Plasmodium falciparum parasites with deletions of the histidine-rich protein 2 and 3 (hrp2 and hrp3) genes evade detection by common rapid diagnostic tests (RDTs) and pose a growing threat to malaria control. While these deletions have emerged in multiple regions globally, the evolutionary forces driving their spread remain unclear. Here, we analyze 1,215 P. falciparum samples collected between 2003 and 2018 in Loreto, Peru. This region experienced a major decline in malaria transmission following the Project for Malaria Control in Andean Border Areas (PAMAFRO) and now harbors a high proportion of hrp2/3 deleted parasites despite limited RDT use. Using molecular inversion probe (MIP) sequencing across > 2,000 genome-wide loci, we observed a marked reduction in genetic diversity, increased clonality, and fixation of parasites with deletions of both hrp2 and hrp3 genes (hrp2-/3-) over time. Identity-by-descent (IBD) analysis revealed rapid expansion of a single hrp2-/3- dominant lineage in the post-PAMAFRO period, consistent with clonal replacement after intense malaria control. Targeted sequencing of the hrp2/3 regions showed conserved deletion breakpoints across three different lineages, indicative of recombination of a common haplotype into distinct genetic backgrounds. To investigate the evolutionary forces driving the fixation of hrp2-/3- in Loreto, we simulated allele frequency trajectories under different selection coefficients. We found that fixation of hrp2-/3- due solely to genetic drift (selection coefficient s = 0) is unlikely; a selection coefficient of s ≥ 0.03 was required for fixation to occur consistently. However, our simulations also indicate that a genetic bottleneck caused by PAMAFRO increased the likelihood of fixation through drift by 4.5- to 17-fold depending on the population. These findings suggest that hrp2-/3- fixation was likely driven by a combination of demographic changes resulting from PAMAFRO and selective advantage unrelated to RDT use. Our results demonstrate how intensive malaria control efforts can reshape parasite populations and underscore the value of expanded genomic surveillance as countries move toward malaria elimination.

In areas progressing toward malaria elimination, distinguishing locally acquired from imported malaria infections is essential for targeted surveillance. We used whole-genome sequencing (WGS) to characterize Plasmodium vivax infections in individuals with reported local travel and non-travelers in three communities in the Peruvian Amazon: Libertad (n = 24), Gamitanacocha (n = 23), and Urco Miraño (n = 9). Among 56 confirmed P. vivax infections, 16 were identified in local travelers. DNA was extracted from whole blood, selectively amplified, and parasite species were confirmed by qPCR. Genetic diversity was assessed using expected heterozygosity and nucleotide diversity, genetic differentiation using Fst, population structure using PCA, DAPC, and ADMIXTURE, and relatedness using IBD analysis. Traveler-associated infections in Libertad showed higher diversity than non-travelers (mean He = 0.432 vs 0.304; mean π = 0.290 vs 0.168). Pairwise Fst showed low differentiation between Libertad and Gamitanacocha (Fst = 0.056), but higher differentiation between Urco Miraño and Libertad (Fst = 0.548) and Gamitanacocha (Fst = 0.773). PCA and DAPC showed clear clustering of Urco Miraño parasites, while Libertad and Gamitanacocha partially overlapped. Traveler-associated infections did not form distinct clusters and mostly remained connected to non-traveler infections. ADMIXTURE indicated greater heterogeneity among travelers, and IBD identified two unconnected traveler-associated infections overall. These findings support integrating genomic and epidemiological data to interpret mobility-associated malaria transmission.

