Comparing malaria risk exposure in rural Cambodia population using GPS tracking and questionnaires.

BACKGROUND: The Great Mekong Subregion has attained a major decline in malaria cases and fatalities over the last years, but residual transmission hotspots remain, supposedly fueled by forest workers and migrant populations. This study aimed to: (i) characterize the fine-scale mobility of forest-goers and understand links between their daily movement patterns and malaria transmission, using parasites detection via real time polymerase chain reaction (RT PCR) and the individual exposure to Anopheles bites by quantification of anti-Anopheles saliva antibodies via enzyme-linked immunosorbent assay; (ii) assess the concordance of questionnaires and Global Positioning System (GPS) data loggers for measuring mobility. METHODS: Two 28 day follow-ups during dry and rainy seasons, including a GPS tracking, questionnaires and health examinations, were performed on male forest goers representing the population at highest risk of infection. Their time spent in different land use categories and demographic data were analyzed in order to understand the risk factors driving malaria in the study area. RESULTS: Malaria risk varied with village forest cover and at a resolution of only a few kilometers: participants from villages outside the forest had the highest malaria prevalence compared to participants from forest fringe's villages. The time spent in a specific environment did not modulate the risk of malaria, in particular the time spent in forest was not associated with a higher probability to detect malaria among forest-goers. The levels of antibody response to Anopheles salivary peptide among participants were significantly higher during the rainy season, in accordance with Anopheles mosquito density variation, but was not affected by sociodemographic and mobility factors. The agreement between GPS and self-reported data was only 61.9% in reporting each kind of visited environment. CONCLUSIONS: In a context of residual malaria transmission which was mainly depicted by P. vivax asymptomatic infections, the implementation of questionnaires, GPS data-loggers and quantification of anti-saliva Anopheles antibodies on the high-risk group were not powerful enough to detect malaria risk factors associated with different mobility behaviours or time spent in various environments. The joint implementation of GPS trackers and questionnaires allowed to highlight the limitations of both methodologies and the benefits of using them together. New detection and follow-up strategies are still called for.

Caveolin-1 protects endothelial cells from extensive expansion of transcellular tunnel by stiffening the plasma membrane.

Large transcellular pores elicited by bacterial mono-ADP-ribosyltransferase (mART) exotoxins inhibiting the small RhoA GTPase compromise the endothelial barrier. Recent advances in biophysical modeling point toward membrane tension and bending rigidity as the minimal set of mechanical parameters determining the nucleation and maximal size of transendothelial cell macroaperture (TEM) tunnels induced by bacterial RhoA-targeting mART exotoxins. We report that cellular depletion of caveolin-1, the membrane-embedded building block of caveolae, and depletion of cavin-1, the master regulator of caveolae invaginations, increase the number of TEMs per cell. The enhanced occurrence of TEM nucleation events correlates with a reduction in cell height due to the increase in cell spreading and decrease in cell volume, which, together with the disruption of RhoA-driven F-actin meshwork, favor membrane apposition for TEM nucleation. Strikingly, caveolin-1 specifically controls the opening speed of TEMs, leading to their dramatic 5.4-fold larger widening. Consistent with the increase in TEM density and width in siCAV1 cells, we record a higher lethality in CAV1 KO mice subjected to a catalytically active mART exotoxin targeting RhoA during staphylococcal bloodstream infection. Combined theoretical modeling with independent biophysical measurements of plasma membrane bending rigidity points toward a specific contribution of caveolin-1 to membrane stiffening in addition to the role of cavin-1/caveolin-1-dependent caveolae in the control of membrane tension homeostasis.

Using serological diagnostics to characterize remaining high-incidence pockets of malaria in forest-fringe Cambodia.

