Population genomic evidence of structured and connected Plasmodium vivax populations under host selection in Latin America.

Pathogen genomic epidemiology has the potential to provide a deep understanding of population dynamics, facilitating strategic planning of interventions, monitoring their impact, and enabling timely responses, and thereby supporting control and elimination efforts of parasitic tropical diseases. Plasmodium vivax, responsible for most malaria cases outside Africa, shows high genetic diversity at the population level, driven by factors like sub-patent infections, a hidden reservoir of hypnozoites, and early transmission to mosquitoes. While Latin America has made significant progress in controlling Plasmodium falciparum, it faces challenges with residual P. vivax. To characterize genetic diversity and population structure and dynamics, we have analyzed the largest collection of P. vivax genomes to date, including 1474 high-quality genomes from 31 countries across Asia, Africa, Oceania, and America. While P. vivax shows high genetic diversity globally, Latin American isolates form a distinctive population, which is further divided into sub-populations and occasional clonal pockets. Genetic diversity within the continent was associated with the intensity of transmission. Population differentiation exists between Central America and the North Coast of South America, vs. the Amazon Basin, with significant gene flow within the Amazon Basin, but limited connectivity between the Northwest Coast and the Amazon Basin. Shared genomic regions in these parasite populations indicate adaptive evolution, particularly in genes related to DNA replication, RNA processing, invasion, and motility - crucial for the parasite's survival in diverse environments. Understanding these population-level adaptations is crucial for effective control efforts, offering insights into potential mechanisms behind drug resistance, immune evasion, and transmission dynamics.

Prevalence and correlates of childhood anemia in the MINA-Brazil birth cohort study.

OBJECTIVE: This study aimed to describe the prevalence and predictors of childhood anemia in an Amazonian population-based birth cohort study. METHODS: Prevalence of maternal anemia was estimated at delivery (hemoglobin [Hb] concentration < 110 g/L) in women participating in the MINA-Brazil birth cohort study and in their children, examined at ages one, two (Hb < 110 g/L), and five (Hb < 115 g/L). Moreover, ferritin, soluble transferrin receptor, and C-reactive protein concentrations were measured in mothers at delivery and in their 1- and 2-year-old children to estimate the prevalence of iron deficiency and its contribution to anemia, while adjusting for potential confounders by multiple Poisson regression analysis (adjusted relative risk [RRa]). RESULTS: The prevalence 95% confidence interval (CI) of maternal anemia, iron deficiency, and iron-deficiency anemia at delivery were 17.3% (14.0-21.0%), 42.6% (38.0-47.2%), and 8.7% (6.3-11.6)%, respectively (n = 462). At one year of age (n = 646), 42.2% (38.7-45.8%) of the study children were anemic, 38.4% (34.6-42.3%) were iron-deficient, and 26.3 (23.0-29.9) had iron-deficiency anemia. At two years of age (n = 761), these values decreased to 12.8% (10.6-15.2%), 18.1% (15.5-21.1%), and 4.1% (2.8-5.7%), respectively; at five years of age (n = 655), 5.2% (3.6-7.2%) were anemic. Iron deficiency (RRa = 2.19; 95%CI: 1.84-2.60) and consumption of ultra-processed foods (UPF) (RRa = 1.56; 95%CI: 1.14-2.13) were significant contributors to anemia at 1 year, after adjusting for maternal schooling. At 2 years, anemia was significantly associated with maternal anemia at delivery (RRa: 1.67; 95%CI: 1.17-2.39), malaria since birth (2.25; 1.30-3.87), and iron deficiency (2.15; 1.47-3.15), after adjusting for children's age and household wealth index. CONCLUSIONS: Anemia continues to be highly prevalent during pregnancy and early childhood in the Amazon. Public health policies should address iron deficiency, UPF intake, maternal anemia, and malaria to prevent and treat anemia in Amazonian children.

Changing Clinical Epidemiology of Plasmodium vivax Malaria as Transmission Decreases: Population-Based Prospective Panel Survey in the Brazilian Amazon.