Undiagnosed acute febrile illness (AFI) remains elevated in the Peruvian Amazon. The contribution of Oropouche virus (OROV) to AFI in Loreto was quantified, and its recent circulation was contextualized using targeted molecular testing and phylogenomic analysis. Using a novel research-use-only OROV real-time quantitative polymerase chain reaction test across seven surveillance sites, 1,000 AFI samples were rescreened in a setting where undiagnosed presentations were common (70% lacked a confirmed etiology at enrollment). Oropouche virus was detected in 7.1% of cases, with nearly all consensus genomes recovered from specimens with a cycle threshold value ≤30, underscoring the importance of early-phase sampling for sequencing success. Positive results occurred at all seven locations, including the remote riverine community of San Lorenzo, indicating broad geographic distribution and supporting ongoing endemic transmission rather than a single localized outbreak. Exposure patterns were consistent with peridomestic, vector-mediated acquisition: animal contact was common (63.4%), whereas contact with symptomatic individuals (12.7%) and recent travel (35.7%) were infrequent, supporting local, not person-to-person or travel-related, transmission. Oropouche virus cases peaked in January to February 2024, temporally overlapping with the seasonal rise in AFIs, indicating synchronous circulation with other endemic pathogens. Phylogenetic arrangement of contemporary Peruvian strains within established South American diversity further supports ongoing local transmission consistent with regional patterns. Together, these findings indicate that OROV accounts for a meaningful portion of AFI in Loreto and that the routine inclusion of OROV testing, paired with genomic surveillance, can close diagnostic gaps, improve situational awareness, and guide targeted public health action.

Understanding the genetic relatedness of Plasmodium vivax recurrences is essential for distinguishing between relapse, reinfection, and recrudescence-a distinction critical for evaluating treatment efficacy and transmission dynamics. We developed P. vivax AmpSeq (PvAmpSeq), an amplicon sequencing assay targeting 11 single-nucleotide polymorphism (SNP)-rich genomic regions. PvAmpSeq was applied to field isolates from a clinical trial in the Solomon Islands and a longitudinal cohort in Peru, and statistical models were applied for the genetic classification of recurrences. In the Solomon Islands trial, where participants received antimalarials at baseline, half of the recurrent infections showed >50% identity-by-descent relatedness to baseline parasites, allowing statistical classification as probable relapses and recrudescences, although with wide uncertainty. In the Peruvian cohort, 68% of the recurrences exhibited <25% relatedness. PvAmpSeq provides high-resolution genotyping to characterize P. vivax recurrences, offering insights into transmission and treatment outcomes. We also discuss the nuances and limitations of available statistical methods for the classification of P. vivax genotyping data.

Plasmodium falciparum populations in the Peruvian Amazon have undergone changes in the last 20 years, coinciding with the implementation of the PAMAFRO malaria control program (2005-2011). In this context, parasites with deletions of the pfhrp2 and pfhrp3 (pfhrp2/3) genes, involved in rapid diagnostic test (RDT) failure, have been increasingly predominant in Peru since 2012. Here, we investigated population genomic processes shaping P. falciparum populations in the Peruvian Amazon, using the PAMAFRO program as a reference point. We analyzed the genomic changes over 102 isolates between 2006 and 2018, grouped in two periods: 33 samples from 2006 to 2011 (Period 1, before PAMAFRO) and 69 from 2012 to 2018 (Period 2, after PAMAFRO). We found a reduction in the parasite population structure complexity, a significant decrease in genetic diversity and a clonal population structure in Period 2. The pfhrp2/3 deletion profiles differed significantly between periods, with the double pfhrp2/3 deletion being predominant in only Period 2 (68%). Importantly, no evidence of positive selection was found on the pfhrp2/3 loci. Together, these results indicate that P. falciparum populations in Peru experienced a bottleneck associated with PAMAFRO, followed by the clonal expansion of the parasites carrying pfhrp2/3 deletions. In contrast to the Horn of Africa, the deletion of the pfhrp2/3 genes were not selected in Peru, and their expansion is better explained by a different evolutionary process, such as genetic drift. These results highlight the importance of population genomic analysis in uncovering the patterns shaping the parasite population in Peru, which impacts malaria diagnostics and other control strategies.

Accurate serological tools are essential for monitoring the transmission of arboviruses with pandemic potential, yet cross-reactivity between closely related viruses hampers diagnostics and surveillance. Here, we develop a high-throughput multiplex serological assay to quantify antibody responses to 28 antigens from nine arboviruses (dengue, Zika, yellow fever, West Nile, Usutu, Japanese encephalitis, chikungunya (CHIKV), Mayaro (MAYV), and O'nyong-nyong virus) and apply it to over 4000 samples from epidemiologically distinct sites on four continents. We implement a flexible analytical method based on Bayesian finite mixture models and Receiver Operating Characteristic analysis to evaluate assay performance and define seropositivity thresholds. As a case study, we resolve cross-reactive and virus-specific responses for CHIKV and the emerging MAYV by combining competitive immunoassays with mathematical modelling of multiplex serological and epidemiological data. This approach yields cross-reactivity-adjusted estimates of local transmission dynamics, in agreement with existing epidemiological evidence, and reveals that CHIKV is more prone to induce cross-reactive antibody responses than MAYV. Our results demonstrate the power of combining multiplex serology with experimental validation and modelling to disentangle exposure histories in the face of serological cross-reactivity. This integrative approach holds promise for improving arbovirus surveillance, particularly in settings with overlapping transmission of multiple viruses and limited diagnostic capacity.

