À la carte: how mosquitoes choose their blood meals.

Mosquitoes are important vectors for human diseases, transmitting pathogens that cause a range of parasitic and viral infections. Mosquito blood-feeding is heterogeneous, meaning that some human hosts are at higher risk of receiving bites than others, and this heterogeneity is multifactorial. Mosquitoes integrate specific cues to locate their hosts, and mosquito attraction differs considerably between individual human hosts. Heterogeneous mosquito biting results from variations in both host attractiveness and availability and can impact transmission of vector-borne diseases. However, the extent and drivers of this heterogeneity and its importance for pathogen transmission remain incompletely understood. Here, we review methods and recent data describing human characteristics that affect host-seeking behavior and host preferences of mosquito disease vectors, and the implications for vector-borne disease transmission.

Tripartite interactions between viruses, parasites, and mosquitoes.

Mosquito-borne diseases have a major impact on global human health. Biological agents that colonize the mosquito vector are increasingly explored as an intervention strategy to prevent vector-borne disease transmission. For instance, the release of mosquitoes carrying the endosymbiotic bacterium Wolbachia effectively reduced dengue virus incidence and disease. Insect-specific viruses are likewise considered as biocontrol agents against vector-borne diseases. While most studies focused on insect-specific viruses as an intervention against arthropod-borne viruses, we here consider whether mosquito-specific viruses may affect the transmission of the malaria-causing Plasmodium parasite by Anopheles mosquitoes. Although there is no direct experimental evidence addressing this question, we found that viral infections in dipteran insects activate some of the immune pathways that are antiparasitic in Anopheles. These findings suggest that indirect virus-parasite interactions could occur and that insect-specific viruses may modulate malaria transmission. Tripartite interactions between viruses, parasites, and Anopheles mosquitoes thus merit further investigation.

Plasmodium falciparum gametocyte production correlates with genetic markers of parasite replication but is not influenced by experimental exposure to mosquito biting.

BACKGROUND: Plasmodium blood-stage parasites balance asexual multiplication with gametocyte development. Few studies link these dynamics with parasite genetic markers in vivo; even fewer in longitudinally monitored infections. Environmental influences on gametocyte formation, such as mosquito exposure, may influence the parasite's investment in gametocyte production. METHODS: We investigated gametocyte production and asexual multiplication in two Plasmodium falciparum infected populations; a controlled human malaria infection (CHMI) study and a 28-day observational study in naturally infected individuals in Burkina Faso with controlled mosquito exposure. We measured gene transcript levels previously related to gametocyte formation (ap2-g, surfin1.2, surfin13.1, gexp-2) or inhibition of asexual multiplication (sir2a) and compared transcript levels to ring-stage parasite and mature gametocyte densities. FINDINGS: Three of the five markers (ap2-g, surfin1.2, surfin13.1) predicted peak gametocytaemia in the CHMI study. An increase in all five markers in natural infections was associated with an increase in mature gametocytes 14 days later; the effect of sir2a on future gametocytes was strongest (fold change = 1.65, IQR = 1.22-2.24, P = 0.004). Mosquito exposure was not associated with markers of gametocyte formation (ap2-g P = 0.277; sir2a P = 0.499) or carriage of mature gametocytes (P = 0.379). INTERPRETATION: All five parasite genetic markers predicted gametocyte formation over a single cycle of gametocyte formation and maturation in vivo; sir2a and ap2-g were most closely associated with gametocyte growth dynamics. We observed no evidence to support the hypothesis that exposure to Anopheles mosquito bites stimulates gametocyte formation. FUNDING: This work was funded by the Bill & Melinda Gates Foundation (INDIE OPP1173572), the European Research Council fellowship (ERC-CoG 864180) and UKRI Medical Research Council (MR/T016272/1) and Wellcome Center (218676/Z/19/Z).

AlbuMAX supplemented media induces the formation of transmission-competent P. falciparum gametocytes.

Asexual blood stage culture of Plasmodium falciparum is routinely performed but reproducibly inducing commitment to and maturation of viable gametocytes remains difficult. Culture media can be supplemented with human serum substitutes to induce commitment but these generally only allow for long-term culture of asexual parasites and not transmission-competent gametocytes due to their different lipid composition. Recent insights demonstrated the important roles lipids play in sexual commitment; elaborating on this we exposed ring stage parasites (20-24 hours hpi) for one day to AlbuMAX supplemented media to trigger induction to gametocytogenesis. We observed a significant increase in gametocytes after AlbuMAX induction compared to serum. We also tested the transmission potential of AlbuMAX inducted gametocytes and found a significant higher oocyst intensity compared to serum. We conclude that AlbuMAX supplemented media induces commitment, allows a more stable and predictable production of transmittable gametocytes than serum alone.