BACKGROUND: Malarial infections are often missed by microscopy, and most parasite carriers are asymptomatic in low-endemicity settings. Whether parasite detectability and its ability to elicit symptoms change as transmission declines remains unclear. METHODS: We performed a prospective panel survey with repeated measurements on the same participants over 12 months to investigate whether Plasmodium vivax detectability by microscopy and risk of symptoms upon infection varied during a community-wide larviciding intervention in the Amazon basin of Brazil that markedly reduced vector density. We screened 1096 to 1400 residents in the intervention site for malaria by microscopy and quantitative TaqMan assays at baseline and twice during intervention. RESULTS: We found that more P vivax infections than expected from their parasite densities measured by TaqMan assays were missed by microscopy as transmission decreased. At lower transmission, study participants appeared to tolerate higher P vivax loads without developing symptoms. We hypothesize that changes in the ratio between circulating parasites and those that accumulate in the bone marrow and spleen, by avoiding peripheral blood microscopy detection, account for decreased parasite detectability and lower risk of symptoms under low transmission. CONCLUSIONS: P vivax infections are more likely to be subpatent and remain asymptomatic as malaria transmission decreases.

Impact of piperaquine resistance in Plasmodium falciparum on malaria treatment effectiveness in The Guianas: a descriptive epidemiological study.

BACKGROUND: Plasmodium falciparum is an apicomplexan parasite responsible for lethal cases of malaria. According to WHO recommendations, P falciparum cases are treated with artemisinin-based combination therapy including dihydroartemisinin-piperaquine. However, the emergence of resistant parasites against dihydroartemisinin-piperaquine was reported in southeast Asia in 2008 and, a few years later, suspected in South America. METHODS: To characterise resistance emergence, a treatment efficacy study was performed on the reported patients infected with P falciparum and treated with dihydroartemisinin-piperaquine in French Guiana (n=6, 2016-18). Contemporary isolates collected in French Guiana were genotyped for P falciparum chloroquine resistance transporter (pfCRT; n=845) and pfpm2 and pfpm3 copy number (n=231), phenotyped using the in vitro piperaquine survival assay (n=86), and analysed through genomic studies (n=50). Additional samples from five Amazonian countries and one outside the region were genotyped (n=1440). FINDINGS: In field isolates, 40 (47%) of 86 (95% CI 35·9-57·1) were resistant to piperaquine in vitro; these phenotypes were more associated with pfCRTC350R (ie, Cys350Arg) and pfpm2 and pfpm3 amplifications (Dunn test, p<0·001). Those markers were also associated with dihydroartemisinin-piperaquine treatment failure (n=3 [50%] of 6). A high prevalence of piperaquine resistance markers was observed in Suriname in 19 (83%) of 35 isolates and in Guyana in 579 (73%) of 791 isolates. The pfCRTC350R mutation emerged before pfpm2 and pfpm3 amplification in a temporal sequence different from southeast Asia, and in the absence of artemisinin partial resistance, suggesting a geographically distinctive epistatic relationship between these genetic markers. INTERPRETATION: The high prevalence of piperaquine resistance markers in parasite populations of the Guianas, and the risk of associated therapeutic failures calls for caution on dihydroartemisinin-piperaquine use in the region. Furthermore, greater attention should be given to potential differences in genotype to phenotype mapping across genetically distinct parasite populations from different continents. FUNDING: Pan American Health Organization and WHO, French Ministry for Research, European Commission, Santé publique France, Agence Nationale de la Recherche, Fundação de Amparo à Pesquisa do Estado do Amazonas, Ministry of Health of Brazil, Oswaldo Cruz Foundation, and National Institutes of Health. TRANSLATIONS: For the French and Portuguese translations of the abstract see Supplementary Materials section.

The structure of a Plasmodium vivax Tryptophan Rich Antigen domain suggests a lipid binding function for a pan-Plasmodium multi-gene family.

Tryptophan Rich Antigens (TRAgs) are encoded by a multi-gene family found in all Plasmodium species, but are significantly expanded in P. vivax and closely related parasites. We show that multiple P. vivax TRAgs are expressed on the merozoite surface and that one, PVP01_0000100 binds red blood cells with a strong preference for reticulocytes. Using X-ray crystallography, we solved the structure of the PVP01_0000100 C-terminal tryptophan rich domain, which defines the TRAg family, revealing a three-helical bundle that is conserved across Plasmodium and has structural homology with lipid-binding BAR domains involved in membrane remodelling. Biochemical assays confirm that the PVP01_0000100 C-terminal domain has lipid binding activity with preference for sulfatide, a glycosphingolipid present in the outer leaflet of plasma membranes. Deletion of the putative orthologue in P. knowlesi, PKNH_1300500, impacts invasion in reticulocytes, suggesting a role during this essential process. Together, this work defines an emerging molecular function for the Plasmodium TRAg family.

