Gametocyte Density Research Articles

Effect of weekly fever-screening and treatment and monthly RDT testing and treatment on the infectious reservoir of malaria parasites in Burkina Faso: a cluster-randomised trial

The majority of Plasmodium spp infections in endemic countries are asymptomatic and a source of onward transmission to mosquitoes. We aimed to examine whether Plasmodium falciparum transmission and malaria burden could be reduced by improving early detection and treatment of infections with active screening approaches. In this 18-month cluster randomised study in Sapone, Burkina Faso, households were enrolled and randomly assigned (1:1:1) to one of three groups: group 1 (control) received standard of care only, group 2 received active weekly, at home, fever screening by a community health worker regardless of symptoms, participants with a fever received a rapid diagnostic test (RDT) and treatment if RDT positive, and group 3 received active weekly fever screening (as in group 2) plus a monthly RDT regardless of symptoms, and treatment if RDT positive. Eligible households had a minimum of threeeligible residents, one in each age group (<5years, 5-15years, and >15years). The primary outcome was parasite prevalence by quantitative PCR (qPCR) in the end-of-study cross-sectional survey. Secondary outcomes included parasite and gametocyte prevalence and density in all three end-of-season cross-sectional surveys, incidence of infection, and the transmissibility of infections to mosquitoes. This trial was registered at ClinicalTrials.gov (NCT03705624) and is completed. A total of 906individuals from 181households were enrolled during two phases, and participated in the study. 412individuals were enrolled between Aug 9and 17, 2018, and participated in phase 1and 494individuals were enrolled between Jan 10and 31, 2019, in phase 2. In the end-of-study cross-sectional survey (conducted between Jan13and21,2020), Pfalciparum prevalence by qPCR was significantly lower in group 3 (29·26%; 79of 270), but not in group 2 (45·66%; 121of 265), when compared with group 1 (48·72%; 133of 273; risk ratio 0·65 [95% CI 0·52-0·81]; p=0·0001). Total parasite and gametocyte prevalence and density were also significantly lower in group3in all surveys. The largest differences were seen at the end of the dry season, with gametocyte prevalence 78·4% and predicted transmission potential 98·2% lower in group 3than in group1. Active monthly RDT testing and treatment can reduce parasite carriage and the infectious reservoir of Pfalciparum to less than 2% when used during the dry season. This insight might inform approaches for malaria control and elimination. Bill & Melinda Gates Foundation, European Research Council, and The Netherlands Organization for ScientificResearch.

Descriptive, Hospital-Based, 10-Year Study of Malaria Transmission in Goa, a Southwest Indian State in the Malaria Elimination Phase.

The South Asia International Center of Excellence for Malaria Research, an NIH-funded collaborative program, investigated the epidemiology of malaria in the Indian state of Goa through health facility-based data collected from the Goa Medical College and Hospital (GMC), the state's largest tertiary healthcare facility, between 2012 and 2021. Our study investigated region-specific spatial and temporal patterns of malaria transmission in Goa and the factors driving such patterns. Over the past decade, the number of malaria cases, inpatients, and deaths at the GMC decreased significantly after a peak in 2014-2015. However, the proportion of severe malaria cases increased over the study period. Also, a trend of decreasing average parasitemia and increasing average gametocyte density suggests a shift toward submicroscopic infections and an increase in transmission commitment characteristic of low-transmission regions. Although transmission occurred throughout the year, 75% of the cases occurred between June and December, overlapping with the monsoon (June-October), which featured rainfall above yearly average, minimal diurnal temperature variation, and high relative humidity. Sociodemographic factors also had a significant association with malaria cases, with cases being more frequent in the 15-50-year-old age group, men, construction workers, and people living in urban areas within the GMC catchment region. Our environmental model of malaria transmission projects almost negligible transmission at the beginning of 2025 (annual parasitic index: 0.0095, 95% CI: 0.0075-0.0114) if the current control measures continue undisrupted.

A simple, field-applicable method to increase the infectivity of wild isolates of Plasmodium falciparum to mosquito vectors