Artemether-lumefantrine with or without single-dose primaquine and sulfadoxine-pyrimethamine plus amodiaquine with or without single-dose tafenoquine to reduce Plasmodium falciparum transmission: a phase 2, single-blind, randomised clinical trial in Ouelessebougou, Mali.

BACKGROUND: Artemether-lumefantrine is widely used for uncomplicated Plasmodium falciparum malaria; sulfadoxine-pyrimethamine plus amodiaquine is used for seasonal malaria chemoprevention. We aimed to determine the efficacy of artemether-lumefantrine with and without primaquine and sulfadoxine-pyrimethamine plus amodiaquine with and without tafenoquine for reducing gametocyte carriage and transmission to mosquitoes. METHODS: In this phase 2, single-blind, randomised clinical trial conducted in Ouelessebougou, Mali, asymptomatic individuals aged 10-50 years with P falciparum gametocytaemia were recruited from the community and randomly assigned (1:1:1:1) to receive either artemether-lumefantrine, artemether-lumefantrine with a single dose of 0·25 mg/kg primaquine, sulfadoxine-pyrimethamine plus amodiaquine, or sulfadoxine-pyrimethamine plus amodiaquine with a single dose of 1·66 mg/kg tafenoquine. All trial staff other than the pharmacist were masked to group allocation. Participants were not masked to group allocation. Randomisation was done with a computer-generated randomisation list and concealed with sealed, opaque envelopes. The primary outcome was the median within-person percent change in mosquito infection rate in infectious individuals from baseline to day 2 (artemether-lumefantrine groups) or day 7 (sulfadoxine-pyrimethamine plus amodiaquine groups) after treatment, assessed by direct membrane feeding assay. All participants who received any trial drug were included in the safety analysis. This study is registered with ClinicalTrials.gov, NCT05081089. FINDINGS: Between Oct 13 and Dec 16, 2021, 1290 individuals were screened and 80 were enrolled and randomly assigned to one of the four treatment groups (20 per group). The median age of participants was 13 (IQR 11-20); 37 (46%) of 80 participants were female and 43 (54%) were male. In individuals who were infectious before treatment, the median percentage reduction in mosquito infection rate 2 days after treatment was 100·0% (IQR 100·0-100·0; n=19; p=0·0011) with artemether-lumefantrine and 100·0% (100·0-100·0; n=19; p=0·0001) with artemether-lumefantrine with primaquine. Only two individuals who were infectious at baseline infected mosquitoes on day 2 after artemether-lumefantrine and none at day 5. By contrast, the median percentage reduction in mosquito infection rate 7 days after treatment was 63·6% (IQR 0·0-100·0; n=20; p=0·013) with sulfadoxine-pyrimethamine plus amodiaquine and 100% (100·0-100·0; n=19; p<0·0001) with sulfadoxine-pyrimethamine plus amodiaquine with tafenoquine. No grade 3-4 or serious adverse events occurred. INTERPRETATION: These data support the effectiveness of artemether-lumefantrine alone for preventing nearly all mosquito infections. By contrast, there was considerable post-treatment transmission after sulfadoxine-pyrimethamine plus amodiaquine; therefore, the addition of a transmission-blocking drug might be beneficial in maximising its community impact. FUNDING: Bill & Melinda Gates Foundation.

Reversible host cell surface remodelling limits immune recognition and maximizes transmission of Plasmodium falciparum gametocytes.

