Biology subjects

Rosenthal, P. J.

Publications and source records attributed to Rosenthal, P. J..

13 recordsLinked to original sources

bioRxiv2026

Whole-genome characterization of Plasmodium falciparum population structure and drug resistance across Uganda and neighboring countries

Uganda has one of the world's highest malaria burdens, but parasite population structure and connectivity have not been characterized nationally with whole-genome data. We analyzed 249 monoclonal Plasmodium falciparum genomes from 26 sentinel sites across Uganda in 2023 and compared them with publicly available genomes from five neighboring countries. Genetic diversity was high and differentiation low in Uganda, but identity-by-descent detected limited structure. This was most pronounced in the low-transmission southwest, where parasites shared ancestry and recent relatedness with isolates from western Tanzania. Selection scans identified signals at loci associated with resistance to artemisinins and their partner drugs, and to chloroquine and antifolates. The strongest signal was at PfPX1 where the PIN haplotype (L1222P, M1701I, D1705N) reached 70% prevalence nationwide, but selection was carried by PIN haplotypes that had additionally acquired D384A, largely confined to northern and eastern Uganda. A second substitution at the same codon, D384Y, formed a haplotype shared across the Tanzanian border. PfKelch13 variants were partitioned across multiple backgrounds, consistent with repeated independent emergence, and C469Y was enriched on the PfPX1 D384A-PIN background. These genomes provide a 2023 baseline for tracking potential mediators of drug resistance and support coordinated genomic surveillance across Uganda and its neighbors.

genomics↗

bioRxiv2026

PfPHAST: Plasmodium falciparum Public Health Amplicon Sequencing Tool, a Streamlined Panel for Malaria Genomic Surveillance

Genomic tools can support malaria control policy through surveillance of Plasmodium falciparum populations, tracking antimalarial drug resistance, pfhrp2/3 deletions that compromise rapid diagnostic tests, and selection at the circumsporozoite protein (PfCSP) vaccine target, as well as through molecular correction of therapeutic efficacy studies (TES). Multiplex Amplicons for Drug, Diagnostic, Diversity, and Differentiation Haplotypes using Targeted Resequencing (MAD4HatTeR), a comprehensive amplicon sequencing panel covering up to 276 targets, supports these applications but is tailored to research rather than routine programmatic use. We developed P. falciparum Public Health Amplicon Sequencing Tool (PfPHAST), a 56-target derivative of MAD4HatTeR spanning drug resistance loci, pfhrp2/3 deletion, PfCSP genotyping, non-falciparum species identification, and 20 high-heterozygosity microhaplotype loci for TES classification. We compared PfPHAST and MAD4HatTeR using laboratory strain controls, including two-strain dilution series and a five-strain mixture, across parasite densities of 100 to 10,000 parasites/L. At matched per-target depth, PfPHAST achieved a higher quality-control pass rate than MAD4HatTeR (94.4% versus 90.0%) and distributed reads more evenly across targets. The panels showed comparable recall and precision for drug resistance codons and microhaplotypes, reaching near-complete recall above 40% within-sample allele frequency (WSAF) at all densities, with reduced sensitivity for minor alleles below 10% WSAF at low parasite density in both panels. Observed and expected WSAF correlated strongly for both panels, and both resolved a five-strain polyclonal mixture, including a 5% minor strain. By concentrating sequencing capacity on targets of greatest programmatic relevance, PfPHAST offers a scalable, lower-cost alternative to comprehensive research panels without sacrificing performance on shared targets, complementing MAD4HatTeR for routine molecular malaria surveillance.

genomics↗

bioRxiv2026

A Plasmodium falciparum PX1 haplotype is associated with reduced susceptibility to artemisinin and lumefantrine