BackgroundThe direct membrane feeding assay (DMFA), whereby gametocyte-infected blood is collected from human donors and from which mosquitoes feed through a membrane, is proving essential for assessing parameters influencing Plasmodium transmission potential in endemic countries. The success of DMFAs is closely tied to gametocyte density in the blood, with relatively high gametocytaemia ensuring optimal infection levels in mosquitoes. As transmission intensity declines with control efforts, the occurrence of asymptomatic individuals with low gametocyte densities, who can significantly contribute to the infectious reservoir, is increasing. This poses a limitation to studies relying on the experimental infection of large numbers of mosquitoes with natural isolates of Plasmodium. A simple, field-applicable method is presented for improving parasite infectivity by concentrating Plasmodium falciparum gametocytes.MethodsAnopheles gambiae received one of the following 5 blood treatments through DMFA: (i) whole blood (WB) samples from naturally-infected donors; (ii) donor blood whose plasma was replaced with the same volume of Plasmodium-naive AB + serum (1:1 control); (iii) plasma replaced with a volume of malaria-naïve AB + serum equivalent to half (1:1/2), or to a quarter (1:1/4), of the initial plasma volume; and (v) donor blood whose plasma was fully removed (RBC). The experiment was repeated 4 times using 4 distinct wild parasite isolates. Seven days post-infection, a total of 1,095 midguts were examined for oocyst presence.ResultsSubstituting plasma with reduced amounts (1:1/2 and 1:1/4) of Plasmodium-naive AB + serum led to a 31% and 17% increase of the mosquito infection rate and to a 85% and 308% increase in infection intensity compared to the 1:1 control, respectively. The full removal of plasma (RBC) reduced the infection rate by 58% and the intensity by 64% compared to the 1:1 control. Reducing serum volumes (1:1/2; 1:1/4 and RBC) had no impact on mosquito feeding rate and survival when compared to the 1:1 control.ConclusionsConcentrating gametocytic blood by replacing natural plasma by lower amount of naive serum can enhance the success of mosquito infection. In an area with low gametocyte density, this simple and practical method of parasite concentration can facilitate studies on human-to-mosquito transmission such as the evaluation of transmission-blocking interventions.

Delayed gametocyte clearance in Plasmodium vivax malaria is associated with polymorphisms in the cytochrome P450 reductase (CPR).

Primaquine (PQ) is the main drug used to eliminate dormant liver stages and prevent relapses in Plasmodium vivax malaria. It also has an effect on the gametocytes of Plasmodium falciparum; however, it is unclear to what extent PQ affects P. vivax gametocytes. PQ metabolism involves multiple enzymes, including the highly polymorphic CYP2D6 and the cytochrome P450 reductase (CPR). Since genetic variability can impact drug metabolism, we conducted an evaluation of the effect of CYP2D6 and CPR variants on PQ gametocytocidal activity in 100 subjects with P. vivax malaria. To determine gametocyte density, we measured the levels of pvs25 transcripts in samples taken before treatment (D0) and 72 hours after treatment (D3). Generalized estimating equations (GEEs) were used to examine the effects of enzyme variants on gametocyte densities, adjusting for potential confounding factors. Linear regression models were adjusted to explore the predictors of PQ blood levels measured on D3. Individuals with the CPR mutation showed a smaller decrease in gametocyte transcript levels on D3 compared to those without the mutation (P = 0.02, by GEE). Consistent with this, higher PQ blood levels on D3 were associated with a lower reduction in pvs25 transcripts. Based on our findings, the CPR variant plays a role in the persistence of gametocyte density in P. vivax malaria. Conceptually, our work points to pharmacogenetics as a non-negligible factor to define potential host reservoirs with the propensity to contribute to transmission in the first days of CQ-PQ treatment, particularly in settings and seasons of high Anopheles human-biting rates.

Monitoring the density of Plasmodium spp. gametocytes in isolates from patient samples in the region of Porto Velho, Rondônia.

Gametocytes are the forms of the malaria parasite that are essential for the continuation of the transmission cycle to the vector Anopheles. This study aimed to evaluate the parasite density of Plasmodium spp gametocytes in samples from patients in the region of Porto Velho, Rondônia. Slides containing patient samples were selected from users who sought out care at the Center for Research in Tropical Medicine (CEPEM) during the period from January to December 2016. Samples of Plasmodium vivax and Plasmodium falciparum were selected for analysis of their respective gametocytes. In parallel, monitoring was performed in cultures of NF54 strain P. falciparum gametocytes. Of 248 thick smear slides (EG) evaluated in double blind, 142 (57.2%) were detected with P. vivax, of this total 47 (18.9%) had gametocytes, 1 (0.4%) with LVC negative diagnosis for gametocytes and 1 (0.4%) Pv + Pf (mixed malaria). Regarding P. falciparum, the total number of samples analyzed was 106 (42.7%), of which 20 (8.0%) had gametocytes detected, 6 (2.4%) LVC negative for gametocyte forms, and 3 (1.2%) Pv + Pf (mixed malaria), Plasmodium malariae species was not detected among the samples. The results showed that P. vivax gametocytes were present in the first days of symptoms, with a higher prevalence in patients with two crosses, a fact that was also observed in patients with P. falciparum regarding the prevalence of gametocytes. Faced with this problem, it is necessary to monitor the fluctuation of gametocytes, since these forms are responsible for continuing the malaria cycle within the mosquito vector.

