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Abstract Background This study investigates the processes regarding changing malaria treatment policies in Vietnam. Moreover, it explores the feasibility of introducing triple artemisinin-based combination therapy (TACT) in Vietnam to support the national malaria control and elimination plan. Methods Data were collected via 12 in-depth interviews with key stakeholders, combined with a review of policy documents. Results TACT is considered as a useful backup strategy in case future treatment failures with current artemisinin-based combination therapy (ACT) would occur. Moreover, TACT is also considered as a promising strategy to prevent the re-establishment of malaria. However, regulatory procedures and implementation timelines for TACT were expected to be lengthy. Therefore, strategies to engage national decision-makers, regulators, and suppliers should be initiated soon, stipulating the benefits of TACT deployment. In Vietnam, a procedure to apply for an import permit without registration that has previously been applied to the introduction of artesunate-pyronaridine was proposed to accelerate the introduction of TACT. Global-level support through the World Health Organization recommendations and prequalification were considered critical for supporting the introduction of TACT in Vietnam. Conclusions Appropriate approach strategies and early stakeholder engagement will be needed to accelerate the introduction of TACT in Vietnam.

Abstract Background Widespread artemisinin resistance in Africa could be catastrophic when drawing parallels with the failure of chloroquine in the 1970s and 1980s. This article explores the role of anti-malarial market characteristics in the emergence and spread of arteminisin resistance in African countries, drawing on perspectives from Burkina Faso. Methods Data were collected through in-depth interviews and focus group discussions. A representative sample of national policy makers, regulators, public and private sector wholesalers, retailers, clinicians, nurses, and community members were purposively sampled. Additional information was also sought via review of policy publications and grey literature on anti-malarial policies and deployment practices in Burkina Faso. Results Thirty seven in-depth interviews and 6 focus group discussions were conducted. The study reveals that the current operational mode of anti-malarial drug markets in Burkina Faso promotes arteminisin resistance emergence and spread. The factors are mainly related to the artemisinin-based combination therapy (ACT) supply chain, to ACT quality, ACT prescription monitoring and to ACT access and misuse by patients. Conclusion Study findings highlight the urgent requirement to reform current characteristics of the anti-malarial drug market in order to delay the emergence and spread of artemisinin resistance in Burkina Faso. Four recommendations for public policy emerged during data analysis: (1) Address the suboptimal prescription of anti-malarial drugs, (2) Apply laws that prohibit the sale of anti-malarials without prescription, (3) Restrict the availability of street drugs, (4) Sensitize the population on the value of compliance regarding correct acquisition and intake of anti-malarials. Funding systems for anti-malarial drugs in terms of availability and accessibility must also be stabilized.

Abstract Background This is a qualitative study to identify implementation challenges for deploying triple artemisinin-based combination therapy (TACT) in the Greater Mekong Subregion (GMS) of Southeast Asia and to explore strategies to overcome these challenges. Methods In-depth interviews were conducted in three countries that have repeatedly been confronted with ACT failures: Cambodia, Vietnam, and Lao PDR. Thirty-nine key stakeholders in the healthcare systems in these countries were interviewed. One participatory workshop was conducted in Cambodia, where scenarios for potential TACT deployment were discussed. Results The results section is organized around four strategic themes that emerged from the data: policy support, data and evidence, logistics and operation, and downstream engagement. The study revealed that countries in the GMS currently rely on ACT to eliminate Plasmodium falciparum malaria by 2025. TACT is, however, considered to be a useful backup strategy in case of future treatment failures and to prevent the re-establishment of malaria. The study showed that a major challenge ahead is to engage decision makers and healthcare providers into deploying TACT, given the low case incidence of falciparum malaria in the GMS. Interview respondents were also skeptical whether healthcare providers would be willing to engage in new therapies for a disease they hardly encounter anymore. Hence, elaborate information dissemination strategies were considered appropriate and these strategies should especially target village malaria workers. Respondents proposed several regulatory and programmatic strategies to anticipate the formation of TACT markets in the GMS. These strategies include early dossier submission to streamline regulatory procedures, early stakeholder engagement strategies to shorten implementation timelines, and inclusion of TACT as second-line therapy to accelerate their introduction in case they are urgently needed. Conclusions This paper presents a qualitative study to identify implementation challenges for deploying TACT in the GMS and to explore strategies to overcome these challenges. The findings could benefit researchers and decision makers in strategizing towards potential future deployment of TACT in the GMS to combat artemisinin and partner drug resistance.

Abstract Increasing levels of artemisinin and partner drug resistance threaten malaria control and elimination globally. Triple artemisinin-based combination therapies (TACTs) which combine artemisinin derivatives with two partner drugs are efficacious and well tolerated in clinical trials, including in areas of multidrug-resistant malaria. Whether early TACT adoption could delay the emergence and spread of antimalarial drug resistance is a question of vital importance. Using two independent individual-based models of Plasmodium falciparum epidemiology and evolution, we evaluated whether introduction of either artesunate-mefloquine-piperaquine or artemether-lumefantrine-amodiaquine resulted in lower long-term artemisinin-resistance levels and treatment failure rates compared with continued ACT use. We show that introduction of TACTs could significantly delay the emergence and spread of artemisinin resistance and treatment failure, extending the useful therapeutic life of current antimalarial drugs, and improving the chances of malaria elimination. We conclude that immediate introduction of TACTs should be considered by policy makers in areas of emerging artemisinin resistance.

