Biology subjects

Volkman, S. K.

Publications and source records attributed to Volkman, S. K..

11 recordsLinked to original sources

bioRxiv2026

Genetic background determines allele-specific Pfkelch13-mediated artemisinin tolerance and persistence in Plasmodium falciparum

Artemisinin-based combination therapies are the frontline treatment for Plasmodium falciparum malaria, but their efficacy is threatened by Artemisinin partial resistance (ARTR). ARTR in Plasmodium falciparum is mediated by Pfkelch13 (k13) mutations but remains rare in West Africa. A critical question is whether short-term drug survival intrinsically predicts long-term evolutionary persistence. Here, using CRISPR-Cas9 editing, and nanopore-assisted allele tracking in a contemporary Senegalese isolate, we evaluated ARTR receptivity using the ring-stage survival assay (RSA), a proxy for clinical resistance. We demonstrate that ARTR survival and drug-free persistence are biologically separable traits. While the WHO-validated Asian I543T mutation conferred the RSA phenotype, it was rapidly counter-selected during drug-free growth. In contrast, emerging African alleles (R561H, M579I) and a regional novel variant (C473S) establish stable resistance without a competitive penalty. These findings reveal that a parasite's genetic background acts as a strict evolutionary filter, uncoupling short-term tolerance from long-term persistence and establishing that genomic context determines allele-specific resistance trajectories.

microbiology↗

bioRxiv2025

Phenotypic assessment and genetic validation of Plasmodium falciparum molecular markers associated with malaria chemoprevention in Senegal

Drug resistance in Plasmodium falciparum threatens to undermine malaria control and elimination efforts. Senegal is a malaria-endemic country that has implemented successive antimalarial and chemopreventive drug-based strategies for two decades. Sulfadoxine-pyrimethamine (SP) is used for chemoprevention in Senegal for intermittent preventive treatment in pregnancy (since 2004) and SP plus amodiaquine (AQ) is used for seasonal malaria chemoprevention (SMC, since 2013). Using whole genome sequence (WGS) data from malaria patient samples from health facilities across Senegal (2006 - 2022), we observed near fixation of Pfdhfr triple mutant and fluctuation in Pfdhps and Pfcrt mutation frequencies over time. It is unclear how these mutations influence drug resistance and fitness phenotypes in natural isolates; therefore, we evaluated natural parasite isolates with different Pfcrt, Pfmdr1, Pfdhps, and Pfdhfr haplotypes. Parasites were culture-adapted and phenotyped for antimalarial drug susceptibility and competitive growth (fitness). Pfcrt CVIET + A220S + Q271E + N326S + R371I and Pfcrt CVIET + A220S + Q271E + I356T + R371I mutants were significantly more resistant to monodesethyl-amodiaquine (md-AQ) compared to Pfcrt wild-type (WT) and Pfcrt CVIET + A220S + Q271E + R371I mutants. Pfdhfr triple mutants were significantly more pyrimethamine (PYR) resistant than Pfdhfr WT and revealed a range of phenotypes, but this was not explained by Pfgch1 copy-number. Pfdhps A437G parasites were significantly more sulfadoxine (SDX) resistant compared to Pfdhps wild-type and Pfdhps S436A mutants, suggesting that A437G is a key mutation for SDX resistance. Competitive growth assays between Pfdhfr-Pfdhps mutants revealed that Pfdhps mutations do not always result in fitness costs. Ongoing phenotypic assessment and genetic validation of these mutations in a Senegalese background is necessary to assess the impact of drug pressure, identify evolving genetic determinants of drug resistance, and provide molecular markers for ongoing surveillance to monitor and guide the use of drug-based interventions. AUTHOR SUMMARYDrug resistance is a major concern for both preventing and treating malaria, especially in Africa where most malaria cases and deaths occur. Since 2013, Senegal has been giving children under 10 years old a combination of sulfadoxine-pyrimethamine plus amodiaquine to prevent malaria during the transmission season, called Seasonal Malaria Chemoprevention (SMC), and plans to continue expanding its use. However, there is evidence from genetic surveillance that drug resistance mutations are present in Senegal which could render this antimalarial drug combination ineffective. Here we use natural P. falciparum isolates obtained from Senegalese patients that represent the extant parasite population to evaluate the consequences of evolving mutations on antimalarial drug resistance and fitness phenotypes. This study is one of the first to use natural parasites to assess the impact of naturally derived mutations on drug resistance and fitness phenotypes. Our results provide evidence that certain combinations of drug resistance mutations impact both parasite drug resistance and fitness, and therefore need to be closely monitored and can inform optimal antimalarial combinations for the prevention or treatment of malaria. This work informs the ongoing evolution of resistance and fitness phenotypes in malaria endemic settings that are introducing new multi first line therapies (MFTs) and SMC interventions that have been used for decades in Senegal. Our approach creates a framework for using genetic surveillance data to form a hypothesis, which can then be phenotypically tested by measuring the resistance and fitness levels of genetically diverse natural parasite isolates.