BACKGROUND: Over the last decades, the number of malaria cases has drastically reduced in Cambodia. As the overall prevalence of malaria in Cambodia declines, residual malaria transmission becomes increasingly fragmented over smaller remote regions. The aim of this study was to get an insight into the burden and epidemiological parameters of Plasmodium infections on the forest-fringe of Cambodia. METHODS: 950 participants were recruited in the province of Mondulkiri in Cambodia and followed up from 2018 to 2020. Whole-blood samples were processed for Plasmodium spp. identification by PCR as well as for a serological immunoassay. A risk factor analysis was conducted for Plasmodium vivax PCR-detected infections throughout the study, and for P. vivax seropositivity at baseline. To evaluate the predictive effect of seropositivity at baseline on subsequent PCR-positivity, an analysis of P. vivax infection-free survival time stratified by serological status at baseline was performed. RESULTS: Living inside the forest significantly increased the odds of P. vivax PCR-positivity by a factor of 18.3 (95% C.I. 7.7-43.5). Being a male adult was also a significant predictor of PCR-positivity. Similar risk profiles were identified for P. vivax seropositivity. The survival analysis showed that serological status at baseline significantly correlated with subsequent infection. Serology is most informative outside of the forest, where 94.0% (95% C.I. 90.7-97.4%) of seronegative individuals survived infection-free, compared to 32.4% (95% C.I.: 22.6-46.6%) of seropositive individuals. CONCLUSION: This study justifies the need for serological diagnostic assays to target interventions in this region, particularly in demographic groups where a lot of risk heterogeneity persists, such as outside of the forest.

Evaluating vector competence for Yellow fever in the Caribbean.

The mosquito-borne disease, Yellow fever (YF), has been largely controlled via mass delivery of an effective vaccine and mosquito control interventions. However, there are warning signs that YF is re-emerging in both Sub-Saharan Africa and South America. Imported from Africa in slave ships, YF was responsible for devastating outbreaks in the Caribbean. In Martinique, the last YF outbreak was reported in 1908 and the mosquito Aedes aegypti was incriminated as the main vector. We evaluated the vector competence of fifteen Ae. aegypti populations for five YFV genotypes (Bolivia, Ghana, Nigeria, Sudan, and Uganda). Here we show that mosquito populations from the Caribbean and the Americas were able to transmit the five YFV genotypes, with YFV strains for Uganda and Bolivia having higher transmission success. We also observed that Ae. aegypti populations from Martinique were more susceptible to YFV infection than other populations from neighboring Caribbean islands, as well as North and South America. Our vector competence data suggest that the threat of re-emergence of YF in Martinique and the subsequent spread to Caribbean nations and beyond is plausible.

Case Series of Primaquine-Induced Haemolytic Events in Controlled Trials with G6PD Screening.

Primaquine for radical cure of Plasmodium vivax malaria poses a potentially life-threatening risk of haemolysis in G6PD-deficient patients. Herein, we review five events of acute haemolytic anaemia following the administration of primaquine in four malaria trials from Indonesia, the Solomon Islands, and Vietnam. Five males aged 9 to 48 years were improperly classified as G6PD-normal by various screening procedures and included as subjects in trials of anti-relapse therapy with daily primaquine. Routine safety monitoring by physical examination, urine inspection, and blood haemoglobin (Hb) assessment were performed in all those trials. Early signs of acute haemolysis, i.e., dark urine and haemoglobin drop >20%, occurred only after day 3 and as late as day 8 of primaquine dosing. All patients were hospitalized and fully recovered, all but one following blood transfusion rescue. Hb nadir was 4.7 to 7.9 g/dL. Hospitalization was for 1 to 7 days. Hb levels returned to baseline values 3 to 10 days after transfusion. Failed G6PD screening procedures in these trials led G6PD-deficient patients to suffer harmful exposures to primaquine. The safe application of primaquine anti-relapse therapy requires G6PD screening and anticipation of its failure with a means of prompt detection and rescue from the typically abrupt haemolytic crisis.

Accelerating towards P. vivax elimination with a novel serological test-and-treat strategy: a modelling case study in Brazil.