Significant number of Plasmodium vivax mono-infections by PCR misidentified as mixed infections (P. vivax/P. falciparum) by microscopy and rapid diagnostic tests: malaria diagnostic challenges in Ethiopia

BackgroundPlasmodium vivax malaria is now recognized as a cause of severe morbidity and mortality, resulting in a substantial negative effect on health especially in endemic countries. Accurate and prompt diagnosis and treatment of P. vivax malaria is vital for the control and elimination of the disease.MethodsA cross-sectional study was conducted from February 2021 to September 2022 at five malaria endemic sites in Ethiopia including Aribaminch, Shewarobit, Metehara, Gambella, and Dubti. A total of 365 samples that were diagnosed positive for P. vivax (mono and mixed infection) using RDT, site level microscopists and expert microscopists were selected for PCR. Statistical analyses were performed to calculate the proportions, agreement (k), frequencies, and ranges among different diagnostic methods. Fisher’s exact tests and correlation test were used to detect associations and relationship between different variables.ResultsOf the 365 samples, 324 (88.8%), 37(10.1%), 2 (0.5%), and 2 (0.5%) were P. vivax (mono), P. vivax/Plasmodium falciparum (mixed), P. falciparum (mono) and negative by PCR, respectively. The overall agreement of rapid diagnostic test (RDT), site level microscopy and expert microscopists result with PCR was 90.41% (k: 0.49), 90.96% (k: 0.53), and 80.27% (k: 0.24). The overall prevalence of sexual (gametocyte) stage P. vivax in the study population was 215/361 (59.6%). The majority of these 215 samples (180; 83.7%) had below 1000 parasites/µl, with only four samples (1.9%) had ≥ 5000 parasites/µl. The gametocyte density was found to be weakly positive but statically significant with asexual parasitaemia (r = 0.31; p < 0.001).ConclusionBoth microscopy and RDT showed moderate agreement with PCR in the detection and identification of P. vivax (mono) and P. vivax/P. falciparum (mixed) infections. Therefore, to achieve malaria elimination goals, strengthening routine malaria diagnostic methods by implementing diagnostic tools with a good performance in detecting and accurately identifying malaria species in clinical settings is recommended.

Malaria rapid diagnostic tests reliably detect asymptomatic Plasmodium falciparum infections in school-aged children that are infectious to mosquitoes

BackgroundAsymptomatic malaria infections (Plasmodium falciparum) are common in school-aged children and represent a disease transmission reservoir as they are potentially infectious to mosquitoes. To detect and treat such infections, convenient, rapid and reliable diagnostic tools are needed. In this study, malaria rapid diagnostic tests (mRDT), light microscopy (LM) and quantitative polymerase chain reaction (qPCR) were used to evaluate their performance detecting asymptomatic malaria infections that are infectious to mosquitoes.MethodsOne hundred seventy asymptomatic school-aged children (6–14 years old) from the Bagamoyo district in Tanzania were screened for Plasmodium spp. infections using mRDT (SD BIOLINE), LM and qPCR. In addition, gametocytes were detected using reverse transcription quantitative polymerase chain reaction (RT-qPCR) for all qPCR-positive children. Venous blood from all P. falciparum positive children was fed to female Anopheles gambiae sensu stricto mosquitoes via direct membrane feeding assays (DMFAs) after serum replacement. Mosquitoes were dissected for oocyst infections on day 8 post-infection.ResultsThe P. falciparum prevalence in study participants was 31.7% by qPCR, 18.2% by mRDT and 9.4% by LM. Approximately one-third (31.2%) of asymptomatic malaria infections were infectious to mosquitoes in DMFAs. In total, 297 infected mosquitoes were recorded after dissections, from which 94.9% (282/297) were derived from infections detected by mRDT and 5.1% (15/297) from subpatent mRDT infections.ConclusionThe mRDT can be used reliably to detect children carrying gametocyte densities sufficient to infect high numbers of mosquitoes. Subpatent mRDT infections contributed marginally to the pool of oocyts-infected mosquitoes.Graphical

Transmission-blocking activity of antimalarials for Plasmodium vivax malaria in Anopheles darlingi.

Malaria is caused by parasite of the genus Plasmodium and is still one of the most important infectious diseases in the world. Several biological characteristics of Plasmodium vivax contribute to the resilience of this species, including early gametocyte production, both of which lead to efficient malaria transmission to mosquitoes. This study evaluated the impact of currently used drugs on the transmission of P. vivax. Participants received one of the following treatments for malaria: i) chloroquine [10 mg/kg on day 1 and 7.5 mg/kg on day 2 and 3] co-administered with Primaquine [0.5 mg/kg/day for 7 days]; ii) Chloroquine [10 mg/kg on day 1 and 7.5 mg/kg on day 2 and 3] co-administered with one-dose of Tafenoquine [300 mg on day 1]; and iii) Artesunate and Mefloquine [100 mg and 200 mg on day 1, 2 and 3] co-administered with Primaquine [0.5 mg/kg/day for 14 days]. Patient blood was collected before treatment and 4 h, 24 h, 48 h and 72 h after treatment. The blood was used to perform a direct membrane feeding assay (DMFA) using Anopheles darlingi mosquitoes. The results showed 100% inhibition of the mosquito infection after 4 h using ASMQ+PQ, after 24 h for the combination of CQ+PQ and 48 h using CQ+TQ. The density of gametocytes declined over time in all treatment groups, although the decline was more rapid in the ASMQ+PQ group. In conclusion, it was possible to demonstrate the transmission-blocking efficacy of the malaria vivax treatment and that ASMQ+PQ acts faster than the two other treatments.