The search for better treatment strategies for mansonellosis: an expert perspective.

INTRODUCTION: Four species of the Mansonella genus infect millions of people across sub-Saharan Africa and Central and South America. Most infections are asymptomatic, but mansonellosis can be associated with nonspecific clinical manifestations such as fever, headache, arthralgia, and ocular lesions (M. ozzardi); pruritus, arthralgia, abdominal pain, angioedema, skin rash, and fatigue (M. perstans and perhaps Mansonella sp. 'DEUX'); and pruritic dermatitis and chronic lymphadenitis (M. perstans). AREAS COVERED: We searched the PubMed and SciELO databases for publications on mansonelliasis in English, Spanish, Portuguese, or French that appeared until 1 May 2023. Literature data show that anthelmintics - single-dose ivermectin for M. ozzardi, repeated doses of mebendazole alone or in combination with diethylcarbamazine (DEC) for M. perstans, and DEC alone for M. streptocerca - are effective against microfilariae. Antibiotics that target Wolbachia endosymbionts, such as doxycycline, are likely to kill adult worms of most, if not all, Mansonella species, but the currently recommended 6-week regimen is relatively impractical. New anthelmintics and shorter antibiotic regimens (e.g. with rifampin) have shown promise in experimental filarial infections and may proceed to clinical trials. EXPERT OPINION: We recommend that human infections with Mansonella species be treated, regardless of any apparent clinical manifestations. We argue that mansonellosis, despite being widely considered a benign infection, may represent a direct or indirect cause of significant morbidity that remains poorly characterized at present.

Multiple lineages of nematode-Wolbachia symbiosis in supergroup F and convergent loss of bacterioferritin in filarial Wolbachia.

The intracellular endosymbiotic proteobacteria Wolbachia have evolved across the phyla nematoda and arthropoda. In Wolbachia phylogeny, supergroup F is the only clade known so far with members from both arthropod and filarial nematode hosts and therefore can provide unique insights into their evolution and biology. In this study, 4 new supergroup F Wolbachia genomes have been assembled using a metagenomic assembly and binning approach, wMoz and wMpe from the human filarial parasites Mansonella ozzardi and Mansonella perstans, and wOcae and wMoviF from the blue mason bee Osmia caerulescens and the sheep ked Melophagus ovinus respectively. A comprehensive phylogenomic analysis revealed two distinct lineages of filarial Wolbachia in supergroup F, indicating multiple horizontal transfer events between arthropod and nematode hosts. The analysis also reveals that the evolution of Wolbachia-filaria symbioses is accompanied by a convergent pseudogenization and loss of the bacterioferritin gene, a phenomenon found to be shared by all filarial Wolbachia, even those outside supergroup F. These observations indicate that differences in heme metabolism might be a key feature distinguishing filarial and arthropod Wolbachia. The new genomes provide a valuable resource for further studies on symbiosis, evolution, and the discovery of new antibiotics to treat mansonellosis.

Plasmodium vivax infection alters the peripheral immunoregulatory network of CD4 T follicular cells and B cells.

Regulatory and effector cell responses to Plasmodium vivax, the most common human malaria parasite outside Africa, remain understudied in naturally infected populations. Here, we describe peripheral CD4+ T- and B-cell populations during and shortly after an uncomplicated P. vivax infection in 38 continuously exposed adult Amazonians. Consistent with previous observations, we found an increased frequency in CD4+ CD45RA- CD25+ FoxP3+ T regulatory cells that express the inhibitory molecule CTLA-4 during the acute infection, with a sustained expansion of CD21- CD27- atypical memory cells within the CD19+ B-cell compartment. Both Th1- and Th2-type subsets of CXCR5+ ICOShi PD-1+ circulating T follicular helper (cTfh) cells, which are thought to contribute to antibody production, were induced during P. vivax infection, with a positive correlation between overall cTfh cell frequency and IgG antibody titers to the P. vivax blood-stage antigen MSP119 . We identified significant changes in cell populations that had not been described in human malaria, such as an increased frequency of CTLA-4+ T follicular regulatory cells that antagonize Tfh cells, and a decreased frequency of circulating CD24hi CD27+ B regulatory cells in response to acute infection. In conclusion, we disclose a complex immunoregulatory network that is critical to understand how naturally acquired immunity develops in P. vivax malaria.