Effective control of falciparum malaria depends on the sustained efficacy of frontline antimalarial drugs, particularly artemether-lumefantrine (AL), the most widely used therapy in Africa. However, the emergence of artemisinin partial resistance and reduced lumefantrine susceptibility in eastern Africa threaten malaria control and elimination. Robust genetic markers of decreased susceptibility to lumefantrine remain elusive, and our understanding of artemisinin resistance is incomplete. We report results of a Plasmodium falciparum genetic cross between a drug-sensitive line and a Ugandan strain exhibiting reduced susceptibility to dihydroartemisinin and lumefantrine. Targeted deep sequencing of progeny pools and 460 recombinant progeny clones derived under drug pressures revealed distinct haplotypic signatures. Drug-selection experiments identified genetic polymorphisms in Plasmodium falciparum px1, encoding a phosphoinositide-binding protein, as the strongest correlates of reduced susceptibility to dihydroartemisinin and lumefantrine. The PX1 PIN haplotype (L1222P, M1701I, D1705N) recently discovered in Ugandan parasites was highly enriched following dihydroartemisinin or lumefantrine treatment of pooled mixtures of genetically diverse Ugandan clinical isolates. This haplotype was associated with reduced susceptibility to dihydroartemisinin and lumefantrine, compared to wild-type sequence, in culture-adapted Ugandan P. falciparum lines. These results confirm that PX1 mutations were selected across geographically distinct Ugandan parasite backgrounds. Long-term competitive fitness assays demonstrated that PX1 mutations confer asexual blood-stage parasites with a growth advantage, potentially explaining a rapid rise of PX1 PIN alleles over the last two decades in Uganda. Overall, our data suggest the PX1 PIN haplotype is a robust marker of reduced AL susceptibility in African P. falciparum, enabling surveillance of emerging drug resistance.

microbiology↗

bioRxiv2025

A novel locus associated with decreased susceptibility of Plasmodium falciparum to lumefantrine and dihydroartemisinin has emerged and spread in Uganda

Malaria control in Uganda is threatened by the emergence of artemisinin partial resistance and reduced lumefantrine susceptibility. To identify loci contributing to decreased drug susceptibility, we assessed signatures of selection in Ugandan whole-genome Plasmodium falciparum sequences. Extended shared haplotypes were seen for Kelch13 C469Y and A675V mutations, but the strongest signal of recent selection was centered on a segment of chromosome 7 encoding the phosphoinositide-binding protein (PX1, PF3D7_0720700). A haplotype, represented by three PX1 mutations (L1222P, M1701I, D1705N) and two deletions (designated PIN), was first seen in 2008 and rapidly increased, reaching prevalence >50% in northern Uganda by 2016 and eastern Uganda by 2023. PIN-carrying parasites showed significantly decreased ex vivo susceptibilities to lumefantrine, mefloquine and dihydroartemisinin. A parasite strain in which px1 was disrupted in vitro showed increased susceptibility to the three drugs. Thus, PX1 polymorphisms appear to impact on the susceptibilities of African malaria parasites to key drugs.

genomics↗

bioRxiv2025

Identifying a next-generation antimalarial trioxolane in a landscape of artemisinin partial resistance