Reducing malaria transmission has been a major pillar of control programmes and is considered crucial for achieving malaria elimination. Gametocytes, the transmissible forms of the P. falciparum parasite, arise during the blood stage of the parasite and develop through 5 morphologically distinct stages. Immature gametocytes (stage I-IV) sequester and develop in the extravascular niche of the bone marrow and possibly spleen. Only mature stage V gametocytes re-enter peripheral circulation to be taken up by mosquitoes for successful onward transmission. We have recently shown that immature, but not mature gametocytes are targets of host immune responses and identified putative target surface antigens. We hypothesize that these antigens play a role in gametocyte sequestration and contribute to acquired transmission-reducing immunity. Here we demonstrate that surface antigen expression, serum reactivity by human IgG, and opsonic phagocytosis by macrophages all show similar dynamics during gametocyte maturation, i.e., on in immature and off in mature gametocytes. Moreover, the switch in surface reactivity coincides with reversal in phosphatidylserine (PS) surface exposure, a marker for red blood cell age and clearance. PS is exposed on the surface of immature gametocytes (as well as in late asexual stages) but is removed from the surface in later gametocyte stages (IV-V). Using parasite reverse genetics and drug perturbations, we confirm that parasite protein export into the host cell and phospholipid scramblase activity are required for the observed surface modifications in asexual and sexual P. falciparum stages. These findings suggest that the dynamic surface remodelling allows (i) immature gametocyte sequestration in bone marrow and (ii) mature gametocyte release into peripheral circulation and immune evasion, therefore contributing to mature gametocyte survival in vivo and onward transmission to mosquitoes. Importantly, blocking scramblase activity during gametocyte maturation results in efficient clearance of mature gametocytes, revealing a potential path for transmission blocking interventions. Our studies have important implications for our understanding of parasite biology and form a starting point for novel intervention strategies to simultaneously reduce parasite burden and transmission.

Household-level effects of seasonal malaria chemoprevention in the Gambia.

BACKGROUND: In 2022 the WHO recommended the discretionary expansion of the eligible age range for seasonal malaria chemoprevention (SMC) to children older than 4 years. Older children are at lower risk of clinical disease and severe malaria so there has been uncertainty about the cost-benefit for national control programmes. However, emerging evidence from laboratory studies suggests protecting school-age children reduces the infectious reservoir for malaria and may significantly impact on transmission. This study aimed to assess whether these effects were detectable in the context of a routinely delivered SMC programme. METHODS: In 2021 the Gambia extended the maximum eligible age for SMC from 4 to 9 years. We conducted a prospective population cohort study over the 2021 malaria transmission season covering 2210 inhabitants of 10 communities in the Upper River Region, and used a household-level mixed modelling approach to quantify impacts of SMC on malaria transmission. RESULTS: We demonstrate that the hazard of clinical malaria in older participants aged 10+ years ineligible for SMC decreases by 20% for each additional SMC round per child 0-9 years in the same household. Older inhabitants also benefit from reduced risk of asymptomatic infections in high SMC coverage households. Spatial autoregression tests show impacts are highly localised, with no detectable spillover from nearby households. CONCLUSIONS: Evidence for the transmission-reducing effects of extended-age SMC from routine programmes implemented at scale has been previously limited. Here we demonstrate benefits to the entire household, indicating such programmes may be more cost-effective than previously estimated.

Seasonal malaria chemoprevention (SMC) is the provision of monthly, preventative, anti-malaria medication to young children at times when they are most at risk of severe disease. Recently the World Health Organisation recommended expanding SMC to children older than 4 years. Older children with malaria typically remain symptomless so the advantages were unclear. However, laboratory evidence suggests this group continues to transmit malaria to others. We conducted a population study in 2021 in 10 communities in the Gambia where SMC was extended to all children up to 9 years of age for the first time. We found household members aged over 9 years were less likely to get clinical disease when most young children in the same household did receive SMC. This suggests an added protection of SMC for those who do not receive it, potentially increasing cost-effectiveness.

Population pharmacokinetics of primaquine and its metabolites in African males.

BACKGROUND: Primaquine (PQ) is the prototype 8-aminoquinoline drug, a class which targets gametocytes and hypnozoites. The World Health Organization (WHO) recommends adding a single low dose of primaquine to the standard artemisinin-based combination therapy (ACT) in order to block malaria transmission in regions with low malaria transmission. However, the haemolytic toxicity is a major adverse outcome of primaquine in glucose-6-phosphate dehydrogenase (G6PD)-deficient subjects. This study aimed to characterize the pharmacokinetic properties of primaquine and its major metabolites in G6PD-deficient subjects. METHODS: A single low-dose of primaquine (0.4-0.5 mg/kg) was administered in twenty-eight African males. Venous and capillary plasma were sampled up to 24 h after the drug administration. Haemoglobin levels were observed up to 28 days after drug administration. Only PQ, carboxy-primaquine (CPQ), and primaquine carbamoyl-glucuronide (PQCG) were present in plasma samples and measured using liquid chromatography mass spectrometry. Drug and metabolites' pharmacokinetic properties were investigated using nonlinear mixed-effects modelling. RESULTS: Population pharmacokinetic properties of PQ, CPQ, and PQCG can be described by one-compartment disposition kinetics with a transit-absorption model. Body weight was implemented as an allometric function on the clearance and volume parameters for all compounds. None of the covariates significantly affected the pharmacokinetic parameters. No significant correlations were detected between the exposures of the measured compounds and the change in haemoglobin or methaemoglobin levels. There was no significant haemoglobin drop in the G6PD-deficient patients after administration of a single low dose of PQ. CONCLUSIONS: A single low-dose of PQ was haematologically safe in this population of G6PD-normal and G6PD-deficient African males without malaria. Trial registration NCT02535767.

