax. Our study shows a low genetic diversity for both species with significant differences in genetic sub-structuring. The high prevalence of CQ-resistance mutations could be a result of indirect selection pressures driven by the P. vivax treatment scheme. These results could be useful for public health authorities to safeguard the progress that Peru has achieved towards malaria elimination.

Epidemiological characteristics of P. vivax asymptomatic infections in the Peruvian Amazon.

Introduction: Herein, we tested the hypothesis that Asymptomatic P. vivax (Pv) infected individuals (Asym) feature different epidemiological, clinical and biochemical characteristics, as well as hematological parameters, potentially predictive of clinical immunity in comparison to symptomatic Pv infected individuals (Sym). Methodology: Between 2018 - 2021, we conducted 11 population screenings (PS, Day 0 (D0)) in 13 different riverine communities around Iquitos city, in the Peruvian Amazon, to identify Pv Sym and Asym individuals. A group of these individuals agreed to participate in a nested case - control study to evaluate biochemical and hematological parameters. Pv Asym individuals did not present common malaria symptoms (fever, headache, and chills), had a positive/negative microscopy result, a positive qPCR result, reported no history of antimalarial treatment during the last month, and were followed-up weekly until Day 21 (D21). Control individuals, had a negative malaria microscopy and qPCR result, no history of antimalarial treatment or malaria infections during the last three years, and no history of comorbidities or chronic infections. Results: From the 2159 individuals screened during PS, data revealed a low but heterogeneous Pv prevalence across the communities (11.4%), where most infections were Asym (66.7%) and submicroscopic (82.9%). A total of 29 Asym, 49 Sym, and 30 control individuals participated in the nested case - control study (n=78). Ten of the individuals that were initially Asym at D0, experienced malaria symptoms during follow up and therefore, were included in the Sym group. 29 individuals remained Asym throughout all follow-ups. High levels of eosinophils were found in Asym individuals in comparison to Sym and controls. Conclusion: For the first-time, key epidemiological, hematological, and biochemical features are reported from Pv Asym infections from the Peruvian Amazon. These results should be considered for the design and reshaping of malaria control measures as the country moves toward malaria elimination.

Assessment of IgG3 as a serological exposure marker for Plasmodium vivax in areas with moderate-high malaria transmission intensity.

A more sensitive surveillance tool is needed to identify Plasmodium vivax infections for treatment and to accelerate malaria elimination efforts. To address this challenge, our laboratory has developed an eight-antigen panel that detects total IgG as serological markers of P. vivax exposure within the prior 9 months. The value of these markers has been established for use in areas with low transmission. In moderate-high transmission areas, there is evidence that total IgG is more long-lived than in areas with low transmission, resulting in poorer performance of these markers in these settings. Antibodies that are shorter-lived may be better markers of recent infection for use in moderate-high transmission areas. Using a multiplex assay, the antibody temporal kinetics of total IgG, IgG1, IgG3, and IgM against 29 P. vivax antigens were measured over 36 weeks following asymptomatic P. vivax infection in Papua New Guinean children (n = 31), from an area with moderate-high transmission intensity. IgG3 declined faster to background than total IgG, IgG1, and IgM. Based on these kinetics, IgG3 performance was then assessed for classifying recent exposure in a cohort of Peruvian individuals (n = 590; age 3-85 years) from an area of moderate transmission intensity. Using antibody responses against individual antigens, the highest performance of IgG3 in classifying recent P. vivax infections in the prior 9 months was to one of the Pv-fam-a proteins assessed (PVX_125728) (AUC = 0.764). Surprisingly, total IgG was overall a better marker of recent P. vivax infection, with the highest individual classification performance to RBP2b1986-2653 (PVX_094255) (AUC = 0.838). To understand the acquisition of IgG3 in this Peruvian cohort, relevant epidemiological factors were explored using a regression model. IgG3 levels were positively associated with increasing age, living in an area with (relatively) higher transmission intensity, and having three or more PCR-detected blood-stage P. vivax infections within the prior 13 months. Overall, we found that IgG3 did not have high accuracy for detecting recent exposure to P. vivax in the Peruvian cohort, with our data suggesting that this is due to the high levels of prior exposure required to acquire high IgG3 antibody levels.

Insights into Plasmodium vivax Asymptomatic Malaria Infections and Direct Skin-Feeding Assays to Assess Onward Malaria Transmission in the Amazon.

Understanding the reservoir and infectivity of Plasmodium gametocytes to vector mosquitoes is crucial to align strategies aimed at malaria transmission elimination. Yet, experimental information is scarce regarding the infectivity of Plasmodium vivax for mosquitoes in diverse epidemiological settings where the proportion of asymptomatically infected individuals varies at a microgeographic scale. We measured the transmissibility of clinical and subclinical P. vivax malaria parasite carriers to the major mosquito vector in the Amazon Basin, Nyssorhynchus darlingi (formerly Anopheles). A total of 105 participants with natural P. vivax malaria infection were recruited from a cohort study in Loreto Department, Peruvian Amazon. Four of 18 asymptomatic individuals with P. vivax positivity by blood smear infected colony-grown Ny. darlingi (22%), with 2.6% (19 of 728) mosquitoes infected. In contrast, 77% (44/57) of symptomatic participants were infectious to mosquitoes with 51% (890 of 1,753) mosquitoes infected. Infection intensity was greater in symptomatic infections (mean, 17.8 oocysts/mosquito) compared with asymptomatic infections (mean, 0.28 oocysts/mosquito), attributed to parasitemia/gametocytemia level. Paired experiments (N = 27) using direct skin-feeding assays and direct membrane mosquito-feeding assays showed that infectivity to mosquitoes was similar for both methods. Longitudinal studies with longer follow-up of symptomatic and asymptomatic parasite infections are needed to determine the natural variations of disease transmissibility.