For over two decades, artemisinin-based combination therapy (ACT) has been the standard of care for the treatment of uncomplicated falciparum malaria. However, artemisinin partial resistance (ART-R) is now prevalent in Southeast Asia and has emerged in eastern Africa, threatening ACT efficacy. Mechanistically, ART-R results from an endocytosis defect that limits concentrations of host-derived free heme in the parasite digestive vacuole, allowing early ring-stage parasites to survive exposure to the artemisinin component of ACT. The artemisinin-inspired 1,2,4-trioxolane artefenomel exhibits an extended pharmacokinetic exposure profile that predicts efficacy against ART-R parasites. Unfortunately, the development of artefenomel was halted recently after almost a decade of clinical trials. Herein, we describe the discovery of RLA-4735 and its single-enantiomer form RLA-5764, next-generation antimalarial trioxolanes that exhibit excellent in vitro potency against Plasmodium falciparum and single-exposure efficacy in a murine P. berghei model, thus retaining many of the favorable pharmacokinetic and pharmacodynamic properties of artefenomel while markedly improving solubility and development potential. In P. falciparum samples collected from patients in Uganda in 2019 and 2023, ex vivo ring-stage survival assays revealed the emergence of the ART-R phenotype over this timeframe, and furthermore demonstrated markedly superior activity of artefenomel and RLA-4735 as compared to dihydroartemisinin (the active metabolite of artemisinin components of ACTs) against ART-R parasites. Overall, our findings suggest a role for next-generation trioxolanes in addressing ART-R, and present a potent new, artefenomel-adjacent chemotype with good potential to deliver new development candidates. Summary SentenceKlope et. al. described the discovery and in vivo characterization of antimalarial endoperoxides effective against artemisinin-resistant parasites as potential development candidates for uncomplicated, blood-stage malaria.

pharmacology and toxicology↗

bioRxiv2024

Placental Malaria Induces a Unique Placental Methylation Profile Associated with Fetal Growth Restriction

BackgroundFetal growth restriction (FGR) is associated with perinatal death and adverse birth outcomes, as well as long-term complications, including increased childhood morbidity, abnormal neurodevelopment, and cardio-metabolic diseases in adulthood. Placental epigenetic reprogramming associated with FGR may mediate these long-term outcomes. Placental malaria (PM), characterized by sequestration of Plasmodium falciparum-infected erythrocytes in placental intervillous space, is the leading global cause of FGR, but its impact on placental epigenetics is unknown. We hypothesized that placental methylomic profiling would reveal common and distinct mechanistic pathways of non-malarial and PM-associated FGR. ResultsWe analyzed placentas from a US cohort with no malaria exposure (n = 12) and a cohort from eastern Uganda, a region with a high prevalence of malaria (n = 12). From each site, 8 cases of FGR (defined as birth weight <10%ile for gestational age by Intergrowth-21 standard curves) and 4 healthy controls with normal weight were analyzed. PM was diagnosed by placental histopathology. We compared the methylation levels of over 850K CpGs of the placentas using Infinium MethylationEPIC v1 microarray. Non-malarial FGR was associated with 65 differentially methylated CpGs (DMCs), whereas PM-FGR was associated with 133 DMCs, compared to their corresponding controls without FGR. One DMC (cg16389901, located in the promoter region of BMP4) was commonly hypomethylated in both groups. We identified 522 DMCs between non-malarial FGR vs. PM-FGR placentas, which was independent of differing geographic location or cellular composition. ConclusionPlacentas with PM-associated FGR have distinct methylation profiles as compared to placentas with non-malarial FGR, suggesting novel epigenetic reprogramming in response to malaria. Larger cohort studies are needed to determine the distinct long-term health outcomes in PM-associated FGR pregnancies.

genetics↗

bioRxiv2023

Distinct transcriptional profiles of maternal and fetal placental macrophages at term are associated with gravidity

Maternal intervillous monocytes (MIMs) and fetal Hofbauer cells (HBCs) are myeloid-derived immune cells at the maternal-fetal interface. Little is known regarding the molecular phenotypes and roles of these distinct monocyte/macrophage populations. Here, we used RNA sequencing to investigate the transcriptional profiles of MIMs and HBCs in six normal term pregnancies. Our analyses revealed distinct transcriptomes of MIMs and HBCs. Genes involved in differentiation and cell organization pathways were more highly expressed in MIMs vs. HBCs. In contrast, HBCs had higher expression of genes involved in inflammatory responses and cell surface receptor signaling. Maternal gravidity influenced monocyte programming, as expression of pro-inflammatory molecules was significantly higher in MIMs from multigravidas compared to primigravidas. In HBCs, multigravidas displayed enrichment of gene pathways involved in cell-cell signaling and differentiation. In summary, our results demonstrated that MIMs and HBCs have highly divergent transcriptional signatures, reflecting their distinct origins, locations, functions, and roles in inflammatory responses. Our data further suggested that maternal gravidity influences the gene signatures of MIMs and HBCs, potentially modulating the interplay between tolerance and trained immunity. The phenomenon of reproductive immune memory may play a novel role in the differential susceptibility of primigravidas to pregnancy complications.