Dramatic resurgence of malaria after 7 years of intensive vector control interventions in Eastern Uganda.

Background: Tororo District, Uganda experienced a dramatic decrease in malaria burden from 2015-19 following 5 years of indoor residual spraying (IRS) with carbamate (Bendiocarb) and then organophosphate (Actellic) insecticides. However, a marked resurgence occurred in 2020, which coincided with a change to a clothianidin-based IRS formulations (Fludora Fusion/SumiShield). To quantify the magnitude of the resurgence, investigate causes, and evaluate the impact of a shift back to IRS with Actellic in 2023, we assessed changes in malaria metrics in regions within and near Tororo District. Methods: Malaria surveillance data from Nagongera Health Center, Tororo District was included from 2011-2023. In addition, a cohort of 667 residents from 84 houses was followed from August 2020 through September 2023 from an area bordering Tororo and neighboring Busia District, where IRS has never been implemented. Cohort participants underwent passive surveillance for clinical malaria and active surveillance for parasitemia every 28 days. Mosquitoes were collected in cohort households every 2 weeks using CDC light traps. Female Anopheles were speciated and tested for sporozoites and phenotypic insecticide resistance. Temporal comparisons of malaria metrics were stratified by geographic regions. Findings: At Nagongera Health Center average monthly malaria cases varied from 419 prior to implementation of IRS; to 56 after 5 years of IRS with Bendiocarb and Actellic; to 1591 after the change in IRS to Fludora Fusion/SumiShield; to 155 after a change back to Actellic. Among cohort participants living away from the border in Tororo, malaria incidence increased over 8-fold (0.36 vs. 2.97 episodes per person year, p<0.0001) and parasite prevalence increased over 4-fold (17% vs. 70%, p<0.0001) from 2021 to 2022 when Fludora Fusion/SumiShield was used. Incidence decreased almost 5-fold (2.97 vs. 0.70, p<0.0001) and prevalence decreased by 39% (70% vs. 43%, p<0.0001) after shifting back to Actellic. There was a similar pattern among those living near the border in Tororo, with increased incidence between 2021 and 2022 (0.93 vs. 2.40, p<0.0001) followed by a decrease after the change to Actellic (2.40 vs. 1.33, p<0.001). Among residents of Busia, malaria incidence did not change significantly over the 3 years of observation. Malaria resurgence in Tororo was temporally correlated with the replacement of An. gambiae s.s. by An. funestus as the primary vector, with a marked decrease in the density of An. funestus following the shift back to IRS with Actellic. In Busia, An. gambiae s.s. remained the primary vector throughout the observation period. Sporozoite rates were approximately 50% higher among An. funestus compared to the other common malaria vectors. Insecticide resistance phenotyping of An. funestus revealed high tolerance to clothianidin, but full susceptibility to Actellic. Conclusions: A dramatic resurgence of malaria in Tororo was temporally associated with a change to clothianidin-based IRS formulations and emergence of An. funestus as the predominant vector. Malaria decreased after a shift back to IRS with Actellic. This study highlights the ability of malaria vectors to rapidly circumvent control efforts and the importance of high-quality surveillance systems to assess the impact of malaria control interventions and generate timely, actionable data.

Geographic Equity and Environmental Sustainability of Conference Models: Results of a Comparative Analysis.

