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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.

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.

The landscape of malaria transmission in the Peruvian Amazon is temporally and spatially heterogeneous, presenting different micro-geographies with particular epidemiologies. Most cases are asymptomatic and escape routine malaria surveillance based on light microscopy (LM). Following the implementation of control programs in this region, new approaches to stratify transmission and direct efforts at an individual and community level are needed. Antibody responses to serological exposure markers (SEM) to Plasmodium vivax have proven diagnostic performance to identify people exposed in the previous 9 months. We measured antibody responses against 8 SEM to identify recently exposed people and determine the transmission dynamics of P. vivax in peri-urban (Iquitos) and riverine (Mazán) communities of Loreto, communities that have seen significant recent reductions in malaria transmission. Socio-demographic, geo-reference, LM and qPCR diagnosis data were collected from two cross-sectional surveys. Spatial and multilevel analyses were implemented to describe the distribution of seropositive cases and the risk factors associated with exposure to P. vivax. Low local transmission was detected by qPCR in both Iquitos (5.3%) and Mazán (2.7%); however, seroprevalence indicated a higher level of (past) exposure to P. vivax in Mazán (56.5%) than Iquitos (38.2%). Age and being male were factors associated with high odds of being seropositive in both sites. Higher antibody levels were found in individuals >15 years old. The persistence of long-lived antibodies in these individuals could overestimate the detection of recent exposure. Antibody levels in younger populations (<15 years old) could be a better indicator of recent exposure to P. vivax. The large number of current and past infections detected by SEMs allows for detailed local epidemiological analyses, in contrast to data from qPCR prevalence surveys which did not produce statistically significant associations. Serological surveillance will be increasingly important in the Peruvian Amazon as malaria transmission is reduced by continued control and elimination efforts.

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.

In the Peruvian Amazon, Plasmodium falciparum and Plasmodium vivax malaria are endemic in rural areas, where microscopy is not available. Malaria rapid diagnostic tests (RDTs) provide quick and accurate diagnosis. However, pfhrp2 gene deletions may limit the use of histidine-rich protein-2 (PfHRP2) detecting RDTs. Further, cross-reactions of P. falciparum with P. vivax-specific test lines and vice versa may impair diagnostic specificity. Thirteen RDT products were evaluated on 179 prospectively collected malaria positive samples. Species diagnosis was performed by microscopy and confirmed by PCR. Pfhrp2 gene deletions were assessed by PCR. Sensitivity for P. falciparum diagnosis was lower for PfHRP2 compared to P. falciparum-specific Plasmodium lactate dehydrogenase (Pf-pLDH)-detecting RDTs (71.6% vs. 98.7%, p<0.001). Most (19/21) false negative PfHRP2 results were associated with pfhrp2 gene deletions (25.7% of 74 P. falciparum samples). Diagnostic sensitivity for P. vivax (101 samples) was excellent, except for two products. In 10/12 P. vivax-detecting RDT products, cross-reactions with the PfHRP2 or Pf-pLDH line occurred at a median frequency of 2.5% (range 0%-10.9%) of P. vivax samples assessed. In two RDT products, two and one P. falciparum samples respectively cross-reacted with the Pv-pLDH line. Two Pf-pLDH/pan-pLDH-detecting RDTs showed excellent sensitivity with few (1.0%) cross-reactions but showed faint Pf-pLDH lines in 24.7% and 38.9% of P. falciparum samples. PfHRP2-detecting RDTs are not suitable in the Peruvian Amazon due to pfhrp2 gene deletions. Two Pf-pLDH-detecting RDTs performed excellently and are promising RDTs for this region although faint test lines are of concern.

