Objectives For the purpose of blocking transmission of Plasmodium falciparum malaria from humans to mosquitoes, a single dose of primaquine is recommended by the WHO as an addition to artemisinin combination therapy. Primaquine clears gametocytes but causes dose-dependent haemolysis in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Evidence is needed to inform the optimal dosing of primaquine for malaria elimination programmes and for the purpose of interrupting the spread of artemisinin-resistant malaria. This study investigates the efficacy and safety of reducing doses of primaquine for clearance of gametocytes in participants with normal G6PD status.
Methods and analysis In this prospective, four-armed randomised placebo-controlled double-blinded trial, children aged 1–10 years, weighing over 10 kg, with haemoglobin ≥8 g/dl and uncomplicated P falciparum malaria are treated with artemether lumefantrine and randomised to receive a dose of primaquine (0.1, 0.4 or 0.75 mg base/kg) or placebo on the third day of treatment. Participants are followed up for 28 days. Gametocytaemia is measured by quantitative nucleic acid sequence-based analysis on days 0, 2, 3, 7, 10 and 14 with a primary endpoint of the number of days to gametocyte clearance in each treatment arm and secondarily the area under the curve of gametocyte density over time. Analysis is for non-inferiority of efficacy compared to the reference dose, 0.75 mg base/kg. Safety is assessed by pair-wise comparisons of the arithmetic mean (±SD) change in haemoglobin concentration per treatment arm and analysed for superiority to placebo and incidence of adverse events. Ethics and dissemination Approval was obtained from the ethical committees of Makerere University School of Medicine, the Ugandan National Council of Science and Technology and the London School of Hygiene and Tropical Medicine.
Results These will be disseminated to inform malaria elimination policy, through peer-reviewed publication and academic presentations.
- Infectious diseases
- Public health
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Single-dose primaquine, administered together with artemisinin combination therapy, blocks transmission of Plasmodium falciparum malaria by clearing gametocytes.
Primaquine, an 8-aminoquinoline, causes dose-dependent haemolysis in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Evidence is lacking on the safety and efficacy of lower doses of primaquine.
This is the protocol of a dose-finding trial being conducted in eastern Uganda.
Dose-finding is a priority for the use of primaquine in malaria elimination programmes and to block the spread of artemisinin-resistant malaria.
This trial is designed to investigate the efficacy and safety of reducing doses of primaquine for gametocytocidal action.
This paper highlights the unique trial design issues that are relevant for investigating the efficacy and safety of antimalarials targeted against the sexual stages of malaria for blocking transmission rather than clinical cure.
Strengths and limitations of this study
For ethical reasons, in this trial, dose-finding is conducted in children with normal G6PD status, but, ultimately, information is needed on the safety of lower doses in people with G6PD deficiency.
This trial measures primaquine's transmission-blocking potential by assessing gametocyte clearance. Endpoints of mosquito transmission at multiple time points could be usefully assessed but on smaller numbers of individuals.
Sustained deployment of vector control measures and accessible, effective drug therapy has reduced the transmission of Plasmodium falciparum in many endemic countries. However, further scaling-up of currently available malaria control measures is unlikely to achieve malaria elimination in most settings.1 Moreover, the emergence of resistance to artemisinin in Southeast Asia,2 ,3 and the development of insecticide resistance and adaptive behaviour in the mosquito vector4––6 present significant threats to the current trend of declining malaria burden. Malaria elimination initiatives and artemisinin-resistance containment strategies both require additional tools that are specifically aimed at reducing the transmission of malarial parasites.7 ,8
