Infectivity of patent Plasmodium falciparum gametocyte carriers to mosquitoes: establishing capacity to investigate the infectious reservoir of malaria in a low-transmission setting in The Gambia.

BACKGROUND: Understanding the human malaria infectious reservoir is important for elimination initiatives. Here, we implemented mosquito membrane feeding experiments to prepare for larger studies to quantify the transmission potential and relative contribution of the human infectious reservoir. METHODS: Patients with clinical malaria attending four health facilities with at least 16 Plasmodium falciparum gametocytes per μL were recruited during the 2018 transmission season. Infectiousness to mosquitoes was assessed by direct membrane feeding assay (DMFA). We compared our results with a Bayesian predictive model to investigate the relationship between infectiousness and gametocyte density and explore the impact of fever on gametocyte infectivity. RESULTS: A total of 3177 suspected malaria cases were screened; 43.3% (1376) had microscopically patent P. falciparum parasites and 3.6% (114) of them had gametocytes. Out of 68 DMFAs, 38 (55.9%) resulted in at least one infected mosquito, with a total of 15.4% (1178/7667) of mosquitoes infected with 1-475 oocysts per gut. The relationship between mosquito infection prevalence and gametocytaemia was similar to other African settings and negatively associated with fever (OR: 0.188, 95% CI 0.0603 to 0.585, p=0.0039). CONCLUSIONS: Among symptomatic malaria patients, fever is strongly associated with transmission failure. Future studies can use DMFA to better understand the human malaria reservoir in settings of low endemicity in The Gambia and inform malaria elimination initiatives

Effect of 4 years of seasonal malaria chemoprevention on the acquisition of antibodies to Plasmodium falciparum antigens in Ouelessebougou, Mali.

BACKGROUND: More than 200 million people live in areas of highly seasonal malaria transmission where Seasonal Malaria Chemoprevention (SMC) with sulfadoxine-pyrimethamine (SP) and amodiaquine (AQ) was recommended in 2012 by WHO. This strategy is now implemented widely and protected more than 19 million children in 2018. It was previously reported that exposure to SMC reduced antibody levels to AMA1, MSP-142 and CSP, but the duration of exposure to SMC up to three 3 years, had no effect on antibody levels to MSP-142 and CSP. METHODS: In 2017, a cross-sectional survey was carried out 1 month after the last dose of SMC had been given to children aged 4-5 years randomly selected from areas where SMC had been given for 2 or 4 years during the malaria transmission season. A total of 461 children were enrolled, 242 children in areas where SMC had been implemented for 4 years and 219 children in areas where SMC had been implemented for 2 years. Antibody extracted from dry blood spots was used to measure IgG levels to the malaria antigens CSP, MSP-142 and AMA1 by ELISA. RESULTS: The prevalence of antibodies to MSP-142 was similar in children who had received SMC for 4 years compared to those who had received SMC for only 2 years (85.1 vs 86.0%, ajusted odd ratio (aOR) = 1.06, 95% confidence intervals (CI 0.62-1.80), p = 0.80). The prevalence of antibodies to AMA-1 and to CSP was not lower in children who received SMC for 4 years compared to those who had received SMC for only 2 years (95.3 vs 88.8%, aOR = 3.16, 95% CI 1.44-6.95, p = 0.004 for AMA-1; and 91.2 vs 81.9%, aOR = 3.14, 95% CI 1.70-5.76, p < 0.001 for CSP). Median antibody levels for anti-MSP-142 IgG were not significatively inferior in children who had received SMC for four rather than 2 years (0.88 (IQR: 0.64-1.15) and 0.95 ((0.68-1.15), respectively), anti-CSP (1.30 (1.00-1.56) and 1.17 (0.87-1.47)), and anti-AMA-1 (1.45 (1.24-1.68) and 1.41 (1.17-1.64)). CONCLUSION: In an area of high seasonal malaria transmission, children who had received SMC for 4 years did not had lower seropositivity or antibody levels to AMA1, MSP-142 and CSP compared to children who had received SMC for only 2 years suggesting that children who have received SMC for 4 years may not be more at risk of malaria after the cessation of SMC than children who have received SMC for a shorter period

Primaquine to reduce transmission of Plasmodium falciparum malaria in Mali : a single-blind, dose-ranging, adaptive randomised phase 2 trial