molecular biology↗

bioRxiv2025

Cellular and molecular basis of PfCoronin function in artemisinin resistance in Plasmodium falciparum

Artemisinin-based therapies are central to malaria treatment, but their efficacy is threatened by the emergence of resistant Plasmodium falciparum strains. While the role of Pfkelch13 mutations in clinical resistance is well established, recent reports from Africa implicate Pfcoronin mutations in treatment failure, adding to the complexity of resistance mechanisms. Here, we show that PfCoronin, a non-essential actin regulator active during the ring-stage, facilitates efficient hemoglobin uptake. We demonstrate that resistance-associated Pfcoronin mutations disrupt PfCoronins interaction with PfActin and its ring-stage localization, leading to impaired endocytosis and reduced hemoglobin acquisition. PfCoronin and PfKelch13 function in distinct cellular regions; mutations in both converge to limit heme availability and artemisinin activation. Although PfCoronin is dispensable for parasite viability, our findings demonstrate that Pfcoronin mutations reduce endocytosis and modulate artemisinin susceptibility during the clinically relevant ring stage--highlighting how non-essential, temporally restricted proteins can shape antimalarial drug response and resistance.

cell biology↗

bioRxiv2024

MalKinID: A Likelihood-Based Model for Identifying Malaria Parasite Genealogical Relationships Using Identity-by-Descent

Pathogen genomics is a powerful tool for tracking infectious disease transmission. In malaria, identity-by-descent (IBD) is used to assess the genetic relatedness between parasites and has been used to study transmission and importation. In theory, IBD can be used to distinguish genealogical relationships to reconstruct transmission history or identify parasites for genotype- to-phenotype quantitative-trait-locus experiments. MalKinID (Malaria Kinship Identifier) is a new likelihood-based classification model designed to identify genealogical relationships among malaria parasites based on genome-wide IBD proportions and IBD segment distributions. MalKinID was calibrated to the genomic data from three laboratory-based genetic crosses (yielding 440 parent-child and 9060 full-sibling comparisons). MalKinID identified lab generated F1 progeny with >80% sensitivity and showed that 0.39 (95% CI 0.28, 0.49) of the second- generation progeny of a NF54 and NHP4026 cross were F1s and 0.56 (0.45, 0.67) were backcrosses of an F1 with the parental NF54 strain. In simulated outcrossed importations, MalKinID accurately reconstructs genealogy history with high precision and sensitivity, with F1- scores exceeding 0.84. However, when importation involves inbreeding, such as during serial co-transmission, the precision and sensitivity of MalKinID declined, with F1-scores of 0.76 (0.56, 0.92) and 0.23 (0.0, 0.4) for PC and FS and <0.05 for second-degree and third-degree relatives. Genealogical inference is most powered 1) when outcrossing is the norm or 2) when multi- sample comparisons based on a predefined pedigree are used. MalKinID lays the foundations for using IBD to track parasite transmission history and for separating progeny for quantitative- trait-locus experiments.