Abstract Background Recent reports of artemisinin partial resistance from Rwanda and Uganda are worrisome and suggest a future policy change to adopt new anti-malarials. This is a case study on the evolution, adoption, and implementation of new anti-malarial treatment policies in Nigeria. The main objective is to provide perspectives to enhance the future uptake of new anti-malarials, with an emphasis on stakeholder engagement strategies. Methods This case study is based on an analysis of policy documents and stakeholders’ perspectives drawn from an empirical study conducted in Nigeria, 2019–2020. A mixed methods approach was adopted, including historical accounts, review of programme and policy documents, and 33 qualitative in-depth interviews and 6 focus group discussions. Results Based on policy documents reviewed, the adoption of artemisinin-based combination therapy (ACT) in Nigeria was swift due to political will, funding and support from global developmental partners. However, the implementation of ACT was met with resistance from suppliers, distributors, prescribers, and end-users, attributed to market dynamics, costs and inadequate stakeholder engagement. Deployment of ACT in Nigeria witnessed increased developmental partner support, robust data generation, ACT case-management strengthening and evidence on anti-malarial use in severe malaria and antenatal care management. A framework for effective stakeholder engagement for the future adoption of new anti-malarial treatment strategies was proposed. The framework covers the pathway from generating evidence on drug efficacy, safety and uptake; to making treatment accessible and affordable to end-users. It addresses which stakeholders to engage with and the content of engagement strategies with key stakeholders at different levels of the transition process. Conclusion Early and staged engagement of stakeholders from global bodies to community level end-users is critical to the successful adoption and uptake of new anti-malarial treatment policies. A framework for these engagements was proposed as a contribution to enhancing the uptake of future anti-malarial strategies.

Plasmodium vivax is a major challenge for malaria control due to its wide geographic distribution, high frequency of submicroscopic infections, and ability to induce relapses due to the latent forms present in the liver (hypnozoites). Deepening our knowledge of parasite biology and its molecular components is key to develop new tools for malaria control and elimination. This study aims to investigate and characterize a P. vivax protein (PvVir14) for its role in parasite biology and its interactions with the immune system. We collected sera or plasma from P.vivax-infected subjects in Brazil (n = 121) and Cambodia (n = 55), and from P. falciparum-infected subjects in Mali (n = 28), to assess antibody recognition of PvVir14. Circulating antibodies against PvVir14 appeared in 61% and 34.5% of subjects from Brazil and Cambodia, respectively, versus none (0%) of the P. falciparum-infected subjects from Mali who have no exposure to P. vivax. IgG1 and IgG3 most frequently contributed to anti-PvVir14 responses. PvVir14 antibody levels correlated with those against other well-characterized sporozoite/liver (PvCSP) and blood stage (PvDBP-RII) antigens, which were recognized by 7.6% and 42% of Brazilians, respectively. Concerning the cellular immune profiling of Brazilian subjects, PvVir14 seroreactive individuals displayed significantly higher levels of circulating atypical (CD21- CD27-) B cells, raising the possibility that atypical B cells may be contribute to the PvVir14 antibody response. When analyzed at a single-cell level, the B cell receptor gene hIGHV3-23 was only seen in subjects with active P.vivax infection where it comprised 20% of V gene usage. Among T cells, CD4+ and CD8+ levels differed (lower and higher, respectively) between subjects with versus without antibodies to PvVir14, while NKT cell levels were higher in those without antibodies. Specific B cell subsets, anti-PvVir14 circulating antibodies, and NKT cell levels declined after treatment of P. vivax. This study provides the immunological characterization of PvVir14, a unique P. vivax protein, and possible association with acute host's immune responses, providing new information of specific host-parasite interaction. Trial registration: TrialClinicalTrials.gov Identifier: NCT00663546 & ClinicalTrials.gov NCT02334462.

Abstract Background Triple Artemisinin-based Combination Therapies (TACTs) are being developed as a response to artemisinin and partner drug resistance in Southeast Asia. However, the desirability, timing and practical feasibility of introducing TACTs in Southeast Asia is subject to debate. This study systematically assesses perspectives of malaria experts towards the introduction of TACTs as first-line treatment for uncomplicated falciparum malaria in Southeast Asia. Methods A two-round Delphi study was conducted. In the first round, 53 malaria experts answered open-ended questions on what they consider the most important advantages, disadvantages, and implementation barriers for introducing TACTs in Southeast Asia. In the second round, the expert panel rated the relevance of each statement on a 5-point Likert scale. Results Malaria experts identified 15 advantages, 15 disadvantages and 13 implementation barriers for introducing TACTs in Southeast Asia in the first round of data collection. In the second round, consensus was reached on 13 advantages (8 perceived as relevant, 5 as not-relevant), 12 disadvantages (10 relevant, 2 not-relevant), and 13 implementation barriers (all relevant). Advantages attributed highest relevance related to the clinical and epidemiological rationale of introducing TACTs. Disadvantages attributed highest relevance related to increased side-effects, unavailability of fixed-dose TACTs, and potential cost increases. Implementation barriers attributed highest relevance related to obtaining timely regulatory approval, timely availability of fixed-dose TACTs, and generating global policy support for introducing TACTs. Conclusions The study provides a structured oversight of malaria experts’ perceptions on the major advantages, disadvantages and implementation challenges for introducing TACTs in Southeast Asia, over current practices of rotating ACTs when treatment failure is observed. The findings can benefit strategic decision making in the battle against drug-resistant malaria.

Transcriptomic analysis of isolates from the malaria parasite (Plasmodium falciparum) in the Greater Mekong Subregion of Southeast Asia identifies gene expression patterns that are correlated with resistance to a common anti-malaria drug, artemisinin.