Background: Plasmodium vivax malaria is challenging to control and eliminate. Treatment with radical cure drugs fails to target the hidden asymptomatic and hypnozoite reservoirs in populations. PvSeroTAT, a novel serological test-and-treat intervention using a serological diagnostic to screen hypnozoite carriers for radical cure eligibility and treatment, could accelerate P. vivax elimination. Methods: Using a previously developed mathematical model of P. vivax transmission adapted to the Brazilian context as a case study for implementation, we evaluate the public health impact of various deployment strategies of PvSeroTAT as a mass campaign. We compare relative reductions in prevalence, cases averted, glucose-6-phosphate dehydrogenase (G6PD) tests, and treatment doses of PvSeroTAT campaigns to strengthened case management alone or mass drug administration (MDA) campaigns across different settings. Findings: Deploying a single round of PvSeroTAT with 80% coverage to treat cases with a high efficacy radical cure regimen with primaquine is predicted to reduce point population prevalence by 22.5% [95% UI: 20.2%-24.8%] in a peri-urban setting with high transmission and by 25.2% [95% UI: 9.6%-42.2%] in an occupational setting with moderate transmission. In the latter example, while a single PvSeroTAT achieves 9.2% less impact on prevalence and averts 300 less cases per 100,000 than a single MDA (25.2% [95% UI: 9.6%-42.2%] point prevalence reduction versus 34.4% [95% UI: 24.9%-44%]), PvSeroTAT requires 4.6 times less radical cure treatments and G6PD tests. Layering strengthened case management and deploying four rounds of PvSeroTAT six months apart is predicted to reduce point prevalence by a mean of 74.1% [95% UI: 61.3%-86.3%] or more in low transmission settings with less than 10 cases per 1000 population. Interpretation: Modelling predicts that mass campaigns with PvSeroTAT are predicted to reduce P. vivax parasite prevalence across a range of transmission settings and require fewer resources than MDA. In combination with strengthened case management, mass campaigns of serological test-and-treat interventions can accelerate towards P. vivax elimination. Funding: This project was funded in part by the Bill and Melinda Gates Foundation and the National Health and Medical Research Council.

Assessing the Combined Public Health Impact of Pharmaceutical Interventions on Pandemic Transmission and Mortality: An Example in SARS CoV-2.

To assess the combined role of anti-viral monoclonal antibodies (mAbs) and vaccines in reducing severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) transmission and mortality in the United States, an agent-based model was developed that accounted for social contacts, movement/travel, disease progression, and viral shedding. The model was calibrated to coronavirus disease 2019 (COVID-19) mortality between October 2020 and April 2021 (aggressive pandemic phase), and projected an extended outlook to estimate mortality during a less aggressive phase (April-August 2021). Simulated scenarios evaluated mAbs for averting infections and deaths in addition to vaccines and aggregated non-pharmaceutical interventions. Scenarios included mAbs as a treatment of COVID-19 and for passive immunity for postexposure prophylaxis (PEP) during a period when variants were susceptible to the mAbs. Rapid diagnostic testing paired with mAbs was evaluated as an early treatment-as-prevention strategy. Sensitivity analyses included increasing mAb supply and vaccine rollout. Allocation of mAbs for use only as PEP averted up to 14% more infections than vaccine alone, and targeting individuals ≥ 65 years averted up to 37% more deaths. Rapid testing for earlier diagnosis and mAb use amplified these benefits. Doubling the mAb supply further reduced infections and mortality. mAbs provided benefits even as proportion of the immunized population increased. Model projections estimated that ~ 42% of expected deaths between April and August 2021 could be averted. Assuming sensitivity to mAbs, their use as early treatment and PEP in addition to vaccines would substantially reduce SARS-CoV-2 transmission and mortality even as vaccination increases and mortality decreases. These results provide a template for informing public health policy for future pandemic preparedness.

Zika vector competence data reveals risks of outbreaks: the contribution of the European ZIKAlliance project.

First identified in 1947, Zika virus took roughly 70 years to cause a pandemic unusually associated with virus-induced brain damage in newborns. Zika virus is transmitted by mosquitoes, mainly Aedes aegypti, and secondarily, Aedes albopictus, both colonizing a large strip encompassing tropical and temperate regions. As part of the international project ZIKAlliance initiated in 2016, 50 mosquito populations from six species collected in 12 countries were experimentally infected with different Zika viruses. Here, we show that Ae. aegypti is mainly responsible for Zika virus transmission having the highest susceptibility to viral infections. Other species play a secondary role in transmission while Culex mosquitoes are largely non-susceptible. Zika strain is expected to significantly modulate transmission efficiency with African strains being more likely to cause an outbreak. As the distribution of Ae. aegypti will doubtless expand with climate change and without new marketed vaccines, all the ingredients are in place to relive a new pandemic of Zika.

Plasmodium vivax malaria serological exposure markers: Assessing the degree and implications of cross-reactivity with P. knowlesi.

