Membrane Feeding Assays Research Articles

The Relative Contribution of Symptomatic and Asymptomatic Plasmodium vivax and Plasmodium falciparum Infections to the Infectious Reservoir in a Low-Endemic Setting in Ethiopia.

The majority of Plasmodium vivax and Plasmodium falciparum infections in low-endemic settings are asymptomatic. The relative contribution to the infectious reservoir of these infections compared to clinical malaria cases is currently unknown. We assessed infectivity of passively recruited symptomatic malaria patients (n = 41) and community-recruited asymptomatic individuals with microscopy-detected (n = 41) and polymerase chain reaction (PCR)-detected infections (n = 82) using membrane feeding assays with Anopheles arabiensis mosquitoes in Adama, Ethiopia. Malaria incidence and prevalence data were used to estimate the contributions of these populations to the infectious reservoir. Overall, 34.9% (29/83) of P. vivax- and 15.1% (8/53) P. falciparum-infected individuals infected ≥1 mosquitoes. Mosquito infection rates were strongly correlated with asexual parasite density for P. vivax (ρ = 0.63; P < .001) but not for P. falciparum (ρ = 0.06; P = .770). Plasmodium vivax symptomatic infections were more infectious to mosquitoes (infecting 46.5% of mosquitoes, 307/660) compared to asymptomatic microscopy-detected (infecting 12.0% of mosquitoes, 80/667; P = .005) and PCR-detected infections (infecting 0.8% of mosquitoes, 6/744; P < .001). Adjusting for population prevalence, symptomatic, asymptomatic microscopy-detected, and PCR-detected infections were responsible for 8.0%, 76.2%, and 15.8% of the infectious reservoir for P. vivax, respectively. For P. falciparum, mosquito infections were sparser and also predominantly from asymptomatic infections. In this low-endemic setting aiming for malaria elimination, asymptomatic infections were highly prevalent and responsible for the majority of onward mosquito infections. The early identification and treatment of asymptomatic infections might accelerate elimination efforts.

What does not kill it makes it weaker: effects of sub-lethal concentrations of ivermectin on the locomotor activity of Anopheles aquasalis

BackgroundMalaria remains a major public health concern. Vector control measures based solely on insecticide treated nets (ITNs) and indoor residual spraying (IRS) have demonstrated not to be feasible for malaria elimination. It has been shown that ivermectin affects several aspects of Anopheles species biology. Along the Latin American seacoast, Anopheles aquasalis Curry plays an important role in malaria transmission. The observation of mosquitoes locomotor activity under laboratory conditions can reveal details of their daily activity rhythms, which is controlled by an endogenous circadian clock that seems to be influenced by external signals, such as light and temperature. In this study, we assessed basal locomotor activity and the effects of ivermectin on locomotor activity of the American malaria vector, An. aquasalis.MethodsAdult females of Anopheles aquasalis used in experiments were three to five days post-emergence. Blood from one single subject was used to provide mosquito meals by membrane feeding assays. Powdered ivermectin compound was used to achieve different concentrations of drug as previously described. Fully engorged mosquitoes were individually placed into glass tubes and provided with 10% sucrose. Each tube was placed into a Locomotor Activity Monitor (LAM). The LAMs were kept inside an incubator under a constant temperature and a 12:12 h light:dark cycle. The average locomotor activity was calculated as the mean number of movements performed per mosquito in the period considered. Intervals of time assessed were adapted from a previous study. One-way ANOVA tests were performed in order to compare means between groups. Additionally, Dunnett’s method was used for post-hoc pairwise means comparisons between each group and control. Stata software version 13 was used for the analysis.ResultsAnopheles aquasalis showed a nocturnal and bimodal pattern for mosquitoes fed both control blood meals and sub-lethal concentrations of ivermectin. In this species, activity peaks occurred at the beginning of the photophase and scotophase in the control group. The nocturnal activity is evident and higher just after the evening peak and maintains basal levels of locomotion throughout the scotophase. In the entire group analysis, locomotor activity means of experimental sets were significantly lower than control for each period of time evaluated. In the survival group, the locomotor activity means of all treatment sets were lower than control mosquitoes for all intervals of time when both the whole period and scotophase were assessed. When the middle of scotophase was evaluated, means were significantly lower for LC15 and LC25, but not LC5. For the beginning of photophase period, significant differences were detected only between control and LC5. When both the photophase and scotophase were assessed alone, no significant differences were found. Mean locomotor activity was significantly lower for dead group when compared to survival group for all experimental sets when whole period, photophase, and scotophase were assessed.ConclusionsIvermectin seems to decrease locomotor activity of An. aquasalis at sub-lethal concentrations. The effects on locomotor activity increase according at higher ivermectin concentrations and are most evident during the whole scotophase as well as in the beginning and in the end of this phase, and sub-lethal effects may still be observed in the photophase. Findings presented in this study demonstrate that sub-lethal ivermectin effects reduce mosquito locomotor activity, which could diminish vectorial capacity and therefore the malaria transmission.

