Biology subjects

Bailey, J. A.

Publications and source records attributed to Bailey, J. A..

At least 19 recordsLinked to original sources

bioRxiv2026

Not so cold after all: tumor infiltrating CD8+ T cells in EBV-positive Burkitt lymphoma are quiescent, not exhausted

Abstract / SummarySurvival outcomes for pediatric Burkitt lymphoma (BL) substantially vary depending on geography (50-90%), which also serves as a proxy for the prevalence of Epstein-Barr virus (EBV) within the tumors. Although BL is considered an immunologically "cold" tumor with few tumor-infiltrating lymphocytes (TILs), their functional status has not been fully evaluated, especially for EBV-positive disease. Here, we characterize the exhaustion and activation profiles of T cells in the tumor microenvironment (TME) of EBV-positive BL using orthogonal methods, single-cell gene expression analysis, spectral flow cytometry, and immuno-histochemistry staining (IHC). We found that CD8+ TILs displayed a mosaic of immune inhibitory gene expression encoding, PD1, TIGIT, LAG3 and HAVCR2/TIM3. IHC validated the expression of PD1 and TIGIT on CD8+ TILs, as well as their respective ligands, PDL-1, PVR, and Nectin-2 on malignant B cells. Despite exhaustion-associated signatures, CD8+ TILs retain cytotoxic potential, expressing granules (i.e. Granzyme A, Perforin) and cytokines (i.e. IFN{gamma}) and demonstrate an increased uptake of metabolites such as glucose, arginine, and methionine. In peripheral blood, pediatric BL patients exhibited a significantly higher abundance of PD1+TIGIT+ CD8+ T cells compared to healthy children. Notably, these circulating T cells from BL patients express significantly lower levels of TOX, suggesting they are not irreversibly dysfunctional. Together, our results indicate that CD8+ T cells both in the TME and in circulation of children with BL are not terminally exhausted but remain poised for functional re-invigoration. These findings support the potential integration of immune checkpoint inhibitors into combination chemotherapeutic regimens to improve outcomes for these children. SignificanceEBV-positive BL tumors contain functional, metabolically active CD8+ T cells. Circulating PD1+TIGIT+CD8+ T cells found in BL patients blood are a biomarker for those in the tumor microenvironment.

immunology↗

bioRxiv2026

Malaria control and the unexpected spread of diagnostic-resistant Plasmodium falciparum in Peru

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.

genetics↗

bioRxiv2026

EBV Type 1 versus Type 2: A determinant of NK cell anti-tumor activity in Burkitt lymphoma

Terminally differentiated CD56negCD16pos NK cells have been described after chronic viral and malaria infections, and in children diagnosed with Burkitt lymphoma (BL). Despite CD56neg NK cells appearing to be poor at direct cytotoxicity, they express high levels of cytotoxic granules (i.e. granzymes, perforin), activation markers, and Fc-{gamma} receptors (CD32 and CD16) that are typically engaged in antibody-dependent cell cytotoxicity (ADCC). In addition, the abundance of CD56neg NK cells strongly correlates with IgG1 and IgG3 plasma levels, which are essential subclasses for ADCC. To determine whether CD56neg NK cells have superior ADCC capacity relative to CD56dim NK cells, we performed ADCC assays using effector cells from pediatric cancer patients and healthy children from malaria endemic regions of Kenya, targeting in vitro rituximab-treated commercial and newly established BL cell lines. We found that CD56neg NK cells were indeed capable of in vitro ADCC, showing a significant increase of CD107a-mediated degranulation in the presence of rituximab; however, they were not as efficient as CD56dim NK cells. Moreover, we found that the ADCC magnitude was significantly lower against EBV-Type 2 (EBV-T2) BL lines compared to EBV-Type 1 (EBV-T1). EBV-T2 tumor cell lines expressed significantly more lytic viral proteins than EBV-T1, making them more sensitive to direct cytotoxicity. Results from this study highlight the importance of assessing inter-patient variation in NK cell profiles in conjunction with ADCC sensitivity and EBV type within tumor cells when evaluating clinical outcomes for NK-mediated immunotherapies. SignificanceEBV type dictates NK cytotoxicity: EBV-T1 BL cells require rituximab for NK killing, while EBV-T2 BL cells are eliminated without antibody assistance, highlighting target-specific immune response to EBV-associated cancers.