Serological markers are a promising tool for surveillance and targeted interventions for Plasmodium vivax malaria. P. vivax is closely related to the zoonotic parasite P. knowlesi, which also infects humans. P. vivax and P. knowlesi are co-endemic across much of South East Asia, making it important to design serological markers that minimize cross-reactivity in this region. To determine the degree of IgG cross-reactivity against a panel of P. vivax serological markers, we assayed samples from human patients with P. knowlesi malaria. IgG antibody reactivity is high against P. vivax proteins with high sequence identity with their P. knowlesi ortholog. IgG reactivity peaks at 7 days post-P. knowlesi infection and is short-lived, with minimal responses 1 year post-infection. We designed a panel of eight P. vivax proteins with low levels of cross-reactivity with P. knowlesi. This panel can accurately classify recent P. vivax infections while reducing misclassification of recent P. knowlesi infections.

Mobility evaluation by GPS tracking in a rural, low-income population in Cambodia.

Global Positioning System (GPS) technology is an effective tool for quantifying individuals' mobility patterns and can be used to understand their influence on infectious disease transmission. In Cambodia, mobility measurements have been limited to questionnaires, which are of limited efficacy in rural environments. In this study, we used GPS tracking to measure the daily mobility of Cambodian forest goers, a population at high risk of malaria, and developed a workflow adapted to local constraints to produce an optimal dataset representative of the participants' mobility. We provide a detailed assessment of the GPS tracking and analysis of the data, and highlight the associated difficulties to facilitate the implementation of similar studies in the future.

Impact of temperature on dengue and chikungunya transmission by the mosquito Aedes albopictus.

The mosquito Aedes albopictus is an invasive species first detected in Europe in Albania in 1979, and now established in 28 European countries. Temperature is a limiting factor in mosquito activities and in the transmission of associated arboviruses namely chikungunya (CHIKV) and dengue (DENV). Since 2007, local transmissions of CHIKV and DENV have been reported in mainland Europe, mainly in South Europe. Thus, the critical question is how far north transmission could occur. In this context, the Albanian infestation by Ae. albopictus is of interest because the species is present up to 1200 m of altitude; this allows using altitude as a proxy for latitude. Here we show that Ae. albopictus can transmit CHIKV at 28 °C as well as 20 °C, however, the transmission of DENV is only observed at 28 °C. We conclude that if temperature is the key environmental factor limiting transmission, then transmission of CHIKV, but not DENV is feasible in much of Europe.

Developing sero-diagnostic tests to facilitate Plasmodium vivax Serological Test-and-Treat approaches: modeling the balance between public health impact and overtreatment.

BACKGROUND: Eliminating Plasmodium vivax will require targeting the hidden liver-stage reservoir of hypnozoites. This necessitates new interventions balancing the benefit of reducing vivax transmission against the risk of over-treating some individuals with drugs which may induce haemolysis. By measuring antibodies to a panel of vivax antigens, a strategy of serological-testing-and-treatment (PvSeroTAT) can identify individuals with recent blood-stage infections who are likely to carry hypnozoites and target them for radical cure. This provides a potential solution to selectively treat the vivax reservoir with 8-aminoquinolines. METHODS: PvSeroTAT can identify likely hypnozoite carriers with ~80% sensitivity and specificity. Diagnostic test sensitivities and specificities ranging 50-100% were incorporated into a mathematical model of vivax transmission to explore how they affect the risks and benefits of different PvSeroTAT strategies involving hypnozoiticidal regimens. Risk was measured as the rate of overtreatment and benefit as reduction of community-level vivax transmission. RESULTS: Across a wide range of combinations of diagnostic sensitivity and specificity, PvSeroTAT was substantially more effective than bloodstage mass screen and treat strategies and only marginally less effective than mass drug administration. The key test characteristic determining of the benefit of PvSeroTAT strategies is diagnostic sensitivity, with higher values leading to more hypnozoite carriers effectively treated and greater reductions in vivax transmission. The key determinant of risk is diagnostic specificity: higher specificity ensures that a lower proportion of uninfected individuals are unnecessarily treated with primaquine. These relationships are maintained in both moderate and low transmission settings (qPCR prevalence 10% and 2%). Increased treatment efficacy and adherence can partially compensate for lower test performance. Multiple rounds of PvSeroTAT with a lower performing test may lead to similar or higher reductions in vivax transmission than fewer rounds with a higher performing test, albeit with higher rate of overtreatment. CONCLUSIONS: At current performance, PvSeroTAT is predicted to be a safe and efficacious option for targeting the hypnozoite reservoir towards vivax elimination. P. vivax sero-diagnostic tests should aim for both high performance and ease of use in the field. The target product profiles informing such development should thus reflect the trade-offs between impact, overtreatment, and ease of programmatic implementation.