The impact of hydropower dam construction on malaria incidence: Space-time analysis in the Brazilian Amazon.

During the first two decades of the 21st century, Brazil carried out massive public investments on infrastructure projects, such as large hydropower dams, with potential impact on population health. Here we characterize local malaria transmission and its potential spread during the construction of three large hydropower dams in the Brazilian Amazon. We focus on Porto Velho (PVH), in Rondônia state, where the Santo Antônio and Jirau dams were built (2008-2013), and Altamira region (ATM), in Pará state, where the construction of the Belo Monte dam took place (2011-2016). Analyzed data cover 4 years before, 6 years during, and 4 years after each dam construction. In total, we utilized malaria case notifications entered into the electronic malaria notification system of the Ministry of Health of Brazil between January 2004 and December 2020 (n = 39,977,167 malaria notifications). First, we used Interrupted Time-Series Analysis (ITSA) to assess temporal changes in malaria notifications in the study sites. Then, we conducted a space-time cluster analysis to investigate the potential of malaria spread from the study sites (sources) to elsewhere (sinks). Finally, we present the sociodemographic characteristics of exported cases over time using multivariate logistic regressions. Our results show that there was no upsurge in malaria cases in the study sites and exported cases did not trigger outbreaks in other localities. Exported malaria infections originating from PVH and ATM were typically found in working age literate males involved in mining, farming or traveling. We suggest that efficient control measures, such as ensuring timely diagnosis and treatment; fostering integrated vector control; promoting health education; and prevention, detection and containment of outbreaks, if properly implemented and sustained, may prevent local and introduced malaria outbreaks during and after hydropower dam construction in the Amazon.

Individual variation in Plasmodium vivax malaria risk: Are repeatedly infected people just unlucky?

Extensive research has examined why some people have frequent Plasmodium falciparum malaria episodes in sub-Saharan Africa while others remain free of disease most of the time. In contrast, malaria risk heterogeneity remains little studied in regions where P. vivax is the dominant species. Are repeatedly infected people in vivax malaria settings such as the Amazon just unlucky? Here, we briefly review evidence that human genetic polymorphism and acquired immunity after repeated exposure to parasites can modulate the risk of P. vivax infection and disease in predictable ways. One-fifth of the hosts account for 80% or more of the community-wide vivax malaria burden and contribute disproportionally to onward transmission, representing a priority target of more intensive interventions to achieve malaria elimination. Importantly, high-risk individuals eventually develop clinical immunity, even in areas with very low or residual malaria transmission, and may constitute a large but silent parasite reservoir.

Prevalence and correlates of childhood anemia in the MINA-Brazil birth cohort study / Prevalência e preditores de anemia na infância no estudo de coorte de nascimentos MINA-Brasil

ABSTRACT OBJECTIVE: This study aimed to describe the prevalence and predictors of childhood anemia in an Amazonian population-based birth cohort study. METHODS: Prevalence of maternal anemia was estimated at delivery (hemoglobin [Hb] concentration < 110 g/L) in women participating in the MINA-Brazil birth cohort study and in their children, examined at ages one, two (Hb < 110 g/L), and five (Hb < 115 g/L). Moreover, ferritin, soluble transferrin receptor, and C-reactive protein concentrations were measured in mothers at delivery and in their 1- and 2-year-old children to estimate the prevalence of iron deficiency and its contribution to anemia, while adjusting for potential confounders by multiple Poisson regression analysis (adjusted relative risk [RRa]). RESULTS: The prevalence 95% confidence interval (CI) of maternal anemia, iron deficiency, and iron-deficiency anemia at delivery were 17.3% (14.0-21.0%), 42.6% (38.0-47.2%), and 8.7% (6.3-11.6)%, respectively (n = 462). At one year of age (n = 646), 42.2% (38.7-45.8%) of the study children were anemic, 38.4% (34.6-42.3%) were iron-deficient, and 26.3 (23.0-29.9) had iron-deficiency anemia. At two years of age (n = 761), these values decreased to 12.8% (10.6-15.2%), 18.1% (15.5-21.1%), and 4.1% (2.8-5.7%), respectively; at five years of age (n = 655), 5.2% (3.6-7.2%) were anemic. Iron deficiency (RRa = 2.19; 95%CI: 1.84-2.60) and consumption of ultra-processed foods (UPF) (RRa = 1.56; 95%CI: 1.14-2.13) were significant contributors to anemia at 1 year, after adjusting for maternal schooling. At 2 years, anemia was significantly associated with maternal anemia at delivery (RRa: 1.67; 95%CI: 1.17-2.39), malaria since birth (2.25; 1.30-3.87), and iron deficiency (2.15; 1.47-3.15), after adjusting for children's age and household wealth index. CONCLUSIONS: Anemia continues to be highly prevalent during pregnancy and early childhood in the Amazon. Public health policies should address iron deficiency, UPF intake, maternal anemia, and malaria to prevent and treat anemia in Amazonian children.