genetics↗

bioRxiv2023

Artemisinin resistance mutations in Pfcoronin impede hemoglobin uptake

Artemisinin (ART) combination therapies have been critical in reducing malaria morbidity and mortality, but these important drugs are threatened by growing resistance associated with mutations in Pfcoronin and Pfkelch13. Here, we describe the mechanism of Pfcoronin-mediated ART resistance. PfCoronin interacts with PfActin and localizes to the parasite plasma membrane (PPM), the digestive vacuole (DV) membrane, and membrane of a newly identified preDV compartment--all structures involved in the trafficking of hemoglobin from the RBC for degradation in the DV. Pfcoronin mutations alter PfActin homeostasis and impair the development and morphology of the preDV. Ultimately, these changes are associated with decreased uptake of red blood cell cytosolic contents by ring-stage Plasmodium falciparum. Previous work has identified decreased hemoglobin uptake as the mechanism of Pfkelch13-mediated ART resistance. This work demonstrates that PfCoronin appears to act via a parallel pathway. For both Pfkelch13-mediated and Pfcoronin-mediated ART resistance, we hypothesize that the decreased hemoglobin uptake in ring stage parasites results in less heme-based activation of the artemisinin endoperoxide ring and reduced cytocidal activity. This study deepens our understanding of ART resistance, as well as hemoglobin uptake and development of the DV in early-stage parasites.

cell biology↗

bioRxiv2021

Antimalarials in mosquitoes overcome Anopheles and Plasmodium resistance to malaria control strategies

The spread of insecticide resistance in Anopheles mosquitoes and drug resistance in Plasmodium parasites is contributing to a global resurgence of malaria, making the generation of control tools that can overcome these issues an urgent public health priority. We recently showed that the transmission of Plasmodium falciparum parasites can be efficiently blocked when exposing Anopheles gambiae females to antimalarials deposited on a treated surface, with no negative consequences on mosquito fitness. Here, we demonstrate this approach can overcome the hurdles of insecticide resistance in mosquitoes and drug resistant in parasites. We show that the transmission-blocking efficacy of mosquito-targeted antimalarials is maintained when field-derived, insecticide resistant Anopheles are exposed to the potent cytochrome b inhibitor atovaquone, demonstrating that this drug escapes insecticide resistance mechanisms that could potentially interfere with its function. Moreover, this approach prevents transmission of field-derived, artemisinin resistant P. falciparum parasites (Kelch13 C580Y mutant), proving that this strategy could be used to prevent the spread of parasite mutations that induce resistance to front-line antimalarials. Atovaquone is also highly effective at limiting parasite development when ingested by mosquitoes in sugar solutions, including in ongoing infections. These data support the use of mosquito-targeted antimalarials as a promising tool to complement and extend the efficacy of current malaria control interventions. Significance StatementEffective control of malaria is hampered by resistance to vector-targeted insecticides and parasite-targeted drugs. This situation is exacerbated by a critical lack of chemical diversity in both interventions and, as such, new interventions are badly needed. Recent laboratory studies have shown that an alternative approach based on treating Anopheles mosquitoes directly with antimalarial compounds can render the vector incapable of transmitting the Plasmodium parasites that cause malaria. While promising, showing that mosquito-targeted antimalarials remain effective against wild parasites and mosquitoes, including drug- and insecticide-resistant populations, respectively, is crucial to the future viability of this approach. In this study, carried out in the US and Burkina Faso, we show that antimalarial exposure is highly effective, even against extremely resistant mosquitoes, and can block transmission of drug-resistant parasites. By combining lab, and field-based studies in this way we have demonstrated that this novel approach can be effective in areas where conventional control measures are no longer as effective.

microbiology↗

bioRxiv2020

Population Genomics of Plasmodium vivax in Panama to Assess the Risk of Case Importation on Malaria Elimination