immunology↗

bioRxiv2025

Dissecting Epstein-Barr Virus Dependence Across Diverse Infected Cell Models

Epstein-Barr virus (EBV) is maintained clonally in a wide array of tumors and cells of lymphoproliferative disorders, yet the degree to which these infected cells continuously depend on EBV remains unresolved. To directly assess EBV dependence, we used two complementary Cas9-based viral eviction strategies: 1) targeting EBNA1 to promote episome dilution during cell division, and 2) targeting repetitive viral genomic regions to rapidly degrade EBV episomes. Using these approaches, we evaluated EBV-loss sensitivity across newly-derived, phenotypically diverse EBV-positive Burkitt lymphoma lines. We also evaluated cell lines from patients with EBV-associated T/NK and epithelial malignancies. We included the Burkitt Lymphoma cell line, Akata-GFP, as our EBV-loss-tolerant control and a lymphoblastoid cell line as our EBV-loss-sensitive control. We observed EBV-loss sensitivity in all the EBV-positive Burkitt Lymphoma cell lines and the chronic active EBV disease cell line. In contrast, the EBV-positive gastric adenocarcinoma lines were markedly less sensitive to EBV loss. EBV latency type did not predict EBV dependence, indicating that viral gene expression programs alone do not dictate reliance on the virus. These findings demonstrate that EBV dependence is prevalent across multiple EBV-associated disease models and is especially pronounced in early-state or newly derived lymphoid malignancies. AUTHOR SUMMARYEpstein-Barr virus (EBV) infects most people worldwide and is linked to many malignancies. Although EBV is present in cancer cells, it is unclear whether these cells require the virus to survive. To answer this question, we used CRISPR/Cas9-based methods to remove EBV from infected cells. Using these tools, we studied many different EBV-positive cell lines, including new Burkitt lymphoma cell lines. We found that most EBV-positive lymphoid cells required EBV for growth. In contrast, EBV-positive gastric cells were more tolerant of viral loss. These results show that many EBV-associated malignancies, especially early or newly established diseases, rely on EBV to survive.

microbiology↗

bioRxiv2025

The Portable Microhaplotype Object and Tools

Structured AbstractO_ST_ABSMotivationC_ST_ABSThe rapid increase in the generation of targeted sequencing data offers immense potential for research, medicine, and public health, however the lack of an established standard for these data has led to disparate solutions for data storage. A widely accepted standard is essential for data sharing, reuse, and the coordinated development of interoperable analysis tools. ResultsWe propose the Portable Microhaplotype Object (PMO), a standardized format for efficiently and losslessly storing phased targeted sequencing data (microhaplotypes). The PMO format is JSON-based, allowing efficient, relational storage of genetic data together with relevant metadata to minimize orphaned data. The format includes required fields and a curated set of optional fields leveraging established ontologies. To facilitate ease of use, we developed pmotools-python, an open-source package for creating, manipulating, and exporting PMO data into common formats. Additionally, we provide a simple web-based app to quickly create PMO files from tabular inputs, making the format accessible to a wide variety of users. Example datasets from Plasmodium, Anopheles, Escherichia coli, and Staphylococcus aureus demonstrate the broad applicability of the approach. PMO will streamline data sharing, foster interoperability, and accelerate the development of harmonized analysis tools. Availability and implementationThe Portable Microhaplotype Object (PMO) project, including the ontology specification, software tools, example datasets, and tutorials, is freely available athttps://plasmogenepi.github.io/PMO_Docs/. Key software components and datasets have archived releases with DOIs to ensure permanence, detailed in the Supplementary Text 1-5. Contactkathrynmmurie@gmail.com or nickjhathaway@gmail.com

bioinformatics↗

bioRxiv2025

COATswga: A Coverage Optimizing and Accurate Toolkit for fast primer design in selective whole genome amplification