Identification of the asymptomatic Plasmodium falciparum and Plasmodium vivax gametocyte reservoir under different transmission intensities.

BACKGROUND: Understanding epidemiological variables affecting gametocyte carriage and density is essential to design interventions that most effectively reduce malaria human-to-mosquito transmission. METHODOLOGY/PRINCIPAL FINDINGS: Plasmodium falciparum and P. vivax parasites and gametocytes were quantified by qPCR and RT-qPCR assays using the same methodologies in 5 cross-sectional surveys involving 16,493 individuals in Brazil, Thailand, Papua New Guinea, and Solomon Islands. The proportion of infections with detectable gametocytes per survey ranged from 44-94% for P. falciparum and from 23-72% for P. vivax. Blood-stage parasite density was the most important predictor of the probability to detect gametocytes. In moderate transmission settings (prevalence by qPCR>5%), parasite density decreased with age and the majority of gametocyte carriers were children. In low transmission settings (prevalence<5%), >65% of gametocyte carriers were adults. Per survey, 37-100% of all individuals positive for gametocytes by RT-qPCR were positive by light microscopy for asexual stages or gametocytes (overall: P. falciparum 178/348, P. vivax 235/398). CONCLUSIONS/SIGNIFICANCE: Interventions to reduce human-to-mosquito malaria transmission in moderate-high endemicity settings will have the greatest impact when children are targeted. In contrast, all age groups need to be included in control activities in low endemicity settings to achieve elimination. Detection of infections by light microscopy is a valuable tool to identify asymptomatic blood stage infections that likely contribute most to ongoing transmission at the time of sampling.

Kinetics of the Severe Acute Respiratory Syndrome Coronavirus 2 Antibody Response and Serological Estimation of Time Since Infection.

BACKGROUND: Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces a complex antibody response that varies by orders of magnitude between individuals and over time. METHODS: We developed a multiplex serological test for measuring antibodies to 5 SARS-CoV-2 antigens and the spike proteins of seasonal coronaviruses. We measured antibody responses in cohorts of hospitalized patients and healthcare workers followed for up to 11 months after symptoms. A mathematical model of antibody kinetics was used to quantify the duration of antibody responses. Antibody response data were used to train algorithms for estimating time since infection. RESULTS: One year after symptoms, we estimate that 36% (95% range, 11%-94%) of anti-Spike immunoglobulin G (IgG) remains, 31% (95% range, 9%-89%) anti-RBD IgG remains, and 7% (1%-31%) of anti-nucleocapsid IgG remains. The multiplex assay classified previous infections into time intervals of 0-3 months, 3-6 months, and 6-12 months. This method was validated using data from a seroprevalence survey in France, demonstrating that historical SARS-CoV-2 transmission can be reconstructed using samples from a single survey. CONCLUSIONS: In addition to diagnosing previous SARS-CoV-2 infection, multiplex serological assays can estimate the time since infection, which can be used to reconstruct past epidemics.

Estimated impact of tafenoquine for Plasmodium vivax control and elimination in Brazil: A modelling study.

