Reversible host cell surface remodelling limits immune recognition and maximizes transmission of Plasmodium falciparum gametocytes.

Reducing malaria transmission has been a major pillar of control programmes and is considered crucial for achieving malaria elimination. Gametocytes, the transmissible forms of the P. falciparum parasite, arise during the blood stage of the parasite and develop through 5 morphologically distinct stages. Immature gametocytes (stage I-IV) sequester and develop in the extravascular niche of the bone marrow and possibly spleen. Only mature stage V gametocytes re-enter peripheral circulation to be taken up by mosquitoes for successful onward transmission. We have recently shown that immature, but not mature gametocytes are targets of host immune responses and identified putative target surface antigens. We hypothesize that these antigens play a role in gametocyte sequestration and contribute to acquired transmission-reducing immunity. Here we demonstrate that surface antigen expression, serum reactivity by human IgG, and opsonic phagocytosis by macrophages all show similar dynamics during gametocyte maturation, i.e., on in immature and off in mature gametocytes. Moreover, the switch in surface reactivity coincides with reversal in phosphatidylserine (PS) surface exposure, a marker for red blood cell age and clearance. PS is exposed on the surface of immature gametocytes (as well as in late asexual stages) but is removed from the surface in later gametocyte stages (IV-V). Using parasite reverse genetics and drug perturbations, we confirm that parasite protein export into the host cell and phospholipid scramblase activity are required for the observed surface modifications in asexual and sexual P. falciparum stages. These findings suggest that the dynamic surface remodelling allows (i) immature gametocyte sequestration in bone marrow and (ii) mature gametocyte release into peripheral circulation and immune evasion, therefore contributing to mature gametocyte survival in vivo and onward transmission to mosquitoes. Importantly, blocking scramblase activity during gametocyte maturation results in efficient clearance of mature gametocytes, revealing a potential path for transmission blocking interventions. Our studies have important implications for our understanding of parasite biology and form a starting point for novel intervention strategies to simultaneously reduce parasite burden and transmission. IMPORTANT: Manuscripts submitted to Review Commons are peer reviewed in a journal-agnostic way.Upon transfer of the peer reviewed preprint to a journal, the referee reports will be available in full to the handling editor.The identity of the referees will NOT be communicated to the authors unless the reviewers choose to sign their report.The identity of the referee will be confidentially disclosed to any affiliate journals to which the manuscript is transferred. GUIDELINES: For reviewers: https://www.reviewcommons.org/reviewers For authors: https://www.reviewcommons.org/authors. CONTACT: The Review Commons office can be contacted directly at: office@reviewcommons.org.

Revisiting the determinants of malaria transmission.

Malaria parasites have coevolved with humans over thousands of years, mirroring their migration out of Africa. They persist to this day, despite continuous elimination efforts worldwide. These parasites can adapt to changing environments during infection of human and mosquito, and when expanding the geographical range by switching vector species. Recent studies in the human malaria parasite, Plasmodium falciparum, identified determinants governing the plasticity of sexual conversion rates, sex ratio, and vector competence. Here we summarize the latest literature revealing environmental, epigenetic, and genetic determinants of malaria transmission.

Sterile protection against P. vivax malaria by repeated blood stage infection in the Aotus monkey model.

The malaria parasite Plasmodium vivax remains a major global public health challenge, and no vaccine is approved for use in humans. Here, we assessed whether P. vivax strain-transcendent immunity can be achieved by repeated infection in Aotus monkeys. Sterile immunity was achieved after two homologous infections, whereas subsequent heterologous challenge provided only partial protection. IgG levels based on P. vivax lysate ELISA and protein microarray increased with repeated infections and correlated with the level of homologous protection. Parasite transcriptional profiles provided no evidence of major antigenic switching upon homologous or heterologous challenge. However, we observed significant sequence diversity and transcriptional differences in the P. vivax core gene repertoire between the two strains used in the study, suggesting that partial protection upon heterologous challenge is due to molecular differences between strains rather than immune evasion by antigenic switching. Our study demonstrates that sterile immunity against P. vivax can be achieved by repeated homologous blood stage infection in Aotus monkeys, thus providing a benchmark to test the efficacy of candidate blood stage P. vivax malaria vaccines.