Background Single low doses of primaquine, when added to artemisinin-based combination therapy, might prevent transmission of Plasmodium falciparum malaria to mosquitoes. We aimed to establish the activity and safety of four low doses of primaquine combined with dihydroartemisinin-piperaquine in male patients in Mali. Methods In this phase 2, single-blind, dose-ranging, adaptive randomised trial, we enrolled boys and men with uncomplicated P falciparum malaria at the Malaria Research and Training Centre (MRTC) field site in Ouelessebougou, Mali. All participants were confirmed positive carriers of gametocytes through microscopy and had normal function of glucose-6-phosphate dehydrogenase (G6PD) on colorimetric quantification In the first phase, participants were randomly assigned (1:1:1) to one of three primaquine doses: 0 mg/kg (control), 0.125 mg/kg, and 0.5 mg/kg. Randomisation was done with a computer-generated randomisation list (in block sizes of six) and concealed with sealed, opaque envelopes. In the second phase, different participants were sequentially assigned (1:1) to 0.25 mg/kg primaquine or 0.0625 mg/kg primaquine. Primaquine tablets were dissolved into a solution and administered orally in a single dose. Participants were also given a 3 day course of dihydroartemisinin-piperaquine, administered by weight (320 mg dihydroartemisinin and 40 mg piperaquine per tablet). Outcome assessors were masked to treatment allocation, but participants were permitted to find out group assignment. Infectivity was assessed through membrane feeding assays, which were optimised through the beginning part of phase one. The primary efficacy endpoint was the mean within-person percentage change in mosquito infectivity 2 days after primaquine treatment in participants who completed the study after optimisation of the infectivity assay, had both a pre-treatment infectivity measurement and at least one follow-up infectivity measurement, and who were given the correct primaquine dose. The safety endpoint was the mean within-person change in haemoglobin concentration during 28 days of study follow-up in participants with at least one follow-up visit. This study is registered with ClinicalTrials.gov, number NCT01743820. Findings Between Jan 2,2013, and Nov 27,2014, we enrolled 81 participants. In the primary analysis sample (n=71), participants in the 0.25 mg/kg primaquine dose group (n=15) and 0.5 mg/kg primaquine dose group (n=14) had significantly lower mean within-person reductions in infectivity at day 2-92.6% (95% CI 78.3-100; p=0.0014) for the 0.25 mg/kg group; and 75.0% (45.7-100; p=0.014) for the 0.5 mg/kg primaquine group compared with those in the control group (n=14; 11.3% [-27.4 to 50.0]). Reductions were not significantly different from control for participants assigned to the 0.0625 mg/kg dose group (n=16; 41.9% [1.4-82.5]; p=0.16) and the 0.125 mg/kg dose group (n=12; 54.9% [13.4-96.3]; p=0.096). No clinically meaningful or statistically significant drops in haemoglobin were recorded in any individual in the haemoglobin analysis (n=70) during follow-up. No serious adverse events were reported and adverse events did not differ between treatment groups. Interpretation A single dose of 0.25 mg/kg primaquine, given alongside dihydroartemisinin-piperaquine, was safe and efficacious for the prevention of P falciparum malaria transmission in boys and men who are not deficient in G6PD. Future studies should assess the safety of single-dose primaquine in G6PD-deficient individuals to define the therapeutic range of primaquine to enable the safe roll-out of community interventions with primaquine.Peer reviewe

Comparison of molecular quantification of Plasmodium falciparum gametocytes by Pfs25 qRT-PCR and QT-NASBA in relation to mosquito infectivity.