BackgroundDespite the transformative nature of next-generation sequencing in genomics, efficiently capturing underrepresented microbial DNA from complex biological mixtures, such as pathogens from host samples, remains a persistent challenge. Unwanted genomes make whole-genome sequencing (WGS) inefficient and expensive without enrichment of the targeted genome. Selective whole genome amplification (sWGA) uses primers that selectively bind to the target genome to preferentially amplify and enrich an entire microbial genome. However, existing sWGA primer design is often complicated and time-consuming, with primers usually having biased amplification producing uneven genome coverage. Developing faster primer design methods that ensure uniform, reliable microbial genome recovery are critical to improving sWGA. MethodsWe developed COATswga, a Coverage Optimizing and Accurate Toolkit for designing sWGA primer sets. The pipeline consists of three key steps: (1) primer discovery, using k-mer counting to identify all candidate primers in the target genomes; (2) filtering, where primers are screened for amplification potential, thermodynamic stability, and specificity; and (3) set formation, which uses a novel interval-based tiling algorithm. COATswga is parallelized for efficiency and supports pre-existing primer set refinement and gap filling. The pipeline was evaluated by designing primer sets for Plasmodium falciparum (strain 3D7) and benchmarking their performance against sets generated using similar pipelines. ResultsCOATswga-designed primers demonstrated superior enrichment based on both qPCR and long-read sequencing. In P. falciparum-spiked human DNA samples at parasitemia levels of 400, 100, and 10 parasites/L, COATswga primers achieved higher amplification efficiency and specificity compared to alternatives. At 100 parasitemia, COATswga yielded [~]99% of reads mapping to the P. falciparum genome with 82.5% of the genome having 5x coverage. Even at 10 parasites/uL, COATswga primers enabled effective amplification of key drug resistance genes (pfcrt and pfmdr1). Additionally, integration with molecular inversion probe (MIP) genotyping showed that COATswga greatly improved sequencing depth and target recovery in low-density infections compared to established sWGA primers. ConclusionCOATswga provides a robust and flexible solution for designing sWGA primers to reliably recover targeted genomes from complex samples. The new P. falciparum primer set performs well in low-parasitemia samples allowing more extensive application of sWGA in falciparum malaria genomics.

bioinformatics↗

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↗

bioRxiv2024

Loss of developmentally derived Irf8+ macrophages promotes hyperinnervation and arrhythmia in the adult zebrafish heart

Recent developments in cardiac macrophage biology have broadened our understanding of the critical functions of macrophages in the heart. As a result, there is further interest in understanding the independent contributions of distinct subsets of macrophage to cardiac development and function. Here, we demonstrate that genetic loss of interferon regulatory factor 8 (Irf8)-positive embryonic-derived macrophages significantly disrupts cardiac conduction, chamber function, and innervation in adult zebrafish. At 4 months post-fertilization (mpf), homozygous irf8st96/st96 mutants have significantly shortened atrial action potential duration and significant differential expression of genes involved in cardiac contraction. Functional in vivo assessments via electro- and echocardiograms at 12 mpf reveal that irf8 mutants are arrhythmogenic and exhibit diastolic dysfunction and ventricular stiffening. To identify the molecular drivers of the functional disturbances in irf8 null zebrafish, we perform single cell RNA sequencing and immunohistochemistry, which reveal increased leukocyte infiltration, epicardial activation, mesenchymal gene expression, and fibrosis. Irf8 null hearts are also hyperinnervated and have aberrant axonal patterning, a phenotype not previously assessed in the context of cardiac macrophage loss. Gene ontology analysis supports a novel role for activated epicardial-derived cells (EPDCs) in promoting neurogenesis and neuronal remodeling in vivo. Together, these data uncover significant cardiac abnormalities following embryonic macrophage loss and expand our knowledge of critical macrophage functions in heart physiology and governing homeostatic heart health.

systems biology↗

bioRxiv2024

Population genomics of Plasmodium ovale species in sub-Saharan Africa

Plasmodium ovale curtisi (Poc) and Plasmodium ovale wallikeri (Pow) are relapsing malaria parasites endemic to Africa and Asia that were previously thought to represent a single species. Amid increasing detection of ovale malaria in sub-Saharan Africa, we performed a population genomic study of both species across the continent. We conducted whole-genome sequencing of 25 isolates from Central and East Africa and analyzed them alongside 20 previously published African genomes. Isolates were predominantly monoclonal (43/45), with their genetic similarity aligning with geography. Pow showed lower average nucleotide diversity (1.8x10-4) across the genome compared to Poc (3.0x10-4) (p < 0.0001). Signatures of selective sweeps involving the dihydrofolate reductase gene were found in both species, as were signs of balancing selection at the merozoite surface protein 1 gene. Differences in the nucleotide diversity of Poc and Pow may reflect unique demographic history, even as similar selective forces facilitate their resilience to malaria control interventions.

genomics↗

bioRxiv2023

Single cell transcriptional changes across the blood stages of artemisinin resistant K13C580Y mutant Plasmodium falciparum upon dihydroartemisinin exposure