Influence of the sickle cell trait on Plasmodium falciparum infectivity from naturally infected gametocyte carriers

BackgroundSickle cell trait (SCT) refers to the carriage of one abnormal copy of the β-globin gene, the HbS allele. SCT offers protection against malaria, controlling parasite density and preventing progression to symptomatic malaria. However, it remains unclear whether SCT also affects transmission stages and mosquito infection parameters. Deciphering the impact of the SCT on human to mosquito malaria transmission is key to understanding mechanisms that maintain the trait in malaria endemic areas.MethodsThe study was conducted from June to July 2017 among asymptomatic children living in the locality of Mfou, Cameroon. Blood samples were collected from asymptomatic children to perform malaria diagnosis by microscopy, Plasmodium species by PCR and hemoglobin typing by RFLP. Infectiousness of gametocytes to mosquitoes was assessed by membrane feeding assays using blood from gametocyte carriers of HbAA and HbAS genotypes. A zero-inflated model was fitted to predict distribution of oocysts in mosquitoes according to hemoglobin genotype of the gametocyte source.ResultsAmong the 1557 children enrolled in the study, 314 (20.16%) were of the HbAS genotype. The prevalence of children with P. falciparum gametocytes was 18.47% in HbAS individuals and 13.57% in HbAA, and the difference is significant (χ2 = 4.61, P = 0.032). Multiplicity of infection was lower in HbAS gametocyte carriers (median = 2 genotypes/carrier in HbAS versus 3.5 genotypes/carrier in HbAA, Wilcoxon sum rank test = 188, P = 0.032). Gametocyte densities in the blood donor significantly influenced mosquito infection prevalence in both HbAS and HbAA individuals. The HbAS genotype had no significant effect on mosquito infection outcomes when using immune or naïve serum in feeding assays. In AB replacement feeding experiments, the odds ratio of mosquito infection for HbAA blood as compared to HbAS was 0.56 (95% CI 0.29–1.10), indicating a twice higher risk of infection in mosquitoes fed on gametocyte-containing blood of HbAS genotype.ConclusionPlasmodium transmission stages were more prevalent in SCT individuals. This may reflect the parasite’s enhanced investment in the sexual stage to increase their survival rate when asexual replication is impeded. The public health impact of our results points the need for intensive malaria control interventions in areas with high prevalence of HbAS. The similar infection parameters in feeding experiments where mosquitoes received the original serum from the blood donor indicated that immune responses to gametocyte surface proteins occur in both HbAS and HbAA individuals. The higher risk of infection in mosquitoes fed on HbAS blood depleted of immune factors suggests that changes in the membrane properties in HbAS erythrocytes may impact on the maturation process of gametocytes within circulating red blood cells.

Naturally acquired antibodies to gametocyte antigens are associated with reduced transmission of Plasmodium vivax gametocytes to Anopheles arabiensis mosquitoes.

Naturally acquired antibodies may reduce the transmission of Plasmodium gametocytes to mosquitoes. Here, we investigated associations between antibody prevalence and P. vivax infectivity to mosquitoes. A total of 368 microscopy confirmed P. vivax symptomatic patients were passively recruited from health centers in Ethiopia and supplemented with 56 observations from asymptomatic P. vivax parasite carriers. Direct membrane feeding assays (DMFA) were performed to assess mosquito infectivity; for selected feeds these experiments were also performed after replacing autologous plasma with malaria naïve control serum (n=61). The prevalence of antibodies against 6 sexual stage antigens (Pvs47, Pvs48/45, Pvs230, PvsHAP2, Pvs25 and PvCelTOS) and an array of asexual antigens was determined by ELISA and multiplexed bead-based assays. Gametocyte (ρ< 0.42; p = 0.0001) and parasite (ρ = 0.21; p = 0.0001) densities were positively associated with mosquito infection rates. Antibodies against Pvs47, Pvs230 and Pvs25 were associated with 23 and 34% reductions in mosquito infection rates (p<0.0001), respectively. Individuals who showed evidence of transmission blockade in serum-replacement DMFAs (n=8) were significantly more likely to have PvsHAP2 or Pvs47 antibodies. Further studies may demonstrate causality for the observed associations, improve our understanding of the natural transmission of P. vivax and support vaccine development.