Genetic validation of PfFKBP35 as an antimalarial drug target.

Plasmodium falciparum accounts for the majority of over 600,000 malaria-associated deaths annually. Parasites resistant to nearly all antimalarials have emerged and the need for drugs with alternative modes of action is thus undoubted. The FK506-binding protein PfFKBP35 has gained attention as a promising drug target due to its high affinity to the macrolide compound FK506 (tacrolimus). Whilst there is considerable interest in targeting PfFKBP35 with small molecules, a genetic validation of this factor as a drug target is missing and its function in parasite biology remains elusive. Here, we show that limiting PfFKBP35 levels are lethal to P. falciparum and result in a delayed death-like phenotype that is characterized by defective ribosome homeostasis and stalled protein synthesis. Our data furthermore suggest that FK506, unlike the action of this drug in model organisms, exerts its antiproliferative activity in a PfFKBP35-independent manner and, using cellular thermal shift assays, we identify putative FK506-targets beyond PfFKBP35. In addition to revealing first insights into the function of PfFKBP35, our results show that FKBP-binding drugs can adopt non-canonical modes of action - with major implications for the development of FK506-derived molecules active against Plasmodium parasites and other eukaryotic pathogens.

From contigs towards chromosomes: automatic improvement of long read assemblies (ILRA).

Recent advances in long read technologies not only enable large consortia to aim to sequence all eukaryotes on Earth, but they also allow individual laboratories to sequence their species of interest with relatively low investment. Long read technologies embody the promise of overcoming scaffolding problems associated with repeats and low complexity sequences, but the number of contigs often far exceeds the number of chromosomes and they may contain many insertion and deletion errors around homopolymer tracts. To overcome these issues, we have implemented the ILRA pipeline to correct long read-based assemblies. Contigs are first reordered, renamed, merged, circularized, or filtered if erroneous or contaminated. Illumina short reads are used subsequently to correct homopolymer errors. We successfully tested our approach by improving the genome sequences of Homo sapiens, Trypanosoma brucei, and Leptosphaeria spp., and by generating four novel Plasmodium falciparum assemblies from field samples. We found that correcting homopolymer tracts reduced the number of genes incorrectly annotated as pseudogenes, but an iterative approach seems to be required to correct more sequencing errors. In summary, we describe and benchmark the performance of our new tool, which improved the quality of novel long read assemblies up to 1 Gbp. The pipeline is available at GitHub: https://github.com/ThomasDOtto/ILRA.

Phenotypic Screens Identify Genetic Factors Associated with Gametocyte Development in the Human Malaria Parasite Plasmodium falciparum.

Transmission of the deadly malaria parasite Plasmodium falciparum from humans to mosquitoes is achieved by specialized intraerythrocytic sexual forms called gametocytes. Though the crucial regulatory mechanisms leading to gametocyte commitment have recently come to light, networks of genes that control sexual development remain to be elucidated. Here, we report a pooled-mutant screen to identify genes associated with gametocyte development in P. falciparum. Our results categorized genes that modulate gametocyte progression as hypoproducers or hyperproducers of gametocytes, and the in-depth analysis of individual clones confirmed phenotypes in sexual commitment rates and putative functions in gametocyte development. We present a new set of genes that have not been implicated in gametocytogenesis before and demonstrate the potential of forward genetic screens in isolating genes impacting parasite sexual biology, an exciting step toward the discovery of new antimalarials for a globally significant pathogen. IMPORTANCE Blocking human-to-vector transmission is an essential step toward malaria elimination. Gametocytes are solely responsible for achieving this transmission and represent an opportunity for therapeutic intervention. While these falciform-shaped parasite stages were first discovered in the 1880s, our understanding of the genetic determinants responsible for their formation and molecular mechanisms that drive their development is limited. In this work, we developed a scalable screening methodology with piggyBac mutants to identify genes that influence the development of gametocytes in the most lethal human malaria parasite, P. falciparum. By doing so, we lay the foundation for large-scale functional genomic studies specifically designed to address remaining questions about sexual commitment, maturation, and mosquito infection in P. falciparum. Such functional genetic screens will serve to expedite the identification of essential pathways and processes for the development of novel transmission-blocking agents.

