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.

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.

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.

Plasmodium falciparum gametocyte carriage in longitudinally monitored incident infections is associated with duration of infection and human host factors.

Malaria transmission depends on the presence of Plasmodium gametocytes that are the only parasite life stage that can infect mosquitoes. Gametocyte production varies between infections and over the course of infections. Infection duration is highly important for gametocyte production but poorly quantified. Between 2017 and 2019 an all-age cohort of individuals from Tororo, eastern Uganda was followed by continuous passive and routine assessments. We longitudinally monitored 104 incident infections from 98 individuals who were sampled once every 28 days and on any day of symptoms. Among infections that lasted ≥ 3 months, gametocyte appearance was near-universal with 96% of infections having detectable gametocytes prior to clearance. However, most infections were of much shorter duration; 55.7% of asymptomatic infections were detected only once. When considering all asymptomatic infections, regardless of their duration, only 36.3% had detectable gametocytes on at least one time-point prior to parasite clearance. Infections in individuals with sickle-cell trait (HbAS) were more likely to have gametocytes detected (Hazard Rate (HR) = 2.68, 95% CI 1.12, 6.38; p = 0.0231) and had gametocytes detected at higher densities (Density Ratio (DR) = 9.19, 95% CI 2.79, 30.23; p = 0.0002) compared to infections in wildtype (HbAA) individuals. Our findings suggest that a large proportion of incident infections is too short in duration and of too low density to contribute to onward transmission.

Persistence of Plasmodium falciparum HRP-2 antigenaemia after artemisinin combination therapy is not associated with gametocytes.

BACKGROUND: In some settings, sensitive field diagnostic tools may be needed to achieve elimination of falciparum malaria. To this end, rapid diagnostic tests (RDTs) based on the detection of the Plasmodium falciparum protein HRP-2 are being developed with increasingly lower limits of detection. However, it is currently unclear how parasite stages that are unaffected by standard drug treatments may contribute to HRP-2 detectability and potentially confound RDT results even after clearance of blood stage infection. This study assessed the detectability of HRP-2 in periods of post-treatment residual gametocytaemia. METHODS: A cohort of 100 P. falciparum infected, gametocyte positive individuals were treated with or without the gametocytocidal drug primaquine (PQ), alongside standard artemisinin-based combination therapy (ACT), in the context of a randomised clinical trial in Ouelessebougou, Mali. A quantitative ELISA was used to measure levels of HRP-2, and compared time to test negativity using a standard and ultra-sensitive RDT (uRDT) between residual gametocyte positive and negative groups. RESULTS: Time to test negativity was longest by uRDT, followed by ELISA and then standard RDT. No significant difference in time to negativity was found between the treatment groups with and without residual gametocytes: uRDT (HR 0.79 [95% CI 0.52-1.21], p = 0.28), RDT (HR 0.77 [95% CI 0.51-1.15], p = 0.20) or ELISA (HR 0.88 [95% CI 0.59-1.32], p = 0.53). Similarly, no difference was observed when adjusting for baseline asexual parasite density. Quantified levels of HRP-2 over time were similar between groups, with differences attributable to asexual parasite densities. Furthermore, no difference in levels of HRP-2 was found between individuals who were or were not infectious to mosquitoes (OR 1.19 [95% CI 0.98-1.46], p = 0.077). CONCLUSIONS: Surviving sexual stage parasites after standard ACT treatment do not contribute to the persistence of HRP-2 antigenaemia, and appear to have little impact on RDT results.

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

BACKGROUND: Tafenoquine 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. METHODS: In 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. FINDINGS: From 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. INTERPRETATION: Tafenoquine 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. FUNDING: Bill & Melinda Gates Foundation. TRANSLATIONS: For the French, Portuguese, Spanish and Swahili translations of the abstract see Supplementary Materials section.

SARS-CoV-2 RNA in exhaled air of hospitalized COVID-19 patients.