BACKGROUND: Quantifying gametocyte densities in natural malaria infections is important to estimate malaria transmission potential. Two molecular methods (Pfs25 mRNA quantitative reverse transcriptase PCR (qRT-PCR) and Pfs25 mRNA quantitative nucleic acid sequence based amplification (QT-NASBA)) are commonly used to determine gametocyte densities in clinical and epidemiological studies and allow gametocyte detection at densities below the microscopic threshold for detection. Here, reproducibility of these measurements and the association between estimated gametocyte densities and mosquito infection rates were compared. METHODS: To quantify intra- and inter-assay variation of QT-NASBA and qRT-PCR, a series of experiments was performed using culture-derived mature Plasmodium falciparum gametocytes from three different parasite isolates (NF54, NF135, NF166). Pfs25 mRNA levels were also determined in samples from clinical trials in Mali and Burkina Faso using both methods. Agreement between the two methods and association with mosquito infection rates in membrane feeding assays were assessed. RESULTS: Intra- and inter-assay variability was larger in QT-NASBA compared to qRT-PCR, particularly at low gametocyte densities (100 gametocyte per ?L). Samples collected in one of the two transmission studies had extremely low gametocyte densities by both molecular methods, which is suggestive of RNA degradation due to an unknown number of freeze-thaw cycles and illustrates the reliance of molecular gametocyte diagnostics on a reliable cold-chain. CONCLUSIONS: The experiments indicate that both qRT-PCR and QT-NASBA are of value for quantifying mature gametocytes in samples collected in field studies. For both assays, estimated gametocyte densities correlated well with mosquito infection rates. QT-NASBA is less reproducible than qRT-PCR, particularly for low gametocyte densities

Efficacy and safety of primaquine and methylene blue for prevention of Plasmodium falciparum transmission in Mali: a phase 2, single-blind, randomised controlled trial.

BACKGROUND: Primaquine and methylene blue are gametocytocidal compounds that could prevent Plasmodium falciparum transmission to mosquitoes. We aimed to assess the efficacy and safety of primaquine and methylene blue in preventing human to mosquito transmission of P falciparum among glucose-6-phosphate dehydrogenase (G6PD)-normal, gametocytaemic male participants. METHODS: This was a phase 2, single-blind, randomised controlled trial done at the Clinical Research Centre of the Malaria Research and Training Centre (MRTC) of the University of Bamako (Bamako, Mali). We enrolled male participants aged 5-50 years with asymptomatic P falciparum malaria. G6PD-normal participants with gametocytes detected by blood smear were randomised 1:1:1:1 in block sizes of eight, using a sealed-envelope design, to receive either sulfadoxine-pyrimethamine and amodiaquine, sulfadoxine-pyrimethamine and amodiaquine plus a single dose of 0·25 mg/kg primaquine, dihydroartemisinin-piperaquine, or dihydroartemisinin-piperaquine plus 15 mg/kg per day methylene blue for 3 days. Laboratory staff, investigators, and insectary technicians were masked to the treatment group and gametocyte density of study participants. The study pharmacist and treating physician were not masked. Participants could request unmasking. The primary efficacy endpoint, analysed in all infected patients with at least one infectivity measure before and after treatment, was median within-person percentage change in mosquito infectivity 2 and 7 days after treatment, assessed by membrane feeding. This study is registered with ClinicalTrials.gov, number NCT02831023. FINDINGS: Between June 27, 2016, and Nov 1, 2016, 80 participants were enrolled and assigned to the sulfadoxine-pyrimethamine and amodiaquine (n=20), sulfadoxine-pyrimethamine and amodiaquine plus primaquine (n=20), dihydroartemisinin-piperaquine (n=20), or dihydroartemisinin-piperaquine plus methylene blue (n=20) groups. Among participants infectious at baseline (54 [68%] of 80), those in the sulfadoxine-pyrimethamine and amodiaquine plus primaquine group (n=19) had a median 100% (IQR 100 to 100) within-person reduction in mosquito infectivity on day 2, a larger reduction than was noted with sulfadoxine-pyrimethamine and amodiaquine alone (n=12; -10·2%, IQR -143·9 to 56·6; p<0·0001). The dihydroartemisinin-piperaquine plus methylene blue (n=11) group had a median 100% (IQR 100 to 100) within-person reduction in mosquito infectivity on day 2, a larger reduction than was noted with dihydroartemisinin-piperaquine alone (n=12; -6·0%, IQR -126·1 to 86·9; p<0·0001). Haemoglobin changes were similar between gametocytocidal arms and their respective controls. After exclusion of blue urine, adverse events were similar across all groups (59 [74%] of 80 participants had 162 adverse events overall, 145 [90%] of which were mild). INTERPRETATION: Adding a single dose of 0·25 mg/kg primaquine to sulfadoxine-pyrimethamine and amodiaquine or 3 days of 15 mg/kg per day methylene blue to dihydroartemisinin-piperaquine was highly efficacious for preventing P falciparum transmission. Both primaquine and methylene blue were well tolerated. FUNDING: Bill & Melinda Gates Foundation, European Research Council