Malaria incidence in Panama has plateaued in recent years in spite of elimination efforts, with almost all cases caused by Plasmodium vivax. Notwithstanding, overall malaria prevalence remains low (fewer than 1 case per 1000 persons). We used selective whole genome amplification to sequence 96 P. vivax samples from Panama collected between 2007 and 2019 to study the population structure and transmission dynamics of the parasite. Imported cases resulting from increased levels of human migration could threaten malaria elimination prospects, and four of the samples evaluated came from individuals with travel history. We explored patterns of recent common ancestry among the samples and observed that a single highly genetically related lineage was dominant among the samples (47 out of 59 samples with good sequencing coverage), spanning the entire period of the collection (2007-2019) and all regions of the country. We also found a second, smaller clonal lineage of four parasites collected between 2017 and 2019. To explore the regional context of Panamanian P. vivax we conducted principal components analysis and constructed a neighbor-joining tree using these samples and samples collected worldwide from a previous study. Three of the four samples with travel history clustered with samples collected from their suspected country of origin (consistent with importation), while one appears to have been a result of local transmission. The small number of genetically unique Panamanian P. vivax samples clustered with samples collected from Colombia, suggesting they represent the genetically similar ancestral P. vivax population in Panama or were recently imported from Colombia. The low diversity we observe in Panama indicates that this parasite population has been previously subject to a severe bottleneck and may be eligible for elimination. Additionally, while we confirmed that P. vivax is imported to Panama from diverse geographic locations, the lack of impact from imported cases on the overall parasite population genomic profile suggests that onward transmission from such cases is limited and that imported cases may not presently pose a major barrier to elimination. Author SummaryPanama has greatly reduced P. vivax incidence, however, this progress has plateaued. Understanding parasite transmission patterns and identifying imported cases is critical to help Panama and other countries succeed in their elimination efforts. Genomic epidemiology and population genomics can help provide information needed to inform malaria control policy. In this study, we collected 100 Panamanian P. vivax samples from two collection periods (2007-2009 and 2017-2019), of which 59 yielded usable sequencing data. 4 samples had patient travel history data associated with them. We found that the majority of samples belong to a single highly related lineage, termed CL1. This lineage has persisted since at least 2007. We also highlight how genomic epidemiology can be used to spotlight parasites that may be imported as a result of human migration, as well as corroborate or refute the country of origin as suggested by patient travel history. We observe no evidence of outcrossing between these potentially imported parasites and the local Panamanian parasite population, suggesting that imported parasites are not driving ongoing malaria transmission in Panama. The low diversity we observe in Panama indicates that this parasite population has been previously subject to a severe bottleneck and may be eligible for elimination.

microbiology↗

bioRxiv2020

Plasmodium falciparum population genetic complexity influences transcriptional profile and immune recognition of highly related genotypic clusters

As transmission intensity has declined in Senegal, so has the genetic complexity of circulating Plasmodium falciparum parasites, resulting in specific genotypes emerging and persisting over years. We address whether changes in parasite genetic signatures can alter the immune repertoire to variant surface antigens, and whether such responses can influence the expansion or contraction of specific parasite genotypes in the population. We characterize parasites within genotypic clusters, defined as identical by a 24-SNP molecular barcode and a haplotype identifier for other highly polymorphic loci; we measure expression of variant surface antigens (VSA) such as PfEMP-1 by transcript expression typing and expressed var DBL1 sequencing in ex vivo and short-term adapted RNA samples; and we measure IgG responses against VSAs from short-term adapted parasites. We find that parasites within genotypic clusters are genetically identical at other highly polymorphic loci. These parasites express similar Ups var classes and largely the same dominant var DBL1 sequences ex vivo. These parasites are recognized similarly by anti-VSA antibodies after short-term adaptation to culture; however, antibody responses do not correlate with genotype frequencies over time. Both genotype-specific and multiple genotype-reactive surface IgG responses are observed in this population. Parasites with identical genomes are extremely similar in their expression and host antibody recognition of VSAs. Monitoring changes in population-level parasite genomics and transmission dynamics is critical, as fluctuations will influence the breadth of resulting host immune responses to circulating parasite genotypes. These findings suggest shared immune recognition of genetically similar parasites, which has implications for both our understanding of immunity and vaccine development strategies in malaria elimination settings.

microbiology↗

bioRxiv2018

Amplicon deep sequencing of low-density Plasmodium falciparum infections: an evaluation of analysis approaches