We conducted a comparative analysis of in-person, virtual, and hybrid conferences on tuberculosis hosted by Keystone Symposia and examined the number of participants, their country of residence, carbon dioxide equivalents (CO2e) produced, and participant impressions regarding scientific quality. Data were available from three in-person meetings, one virtual meeting, and one hybrid. The virtual conference hosted 2.5-fold more participants compared with the in-person conferences (842 versus an average of 328) from more than double the number of countries (68 versus an average of 33). The virtual conference attracted 4.5-fold more participants from countries with a high burden of tuberculosis, compared with the average in-person conference (209 versus an average of 46). For in-person meetings, an average of 79% of participants were based in high-income countries. For the virtual meeting, 53% of participants were from high-income countries, and 47% from low- and middle-income countries. For the hybrid conference, there were 465 participants from 43 countries, of which 289 attended in person from a total of 20 countries, and 176 participated virtually from 34 countries. Of those who took part in person, 91% were from high-income countries. The average CO2e emissions from an in-person conference was 696 tons of CO2e, with 96.0% from air travel. The virtual meeting produced 0.48 ton of CO2e from electricity usage, a 1,450-fold decrease compared with in-person events. Virtual conferences scored a content quality rating of 87.3% to 90.8% compared with a range of 86.4% to 92.2% for in-person conferences.

Quantification of sporozoite expelling by Anopheles mosquitoes infected with laboratory and naturally circulating P. falciparum gametocytes.

It is currently unknown whether all Plasmodium falciparum-infected mosquitoes are equally infectious. We assessed sporogonic development using cultured gametocytes in the Netherlands and naturally circulating strains in Burkina Faso. We quantified the number of sporozoites expelled into artificial skin in relation to intact oocysts, ruptured oocysts, and residual salivary gland sporozoites. In laboratory conditions, higher total sporozoite burden was associated with shorter duration of sporogony (p<0.001). Overall, 53% (116/216) of infected Anopheles stephensi mosquitoes expelled sporozoites into artificial skin with a median of 136 expelled sporozoites (interquartile range [IQR], 34-501). There was a strong positive correlation between ruptured oocyst number and salivary gland sporozoite load (ρ = 0.8; p<0.0001) and a weaker positive correlation between salivary gland sporozoite load and number of sporozoites expelled (ρ = 0.35; p=0.0002). In Burkina Faso, Anopheles coluzzii mosquitoes were infected by natural gametocyte carriers. Among salivary gland sporozoite positive mosquitoes, 89% (33/37) expelled sporozoites with a median of 1035 expelled sporozoites (IQR, 171-2969). Again, we observed a strong correlation between ruptured oocyst number and salivary gland sporozoite load (ρ = 0.9; p<0.0001) and a positive correlation between salivary gland sporozoite load and the number of sporozoites expelled (ρ = 0.7; p<0.0001). Several mosquitoes expelled multiple parasite clones during probing. Whilst sporozoite expelling was regularly observed from mosquitoes with low infection burdens, our findings indicate that mosquito infection burden is positively associated with the number of expelled sporozoites. Future work is required to determine the direct implications of these findings for transmission potential.

Understanding and maximising the community impact of seasonal malaria chemoprevention in Burkina Faso (INDIE-SMC): study protocol for a cluster randomised evaluation trial.

INTRODUCTION: Seasonal malaria chemoprevention (SMC) involves repeated administrations of sulfadoxine-pyrimethamine plus amodiaquine to children below the age of 5 years during the peak transmission season in areas of seasonal malaria transmission. While highly impactful in reducing Plasmodium falciparum malaria burden in controlled research settings, the impact of SMC on infection prevalence is moderate in real-life settings. It remains unclear what drives this efficacy decay. Recently, the WHO widened the scope for SMC to target all vulnerable populations. The Ministry of Health (MoH) in Burkina Faso is considering extending SMC to children below 10 years old. We aim to assess the impact of SMC on clinical incidence and parasite prevalence and quantify the human infectious reservoir for malaria in this population. METHODS AND ANALYSIS: We will perform a cluster randomised trial in Saponé Health District, Burkina Faso, with three study arms comprising 62 clusters of three compounds: arm 1 (control): SMC in under 5-year-old children, implemented by the MoH without directly observed treatment (DOT) for the full course of SMC; arm 2 (intervention): SMC in under 5-year-old children, with DOT for the full course of SMC; arm 3 (intervention): SMC in under 10-year-old children, with DOT for the full course of SMC. The primary endpoint is parasite prevalence at the end of the malaria transmission season. Secondary endpoints include the impact of SMC on clinical incidence. Factors affecting SMC uptake, treatment adherence, drug concentrations, parasite resistance markers and transmission of parasites will be determined. ETHICS AND DISSEMINATION: The London School of Hygiene & Tropical Medicine's Ethics Committee (29193) and the Burkina Faso National Medical Ethics Committee (Deliberation No 2023-05-104) approved this study. The findings will be presented to the community; disease occurrence data and study outcomes will also be shared with the Burkina Faso MoH. Findings will be published irrespective of their results. TRIAL REGISTRATION NUMBER: NCT05878366.