immunology↗

bioRxiv2021

Identification, Characterization and Synthesis of Natural Parasitic Cysteine Protease Inhibitors -- More Potent Falcitidin Analogs

Protease inhibitors represent a promising therapeutic option for the treatment of parasitic diseases such as malaria and human African trypanosomiasis. Falcitidin was the first member of a new class of inhibitors of falcipain-2, a cysteine protease of the malaria parasite Plasmodium falciparum. Using a metabolomics dataset of 25 Chitinophaga strains for molecular networking enabled identification of over 30 natural analogs of falcitidin. Based on MS/MS spectra, they vary in their amino acid chain length, sequence, acyl residue, and C-terminal functionalization; therefore, they were grouped into the four falcitidin peptide families A-D. The isolation, characterization and absolute structure elucidation of two falcitidin-related pentapeptide aldehyde analogs by extensive MS/MS spectrometry and NMR spectroscopy in combination with advanced Marfeys analysis was in agreement with the in silico analysis of the corresponding biosynthetic gene cluster. Total synthesis of chosen pentapeptide analogs followed by in vitro testing against a panel of proteases revealed selective parasitic cysteine protease inhibition and additionally low-micromolar inhibition of -chymotrypsin. The pentapeptides investigated here showed superior inhibitory activity compared to falcitidin.

microbiology↗

bioRxiv2021

Preclinical characterization and target validation of the antimalarial pantothenamide MMV693183

Drug resistance and a dire lack of transmission-blocking antimalarials hamper malaria elimination. Here, we present the pantothenamide MMV693183 as a first-in-class acetyl-CoA synthetase (ACS) inhibitor to enter preclinical development. Our studies demonstrated attractive drug-like properties and in vivo efficacy in a humanized mouse model of Plasmodium falciparum infection. The compound showed exceptional in vitro activity against P. falciparum and P. vivax clinical isolates, and potently blocked P. falciparum transmission to Anopheles mosquitoes. Genetic and biochemical studies identified ACS as the target of the MMV693183-derived antimetabolite, CoA-MMV693183. MMV693183 was well adsorbed after oral administration in mice, rats and dogs. Pharmacokinetic - pharmacodynamic modelling predicted that a single 30 mg oral dose is sufficient to cure a malaria infection in humans. In conclusion, the ACS-targeting compound MMV693183 represents a promising addition to the portfolio of antimalarials in (pre)clinical development with a novel mode of action for the treatment of malaria and blocking transmission.

microbiology↗

bioRxiv2021

P. falciparum K13 mutations present varying degrees of artemisinin resistance and reduced fitness in African parasites

The emergence of artemisinin (ART) resistance in Plasmodium falciparum parasites, driven by K13 mutations, has led to widespread antimalarial treatment failure in Southeast Asia. In Africa, our genotyping of 3,299 isolates confirms the emergence of the K13 R561H variant in Rwanda and reveals the continuing dominance of wild-type K13 across 11 countries. We show that this mutation, along with M579I and C580Y, confers varying degrees of in vitro ART resistance in African parasites. C580Y and M579I cause substantial fitness costs, which may counter-select against their dissemination in high-transmission settings. We also define the impact of multiple K13 mutations on ART resistance and fitness in multiple Southeast Asian strains. ART susceptibility is unaltered upon editing point mutations in ferrodoxin or mdr2, earlier resistance markers. These data point to the lack of an evident biological barrier to mutant K13 mediating ART resistance in Africa, while identifying their detrimental impact on parasite growth.