Functional Characterization and Comparison of Plasmodium falciparum Proteins as Targets of Transmission-blocking Antibodies.

Plasmodium falciparum malaria continues to evade control efforts, utilizing highly specialized sexual-stages to transmit infection between the human host and mosquito vector. In a vaccination model, antibodies directed to sexual-stage antigens, when ingested in the mosquito blood meal, can inhibit parasite growth in the midgut and consequently arrest transmission. Despite multiple datasets for the Plasmodium sexual-stage transcriptome and proteome, there have been no rational screens to identify candidate antigens for transmission-blocking vaccine (TBV) development. This study characterizes 12 proteins from across the P. falciparum sexual-stages as possible TBV targets. Recombinant proteins are heterologously expressed as full-length ectodomains in a mammalian HEK293 cell system. The proteins recapitulate native parasite epitopes as assessed by indirect fluorescence assay and a proportion exhibits immunoreactivity when tested against sera from individuals living in malaria-endemic Burkina Faso and Mali. Purified IgG generated to the mosquito-stage parasite antigen enolase demonstrates moderate inhibition of parasite development in the mosquito midgut by the ex vivo standard membrane feeding assay. The findings support the use of rational screens and comparative functional assessments in identifying proteins of the P. falciparum transmission pathway and establishing a robust pre-clinical TBV pipeline.

Comparative assessment of An. gambiae and An. stephensi mosquitoes to determine transmission-reducing activity of antibodies against P. falciparum sexual stage antigens

BackgroundWith the increasing interest in vaccines to interrupt malaria transmission, there is a demand for harmonization of current methods to assess Plasmodium transmission in laboratory settings. Potential vaccine candidates are currently tested in the standard membrane feeding assay (SMFA) that commonly relies on Anopheles stephensi mosquitoes. Other mosquito species including Anopheles gambiae are the dominant malaria vectors for Plasmodium falciparum in sub-Saharan Africa.MethodsUsing human serum and monoclonal pre-fertilization (anti-Pfs48/45) and post-fertilization (anti-Pfs25) antibodies known to effectively inhibit sporogony, we directly compared SMFA based estimates of transmission-reducing activity (TRA) for An. stephensi and An. gambiae mosquitoes.ResultsIn the absence of transmission-reducing antibodies, average numbers of oocysts were similar between An. gambiae and An. stephensi. Antibody-mediated TRA was strongly correlated between both mosquito species, and absolute TRA estimates for pre-fertilisation monoclonal antibodies (mAb) showed no significant difference between the two species. TRA estimates for IgG of naturally exposed individuals and partially effective concentrations of anti-Pfs25 mAb were higher for An. stephensi than for An. gambiae.ConclusionOur findings support the use of An. stephensi in the SMFA for target prioritization. As a vaccine moves through product development, better estimates of TRA and transmission-blocking activity (TBA) may need to be obtained in epidemiologically relevant parasite-species combination.

N-Terminal Pfs230 Domain Produced in Baculovirus as a Biological Active Transmission-Blocking Vaccine Candidate.

