Eziefula AC Staedke SG Yeung S Webb E Kamya M WhiteNJ Bousema T Drakeley C (2013) Study protocol for a randomisedcontrolled double-blinded trial of the dose-dependent efficacy andsafety of primaquine for clearance of gametocytes in children withuncomplicated falciparum malaria in Uganda BMJ Open 3 (3)ISSN 2044-6055 DOI 101136bmjopen-2013-002759

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Study protocol for a randomisedcontrolled double-blinded trial of thedose-dependent efficacy and safety ofprimaquine for clearance of gametocytesin children with uncomplicatedfalciparum malaria in Uganda

Alice Chijioke Eziefula1 Sarah G Staedke1 Shunmay Yeung1 Emily Webb2

Moses Kamya3 Nicholas J White4 Teun Bousema15 Chris Drakeley1

To cite Eziefula ACStaedke SG Yeung S et alStudy protocol for arandomised controlleddouble-blinded trial of thedose-dependent efficacy andsafety of primaquine forclearance of gametocytes inchildren with uncomplicatedfalciparum malaria in UgandaBMJ Open 20133e002759doi101136bmjopen-2013-002759

Prepublication history forthis paper are availableonline To view these filesplease visit the journal online(httpdxdoiorg101136bmjopen-2013-002759)

Received 19 February 2013Accepted 25 February 2013

This final article is availablefor use under the terms ofthe Creative CommonsAttribution Non-Commercial20 Licence seehttpbmjopenbmjcom

For numbered affiliations seeend of article

Correspondence toDr Alice C Eziefulachieziefulagmailcom

ABSTRACTObjectives For the purpose of blocking transmissionof Plasmodium falciparum malaria from humans tomosquitoes a single dose of primaquine isrecommended by the WHO as an addition to artemisinincombination therapy Primaquine clears gametocytes butcauses dose-dependent haemolysis in individuals withglucose-6-phosphate dehydrogenase (G6PD) deficiencyEvidence is needed to inform the optimal dosing ofprimaquine for malaria elimination programmes and forthe purpose of interrupting the spread of artemisinin-resistant malaria This study investigates the efficacy andsafety of reducing doses of primaquine for clearance ofgametocytes in participants with normal G6PD statusMethods and analysis In this prospective four-armed randomised placebo-controlled double-blindedtrial children aged 1ndash10 years weighing over 10 kg withhaemoglobin ge8 gdl and uncomplicated P falciparummalaria are treated with artemether lumefantrine andrandomised to receive a dose of primaquine (01 04 or075 mg basekg) or placebo on the third day oftreatment Participants are followed up for 28 daysGametocytaemia is measured by quantitative nucleic acidsequence-based analysis on days 0 2 3 7 10 and 14with a primary endpoint of the number of days togametocyte clearance in each treatment arm andsecondarily the area under the curve of gametocytedensity over time Analysis is for non-inferiority ofefficacy compared to the reference dose 075 mgbasekg Safety is assessed by pair-wise comparisonsof the arithmetic mean (plusmnSD) change in haemoglobinconcentration per treatment arm and analysed forsuperiority to placebo and incidence of adverse eventsEthics and dissemination Approval was obtained from theethical committees of Makerere University School ofMedicine the Ugandan National Council of Science andTechnology and the London School of Hygiene andTropical MedicineResults These will be disseminated to inform malariaelimination policy through peer-reviewed publication andacademic presentations

BACKGROUNDSustained deployment of vector control mea-sures and accessible effective drug therapy has

ARTICLE SUMMARY

Article focus Single-dose primaquine administered together

with artemisinin combination therapy blockstransmission of Plasmodium falciparum malariaby clearing gametocytes

Primaquine an 8-aminoquinoline causes dose-dependent haemolysis in individuals withglucose-6-phosphate dehydrogenase (G6PD)deficiency Evidence is lacking on the safetyand efficacy of lower doses of primaquine

This is the protocol of a dose-finding trial beingconducted in eastern Uganda

Key messages Dose-finding is a priority for the use of prima-

quine in malaria elimination programmes and toblock the spread of artemisinin-resistant malaria

This trial is designed to investigate the efficacyand safety of reducing doses of primaquine forgametocytocidal action

This paper highlights the unique trial designissues that are relevant for investigating the effi-cacy and safety of antimalarials targeted againstthe sexual stages of malaria for blocking trans-mission 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 statusbut ultimately information is needed on the safetyof lower doses in people with G6PD deficiency

This trial measures primaquinersquos transmission-blocking potential by assessing gametocyteclearance Endpoints of mosquito transmission atmultiple time points could be usefully assessedbut on smaller numbers of individuals