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

Mass Drug Administration With High-Dose Ivermectin and Dihydroartemisinin-Piperaquine for Malaria Elimination in an Area of Low Transmission With High Coverage of Malaria Control Interventions: Protocol for the MASSIV Cluster Randomized Clinical Trial

Background: With a decline in malaria burden, innovative interventions and tools are required to reduce malaria transmission further. Mass drug administration (MDA) of artemisinin-based combination therapy (ACT) has been identified as a potential tool to further reduce malaria transmission, where coverage of vector control interventions is already high. However, the impact is limited in time. Combining an ACT with an endectocide treatment that is able to reduce vector survival, such as ivermectin (IVM), could increase the impact of MDA and offer a new tool to reduce malaria transmission. Objective: The study objective is to evaluate the impact of MDA with IVM plus dihydroartemisinin-piperaquine (DP) on malaria transmission in an area with high coverage of malaria control interventions. Methods: The study is a cluster randomized trial in the Upper River Region of The Gambia and included 32 villages (16 control and 16 intervention). A buffer zone of ~2 km was created around all intervention clusters. MDA with IVM plus DP was implemented in all intervention villages and the buffer zones; control villages received standard malaria interventions according to the Gambian National Malaria Control Program plans. Results: The MDA campaigns were carried out from August to October 2018 for the first year and from July to September 2019 for the second year. Statistical analysis will commence once the database is completed, cleaned, and locked. Conclusions: This is the first cluster randomized clinical trial of MDA with IVM plus DP. The results will provide evidence on the impact of MDA with IVM plus DP on malaria transmission. Trial Registration: ClinicalTrials.gov NCT03576313; https://clinicaltrials.gov/ct2/show/NCT03576313 International Registered Report Identifier (IRRID): DERR1-10.2196/2090

Maintaining Plasmodium falciparum gametocyte infectivity during blood collection and transport for mosquito feeding assays in the field.

BACKGROUND: Mosquito feeding assays using venous blood are commonly used for evaluating the transmission potential of malaria infected individuals. To improve the accuracy of these assays, care must be taken to prevent premature activation or inactivation of gametocytes before they are fed to mosquitoes. This can be challenging in the field where infected individuals and insectary facilities are sometimes very far apart. In this study, a simple, reliable, field applicable method is presented for storage and transport of gametocyte infected blood using a thermos flask. METHODS: The optimal storage conditions for maintaining the transmissibility of gametocytes were determined initially using cultured Plasmodium falciparum gametocytes in standard membrane feeding assays (SMFAs). The impact of both the internal thermos water temperature (35.5 to 37.8 °C), and the external environmental temperature (room temperature to 42 °C) during long-term (4 h) storage, and the impact of short-term (15 min) temperature changes (room temp to 40 °C) during membrane feeding assays was assessed. The optimal conditions were then evaluated in direct membrane feeding assays (DMFAs) in Burkina Faso and The Gambia where blood from naturally-infected gametocyte carriers was offered to mosquitoes immediately and after storage in thermos flasks. RESULTS: Using cultured gametocytes in SMFAs it was determined that an internal thermos water temperature of 35.5 °C and storage of the thermos flask between RT (~ 21.3 °C) and 32 °C was optimal for maintaining transmissibility of gametocytes for 4 h. Short-term storage of the gametocyte infected blood for 15 min at temperatures up to 40 °C (range: RT, 30 °C, 38 °C and 40 °C) did not negatively affect gametocyte infectivity. Using samples from natural gametocyte carriers (47 from Burkina Faso and 16 from The Gambia), the prevalence of infected mosquitoes and the intensity of oocyst infection was maintained when gametocyte infected blood was stored in a thermos flask in water at 35.5 °C for up to 4 h. CONCLUSIONS: This study determines the optimal long-term (4 h) storage temperature for gametocyte infected blood and the external environment temperature range within which gametocyte infectivity is unaffected. This will improve the accuracy, reproducibility, and utility of DMFAs in the field, and permit reliable comparative assessments of malaria transmission epidemiology in different settings