Plasmodium falciparum adapts its investment into replication versus transmission according to the host environment.

The malaria parasite life cycle includes asexual replication in human blood, with a proportion of parasites differentiating to gametocytes required for transmission to mosquitoes. Commitment to differentiate into gametocytes, which is marked by activation of the parasite transcription factor ap2-g, is known to be influenced by host factors but a comprehensive model remains uncertain. Here, we analyze data from 828 children in Kilifi, Kenya with severe, uncomplicated, and asymptomatic malaria infection over 18 years of falling malaria transmission. We examine markers of host immunity and metabolism, and markers of parasite growth and transmission investment. We find that inflammatory responses associated with reduced plasma lysophosphatidylcholine levels are associated with markers of increased investment in parasite sexual reproduction (i.e. transmission investment) and reduced growth (i.e. asexual replication). This association becomes stronger with falling transmission and suggests that parasites can rapidly respond to the within-host environment, which in turn is subject to changing transmission.

The acquisition of humoral immune responses targeting Plasmodium falciparum sexual stages in controlled human malaria infections.

Individuals infected with P. falciparum develop antibody responses to intra-erythrocytic gametocyte proteins and exported gametocyte proteins present on the surface of infected erythrocytes. However, there is currently limited knowledge on the immunogenicity of gametocyte antigens and the specificity of gametocyte-induced antibody responses. In this study, we assessed antibody responses in participants of two controlled human malaria infection (CHMI) studies by ELISA, multiplexed bead-based antibody assays and protein microarray. By comparing antibody responses in participants with and without gametocyte exposure, we aimed to disentangle the antibody response induced by asexual and sexual stage parasites. We showed that after a single malaria infection, a significant anti-sexual stage humoral response is induced in malaria-naïve individuals, even after exposure to relatively low gametocyte densities (up to ~1,600 gametocytes/mL). In contrast to antibody responses to well-characterised asexual blood stage antigens that were detectable by day 21 after infection, responses to sexual stage antigens (including transmission blocking vaccine candidates Pfs48/45 and Pfs230) were only apparent at 51 days after infection. We found antigens previously associated with early gametocyte or anti-gamete immunity were highly represented among responses linked with gametocyte exposure. Our data provide detailed insights on the induction and kinetics of antibody responses to gametocytes and identify novel antigens that elicit antibody responses exclusively in individuals with gametocyte exposure. Our findings provide target identification for serological assays for surveillance of the malaria infectious reservoir, and support vaccine development by describing the antibody response to leading vaccine antigens after primary infection.

Comparison of PvLAP5 and Pvs25 qRT-PCR assays for the detection of Plasmodium vivax gametocytes in field samples preserved at ambient temperature from remote malaria endemic regions of Panama.