Antimalarial drugs are designed primarily to target the asexual stages of the parasite that cause morbidity and mortality. The effect of antimalarial drugs on gametocytes, the transmission stages, has for decades been seen as ancillary. P falciparum gametocytes undergo complex development that is characterised by five morphologically distinct stages of maturation.9 The immature gametocyte stages (I–IV) are sequestered in the reticuloendothelial system and bone marrow.10––12 Mature stage V gametocytes typically appear approximately 12 days after the onset of patent asexual bloodstream infection, and are the only gametocyte stage that circulates in the peripheral blood and is infective to biting female Anopheles mosquitoes.13 ,14 The majority of antimalarial drugs, including artemisinins, lumefantrine and piperaquine, have some efficacy against immature gametocytes.15 ,16 These drugs have the potential to reduce transmission at a population level because asexual parasites are cleared, preventing de novo development of gametocytes, and fewer of the immature gametocytes that are present upon initiation of treatment survive to maturity. However, the vast majority of symptomatic cases have measurable and transmissible levels of mature gametocytes at presentation.17 ,18 These persist after treatment with all antimalarials that are currently implemented as first-line treatment, including artemisinin combination therapy (ACT). Gametocytes that persist after ACT have repeatedly been shown to be infectious to mosquitoes.17 ,19 ,20 This post-treatment gametocyte carriage frequently occurs at low densities, commonly below the microscopic threshold for detection,21 ,22 but is sufficiently high for efficient mosquito infection.17 ,23
The only class of drugs that are effective against mature P falciparum gametocytes is the 8-aminoquinolines. Primaquine is the most widely available drug in this class. The exact mechanism for this gametocytocidal activity is unknown, but it is probably dependent on oxidative damage to the intraerythrocytic parasite by primaquine metabolites.24 Primaquine as a single dose of 0.75 mg base/kg added to standard ACT has superior gametocytocidal activity to ACT alone.25––27 All doses of primaquine described hereafter refer to the dose of primaquine base per unit weight. There are indications that doses of primaquine lower than 0.75 mg/kg may be equally efficacious. A Thai study showed that both 0.5 and 0.25 mg/kg of primaquine administered with ACT to adults infected with malaria effectively and indistinguishably reduced the proportion of mosquitoes that became infected after a blood meal.28 In small numbers of adults, total doses of 30 mg and 15 mg have shown comparable efficacy to a 45 mg dose in reducing mosquito infection rates.29 ,30
The efficacy of primaquine when given as a single low dose is important in the light of concerns over the haematological safety of primaquine. There is conclusive evidence for primaquine-induced haemolysis in glucose-6-phosphate dehydrogenase (G6PD) deficient individuals.31 ,32 G6PD deficient individuals are vulnerable to oxidative stress because their erythrocytes do not have alternative pathways for G6PD-dependent nicotinamide adenine dinucleotide phosphate production, which is essential to maintain antioxidant defences. There is conflicting evidence on the risk of haemolysis after a single dose of primaquine. A single dose of 45 mg primaquine administered to a Vanuatan adult caused life-threatening haemolysis.33 In G6PD-deficient Tanzanian children, the mean fall in haemoglobin after a single dose of 0.75 mg/kg primaquine was 2.5 g/dl (95% CI 1.2 to 3.8 g/dl), though no associated severe adverse events were recorded and haemolysis was transient.34 On the other hand, primaquine was reported to be well tolerated when 0.75 mg/kg was given without prior G6PD testing in large studies in Myanmar, Sudan, Russia, Cambodia and China.27 ,31 ,35 ,36
Because primaquine-induced haemolysis is dose-dependent,29 and because gametocytocidal efficacy may be retained with primaquine doses lower than 0.75 mg/kg, the WHO-recommended dose in its 2010 Guidelines for the Treatment of Malaria, dose-finding studies are needed urgently. This trial tests the hypothesis that lower doses of primaquine have a substantially lower risk of, or an absence of, adverse effects but that their gametocytocidal efficacy is retained.
Methods and analysis
The study is a prospective, randomised, parallel arm, placebo-controlled, double-blinded clinical trial of reducing doses of primaquine administered with artemether lumefantrine (AL) for the treatment of uncomplicated clinical P falciparum malaria infection in children aged 1–10 years of age. The study uses a non-inferiority design to evaluate the efficacy and a superiority design to evaluate the safety of 0.1 and 0.4 mg/kg primaquine compared with 0.75 mg/kg when added to AL.
To evaluate the efficacy of 0.1, 0.4 and 0.75 mg/kg primaquine when administered together with the fifth dose of AL as measured by gametocyte prevalence and density.
To evaluate the safety of 0.1, 0.4 and 0.75 mg/kg primaquine when administered together with the fifth dose of AL as measured by change in mean haemoglobin, prevalence of severe anaemia (haemoglobin <5 g/dl) and evidence of black urine (haemoglobinuria).
To assess the safety of different doses of 0.1, 0.4 and 0.75 mg/kg primaquine when administered together with the fifth dose of AL as measured by prevalence/incidence of adverse events and tolerability.