BACKGROUND: Despite recent intensification of control measures, Plasmodium vivax poses a major challenge for malaria elimination efforts. Liver-stage hypnozoite parasites that cause relapsing infections can be cleared with primaquine; however, poor treatment adherence undermines drug effectiveness. Tafenoquine, a new single-dose treatment, offers an alternative option for preventing relapses and reducing transmission. In 2018, over 237,000 cases of malaria were reported to the Brazilian health system, of which 91.5% were due to P. vivax. METHODS AND FINDINGS: We evaluated the impact of introducing tafenoquine into case management practices on population-level transmission dynamics using a mathematical model of P. vivax transmission. The model was calibrated to reflect the transmission dynamics of P. vivax endemic settings in Brazil in 2018, informed by nationwide malaria case reporting data. Parameters for treatment pathways with chloroquine, primaquine, and tafenoquine with glucose-6-phosphate dehydrogenase deficiency (G6PDd) testing were informed by clinical trial data and the literature. We assumed 71.3% efficacy for primaquine and tafenoquine, a 66.7% adherence rate to the 7-day primaquine regimen, a mean 5.5% G6PDd prevalence, and 8.1% low metaboliser prevalence. The introduction of tafenoquine is predicted to improve effective hypnozoite clearance among P. vivax cases and reduce population-level transmission over time, with heterogeneous levels of impact across different transmission settings. According to the model, while achieving elimination in only few settings in Brazil, tafenoquine rollout in 2021 is estimated to improve the mean effective radical cure rate from 42% (95% uncertainty interval [UI] 41%-44%) to 62% (95% UI 54%-68%) among clinical cases, leading to a predicted 38% (95% UI 7%-99%) reduction in transmission and over 214,000 cumulative averted cases between 2021 and 2025. Higher impact is predicted in settings with low transmission, low pre-existing primaquine adherence, and a high proportion of cases in working-aged males. High-transmission settings with a high proportion of cases in children would benefit from a safe high-efficacy tafenoquine dose for children. Our methodological limitations include not accounting for the role of imported cases from outside the transmission setting, relying on reported clinical cases as a measurement of community-level transmission, and implementing treatment efficacy as a binary condition. CONCLUSIONS: In our modelling study, we predicted that, provided there is concurrent rollout of G6PDd diagnostics, tafenoquine has the potential to reduce P. vivax transmission by improving effective radical cure through increased adherence and increased protection from new infections. While tafenoquine alone may not be sufficient for P. vivax elimination, its introduction will improve case management, prevent a substantial number of cases, and bring countries closer to achieving malaria elimination goals.

Single-cell RNA sequencing reveals developmental heterogeneity among Plasmodium berghei sporozoites.

In the malaria-causing parasite's life cycle, Plasmodium sporozoites must travel from the midgut of a mosquito to the salivary glands before they can infect a mammalian host. However, only a fraction of sporozoites complete the journey. Since salivary gland invasion is required for transmission of sporozoites, insights at the molecular level can contribute to strategies for malaria prevention. Recent advances in single-cell RNA sequencing provide an opportunity to assess sporozoite heterogeneity at a resolution unattainable by bulk RNA sequencing methods. In this study, we use a droplet-based single-cell RNA sequencing workflow to analyze the transcriptomes of over 8000 Plasmodium berghei sporozoites derived from the midguts and salivary glands of Anopheles stephensi mosquitoes. The detection of known marker genes confirms the successful capture and sequencing of samples composed of a mixed population of sporozoites. Using data integration, clustering, and trajectory analyses, we reveal differences in gene expression profiles of individual sporozoites, and identify both annotated and unannotated markers associated with sporozoite development. Our work highlights the utility of a high-throughput workflow for the transcriptomic profiling of Plasmodium sporozoites, and provides new insights into gene usage during the parasite's development in the mosquito.

Risk of yellow fever virus transmission in the Asia-Pacific region.

Historically endemic to Sub-Saharan Africa and South America, yellow fever is absent from the Asia-Pacific region. Yellow fever virus (YFV) is mainly transmitted by the anthropophilic Aedes mosquitoes whose distribution encompasses a large belt of tropical and sub tropical regions. Increasing exchanges between Africa and Asia have caused imported YFV incidents in non-endemic areas, which are threatening Asia with a new viral emergence. Here, using experimental infections of field-collected mosquitoes, we show that Asian-Pacific Aedes mosquitoes are competent vectors for YFV. We observe that Aedes aegypti populations from Singapore, Taiwan, Thailand, and New Caledonia are capable of transmitting YFV 14 days after oral infections, with a number of viral particles excreted from saliva reaching up to 23,000 viral particles. These findings represent the most comprehensive assessment of vector competence and show that Ae. aegypti mosquitoes from the Asia-Pacific region are highly competent to YFV, corroborating that vector populations are seemingly not a brake to the emergence of yellow fever in the region.

Forest malaria in Cambodia: the occupational and spatial clustering of Plasmodium vivax and Plasmodium falciparum infection risk in a cross-sectional survey in Mondulkiri province, Cambodia.