BACKGROUND: As the elimination of malaria in Mesoamerica progresses, detection of Plasmodium vivax using light microscopy (LM) becomes more difficult. Highly sensitive molecular tools have been developed to help determine the hidden reservoir of malaria transmission in low transmission settings. In this study we compare the performance of PvLAP5 and Pvs25 qRT-PCR assays to LM for the detection of Plasmodium vivax gametocytes in field samples preserved at ambient temperature from malaria endemic regions of Panama. METHODS: For this purpose, we collected a total of 83 malaria field samples during 2017-2020 preserved in RNAprotect (RNAp) of which 63 (76%) were confirmed P. vivax by LM and selected for further analysis. Additionally, 16 blood samples from local healthy malaria smear negative volunteers, as well as, from 15 malaria naïve lab-bred Aotus monkeys were used as controls. To optimize the assays, we first determined the minimum blood volume sufficient for detection of PvLAP5 and Pv18SrRNA using P. vivax infected Aotus blood that was preserved in RNAp and kept either at ambient temperature for up to 8 days before freezing or was snap-frozen at -80° Celsius at the time of bleeding. We then compared the mean differences in gametocyte detection rates of both qRT-PCR assays to LM and performed a multivariate correlation analysis of study variables. Finally, we determined the sensitivity (Se) and specificity (Sp) of the assays at detecting gametocytes compared to LM. RESULTS: Blood volume optimization indicated that a blood volume of at least 60 µL was sufficient for detection of PvLAP5 and Pv18SrRNA and no significant differences were found between RNA storage conditions. Both PvLAP5 and Pvs25 qRT-PCR assays showed a 37-39% increase in gametocyte detection rate compared to LM respectively. Strong positive correlations were found between gametocytemia and parasitemia and both PvLAP5 and Pvs25 gametocyte markers. However, no significant differences were detected in the Se and Sp of the Pvs25 and PvLAP5 qRT-PCR assays, even though data from control samples suggested Pvs25 to be more abundant than PvLAP5. CONCLUSIONS: This study shows that the PvLAP5 qRT-PCR assay is as Se and Sp as the gold standard Pvs25 assay and is at least 37% more sensitive than LM at detecting P. vivax gametocytes in field samples preserved in RNAp at ambient temperature from malaria endemic regions of Panama. AUTHOR SUMMARY: Plasmodium vivax is one of the five species of malaria (P. falciparum, P. malariae, P. ovale and P. knowlesi) that are transmitted to man by the bite of female anopheles mosquitoes. It causes ~14.3 million cases mainly in Southeast Asia, India, the Western Pacific and the Americas annually. In the Americas, malaria remains a major problem in underdeveloped areas and indigenous communities in the Amazon region and eastern Panama, where it is endemic and difficult to eliminate. As malaria elimination progresses, detection of P. vivax by light microscopy (LM) becomes more difficult. Therefore, highly sensitive molecular tools have been developed that use genetic markers for the parasite to help determine the hidden reservoir of malaria transmission. This study compares the performance of two molecular assays based on the genetic markers of mature gametocytes PvLAP5 and Pvs25 with LM. The study shows that the PvLAP5 qRT-PCR assay is as sensitive and specific as the gold standard Pvs25 assay and is at least 37% more sensitive than LM at detecting P. vivax gametocytes. These data suggest that the PvLAP5 qRT-PCR assay can be a useful tool to help determine the hidden reservoir of transmission in endemic foci approaching elimination.

Host cell maturation modulates parasite invasion and sexual differentiation in Plasmodium berghei.

Malaria remains a global health problem causing more than 400,000 deaths annually. Plasmodium parasites, the causative agents of malaria, replicate asexually in red blood cells (RBCs) of their vertebrate host, while a subset differentiates into sexual stages (gametocytes) for mosquito transmission. Parasite replication and gametocyte maturation in the erythropoietic niches of the bone marrow and spleen contribute to pathogenesis and drive transmission, but the mechanisms underlying this organ enrichment remain unknown. Here, we performed a comprehensive analysis of rodent P. berghei infection by flow cytometry and single-cell RNA sequencing. We identified CD71 as a host receptor for reticulocyte invasion and found that parasites metabolically adapt to the host cell environment. Transcriptional analysis and functional assays further revealed a nutrient-dependent tropism for gametocyte formation in reticulocytes. Together, we provide a thorough characterization of host-parasite interactions in erythropoietic niches and define host cell maturation state as the key driver of parasite adaptation.

Raman spectroscopic analysis of skin as a diagnostic tool for Human African Trypanosomiasis.