Participants and enrolment
The study is conducted at Walukuba Health Centre IV in Walukuba subcounty, Jinja district, in eastern Uganda. In this area, malaria transmission is year-round with two seasonal peaks. The entomological inoculation rate (EIR) was estimated at 7 infectious bites per person per year in Walukuba.37 Study participants are recruited from children attending the Health Centre IV with suspected malaria (figure 1). Inclusion criteria are age 1–10 years, weight over 10 kg, fever (tympanic temperature >38°C) or history of fever in the last 24 h, P falciparum mono-infection with a parasite density <5 00 000/µl and normal G6PD enzyme function. Exclusion criteria are evidence of severe illness/danger signs, known allergy to study medications, haemoglobin <8 g/dl, started menstruation, pregnancy or breastfeeding, antimalarials taken within the last 2 days, primaquine taken within the last 4 weeks and blood transfusion within the last 90 days.
The fluorescent spot test38 is used for G6PD screening. This test has a cut-off of approximately 20% enzyme function, below that, there is no fluorescence. The WHO classification defines severe G6PD deficiency as 10% enzyme function.39
Randomisation, blinding and intervention
After enrolment (day 0), participants are randomised to a treatment arm stratified by gender (figure 2). The study pharmacist selects sequential opaque envelopes (from either the male or the female pile). Each envelope contains a predetermined treatment assignment code. The study pharmacist is the only member of the clinic team not blinded to the treatment arm and is not involved in assessing patients or assigning outcomes. All study site staff who administer drugs, assess patients and process laboratory samples do not have access to the randomisation code breaker.
All participants receive a 3 day course of artemether lumefantrine according to Ugandan national treatment guidelines for uncomplicated malaria. Participants are randomised to receive a placebo or a dose of 0.1, 0.4 or 0.75 mg/kg primaquine in addition to the AL treatment. The dose of primaquine/placebo is given at the same time as the fifth dose of AL, in the morning of day 2. To preserve the accuracy of lower weight-based doses, all primaquine doses are administered in aqueous solution and measured using a sterile syringe. The placebo is aqueous solution alone. All doses including placebo are mixed with glucose-based syrup that masks the colour and taste of primaquine. All treatments are directly observed. A snack with approximately 5 g of fat is administered prior to both AL and primaquine administration to optimise absorption of AL and minimise gastrointestinal side effects with primaquine. Participants are observed for 30 minutes; treatment is readministered in any case of vomiting within this period. Repeated vomiting (>3 times) leads to exclusion from the study and treatment as complicated malaria according to national guidelines.
Study participants are reviewed on days 0, 1, 2, 3, 7, 10, 14, 21 and 28 after enrolment or on any day of illness. On each of the scheduled visit days they are assessed clinically with standardised adverse event recording and blood samples are taken for microscopical detection of asexual parasites and gametocytes, molecular detection of gametocytes and haemoglobin measurements (table 1).
Blood smears from all visits are Giemsa-stained and 100 microscopic fields are screened for asexual parasites on days 0, 1, 2, 3, 7, 10, 14, 21 and 28. Asexual parasites are counted against 200 white blood cells (WBC) or, if fewer than 10 parasites are observed per 200 WBC, against 500 WBC. Gametocytes are recorded if observed during this screening process. On day 0, 100 microscopic fields are reread for gametocytes specifically. If gametocytes are observed, they are quantified against 500 WBC. All microscopy readings are performed by two independent microscopists, if they disagree on prevalence or if density results differ by more than 25%, a third reading is requested.
Gametocytes are quantified on days 0, 2, 3, 7, 10 and 14 using quantitative real-time nucleic acid sequence-based analysis (QT-NASBA), detecting and quantifying Pfs25 mRNA. One hundred microlitres of finger prick blood is mixed with 900 µl L6 guanidine buffer (Severn Biotech, UK) and stored at −80°C until automatic nucleic acid extraction by MagNAPure (Roche) using commercial high-yield kits. The Pfs25 QT-NASBA is specific for mature gametocytes with a sensitivity of 0.01–0.1 gametocytes/µl of blood when 50 µl blood samples are used for RNA extraction.40
Haemoglobin is measured on days 0, 1, 2, 3, 7, 10, 14, 21 and 28 using HemoCue 201+ photometers (HemoCue; Angelholm, Sweden). At each follow-up visit, study staff assess participants in an objective manner according to a clinical record form and assessment for adverse events is conducted in a prospective, systematic fashion during all visits, including the enrolment visit (eg, vomiting post-AL). All data are double-entered in real time.
A protocol was developed in order to standardise the detection, investigation and management of severe haemolysis in this trial (figures 3 and 4). A Data Safety Monitoring Board (DSMB) has been installed; clinically relevant haemolytic events, hospital admissions, blood transfusions and deaths are reported within 72 h to this DSMB.