ABSTRACTTransmission-blocking vaccines have the potential to accelerate malaria parasite elimination by inducing antibodies that block parasite transmission from humans to mosquitoes. Pfs230, a gametocyte surface protein involved in gamete function, has long been a promising candidate. Due to the large size (3,135 amino acids), complex domains, and repeating 6-cysteine (6-Cys) motifs with a multitude of disulfide bonds, the feasibility of expression of a full-length protein has been difficult. A priority focus, therefore, has been on the generation of single domains, including N-terminal fragments. Here we utilized a heterologous expression system, baculovirus, to produce an N-terminal domain of Pfs230 (Pfs230C1). Pfs230C1 (amino acids 443 to 731) with a polyhistidine affinity tag was expressed in Super Sf9 cells. Since the native host lacks glycosylation machinery, a single N585Q mutation was made to eliminate potential N-linked glycosylation. The expressed protein, purified by nickel affinity, ion exchange, and size exclusion chromatography to >90% purity, was present in monomeric form with an observed mass of 33,510 Da (matching oxidized form). Peptide mapping and disulfide analysis confirmed the proper formation of predicted disulfide bonds. Antibodies, generated against Pfs230C1 in mice, bound to the gametocyte in an immunofluorescence assay (IFA) and demonstrated functional activity in both the standard membrane feeding assay (SMFA) and the exflagellation assay (EXA). The biochemical, biophysical, and immunological results reported herein support the continued advancement of an N-terminal Pfs230 antigen (Pfs230C1) as a component of a transmission-blocking vaccine. Our results also support the continued use of the scalable baculovirus expression system for the generation of complex Plasmodium proteins.

Do the venous blood samples replicate malaria parasite densities found in capillary blood? A field study performed in naturally-infected asymptomatic children in Cameroon

BackgroundThe measure of new drug- or vaccine-based approaches for malaria control is based on direct membrane feeding assays (DMFAs) where gametocyte-infected blood samples are offered to mosquitoes through an artificial feeder system. Gametocyte donors are identified by the microscopic detection and quantification of malaria blood stages on blood films prepared using either capillary or venous blood. However, parasites are known to sequester in the microvasculature and this phenomenon may alter accurate detection of parasites in blood films. The blood source may then impact the success of mosquito feeding experiments and investigations are needed for the implementation of DMFAs under natural conditions.MethodsThick blood smears were prepared from blood obtained from asymptomatic children attending primary schools in the vicinity of Mfou (Cameroon) over four transmission seasons. Parasite densities were determined microscopically from capillary and venous blood for 137 naturally-infected gametocyte carriers. The effect of the blood source on gametocyte and asexual stage densities was then assessed by fitting cumulative link mixed models (CLMM). DMFAs were performed to compare the infectiousness of gametocytes from the different blood sources to mosquitoes.ResultsPrevalence of Plasmodium falciparum asexual stages among asymptomatic children aged from 4 to 15 years was 51.8% (2116/4087). The overall prevalence of P. falciparum gametocyte carriage was 8.9% and varied from one school to another. No difference in the density of gametocyte and asexual stages was found between capillary and venous blood. Attempts to perform DMFAs with capillary blood failed.ConclusionsPlasmodium falciparum malaria parasite densities do not differ between capillary and venous blood in asymptomatic subjects for both gametocyte and trophozoite stages. This finding suggests that the blood source should not interfere with transmission efficiency in DMFAs.

Evaluation of two lead malaria transmission blocking vaccine candidate antibodies in natural parasite-vector combinations

Transmission blocking vaccines (TBV) which aim to control malaria by inhibiting human-to-mosquito transmission show considerable promise though their utility against naturally circulating parasites remains unknown. The efficacy of two lead candidates targeting Pfs25 and Pfs230 antigens to prevent onwards transmission of naturally occurring parasites to a local mosquito strain is assessed using direct membrane feeding assays and murine antibodies in Burkina Faso. The transmission blocking activity of both candidates depends on the level of parasite exposure (as assessed by the mean number of oocysts in control mosquitoes) and antibody titers. A mathematical framework is devised to allow the efficacy of different candidates to be directly compared and determine the minimal antibody titers required to halt transmission in different settings. The increased efficacy with diminishing parasite exposure indicates that the efficacy of vaccines targeting either Pfs25 or Pfs230 may increase as malaria transmission declines. This has important implications for late-stage candidate selection and assessing how they can support the drive for malaria elimination.

Optimizing Direct Membrane and Direct Skin Feeding Assays for Plasmodium falciparum Transmission-Blocking Vaccine Trials in Bancoumana, Mali.

