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.

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.

Epidemiology of COVID-19 after Emergence of SARS-CoV-2 Gamma Variant, Brazilian Amazon, 2020-2021.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Gamma variant has been hypothesized to cause more severe illness than previous variants, especially in children. Successive SARS-CoV-2 IgG serosurveys in the Brazilian Amazon showed that age-specific attack rates and proportions of symptomatic SARS-CoV-2 infections were similar before and after Gamma variant emergence.

Cohort profile: the Mâncio Lima cohort study of urban malaria in Amazonian Brazil.

PURPOSE: This population-based open cohort study aims to investigate biological and sociodemographic drivers of malaria transmission in the main urban hotspot of Amazonian Brazil. PARTICIPANTS: Nearly 20% of the households in the northwestern town of Mâncio Lima were randomly selected and 2690 participants were enrolled since April 2018. Sociodemographic, housing quality, occupational, behavioural and morbidity information and travel histories were collected during consecutive study visits. Blood samples from participants>3 months old were used for malaria diagnosis and human genetic studies; samples from participants with laboratory-confirmed malaria have been cryopreserved for genetic and phenotypic characterisation of parasites. Serology was introduced in 2020 to measure the prevalence and longevity of SARS-CoV-2 IgG antibodies. FINDINGS TO DATE: Malaria prevalence rates were low (up to 1.0% for Plasmodium vivax and 0.6% for P. falciparum) during five consecutive cross-sectional surveys between April-May 2018 and October-November 2020; 63% of infections diagnosed by microscopy were asymptomatic. Malaria risk is heterogeneously distributed, with 20% study participants contributing 86% of the overall burden of P. vivax infection. Adult males are at greatest risk of infection and human mobility across the urban-rural interface may contribute to sustained malaria transmission. Local P. vivax parasites are genetically diverse and fragmented into discrete inbred lineages that remain stable across space and time. FUTURE PLANS: Two follow-up visits, with similar study protocols, are planned in 2021. We aim to identify high-risk individuals that fuel onwards malaria transmission and represent a priority target for more intensive and effective control interventions. TRIAL REGISTRATION NUMBER: NCT03689036.

Ongoing host-shift speciation in Plasmodium simium.

Plasmodium simium, a malaria parasite that infects platyrrhine monkeys and humans in the New World, is nearly identical to Plasmodium vivax. Recent genomic comparative analyses of these sister species have identified elevated divergence in a gene that may underlie P. simium adaptation to non-human primates during its gradual speciation process.

Monthly biological larviciding associated with a tenfold decrease in larval density in fish farming ponds and reduced community-wide malaria incidence in northwestern Brazil.

BACKGROUND: Larvicides are typically applied to fixed and findable mosquito breeding sites, such as fish farming ponds used in commercial aquaculture, to kill immature forms and thereby reduce the size of adult malaria vector populations. However, there is little evidence suggesting that larviciding may suppress community-wide malaria transmission outside Africa. Here, we tested whether the biological larvicide VectoMax FG applied at monthly intervals to fish farming ponds can reduce malaria incidence in Amazonian Brazil. METHODS: This study was carried out in Vila Assis Brasil (VAB; population 1700), a peri-urban malaria hotspot in northwestern Brazil with a baseline annual parasite incidence of 553 malaria cases per 1000 inhabitants. The intervention consisted of monthly treatments with 20 kg/ha of VectoMax FG of all water-filled fish ponds in VAB (n ranging between 167 and 170) with a surface area between 20 and 8000 m2, using knapsack power mistblowers. We used single-group interrupted time-series analysis to compare monthly larval density measurements in fish ponds during a 14-month pre-intervention period (September 2017-October 2018), with measurements made during November 2018-October 2019 and shortly after the 12-month intervention (November 2019). We used interrupted time-series analysis with a comparison group to contrast the malaria incidence trends in VAB and nearby nonintervention localities before and during the intervention. RESULTS: Average larval densities decreased tenfold in treated fish farming ponds, from 0.467 (95% confidence interval [CI], 0.444-0.490) anopheline larvae per dip pre-intervention (September 2017-October 2018) to 0.046 (95% CI, 0.041-0.051) larvae per dip during (November 2018-October 2019) and shortly after the intervention (November 2019). Average malaria incidence rates decreased by 0.08 (95% CI, 0.04-0.11) cases per 100 person-months (P < 0.0001) during the intervention in VAB and remained nearly unchanged in comparison localities. We estimate that the intervention averted 24.5 (95% CI, 6.2-42.8) malaria cases in VAB between January and December 2019. CONCLUSIONS: Regular larviciding is associated with a dramatic decrease in larval density and a modest but significant decrease in community-wide malaria incidence. Larviciding may provide a valuable complementary vector control strategy in commercial aquaculture settings across the Amazon.