Human African Trypanosomiasis (HAT) has been responsible for several deadly epidemics throughout the 20th century, but a renewed commitment to disease control has significantly reduced new cases and motivated a target for the elimination of Trypanosoma brucei gambiense-HAT by 2030. However, the recent identification of latent human infections, and the detection of trypanosomes in extravascular tissues hidden from current diagnostic tools, such as the skin, has added new complexity to identifying infected individuals. New and improved diagnostic tests to detect Trypanosoma brucei infection by interrogating the skin are therefore needed. Recent advances have improved the cost, sensitivity and portability of Raman spectroscopy technology for non-invasive medical diagnostics, making it an attractive tool for gambiense-HAT detection. The aim of this work was to assess and develop a new non-invasive diagnostic method for T. brucei through Raman spectroscopy of the skin. Infections were performed in an established murine disease model using the animal-infective Trypanosoma brucei brucei subspecies. The skin of infected and matched control mice was scrutinized ex vivo using a confocal Raman microscope with 532 nm excitation and in situ at 785 nm excitation with a portable field-compatible instrument. Spectral evaluation and Principal Component Analysis confirmed discrimination of T. brucei-infected from uninfected tissue, and a characterisation of biochemical changes in lipids and proteins in parasite-infected skin indicated by prominent Raman peak intensities was performed. This study is the first to demonstrate the application of Raman spectroscopy for the detection of T. brucei by targeting the skin of the host. The technique has significant potential to discriminate between infected and non-infected tissue and could represent a unique, non-invasive diagnostic tool in the goal for elimination of gambiense-HAT as well as for Animal African Trypanosomiasis (AAT).

Total parasite biomass but not peripheral parasitaemia is associated with endothelial and haematological perturbations in Plasmodium vivax patients.

Plasmodium vivax is the major cause of human malaria in the Americas. How P. vivax infection can lead to poor clinical outcomes, despite low peripheral parasitaemia, remains a matter of intense debate. Estimation of total P. vivax biomass based on circulating markers indicates existence of a predominant parasite population outside of circulation. In this study, we investigate associations between both peripheral and total parasite biomass and host response in vivax malaria. We analysed parasite and host signatures in a cohort of uncomplicated vivax malaria patients from Manaus, Brazil, combining clinical and parasite parameters, multiplexed analysis of host responses, and ex vivo assays. Patterns of clinical features, parasite burden, and host signatures measured in plasma across the patient cohort were highly heterogenous. Further data deconvolution revealed two patient clusters, here termed Vivaxlow and Vivaxhigh. These patient subgroups were defined based on differences in total parasite biomass but not peripheral parasitaemia. Overall Vivaxlow patients clustered with healthy donors and Vivaxhigh patients showed more profound alterations in haematological parameters, endothelial cell (EC) activation, and glycocalyx breakdown and levels of cytokines regulating different haematopoiesis pathways compared to Vivaxlow. Vivaxhigh patients presented more severe thrombocytopenia and lymphopenia, along with enrichment of neutrophils in the peripheral blood and increased neutrophil-to-lymphocyte ratio (NLCR). When patients' signatures were combined, high association of total parasite biomass with a subset of markers of EC activation, thrombocytopenia, and lymphopenia severity was observed. Finally, machine learning models defined a combination of host parameters measured in the circulation that could predict the extent of parasite infection outside of circulation. Altogether, our data show that total parasite biomass is a better predictor of perturbations in host homeostasis in P. vivax patients than peripheral parasitaemia. This supports the emerging paradigm of a P. vivax tissue reservoir, particularly in the haematopoietic niche of bone marrow and spleen.

A prenylated dsRNA sensor protects against severe COVID-19.

Inherited genetic factors can influence the severity of COVID-19, but the molecular explanation underpinning a genetic association is often unclear. Intracellular antiviral defenses can inhibit the replication of viruses and reduce disease severity. To better understand the antiviral defenses relevant to COVID-19, we used interferon-stimulated gene (ISG) expression screening to reveal that 2'-5'-oligoadenylate synthetase 1 (OAS1), through ribonuclease L, potently inhibits severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We show that a common splice-acceptor single-nucleotide polymorphism (Rs10774671) governs whether patients express prenylated OAS1 isoforms that are membrane-associated and sense-specific regions of SARS-CoV-2 RNAs or if they only express cytosolic, nonprenylated OAS1 that does not efficiently detect SARS-CoV-2. In hospitalized patients, expression of prenylated OAS1 was associated with protection from severe COVID-19, suggesting that this antiviral defense is a major component of a protective antiviral response.