Malaria transmission-blocking vaccines (TBV) have been evaluated in field trials in Mali since 2013. However, the assays currently used to measure serum antibody TB activity (TBA) after vaccination are highly variable, in part due to the lack of optimization and standardization for field assays in which mosquitoes feed on gametocytemic blood. Herein, we report a study conducted in Bancoumana village, Mali, where we identify and optimize the parameters that contribute to successful mosquito feeding outcomes in both direct skin feeds (DSFs) and direct membrane feeding assays (DMFA). These parameters include: 1) mosquito age, 2) duration of mosquito starvation prior to feeding, 3) membrane selection for DMFA, 4) anatomical location of DSF feeding (arm, calf, and ankle), and 5) time of day for DSF (dawn or dusk). We found that younger mosquitoes were significantly associated with higher feeding, survival, and infection rates. Longer starvation times were positively, but not significantly, associated with higher infection rates, but were negatively associated with feeding and survival. Membrane type and body location did not affect infection outcome significantly. Although dusk was found to be associated with higher infection rates, this may be confounded by the time from positive blood smear. Based on these findings, we make specific recommendations for optimal feeding parameters in the different assay types to maximize the chance of detecting parasite transmission in a standardized manner.

The fibrinogen-like domain of FREP1 protein is a broad-spectrum malaria transmission-blocking vaccine antigen

FREP1 in mosquito midguts facilitates Plasmodium falciparum parasite transmission. The fibrinogen-like (FBG) domain of FREP1 is highly conserved (>90% identical) among Anopheles species from different continents, suggesting that anti-FBG antibodies may block malaria transmission to all anopheline mosquitoes. Using standard membrane-feeding assays, anti-FREP1 polyclonal antibodies significantly blocked transmission of Plasmodium berghei and Plasmodium vivax to Anopheles gambiae and Anopheles dirus, respectively. Furthermore, in vivo studies of mice immunized with FBG achieved >75% blocking efficacy of P. berghei to A. gambiae without triggering immunopathology. Anti-FBG serum also reduced >81% of P. falciparum infection to A. gambiae Finally, we showed that FBG interacts with Plasmodium gametocytes and ookinetes, revealing the molecular mechanism of its antibody transmission-blocking activity. Collectively, our data support that FREP1-mediated Plasmodium transmission to mosquitoes is a conserved pathway and that targeting the FBG domain of FREP1 will limit the transmission of multiple Plasmodium species to multiple Anopheles species.

New adenovirus-based vaccine vectors targeting Pfs25 elicit antibodies that inhibit Plasmodium falciparum transmission

BackgroundAn effective malaria transmission-blocking vaccine (TBV) would be a major advance in the current efforts to eliminate and, ultimately, eradicate malaria. Antibodies against Plasmodium falciparum surface protein, Pfs25, are known to block parasite development in the mosquito vector. However, in initial clinical trials the limited immunogenicity of recombinant Pfs25 protein-in-adjuvant vaccines has been a challenge.MethodsNovel human adenovirus type 5 (Ad5) vectors were used in heterologous prime boost vaccination strategies to augment the immune response against Pfs25. Specifically, an Ad5 vector that directs expression of full-length, membrane-bound Pfs25 was used as a priming immunization followed by a boost with Ad5 viral particles displaying only the Pfs25 epitope targeted by transmission-blocking antibodies 4B7 and 1D2 (Pfs25 aa 122–134) in hypervariable region 5 of the hexon capsid protein.ResultsThis heterologous prime-boost vaccine strategy induced antibodies that significantly inhibit P. falciparum transmission to mosquitoes in a standard membrane-feeding assay. Further, immunized mice generated a robust anti-Pfs25 antibody response characterized by higher titer, higher relative avidity and a broader IgG subclass profile than observed with a homologous prime-boost with recombinant Pfs25/alum.ConclusionThe data suggest that focusing the immune response against defined epitopes displayed on the viral capsid is an effective strategy for transmission-blocking vaccine development.

Single dose primaquine to reduce gametocyte carriage and Plasmodium falciparum transmission in Cambodia: An open-label randomized trial.

