Priscila Thihara Rodrigues

Background: A promoter variant commonly found in sub-Saharan Africans and their descendants disrupts Duffy antigen (Fy) expression on erythrocytes, leading to the Fy-negative phenotype, and confers partial resistance to blood-stage Plasmodium vivax infection. In addition, the 125G→A substitution, rare in Africans, defines the Fya/Fyb polymorphism that can modulate vivax malaria risk in Amazonians. The combined effect of these FY polymorphisms on P. vivax infection risk remains little explored at the population level. Methods: We studied a household-based random sample of 1737 Amazonians, with a well-balanced distribution of FY alleles, who were exposed to P. vivax transmission and contributed 7878.9 person-years of follow-up. We fitted a multivariable zero-inflated negative binomial model to incidence data, assuming that zero counts could arise from individuals at risk who remained uninfected over 5 years of follow-up (“sampling zeroes”) or from not-at-risk individuals (“structural zeroes”). Results: Plasmodium vivax infections were heterogeneously distributed in the population, with 0 to 11 cases per person (average incidence, 25.8 cases/100 person-years at risk). We show that Fy-negativity remains a major malaria resistance trait in Amazonians and contributes significantly to the “structural zeroes” observed in P. vivax incidence data. Moreover, the differences in P. vivax infection risk associated with the Fya/Fyb polymorphism observed among young participants were attenuated with increasing age, most likely because more susceptible Fy(b+) individuals develop clinical immunity faster than less susceptible Fy(a+) individuals. Conclusions: FY polymorphism appears to modulate the rate at which immunity to P. vivax develops in Amazonians, with clear clinical and public health implications.

Background: Brazil's progress toward malaria elimination has stalled and 163 000 new cases (more than 80% caused by Plasmodium vivax) were recorded in the Brazilian Amazon in 2023. We hypothesize that human mobility continues to disperse parasites from hotspots to areas with decreasing endemicity. Methods: We analyzed 5.5 million malaria case notifications between 2003 and 2023 to describe malaria case mobility and identify sources and sinks of P. vivax in the Brazilian Amazon. We leveraged whole-genome sequence data from 408 P. vivax isolates sampled from across South America to characterize parasite gene flow and infer likely regional routes of parasite dispersal. Results: We found that nearly one-third of the P. vivax infections diagnosed in residents in the Brazilian Amazon over 21 years were acquired outside the locality or municipality of residence, but only 1.7% were imported from other countries in South America, mostly from the Guiana Shield. We show that large cities with residual malaria transmission—such as Manaus and Porto Velho—are receptive parasite sinks surrounded by high-risk source rural localities. Although the genetic relatedness of parasites tended to decrease with geographic distance, parasites from sites more than 1000 km apart often remained genetically connected. Conclusions: Understanding parasite source-sink dynamics on different geographic scales is crucial to target high-risk mobile populations and source localities along with receptive sinks within low-transmission municipalities, with the goal of eliminating malaria transmission and preventing its reintroduction into malaria-free areas.

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.

Plasmodium vivax is a neglected human malaria parasite that causes significant morbidity in the Americas, the Middle East, Asia, and the Western Pacific. Population genomic approaches remain little explored to map local and regional transmission pathways of P. vivax across the main endemic sites in the Americas, where great progress has been made towards malaria elimination over the past decades. We analyze 38 patient-derived P. vivax genome sequences from Mâncio Lima (ML)–the Amazonian malaria hotspot next to the Brazil-Peru border—and 24 sequences from two other sites in Acre State, Brazil, a country that contributes 23% of malaria cases in the Americas. We show that the P. vivax population of ML is genetically diverse (π = 4.7 × 10⁻⁴), with a high polymorphism particularly in genes encoding proteins putatively involved in red blood cell invasion. Paradoxically, however, parasites display strong genome-wide linkage disequilibrium, being fragmented into discrete lineages that are remarkably stable across time and space, with only occasional recombination between them. Using identity-by-descent approaches, we identified a large cluster of closely related sequences that comprises 16 of 38 genomes sampled in ML over 26 months. Importantly, we found significant ancestry sharing between parasites at a large geographic distance, consistent with substantial gene flow between regional P. vivax populations. We have characterized the sustained expansion of highly inbred P. vivax lineages in a malaria hotspot that can seed regional transmission. Potential source populations in hotspots represent a priority target for malaria elimination in the Amazon. Plasmodium vivax is the geographically most widespread human malaria parasite and causes 80% of the malaria burden in the Americas. Here we use whole-genome sequencing to explore levels of parasite relatedness and infer P. vivax transmission networks in the upper Juruá Valley, the main transmission hotspot in Amazonian Brazil. We characterize a genetically diverse population that displays significant linkage disequilibrium, consistent with the local circulation of highly inbred but genetically distant parasite lineages. Noteworthy, these discrete lineages remain stable over time and share recent ancestry with parasites at a large geographic distance. These results illustrate the power of genomic epidemiology approaches to map potential source parasite populations and prioritize areas for targeted control interventions to eliminate residual P. vivax transmission in the Amazon and similar endemic settings worldwide.