Artemisinin-based therapies have been central to malaria control, but partial resistance in Plasmodium falciparum, driven by mutations in the Kelch13 (K13) protein, threatens these gains. To investigate the molecular basis of this resistance, we applied single-cell RNA sequencing to coisogenic parasite lines, K13 wild-type (K13C580) and the artemisinin-resistant mutant (K13580Y), following a 6 hour pulse of dihydroartemisinin (DHA). This approach enabled high-resolution profiling across intraerythrocytic stages. Both lines exhibited stage-specific transcriptional responses, with pronounced changes in ring and trophozoite stages. Using Manifold Enhancement of Latent Dimensions (MELD), a computational framework for quantifying transcriptional perturbation, DHA-treatment induces stage-specific differences in protein export and metabolic pathways in K13C580 and K13580Y parasites, relating to an altered metabolic stress response state. GARP, a potential therapeutic target, was highly differentially expressed in untreated ring stages of K13580Y and K13C580. Functional assays confirmed that anti-GARP antibodies retained efficacy against K13580Y, supporting its potential as a therapeutic target. These findings provide a comprehensive view of the cellular responses related to artemisinin resistance, identify molecular features of pathogenesis, and highlight surface proteins like GARP as promising intervention targets. This work underscores the power of single-cell approaches to dissect drug responses and guide strategies to overcome resistant parasites.

microbiology↗

bioRxiv2023

Bloodmeal metabarcoding of the argasid tick (Ornithodoros turicata Duges) reveals extensive vector-host associations

Molecular methods to understand host feeding patterns of arthropod vectors are critical to assess exposure risk to vector-borne disease and unveil complex ecological interactions. We build on our prior work discovering the utility of PCR-Sanger sequencing bloodmeal analysis that work remarkably well for soft ticks (Acari: Argasidae), unlike for hard ticks (Acari: Ixodidae), thanks to their unique physiology that retains vertebrate DNA from prior bloodmeals viable for years. Here, we capitalize on this feature and apply bloodmeal metabarcoding using amplicon deep sequencing to identify multiple host species in individual Ornithodoros turicata soft ticks collected from two natural areas in Texas, United States. Of 788 collected O. turicata, 394 were evaluated for bloodmeal source via metabarcoding, revealing 27 different vertebrate host species (17 mammals, 5 birds, 1 reptile, and 4 amphibians) fed upon by 274 soft ticks. Information on multiple hosts for individual O. turicata was derived from 168 of these (61%). Metabarcoding revealed more mixed vertebrate bloodmeals in O. turicata previously processed using Sanger sequencing. These data reveal wide host range of O. turicata and demonstrate the value of bloodmeal metabarcoding for understanding the ecology for known and potential tick-borne pathogens circulating among humans, domestic animals and wildlife such as relapsing fever caused by Borrelia turicatae. Our results also document, for the first time an off-host soft tick collected to have evidence of prior feeding on wild pig which is a critical observation in the context of the threat of enzootic transmission of African swine fever virus if it were introduced to the US. This research enhances our understanding of vector-host associations and offers a promising perspective for biodiversity monitoring and disease control strategies.

ecology↗

bioRxiv2022

Non-allelic homologous recombination driven translocation explains histidine-rich protein 3 deletion mechanism in Plasmodium falciparum

Most malaria rapid diagnostic tests (RDTs) detect Plasmodium falciparum histidine-rich protein 2 (PfHRP2) and PfHRP3, but deletions of pfhrp2 and phfrp3 genes make parasites undetectable by RDTs. We analyzed 19,313 public whole-genome-sequenced P. falciparum field samples to understand these deletions better. Pfhrp2 deletion only occurred by chromosomal breakage with subsequent telomere healing. Pfhrp3 deletions involved loss from pfhrp3 to the telomere and showed 3 patterns: no other associated rearrangement with evidence of telomere healing at breakpoint (Asia; Pattern 13-TARE1); associated with duplication of a chromosome 5 segment containing multidrug-resistant-1 gene (Asia; Pattern 13-5++); and most commonly, associated with duplication of a chromosome 11 segment (Americas/Africa; Pattern 13-11++). We confirmed a 13-11 hybrid chromosome with long-read sequencing, consistent with a translocation product arising from recombination between large interchromosomal ribosome-containing segmental duplications. Within most 13-11++ parasites, the duplicated chromosome 11 segments were identical. Across parasites, multiple distinct haplotype groupings were consistent with emergence due to clonal expansion of progeny from intrastrain meiotic recombination. Together, these observations suggest negative selection normally removes 13-11++ pfhrp3 deletions, and specific conditions are needed for their emergence and spread including low transmission, findings that can help refine surveillance strategies.

genomics↗

bioRxiv2022

Expression Microdissection for use in qPCR based analysis of miRNA in a single cell type

