Marcelo Urbano Ferreira

Background: Malaria elimination in Latin America (LATAM) is challenged by heterogeneous Plasmodium vivax transmission intensity, yet regional genomic evidence from malaria molecular surveillance (MMS) remains scarce. We aimed to generate regional genomic evidence to inform malaria control. Methods: We analyzed 578 P. vivax isolates collected in seven LATAM countries (2010–2024) using the Pv AmpliSeq Peru targeted sequencing assay. We investigated genetic diversity, complexity of infection, population structure, relatedness and connectivity, and candidate drug-resistance variants diversity. Findings: Genetic diversity (He: 0.15–0.39) and polyclonality (0–33%) varied across LATAM. Parasite populations showed country-level and subnational structure, including marked differentiation among Loreto, Amazonas and Tumbes in Peru (FST: 0.23–0.62). Country-level connectivity was limited but occurred across the Peru–Ecuador and Peru–Brazil borders. Highly related pairs (≥90% IBD) predominated in Amazonas (80.2%), Venezuela (75.4%) and Tumbes (56.9%). At lower thresholds, parasite connections among Venezuela, Colombia, Ecuador and Tumbes, geographically coincided with the regional migration corridor from Venezuela to Peru. Candidate pvmdr1 variants for reduced chloroquine susceptibility were uncommon (3.8–5.4%), whereas antifolate resistance mutations were widespread (42.8–80.6%). Interpretation: The geographic structure suggests that P. vivax transmission is mainly maintained locally, while connectivity in some border areas indicates parasite movement between countries by human mobility. Control strategies should be tailored to local transmission settings and supported by coordinated cross-border surveillance and longitudinal sentinel MMS, particularly in hotspots and border areas.

Introduction: A combination of mefloquine associated with artesunate (AS-MQ) was the first artemisinin-based combination therapy (ACT) to be used widely for acute uncomplicated P. falciparum malaria. Methods: Individual patient data from 31 studies of patients with uncomplicated falciparum malaria treated with various AS-MQ regimens (target mefloquine dose 25 mg/kg), conducted in Asia, Africa and South America, were pooled and analysed to investigate the effects of MQ mg/kg dosing on malaria recurrence and other clinical, parasitological and tolerability endpoints. Results: A total of 6,761 patients were enrolled in clinical studies conducted between 1995 and 2018; the majority (74%) were from Asia. The median age of study participants was 18 years (interquartile range IQR 8–30 years), of whom 13.5% (915/6761) were aged less than 5 years old. Participants received an estimated median [range] total mg/kg dose of mefloquine and artesunate of 25 [6.8–60] and 12 [3.6–33.3] respectively, and 1,572 (23.4%) participants were treated with the coformulation. The PCR-corrected recrudescence rate 42 days after any ASMQ treatment was 2.4% for patients enrolled in Asia and 2.5% in Africa, before artemisinin resistance emerged. Corresponding rates in children aged 1 to < 5 years were 2.9% and 3.3%. After adjusting for background artemisinin resistance, the hazard of recrudescence was higher in children aged 1 to < 5 years than in adults in Asia, but not in Africa (Asia, HR 3.01, 95%CI 1.60–5.64, p < 0.001; Africa HR 5.18, 95% CI 0.61–44.28, p = 0.133). There was only one recrudescent infection in South America. A significant MQ dose–effect was observed in Asia in patients treated with 3-dose regimens (AHR 0.89, 95% CI 0.83–0.96, p = 0.003 for 1 mg/kg increase in dose). Vomiting rates within an hour of dosing were highest in children 1- 5 years of age (n = 140) at 3.4% doses (13/378) compared to 1.7% (20/1,168) in children 5–11 years (n = 476), and 1.1% (47/4,191) in patients 12 years of age or older (n = 2027) (p < 0.001, test for trend). Conclusion: AS-MQ administered over three days is a highly efficacious ACT in low to moderate transmission areas without artemisinin resistance. While further optimisation of the mefloquine dose in the coformulation in children aged 1–5 years could be considered, dose-related early vomiting is highest in this age group and may preclude this.