Knowledge about contagiousness is key to accurate management of hospitalized COVID-19 patients. Epidemiological studies suggest that in addition to transmission through droplets, aerogenic SARS-CoV-2 transmission contributes to the spread of infection. However, the presence of virus in exhaled air has not yet been sufficiently demonstrated. In pandemic situations low tech disposable and user-friendly bedside devices are required, while commercially available samplers are unsuitable for application in patients with respiratory distress. We included 49 hospitalized COVID-19 patients and used a disposable modular breath sampler to measure SARS-CoV-2 RNA load in exhaled air samples and compared these to SARS-CoV-2 RNA load of combined nasopharyngeal throat swabs and saliva. Exhaled air sampling using the modular breath sampler has proven feasible in a clinical COVID-19 setting and demonstrated viral detection in 25% of the patients.

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 (ß = 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.

A Cohort Study on the Duration of Plasmodium falciparum Infections During the Dry Season in The Gambia.

BACKGROUND: In areas where Plasmodium falciparum malaria is seasonal, a dry season reservoir of blood-stage infection is essential for initiating transmission during the following wet season. METHODS: In The Gambia, a cohort of 42 individuals with quantitative polymerase chain reaction-positive P falciparum infections at the end of the transmission season (December) were followed monthly until the end of the dry season (May) to evaluate infection persistence. The influence of human host and parasitological factors was investigated. RESULTS: A large proportion of individuals infected at the end of the wet season had detectable infections until the end of the dry season (40.0%; 16 of 40). At the start of the dry season, the majority of these persistent infections (82%) had parasite densities >10 p/µL compared to only 5.9% of short-lived infections. Persistent infections (59%) were also more likely to be multiclonal than short-lived infections (5.9%) and were associated with individuals having higher levels of P falciparum-specific antibodies (P = .02). CONCLUSIONS: Asymptomatic persistent infections were multiclonal with higher parasite densities at the beginning of the dry season. Screening and treating asymptomatic infections during the dry season may reduce the human reservoir of malaria responsible for initiating transmission in the wet season.

Human antibodies against noncircumsporozoite proteins block Plasmodium falciparum parasite development in hepatocytes.

Sporozoite-based approaches currently represent the most effective vaccine strategies for induction of sterile protection against Plasmodium falciparum (Pf) malaria. Clinical development of subunit vaccines is almost exclusively centered on the circum-sporozoite protein (CSP), an abundantly expressed protein on the sporozoite membrane. Anti-CSP antibodies are able to block sporozoite invasion and development in human hepatocytes and subsequently prevent clinical malaria. Here, we have investigated whether sporozoite-induced human antibodies with specificities different from CSP can reduce Pf-liver stage development. IgG preparations were obtained from 12 volunteers inoculated with a protective immunization regime of whole sporozoites under chloroquine prophylaxis. These IgGs were depleted for CSP specificity by affinity chromatography. Recovered non-CSP antibodies were tested for sporozoite membrane binding and for functional inhibition of sporozoite invasion of a human hepatoma cell line and hepatocytes both in vitro and in vivo. Postimmunization IgGs depleted for CS specificity of 9 of 12 donors recognized sporozoite surface antigens. Samples from 5 of 12 donors functionally reduced parasite-liver cell invasion or development using the hepatoma cell line HC-04 and FRG-huHep mice containing human liver cells. The combined data provide clear evidence that non-CSP proteins, as yet undefined, do represent antibody targets for functional immunity against Pf parasites responsible for malaria.

Pyronaridine-artesunate or dihydroartemisinin-piperaquine combined with single low-dose primaquine to prevent Plasmodium falciparum malaria transmission in Ouélessébougou, Mali: a four-arm, single-blind, phase 2/3, randomised trial.