physiology↗

bioRxiv2021

Plasmodium falciparum K13 mutations in Africa and Asia present varying degrees of artemisinin resistance and an elevated fitness cost in African parasites

The emergence of artemisinin (ART) resistance in Plasmodium falciparum parasites has led to increasing rates of treatment failure with first-line ART-based combination therapies (ACTs) in Southeast Asia. In this region, select mutations in K13 can result in delayed parasite clearance rates in vivo and enhanced survival in the ring-stage survival assay (RSA) in vitro. Our genotyping of 3,299 P. falciparum isolates across 11 sub-Saharan countries reveals the continuing dominance of wild-type K13 and confirms the emergence of a K13 R561H variant in Rwanda. Using gene editing, we provide definitive evidence that this mutation, along with M579I and C580Y, can confer variable degrees of in vitro ART resistance in African P. falciparum strains. C580Y and M579I were both associated with substantial fitness costs in African parasites, which may counter-select against their dissemination in high-transmission settings. We also report the impact of multiple K13 mutations, including the predominant variant C580Y, on RSA survival rates and fitness in multiple Southeast Asian strains. No change in ART susceptibility was observed upon editing point mutations in ferrodoxin or mdr2, earlier associated with ART resistance in Southeast Asia. These data point to the lack of an evident biological barrier to mutant K13 mediating ART resistance in Africa, while identifying their detrimental impact on parasite growth.

microbiology↗

bioRxiv2019

The Impact of Antimalarial Resistance on the Genetic Structure of Plasmodium falciparum in the DRC

The Democratic Republic of the Congo (DRC) harbors 11% of global malaria cases, yet little is known about the spatial and genetic structure of the parasite population in that country. We sequenced 2537 Plasmodium falciparum infections, including a nationally representative population sample from DRC and samples from surrounding countries, using molecular inversion probes - a novel high-throughput genotyping tool. We identified an east-west divide in haplotypes known to confer resistance to chloroquine and sulfadoxine-pyrimethamine. Furthermore, we identified highly related parasites over large geographic distances, indicative of gene flow and migration. Our results were consistent with a background of isolation by distance combined with the effects of selection for antimalarial drug resistance. This study provides a high-resolution view of parasite genetic structure across a large country in Africa and provides a baseline to study how implementation programs may impact parasite populations.

genomics↗

bioRxiv2018

Etiology of fever in Ugandan children: identification of microbial pathogens using metagenomic next-generation sequencing and IDseq, a platform for unbiased metagenomic analysis

BackgroundFebrile illness is a major burden in African children, and non-malarial causes of fever are uncertain. We built and employed IDseq, a cloud-based, open access, bioinformatics platform and service to identify microbes from metagenomic next-generation sequencing of tissue samples. In this pilot study, we evaluated blood, nasopharyngeal, and stool specimens from 94 children (aged 2-54 months) with febrile illness admitted to Tororo District Hospital, Uganda.\n\nResultsThe most common pathogens identified were Plasmodium falciparum (51.1% of samples) and parvovirus B19 (4.4%) from blood; human rhinoviruses A and C (40%), respiratory syncytial virus (10%), and human herpesvirus 5 (10%) from nasopharyngeal swabs; and rotavirus A (50% of those with diarrhea) from stool. Among other potential pathogens, we identified one novel orthobunyavirus, tentatively named Nyangole virus, from the blood of a child diagnosed with malaria and pneumonia, and Bwamba orthobunyavirus in the nasopharynx of a child with rash and sepsis. We also identified two novel human rhinovirus C species.\n\nConclusionsThis exploratory pilot study demonstrates the utility of mNGS and the IDseq platform for defining the molecular landscape of febrile infectious diseases in resource limited areas. These methods, supported by a robust data analysis and sharing platform, offer a new tool for the surveillance, diagnosis, and ultimately treatment and prevention of infectious diseases.

genomics↗