BackgroundSingle low dose primaquine (SLD PQ, 0.25mg/kg) is recommended in combination with artemisinin-based combination therapy (ACT) as a gametocytocide to prevent Plasmodium falciparum transmission in areas threatened by artemisinin resistance. To date, no randomized controlled trials have measured primaquine’s effect on infectiousness to Anopheline mosquitoes in Southeast Asia.MethodsCambodian adults with uncomplicated falciparum malaria were randomized to receive a single 45mg dose of primaquine (equivalent to three SLD PQ) or no primaquine after the third dose of dihydroartemisin-piperaquine (DHP) therapy. A membrane-feeding assay measured infectiousness to Anopheles dirus on days 0, 3, 7, and 14 of blood-stage therapy. Gametocytemia was evaluated by microscopy and reverse-transcriptase PCR.ResultsPrior to trial halt for poor DHP treatment efficacy, 101 participants were randomized and 50 received primaquine. Overall microscopic gametocyte prevalence was low (9%), but gametocytemic subjects given primaquine were gametocyte-free by day 14, and significantly less likely to harbor gametocytes by day 7 compared to those treated with DHP-alone, who remained gametocytemic for a median of two weeks. Only one infectious subject was randomized to the primaquine group, precluding assessment of transmission-blocking efficacy. However, he showed a two-fold reduction in oocyst density of infected mosquitoes less than 24 hours after primaquine dosing. In the DHP-alone group, four subjects remained infectious through day 14, infecting roughly the same number of mosquitoes pre and post-treatment. Overall, microscopic gametocytemia was an excellent predictor of infectiousness, and performed better than submicroscopic gametocytemia post-treatment, with none of 474 mosquitoes infected post-treatment arising from submicroscopic gametocytes.ConclusionsIn a setting of established ACT resistance, a single dose of 45mg primaquine added to DHP rapidly and significantly reduced gametocytemia, while DHP-alone failed to reduce gametocytemia and prevent malaria transmission to mosquitoes. Continued efforts to make single dose primaquine widely available are needed to help achieve malaria elimination.

The need to compare: assessing the level of agreement of three high-throughput assays against Plasmodium falciparum mature gametocytes

Whole-cell High-Throughput Screening (HTS) is a key tool for the discovery of much needed malaria transmission blocking drugs. Discrepancies in the reported outcomes from various HTS Plasmodium falciparum gametocytocidal assays hinder the direct comparison of data and ultimately the interpretation of the transmission blocking potential of hits. To dissect the underlying determinants of such discrepancies and assess the impact that assay-specific factors have on transmission-blocking predictivity, a 39-compound subset from the Medicines for Malaria Venture Malaria Box was tested in parallel against three distinct mature stage gametocytocidal assays, under strictly controlled parasitological, chemical, temporal and analytical conditions resembling the standard membrane feeding assay (SMFA). Apart from a few assay-specific outliers, which highlighted the value of utilizing multiple complementary approaches, good agreement was observed (average ΔpIC50 of 0.12 ± 0.01). Longer compound incubation times improved the ability of the least sensitive assay to detect actives by 2-fold. Finally, combining the number of actives identified by any single assay with those obtained at longer incubation times yielded greatly improved outcomes and agreement with SMFA. Screening compounds using extended incubation times and using multiple in vitro assay technologies are valid approaches for the efficient identification of biologically relevant malaria transmission blocking hits.

Optimization of an artificial tick feeding assay for Dermacentor reticulatus

BackgroundThe development of standardized in vitro feeding methods for ixodid ticks has been hampered by their complex feeding behaviour and the long duration of their blood meal. In this study, we aimed to optimize several parameters for the in vitro feeding of adult Dermacentor reticulatus.MethodsTicks were fed on heparinized bovine blood collected at a slaughterhouse, using a modified silicone membrane feeding assay. Effects on tick feeding and fecundity of different blood meal treatments (freezing, irradiation, addition of antibiotics), ambient conditions (increased CO2 concentration) and phagostimulant use (addition of 2 g/l and 4 g/l glucose to the blood meal) were systematically evaluated.ResultsAlthough fungal growth occurred more frequent in feeding units of ticks fed on defrosted blood, the attachment rate, engorgement mass and fecundity of females fed on defrosted blood did not significantly differ from that of ticks fed on fresh blood. A reduction in the fecundity of female D. reticulatus ticks was observed when ticks were fed with gamma-irradiated blood or untreated blood compared to blood treated with gentamycin. Both the engorgement mass and fecundity increased when ticks were fed at a 5% CO2 level. A non-significant increase in the engorgement mass and engorgement rate of D. reticulatus was observed when blood was supplemented with 4 g glucose per litre compared to 2 g/l.ConclusionAn artificial feeding method was adapted for the feeding of adult D. reticulatus ticks. Of all parameters tested, only the artificial feeding at 5% CO2 levels resulted in a significant increase (P < 0.05) in the engorgement mass and fecundity of female D. reticulatus ticks. The supplementation of blood with antibiotics resulted in a significantly higher tick fecundity in comparison to ticks fed with untreated or irradiated blood.