Point-of-care assays have greatly increased access to diagnostic information and improved healthcare outcomes globally, especially in the case of tropical diseases in rural settings. Increased recognition of the impact of these tools and increased funding, along with advances in technology have led to a surge in development of new assays. However, many new tools fail to fulfill their intended purpose due to a lack of clinical impact, operational feasibility, and input from envisioned operators. To be successful, they must fit into existing clinical decision-making models and be designed in collaboration with end users. We describe a case study of the development of a new low-cost sensor for antimalarial drugs, from initial planning through collection and incorporation of design feedback to final assay design. The assay uses an aptamer-based sensor to detect antimalarial drugs from patient samples for tracking antimalarial use in Southeast Asia, a region with a long history of emerging antimalarial drug resistance. Design and use-case input was collected from malaria control experts, researchers, and healthcare workers to develop target product profiles. Data was collected via surveys and in-person interviews during assay development and ultimately informed a change in assay format. This aptamer sensor platform can be easily adapted to detect other small molecule and protein targets and the design process described here can serve as a model for the development of effective new assays to improve access to healthcare technology.

We describe the MalariaGEN Pf7 data resource, the seventh release of Plasmodium falciparum genome variation data from the MalariaGEN network. It comprises over 20,000 samples from 82 partner studies in 33 countries, including several malaria endemic regions that were previously underrepresented. For the first time we include dried blood spot samples that were sequenced after selective whole genome amplification, necessitating new methods to genotype copy number variations. We identify a large number of newly emerging crt mutations in parts of Southeast Asia, and show examples of heterogeneities in patterns of drug resistance within Africa and within the Indian subcontinent. We describe the profile of variations in the C-terminal of the csp gene and relate this to the sequence used in the RTS,S and R21 malaria vaccines. Pf7 provides high-quality data on genotype calls for 6 million SNPs and short indels, analysis of large deletions that cause failure of rapid diagnostic tests, and systematic characterisation of six major drug resistance loci, all of which can be freely downloaded from the MalariaGEN website.

Abstract Malaria remains a major cause of morbidity and mortality in Africa, particularly in children under five years of age. Availability of effective anti-malarial drug treatment is a cornerstone for malaria control and eventual malaria elimination. Artemisinin-based combination therapy (ACT) is worldwide the first-line treatment for uncomplicated falciparum malaria, but the ACT drugs are starting to fail in Southeast Asia because of drug resistance. Resistance to artemisinins and their partner drugs could spread from Southeast Asia to Africa or emerge locally, jeopardizing the progress made in malaria control with the increasing deployment of ACT in Africa. The development of triple artemisinin-based combination therapy (TACT) could contribute to mitigating the risks of artemisinin and partner drug resistance on the African continent. However, there are pertinent ethical and practical issues that ought to be taken into consideration. In this paper, the most important ethical tensions, some implementation practicalities and preliminary thoughts on addressing them are discussed. The discussion draws upon data from randomized clinical studies using TACT combined with ethical principles, published literature and lessons learned from the introduction of artemisinin-based combinations in African markets.

The emergence and spread of artemisinin resistance has compromised antimalarial efficacy. Here, Mok et al. apply quantitative transcriptomics, proteomics, and metabolomics to provide evidence that K13 mutations alter multiple aspects of the parasite’s intra-erythrocytic development to enhance survival following artemisinin treatment.

This report describes the MalariaGEN Pv4 dataset, a new release of curated genome variation data on 1,895 samples of Plasmodium vivax collected at 88 worldwide locations between 2001 and 2017. It includes 1,370 new samples contributed by MalariaGEN and VivaxGEN partner studies in addition to previously published samples from these and other sources. We provide genotype calls at over 4.5 million variable positions including over 3 million single nucleotide polymorphisms (SNPs), as well as short indels and tandem duplications. This enlarged dataset highlights major compartments of parasite population structure, with clear differentiation between Africa, Latin America, Oceania, Western Asia and different parts of Southeast Asia. Each sample has been classified for drug resistance to sulfadoxine, pyrimethamine and mefloquine based on known markers at the dhfr, dhps and mdr1 loci. The prevalence of all of these resistance markers was much higher in Southeast Asia and Oceania than elsewhere. This open resource of analysis-ready genome variation data from the MalariaGEN and VivaxGEN networks is driven by our collective goal to advance research into the complex biology of P. vivax and to accelerate genomic surveillance for malaria control and elimination.

IntroductionUnderstanding the human immune response to Plasmodium falciparum gametocytes and its association with gametocytemia is essential for understanding the transmission of malaria as well as progressing transmission blocking vaccine candidates.MethodsIn a multi-national clinical efficacy trial of artemisinin therapies (13 sites of varying transmission over South-East Asia and Africa), we measured Immunoglobulin G (IgG) responses to recombinant P. falciparum gametocyte antigens expressed on the gametocyte plasma membrane and leading transmission blocking vaccine candidates Pfs230 (Pfs230c and Pfs230D1M) and Pfs48/45 at enrolment in 1,114 participants with clinical falciparum malaria. Mixed effects linear and logistic regression were used to determine the association between gametocyte measures (gametocytemia and gametocyte density) and antibody outcomes at enrolment.ResultsMicroscopy detectable gametocytemia was observed in 11% (127/1,114) of participants at enrolment, and an additional 9% (95/1,114) over the follow-up period (up to day 42) (total 20% of participants [222/1,114]). IgG levels in response to Pfs230c, Pfs48/45 and Pfs230D1M varied across study sites at enrolment (p < 0.001), as did IgG seroprevalence for anti-Pfs230c and D1M IgG (p < 0.001), but not for anti-Pfs48/45 IgG (p = 0.159). In adjusted analyses, microscopy detectable gametocytemia at enrolment was associated with an increase in the odds of IgG seropositivity to the three gametocyte antigens (Pfs230c OR [95% CI], p: 1.70 [1.10, 2.62], 0.017; Pfs48/45: 1.45 [0.85, 2.46], 0.174; Pfs230D1M: 1.70 [1.03, 2.80], 0.037), as was higher gametocyte density at enrolment (per two-fold change in gametocyte density Pfs230c OR [95% CI], p: 1.09 [1.02, 1.17], 0.008; Pfs48/45: 1.05 [0.98, 1.13], 0.185; Pfs230D1M: 1.07 [0.99, 1.14], 0.071).ConclusionPfs230 and Pfs48/45 antibodies are naturally immunogenic targets associated with patent gametocytemia and increasing gametocyte density across multiple malaria endemic settings, including regions with emerging artemisinin-resistant P. falciparum.