The study protocol and informed consent forms were approved by the Makerere University School of Medicine Research Ethics Committee (protocol 2011–210), the Uganda National Council of Science and Technology (protocol HS1056) and the London School of Hygiene and Tropical Medicine research ethics committee (protocol 5987). The Ugandan National Drug Authority approved the protocol and importation of primaquine for the purposes of the study. The DSMB and Trial Advisory Committee for the study agreed to meet at predetermined stages of the study. Before the study began, local community stakeholders (including village health team and local council members) in Walukuba were consulted and a community advisory board meeting was held.
For efficacy, the sample size calculation is based on non-inferiority of each of the two test dose arms to the comparator arm, the WHO-recommended dose of primaquine, 0.75 mg/kg. The primary outcome measure is number of days to gametocyte clearance. The addition of primaquine (0.75 mg/kg) to ACT in Tanzania reduced the time to gametocyte clearance from 28.6 to 6.3 days (SD 6 days).41 Allowing for a 10% loss to follow-up, a sample size of 120/arm will provide over 80% power at the 0.05 significance level to detect non-inferiority to the standard arm with a non-inferiority margin of 2.5 days, which was considered to be a clinically relevant reduction in gametocyte clearance time. This sample size also allows for an analysis of superiority of the efficacy of the two test dose arms to placebo.
For safety, the sample size calculation is based on superiority of each of the two test dose arms to the comparator arm (0.75 mg/kg). For this comparator arm, Shekalaghe et al34 found an overall mean absolute drop in haemoglobin by day 7 after treatment of 0.6 g/dl (SD 1.5). Therefore, with 80% power and at the 0.05 significance level, a sample size of 99 would be required to detect a difference in mean maximal drop in haemoglobin between treatment groups of 0.6 g/dl.
Data will be double entered in Microsoft Access and imported into Stata V.12.0 (Statacorp Ltd, Texas, USA). All efficacy analyses will be based on gametocyte detection by Pfs25 QT-NASBA. Gametocyte density on days 7, 10 and 14 will be compared with the comparator arm (0.75 mg primaquine/kg) by χ2 test. The mean duration of gametocyte carriage and 95% CI will be estimated in each treatment arm and compared with the comparator arm using a previously validated mathematical model.42 The area under the curve of gametocyte density over time will be calculated using the method described by Mendez et al43 For individuals who are gametocyte positive at enrolment, Kaplan-Meier survival analysis will be used to compare the decline in gametocyte prevalence.
The primary safety outcome, mean maximal fall in haemoglobin concentration during 28 days of follow-up will be assessed for each treatment arm. Pair-wise comparisons will be made between each of the treatment arms and compared with the comparator arm using unpaired t tests.
In the 2010 edition of the Guidelines for the Treatment of Malaria, the WHO recommends that a single dose of 0.75 mg/kg primaquine is added to ACT in malaria elimination programmes and for epidemic control, provided the risks of haemolysis in G6PD-deficient patients are considered. This guidance was recently updated to recommend a lower dose of 0.25 mg/kg primaquine without G6PD testing for new malaria elimination programmes and to prevent the spread of artemisinin resistance.31 The revision was based largely on grey literature and historical data rather than on recent clinical trials and few of the data are in the public domain.44 There have been no formal dose-finding studies using contemporary tools and standards for the measurement of drug efficacy and safety for the combination of ACTs and primaquine. In the current study, we aim to provide these urgently needed efficacy data and provide safety data for individuals with normal G6PD enzyme function.