ASSESSING THE COMMITMENT, MATURATION AND INFECTIVITY OF SEXUAL STAGES OF MALARIA PARASITES IN SCHOOLCHILDREN LIVING IN A HIGH MALARIA TRANSMISSION AREA OF BURKINA FASO

BackgroundWith the move towards malaria elimination, it becomes essential to understand the contribution of asymptomatic parasite carriers to disease transmission. However, the dynamics of infection and gametocyte development are poorly...

Immunogenicity and malaria transmission reducing potency of Pfs48/45 and Pfs25 encoded by DNA vaccines administered by intramuscular electroporation

Pfs48/45 and Pfs25 are leading candidates for the development of Plasmodium falciparum transmission blocking vaccines (TBV). Expression of Pfs48/45 in the erythrocytic sexual stages and presentation to the immune system during infection in the human host also makes it ideal for natural boosting. However, it has been challenging to produce a fully folded, functionally active Pfs48/45, using various protein expression platforms. In this study, we demonstrate that full-length Pfs48/45 encoded by DNA plasmids is able to induce significant transmission reducing immune responses. DNA plasmids encoding Pfs48/45 based on native (WT), codon optimized (SYN), or codon optimized and mutated (MUT1 and MUT2), to prevent any asparagine (N)-linked glycosylation were compared with or without intramuscular electroporation (EP). EP significantly enhanced antibody titers and transmission blocking activity elicited by immunization with SYN Pfs48/45 DNA vaccine. Mosquito membrane feeding assays also revealed improved functional immunogenicity of SYN Pfs48/45 (N-glycosylation sites intact) as compared to MUT1 or MUT2 Pfs48/45 DNA plasmids (all N-glycosylation sites mutated). Boosting with recombinant Pfs48/45 protein after immunization with each of the different DNA vaccines resulted in significant boosting of antibody response and improved transmission reducing capabilities of all four DNA vaccines. Finally, immunization with a combination of DNA plasmids (SYN Pfs48/45 and SYN Pfs25) also provides support for the possibility of combining antigens targeting different life cycle stages in the parasite during transmission through mosquitoes.

Efficacy and Safety of High-Dose Ivermectin for Reducing Malaria Transmission (IVERMAL): Protocol for a Double-Blind, Randomized, Placebo-Controlled, Dose-Finding Trial in Western Kenya.