RESUMO OBJETIVO: O objetivo deste estudo foi descrever a prevalência e os preditores de anemia na infância em um estudo de coorte de nascimentos de base populacional amazônica. MÉTODOS: Estimou-se a prevalência de anemia materna no parto (concentração de hemoglobina [Hb] < 110 g/L) em mulheres participantes do estudo de coorte de nascimentos MINA-Brasil e em seus filhos, examinados nas idades um, dois (Hb < 110 g/L) e cinco anos (Hb < 115 g/L). Além disso, as concentrações de ferritina, receptor solúvel de transferrina e proteína C reativa foram medidas em mães no parto e em seus filhos de 1 e 2 anos de idade para estimar a prevalência de deficiência de ferro e sua contribuição para anemia, ajustando para potenciais fatores de confusão por análise de regressão múltipla de Poisson (risco relativo ajustado [RRa]). RESULTADOS: As prevalências com intervalo de confiança (IC) de 95% de anemia materna, deficiência de ferro e anemia ferropriva no parto foram de 17,3% (14,0-21,0%), 42,6% (38,0-47,2%) e 8,7% (6,3-11,6%), respectivamente (n = 462). No primeiro ano de idade (n = 646), 42,2% (38,7-45,8%) das crianças estudadas eram anêmicas, 38,4% (34,6-42,3%) eram deficientes em ferro e 26,3 (23,0-29,9%) tinham anemia ferropriva. Aos dois anos de idade (n = 761), esses valores diminuíram para 12,8% (10,6-15,2%), 18,1% (15,5-21,1%) e 4,1% (2,8-5,7%), respectivamente; aos cinco anos de idade (n = 655), 5,2% (3,6-7,2%) eram anêmicos. A deficiência de ferro (RRa = 2,19, IC95%: 1,84-2,60) e consumo de alimentos ultraprocessados (AUP) (RRa = 1,56, IC95%: 1,14-2,13) foram contribuintes significantes para anemia no 1° ano de idade, após ajuste para escolaridade materna. Aos 2 anos, a anemia associou-se significativamente à anemia materna no parto (RRa = 1,67; IC95%: 1,17-2,39), malária desde o nascimento (2,25; 1,30-3,87) e deficiência de ferro (2,15; 1,47-3,15), após ajuste para idade das crianças e índice de riqueza familiar. CONCLUSÕES: A anemia continua sendo altamente prevalente durante a gravidez e a primeira infância na Amazônia. Políticas públicas de saúde devem abordar a deficiência de ferro, o consumo de AUP, a anemia materna e a malária para prevenir e tratar a anemia em crianças amazônicas.

A molecular barcode and web-based data analysis tool to identify imported Plasmodium vivax malaria.

Traditionally, patient travel history has been used to distinguish imported from autochthonous malaria cases, but the dormant liver stages of Plasmodium vivax confound this approach. Molecular tools offer an alternative method to identify, and map imported cases. Using machine learning approaches incorporating hierarchical fixation index and decision tree analyses applied to 799 P. vivax genomes from 21 countries, we identified 33-SNP, 50-SNP and 55-SNP barcodes (GEO33, GEO50 and GEO55), with high capacity to predict the infection's country of origin. The Matthews correlation coefficient (MCC) for an existing, commonly applied 38-SNP barcode (BR38) exceeded 0.80 in 62% countries. The GEO panels outperformed BR38, with median MCCs > 0.80 in 90% countries at GEO33, and 95% at GEO50 and GEO55. An online, open-access, likelihood-based classifier framework was established to support data analysis (vivaxGEN-geo). The SNP selection and classifier methods can be readily amended for other use cases to support malaria control programs.