Susceptibility of primary, secondary and suspected vectors to Plasmodium vivax and Plasmodium falciparum infection in Ethiopia

BackgroundInsecticide-based vector control interventions in combination with case management with artemisinin-based combination therapy has reduced malaria incidence and prevalence worldwide. Current control methods focus on the primary malaria vectors, Anopheles gambiae sensu lato (s.l.) and the An. funestus group; however, the impact of secondary and suspected vectors has been either sidelined or received limited attention. Defining the susceptibility of secondary, suspected vector species to different parasites in time and space is essential for efficient malaria control and elimination programs. The aim of this study was to assess the susceptibility of An. gambiae s.l., An. coustani complex and An. pharoensis to Plasmodium vivax and P. falciparum infection in Ethiopia.MethodsLarvae of Anopheles spp. were collected from different aquatic habitats and reared to adults under laboratory conditions, with the temperature and humidity maintained at 27 ± 1 °C and 75 ± 5%, respectively. Adult female mosquitoes were identified to species as An. gambiae s.l., An. coustani complex and An. pharoensis. Females of these three Anopheles spp. were allowed to feed in parallel feeding assays on infected blood containing the same gametocytes isolated from P. falciparum and P. vivax gametocyte-positive patients by indirect membrane feeding assays. All blood-fed mosquitoes were held under laboratory conditions. After 7 days, all surviving mosquitoes were dissected to detect mid-gut oocyst and enumerated under a microscope.ResultsOf 5915 female Anopheles mosquitoes exposed to gametocyte-infected blood, 2106 (35.6%)s fed successfully in the 32 independent infection experiments. There was a significant variation in feeding rates among An. gambiae s.l., An. pharoensis and An. coustani complex (G-test = 48.43, P = 3.049e-11). All three exposed mosquito species were receptive to P. vivax and P. falciparum infection development. The percentage of infected mosquitoes following feeding on an infected blood meal was significantly different among species (G-test = 6.49, P = 0.03886). The median infection intensity (II) for An. coustani complex, An. gambiae s.l. and An. pharoensis was 1.16, 2.00 and 1.25, respectively. Although the proportion of infected mosquitoes significantly differed in terms of II, infection rate (IR) and mean oocyst density among the species, mean oocyst density and IR were highly correlated with gametocyte density in all tests (P < 0.001).ConclusionPrimary, secondary and suspected vectors were experimentally susceptible to both P. vivax and P. falciparum infection. An effective malaria elimination program might include surveillance and control tools which target secondary and suspected vectors that might play an outdoor transmission role, possibly resulting in reduced focal malaria transmission.Graphical Comparison of the three species’ mean infection rates with standard deviation

The acquisition of humoral immune responses targeting Plasmodium falciparum sexual stages in controlled human malaria infections.

Individuals infected with P. falciparum develop antibody responses to intra-erythrocytic gametocyte proteins and exported gametocyte proteins present on the surface of infected erythrocytes. However, there is currently limited knowledge on the immunogenicity of gametocyte antigens and the specificity of gametocyte-induced antibody responses. In this study, we assessed antibody responses in participants of two controlled human malaria infection (CHMI) studies by ELISA, multiplexed bead-based antibody assays and protein microarray. By comparing antibody responses in participants with and without gametocyte exposure, we aimed to disentangle the antibody response induced by asexual and sexual stage parasites. We showed that after a single malaria infection, a significant anti-sexual stage humoral response is induced in malaria-naïve individuals, even after exposure to relatively low gametocyte densities (up to ~1,600 gametocytes/mL). In contrast to antibody responses to well-characterised asexual blood stage antigens that were detectable by day 21 after infection, responses to sexual stage antigens (including transmission blocking vaccine candidates Pfs48/45 and Pfs230) were only apparent at 51 days after infection. We found antigens previously associated with early gametocyte or anti-gamete immunity were highly represented among responses linked with gametocyte exposure. Our data provide detailed insights on the induction and kinetics of antibody responses to gametocytes and identify novel antigens that elicit antibody responses exclusively in individuals with gametocyte exposure. Our findings provide target identification for serological assays for surveillance of the malaria infectious reservoir, and support vaccine development by describing the antibody response to leading vaccine antigens after primary infection.

Transmission Blocking Activity of Low-dose Tafenoquine in Healthy Volunteers Experimentally Infected With Plasmodium falciparum.