BackgroundInnovative approaches are needed to complement existing tools for malaria elimination. Ivermectin is a broad spectrum antiparasitic endectocide clinically used for onchocerciasis and lymphatic filariasis control at single doses of 150 to 200 mcg/kg. It also shortens the lifespan of mosquitoes that feed on individuals recently treated with ivermectin. However, the effect after a 150 to 200 mcg/kg oral dose is short-lived (6 to 11 days). Modeling suggests higher doses, which prolong the mosquitocidal effects, are needed to make a significant contribution to malaria elimination. Ivermectin has a wide therapeutic index and previous studies have shown doses up to 2000 mcg/kg (ie, 10 times the US Food and Drug Administration approved dose) are well tolerated and safe; the highest dose used for onchocerciasis is a single dose of 800 mcg/kg.ObjectiveThe aim of this study is to determine the safety, tolerability, and efficacy of ivermectin doses of 0, 300, and 600 mcg/kg/day for 3 days, when provided with a standard 3-day course of the antimalarial dihydroartemisinin-piperaquine (DP), on mosquito survival.MethodsThis is a double-blind, randomized, placebo-controlled, parallel-group, 3-arm, dose-finding trial in adults with uncomplicated malaria. Monte Carlo simulations based on pharmacokinetic modeling were performed to determine the optimum dosing regimens to be tested. Modeling showed that a 3-day regimen of 600 mcg/kg/day achieved similar median (5 to 95 percentiles) maximum drug concentrations (Cmax) of ivermectin to a single of dose of 800 mcg/kg, while increasing the median time above the lethal concentration 50% (LC50, 16 ng/mL) from 1.9 days (1.0 to 5.7) to 6.8 (3.8 to 13.4) days. The 300 mcg/kg/day dose was chosen at 50% of the higher dose to allow evaluation of the dose response. Mosquito survival will be assessed daily up to 28 days in laboratory-reared Anopheles gambiae s.s. populations fed on patients’ blood taken at days 0, 2 (Cmax), 7 (primary outcome), 10, 14, 21, and 28 after the start of treatment. Safety outcomes include QT-prolongation and mydriasis. The trial will be conducted in 6 health facilities in western Kenya and requires a sample size of 141 participants (47 per arm). Sub-studies include (1) rich pharmacokinetics and (2) direct skin versus membrane feeding assays.ResultsRecruitment started July 20, 2015. Data collection was completed July 2, 2016. Unblinding and analysis will commence once the database has been completed, cleaned, and locked.ConclusionsHigh-dose ivermectin, if found to be safe and well tolerated, might offer a promising new tool for malaria elimination.

A Malaria Transmission-Blocking (+)-Usnic Acid Derivative Prevents Plasmodium Zygote-to-Ookinete Maturation in the Mosquito Midgut.

The evolution of drug resistance is a recurrent problem that has plagued efforts to treat and control malaria. Recent emergence of artemisinin resistance in Southeast Asia underscores the need to develop novel antimalarials and identify new targetable pathways in Plasmodium parasites. Transmission-blocking approaches, which typically target gametocytes in the host bloodstream or parasite stages in the mosquito gut, are recognized collectively as a strategy that when used in combination with antimalarials that target erythrocytic stages will not only cure malaria but will also prevent subsequent transmission. We tested four derivatives of (+)-usnic acid, a metabolite isolated from lichens, for transmission-blocking activity against Plasmodium falciparum using the standard membrane feeding assay. For two of the derivatives, BT37 and BT122, we observed a consistent dose-response relationship between concentration in the blood meal and oocyst intensity in the midgut. To explore their mechanism of action, we used the murine model Plasmodium berghei and found that both derivatives prevent ookinete maturation. Using fluorescence microscopy, we demonstrated that in the presence of each compound zygote vitality was severely affected, and those that did survive failed to elongate and mature into ookinetes. The observed phenotypes were similar to those described for mutants of specific kinases (NEK2/NEK4) and of inner membrane complex 1 (IMC1) proteins, which are all vital to the zygote-to-ookinete transition. We discuss the implications of our findings and our high-throughput screening approach to identifying next generation, transmission-blocking antimalarials based on the scaffolds of these (+)-usnic acid derivatives.

Assessing Gametocyte Carriage in Treated Asymptomatic Falciparum Carriers in Africa

Assessing Gametocyte Carriage in Treated Asymptomatic Falciparum Carriers in Africa

Similar trends of susceptibility in Anopheles arabiensis and Anopheles pharoensis to Plasmodium vivax infection in Ethiopia.

