Plasmodium vivax Strains Use Alternative Pathways for Invasion.

Plasmodium vivax has 2 invasion ligand/host receptor pathways (P. vivax Duffy-binding protein/Duffy antigen receptor for chemokines [DARC] and P. vivax reticulocyte binding protein 2b/transferrin receptor [TfR1]) that are promising targets for therapeutic intervention. We optimized invasion assays with isogenic cultured reticulocytes. Using a receptor blockade approach with multiple P. vivax isolates, we found that all strains utilized both DARC and TfR1, but with significant variation in receptor usage. This suggests that P. vivax, like Plasmodium falciparum, uses alternative invasion pathways, with implications for pathogenesis and vaccine development.

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.

Generation of an immortalized erythroid progenitor cell line from peripheral blood: A model system for the functional analysis of Plasmodium spp. invasion.

Malaria pathogenesis is caused by the replication of Plasmodium parasites within the red blood cells (RBCs) of the vertebrate host. This selective pressure has favored the evolution of protective polymorphisms in erythrocyte proteins, a subset of which serve as cognate receptors for parasite invasion ligands. Recently, the generation of RBCs from immortalized hematopoietic stem cells (HSCs) has offered a more tractable system for genetic manipulation and long-term in vitro culture, enabling elucidation of the functional determinants of host susceptibility in vitro. Here we report the generation of an immortalized erythroid progenitor cell line (EJ cells) from as few as 100 000 peripheral blood mononuclear cells. It offers a robust method for the creation of customized model systems from small volumes of peripheral blood. The EJ cell differentiation mirrored erythropoiesis of primary HSCs, yielding orthochromatic erythroblasts and enucleated RBCs after eight days (ejRBCs). The ejRBCs supported invasion by both P. vivax and P. falciparum. To demonstrate the genetic tractability of this system, we used CRISPR/Cas9 to disrupt the Duffy Antigen/Receptor for Chemokines (DARC) gene, which encodes the canonical receptor of P. vivax in humans. Invasion of P. vivax into this DARC-knockout cell line was strongly inhibited providing direct genetic evidence that P. vivax requires DARC for RBC invasion. Further, genetic complementation of DARC restored P. vivax invasion. Taken together, the peripheral blood immortalization method presented here offers the capacity to generate biologically representative model systems for studies of blood-stage malaria invasion from the peripheral blood of donors harboring unique genetic backgrounds, or rare polymorphisms.

Enhanced Ex Vivo Plasmodium vivax Intraerythrocytic Enrichment and Maturation for Rapid and Sensitive Parasite Growth Assays.

Plasmodium vivax chloroquine resistance has been documented in nearly every region where this malaria-causing parasite is endemic. Unfortunately, P. vivax resistance surveillance and drug discovery are challenging due to the low parasitemias of patient isolates and poor parasite survival through ex vivo maturation that reduce the sensitivity and scalability of current P. vivax antimalarial assays. Using cryopreserved patient isolates from Brazil and fresh patient isolates from India, we established a robust enrichment method for P. vivax parasites. We next performed a medium screen for formulations that enhance ex vivo survival. Finally, we optimized an isotopic metabolic labeling assay for measuring P. vivax maturation and its sensitivity to antimalarials. A KCl Percoll density gradient enrichment method increased parasitemias from small-volume ex vivo isolates by an average of >40-fold. The use of Iscove's modified Dulbecco's medium for P. vivax ex vivo culture approximately doubled the parasite survival through maturation. Coupling these with [3H]hypoxanthine metabolic labeling permitted sensitive and robust measurements of parasite maturation, which was used to measure the sensitivities of Brazilian P. vivax isolates to chloroquine and several novel antimalarials. These techniques can be applied to rapidly and robustly assess the P. vivax isolate sensitivities to antimalarials for resistance surveillance and drug discovery.

Additional PfCRT mutations driven by selective pressure for improved fitness can result in the loss of piperaquine resistance and altered Plasmodium falciparum physiology.