Longitudinal IgG antibody responses to Plasmodium vivax blood-stage antigens during and after acute vivax malaria in individuals living in the Brazilian Amazon.

BACKGROUND: To make progress towards malaria elimination, a highly effective vaccine targeting Plasmodium vivax is urgently needed. Evaluating the kinetics of natural antibody responses to vaccine candidate antigens after acute vivax malaria can inform the design of serological markers of exposure and vaccines. METHODOLOGY/PRINCIPAL FINDINGS: The responses of IgG antibodies to 9 P. vivax vaccine candidate antigens were evaluated in longitudinal serum samples from Brazilian individuals collected at the time of acute vivax malaria and 30, 60, and 180 days afterwards. Antigen-specific IgG correlations, seroprevalence, and half-lives were determined for each antigen using the longitudinal data. Antibody reactivities against Pv41 and PVX_081550 strongly correlated with each other at each of the four time points. The analysis identified robust responses in terms of magnitude and seroprevalence against Pv41 and PvGAMA at 30 and 60 days. Among the 8 P. vivax antigens demonstrating >50% seropositivity across all individuals, antibodies specific to PVX_081550 had the longest half-life (100 days; 95% CI, 83-130 days), followed by PvRBP2b (91 days; 95% CI, 76-110 days) and Pv12 (82 days; 95% CI, 64-110 days). CONCLUSION/SIGNIFICANCE: This study provides an in-depth assessment of the kinetics of antibody responses to key vaccine candidate antigens in Brazilians with acute vivax malaria. Follow-up studies are needed to determine whether the longer-lived antibody responses induced by natural infection are effective in controlling blood-stage infection and mediating clinical protection.

Evidence-Based Malaria Control and Elimination in the Amazon: Input from the International Center of Excellence in Malaria Research Network in Peru and Brazil.

Malaria remains endemic in 17 countries in the Americas, where 723,000 cases were reported in 2019. The majority (> 90%) of the regional malaria burden is found within the Amazon Basin, which includes nine countries and territories in South America. Locally generated evidence is critical to provide information to public health decision makers upon which the design of efficient and regionally directed malaria control and elimination programs can be built. Plasmodium vivax is the predominant malaria parasite in the Amazon Basin. This parasite species appears to be more resilient to malaria control strategies worldwide. Asymptomatic Plasmodium infections constitute a potentially infectious reservoir that is typically missed by routine microscopy-based surveillance and often remains untreated. The primary Amazonian malaria vector, Nyssorhynchus (formerly Anopheles) darlingi, has changed its behavior to feed and rest predominantly outdoors, reducing the efficiency of core vector control measures such as indoor residual spraying and distribution of long-lasting insecticide-treated bed nets. We review public health implications of recent field-based research carried out by the Amazonia International Center of Excellence in Malaria Research in Peru and Brazil. We discuss the relative role of traditional and novel tools and strategies for better malaria control and elimination across the Amazon, including improved diagnostic methods, new anti-relapse medicines, and biological larvicides, and emphasize the need to integrate research and public health policymaking.

Malaria Resilience in South America: Epidemiology, Vector Biology, and Immunology Insights from the Amazonian International Center of Excellence in Malaria Research Network in Peru and Brazil.

The 1990s saw the rapid reemergence of malaria in Amazonia, where it remains an important public health priority in South America. The Amazonian International Center of Excellence in Malaria Research (ICEMR) was designed to take a multidisciplinary approach toward identifying novel malaria control and elimination strategies. Based on geographically and epidemiologically distinct sites in the Northeastern Peruvian and Western Brazilian Amazon regions, synergistic projects integrate malaria epidemiology, vector biology, and immunology. The Amazonian ICEMR's overarching goal is to understand how human behavior and other sociodemographic features of human reservoirs of transmission-predominantly asymptomatically parasitemic people-interact with the major Amazonian malaria vector, Nyssorhynchus (formerly Anopheles) darlingi, and with human immune responses to maintain malaria resilience and continued endemicity in a hypoendemic setting. Here, we will review Amazonian ICEMR's achievements on the synergies among malaria epidemiology, Plasmodium-vector interactions, and immune response, and how those provide a roadmap for further research, and, most importantly, point toward how to achieve malaria control and elimination in the Americas.