BACKGROUND: Pyronaridine-artesunate is the most recently licensed artemisinin-based combination therapy. WHO has recommended that a single low dose of primaquine could be added to artemisinin-based combination therapies to reduce Plasmodium falciparum transmission in areas aiming for elimination of malaria or areas facing artemisinin resistance. We aimed to determine the efficacy of pyronaridine-artesunate and dihydroartemisinin-piperaquine with and without single low-dose primaquine for reducing gametocyte density and transmission to mosquitoes. METHODS: We conducted a four-arm, single-blind, phase 2/3, randomised trial at the Ouélessébougou Clinical Research Unit of the Malaria Research and Training Centre of the University of Bamako (Bamako, Mali). Participants were aged 5-50 years, with asymptomatic P falciparum malaria mono-infection and gametocyte carriage on microscopy, haemoglobin density of 9·5 g/dL or higher, bodyweight less than 80 kg, and no use of antimalarial drugs over the past week. Participants were randomly assigned (1:1:1:1) to one of four treatment groups: pyronaridine-artesunate, pyronaridine-artesunate plus primaquine, dihydroartemisinin-piperaquine, or dihydroartemisinin-piperaquine plus primaquine. Treatment allocation was concealed to all study staff other than the trial pharmacist and treating physician. Dihydroartemisinin-piperaquine and pyronaridine-artesunate were administered as per manufacturer guidelines over 3 days; primaquine was administered as a single dose in oral solution according to bodyweight (0·25 mg/kg; in 1 kg bands). The primary endpoint was percentage reduction in mosquito infection rate (percentage of mosquitoes surviving to dissection that were infected with P falciparum) at 48 h after treatment compared with baseline (before treatment) in all treatment groups. Data were analysed per protocol. This trial is now complete, and is registered with ClinicalTrials.gov, NCT04049916. FINDINGS: Between Sept 10 and Nov 19, 2019, 1044 patients were assessed for eligibility and 100 were enrolled and randomly assigned to one of the four treatment groups (n=25 per group). Before treatment, 66 (66%) of 100 participants were infectious to mosquitoes, with a median of 15·8% (IQR 5·4-31·9) of mosquitoes becoming infected. In individuals who were infectious before treatment, the median percentage reduction in mosquito infection rate 48 h after treatment was 100·0% (IQR 100·0 to 100·0) for individuals treated with pyronaridine-artesunate plus primaquine (n=18; p<0·0001) and dihydroartemisinin-piperaquine plus primaquine (n=15; p=0·0001), compared with -8·7% (-54·8 to 93·2) with pyronaridine-artesunate (n=17; p=0·88) and 50·4% (13·8 to 70·9) with dihydroartemisinin-piperaquine (n=16; p=0·13). There were no serious adverse events, and there were no significant differences between treatment groups at any point in the frequency of any adverse events (Fisher's exact test p=0·96) or adverse events related to study drugs (p=0·64). The most common adverse events were headaches (40 events in 32 [32%] of 100 participants), rhinitis (31 events in 30 [30%]), and respiratory infection (20 events in 20 [20%]). INTERPRETATION: These data support the use of single low-dose primaquine as an effective supplement to dihydroartemisinin-piperaquine and pyronaridine-artesunate for blocking P falciparum transmission. The new pyronaridine-artesunate plus single low-dose primaquine combination is of immediate relevance to regions in which the containment of partial artemisinin and partner-drug resistance is a growing concern and in regions aiming to eliminate malaria. FUNDING: The Bill & Melinda Gates Foundation. TRANSLATIONS: For the French, Spanish and Swahilil translations of the abstract see Supplementary Materials section.

The effect of anticoagulants in blood collection tubes on Plasmodium falciparum transmission in direct membrane feeding assays.

BACKGROUND: Direct membrane feeding assays assess the transmission potential of malaria-infected individuals using whole blood collected in anticoagulant vacutainers. METHODS: The potential inhibitory effect of four commonly used anticoagulants on gametocyte infectivity to mosquitoes was assessed in standard membrane feeding assays with cultured Plasmodium falciparum. RESULTS: Infection burden in mosquitoes was significantly reduced when blood was collected in sodium citrate and EDTA. Transmission was highest when blood was collected in lithium heparin and sodium heparin, although a concentration-dependent inhibition of mosquito infection was also observed. CONCLUSIONS: Although anticoagulants can reduce transmission efficiency, lithium heparin and sodium heparin are the best anticoagulants for evaluating malaria transmission.

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.

SARS-CoV-2 mucosal antibody development and persistence and their relation to viral load and COVID-19 symptoms.

Although serological studies have shown that antibodies against SARS-CoV-2 play an important role in protection against (re)infection, the dynamics of mucosal antibodies during primary infection and their potential impact on viral load and the resolution of disease symptoms remain unclear. During the first pandemic wave, we assessed the longitudinal nasal antibody response in index cases with mild COVID-19 and their household contacts. Nasal and serum antibody responses were analysed for up to nine months. Higher nasal receptor binding domain and spike protein-specific antibody levels at study inclusion were associated with lower viral load. Older age was correlated with more frequent COVID-19 related symptoms. Receptor binding domain and spike protein-specific mucosal antibodies were associated with the resolution of systemic, but not respiratory symptoms. Finally, receptor binding domain and spike protein-specific mucosal antibodies remained elevated up to nine months after symptom onset.