IMPORTANCE: Our study leverages gene editing techniques in Plasmodium falciparum asexual blood stage parasites to profile novel mutations in mutant PfCRT, an important mediator of piperaquine resistance, which developed in Southeast Asian field isolates or in parasites cultured for long periods of time. We provide evidence that increased parasite fitness of these lines is the primary driver for the emergence of these PfCRT variants. These mutations differentially impact parasite susceptibility to piperaquine and chloroquine, highlighting the multifaceted effects of single point mutations in this transporter. Molecular features of drug resistance and parasite physiology were examined in depth using proteoliposome-based drug uptake studies and peptidomics, respectively. Energy minimization calculations, showing how these novel mutations might impact the PfCRT structure, suggested a small but significant effect on drug interactions. This study reveals the subtle interplay between antimalarial resistance, parasite fitness, PfCRT structure, and intracellular peptide availability in PfCRT-mediated parasite responses to changing drug selective pressures.

Rethinking integrated service delivery for malaria.

Despite worldwide efforts and much progress toward malaria control, declines in malaria morbidity and mortality have hit a plateau. While many nations achieved significant malaria suppression or even elimination, success has been uneven, and other nations have made little headway-or even lost ground in this battle. These alarming trends threaten to derail the attainment of global targets for malaria control. Among the challenges impeding success in malaria reduction, many strategies center malaria as a set of technical problems in commodity development and delivery. Yet, this narrow perspective overlooks the importance of strong health systems and robust healthcare delivery. This paper argues that strategies that move the needle on health services and behaviors offer a significant opportunity to achieve malaria control through a comprehensive approach that integrates malaria with broader health services efforts. Indeed, malaria may serve as the thread that weaves integrated service delivery into a path forward for universal health coverage. Using key themes identified by the "Rethinking Malaria in the Context of COVID-19" effort through engagement with key stakeholders, we provide recommendations for pursuing integrated service delivery that can advance malaria control via strengthening health systems, increasing visibility and use of high-quality data at all levels, centering issues of equity, promoting research and innovation for new tools, expanding knowledge on effective implementation strategies for interventions, making the case for investing in malaria among stakeholders, and engaging impacted communities and nations.

Re-Envisioning Anti-Apicomplexan Parasite Drug Discovery Approaches.

Parasites of the phylum Apicomplexa impact humans in nearly all parts of the world, causing diseases including to toxoplasmosis, cryptosporidiosis, babesiosis, and malaria. Apicomplexan parasites have complex life cycles comprised of one or more stages characterized by rapid replication and biomass amplification, which enables accelerated evolutionary adaptation to environmental changes, including to drug pressure. The emergence of drug resistant pathogens is a major looming and/or active threat for current frontline chemotherapies, especially for widely used antimalarial drugs. In fact, resistant parasites have been reported against all modern antimalarial drugs within 15 years of clinical introduction, including the current frontline artemisinin-based combination therapies. Chemotherapeutics are a major tool in the public health arsenal for combatting the onset and spread of apicomplexan diseases. All currently approved antimalarial drugs have been discovered either through chemical modification of natural products or through large-scale screening of chemical libraries for parasite death phenotypes, and so far, none have been developed through a gene-to-drug pipeline. However, the limited duration of efficacy of these drugs in the field underscores the need for new and innovative approaches to discover drugs that can counter rapid resistance evolution. This review details both historical and current antimalarial drug discovery approaches. We also highlight new strategies that may be employed to discover resistance-resistant drug targets and chemotherapies in order to circumvent the rapid evolution of resistance in apicomplexan parasites.

Monitoring Plasmodium vivax resistance to antimalarials: Persisting challenges and future directions.