Abstract Plasmodium vivax gene regulation remains difficult to study due to the lack of a robust in vitro culture method, low parasite densities in peripheral circulation and asynchronous parasite development. We adapted an RNA-seq protocol “DAFT-seq” to sequence the transcriptome of four P. vivax field isolates that were cultured for a short period ex vivo before using a density gradient for schizont enrichment. Transcription was detected from 78% of the PvP01 reference genome, despite being schizont-enriched samples. This extensive data was used to define thousands of 5′ and 3′ untranslated regions, some of which overlapped with neighbouring transcripts, and to improve the gene models of 352 genes, including identifying 20 novel gene transcripts. This dataset has also significantly increased the known amount of heterogeneity between P. vivax schizont transcriptomes from individual patients. The majority of genes found to be differentially expressed between the isolates lack Plasmodium falciparum homologs and are predicted to be involved in host-parasite interactions, with an enrichment in reticulocyte binding proteins, merozoite surface proteins and exported proteins with unknown function. An improved understanding of the diversity within P. vivax transcriptomes will be essential for the prioritisation of novel vaccine targets.

Abstract Background Long regarded as an epicenter of drug-resistant malaria, Southeast Asia continues to provide new challenges to the control of Plasmodium falciparum malaria. Recently, resistance to the artemisinin combination therapy partner drug piperaquine has been observed in multiple locations across Southeast Asia. Genetic studies have identified single nucleotide polymorphisms as well as copy number variations in the plasmepsin 2 and plasmepsin 3 genes, which encode haemoglobin-degrading proteases that associate with clinical and in vitro piperaquine resistance. Results To accurately and quickly determine the presence of copy number variations in the plasmepsin 2/3 genes in field isolates, this study developed a quantitative PCR assay using TaqMan probes. Copy number estimates were validated using a separate SYBR green-based quantitative PCR assay as well as a novel PCR-based breakpoint assay to detect the hybrid gene product. Field samples from 2012 to 2015 across three sites in Cambodia were tested using DNA extracted from dried blood spots and whole blood to monitor the extent of plasmepsin 2/3 gene amplifications, as well as amplifications in the multidrug resistance transporter 1 gene (pfmdr1), a marker of mefloquine resistance. This study found high concordance across all methods of copy number detection. For samples derived from dried blood spots, a success rate greater than 80% was found in each assay, with more recent samples performing better. Evidence of extensive plasmepsin 2/3 copy number amplifications was observed in Pursat (94%, 2015) (Western Cambodia) and Preah Vihear (87%, 2014) (Northern Cambodia), and lower levels in Ratanakiri (16%, 2014) (Eastern Cambodia). A shift was observed from two copies of plasmepsin 2 in Pursat in 2013 to three copies in 2014–2015 (25% to 64%). Pfmdr1 amplifications were absent in all samples from Preah Vihear and Ratanakiri in 2014 and absent in Pursat in 2015. Conclusions The multiplex TaqMan assay is a robust tool for monitoring both plasmepsin 2/3 and pfmdr1 copy number variations in field isolates, and the SYBR-green and breakpoint assays are useful for monitoring plasmepsin 2/3 amplifications. This study shows increasing levels of plasmepsin 2 copy numbers across Cambodia from 2012 to 2015 and a complete reversion of multicopy pfmdr1 parasites to single copy parasites in all study locations.

Background Artemisinin-based combination therapies (ACTs) are the recommended treatment for uncomplicated Plasmodium falciparum malaria in all malaria endemic countries. Artemisinin resistance, partner drug resistance, and subsequent ACT failure are widespread in Southeast Asia. The more recent independent emergence of artemisinin resistance in Africa is alarming. In response, triple artemisinin-based combination therapies (TACTs) are being developed to mitigate the risks associated with increasing drug resistance. Since ACTs are still effective in Africa, where malaria is mainly a paediatric disease, the potential deployment of TACTs raises important ethical questions. This paper presents an analysis of stakeholders’ perspectives regarding key ethical considerations to be considered in the deployment of TACTs in Africa provided they are found to be safe, well-tolerated and effective for the treatment of uncomplicated malaria. Methods We conducted a qualitative study in Burkina Faso and Nigeria assessing stakeholders’ (policy makers, suppliers and end-users) perspectives on ethical issues regarding the potential future deployment of TACTs through 68 in-depth interviews and 11 focus group discussions. Findings Some respondents suggested that there should be evidence of local artemisinin resistance before they consider deploying TACTs, while others suggested that TACTs should be deployed to protect the efficacy of current ACTs. Respondents suggested that additional side effects of TACTs compared to ACTs should be minimal and the cost of TACTs to end-users should not be higher than the cost of current ACTs. There was some disagreement among respondents regarding whether patients should have a choice of treatment options between ACTs and TACTs or only have TACTs available, while ACTs are still effective. The study also suggests that community, public and stakeholder engagement activities are essential to support the introduction and effective uptake of TACTs. Conclusion Addressing ethical issues regarding TACTs and engaging early with stakeholders will be important for their potential deployment in Africa.