Sources of persistent malaria transmission in a setting with effective malaria control in eastern Uganda: a longitudinal, observational cohort study.

BACKGROUND: Symptomatic malaria cases reflect only a small proportion of all Plasmodium spp infections. Many infected individuals are asymptomatic, and persistent asymptomatic Plasmodium falciparum infections are common in endemic settings. We aimed to quantify the contribution of symptomatic and asymptomatic infections to P falciparum transmission in Tororo, Uganda. METHODS: We did a longitudinal, observational cohort study in Tororo district, Uganda. We recruited participants of all ages from randomly selected households within this district. Participants were eligible if the selected household had no more than nine permanent residents and at least two members younger than 10 years, and the household was their primary residence, and they agreed to come to the study clinic for any fever episode and avoid antimalarial medications outside the study. Participants were followed-up by continuous passive surveillance for the incidence of symptomatic infections; routine assessments (ie, standardised clinical evaluation and blood samples) were done at baseline and at routine visits every 4 weeks for 2 years. P falciparum parasite density, gametocyte density, and genetic composition were determined molecularly using quantitative PCR (qPCR), quantitative reverse transcriptase PCR (qRT-PCR), and amplicon deep sequencing, respectively. Membrane feeding assays were also done to assess infectivity to mosquitoes. The contribution of different populations to the infectious reservoir was estimated for symptomatic infections, asymptomatic but microscopically detected infections, and asymptomatic but qPCR-detected infections; and for age groups younger than 5 years, 5-15 years, and 16 years or older. FINDINGS: Between Oct 4, 2017, and Oct 31, 2019, 531 individuals were enrolled from 80 randomly selected households and were followed-up for 2 years. At baseline, P falciparum was detected in 28 (5·3%) of 531 participants by microscopy and an additional 64 (12·1%) by qPCR and declined thereafter. In 538 mosquito feeding experiments on 107 individuals, 446 (1·2%) of 37 404 mosquitoes became infected, with mosquito infection rates being strongly associated with gametocyte densities (ß=2·11, 95% CI 1·62-2·67; p<0·0001). Considering both transmissibility of infections and their relative frequency, the estimated human infectious reservoir consisted primarily of asymptomatic microscopy-detected infections (83·8%), followed by asymptomatic submicroscopic infections (15·6%), and symptomatic infections (0·6%). Children aged 5-15 years accounted for more than half of the infectious reservoir (58·7%); individuals younger than 5 years (25·8%) and those 16 years or older (15·6%) contributed less. Samples from four children contribued to 279 (62·6%) of 446 infected mosquitoes after multiple mosquito-feeding assays. INTERPRETATION: Individuals with asymptomatic infections were important drivers of malaria transmission. School-aged children contributed to more than half of all mosquito infections, with a small minority of asymptomatic children being highly infectious. Demographically targeted interventions, aimed at school-aged children, could further reduce transmission in areas under effective vector control. FUNDING: US National Institutes of Health, Bill & Melinda Gates Foundation, and the European Research Council.

G6PD Polymorphisms and Hemolysis After Antimalarial Treatment With Low Single-Dose Primaquine: A Pooled Analysis of Six African Clinical Trials.