Background:Human mobility is increasingly recognized as a key factor influencing malaria transmission dynamics, particularly in low-transmission settings approaching elimination. This study aimed to assess mobility patterns and their association with malaria risk in two hypoendemic communities in the Peruvian Amazon. Method:A longitudinal study was conducted in the communities of Libertad and Urcomiraño (Mazán River basin). Monthly population screenings were combined with weekly active and passive case detection. A total of 678 individuals were enrolled. Mobility patterns were assessed through structured questionnaires, and social network analysis was used to characterize travel connections. Log-binomial regression analysis was applied to identify risk factors associated with malaria infection. Result:Internally, mobile individuals in Libertad showed a higher malaria incidence (>32.47 cases per 1,000 person-months) than those in Urcomiraño (<10.15 cases per 1,000 person-months). Travel networks were mainly connected to Mazan district and Iquitos city, followed by local streams such as Armas and Arahuana. Mobility was primarily driven by family, administrative and occupational activities. Male sex (PR = 2.15, 95% CI: 1.37 - 3.37) and age ≥15 years (PR = 1.98, 95% CI: 1.24 - 3.19) were significantly associated with malaria infection (p-value < 0.05). Conclusion:Internally mobile populations represent a key high-risk group sustaining malaria transmission in hypoendemic settings. Targeted interventions focusing on mobile individuals should be integrated into malaria elimination strategies in the Peruvian Amazon and similar endemic regions.

Malaria in South America remains a serious public health problem. Anopheles (Nyssorhynchus) darlingi is the most important malaria vector across tropical Latin America. Vector-targeted disease control efforts require a thorough understanding of mosquito demographic and evolutionary patterns. We present and analyze whole genomes of 1094 An. darlingi (median depth 18x) from six South American countries. We observe deep geographic population structure, high genetic diversity including 13 putative segregating inversions, and no evidence for sympatric cryptic taxa despite high interpopulation divergence. Strong signals of selection are plausibly driven by insecticides, especially on cytochrome P450 genes. Our results will facilitate effective mosquito surveillance and control while highlighting ongoing challenges that a diverse vector poses for malaria elimination in the Western hemisphere.

The Plasmodium falciparum phenotypic characteristics (cytoadherence and rosetting), and the concentration of Histidine Rich Protein 2 (HRP2) in serum are associated with severe malaria in regions like Africa and Asia. Also, HRP2 has recently been associated as a P. falciparum virulence factor. Parasites lacking pfhrp2 gene were first reported in the Peruvian Amazon, however, there are not yet reports of the study of their phenotypes. For this research we decided to characterize the phenotype and genotype of P. falciparum field isolates from the Peruvian Amazon with different pfhrp2 patterns (positive or negative) cultivated in vitro. P. falciparum field isolates (n = 5) from the Peruvian Amazon region were isolated and cultured in vitro. Three isolates had a positive result or pfhrp2 positive pattern and two of them were negatives (pfhrp2 negative pattern) by HRP2 Rapid diagnostic test. Then, these results were confirmed by conventional PCR. The following phenotypic characterization assays were tested: (i) Cytoadherence capacity (evaluated against Chondroitin Sulfate A (CSA) protein and Human umbilical vein endothelial cells (HUVEC cells)), and (ii) rosetting formation. Genotypic characterization assays were carried out by PCR (gene detection) and qPCR assays for pfhrp2 and pfhrp3 gene expression. Additionally, we evaluate the pfhrp2/3 genes and flanking genes stability using long-term cultures of these field isolates during a year. Two of the three isolates with pfhrp2 positive pattern showed rosetting formation (R + , CS-, Hu-) and cytoadherence to CSA protein (R-, CS + , Hu-), on the other hand the two field isolates with pfhrp2 negative pattern showed adhesion to HUVEC cells (R-, CS-, Hu +). 3D7 strain was used for normalization during qPCR assays. We identified that pfhrp2 gene expression levels were higher in the field isolates with pfhrp2 positive pattern, while pfhrp3 gene expression levels were similar or lower. All cultured field isolates showed genomic stability of pfhrp2 gene and flanking genes during the long-term in vitro culture. However, one field isolate showed two pfhrp3 patterns during the culture due to the presence of two parasite genotypes populations, as corroborated by microsatellite markers. P. falciparum Peruvian field isolates with pfhrp2 positive pattern showed cytoadherence to Chondroitin sulfate A protein (R-, CS + , Hu-) or positive characteristics for rosetting (R + , CS-, Hu-); pfhrp2 negative parasites showed cytoadherence to HUVEC cells (R-, CS-, Hu +). In this small set of Peruvian field isolates, a clear cytoadherence and resolution profile was observed for the different pfhrp2 patterns. These findings should be interpreted as preliminary and require further verification in larger isolated panels.