Evaluation of splenic accumulation and colocalization of immature reticulocytes and Plasmodium vivax in asymptomatic malaria: A prospective human splenectomy study.

BACKGROUND: A very large biomass of intact asexual-stage malaria parasites accumulates in the spleen of asymptomatic human individuals infected with Plasmodium vivax. The mechanisms underlying this intense tropism are not clear. We hypothesised that immature reticulocytes, in which P. vivax develops, may display high densities in the spleen, thereby providing a niche for parasite survival. METHODS AND FINDINGS: We examined spleen tissue in 22 mostly untreated individuals naturally exposed to P. vivax and Plasmodium falciparum undergoing splenectomy for any clinical indication in malaria-endemic Papua, Indonesia (2015 to 2017). Infection, parasite and immature reticulocyte density, and splenic distribution were analysed by optical microscopy, flow cytometry, and molecular assays. Nine non-endemic control spleens from individuals undergoing spleno-pancreatectomy in France (2017 to 2020) were also examined for reticulocyte densities. There were no exclusion criteria or sample size considerations in both patient cohorts for this demanding approach. In Indonesia, 95.5% (21/22) of splenectomy patients had asymptomatic splenic Plasmodium infection (7 P. vivax, 13 P. falciparum, and 1 mixed infection). Significant splenic accumulation of immature CD71 intermediate- and high-expressing reticulocytes was seen, with concentrations 11 times greater than in peripheral blood. Accordingly, in France, reticulocyte concentrations in the splenic effluent were higher than in peripheral blood. Greater rigidity of reticulocytes in splenic than in peripheral blood, and their higher densities in splenic cords both suggest a mechanical retention process. Asexual-stage P. vivax-infected erythrocytes of all developmental stages accumulated in the spleen, with non-phagocytosed parasite densities 3,590 times (IQR: 2,600 to 4,130) higher than in circulating blood, and median total splenic parasite loads 81 (IQR: 14 to 205) times greater, accounting for 98.7% (IQR: 95.1% to 98.9%) of the estimated total-body P. vivax biomass. More reticulocytes were in contact with sinus lumen endothelial cells in P. vivax- than in P. falciparum-infected spleens. Histological analyses revealed 96% of P. vivax rings/trophozoites and 46% of schizonts colocalised with 92% of immature reticulocytes in the cords and sinus lumens of the red pulp. Larger splenic cohort studies and similar investigations in untreated symptomatic malaria are warranted. CONCLUSIONS: Immature CD71+ reticulocytes and splenic P. vivax-infected erythrocytes of all asexual stages accumulate in the same splenic compartments, suggesting the existence of a cryptic endosplenic lifecycle in chronic P. vivax infection. Findings provide insight into P. vivax-specific adaptions that have evolved to maximise survival and replication in the spleen.

Higher gametocyte production and mosquito infectivity in chronic compared to incident Plasmodium falciparum infections.

Plasmodium falciparum gametocyte kinetics and infectivity may differ between chronic and incident infections. In the current study, we assess parasite kinetics and infectivity to mosquitoes among children (aged 5-10 years) from Burkina Faso with (a) incident infections following parasite clearance (n = 48) and (b) chronic asymptomatic infections (n = 60). In the incident infection cohort, 92% (44/48) of children develop symptoms within 35 days, compared to 23% (14/60) in the chronic cohort. All individuals with chronic infection carried gametocytes or developed them during follow-up, whereas only 35% (17/48) in the incident cohort produce gametocytes before becoming symptomatic and receiving treatment. Parasite multiplication rate (PMR) and the relative abundance of ap2-g and gexp-5 transcripts are positively associated with gametocyte production. Antibody responses are higher and PMR lower in chronic infections. The presence of symptoms and sexual stage immune responses are associated with reductions in gametocyte infectivity to mosquitoes. We observe that most incident infections require treatment before the density of mature gametocytes is sufficient to infect mosquitoes. In contrast, chronic, asymptomatic infections represent a significant source of mosquito infections. Our observations support the notion that malaria transmission reduction may be expedited by enhanced case management, involving both symptom-screening and infection detection.