Interacting Epidemics in Amazonian Brazil: Prior Dengue Infection Associated With Increased Coronavirus Disease 2019 (COVID-19) Risk in a Population-Based Cohort Study.

BACKGROUND: Immunity after dengue virus (DENV) infection has been suggested to cross-protect from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and mortality. METHODS: We tested whether serologically proven prior DENV infection diagnosed in September-October 2019, before the coronavirus disease 2019 (COVID-19) pandemic, reduced the risk of SARS-CoV-2 infection and clinically apparent COVID-19 over the next 13 months in a population-based cohort in Amazonian Brazil. Mixed-effects multiple logistic regression analysis was used to identify predictors of infection and disease, adjusting for potential individual and household-level confounders. Virus genomes from 14 local SARS-CoV-2 isolates were obtained using whole-genome sequencing. RESULTS: Anti-DENV immunoglobulin G (IgG) was found in 37.0% of 1285 cohort participants (95% confidence interval [CI]: 34.3% to 39.7%) in 2019, with 10.4 (95% CI: 6.7-15.5) seroconversion events per 100 person-years during the follow-up. In 2020, 35.2% of the participants (95% CI: 32.6% to 37.8%) had anti-SARS-CoV-2 IgG and 57.1% of the 448 SARS-CoV-2 seropositives (95% CI: 52.4% to 61.8%) reported clinical manifestations at the time of infection. Participants aged >60 years were twice more likely to have symptomatic COVID-19 than children under 5 years. Locally circulating SARS-CoV-2 isolates were assigned to the B.1.1.33 lineage. Contrary to the cross-protection hypothesis, prior DENV infection was associated with twice the risk of clinically apparent COVID-19 upon SARS-CoV-2 infection, with P values between .025 and .039 after adjustment for identified confounders. CONCLUSIONS: Higher risk of clinically apparent COVID-19 among individuals with prior dengue has important public health implications for communities sequentially exposed to DENV and SARS-CoV-2 epidemics.

Plasmodium simium: Population Genomics Reveals the Origin of a Reverse Zoonosis.

BACKGROUND: The population history of Plasmodium simium, which causes malaria in sylvatic Neotropical monkeys and humans along the Atlantic Coast of Brazil, remains disputed. Genetically diverse P vivax populations from various sources, including the lineages that founded the species P simium, are thought to have arrived in the Americas in separate migratory waves. METHODS: We use population genomic approaches to investigate the origin and evolution of P simium. RESULTS: We find a minimal genome-level differentiation between P simium and present-day New World P vivax isolates, consistent with their common geographic origin and subsequent divergence on this continent. The meagre genetic diversity in P simium samples from humans and monkeys implies a recent transfer from humans to non-human primates - a unique example of malaria as a reverse zoonosis of public health significance. Likely genomic signatures of P simium adaptation to new hosts include the deletion of >40% of a key erythrocyte invasion ligand, PvRBP2a, which may have favored more efficient simian host cell infection. CONCLUSIONS: New World P vivax lineages that switched from humans to platyrrhine monkeys founded the P simium population that infects nonhuman primates and feeds sustained human malaria transmission in the outskirts of major cities.

Human mobility and urban malaria risk in the main transmission hotspot of Amazonian Brazil.

Malaria in the Amazon is often perceived as an exclusively rural disease, but transmission has been increasingly documented within and near urban centers. Here we explore patterns and causes of urban-to-rural mobility, which places travelers at risk of malaria in Mâncio Lima, the main malaria hotspot in northwestern Brazil. We also analyze rural-to-urban mobility caused by malaria treatment seeking, which poses an additional risk of infection to urban residents. We show that the rural localities most frequently visited by urban residents-typically farming settlements in the vicinity of the town-are those with the most intense malaria transmission and also the most frequent source localities of imported malaria cases diagnosed in the town. The most mobile urban residents are typically poor males 16 to 60-years old from multi-sited households who lack a formal job. Highly mobile residents represent a priority target for more intensive and effective malaria control interventions, that cannot be readily delivered to the entire community, in this and similar urbanized endemic settings across the Amazon.