Background: National Malaria Control Programmes (NMCPs) currently make limited use of parasite genetic data. We have developed GenRe-Mekong, a platform for genetic surveillance of malaria in the Greater Mekong Subregion (GMS) that enables NMCPs to implement large-scale surveillance projects by integrating simple sample collection procedures in routine public health procedures. Methods: Samples from symptomatic patients are processed by SpotMalaria, a high-throughput system that produces a comprehensive set of genotypes comprising several drug resistance markers, species markers and a genomic barcode. GenRe-Mekong delivers Genetic Report Cards, a compendium of genotypes and phenotype predictions used to map prevalence of resistance to multiple drugs. Results: GenRe-Mekong has worked with NMCPs and research projects in eight countries, processing 9623 samples from clinical cases. Monitoring resistance markers has been valuable for tracking the rapid spread of parasites resistant to the dihydroartemisinin-piperaquine combination therapy. In Vietnam and Laos, GenRe-Mekong data have provided novel knowledge about the spread of these resistant strains into previously unaffected provinces, informing decision-making by NMCPs. Conclusions: GenRe-Mekong provides detailed knowledge about drug resistance at a local level, and facilitates data sharing at a regional level, enabling cross-border resistance monitoring and providing the public health community with valuable insights. The project provides a rich open data resource to benefit the entire malaria community. Funding: The GenRe-Mekong project is funded by the Bill and Melinda Gates Foundation (OPP11188166, OPP1204268). Genotyping and sequencing were funded by the Wellcome Trust (098051, 206194, 203141, 090770, 204911, 106698/B/14/Z) and Medical Research Council (G0600718). A proportion of samples were collected with the support of the UK Department for International Development (201900, M006212), and Intramural Research Program of the National Institute of Allergy and Infectious Diseases.

MalariaGEN is a data-sharing network that enables groups around the world to work together on the genomic epidemiology of malaria. Here we describe a new release of curated genome variation data on 7,000 Plasmodium falciparum samples from MalariaGEN partner studies in 28 malaria-endemic countries. High-quality genotype calls on 3 million single nucleotide polymorphisms (SNPs) and short indels were produced using a standardised analysis pipeline. Copy number variants associated with drug resistance and structural variants that cause failure of rapid diagnostic tests were also analysed. Almost all samples showed genetic evidence of resistance to at least one antimalarial drug, and some samples from Southeast Asia carried markers of resistance to six commonly-used drugs. Genes expressed during the mosquito stage of the parasite life-cycle are prominent among loci that show strong geographic differentiation. By continuing to enlarge this open data resource we aim to facilitate research into the evolutionary processes affecting malaria control and to accelerate development of the surveillance toolkit required for malaria elimination.

Abstract Background Mosquito-borne arboviruses, like dengue virus, continue to cause significant global morbidity and mortality, particularly in Southeast Asia. When the infectious mosquitoes probe into human skin for a blood meal, they deposit saliva containing a myriad of pharmacologically active compounds, some of which alter the immune response and influence host receptivity to infection, and consequently, the establishment of the virus. Previous reports have highlighted the complexity of mosquito vector-derived factors and immunity in the success of infection. Cumulative evidence from animal models and limited data from humans have identified various vector-derived components, including salivary components, that are co-delivered with the pathogen and play an important role in the dissemination of infection. Much about the roles and effects of these vector-derived factors remain to be discovered. Methods/Design We describe a longitudinal, pagoda (community)-based pediatric cohort study to evaluate the burden of dengue virus infection and document the immune responses to salivary proteins of Aedes aegypti, the mosquito vector of dengue, Zika, and chikungunya viruses. The study includes community-based seroprevalence assessments in the peri-urban town of Chbar Mon in Kampong Speu Province, Cambodia. The study aims to recruit 771 children between the ages of 2 and 9 years for a three year period of longitudinal follow-up, including twice per year (rainy and dry season) serosurveillance for dengue seroconversion and Ae. aegypti salivary gland homogenate antibody intensity determinations by ELISA assays. Diagnostic tests for acute dengue, Zika and chikungunya viral infections will be performed by RT-PCR. Discussion This study will serve as a foundation for further understanding of mosquito saliva immunity and its impact on Aedes-transmitted arboviral diseases endemic to Cambodia. Trial registration NCT03534245 registered on 23 May 2018.

Introduction: According to the World Malaria Report 2019, Africa accounts for 94% of the global malaria deaths. While malaria prevalence and mortality have declined over the years, recent reports suggest that these gains may stand the risk of being reversed if resistance to Artemisinin Combination Therapies (ACTs) spreads from Southeast Asia to Africa. Efforts are being made to develop new treatments that will address the looming threat of ACT resistance, including the development of triple artemisinin combination therapies (TACTs). The proposed study seeks to explore the views of stakeholders on the key ethical, regulatory and market-related issues that should be considered in the potential introduction of triple artemisinin combination therapies (TACTs) in Africa. Methods: The study employed qualitative research methods involving in-depth interviews and focus group discussions (FGDs) with stakeholders, who will be directly affected by the potential deployment of triple artemisinin combination treatments, as regulators, suppliers and end-users. Participants will be purposively selected and will include national regulatory authorities, national malaria control programs, clinicians, distributors and retailers as well as community members in selected districts in Burkina Faso and Nigeria. Discussion: The proposed study is unique in being one of the first studies that seeks to understand the ethical, social, regulatory and market position issues prior to the development of a prospective antimalarial medicine.