Adipose tissue parasite sequestration drives leptin production in mice and correlates with human cerebral malaria.

Circulating levels of the adipokine leptin are linked to neuropathology in experimental cerebral malaria (ECM), but its source and regulation mechanism remain unknown. Here, we show that sequestration of infected red blood cells (iRBCs) in white adipose tissue (WAT) microvasculature increased local vascular permeability and leptin production. Mice infected with parasite strains that fail to sequester in WAT displayed reduced leptin production and protection from ECM. WAT sequestration and leptin induction were lost in CD36KO mice; however, ECM susceptibility revealed sexual dimorphism. Adipocyte leptin was regulated by the mechanistic target of rapamycin complex 1 (mTORC1) and blocked by rapamycin. In humans, although Plasmodium falciparum infection did not increase circulating leptin levels, iRBC sequestration, tissue leptin production, and mTORC1 activity were positively correlated with CM in pediatric postmortem WAT. These data identify WAT sequestration as a trigger for leptin production with potential implications for pathogenesis of malaria infection, prognosis, and treatment.

Plasmodium falciparum Gametocyte Density and Infectivity in Peripheral Blood and Skin Tissue of Naturally Infected Parasite Carriers in Burkina Faso.

BACKGROUND: Plasmodium falciparum transmission depends on mature gametocytes that can be ingested by mosquitoes taking a blood meal on human skin. Although gametocyte skin sequestration has long been hypothesized as important contributor to efficient malaria transmission, this has never been formally tested. METHODS: In naturally infected gametocyte carriers from Burkina Faso, we assessed infectivity to mosquitoes by direct skin feeding and membrane feeding. We directly quantified male and female gametocytes and asexual parasites in finger-prick and venous blood samples, skin biopsy samples, and in of mosquitoes that fed on venous blood or directly on skin. Gametocytes were visualized in skin tissue with confocal microscopy. RESULTS: Although more mosquitoes became infected when feeding directly on skin then when feeding on venous blood (odds ratio, 2.01; 95% confidence interval, 1.21-3.33; P = .007), concentrations of gametocytes were not higher in the subdermal skin vasculature than in other blood compartments; only sparse gametocytes were observed in skin tissue. DISCUSSION: Our data strongly suggest that there is no significant skin sequestration of P. falciparum gametocytes. Gametocyte densities in peripheral blood are thus informative for predicting onward transmission potential to mosquitoes and can be used to target and monitor malaria elimination initiatives.

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 59 P. vivax samples from Panama. The P. vivax samples were collected from two periods (2007-2009 and 2017-2019) 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 highly genetically related lineage (termed CL1) 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 (termed CL2) 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 Panamanian P. vivax samples not belonging to either CL1 or CL2 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.

Keras R-CNN: library for cell detection in biological images using deep neural networks.

BACKGROUND: A common yet still manual task in basic biology research, high-throughput drug screening and digital pathology is identifying the number, location, and type of individual cells in images. Object detection methods can be useful for identifying individual cells as well as their phenotype in one step. State-of-the-art deep learning for object detection is poised to improve the accuracy and efficiency of biological image analysis. RESULTS: We created Keras R-CNN to bring leading computational research to the everyday practice of bioimage analysts. Keras R-CNN implements deep learning object detection techniques using Keras and Tensorflow ( https://github.com/broadinstitute/keras-rcnn ). We demonstrate the command line tool's simplified Application Programming Interface on two important biological problems, nucleus detection and malaria stage classification, and show its potential for identifying and classifying a large number of cells. For malaria stage classification, we compare results with expert human annotators and find comparable performance. CONCLUSIONS: Keras R-CNN is a Python package that performs automated cell identification for both brightfield and fluorescence images and can process large image sets. Both the package and image datasets are freely available on GitHub and the Broad Bioimage Benchmark Collection.