Background: Despite continued control efforts, malaria due to Plasmodium vivax and Plasmodium falciparum remains endemic across the Amazon Basin. Here, we sought to understand how malaria parasites spread between sources and sinks within the upper Juruá Valley region of northwestern Brazil, the country's main residual transmission hotspot, to inform more effective control and elimination strategies. Methods: We combined the analysis of nearly 70 000 case notifications between 2018 and 2021 with high-resolution molecular genotyping data from 207 P. vivax and 117 P. falciparum isolates to map malaria parasite movement across the Juruá Valley hotspot. We introduce the C50 metric to estimate parasite connectivity within clusters of genetically related parasites, or molecular clusters. Results: We found that 16%–18% of infections were acquired outside participants’ localities of residence. Genotyping of P. vivax and P. falciparum revealed large molecular clusters comprising parasites of each species collected across rural–urban boundaries over 4 years, with some pairs of genetically related parasites originating from distant localities within Juruá Valley. We found a large variation in C50 values, with a few parasites serving as main hubs within molecular clusters. Imported P. vivax isolates were significantly more connected than locally acquired infections. Conclusions: We suggest that the combined use of routine epidemiological surveillance data and molecular genotyping can help to map human mobility and parasite lineage dispersal across space and time, aiming to inform specific interventions tailored for local populations that move malaria parasites between source and sink localities in residual transmission settings in the Amazon.

Plasmodium vivax ‐associated anemia is a multifactorial clinical outcome, in which the destruction of uninfected red blood cells (uRBCs) plays a major role in disease development and severity. Here, we identified autoantibody targets within the extracellular loops of Band 3 (B3), a major erythrocyte transmembrane protein. In silico epitope prediction identified six of the seven potentially immunogenic extracellular domains of B3. Patients with P. vivax infection who were anemic ( n = 79) and non‐anemic ( n = 95), and 40 healthy donors from a non‐endemic area were included as negative controls. In addition, 15 anemic and 15 non‐anemic infected individuals were also analyzed immediately before treatment (D0) and on Days 7, 14, and 28 after antimalarial therapy. Synthetic peptides corresponding to the six B3 domains were robustly recognized by autoantibodies from P. vivax ‐infected patients. Anemic individuals exhibited prominent autoantibody responses, particularly against loops 4 and 6. IgG3 was the predominant subclass targeting these loops and remained detectable up to 28 days post‐treatment, suggesting a possible role in the persistence of anemia even after parasite clearance. In contrast, IgG1 was the principal subclass recognizing the peptides corresponding to loops 1, 2, and 3, indicating a significant response to senescent domains during P. vivax infection. Analysis of IgG subclass against full‐length B3 confirmed the presence of both IgG1 and IgG3 responses. The pro‐inflammatory properties of these IgG subclasses support the hypothesis that anti‐B3 autoantibodies contribute to the immunopathogenesis of P. vivax ‐related anemia. Collectively, these findings identify potential targets for future mechanistic investigations and therapeutic intervention strategies.

Background: Acute febrile illnesses in sub‐Saharan Africa are often attributed to malaria, yet many patients test negative for malaria parasites. The aetiology of nonmalarial fevers remains understudied. Here, we examine likely causes of febrile illnesses and their association with poor clinical outcomes in Angola. Methods: In September–October 2023 and May–June 2024 we enrolled 849 febrile patients with ages between 2 months and 94 years in two tertiary hospitals of Lubango city that serve 7 million people in southern Angola. We used clinical assessments and locally available laboratory diagnostics to assign likely causes of fever and applied logistic regression analysis to identify factors independently associated with an increased risk of hospital admission and death. Results: Overall, 67.6% of the patients tested negative for malaria by microscopy, with upper (13.0%) and lower (19.5%) respiratory infections and gastrointestinal infections (16.7%) being leading causes of nonmalarial fever. The frequencies of gastrointestinal, upper respiratory, and genitourinary infections varied markedly with age, and malaria predominated at the beginning of the dry season. Malaria and lower respiratory infections were associated with increased risk of hospitalization (odds ratio [OR] = 3.20 and 5.17, p < 0.0001), while malaria and central nervous system infections were linked to significantly higher mortality (OR = 1.90 and 6.30, p < 0.0001). Empirical antibiotic use was widespread, even without clinical or laboratory evidence of bacterial infection. Conclusion: Most fevers in southern Angola are of nonmalarial origin, underscoring the need for better diagnostic strategies to guide specific treatment and reduce unnecessary antibiotic use.