BackgroundAround half of the global population is living in areas at risk of malaria infection. Plasmodium vivax malaria has become increasingly prevalent and responsible for a high health and socio-economic burden in Ethiopia. The availability of gametocyte carriers and mosquito species susceptible to P. vivax infection are vital for malaria transmission. Determining the susceptibility of vector species to parasite infection in space and time is important in vector control programs. This study assesses the susceptibility of Anopheles arabiensis, An. pharoensis and An. coustani group to Plasmodium vivax infection in Ethiopia.MethodsLarvae of An. arabiensis, An. pharoensis and An. coustani group were collected from an array of breeding sites and reared to adult under controlled conditions. Batches of adult female mosquitoes of the three species were allowed to feed in parallel on the same infected blood with gametocytes drawn from Plasmodium vivax infected patients by Direct Membrane Feeding Assays (DMFA). Fed mosquitoes were kept in an incubator under controlled laboratory conditions. Seven days after each feeding assay, mosquitoes were dissected for midgut oocyst microscopy and enumeration. Data were analysed using R statistical software package version 3.1.0.ResultsOver all, 8,139 adult female mosquitoes were exposed to P. vivax infection. Of the exposed mosquitoes 16.64 % (95 % CI: 1,354–8,139) were properly fed and survived until dissection. The infection rate in An. arabiensis and An. pharoensis was 31.72 % (95 % CI: 28.35–35.08) and 28.80 % (95 % CI: 25.31–32.28), respectively. The intensity of infection for An. arabiensis and An. pharoensis was 2.5 (95 % CI: 1.9–3.2) and 1.4 (95 % CI: 1.1–1.8), respectively. Gametocyte density was positively correlated to infection rate and intensity of infection in An. arabiensis as well as An. pharoensis. No An. coustani group mosquitoes were found infected, though almost four hundred mosquitoes were successfully fed and dissected. All groups received blood from the same infected blood source containing gametocytes in parallel. There was no significant difference in susceptibility rates between An. arabiensis and An. pharoensis (P = 0.215).Conclusions Anopheles arabiensis and An. pharoensis showed similar susceptibility to P. vivax infection. However, An. coustani group was not permissive for the development of P. vivax parasites.

Filling gaps on ivermectin knowledge: effects on the survival and reproduction of Anopheles aquasalis, a Latin American malaria vector.

BackgroundStrategies designed to advance towards malaria elimination rely on the detection and treatment of infections, rather than fever, and the interruption of malaria transmission between mosquitoes and humans. Mass drug administration with anti-malarials directed at eliminating parasites in blood, either to entire populations or targeting only those with malaria infections, are considered useful strategies to progress towards malaria elimination, but may be insufficient if applied on their own. These strategies assume a closer contact with populations, so incorporating a vector control intervention tool to those approaches could significantly enhance their efficacy. Ivermectin, an endectocide drug efficacious against a range of Anopheles species, could be added to other drug-based interventions. Interestingly, ivermectin could also be useful to target outdoor feeding and resting vectors, something not possible with current vector control tools, such as impregnated bed nets or indoor residual spraying (IRS).ResultsAnopheles aquasalis susceptibility to ivermectin was assessed. In vivo assessments were performed in six volunteers, being three men and three women. The effect of ivermectin on reproductive fitness and mosquito survivorship using membrane feeding assay (MFA) and direct feeding assay (DFA) was assessed and compared. The ivermectin lethal concentration (LC) values were LC50 = 47.03 ng/ml [44.68–49.40], LC25 = 31.92 ng/ml [28.60–34.57] and LC5 = 18.28 ng/ml [14.51–21.45]. Ivermectin significantly reduced the survivorship of An. aquasalis blood-fed 4 h post-ingestion (X2 [N = 880] = 328.16, p < 0.001), 2 days post-ingestion (DPI 2) (X2 [N = 983] = 156.75, p < 0.001), DPI 7 (X2 [N = 935] = 31.17, p < 0.001) and DPI 14 (X2 [N = 898] = 38.63, p < 0.001) compared to the blood fed on the untreated control. The average number of oviposited eggs per female was significantly lower in LC5 group (22.44 [SD = 3.38]) than in control (34.70 [SD = 12.09]) (X2 [N = 199] = 10.52, p < 0.001) as well as the egg hatch rate (LC5 = 74.76 [SD = 5.48]) (Control = 81.91 [SD = 5.92]) (X2 [N = 124] = 64.24, p < 0.001). However, no differences were observed on the number of pupae that developed from larvae (Control = 34.19 [SD = 10.42) and group (LC5 = 33.33 [SD = 11.97]) (X2 [N = 124] = 0.96, p > 0.05).ConclusionsIvermectin drug reduces mosquito survivorship when blood fed on volunteer blood from 4 h to 14 days post-ingestion controlling for volunteers’ gender. Ivermectin at mosquito sub-lethal concentrations (LC5) reduces fecundity and egg hatch rate but not the number of pupae that developed from larvae. DFA had significantly higher effects on mosquito survival compared to MFA. The findings are presented and discussed through the prism of malaria elimination in the Amazon region.