The relative contribution of imported vs. locally acquired infections to urban malaria burden remains largely unexplored in Latin America, the most urbanised region in the developing world. Here we use a simple molecular epidemiology framework to examine the transmission dynamics of Plasmodium vivax in Mâncio Lima, the Amazonian municipality with the highest malaria incidence rate in Brazil. We prospectively genotyped 177 P. vivax infections diagnosed in urban residents between June 2014 and July 2015 and showed that local parasites are structured into several lineages of closely related microsatellite haplotypes, with the largest genetic cluster comprising 32% of all infections. These findings are very unlikely under the hypothesis of multiple independent imports of parasite strains from the rural surroundings. Instead, the presence of an endemic near-clonal parasite lineage circulating over 13 consecutive months is consistent with a local P. vivax transmission chain in the town, with major implications for malaria elimination efforts in this and similar urban environments across the Amazon.

Background: Plasmodium falciparum(Pf) whole-organism sporozoite vaccines have been shown to provide significant protection against controlled human malaria infection (CHMI) in clinical trials. Initial CHMI studies showed significantly higher durable protection against homologous than heterologous strains, suggesting the presence of strain-specific vaccine-induced protection. However, interpretation of these results and understanding of their relevance to vaccine efficacy have been hampered by the lack of knowledge on genetic differences between vaccine and CHMI strains, and how these strains are related to parasites in malaria endemic regions.Methods: Whole genome sequencing using long-read (Pacific Biosciences) and short-read (Illumina) sequencing platforms was conducted to generate de novo genome assemblies for the vaccine strain, NF54, and for strains used in heterologous CHMI (7G8 from Brazil, NF166.C8 from Guinea, and NF135.C10 from Cambodia). The assemblies were used to characterize sequences in each strain relative to the reference 3D7 (a clone of NF54) genome. Strains were compared to each other and to a collection of clinical isolates (sequenced as part of this study or from public repositories) from South America, sub-Saharan Africa, and Southeast Asia.Results: While few variants were detected between 3D7 and NF54, we identified tens of thousands of variants between NF54 and the three heterologous strains. These variants include SNPs, indels, and small structural variants that fall in regulatory and immunologically important regions, including transcription factors (such as PfAP2-L and PfAP2-G) and pre-erythrocytic antigens that may be key for sporozoite vaccine-induced protection. Additionally, these variants directly contributed to diversity in immunologically important regions of the genomes as detected through in silico CD8⁺T cell epitope predictions. Of all heterologous strains, NF135.C10 had the highest number of unique predicted epitope sequences when compared to NF54. Comparison to global clinical isolates revealed that these four strains are representative of their geographic origin despite long-term culture adaptation; of note, NF135.C10 is from an admixed population, and not part of recently formed subpopulations resistant to artemisinin-based therapies present in the Greater Mekong Sub-region.Conclusions: These results will assist in the interpretation of vaccine efficacy of whole-organism vaccines against homologous and heterologous CHMI.