MalariaGEN is a data-sharing network that enables groups around the world to work together on the genomic epidemiology of malaria. Here we describe a new release of curated genome variation data on 7,000 Plasmodium falciparum samples from MalariaGEN partner studies in 28 malaria-endemic countries. High-quality genotype calls on 3 million single nucleotide polymorphisms (SNPs) and short indels were produced using a standardised analysis pipeline. Copy number variants associated with drug resistance and structural variants that cause failure of rapid diagnostic tests were also analysed. Almost all samples showed genetic evidence of resistance to at least one antimalarial drug, and some samples from Southeast Asia carried markers of resistance to six commonly-used drugs. Genes expressed during the mosquito stage of the parasite life-cycle are prominent among loci that show strong geographic differentiation. By continuing to enlarge this open data resource we aim to facilitate research into the evolutionary processes affecting malaria control and to accelerate development of the surveillance toolkit required for malaria elimination.

IntroductionTriple artemisinin-based combination therapies (TACTs) are being developed as a response to artemisinin and partner drug resistance in the treatment of falciparum malaria in Southeast Asia. In African countries, where current artemisinin-based combination therapies (ACTs) are still effective, TACTs have the potential to benefit the larger community and future patients by mitigating the risk of drug resistance. This study explores the extent to which the antimalarial drug markets in African countries are ready for a transition to TACTs.MethodsA qualitative study was conducted in Nigeria and Burkina Faso and comprised in-depth interviews (n = 68) and focus group discussions (n = 11) with key actor groups in the innovation system of antimalarial therapies.ResultsEvidence of ACT failure in African countries and explicit support for TACTs by the World Health Organization (WHO) and international funders were perceived important determinants for the market prospects of TACTs in Nigeria and Burkina Faso. At the country level, slow regulatory and implementation procedures were identified as potential barriers towards rapid TACTs deployment. Integrating TACTs in public sector distribution channels was considered relatively straightforward. More challenges were expected for integrating TACTs in private sector distribution channels, which are characterized by patient demand and profit motives. Finally, several affordability and acceptability issues were raised for which ACTs were suggested as a benchmark.ConclusionThe market prospects of TACTs in Nigeria and Burkina Faso will depend on the demonstration of the added value of TACTs over ACTs, their advocacy by the WHO, the inclusion of TACTs in financial and regulatory arrangements, and their alignment with current distribution and deployment practices. Further clinical, health-economic and feasibility studies are required to inform decision makers about the broader implications of a transition to TACTs in African counties. The recent reporting of artemisinin resistance and ACT failure in Africa might change important determinants of the market readiness for TACTs.

BackgroundThere is a high risk of Plasmodium vivax parasitaemia following treatment of falciparum malaria. Our study aimed to quantify this risk and the associated determinants using an individual patient data meta-analysis in order to identify populations in which a policy of universal radical cure, combining artemisinin-based combination therapy (ACT) with a hypnozoitocidal antimalarial drug, would be beneficial.Methods and findingsA systematic review of Medline, Embase, Web of Science, and the Cochrane Database of Systematic Reviews identified efficacy studies of uncomplicated falciparum malaria treated with ACT that were undertaken in regions coendemic for P. vivax between 1 January 1960 and 5 January 2018. Data from eligible studies were pooled using standardised methodology. The risk of P. vivax parasitaemia at days 42 and 63 and associated risk factors were investigated by multivariable Cox regression analyses. Study quality was assessed using a tool developed by the Joanna Briggs Institute. The study was registered in the International Prospective Register of Systematic Reviews (PROSPERO: CRD42018097400). In total, 42 studies enrolling 15,341 patients were included in the analysis, including 30 randomised controlled trials and 12 cohort studies. Overall, 14,146 (92.2%) patients had P. falciparum monoinfection and 1,195 (7.8%) mixed infection with P. falciparum and P. vivax. The median age was 17.0 years (interquartile range [IQR] = 9.0-29.0 years; range = 0-80 years), with 1,584 (10.3%) patients younger than 5 years. 2,711 (17.7%) patients were treated with artemether-lumefantrine (AL, 13 studies), 651 (4.2%) with artesunate-amodiaquine (AA, 6 studies), 7,340 (47.8%) with artesunate-mefloquine (AM, 25 studies), and 4,639 (30.2%) with dihydroartemisinin-piperaquine (DP, 16 studies). 14,537 patients (94.8%) were enrolled from the Asia-Pacific region, 684 (4.5%) from the Americas, and 120 (0.8%) from Africa. At day 42, the cumulative risk of vivax parasitaemia following treatment of P. falciparum was 31.1% (95% CI 28.9-33.4) after AL, 14.1% (95% CI 10.8-18.3) after AA, 7.4% (95% CI 6.7-8.1) after AM, and 4.5% (95% CI 3.9-5.3) after DP. By day 63, the risks had risen to 39.9% (95% CI 36.6-43.3), 42.4% (95% CI 34.7-51.2), 22.8% (95% CI 21.2-24.4), and 12.8% (95% CI 11.4-14.5), respectively. In multivariable analyses, the highest rate of P. vivax parasitaemia over 42 days of follow-up was in patients residing in areas of short relapse periodicity (adjusted hazard ratio [AHR] = 6.2, 95% CI 2.0-19.5; p = 0.002); patients treated with AL (AHR = 6.2, 95% CI 4.6-8.5; p < 0.001), AA (AHR = 2.3, 95% CI 1.4-3.7; p = 0.001), or AM (AHR = 1.4, 95% CI 1.0-1.9; p = 0.028) compared with DP; and patients who did not clear their initial parasitaemia within 2 days (AHR = 1.8, 95% CI 1.4-2.3; p < 0.001). The analysis was limited by heterogeneity between study populations and lack of data from very low transmission settings. Study quality was high.ConclusionsIn this meta-analysis, we found a high risk of P. vivax parasitaemia after treatment of P. falciparum malaria that varied significantly between studies. These P. vivax infections are likely attributable to relapses that could be prevented with radical cure including a hypnozoitocidal agent; however, the benefits of such a novel strategy will vary considerably between geographical areas.