Humanized mice for investigating sustained Plasmodium vivax blood-stage infections and transmission.

Plasmodium vivax is the most widespread human malaria parasite. Due to the presence of extravascular reservoirs and relapsing infections from dormant liver stages, P. vivax is particularly difficult to control and eliminate. Experimental research is hampered by the inability to maintain P. vivax cultures in vitro, due to its tropism for immature red blood cells (RBCs). Here, we describe a new humanized mice model that can support efficient human erythropoiesis and maintain long-lasting multiplication of inoculated cryopreserved P. vivax parasites and their sexual differentiation, including in bone marrow. Mature gametocytes were transmitted to Anopheles mosquitoes, which led to the formation of salivary gland sporozoites. Importantly, blood-stage P. vivax parasites were maintained after the secondary transfer of fresh or frozen infected bone marrow cells to naïve chimeras. This model provides a unique tool for investigating, in vivo, the biology of intraerythrocytic P. vivax.

SARS-CoV-2 seropositivity and COVID-19 among 5 years-old Amazonian children and their association with poverty and food insecurity.

BACKGROUND: The epidemiology of childhood SARS-CoV-2 infection and COVID-19-related illness remains little studied in high-transmission tropical settings, partly due to the less severe clinical manifestations typically developed by children and the limited availability of diagnostic tests. To address this knowledge gap, we investigate the prevalence and predictors of SARS-CoV-2 infection (either symptomatic or not) and disease in 5 years-old Amazonian children. METHODOLOGY/PRINCIPAL FINDINGS: We retrospectively estimated SARS-CoV-2 attack rates and the proportion of infections leading to COVID-19-related illness among 660 participants in a population-based birth cohort study in the Juruá Valley, Amazonian Brazil. Children were physically examined, tested for SARS-CoV-2 IgG and IgM antibodies, and had a comprehensive health questionnaire administered during a follow-up visit at the age of 5 years carried out in January or June-July 2021. We found serological evidence of past SARS-CoV-2 infection in 297 (45.0%; 95% confidence interval [CI], 41.2-48.9%) of 660 cohort participants, but only 15 (5.1%; 95% CI, 2.9-8.2%) seropositive children had a prior medical diagnosis of COVID-19 reported by their mothers or guardians. The period prevalence of clinically apparent COVID-19, defined as the presence of specific antibodies plus one or more clinical symptoms suggestive of COVID-19 (cough, shortness of breath, and loss of taste or smell) reported by their mothers or guardians since the pandemic onset, was estimated at 7.3% (95% CI, 5.4-9.5%). Importantly, children from the poorest households and those with less educated mothers were significantly more likely to be seropositive, after controlling for potential confounders by mixed-effects multiple Poisson regression analysis. Likewise, the period prevalence of COVID-19 was 1.8-fold (95%, CI 1.2-2.6-fold) higher among cohort participants exposed to food insecurity and 3.0-fold (95% CI, 2.8-3.5-fold) higher among those born to non-White mothers. Finally, children exposed to household and family contacts who had COVID-19 were at an increased risk of being SARS-CoV-2 seropositive and-even more markedly-of having had clinically apparent COVID-19 by the age of 5 years. CONCLUSIONS/SIGNIFICANCE: Childhood SARS-CoV-2 infection and COVID-19-associated illness are substantially underdiagnosed and underreported in the Amazon. Children in the most socioeconomically vulnerable households are disproportionately affected by SARS-CoV-2 infection and disease.

Relative contribution of low-density and asymptomatic infections to Plasmodium vivax transmission in the Amazon: pooled analysis of individual participant data from population-based cross-sectional surveys.