Population genomics reveals the expansion of highly inbred Plasmodium vivax lineages in the main malaria hotspot of Brazil.

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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.

Strains used in whole organism Plasmodium falciparum vaccine trials differ in genome structure, sequence, and immunogenic potential.

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.

Monitoring the Efficacy of Chloroquine-Primaquine Therapy for Uncomplicated Plasmodium vivax Malaria in the Main Transmission Hot Spot of Brazil.

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 hot spots across the Amazon Basin of Brazil, where CQ is routinely coadministered 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 coadministration 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. (This study has been registered at ClinicalTrials.gov under identifier NCT02691910.).

Human migration and the spread of malaria parasites to the New World.

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.

Genome-wide diversity and differentiation in New World populations of the human malaria parasite Plasmodium vivax.

BACKGROUND: The Americas were the last continent colonized by humans carrying malaria parasites. Plasmodium falciparum from the New World shows very little genetic diversity and greater linkage disequilibrium, compared with its African counterparts, and is clearly subdivided into local, highly divergent populations. However, limited available data have revealed extensive genetic diversity in American populations of another major human malaria parasite, P. vivax. METHODS: We used an improved sample preparation strategy and next-generation sequencing to characterize 9 high-quality P. vivax genome sequences from northwestern Brazil. These new data were compared with publicly available sequences from recently sampled clinical P. vivax isolates from Brazil (BRA, total n = 11 sequences), Peru (PER, n = 23), Colombia (COL, n = 31), and Mexico (MEX, n = 19). PRINCIPAL FINDINGS/CONCLUSIONS: We found that New World populations of P. vivax are as diverse (nucleotide diversity π between 5.2 × 10-4 and 6.2 × 10-4) as P. vivax populations from Southeast Asia, where malaria transmission is substantially more intense. They display several non-synonymous nucleotide substitutions (some of them previously undescribed) in genes known or suspected to be involved in antimalarial drug resistance, such as dhfr, dhps, mdr1, mrp1, and mrp-2, but not in the chloroquine resistance transporter ortholog (crt-o) gene. Moreover, P. vivax in the Americas is much less geographically substructured than local P. falciparum populations, with relatively little between-population genome-wide differentiation (pairwise FST values ranging between 0.025 and 0.092). Finally, P. vivax populations show a rapid decline in linkage disequilibrium with increasing distance between pairs of polymorphic sites, consistent with very frequent outcrossing. We hypothesize that the high diversity of present-day P. vivax lineages in the Americas originated from successive migratory waves and subsequent admixture between parasite lineages from geographically diverse sites. Further genome-wide analyses are required to test the demographic scenario suggested by our data.

No Clinical or Molecular Evidence of Plasmodium falciparum Resistance to Artesunate-Mefloquine in Northwestern Brazil.

We evaluated the clinical efficacy of artesunate-mefloquine (ASMQ) fixed-dose combination to treat uncomplicated malaria in Juruá Valley, the main Plasmodium falciparum transmission hotspot in Brazil. Between November 2010 and February 2013, we enrolled 162 patients aged 4-73 years, with fever or a history of fever, and a single-species P. falciparum infection confirmed by microscopy and polymerase chain reaction (PCR). All 154 patients who completed the 42-day follow-up presented an adequate clinical and parasitologic response. ASMQ was well tolerated and no adverse event caused treatment interruption. Gametocytes were detected in 46.3% patients; 35.2% had gametocytes at enrollment, whereas others developed patent gametocytemia 1-14 days after starting ASMQ. By day 3 of treatment, all subjects had cleared asexual parasitemia, but parasite DNA remained PCR detectable in 37.6% of them. Day-3 PCR positivity was associated with prolonged gametocyte carriage. We found no molecular evidence of resistance to either MQ (pfmdr1 gene amplification) or AS (mutations in selected kelch13 gene domains known to be associated with AS resistance) in the local P. falciparum population. These results strongly support the use of ASMQ as a first-line regimen to treat uncomplicated P. falciparum malaria in northwestern Brazil, but underscore the need for gametocytocidal drugs to reduce the transmission potential of ASMQ-treated patients (ClinicalTrials.gov number NCT01144702).