We present two genome assemblies, each generated from individual female Anopheles ( Nyssorhynchus) darlingi (the malaria mosquito; Arthropoda; Insecta; Diptera; Culicidae), from wild populations in French Guiana and Peru. The genome sequences are approximately 180 megabases in span. The majority of each assembly is scaffolded into three chromosomal pseudomolecules with the X sex chromosome assembled. The complete mitochondrial genomes were also assembled and are both 15.4 kilobases in length. The assemblies differ by two inversions in chromosome arm 2R.

Assessing parasite population genetic diversity and structure in remote areas is essential for understanding malaria transmission and guiding interventions toward elimination. This study monitored the genetic diversity and population structure of Plasmodium vivax as part of a longitudinal surveillance in Santa Emilia, a hard-to-reach community in Loreto, Peru. A total of 221 of 3,434 P. vivax samples collected through active and passive case detection between 2015 and 2016 were genotyped using 16 neutral microsatellites. Additionally, 139 genotyped samples from 2013, previously reported, were included for comparison. Malaria prevalence (microscopic and submicroscopic), genetic diversity, population differentiation, structure, bottleneck analysis, and relatedness between years were evaluated. We found 56% P. vivax prevalence by quantitative real-time polymerase chain reaction, with 44% submicroscopic infections in 2015 and 2016. Genetic diversity and population differentiation were high between 2013, 2015, and 2016. Parasites from 2015 to 2016 had a lower Jost D. In 2013 and 2015, more than 40% of infections were polyclonal infections, but only 29% were polyclonal infections in 2016. Moderate linkage disequilibrium was found over time. Four populations were detected in 2013, 2015, and 2016, with increasing admixture in 2015-2016. Genetically related parasites with clonal expansion suggest that there was no recent bottleneck. Santa Emilia has a persistent high genetic diversity and structured, temporally differentiated clonal populations over the time periods of the study. This analysis highlights the complexity of parasite dynamics in this remote area of malaria transmission, making it a challenging area for the malaria elimination plan in Peru.

Background While the global burden of malaria cases has decreased over the last two decades, the disease remains a major international threat, even on the rise in many regions. More than 85% of Peruvian malaria cases are in the Amazonian region of Loreto. Internal mobility primarily related to occupation is thought to be primarily responsible for maintaining endemicity and introducing and reintroducing malaria parasites into areas of anophelism, a challenge for malaria eradication. This study focuses on identifying the sources of malaria transmission and patterns of human mobility in order to understand the movement and transmission of the parasite. Methods The assessment of connectivity produced by human mobility was evaluated in three districts of Loreto, through 10 cross-sectional population screening from 2018 to 2020. We used social network analysis (SNA) to obtain weighted and unweighted degrees of connectivity and explore its variability by socio-demographic characteristics. In addition, we integrated travel history and malaria incidence data to estimate parasite connectivity due to internal human mobility between locations. Finally, we used logistic multivariate regressions to explore the factors associated with Plasmodium spp. infection in mobile individuals. Results We found that internal human mobility results in high connectivity between communities from the Mazan, Iquitos, and San Juan Bautista districts. We identified nearby destinations that may act as sinks or sources for malaria transmission, including densely populated towns and rural campsites. In addition, we found that being a male, traveling to rural campsites, and working outdoors are associated with Plasmodium spp. infection in travelers from the Mazan district. Conclusions We provide compelling evidence about how human mobility connects rural communities in the Peruvian Amazon. Using SNA, we uncovered district-specific patterns and destinations, providing further evidence of human mobility heterogeneity in the region. To address the challenge of human mobility and malaria in this setting, geographic heterogeneity of malaria transmission must be considered.