BackgroundDeep sequencing of targeted genomic regions is becoming a common tool for understanding the dynamics and complexity of Plasmodium infections, but its lower limit of detection is currently unknown. Here, a new amplicon analysis tool, the Parallel Amplicon Sequencing Error Correction (PASEC) pipeline, is used to evaluate the performance of amplicon sequencing on low-density Plasmodium DNA samples. Illumina-based sequencing of two P. falciparum genomic regions (CSP and SERA2) was performed on two types of samples: in vitro DNA mixtures mimicking low-density infections (1-200 genomes/l) and extracted blood spots from a combination of symptomatic and asymptomatic individuals (44-653,080 parasites/l). Three additional analysis tools--DADA2, HaplotypR, and SeekDeep--were applied to both datasets and the precision and sensitivity of each tool were evaluated. ResultsAmplicon sequencing can contend with low-density samples, showing reasonable detection accuracy down to a concentration of 5 Plasmodium genomes/l. Due to increased stochasticity and background noise, however, all four tools showed reduced sensitivity and precision on samples with very low parasitemia (<5 copies/l) or low read count (<100 reads per amplicon). PASEC could distinguish major from minor haplotypes with an accuracy of 90% in samples with at least 30 Plasmodium genomes/l, but only 61% at low Plasmodium concentrations (<5 genomes/l) and 46% at very low read counts (<25 reads per amplicon). The four tools were additionally used on a panel of extracted parasite-positive blood spots from natural malaria infections. While all four identified concordant patterns of complexity of infection (COI) across four sub-Saharan African countries, the COI values obtained for individual samples differed in some cases. ConclusionsAmplicon deep sequencing can be used to determine the complexity and diversity of low-density Plasmodium infections. Despite differences in their approach, four state-of-the-art tools resolved known haplotype mixtures with similar sensitivity and precision. Researchers can therefore choose from multiple robust approaches for analyzing amplicon data, however, error filtration approaches should not be uniformly applied across samples of varying parasitemia. Samples with very low parasitemia and very low read count have higher false positive rates and call for read count thresholds that are higher than current recommendations.

microbiology↗

bioRxiv2017

De Novo Mutations Resolve Disease Transmission Pathways in Clonal Malaria

Detecting de novo mutations in viral and bacterial pathogens enables researchers to reconstruct detailed networks of disease transmission and is a key technique in genomic epidemiology. However these techniques have not yet been applied to the malaria parasite, Plasmodium falciparum, in which a larger genome, slower generation times, and a complex life cycle make them difficult to implement. Here we demonstrate the viability of de novo mutation studies in P. falciparum for the first time. Using a set of clinical samples and novel methods of sequencing, library preparation, and genotyping, we have genotyped low-complexity regions of the genome with a high degree of accuracy. Despite its slower evolutionary rate compared to bacterial or viral species, de novo mutation can be detected in P. falciparum across timescales of just 1-2 years and evolutionary rates in low-complexity regions of the genome can be up to twice that detected in the rest of the genome. The increased mutation rate allows the identification of separate clade expansions that cannot be found using previous genomic epidemiology approaches and could be a crucial tool for mapping residual transmission patterns in disease elimination campaigns and reintroduction scenarios.

genomics↗

bioRxiv2017

Within-infection diversity of Plasmodium falciparum antigens reflects host-mediated selection

Host immunity exerts strong selection on pathogens, but it does not act in a uniform manner across individual hosts. By providing a direct approach for understanding host-specific selection pressures, patterns of intra-host pathogen diversity complement population genetic analyses performed on broad geographic scales. Here, we perform a combined analysis of inter- and intra-host diversity for the malaria parasite Plasmodium falciparum with haplotype sequences of three antigens sampled from over 4,500 natural infections in sub-Saharan Africa using targeted deep sequencing. We find that multi-strain infections in young children contain non-random combinations of parasite genotypes, and identify individual amino acid positions that may contribute to strain-specific blocking of infections. These results demonstrate for the first time that natural host defenses to Plasmodium detectably impact which infections proceed to the blood stage within a given host. This selection partially explains the extreme amino acid diversity observed at many parasite antigens and suggests that vaccines targeting such proteins should account for the impact of allele-specific immunity.

evolutionary biology↗