Blocking the transmission of parasites from humans to mosquitoes is a key component of malaria control. Tafenoquine exhibits activity against all stages of the malaria parasite and may have utility as a transmission blocking agent. We aimed to characterize the transmission blocking activity of low-dose tafenoquine. Healthy adults were inoculated with Plasmodium falciparum 3D7-infected erythrocytes on day 0. Piperaquine was administered on days 9 and 11 to clear asexual parasitemia while allowing gametocyte development. A single 50-mg oral dose of tafenoquine was administered on day 25. Transmission was determined by enriched membrane feeding assays predose and at 1, 4, and 7 days postdose. Artemether-lumefantrine was administered following the final assay. Outcomes were the reduction in mosquito infection and gametocytemia after tafenoquine and safety parameters. Six participants were enrolled, and all were infective to mosquitoes before tafenoquine, with a median 86% (range, 22-98) of mosquitoes positive for oocysts and 57% (range, 4-92) positive for sporozoites. By day 4 after tafenoquine, the oocyst and sporozoite positivity rate had reduced by a median 35% (interquartile range [IQR]: 16-46) and 52% (IQR: 40-62), respectively, and by day 7, 81% (IQR 36-92) and 77% (IQR 52-98), respectively. The decline in gametocyte density after tafenoquine was not significant. No significant participant safety concerns were identified. Low-dose tafenoquine (50 mg) reduces P. falciparum transmission to mosquitoes, with a delay in effect.

Evaluation of sustainable susceptibility to Plasmodium vivax infection among colonized Anopheles darlingi and Anopheles deaneorum

BackgroundThe colonization of mosquitoes susceptible to Plasmodium vivax via direct membrane feeding assay (DMFA) has the potential to significantly advance our knowledge of P. vivax biology, vector-parasite interaction and transmission-blocking vaccine research. Anopheles darlingi and Anopheles deaneorum are important vectors of malaria in the Western Brazilian Amazon. Since 2018, well-established colonies of these species have been maintained in order to mass produce mosquitoes destined for P. vivax infection. Plasmodium susceptibility was confirmed when the colonies were established, but susceptibility needs to be maintained for these colonies to remain good models for pathogen transmission. Thus, the susceptibility was assessed of colonized mosquitoes to P. vivax isolates circulating in the Western Amazon.MethodsLaboratory-reared mosquitoes from F10-F25 generations were fed on P. vivax blood isolates via DMFA. Susceptibility was determined by prevalence and intensity of infection as represented by oocyst load seven days after blood feeding, and sporozoite load 14 days after blood feeding. The effect of infection on mosquito survival was evaluated from initial blood feeding until sporogonic development and survival rates were compared between mosquitoes fed on infected and uninfected blood. Correlation was calculated between gametocytaemia and prevalence/intensity of infection, and between oocyst and sporozoite load.ResultsSignificant differences were found in prevalence and intensity of infection between species. Anopheles darlingi showed a higher proportion of infected mosquitoes and higher oocyst and sporozoite intensity than An. deaneorum. Survival analysis showed that An. deaneorum survival decreased drastically until 14 days post infection (dpi). Plasmodium vivax infection decreased survival in both species relative to uninfected mosquitoes. No correlation was observed between gametocytaemia and prevalence/intensity of infection, but oocyst and sporozoite load had a moderate to strong correlation.ConclusionsColonized An. darlingi make excellent subjects for modelling pathogen transmission. On the other hand, An. deaneorum could serve as a model for immunity studies due the low susceptibility under current colonized conditions. In the application of DMFA, gametocyte density is not a reliable parameter for predicting mosquito infection by P. vivax, but oocyst intensity should be used to schedule sporozoite experiments.

Quantifying Reductions in Plasmodium falciparum Infectivity to Mosquitos: A Sample Size Calculator to Inform Clinical Trials on Transmission-Reducing Interventions.

Malaria transmission depends on the presence of mature Plasmodium transmission stages (gametocytes) that may render blood-feeding Anopheles mosquitos infectious. Transmission-blocking antimalarial drugs and vaccines can prevent transmission by reducing gametocyte densities or infectivity to mosquitos. Mosquito infection outcomes are thereby informative biological endpoints of clinical trials with transmission blocking interventions. Nevertheless, trials are often primarily designed to determine intervention safety; transmission blocking efficacy is difficult to incorporate in sample size considerations due to variation in infection outcomes and considerable inter-study variation. Here, we use clinical trial data from studies in malaria naive and naturally exposed study participants to present an online sample size calculator tool. This sample size calculator allows studies to be powered to detect reductions in the proportion of infected mosquitos or infection burden (oocyst density) in mosquitos. The utility of this online tool is illustrated using trial data with transmission blocking malaria drugs.

Asymptomatic School-Aged Children Are Important Drivers of Malaria Transmission in a High Endemicity Setting in Uganda.

Achieving malaria elimination requires a better understanding of the transmissibility of human infections in different transmission settings. This study aimed to characterize the human infectious reservoir in a high endemicity setting in eastern Uganda, using gametocyte quantification and mosquito feeding assays. In asymptomatic infections, gametocyte densities were positively associated with the proportion of infected mosquitoes (β = 1.60; 95% CI, 1.32–1.92; P < .0001). Combining transmissibility and abundance in the population, symptomatic and asymptomatic infections were estimated to contribute to 5.3% and 94.7% of the infectious reservoir, respectively. School-aged children (5–15 years old) contributed to 50.4% of transmission events and were important drivers of malaria transmission.