Malaria rapid diagnostic tests (RDTs) offer significant potential to improve the diagnosis of malaria, and are playing an increasing role in malaria case management, control and elimination. Peru, along with other South American countries, is moving to introduce malaria RDTs as components of malaria control programmes supported by the Global Fund for AIDS, TB and malaria. The selection of the most suitable malaria RDTs is critical to the success of the programmes. Eight of nine microscopy positive P. falciparum samples collected in Iquitos, Peru tested negative or weak positive using HRP2-detecting RDTs. These samples were tested for the presence of pfhrp2 and pfhrp3 and their flanking genes by PCR, as well as the presence of HRP proteins by ELISA. To investigate for geographic extent of HRP-deleted parasites and their temporal occurrence a retrospective study was undertaken on 148 microscopy positive P. falciparum samples collected in different areas of the Amazon region of Peru. Eight of the nine isolates lacked the pfhrp2 and/or pfhrp3 genes and one or both flanking genes, and the absence of HRP was confirmed by ELISA. The retrospective study showed that 61 (41%) and 103 (70%) of the 148 samples lacked the pfhrp2 or pfhrp3 genes respectively, with 32 (21.6%) samples lacking both hrp genes. This is the first documentation of P. falciparum field isolates lacking pfhrp2 and/or pfhrp3. The high frequency and wide distribution of different parasites lacking pfhrp2 and/or pfhrp3 in widely dispersed areas in the Peruvian Amazon implies that malaria RDTs targeting HRP2 will fail to detect a high proportion of P. falciparum in malaria-endemic areas of Peru and should not be used. RDTs detecting parasite LDH or aldolase and quality microscopy should be use for malaria diagnosis in this region. There is an urgent need for investigation of the abundance and geographic distribution of these parasites in Peru and neighbouring countries.

Interest in larval source management (LSM) as an adjunct intervention to control and eliminate malaria transmission has recently increased mainly because long-lasting insecticidal nets (LLINs) and indoor residual spray (IRS) are ineffective against exophagic and exophilic mosquitoes. In Amazonian Peru, the identification of the most productive, positive water bodies would increase the impact of targeted mosquito control on aquatic life stages. The present study explores the use of unmanned aerial vehicles (drones) for identifying Nyssorhynchus darlingi (formerly Anopheles darlingi) breeding sites with high-resolution imagery (~0.02m/pixel) and their multispectral profile in Amazonian Peru. Our results show that high-resolution multispectral imagery can discriminate a profile of water bodies where Ny. darlingi is most likely to breed (overall accuracy 86.73%- 96.98%) with a moderate differentiation of spectral bands. This work provides proof-of-concept of the use of high-resolution images to detect malaria vector breeding sites in Amazonian Peru and such innovative methodology could be crucial for LSM malaria integrated interventions.

Background Rapid diagnostic tests (RDTs) are today the most widely used method for malaria diagnosis and are recommended, alongside microscopy, for the confirmation of suspected cases before the administration of anti-malarial treatment. The diagnostic performance of RDTs, as compared to microscopy or PCR is well described but the actual analytical sensitivity of current best-in-class tests is poorly documented. This value is however a key performance indicator and a benchmark value needed to developed new RDTs of improved sensitivity. Methods Thirteen RDTs detecting either the Plasmodium falciparum histidine rich protein 2 (HRP2) or the plasmodial lactate dehydrogenase (pLDH) antigens were selected from the best performing RDTs according to the WHO–FIND product testing programme. The analytical sensitivity of these products was evaluated using a range of reference materials including P. falciparum and Plasmodium vivax whole parasite samples as well as recombinant proteins. Results The best performing HRP2-based RDTs could detect all P. falciparum cultured samples at concentrations as low as 0.8 ng/mL of HRP2. The limit of detection of the best performing pLDH-based RDT specifically detecting P. vivax was 25 ng/mL of pLDH. Conclusion The analytical sensitivity of P. vivax and Pan pLDH-based RDTs appears to vary considerably from product to product, and improvement of the limit-of-detection for P. vivax detecting RDTs is needed to match the performance of HRP2 and Pf pLDH-based RDTs for P. falciparum . Different assays using different reference materials produce different values for antigen concentration in a given specimen, highlighting the need to establish universal reference assays.

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. 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. 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. 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.

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. 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. 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.