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

Kattenberg, Johanna Helena; Fernández-Miñope, Carlos; Dijk, Norbert J. van; Allcca, Lidia Llacsahuanga; Guetens, Pieter; Valdivia, Hugo O.; Geertruyden, Jean-Pierre Van; Rovira-Vallbona, Eduard; Monsieurs, Pieter; Delgado-Ratto, Christopher; Gamboa, Dionicia; Rosanas-Urgell, Anna

doi:10.1128/spectrum.00960-22

Cited by 23 publications

(65 citation statements)

References 127 publications

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“…To design a SNP barcode with in-country resolution in Peru, raw whole genome sequencing data (Fast Q files) of P. vivax isolates from Peru generated in-house (n=30 from [23, 24]) was combined with online available Peruvian P. vivax genomes (n=100 from [25–28]) and jointly genotyped after variant calling as described elsewhere [17]. Briefly, FASTQ files were aligned to the PvP01 reference genome version 46 from PlasmoDB [29] and variants were called using GATK4 HaplotypeCaller.…”

Section: Methodsmentioning

confidence: 99%

“…Previously, we have developed a highly-multiplexed amplicon sequencing (AmpliSeq) assay for malaria genomic surveillance of P. falciparum in Peru (Pf AmpliSeq Peru, [22]) and P. vivax in Vietnam (Pv AmpliSeq v1 Vietnam, [23]). Our assays include validated and candidate genes associated with antimalarial resistance, SNP-barcodes to serve different functions, i.e.…”

Section: Introductionmentioning

confidence: 99%

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Plasmodium vivaxgenomic surveillance in the Peruvian Amazon with Pv AmpliSeq assay

Kattenberg,

Cabrera-Sosa,

Figueroa-Idelfonso

et al. 2023

Preprint

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BackgroundPlasmodium vivaxis 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 FindingsHere, 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 heterogenousP. vivaxpopulation with high diversity and gene flow in peri-urban areas of Maynas province (Loreto region) with a temporal drift. 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 clonalP. vivaxcluster was identified, with distinct haplotypes in drug resistant genes andama1, more similar to Brazilian isolates, likely representing an introduction ofP. vivaxfrom Brazil at that time. To test its applicability for Latin America, we evaluated the SNP Peru barcode inP. vivaxgenomes from the region and demonstrated the capacity to capture local population clustering at within-country level.Conclusions/SignificanceTogether this data shows thatP. vivaxtransmission 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.Author SummaryLatin America is aiming towards malaria elimination. Genomic surveillance is crucial for a country’s malaria strategy, aiding in understanding and stopping the spread of the disease. While widely used for another malaria species (Plasmodium falciparum), limited tools exist for trackingP. vivax, a significant player in malaria-endemic areas outside of Africa, and the primary cause of malaria in Latin America.In this study, we used a new tool, Pv AmpliSeq v2 Peru assay, to examine the genetic makeup of malaria parasites in the Peruvian Amazon. This tool helps us see how the parasites from different areas are connected and tracks markers that could indicate resistance to drugs. We found that the parasites from remote areas in the Amazon were genetically different from parasites in areas surrounding the main city of Iquitos, and parasites in a remote border community were genetically more similar to Brazilian parasites.We also show that the Pv AmpliSeq v2 Peru assay can be used to study parasites from other countries in Latin America, highlighting the broader application in the region. Considering that parasites are not constrained by borders and can easily spread between neighboring countries, a regional approach can be crucial for malaria elimination.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Plasmodium vivaxgenomic surveillance in the Peruvian Amazon with Pv AmpliSeq assay

Kattenberg,

Cabrera-Sosa,

Figueroa-Idelfonso

et al. 2023

Preprint

Self Cite

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“…However, for pathogens with larger genomes such as the malaria parasite, Plasmodium falciparum 2 , targeted genotyping approaches are more affordable and scalable. Panels of single nucleotide polymorphisms (SNPs) referred as "SNP barcodes" have been increasingly used for malaria genomic surveillance [3][4][5][6][7][8] . For malaria genomic surveillance to reach its full potential however, assessment of the capabilities and resolution of these genotyping tools is essential 9 .…”