Piperaquine (PPQ) resistance of Plasmodium is an increasing problem. Here, Bopp et al. find a bimodal dose−response curve of Cambodian isolates exposed to PPQ, with the area under the curve correlating with in vitro PPQ resistance, and show the importance of Plasmepsin II–III copy number to PPQ resistance.

Abstract Background Artemisinin resistance is associated with delayed parasite clearance half-life in vivo and correlates with ring-stage survival under dihydroartemisinin in vitro. Both phenotypes are associated with mutations in the PF3D7_1343700 pfkelch13 gene. Recent spread of artemisinin resistance and emerging piperaquine resistance in Southeast Asia show that artemisinin combination therapy, such as dihydroartemisinin–piperaquine, are losing clinical effectiveness, prompting investigation of drug resistance mechanisms and development of strategies to surmount emerging anti-malarial resistance. Methods Sixty-eight parasites isolates with in vivo clearance data were obtained from two Tracking Resistance to Artemisinin Collaboration study sites in Cambodia, culture-adapted, and genotyped for pfkelch13 and other mutations including pfmdr1 copy number; and the RSA0–3h survival rates and response to antimalarial drugs in vitro were measured for 36 of these isolates. Results Among these 36 parasites one isolate demonstrated increased ring-stage survival for a PfKelch13 mutation (D584V, RSA0–3h = 8%), previously associated with slow clearance but not yet tested in vitro. Several parasites exhibited increased ring-stage survival, yet lack pfkelch13 mutations, and one isolate showed evidence for piperaquine resistance. Conclusions This study of 68 culture-adapted Plasmodium falciparum clinical isolates from Cambodia with known clearance values, associated the D584V PfKelch13 mutation with increased ring-stage survival and identified parasites that lack pfkelch13 mutations yet exhibit increased ring-stage survival. These data suggest mutations other than those found in pfkelch13 may be involved in conferring artemisinin resistance in P. falciparum. Piperaquine resistance was also detected among the same Cambodian samples, consistent with reports of emerging piperaquine resistance in the field. These culture-adapted parasites permit further investigation of mechanisms of both artemisinin and piperaquine resistance and development of strategies to prevent or overcome anti-malarial resistance.

Plasmodium vivax malaria incidence has increased in Latin America and Asia and is responsible for nearly 74.1% of malaria cases in Latin America. Immune responses to P. vivax are less well characterized than those to P. falciparum, partly because P. vivax is more difficult to cultivate in the laboratory. While antibodies are known to play an important role in P. vivax disease control, few studies have evaluated responses to P. vivax sexual stage antigens. We collected sera or plasma samples from P. vivax-infected subjects from Brazil (n = 70) and Cambodia (n = 79) to assess antibody responses to domain 1 of the gametocyte/gamete stage protein Pvs230 (Pvs230D1M). We found that 27.1% (19/70) and 26.6% (21/79) of subjects from Brazil and Cambodia, respectively, presented with detectable antibody responses to Pvs230D1M antigen. The most frequent subclasses elicited in response to Pvs230D1M were IgG1 and IgG3. Although age did not correlate significantly with Pvs230D1M antibody levels overall, we observed significant differences between age strata. Hemoglobin concentration inversely correlated with Pvs230D1M antibody levels in Brazil, but not in Cambodia. Additionally, we analyzed the antibody response against Pfs230D1M, the P. falciparum ortholog of Pvs230D1M. We detected antibodies to Pfs230D1M in 7.2 and 16.5% of Brazilian and Cambodian P. vivax-infected subjects. Depletion of Pvs230D1M IgG did not impair the response to Pfs230D1M, suggesting pre-exposure to P. falciparum, or co-infection. We also analyzed IgG responses to sporozoite protein PvCSP (11.4 and 41.8% in Brazil and Cambodia, respectively) and to merozoite protein PvDBP-RII (67.1 and 48.1% in Brazil and Cambodia, respectively), whose titers also inversely correlated with hemoglobin concentration only in Brazil. These data establish patterns of seroreactivity to sexual stage Pvs230D1M and show similar antibody responses among P. vivax-infected subjects from regions of differing transmission intensity in Brazil and Cambodia.

BACKGROUND:Plasmodium vivax causes the majority of malaria episodes outside Africa, but remains a relatively understudied pathogen. The pathology of P. vivax infection depends critically on the parasite's ability to recognize and invade human erythrocytes. This invasion process involves an interaction between P. vivax Duffy Binding Protein (PvDBP) in merozoites and the Duffy antigen receptor for chemokines (DARC) on the erythrocyte surface. Whole-genome sequencing of clinical isolates recently established that some P. vivax genomes contain two copies of the PvDBP gene. The frequency of this duplication is particularly high in Madagascar, where there is also evidence for P. vivax infection in DARC-negative individuals. The functional significance and global prevalence of this duplication, and whether there are other copy number variations at the PvDBP locus, is unknown. METHODOLOGY/PRINCIPAL FINDINGS:Using whole-genome sequencing and PCR to study the PvDBP locus in P. vivax clinical isolates, we found that PvDBP duplication is widespread in Cambodia. The boundaries of the Cambodian PvDBP duplication differ from those previously identified in Madagascar, meaning that current molecular assays were unable to detect it. The Cambodian PvDBP duplication did not associate with parasite density or DARC genotype, and ranged in prevalence from 20% to 38% over four annual transmission seasons in Cambodia. This duplication was also present in P. vivax isolates from Brazil and Ethiopia, but not India. CONCLUSIONS/SIGNIFICANCE:PvDBP duplications are much more widespread and complex than previously thought, and at least two distinct duplications are circulating globally. The same duplication boundaries were identified in parasites from three continents, and were found at high prevalence in human populations where DARC-negativity is essentially absent. It is therefore unlikely that PvDBP duplication is associated with infection of DARC-negative individuals, but functional tests will be required to confirm this hypothesis.