Anti-Gametocyte Antigen Humoral Immunity and Gametocytemia During Treatment of Uncomplicated Falciparum Malaria: A Multi-National Study.

IntroductionUnderstanding the human immune response to Plasmodium falciparum gametocytes and its association with gametocytemia is essential for understanding the transmission of malaria as well as progressing transmission blocking vaccine candidates.MethodsIn a multi-national clinical efficacy trial of artemisinin therapies (13 sites of varying transmission over South-East Asia and Africa), we measured Immunoglobulin G (IgG) responses to recombinant P. falciparum gametocyte antigens expressed on the gametocyte plasma membrane and leading transmission blocking vaccine candidates Pfs230 (Pfs230c and Pfs230D1M) and Pfs48/45 at enrolment in 1,114 participants with clinical falciparum malaria. Mixed effects linear and logistic regression were used to determine the association between gametocyte measures (gametocytemia and gametocyte density) and antibody outcomes at enrolment.ResultsMicroscopy detectable gametocytemia was observed in 11% (127/1,114) of participants at enrolment, and an additional 9% (95/1,114) over the follow-up period (up to day 42) (total 20% of participants [222/1,114]). IgG levels in response to Pfs230c, Pfs48/45 and Pfs230D1M varied across study sites at enrolment (p < 0.001), as did IgG seroprevalence for anti-Pfs230c and D1M IgG (p < 0.001), but not for anti-Pfs48/45 IgG (p = 0.159). In adjusted analyses, microscopy detectable gametocytemia at enrolment was associated with an increase in the odds of IgG seropositivity to the three gametocyte antigens (Pfs230c OR [95% CI], p: 1.70 [1.10, 2.62], 0.017; Pfs48/45: 1.45 [0.85, 2.46], 0.174; Pfs230D1M: 1.70 [1.03, 2.80], 0.037), as was higher gametocyte density at enrolment (per two-fold change in gametocyte density Pfs230c OR [95% CI], p: 1.09 [1.02, 1.17], 0.008; Pfs48/45: 1.05 [0.98, 1.13], 0.185; Pfs230D1M: 1.07 [0.99, 1.14], 0.071).Conclusion Pfs230 and Pfs48/45 antibodies are naturally immunogenic targets associated with patent gametocytemia and increasing gametocyte density across multiple malaria endemic settings, including regions with emerging artemisinin-resistant P. falciparum.

Effectiveness of Artemether Lumefantrine and Dihydroartemisinin Piperaquine in Clearance of Gametocytes in Uncomplicated Plasmodium falciparum Malaria in Tiwi Kenya

Background: Over 80 countries worldwide have now implemented WHO recommendations to use Artemisinin-Based Combination Therapy as a first-line treatment for Plasmodium falciparum malaria. The sexual stage of P. falciparum is responsible for the transmission of malarial parasites to infectious mosquitoes. Studies on gametocytes are generally based on microscopic detection, which is not sensitive, and there is a need for more sensitive molecular techniques that can detect and quantify gametocytes at densities as low as 0.02 to 0.1 gametocytes per micro-litre. The objective of this study was to determine the clearance rates of gametocytes after AL and DHA&amp;P in uncomplicated P. falciparum and to compare the effectiveness of microscopy and reverse transcriptase-polymerase chain reaction in gametocyte detection. Methods: In a randomised controlled clinical trial of samples collected, gametocyte densities were quantified by microscopy by counting against 500 leukocytes in the thick smear converted to numbers of parasites per micro-litre by assuming a standard count of 800 leukocytes per micro-litre of blood after staining with 10% Giemsa stain and by reverse transcriptase-polymerase chain reaction using primers specific to the pfs25 gene. Results: There was no significant difference between the drug’s gametocyte clearance (p&lt;.082). The drugs cleared gametocytes in infected patients by day 28 as detected by microscopy. There was a significant difference in the detection of gametocytes by RT-PCR and microscopy (p&lt;.001). Conclusion: This study showed that Artemether-Lumefantrine and Dihydroartemisinin piperaquine have gametocytocidal effects on P. falciparum and the study on the clearance of gametocytes using both artemether-lumefantrine and Dihydroartemisinin piperaquine may be carried out using a larger sample size for policy implementation. The reverse transcriptase-polymerase chain reaction is more effective than microscopy in detecting low levels of gametocytes and the pfs25 gene can be used in the detection of gametocytes in the field to monitor the clearance of gametocytes.