Section: Mainmentioning

confidence: 99%

“…Alternative approaches are urgently needed to estimate genome-wide relatedness and define population structure at a similar resolution to WGS but for a fraction of the cost. Previous approaches have included panels of 10-14 polymorphic microsatellites [10], or barcodes comprising 24 [29], 28 [3], 54 [30] or 101 single nucleotide polymorphisms (SNPs) [6]. However, these genotyping tools lacked the required accuracy or spatial resolution to define local parasite populations, especially where the barcodes contained too few SNPs to accurately capture genome-wide relatedness [16,[31][32][33].…”

Section: Introductionmentioning

confidence: 99%

Plasmodium falciparumpopulations, transmission dynamics and infection origins across Papua New Guinea

Harrison,

Mehra,

Razook

et al. 2023

Preprint

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The successful implementation of pathogen genomic surveillance demands rapid, low-cost genotyping solutions for tracking infections. Here we demonstrate the capacity of single nucleotide polymorphism (SNP) barcodes to generate practical information for malaria surveillance and control. The study was conducted in Papua New Guinea (PNG), a country with a wide range of malaria transmission intensities. A panel of 191 candidate SNPs was selected from 5786 SNPs with minor allele frequency greater than 0.1, identified amongst 91Plasmodium falciparumgenomes from three provinces of PNG. We then genotyped 772P. falciparumisolates from a 2008 nationwide malaria indicator survey and 31 clinical infections from an outbreak of unknown origin. We assessed the performance of SNP panels with different allele frequency characteristics, and measured population diversity, structure and connectivity using both whole genome data and the SNP barcode. The full SNP barcode captured similar patterns of population structure evident with 5786 ‘whole genome’ SNPs. Geographically informative SNPs (iSNPs,FST>0.05) show increased population clustering compared to the full barcode whilst randomly selected SNPs (rSNPs) and SNPs with similar allele frequencies (FST<0.05) amongst different countries (universal, uSNPs) or local PNG populations (balanced, bSNPs) indicated little clustering. Applied to samples from all endemic areas of PNG, this barcode identified variable transmission dynamics, and eight major populations. Genetic diversity was high throughout most areas, however, in the southern region, isolates were either closely related, suggesting highly inbred or near-clonal populations; or, they shared ancestry with other parasite populations, consistent with importation. Applied to outbreak samples, only the full barcode, the iSNPs and bSNPs distinguished between locally acquired and imported infections. The full barcode contains more than 100 SNPs prevalent in other endemic regions, allowing the transfer of this tool to other settings. SNP barcodes must be validated in local settings to ensure they capture the diversity and population structure of the target population. Subsets of geographically informative SNPs will be essential for predicting geographic origins but may bias analyses of population structure and gene flow if used alone.AUTHOR SUMMARYPathogen genomic surveillance is a sensitive approach for mapping pathogen transmission dynamics to support decisions about how to prevent and control infectious diseases. High throughput genotyping tools known as single nucleotide polymorphism (SNP) barcodes are used to measure relationships between individuals and connectivity between populations, however, the barcode design may influence these results. We used whole genome sequences from the malaria parasitePlasmodium falciparumto design a barcode that captures the diversity both within and between parasite populations of Papua New Guinea (PNG), where transmission is variable amongst provinces. By investigating the performance of different panels of SNPs, we show that validation for use in the target population is crucial to correctly identifying population genetic structure. Applying the validated SNP barcode on hundreds of samples from all endemic provinces of PNG, high levels of variability in local transmission dynamics, and regions of population subdivision were observed. Some geographic areas show evidence of interrupted transmission, and the substantial genetic differentiation between the northern, eastern and island populations, presents an opportunity to design targeted, subnational control efforts. Application of the barcode to outbreak samples classified cases into imported and locally acquired infections, with substantial local transmission indicating control efforts were not sufficient to prevent the spread of infections. SNP barcodes are useful tools that can be used to supplement existing malaria surveillance tools however careful validation of their effectiveness in different settings is recommended.