Nature Communications 6: Article number: 8614 (2015); Published: 20 October 2015; Updated: 8 January 2016. In the first paragraph of the Results section in this Article, the An. gambiae G3 line is incorrectly stated to have been artificially selected for permissiveness to infection by multiple P. falciparum strains.

α-Thalassemia results from decreased production of α-globin chains that make up part of hemoglobin tetramers (Hb; α(2)β(2)) and affects up to 50% of individuals in some regions of sub-Saharan Africa. Heterozygous (-α/αα) and homozygous (-α/-α) genotypes are associated with reduced risk of severe Plasmodium falciparum malaria, but the mechanism of this protection remains obscure. We hypothesized that α-thalassemia impairs the adherence of parasitized red blood cells (RBCs) to microvascular endothelial cells (MVECs) and monocytes--two interactions that are centrally involved in the pathogenesis of severe disease.We obtained P. falciparum isolates directly from Malian children with malaria and used them to infect αα/αα (normal), -α/αα and -α/-α RBCs. We also used laboratory-adapted P. falciparum clones to infect -/-α RBCs obtained from patients with HbH disease. Following a single cycle of parasite invasion and maturation to the trophozoite stage, we tested the ability of parasitized RBCs to bind MVECs and monocytes. Compared to parasitized αα/αα RBCs, we found that parasitized -α/αα, -α/-α and -/-α RBCs showed, respectively, 22%, 43% and 63% reductions in binding to MVECs and 13%, 33% and 63% reductions in binding to monocytes. α-Thalassemia was associated with abnormal display of P. falciparum erythrocyte membrane protein 1 (PfEMP1), the parasite's main cytoadherence ligand and virulence factor, on the surface of parasitized RBCs.Parasitized α-thalassemic RBCs show PfEMP1 display abnormalities that are reminiscent of those on the surface of parasitized sickle HbS and HbC RBCs. Our data suggest a model of malaria protection in which α-thalassemia ameliorates the pro-inflammatory effects of cytoadherence. Our findings also raise the possibility that other unstable hemoglobins such as HbE and unpaired α-globin chains (in the case of β-thalassemia) protect against life-threatening malaria by a similar mechanism.

In Africa, infant susceptibility to Plasmodium falciparum malaria increases substantially as fetal hemoglobin (HbF) and maternal immune IgG disappear from circulation. During the first few months of life, however, resistance to malaria is evidenced by extremely low parasitemias, the absence of fever, and the almost complete lack of severe disease. This resistance has previously been attributed in part to poor parasite growth in HbF-containing red blood cells (RBCs). A specific role for maternal immune IgG in infant resistance to malaria has been hypothesized but not yet identified.We found that P. falciparum parasites invade and develop normally in fetal (cord blood, CB) RBCs, which contain up to 95% HbF. However, these parasitized CB RBCs are impaired in their binding to human microvascular endothelial cells (MVECs), monocytes, and nonparasitized RBCs--cytoadherence interactions that have been implicated in the development of high parasite densities and the symptoms of malaria. Abnormal display of the parasite's cytoadherence antigen P. falciparum erythrocyte membrane protein-1 (PfEMP-1) on CB RBCs accounts for these findings and is reminiscent of that on HbC and HbS RBCs. IgG purified from the plasma of immune Malian adults almost completely abolishes the adherence of parasitized CB RBCs to MVECs.Our data suggest a model of malaria protection in which HbF and maternal IgG act cooperatively to impair the cytoadherence of parasitized RBCs in the first few months of life. In highly malarious areas of Africa, an infant's contemporaneous expression of HbC or HbS and development of an immune IgG repertoire may effectively reconstitute the waning protective effects of HbF and maternal immune IgG, thereby extending the malaria resistance of infancy into early childhood.

The diversity in the Plasmodium falciparum genome can be used to explore parasite population dynamics, with practical applications to malaria control. The ability to identify the geographic origin and trace the migratory patterns of parasites with clinically important phenotypes such as drug resistance is particularly relevant. With increasing single-nucleotide polymorphism (SNP) discovery from ongoing Plasmodium genome sequencing projects, a demand for high SNP and sample throughput genotyping platforms for large-scale population genetic studies is required. Low parasitaemias and multiple clone infections present a number of challenges to genotyping P. falciparum. We addressed some of these issues using a custom 384-SNP Illumina GoldenGate assay on P. falciparum DNA from laboratory clones (long-term cultured adapted parasite clones), short-term cultured parasite isolates and clinical (non-cultured isolates) samples from East and West Africa, Southeast Asia and Oceania. Eighty percent of the SNPs (n = 306) produced reliable genotype calls on samples containing as little as 2 ng of total genomic DNA and on whole genome amplified DNA. Analysis of artificial mixtures of laboratory clones demonstrated high genotype calling specificity and moderate sensitivity to call minor frequency alleles. Clear resolution of geographically distinct populations was demonstrated using Principal Components Analysis (PCA), and global patterns of population genetic diversity were consistent with previous reports. These results validate the utility of the platform in performing population genetic studies of P. falciparum.