Emerging antimalarial drug resistance may undermine current efforts to control and eliminate Plasmodium vivax, the most geographically widespread yet neglected human malaria parasite. Endemic countries are expected to assess regularly the therapeutic efficacy of antimalarial drugs in use in order to adjust their malaria treatment policies, but proper funding and trained human resources are often lacking to execute relatively complex and expensive clinical studies, ideally complemented by ex vivo assays of drug resistance. Here we review the challenges for assessing in vivo P. vivax responses to commonly used antimalarials, especially chloroquine and primaquine, in the presence of confounding factors such as variable drug absorption, metabolism and interaction, and the risk of new infections following successful radical cure. We introduce a simple modeling approach to quantify the relative contribution of relapses and new infections to recurring parasitemias in clinical studies of hypnozoitocides. Finally, we examine recent methodological advances that may render ex vivo assays more practical and widely used to confirm P. vivax drug resistance phenotypes in endemic settings and review current approaches to the development of robust genetic markers for monitoring chloroquine resistance in P. vivax populations.

Plasmodium vivax infection compromises reticulocyte stability.

The structural integrity of the host red blood cell (RBC) is crucial for propagation of Plasmodium spp. during the disease-causing blood stage of malaria infection. To assess the stability of Plasmodium vivax-infected reticulocytes, we developed a flow cytometry-based assay to measure osmotic stability within characteristically heterogeneous reticulocyte and P. vivax-infected samples. We find that erythroid osmotic stability decreases during erythropoiesis and reticulocyte maturation. Of enucleated RBCs, young reticulocytes which are preferentially infected by P. vivax, are the most osmotically stable. P. vivax infection however decreases reticulocyte stability to levels close to those of RBC disorders that cause hemolytic anemia, and to a significantly greater degree than P. falciparum destabilizes normocytes. Finally, we find that P. vivax new permeability pathways contribute to the decreased osmotic stability of infected-reticulocytes. These results reveal a vulnerability of P. vivax-infected reticulocytes that could be manipulated to allow in vitro culture and develop novel therapeutics.

Plasmodium vivax transcriptional profiling of low input cryopreserved isolates through the intraerythrocytic development cycle.

Approximately one-third of the global population is at risk of Plasmodium vivax infection, and an estimated 7.51 million cases were reported in 2017. Although, P. vivax research is currently limited by the lack of a robust continuous in vitro culture system for this parasite, recent work optimizing short-term ex vivo culture of P. vivax from cryopreserved isolates has facilitated quantitative assays on synchronous parasites. Pairing this improved culture system with low-input Smart-seq2 RNAseq library preparation, we sought to determine whether transcriptional profiling of P. vivax would provide insight into the differential survival of parasites in different culture media. To this end we probed the transcriptional signature of three different ex vivo P. vivax samples in four different culture media using only 1000 cells for each time point taken during the course of the intraerythrocytic development cycle (IDC). Using this strategy, we achieved similar quality transcriptional data to previously reported P. vivax transcriptomes. We found little effect with varying culture media on parasite transcriptional signatures, identified many novel gametocyte-specific genes from transcriptomes of FACS-isolated gametocytes, and determined invasion ligand expression in schizonts in biological isolates and across the IDC. In total, these data demonstrate the feasibility and utility of P. vivax RNAseq-based transcriptomic studies using minimal biomass input to maximize experimental capacity.

Molecular and cellular interactions defining the tropism of Plasmodium vivax for reticulocytes.

Plasmodium vivax is uniquely restricted to invading reticulocytes, the youngest of red blood cells. Parasite invasion relies on the sequential deployment of multiple parasite invasion ligands. Correct targeting of the host reticulocyte is mediated by two families of invasion ligands: the reticulocyte binding proteins (RBPs) and erythrocyte binding proteins (EBPs). The Duffy receptor has long been established as a key determinant for P. vivax invasion. However, recently, the RBP protein PvRBP2b has been shown to bind to transferrin receptor, which is expressed on reticulocytes but lost on normocytes, implicating the ligand-receptor in the reticulocyte tropism of P. vivax. Furthermore there is increasing evidence for P. vivax growth and sexual development in reticulocyte-enriched tissues such as the bone marrow.