Dynamics of asymptomatic Plasmodium falciparum and Plasmodium vivax infections and their infectiousness to mosquitoes in a low transmission setting of Ethiopia: a longitudinal observational study.

OBJECTIVE: A 15-month longitudinal study was conducted to determine the duration and infectivity of asymptomatic qPCR-detected Plasmodium falciparum and Plasmodium vivax infections in Ethiopia. METHOD: Total parasite and gametocyte kinetics were determined by molecular methods; infectivity to Anopheles arabiensis mosquitoes by repeated membrane feeding assays. Infectivity results were contrasted with passively recruited symptomatic malaria cases. RESULTS: For P. falciparum and P. vivax infections detected at enrolment, median durations of infection were 37 days (95% confidence interval [CI], 15-93) and 60 days (95% CI, 18-213), respectively. P. falciparum and P. vivax parasite densities declined over the course of infections. From 47 feeding assays on 22 asymptomatic P. falciparum infections, 6.4% (3/47) were infectious and these infected 1.8% (29/1579) of mosquitoes. No transmission was observed in feeding assays on asymptomatic P. vivax mono-infections (0/56); one mixed-species infection was highly infectious. Among the symptomatic cases, 4.3% (2/47) of P. falciparum and 73.3% (53/86) of P. vivax patients were infectious to mosquitoes. CONCLUSION: The majority of asymptomatic infections were of short duration and low parasite density. Only a minority of asymptomatic individuals were infectious to mosquitoes. This contrasts with earlier findings and is plausibly due to the low parasite densities in this population.

Asymptomatic Plasmodium falciparum carriage at the end of the dry season is associated with subsequent infection and clinical malaria in Eastern Gambia.

BACKGROUND: Chronic carriage of asymptomatic low-density Plasmodium falciparum parasitaemia in the dry season may support maintenance of acquired immunity that protects against clinical malaria. However, the relationship between chronic low-density infections and subsequent risk of clinical malaria episodes remains unclear. METHODS: In a 2-years study (December 2014 to December 2016) in eastern Gambia, nine cross-sectional surveys using molecular parasite detection were performed in the dry and wet season. During the 2016 malaria transmission season, passive case detection identified episodes of clinical malaria. RESULTS: Among the 5256 samples collected, 444 (8.4%) were positive for P. falciparum. A multivariate model identified village of residence, male sex, age ≥ 5 years old, anaemia, and fever as independent factors associated with P. falciparum parasite carriage. Infections did not cluster over time within the same households or recurred among neighbouring households. Asymptomatic parasite carriage at the end of dry season was associated with a higher risk of infection (Hazard Ratio, HR = 3.0, p < 0.0001) and clinical malaria (HR = 1.561, p = 0.057) during the following transmission season. Age and village of residence were additional predictors of infection and clinical malaria during the transmission season. CONCLUSION: Chronic parasite carriage during the dry season is associated with an increased risk of malaria infection and clinical malaria. It is unclear whether this is due to environmental exposure or to other factors.

Revisiting the Plasmodium sporozoite inoculum and elucidating the efficiency with which malaria parasites progress through the mosquito.

Malaria is initiated when infected anopheline mosquitoes inoculate sporozoites as they probe for blood. It is thought that all infected mosquitoes are equivalent in terms of their infectious potential, with parasite burden having no role in transmission success. In this study, using mosquitoes harboring the entire range of salivary gland sporozoite loads observed in the field, we demonstrate a strong and highly significant correlation between mosquito parasite burden and inoculum size. We then link the inoculum data to oocyst counts, the most commonly-used metric to assess mosquito infection in the field, and determine the efficiency with which oocyst sporozoites enter mosquito salivary glands. Taken together our data support the conclusion that mosquitoes with higher parasite burdens are more likely to initiate infection and contribute to onward transmission. Overall these data may account for some of the unexplained heterogeneity in transmission and enable more precise benchmarks for transmission-blocking interventions.