We present two genome assemblies, each generated from individual female Anopheles (Nyssorhynchus) darlingi (the malaria mosquito; Arthropoda; Insecta; Diptera; Culicidae), from wild populations in French Guiana and Peru. The genome sequences are approximately 180 megabases in span. The majority of each assembly is scaffolded into three chromosomal pseudomolecules with the X sex chromosome assembled. The complete mitochondrial genomes were also assembled and are both 15.4 kilobases in length. The assemblies differ by two inversions in chromosome arm 2R.

The Global Technical Strategy for Malaria 2016–2030 targets eliminating malaria from at least 35 countries and reducing case incidence by 90% globally. The importation of parasites due to human mobilization poses a significant obstacle to achieve malaria elimination as it can undermine the effectiveness of local interventions. Gaining a comprehensive understanding of parasite importation is essential to support control efforts and advance progress toward elimination. Parasite genetic data is widely used to investigate the spatial and temporal dynamics of imported infections. In this context, this systematic review aimed to aggregate evidence on the application of parasite genetic data for mapping imported malaria and the analytical methods used to analyze it. We discuss the advantages and limitations of the genetic approaches employed and propose a suitable type of genetic data along with an analytical framework to discriminate imported malaria infections from local infections. The findings offer potential actionable insights for national control programs, enabling them select the most effective methods for detecting imported cases. This also may aid in the evaluation and refinement of elimination programs by identifying high-risk areas and enabling the targeted allocation of resources to these regions.

We describe the Pf8 data resource, the latest MalariaGEN release of curated genome variation data on over 33,000 Plasmodium falciparum samples from 99 partner studies and 122 locations over more than 50 years. This release provides open access to raw sequencing data and genotypes at over 12 million genomic positions. For the first time, it includes copy-number variation (CNV) calls in the drug-resistance associated genes gch1 and crt. As in Pf7, CNV calls are provided for mdr1 and plasmepsin2/3, along with calls for deletion in hrp2 and hrp3, genes associated with rapid diagnostic test failures. This data resource additionally features derived datasets, interactive web applications for exploring patterns of drug resistance and variation in over 5,000 genes, an updated Python package providing methods for accessing and analysing the data, and open access analysis notebooks that can be used as starting points for further analyses. In addition, informative example analyses show contrasting profiles of the decline of chloroquine resistance-associated mutations in Africa, and variation in copy number variation across 10 distinct sub-populations. To the best of our knowledge, Pf8 is the largest open data set of genome variation in any eukaryotic species, making it an invaluable foundational resource for understanding evolution, including that of pathogens.

In the Loreto region of Amazonian Peru, the primary malaria vector is Nyssorhynchus (or Anopheles) darlingi. The present year-long study sought to identify specific landscapes with the greatest risk of Plasmodium transmission, incorporating epidemiological data, landscape stratification models, satellite imagery, and vector biology. A monthly longitudinal cohort study and population screening of all residents of the focal village Santa Rita detected, among 353 inhabitants enrolled, a ratio of 17:1 submicroscopic/ microscopic malaria infections. Microscopy-detected human infections were seasonal, generally peaking with rising river levels and increasing rainfall, but submicroscopic infections, detected by qPCR, were perennial. Despite high mosquito net usage, inhabitants with outdoor occupations had significantly higher P. vivax infection rates compared with those with indoor occupations or those not working (35% vs. 21%, p = 0.012). A spatial grid subdivided the study area into anopheline mosquito sampling units where mosquitoes were collected monthly. Plasmodium-infected specimens of Ny. darlingi were detected in each of the 5 landscapes that were determined by a cluster analysis. A putative secondary vector, Anopheles near costai G1, was infected with Plasmodium vivax and Plasmodium malariae, reported herein for the first time in Peru. Overall, the most pathogenic landscape was degraded forest