Plasmodium vivax Diversity and Population Structure across Four Continents.

Plasmodium vivax is the geographically most widespread human malaria parasite. To analyze patterns of microsatellite diversity and population structure across countries of different transmission intensity, genotyping data from 11 microsatellite markers was either generated or compiled from 841 isolates from four continents collected in 1999-2008. Diversity was highest in South-East Asia (mean allelic richness 10.0-12.8), intermediate in the South Pacific (8.1-9.9) Madagascar and Sudan (7.9-8.4), and lowest in South America and Central Asia (5.5-7.2). A reduced panel of only 3 markers was sufficient to identify approx. 90% of all haplotypes in South Pacific, African and SE-Asian populations, but only 60-80% in Latin American populations, suggesting that typing of 2-6 markers, depending on the level of endemicity, is sufficient for epidemiological studies. Clustering analysis showed distinct clusters in Peru and Brazil, but little sub-structuring was observed within Africa, SE-Asia or the South Pacific. Isolates from Uzbekistan were exceptional, as a near-clonal parasite population was observed that was clearly separated from all other populations (FST>0.2). Outside Central Asia FST values were highest (0.11-0.16) between South American and all other populations, and lowest (0.04-0.07) between populations from South-East Asia and the South Pacific. These comparisons between P. vivax populations from four continents indicated that not only transmission intensity, but also geographical isolation affect diversity and population structure. However, the high effective population size results in slow changes of these parameters. This persistency must be taken into account when assessing the impact of control programs on the genetic structure of parasite populations.

Tracking malaria parasites in the eradication era.

As more endemic countries enter the elimination phase, the detection of sporadic malaria infections or outbreaks elicits many questions: (i) are the infections locally acquired or imported? (ii) If imported, where do they come from? (iii) Do outbreak strains have a single or multiple geographic origins? New molecular barcoding methods provide ways to analyze clinical malaria parasite samples and answer these and other crucial questions.

Using mitochondrial genome sequences to track the origin of imported Plasmodium vivax infections diagnosed in the United States.

Although the geographic origin of malaria cases imported into the United States can often be inferred from travel histories, these histories may be lacking or incomplete. We hypothesized that mitochondrial haplotypes could provide region-specific molecular barcodes for tracing the origin of imported Plasmodium vivax infections. An analysis of 348 mitochondrial genomes from worldwide parasites and new sequences from 69 imported malaria cases diagnosed across the United States allowed for a geographic assignment of most infections originating from the Americas, southeast Asia, east Asia, and Melanesia. However, mitochondrial lineages from Africa, south Asia, central Asia, and the Middle East, which altogether contribute the vast majority of imported malaria cases in the United States, were closely related to each other and could not be reliably assigned to their geographic origins. More mitochondrial genomes are required to characterize molecular barcodes of P. vivax from these regions.

Higher microsatellite diversity in Plasmodium vivax than in sympatric Plasmodium falciparum populations in Pursat, Western Cambodia.

Previous microsatellite analyses of sympatric populations of Plasmodium vivax and Plasmodium falciparum in Brazil revealed higher diversity in the former species. However, it remains unclear whether regional species-specific differences in prevalence and transmission levels might account for these findings. Here, we examine sympatric populations of P. vivax (n=87) and P. falciparum (n=164) parasites from Pursat province, Western Cambodia, where both species are similarly prevalent. Using 10 genome-wide microsatellites for P. falciparum and 13 for P. vivax, we found that the P. vivax population was more diverse than the sympatric P. falciparum population (average virtual heterozygosity [HE], 0.87 vs. 0.66, P=0.003), with more multiple-clone infections (89.6% vs. 47.6%) and larger mean number of alleles per marker (16.2 vs. 11.1, P=0.07). Both populations showed significant multi-locus linkage disequilibrium suggestive of a predominantly clonal mode of parasite reproduction. The higher microsatellite diversity found in P. vivax isolates, compared to sympatric P. falciparum isolates, does not necessarily result from local differences in transmission level and may reflect differences in population history between species or increased mutation rates in P. vivax.