BACKGROUND: After a marked reduction in malaria burden in Cambodia over the last decades, case numbers increased again in 2017-2018. In light of the national goal of malaria elimination by 2025, remaining pockets of high risk need to be well defined and strategies well-tailored to identify and target the persisting burden cost-effectively. This study presents species-specific prevalence estimates and risk stratification for a remote area in Cambodia. METHODS: A cross-sectional survey was conducted in 17 villages in the high-incidence province Mondulkiri in the dry season (December 2017 to April 2018). 4200 randomly selected participants (2-80 years old) were tested for Plasmodium infection by PCR. Risk of infection was associated with questionnaire-derived covariates and spatially stratified based on household GPS coordinates. RESULTS: The prevalence of PCR-detectable Plasmodium infection was 8.3% (349/4200) and was more than twice as high for Plasmodium vivax (6.4%, 268) than for Plasmodium falciparum (3.0%, 125, p < 0.001). 97.8% (262/268) of P. vivax and 92.8% (116/125, p < 0.05) of P. falciparum infections were neither accompanied by symptoms at the time of the interview nor detected by microscopy or RDT. Recent travels to forest sites (aOR 2.17, p < 0.01) and forest work (aOR 2.88, p < 0.001) were particularly strong risk factors and risk profiles for both species were similar. Large village-level differences in prevalence of Plasmodium infection were observed, ranging from 0.6% outside the forest to 40.4% inside. Residing in villages at the forest fringe or inside the forest compared to outside was associated with risk of infection (aOR 2.14 and 12.47, p < 0.001). Villages inside the forest formed spatial hotspots of infection despite adjustment for the other risk factors. CONCLUSIONS: Persisting pockets of high malaria risk were detected in forested areas and in sub-populations engaging in forest-related activities. High levels of asymptomatic infections suggest the need of better case detection plans and the predominance of P. vivax the implementation of radical cure. In villages inside the forest, within-village exposure was indicated in addition to risk due to forest activities. Village-level stratification of targeted interventions based on forest proximity could render the elimination efforts more cost-effective and successful.

Development and validation of serological markers for detecting recent Plasmodium vivax infection.

A major gap in the Plasmodium vivax elimination toolkit is the identification of individuals carrying clinically silent and undetectable liver-stage parasites, called hypnozoites. This study developed a panel of serological exposure markers capable of classifying individuals with recent P. vivax infections who have a high likelihood of harboring hypnozoites. We measured IgG antibody responses to 342 P. vivax proteins in longitudinal clinical cohorts conducted in Thailand and Brazil and identified candidate serological markers of exposure. Candidate markers were validated using samples from year-long observational cohorts conducted in Thailand, Brazil and the Solomon Islands and antibody responses to eight P. vivax proteins classified P. vivax infections in the previous 9 months with 80% sensitivity and specificity. Mathematical models demonstrate that a serological testing and treatment strategy could reduce P. vivax prevalence by 59-69%. These eight antibody responses can serve as a biomarker, identifying individuals who should be targeted with anti-hypnozoite therapy.

Plasmodium vivax spleen-dependent genes encode antigens associated with cytoadhesion and clinical protection.

Plasmodium vivax, the most widely distributed human malaria parasite, causes severe clinical syndromes despite low peripheral blood parasitemia. This conundrum is further complicated as cytoadherence in the microvasculature is still a matter of investigations. Previous reports in Plasmodium knowlesi, another parasite species shown to infect humans, demonstrated that variant genes involved in cytoadherence were dependent on the spleen for their expression. Hence, using a global transcriptional analysis of parasites obtained from spleen-intact and splenectomized monkeys, we identified 67 P. vivax genes whose expression was spleen dependent. To determine their role in cytoadherence, two Plasmodium falciparum transgenic lines expressing two variant proteins pertaining to VIR and Pv-FAM-D multigene families were used. Cytoadherence assays demonstrated specific binding to human spleen but not lung fibroblasts of the transgenic line expressing the VIR14 protein. To gain more insights, we expressed five P. vivax spleen-dependent genes as recombinant proteins, including members of three different multigene families (VIR, Pv-FAM-A, Pv-FAM-D), one membrane transporter (SECY), and one hypothetical protein (HYP1), and determined their immunogenicity and association with clinical protection in a prospective study of 383 children in Papua New Guinea. Results demonstrated that spleen-dependent antigens are immunogenic in natural infections and that antibodies to HYP1 are associated with clinical protection. These results suggest that the spleen plays a major role in expression of parasite proteins involved in cytoadherence and can reveal antigens associated with clinical protection, thus prompting a paradigm shift in P. vivax biology toward deeper studies of the spleen during infections.