Single low-dose tafenoquine combined with dihydroartemisinin–piperaquine to reduce Plasmodium falciparum transmission in Ouelessebougou, Mali: a phase 2, single-blind, randomised clinical trial

SummaryBackgroundTafenoquine was recently approved as a prophylaxis and radical cure for Plasmodium vivax infection, but its Plasmodium falciparum transmission-blocking efficacy is unclear. We aimed to establish the efficacy and safety of three single low doses of tafenoquine in combination with dihydroartemisinin–piperaquine for reducing gametocyte density and transmission to mosquitoes.MethodsIn this four-arm, single-blind, phase 2, randomised controlled trial, participants were recruited at the Clinical Research Unit of the Malaria Research and Training Centre of the University of Bamako in Mali. Eligible participants were aged 12–50 years, with asymptomatic P falciparum microscopy-detected gametocyte carriage, had a bodyweight of 80 kg or less, and had no clinical signs of malaria defined by fever. Participants were randomly assigned (1:1:1:1) to standard treatment with dihydroartemisinin–piperaquine, or dihydroartemisinin–piperaquine plus a single dose of tafenoquine (in solution) at a final dosage of 0·42 mg/kg, 0·83 mg/kg, or 1·66 mg/kg. Randomisation was done with a computer-generated randomisation list and concealed with sealed, opaque envelopes. Dihydroartemisinin–piperaquine was administered as oral tablets over 3 days (day 0, 1, and 2), as per manufacturer instructions. A single dose of tafenoquine was administered as oral solution on day 0 in parallel with the first dose of dihydroartemisinin–piperaquine. Tafenoquine dosing was based on bodyweight to standardise efficacy and risk variance. The primary endpoint, assessed in the per-protocol population, was median percentage change in mosquito infection rate 7 days after treatment compared with baseline. Safety endpoints included frequency and incidence of adverse events. The final follow-up visit was on Dec 23, 2021; the trial is registered with ClinicalTrials.gov, NCT04609098.FindingsFrom Oct 29 to Nov 25, 2020, 1091 individuals were screened for eligibility, 80 of whom were enrolled and randomly assigned (20 per treatment group). Before treatment, 53 (66%) individuals were infectious to mosquitoes, infecting median 12·50% of mosquitoes (IQR 3·64–35·00). Within-group reduction in mosquito infection rate on day 7 was 79·95% (IQR 57·15–100; p=0·0005 for difference from baseline) following dihydroartemisinin–piperaquine only, 100% (98·36–100; p=0·0005) following dihydroartemisinin–piperaquine plus tafenoquine 0·42 mg/kg, 100% (100–100; p=0·0001) following dihydroartemisinin–piperaquine plus tafenoquine 0·83 mg/kg, and 100% (100–100; p=0·0001) following dihydroartemisinin–piperaquine plus tafenoquine 1·66 mg/kg. 55 (69%) of 80 participants had a total of 94 adverse events over the course of the trial; 86 (92%) adverse events were categorised as mild, seven (7%) as moderate, and one (1%) as severe. The most common treatment-related adverse event was mild or moderate headache, which occurred in 15 (19%) participants (dihydroartemisinin–piperaquine n=2; dihydroartemisinin–piperaquine plus tafenoquine 0·42 mg/kg n=6; dihydroartemisinin–piperaquine plus tafenoquine 0·83 mg/kg n=3; and dihydroartemisinin–piperaquine plus tafenoquine 1·66 mg/kg n=4). No serious adverse events occurred. No significant differences in the incidence of all adverse events (p=0·73) or treatment-related adverse events (p=0·62) were observed between treatment groups.InterpretationTafenoquine was well tolerated at all doses and accelerated P falciparum gametocyte clearance. All tafenoquine doses showed improved transmission reduction at day 7 compared with dihydroartemisinin–piperaquine alone. These data support the case for further research on tafenoquine as a transmission-blocking supplement to standard antimalarials.FundingBill & Melinda Gates Foundation.TranslationsFor the French, Portuguese, Spanish and Swahili translations of the abstract see Supplementary Materials section.

Persistent Plasmodium falciparum infections enhance transmission-reducing immunity development

Subclinical infections that serve as reservoir populations to drive transmission remain a hurdle to malaria control. Data on infection dynamics in a geographical area is required to strategically design and implement malaria interventions. In a longitudinal cohort, we monitored Plasmodium falciparum infection prevalence and persistence, and anti-parasite immunity to gametocyte and asexual antigens for 10 weeks. Of the 100 participants, only 11 were never infected, whilst 16 had persistent infections detected by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), and one participant had microscopic parasites at all visits. Over 70% of the participants were infected three or more times, and submicroscopic gametocyte prevalence was high, ≥ 48% of the parasite carriers. Naturally induced responses against recombinant Pfs48/45.6C, Pfs230proC, and EBA175RIII–V antigens were not associated with either infection status or gametocyte carriage, but the antigen-specific IgG titers inversely correlated with parasite and gametocyte densities consistent with partial immunity. Longitudinal analysis of gametocyte diversity indicated at least four distinct clones circulated throughout the study period. The high prevalence of children infected with distinct gametocyte clones coupled with marked variation in infection status at the individual level suggests ongoing transmission and should be targeted in malaria control programs.