Background: Low-density and asymptomatic Plasmodium vivax infections remain largely undetected and untreated and may contribute significantly to malaria transmission in the Amazon. Methods: We analysed individual participant data from population-based surveys that measured P vivax prevalence by microscopy and polymerase chain reaction (PCR) between 2002 and 2015 and modelled the relationship between parasite density and infectiousness to vectors using membrane feeding assay data. We estimated the proportion of sub-patent (i.e., missed by microscopy) and asymptomatic P vivax infections and examined how parasite density relates to clinical manifestations and mosquito infection in Amazonian settings. Findings: We pooled 24,986 observations from six sites in Brazil and Peru. P vivax was detected in 6·8% and 2·1% of them by PCR and microscopy, respectively. 58·5% to 92·6% of P vivax infections were asymptomatic and 61·2% to 96·3% were sub-patent across study sites. P vivax density thresholds associated with clinical symptoms were one order of magnitude higher in children than in adults. We estimate that sub-patent parasite carriers are minimally infectious and contribute 12·7% to 24·9% of the community-wide P vivax transmission, while asymptomatic carriers are the source of 28·2% to 79·2% of mosquito infections. Interpretation: Asymptomatic P vivax carriers constitute a vast infectious reservoir that, if targeted by malaria elimination strategies, could substantially reduce malaria transmission in the Amazon. Infected children may remain asymptomatic despite high parasite densities that elicit clinical manifestations in adults. Funding: US National Institutes of Health, Fundação de Amparo à Pesquisa do Estado de São Paulo, and Belgium Development Cooperation.

High-throughput Plasmodium falciparum hrp2 and hrp3 gene deletion typing by digital PCR to monitor malaria rapid diagnostic test efficacy.

Most rapid diagnostic tests for Plasmodium falciparum malaria target the Histidine-Rich Proteins 2 and 3 (HRP2 and HRP3). Deletions of the hrp2 and hrp3 genes result in false-negative tests and are a threat for malaria control. A novel assay for molecular surveillance of hrp2/hrp3 deletions was developed based on droplet digital PCR (ddPCR). The assay quantifies hrp2, hrp3, and a control gene with very high accuracy. The theoretical limit of detection was 0.33 parasites/µl. The deletion was reliably detected in mixed infections with wild-type and hrp2-deleted parasites at a density of >100 parasites/reaction. For a side-by-side comparison with the conventional nested PCR (nPCR) assay, 248 samples were screened in triplicate by ddPCR and nPCR. No deletions were observed by ddPCR, while by nPCR hrp2 deletion was observed in 8% of samples. The ddPCR assay was applied to screen 830 samples from Kenya, Zanzibar/Tanzania, Ghana, Ethiopia, Brazil, and Ecuador. Pronounced differences in the prevalence of deletions were observed among sites, with more hrp3 than hrp2 deletions. In conclusion, the novel ddPCR assay minimizes the risk of false-negative results (i.e., hrp2 deletion observed when the sample is wild type), increases sensitivity, and greatly reduces the number of reactions that need to be run.

Prolonged Breastfeeding and the Risk of Plasmodium vivax Infection and Clinical Malaria in Early Childhood: A Birth Cohort Study.

BACKGROUND: Relatively few Amazonian infants have clinical malaria diagnosed, treated and notified before their first birthday, either because they are little exposed to an infection or remain asymptomatic once infected. Here we measure the proportion of children who have experienced Plasmodium vivax infection and malaria by 2 years of age in the main transmission hotspot of Amazonian Brazil. METHODS: We measured IgG antibodies to 3 blood-stage P. vivax antigens at the 1- and 2-year follow-up assessment of 435 participants in a population-based birth cohort. Children's malaria case notifications were retrieved from the electronic database of the Ministry of Health. We used multiple Poisson regression models to identify predictors of serologically proven P. vivax infection and clinical vivax malaria during the first 2 years of life. RESULTS: Overall, 23 [5.3%; 95% confidence interval (CI): 3.5-7.8%) children had antibodies to ≥2 antigens detected during at least one follow-up assessment, consistent with past P. vivax infection(s). Fifteen (3.4%; 95% CI: 2.1-5.6%) children had clinical vivax episodes notified during the first 2 years of life; 7 of them were seronegative. We estimate that half of the infections remained unnotified. Children born to women who experienced P. vivax infection during pregnancy were more likely to be infected and develop clinical vivax malaria, while those breast-fed for ≥12 months had their risk of being P. vivax -seropositive (which we take as evidence of blood-stage P. vivax infection during the first 2 years of life) decreased by 79.8% (95% CI: 69.3-86.7%). CONCLUSION: P. vivax infections in early childhood are underreported in the Amazon, are associated with anemia at 2 years of age, and appear to be partially prevented by prolonged breastfeeding.