Primaquine (PQ) is an antimalarial drug with the potential to reduce malaria transmission due to its capacity to clear mature Plasmodium falciparum gametocytes in the human host. However, the large-scale roll-out of PQ has to be counterbalanced by the additional risk of drug-induced hemolysis in individuals suffering from Glucose-6-phospate dehydrogenase (G6PD) deficiency, a genetic condition determined by polymorphisms on the X-linked G6PD gene. Most studies on G6PD deficiency and PQ-associated hemolysis focused on the G6PD A- variant, a combination of the two single nucleotide changes G202A (rs1050828) and A376G (rs1050829), although other polymorphisms may play a role. In this study, we tested the association of 20 G6PD single nucleotide polymorphisms (SNPs) with hemolysis measured seven days after low single dose of PQ given at the dose of 0.1 mg/kg to 0.75 mg/kg in 957 individuals from 6 previously published clinical trials investigating the safety and efficacy of this drug spanning five African countries. After adjusting for inter-study effects, age, gender, baseline hemoglobin level, PQ dose, and parasitemia at screening, our analysis showed putative association signals from the common G6PD mutation, A376G [-log10(p-value) = 2.44] and two less-known SNPs, rs2230037 [-log10(p-value] = 2.60), and rs28470352 [-log10(p-value) = 2.15]; A376G and rs2230037 were in very strong linkage disequilibrium with each other (R 2 = 0.978). However, when the effects of these SNPs were included in the same regression model, the subsequent associations were in the borderline of statistical significance. In conclusion, whilst a role for the A- variant is well established, we did not observe an important additional role for other G6PD polymorphisms in determining post-treatment hemolysis in individuals treated with low single-dose PQ.

Higher gametocyte production and mosquito infectivity in chronic compared to incident Plasmodium falciparum infections.

Plasmodium falciparum gametocyte kinetics and infectivity may differ between chronic and incident infections. In the current study, we assess parasite kinetics and infectivity to mosquitoes among children (aged 5-10 years) from Burkina Faso with (a) incident infections following parasite clearance (n = 48) and (b) chronic asymptomatic infections (n = 60). In the incident infection cohort, 92% (44/48) of children develop symptoms within 35 days, compared to 23% (14/60) in the chronic cohort. All individuals with chronic infection carried gametocytes or developed them during follow-up, whereas only 35% (17/48) in the incident cohort produce gametocytes before becoming symptomatic and receiving treatment. Parasite multiplication rate (PMR) and the relative abundance of ap2-g and gexp-5 transcripts are positively associated with gametocyte production. Antibody responses are higher and PMR lower in chronic infections. The presence of symptoms and sexual stage immune responses are associated with reductions in gametocyte infectivity to mosquitoes. We observe that most incident infections require treatment before the density of mature gametocytes is sufficient to infect mosquitoes. In contrast, chronic, asymptomatic infections represent a significant source of mosquito infections. Our observations support the notion that malaria transmission reduction may be expedited by enhanced case management, involving both symptom-screening and infection detection.

Persistence of mRNA indicative of Plasmodium falciparum ring-stage parasites 42 days after artemisinin and non-artemisinin combination therapy in naturally infected Malians.

BACKGROUND: Malaria control in sub-Saharan Africa relies upon prompt case management with artemisinin-based combination therapy (ACT). Ring-stage parasite mRNA, measured by sbp1 quantitative reverse-transcriptase PCR (qRT-PCR), was previously reported to persist after ACT treatment and hypothesized to reflect temporary arrest of the growth of ring-stage parasites (dormancy) following exposure to artemisinins. Here, the persistence of ring-stage parasitaemia following ACT and non-ACT treatment was examined. METHODS: Samples were used from naturally infected Malian gametocyte carriers who received dihydroartemisinin-piperaquine (DP) or sulfadoxine-pyrimethamine (SP-AQ) with or without gametocytocidal drugs. Gametocytes and ring-stage parasites were quantified by qRT-PCR during 42 days of follow-up. RESULTS: At baseline, 89% (64/73) of participants had measurable ring-stage parasite mRNA. Following treatment, the proportion of ring-stage parasite-positive individuals and estimated densities declined for all four treatment groups. Ring-stage parasite prevalence and density was generally lower in arms that received DP compared to SP-AQ. This finding was most apparent days 1, 2, and 42 of follow-up (p < 0.01). Gametocytocidal drugs did not influence ring-stage parasite persistence. Ring-stage parasite density estimates on days 14 and 28 after initiation of treatment were higher among individuals who subsequently experienced recurrent parasitaemia compared to those who remained free of parasites until day 42 after initiation of treatment (pday 14 = 0.011 and pday 28 = 0.068). No association of ring-stage persistence with gametocyte carriage was observed. CONCLUSIONS: The current findings of lower ring-stage persistence after ACT without an effect of gametocytocidal partner drugs affirms the use of sbp1 as ring-stage marker. Lower persistence of ring-stage mRNA after ACT treatment suggests the marker may not reflect dormant parasites whilst it was predictive of re-appearance of parasitaemia.