Target-agnostic identification of human antibodies to Plasmodium falciparum sexual forms reveals cross stage recognition of glutamate-rich repeats.

Circulating sexual stages of Plasmodium falciparum (Pf) can be transmitted from humans to mosquitoes, thereby furthering the spread of malaria in the population. It is well established that antibodies (Abs) can efficiently block parasite transmission. In search for naturally acquired Ab targets on sexual stages, we established an efficient method for target-agnostic single B cell activation followed by high-throughput selection of human monoclonal antibodies (mAbs) reactive to sexual stages of Pf in the form of gamete and gametocyte extract. We isolated mAbs reactive against a range of Pf proteins including well-established targets Pfs48/45 and Pfs230. One mAb, B1E11K, was cross-reactive to various proteins containing glutamate-rich repetitive elements expressed at different stages of the parasite life cycle. A crystal structure of two B1E11K Fab domains in complex with its main antigen, RESA, expressed on asexual blood stages, showed binding of B1E11K to a repeating epitope motif in a head-to-head conformation engaging in affinity-matured homotypic interactions. Thus, this mode of recognition of Pf proteins, previously described only for PfCSP, extends to other repeats expressed across various stages. The findings augment our understanding of immune-pathogen interactions to repeating elements of the Plasmodium parasite proteome and underscore the potential of the novel mAb identification method used to provide new insights into the natural humoral immune response against Pf . Impact Statement: A naturally acquired human monoclonal antibody recognizes proteins expressed at different stages of the Plasmodium falciparum lifecycle through affinity-matured homotypic interactions with glutamate-rich repeats.

Susceptibility of Anopheles gambiae to Natural Plasmodium falciparum Infection: A Comparison between the Well-Established Anopheles gambiae s.s Line and a Newly Established Ugandan Anopheles gambiae s.s. Line.

Much of our understanding of malaria transmission comes from mosquito feeding assays using Anopheles mosquitoes from colonies that are well adapted to membrane feeding. This raises the question whether results from colony mosquitoes lead to overestimates of outcomes in wild Anopheles mosquitoes. We successfully established an Anopheles colony using progeny of wild Anopheles gambiae s.s. mosquitoes (Busia mosquitoes) and directly compared their susceptibility to infection with Plasmodium falciparum with the widely used An. gambiae s.s. mosquitoes (Kisumu mosquitoes) using gametocyte-infected Ugandan donor blood. The proportion of infectious feeds did not differ between Busia (71.8%, 23/32) and Kisumu (68.8%, 22/32, P = 1.00) mosquitoes. When correcting for random effects of donor blood, we observed a 23% higher proportion of infected Busia mosquitoes than infected Kisumu mosquitoes (RR, 1.23; 95% CI, 1.10-1.38, P < 0.001). This study suggests that feeding assays with Kisumu mosquitoes do not overestimate outcomes in wild An. gambiae s.s. mosquitoes, the mosquito species most relevant to malaria transmission in Uganda.

Development of WHO Recommendations for the Final Phase of Elimination and Prevention of Re-Establishment of Malaria.

The WHO recommends that all affected countries work toward the elimination of malaria, even those still experiencing a high burden of disease. However, malaria programs in the final phase of elimination or those working to prevent re-establishment of transmission after elimination could benefit from specific evidence-based recommendations for these settings as part of comprehensive and quality-controlled malaria guidelines. The WHO convened an external guideline development group to formulate recommendations for interventions to reduce or prevent malaria transmission in areas with very low- to low-transmission levels and those that have eliminated malaria. In addition, several interventions that could be deployed in higher burden areas to accelerate elimination, such as mass drug administration, were reviewed. Systematic reviews were conducted that synthesized and evaluated evidence for the benefits and harms of public health interventions and summarized critical contextual factors from a health systems perspective. A total of 12 recommendations were developed, with five related to mass interventions that could be deployed at higher transmission levels and seven that would be most appropriate for programs in areas close to elimination or those working to prevent re-establishment of transmission. Four chemoprevention, two active case detection, and one vector control interventions were positively recommended, whereas two chemoprevention and three active case detection interventions were not recommended by the WHO. None of the recommendations were classified as strong given the limited and low-quality evidence base. Approaches to conducting higher quality research in very low- to low-transmission settings to improve the strength of WHO recommendations are discussed.