Emerging Plasmodium vivax resistance to chloroquine (CQ) may undermine malaria elimination efforts in South America. CQ-resistant P. vivax has been found in the major port city of Manaus, but not in the main malaria hotspots across the Amazon Basin of Brazil, where CQ is routinely co-administered with primaquine (PQ) for radical cure of vivax malaria. Here we randomly assigned 204 uncomplicated vivax malaria patients from Juruá Valley, northwestern Brazil, to receive either sequential (Arm 1) or concomitant (Arm 2) CQ-PQ treatment. Because PQ may synergize the blood schizontocidal effect of CQ and mask low-level CQ resistance, we monitored CQ-only efficacy in Arm 1 subjects, who had PQ administered only at the end of the 28-day follow up. We found adequate clinical and parasitological responses in all subjects assigned to Arm 2. However, 2.2% of Arm 1 patients had microscopy-detected parasite recrudescences at day 28. When PCR-detected parasitemias at day 28 were considered, response rates decreased to 92.1% and 98.8% in Arms 1 and 2, respectively. Therapeutic CQ levels were documented in 6 of 8 recurrences, consistent with true CQ resistance in vivo. In contrast, ex-vivo assays provided no evidence of CQ resistance in 49 local P. vivax isolates analyzed. CQ-PQ co-administration was not found to potentiate the antirelapse efficacy of PQ over 180 days of surveillance; however, we suggest that larger studies are needed to examine whether and how CQ-PQ interactions, e.g. CQ-mediated inhibition of PQ metabolism, modulate radical cure efficacy in different P. vivax-infected populations.

We examined the mitogenomes of a large global collection of human malaria parasites to explore how and when Plasmodium falciparum and P. vivax entered the Americas. We found evidence of a significant contribution of African and South Asian lineages to present-day New World malaria parasites with additional P. vivax lineages appearing to originate from Melanesia that were putatively carried by the Australasian peoples who contributed genes to Native Americans. Importantly, mitochondrial lineages of the P. vivax-like species P. simium are shared by platyrrhine monkeys and humans in the Atlantic Forest ecosystem, but not across the Amazon, which most likely resulted from one or a few recent human-to-monkey transfers. While enslaved Africans were likely the main carriers of P. falciparum mitochondrial lineages into the Americas after the conquest, additional parasites carried by Australasian peoples in pre-Columbian times may have contributed to the extensive diversity of extant local populations of P. vivax.

The transmission of malaria in the extra-Amazonian regions of Brazil, although interrupted in the 1960s, has persisted to the present time in some areas of dense Atlantic Forest, with reports of cases characterized by particular transmission cycles and clinical presentations. Bromeliad-malaria, as it is named, is particularly frequent in the state of Espírito Santo, with Plasmodium vivax being the parasite commonly recognized as the aetiologic agent of human infections. With regard to the spatial and temporal distances between cases reported in this region, the transmission cycle does not fit the traditional malaria cycle. The existence of a zoonosis, with infected simians participating in the epidemiology, is therefore hypothesized. In the present study, transmission of bromeliad-malaria in Espírito Santo is investigated, based on the complete mitochondrial genome of DNA extracted from isolates of Plasmodium species, which had infected humans, a simian from the genus Allouata, and Anopheles mosquitoes. Plasmodium vivax/simium was identified in the samples by both nested PCR and real-time PCR. After amplification, the mitochondrial genome was completely sequenced and compared with a haplotype network which included all sequences of P. vivax/simium mitochondrial genomes sampled from humans and simians from all regions in Brazil. The haplotype network indicates that humans and simians from the Atlantic Forest become infected by the same haplotype, but some isolates from humans are not identical to the simian isolate. In addition, the plasmodial DNA extracted from mosquitoes revealed sequences different from those obtained from simians, but similar to two isolates from humans. These findings strengthen support for the hypothesis that in the Atlantic Forest, and especially in the state with the highest frequency of bromeliad-malaria in Brazil, parasites with similar molecular backgrounds are shared by humans and simians. The recognized identity between P. vivax and P. simium at the species level, the sharing of haplotypes, and the participation of the same vector in transmitting the infection to both host species indicate interspecies transference of the parasites. However, the intensity, frequency and direction of this transfer remain to be clarified.