Cell-specific microRNA (miRNA) expression estimates are important in characterizing the localization of miRNA signaling within tissues. Much of this data is obtained from cultured cells, a process known to significantly alter miRNA expression levels. Thus, our knowledge of in vivo cell miRNA expression estimates is poor. We previously demonstrated expression microdissection-miRNA-sequencing (xMD-miRNA-seq) as a means to acquire in vivo estimates, directly from formalin fixed tissues, albeit with limited yield. Here we optimized each step of the xMD process including tissue retrieval, tissue transfer, film preparation, and RNA isolation to increase RNA yields and ultimately show strong enrichment for in vivo miRNA expression by qPCR array. These method improvements, including the development of a non-crosslinked ethylene vinyl acetate (EVA) membrane, resulted in a 23-45 fold increase in miRNA yield, depending on cell type. By qPCR, miR-200a was increased 14-fold in xMD-derived small intestine epithelial cells, with a concurrent 336-fold reduction in miR-143, relative to the matched non-dissected duodenal tissue. xMD is now an optimized method to obtain robust in vivo miRNA expression estimates from cells.

genomics↗

bioRxiv2022

coiaf: directly estimating complexity of infection with allele frequencies

In malaria, individuals are often infected with different parasite strains; the complexity of infection (COI) is defined as the number of genetically distinct parasite strains in an individual. Changes in the mean COI in a population have been shown to be informative of changes in transmission intensity with a number of probabilistic likelihood and Bayesian models now developed to estimate the COI. However, rapid, direct measures based on heterozygosity or FwS do not properly represent the COI. In this work, we present two new methods that use easily calculated measures to directly estimate the COI from allele frequency data. Using a simulation framework, we show that our methods are computationally efficient and comparably accurate to current methods in the literature. Through a sensitivity analysis, we characterize how the bias and accuracy of our two methods are impacted by the distribution of parasite densities and the assumed sequencing depth and number of sampled loci. We further estimate the COI globally from Plasmodium falciparum sequencing data using our developed methods and compare the results against the literature. We show significant differences in estimated COI globally between continents and a weak relationship between malaria prevalence and COI. Author summaryComputational models, used in conjunction with rapidly advancing sequencing technologies, are increasingly being used to help inform surveillance efforts and understand the epidemiological dynamics of malaria. One such important metric, the complexity of infection (COI), indirectly quantifies the level of transmission. Existing "gold-standard" COI measures rely on complex probabilistic likelihood and Bayesian models. As an alternative, we have developed the statistics and software package coiaf, which features two rapid, direct measures to estimate of the number of genetically distinct parasite strains in an individual (the COI). Our methods were evaluated using simulated data and subsequently compared to current "state-of-the-art" methods, yielding comparable results. Lastly, we examined the distribution of the COI in several locations across the world, identifying significant differences in COI between continents. coiaf, therefore, provides a new, promising framework for rapidly characterizing polyclonal infections.

genomics↗

bioRxiv2020

Single-cell characterization of transcriptomic heterogeneity in lymphoblastoid cell lines

Lymphoblastoid Cell Lines (LCLs) are generated by transforming primary B cells with Epstein-Barr Virus (EBV) and are used extensively as model systems in viral oncology, immunology, and human genetics research. In this study, we characterized single-cell transcriptomic profiles of five LCLs and present a simple discrete-time simulation to explore the influence of stochasticity on LCL clonal evolution. Single-cell RNA sequencing revealed substantial phenotypic heterogeneity within and across LCLs with respect to immunoglobulin isotype; virus-modulated host pathways involved in survival, proliferation, and differentiation; viral replication state; and oxidative stress. This heterogeneity is likely attributable to intrinsic variance in primary B cells and host-pathogen dynamics. Stochastic simulations demonstrate that initial primary cell heterogeneity, random sampling, time in culture, and even mild differences in phenotype-specific fitness can contribute substantially to dynamic diversity in populations of nominally clonal cells.

immunology↗

bioRxiv2020

Describing the current status of Plasmodium falciparum population structure and drug resistance within mainland Tanzania using molecular inversion probes