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“…2). Genetic loci were selected according to a 24 SNP panel [20], a 101 SNP panel [21], and 3 recently developed amplicon sequencing panels consisting of 128 [13], 165 [22], and 233 [14] diverse microhaplotypes respectively. These simulations were done at moderate false positive and false negative rates of .01 and .1 respectively.…”

Section: Estimation Of Multiplicity Of Infection Within-host Relatedn...mentioning

confidence: 99%

MOIRE: A software package for the estimation of allele frequencies and effective multiplicity of infection from polyallelic data

Murphy,

Greenhouse

2023

Preprint

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Malaria parasite genetic data can provide insight into parasite phenotypes, evolution, and transmission. However, estimating key parameters such as allele frequencies, multiplicity of infection (MOI), and within-host relatedness from genetic data has been challenging, particularly in the presence of multiple related coinfecting strains. Existing methods often rely on single nucleotide polymorphism (SNP) data and do not account for within-host relatedness. In this study, we introduce a Bayesian approach called MOIRE (Multiplicity Of Infection and allele frequency REcovery), designed to estimate allele frequencies, MOI, and within-host relatedness from genetic data subject to experimental error. Importantly, MOIRE is flexible in accommodating both polyallelic and SNP data, making it adaptable to diverse genotyping panels. We also introduce a novel metric, the effective MOI (eMOI), which integrates MOI and within-host relatedness, providing a robust and interpretable measure of genetic diversity. Using extensive simulations and real-world data from a malaria study in Namibia, we demonstrate the superior performance of MOIRE over naive estimation methods, accurately estimating MOI up to 7 with moderate sized panels of diverse loci (e.g. microhaplotypes). MOIRE also revealed substantial heterogeneity in population mean MOI and mean relatedness across health districts in Namibia, suggesting detectable differences in transmission dynamics. Notably, eMOI emerges as a portable metric of within-host diversity, facilitating meaningful comparisons across settings, even when allele frequencies or genotyping panels are different. MOIRE represents an important addition to the analysis toolkit for malaria population dynamics. Compared to existing software, MOIRE enhances the accuracy of parameter estimation and enables more comprehensive insights into within-host diversity and population structure. Additionally, MOIRE’s adaptability to diverse data sources and potential for future improvements make it a valuable asset for research on malaria and other organisms, such as other eukaryotic pathogens. MOIRE is available as an R package athttps://eppicenter.github.io/moire/.Author SummaryRecent advances in amplicon sequencing have enabled reliable high throughput generation of polyallelic data from malaria causing parasites. However, few methods exist to analyze these information rich genetic markers in the context of multiply infected individuals. To address this gap in analyzing data from polyclonal samples, we have developed MOIRE, an R package implementing a Bayesian approach to simultaneously estimate allele frequencies, multiplicity of infection (MOI), and within-host relatedness from noisy polyallelic data. We also introduce a new metric of within-host diversity, the effective MOI (eMOI), a continuous metric that integrates MOI and within-host relatedness into a single value that can be readily compared. We evaluated our method on simulated data from idealized high diversity genetic loci, as well as on recently developed high diversity amplicon sequencing panels. We also evaluated our method on real world data collected from 4 health districts across Namibia, demonstrating our ability to recover heterogeneity in multiplicity of infection as well as within-host relatedness, suggesting detectable differences in transmission dynamics. MOIRE serves as a new tool to better understand transmission dynamics and provide fundamental quantities such as population allele frequencies for downstream analysis.

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

Cited by 23 publications

References 127 publications

Plasmodium vivaxgenomic surveillance in the Peruvian Amazon with Pv AmpliSeq assay

Plasmodium vivaxgenomic surveillance in the Peruvian Amazon with Pv AmpliSeq assay

Plasmodium falciparumpopulations, transmission dynamics and infection origins across Papua New Guinea

MOIRE: A software package for the estimation of allele frequencies and effective multiplicity of infection from polyallelic data

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