Relatively few trials have been designed specifically to test gametocytocidal drugs in vivo. Standardised protocols and trial designs for assessing the efficacy of drugs targeted against asexual parasites45 ,46 are not suitable to assess gametocytocidal drugs, where the main outcome is transmission-blocking activity rather than clinical or parasitological cure. There is no agreement on the best tools to quantify gametocyte carriage. Many trials have used microscopy to measure gametocytes26––28 ,47 ,48 while it has been known for decades that microscopy is notoriously insensitive for detecting gametocytes.49 Gametocytes typically circulate at densities that are ≤1% of asexual parasite densities.16 ,50 Nevertheless, gametocytes are often simply recorded while screening for asexual parasites. If slides are specifically read for gametocytes, the number of microscopic fields that is screened is mostly the same as that for asexual parasites.51 As a consequence, gametocytes measured microscopically by routine underestimate the total gametocyte prevalence by up to 10-fold.16 ,17 ,21 ,22 In the current study, gametocytes are quantified with the most widely used quantitative molecular gametocyte detection method, QT-NASBA that has an estimated sensitivity of 0.01–0.1 gametocytes/µl blood in the blood sample taken.40 The use of this sensitive molecular method will increase the power of our efficacy estimates since up to 90% of symptomatic malaria patients may harbour (submicroscopic) gametocyte densities prior to the initiation of treatment.16
Gametocyte density is associated with the likelihood of mosquito infection and some of the lowest gametocyte densities may therefore be unlikely to result in mosquito infections. In general, there are limitations to which gametocyte prevalence or density can be used to predict mosquito infection rates. The fitness or infectivity of gametocytes is variable, especially after treatment.19 ,52 ,53 Very early studies demonstrated that primaquine may render gametocytes non-infectious several days before they are cleared from the circulation.30 ,54 ,55 The only approach to directly measure transmission-blocking potential involves assessing the infectiousness of the participant's blood to mosquitoes using the membrane feeding assay or direct skin feeding assays,56 the latter being described by early malariologists.57 ,58 However, the capacity for mosquito feeding assays is not widely available and repeated assessments of infectiousness on the same patients have never been performed as part of clinical trials. This is partly because of ethical concerns related to repeated venous bleeding in young children, and partly because of the complexity of mosquito husbandry when large numbers of mosquitoes are required for robust transmission estimates.59 In the absence of biomarkers, using the prevalence and density of gametocytes after treatment is the most pragmatic approach to assess the transmission-blocking efficacy of drugs across a variety of malaria endemic settings.
To assess the safety of the 8-aminoquinoline drugs, there must be a clear definition of the risk of haemolysis and how it should be measured.31 ,60 The safety profile may best be defined by the incidence of endpoints that could compromise health, such as signs of severe haemolysis, and the need for interventions such as haematinic drug administration, hospitalisation or blood transfusion. These events, however, are rare and changes in haemoglobin concentration may be a more sensitive primary safety outcome for standard clinical trials. In a recent Cochrane review of randomised controlled trials of primaquine's efficacy, only one trial25 was found to have measured the haemoglobin concentration to assess safety.61 In this current study, clinically relevant safety endpoints have been selected and a standardised procedure is in place for the investigation and management of severe haemolysis. A shortcoming of the current study is that safety data are most urgently needed in the most vulnerable group, G6PD-deficient individuals. For ethical reasons this group was excluded. The authors consider that the priority is first to determine the minimal effective dose in a G6PD normal population before G6PD-deficient individuals are exposed to this low dose of primaquine to assess safety.
The ultimate evidence for a beneficial role of primaquine in reducing malaria transmission would come from trials assessing the effect of the drug on measures of community-level transmission. Once a safe and efficacious dose of primaquine in combination with ACTs is established, the next step involves designing these community trials. Treatment of symptomatic cases could play an important role in reducing the spread of (resistant) malaria strains from symptomatic patients.62 However, because of the large pool of asymptomatic parasite carriers in all endemic settings63 and their importance in defining transmission potential, any effect of primaquine on community-wide transmission will be limited if administration is restricted to symptomatic cases. Other strategies such as pro-active screening and treatment and (focal) mass drug administration may have a larger impact in some settings.64 This trial forms the starting point for defining the optimal dose of primaquine for use in transmission-blocking interventions.
The authors would like to thank staff at the Infectious Diseases Research Collaboration (IDRC) in Uganda, notably, Mr Moses Kiggundu, Dr Catherine Maiteki-Sebuguzi, the members of the Programme for Resistance, Immunology, Surveillance and Modelling of Malaria in Uganda (PRISM) and the ACT PRIME and PROCESS research teams and also Ms Carolynne Stanley for logistical support at the London School of Hygiene and Tropical Medicine.
Contributors ACE, SGS, SY, NJW, TB and CD have conceived and designed the study and participated in logistical planning together with EW and MK. EW provided the statistical support for the sample size estimates and the design of the statistical analysis. TB provided the expertise for submicroscopic gametocyte measurement. ACE organised the ethical applications, community sensitisation and study implementation and wrote the manuscript together with TB and CD. All authors have read and approved the final manuscript.
Funding This study is funded by ACE's Clinical Fellowship from the Wellcome Trust of Great Britain (grant #090558/Z/09/Z).
Competing interests None.
Ethics approval Makerere University School of Medicine Research Ethics Committee (protocol 2011-210), the Uganda National Council of Science and Technology (protocol HS1056) and the London School of Hygiene and Tropical Medicine research ethics committee (protocol 5987).
Provenance and peer review Not commissioned; externally peer reviewed.
Trial status Recruitment began on 6 December 2011. The trial is going on .
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