High-throughput Plasmodium genomic data is increasingly useful in assessing prevalence of clinically important mutations and malaria transmission patterns. Understanding parasite diversity is important for identification of specific human or parasite populations that can be targeted by control programs, and to monitor the spread of mutations associated with drug resistance. An up-to-date understanding of regional parasite population dynamics is also critical to monitor the impact of control efforts. However, this data is largely absent from high-burden nations in Africa, and to date, no such analysis has been conducted for malaria parasites in Tanzania country-wide. To this end, over 1,000 P. falciparum clinical isolates were collected in 2017 from 13 sites in seven administrative regions across Tanzania, and parasites were genotyped at 1,800 variable positions genome-wide using molecular inversion probes. Population structure was detectable among Tanzanian P. falciparum parasites, roughly separating parasites from the northern and southern districts and identifying genetically admixed populations in the north. Isolates from geographically close districts were more likely to be genetically related compared to parasites sampled from more distant districts. Known drug resistance mutations were seen at increased frequency in northern districts, and additional variants with undetermined significance for antimalarial resistance also varied by geography. Malaria Indicator Survey (2017) data corresponded with genetic findings, including average region-level complexity-of-infection and malaria prevalence estimates. The parasite populations identified here provide important information on extant spatial patterns of genetic diversity of Tanzanian parasites, to which future surveys of genetic relatedness can be compared. SIGNIFICANCEDocumenting dynamics of malaria parasite genomics in high-transmission settings at scale in sub-Saharan Africa is critical for policy and decision making to support ongoing malaria elimination initiatives. Using molecular inversion probes, we genotyped over 1,000 Tanzanian Plasmodium falciparum samples collected country-wide in 2017 at hundreds of variable polymorphic positions across the genome. Frequencies of known drug resistance mutations were higher in northern districts of the country compared to the south. Results also showed a distinct isolation-by-distance pattern (whereby increasing geographic distance was correlated with decreasing genetic relatedness), as well as signals of higher genetic sharing between several southern districts. These results provide, for the first time, a picture of current within-country diversity of Tanzanian P. falciparum populations.

genomics↗

bioRxiv2020

Sensitive, highly multiplexed sequencing of microhaplotypes from the Plasmodium falciparum heterozygome

BackgroundTargeted next generation sequencing offers the potential for consistent, deep coverage of information rich genomic regions to characterize polyclonal Plasmodium falciparum infections. However, methods to identify and sequence these genomic regions are currently limited. MethodsA bioinformatic pipeline and multiplex methods were developed to identify and simultaneously sequence 100 targets and applied to dried blood spot (DBS) controls and field isolates from Mozambique. For comparison, WGS data were generated for the same controls. ResultsUsing publicly available genomes, 4465 high diversity genomic regions suited for targeted sequencing were identified, representing the P. falciparum heterozygome. For this study, 93 microhaplotypes with high diversity (median HE = 0.7) were selected along with 7 drug resistance loci. The sequencing method achieved very high coverage (median 99%), specificity (99.8%) and sensitivity (90% for haplotypes with 5% within sample frequency in DBS with 100 parasites/{micro}L). In silico analyses revealed that microhaplotypes provided much higher resolution to discriminate related from unrelated polyclonal infections than biallelic SNP barcodes. DiscussionThe bioinformatic and laboratory methods outlined here provide a flexible tool for efficient, low-cost, high throughput interrogation of the P. falciparum genome, and can be tailored to simultaneously address multiple questions of interest in various epidemiological settings.

molecular biology↗

bioRxiv2019

Falciparum malaria from coastal Tanzania and Zanzibar remains highly connected despite effective control efforts on the archipelago

BackgroundTanzanias Zanzibar archipelago has made significant gains in malaria control over the last decade and is a target for malaria elimination. Despite consistent implementation of effective tools since 2002, elimination has not been achieved. Importation of parasites from outside of the archipelago is thought to be an important cause of malarias persistence, but this paradigm has not been studied using modern genetic tools. MethodsWe used whole-genome sequencing (WGS) to investigate the impact of importation, employing population genetic analyses of Plasmodium falciparum isolates from both the archipelago and mainland Tanzania. We assessed ancestry, levels of genetic diversity and differentiation, patterns of relatedness, and patterns of selection between these two populations by leveraging recent advances in deconvolution of genomes from polyclonal malaria infections. ResultsWe identified significant decreases in the effective population sizes in both populations in the timeframe of decreasing malaria transmission in Tanzania. Identity by descent analysis showed that parasites in the two populations shared large sections of their genomes, on the order of 5 cM, suggesting shared ancestry within the last 10 generations. Even with limited sampling,, we demonstrate a pair of isolates between the mainland and Zanzibar that are related at the expected level of half-siblings, consistent with recent importation ConclusionsThese findings suggest that importation plays an increasing role for malaria incidence on Zanzibar and demonstrate the value of genomic approaches for identifying corridors of parasite movement to the island.

genetics↗