Background: Plasmodium vivax is the most widespread malaria parasite, and its cocirculation with Plasmodium falciparum often leads to underdiagnosis and underestimated transmission. We investigated whether underdiagnosis contributes to the selection and spread of drug-resistant parasites in coendemic areas of the Brazilian Amazon. Methods: We enrolled 727 confirmed malaria cases from three Brazilian states, primarily from Roraima in the Guiana Shield, a region characterized by cocirculation of P. vivax and P. falciparum, intense cross-border mobility, illegal mining activities, and informal antimalarial drug use. To examine indirect P. vivax exposure to antimalarial targeting P. falciparum, we evaluated pvmdr1 copy number variation (CNV). Results: In Roraima, molecular diagnosis revealed frequent misclassification of mixed infections, with P. vivax often missed by microscopy due to higher P. falciparum densities, demonstrating the limitations of microscopy in accurately detecting mixed infections. The pvmdr1 CNV exhibited significant geographic variation. While nearly all isolates from the western Amazon carried a single copy, pvmdr1 amplification was detected in 60% of Roraima isolates. In addition to this spatial heterogeneity, temporal analysis revealed significant shifts in amplification patterns over the years, rising to over 70% after 2020. Conclusions: These findings emphasize the critical importance of accurate diagnosis and rational use of antimalarials, as inadequate or unintended drug exposure may inadvertently drive the selection and spread of parasite strains with reduced drug susceptibility.

Background: Primaquine (PQ) is the only widely available drug that eliminates latent hypnozoites, thereby preventing relapses of Plasmodium vivax malaria. Because PQ biotransformation mediated by the cytochrome P-450 (CYP) isoenzyme CYP2D6 is required for therapeutic activity, patients with reduced CYP2D6 activity can experience relapse despite treatment. The minimum level of CYP2D6 activity for adequate anti-relapse PQ efficacy is unknown. Methods and findings: We conducted a retrospective, population-based cohort study in the main malaria hotspot of Brazil. We fitted time-to-event data from cohort participants who experienced vivax malaria, using Cox proportional hazards models, to explore how genotype-determined CYP2D6 activity, expressed as activity scores (AS), modulates the risk of P. vivax recurrence within 6 months after treatment with chloroquine and PQ (total dose, 3.5 mg/kg). We analyzed community-wide P. vivax malaria incidence data, using a multivariable negative binomial regression model, to quantify the impact of reduced CYP2D6 activity on the overall risk of vivax malaria, whether from relapses or new infections. Among 466 P. vivax -infected cohort participants , those with null/low CYP2D6 activity (AS ≤ 0.25), but not participants with intermediate CYP2D6 activity (AS from 0.5 to 1.0), had twice the risk of recurrence compared to an AS > 1.0 (hazard ratio = 2.22, P = 0.004). However, vivax malaria incidence did not differ significantly across CYP2D6 activity levels during 5 years of follow-up of 997 Amazonians exposed to intense transmission (mean, 26.6 vivax malaria cases/100 person-years). These findings suggest that the excess of relapses among people with severely reduced CYP2D6 activity adds relatively little to the overall burden of vivax malaria episodes. Conclusion: Amazonians with an AS ≤ 0.25, but not necessarily those with intermediate CYP2D6 activity, have a greater risk of recurrence after a PQ-treated P. vivax infection and require alternative relapse suppression regimens for the radical cure of vivax malaria.

Malaria is a highly prevalent infectious disease caused by Plasmodium parasites. Plasmodium intraerythrocytic replication leads to hemolysis-driven intermittent febrile crises in patients. In addition, the lysis of unparasitized red blood cells contributes to anemia and endotoxemia. Because thrombocytopenia is an important feature of vivax and severe falciparum malaria, we hypothesized that increased hemolysis in malaria contributes to severe thrombocytopenia by releasing endogenous and parasite toxins (ie, heme and hemozoin) capable of inducing programmed cell death in platelets. Using complementary biochemical, ultrastructural, pharmacological, and molecular approaches, we examined response to stress and cell death pathways that were elevated in the transcriptome of platelets during vivax malaria and evaluated markers of hemolysis that correlated with thrombocytopenia. We found that heme in plasma from thrombocytopenic vivax malaria, but not nonthrombocytopenic vivax or falciparum malaria, induced platelet cell death ex vivo. Platelet stimulation with heme and hemozoin induced apoptotic and necrotic cell death features, with stronger necrosis triggered by hemozoin. Heme and hemozoin activated apoptotic caspases, but only heme induced calpain-dependent BcL-xL degradation, which was not required for platelet apoptosis. We unmasked a caspase-independent intrinsic apoptosis program mechanism depending on the endoplasmic reticulum stress sensor and unfolded protein response trigger IRE1α. Regarding necrosis, we observed inflammasome activation but not pyroptosis. Instead, we distinguished a necrotic cell death feature consistent with ferroptosis, dependent on lipid peroxidation and regulated by DGAT1/2 enzymes, which was the main pathway for hemozoin-induced thrombocytopenia in vitro. Our results identify novel pathways of regulated cell death in platelets that were associated with thrombocytopenia in malaria and may have potential implications for other hemolytic disorders.