Eziefula AC Staedke SG Yeung S et al BMJ Open 20133e002759 doi101136bmjopen-2013-002759 1

Open Access Protocol

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reduced the transmission of Plasmodium falciparum in manyendemic countries However further scaling-up of cur-rently available malaria control measures is unlikely toachieve malaria elimination in most settings1 Moreoverthe emergence of resistance to artemisinin in SoutheastAsia2 3 and the development of insecticide resistance andadaptive behaviour in the mosquito vector4ndash6 present sig-nificant threats to the current trend of declining malariaburden Malaria elimination initiatives and artemisinin-resistance containment strategies both require additionaltools that are specifically aimed at reducing the transmis-sion of malarial parasites7 8

Antimalarial drugs are designed primarily to targetthe asexual stages of the parasite that cause morbidityand mortality The effect of antimalarial drugs on game-tocytes the transmission stages has for decades beenseen as ancillary P falciparum gametocytes undergocomplex development that is characterised by five mor-phologically distinct stages of maturation9 The imma-ture gametocyte stages (IndashIV) are sequestered in thereticuloendothelial system and bone marrow10ndash12

Mature stage V gametocytes typically appear approxi-mately 12 days after the onset of patent asexual blood-stream infection and are the only gametocyte stage thatcirculates in the peripheral blood and is infective tobiting female Anopheles mosquitoes13 14 The majority ofantimalarial drugs including artemisinins lumefantrineand piperaquine have some efficacy against immaturegametocytes15 16 These drugs have the potential toreduce transmission at a population level becauseasexual parasites are cleared preventing de novo devel-opment of gametocytes and fewer of the immaturegametocytes that are present upon initiation of treat-ment survive to maturity However the vast majority ofsymptomatic cases have measurable and transmissiblelevels of mature gametocytes at presentation17 18 Thesepersist after treatment with all antimalarials that are cur-rently implemented as first-line treatment includingartemisinin combination therapy (ACT) Gametocytesthat persist after ACT have repeatedly been shown to beinfectious to mosquitoes17 19 20 This post-treatmentgametocyte carriage frequently occurs at low densitiescommonly below the microscopic threshold for detec-tion21 22 but is sufficiently high for efficient mosquitoinfection17 23

The only class of drugs that are effective against matureP falciparum gametocytes is the 8-aminoquinolinesPrimaquine is the most widely available drug in this classThe exact mechanism for this gametocytocidal activity isunknown but it is probably dependent on oxidativedamage to the intraerythrocytic parasite by primaquinemetabolites24 Primaquine as a single dose of 075 mgbasekg added to standard ACT has superior gametocyto-cidal activity to ACT alone25ndash27 All doses of primaquinedescribed hereafter refer to the dose of primaquine baseper unit weight There are indications that doses ofprimaquine lower than 075 mgkg may be equally effica-cious A Thai study showed that both 05 and 025 mgkg

of primaquine administered with ACT to adults infectedwith malaria effectively and indistinguishably reduced theproportion of mosquitoes that became infected after ablood meal28 In small numbers of adults total doses of30 mg and 15 mg have shown comparable efficacy to a45 mg dose in reducing mosquito infection rates29 30

The efficacy of primaquine when given as a single lowdose is important in the light of concerns over thehaematological safety of primaquine There is conclusiveevidence for primaquine-induced haemolysis inglucose-6-phosphate dehydrogenase (G6PD) deficientindividuals31 32 G6PD deficient individuals are vulner-able to oxidative stress because their erythrocytes do nothave alternative pathways for G6PD-dependent nicotina-mide adenine dinucleotide phosphate productionwhich is essential to maintain antioxidant defencesThere is conflicting evidence on the risk of haemolysisafter a single dose of primaquine A single dose of45 mg primaquine administered to a Vanuatan adultcaused life-threatening haemolysis33 In G6PD-deficientTanzanian children the mean fall in haemoglobin aftera single dose of 075 mgkg primaquine was 25 gdl(95 CI 12 to 38 gdl) though no associated severeadverse events were recorded and haemolysis wastransient34 On the other hand primaquine wasreported to be well tolerated when 075 mgkg was givenwithout prior G6PD testing in large studies in MyanmarSudan Russia Cambodia and China27 31 35 36

Because primaquine-induced haemolysis is dose-dependent29 and because gametocytocidal efficacy maybe retained with primaquine doses lower than075 mgkg the WHO-recommended dose in its 2010Guidelines for the Treatment of Malaria dose-findingstudies are needed urgently This trial tests the hypoth-esis that lower doses of primaquine have a substantiallylower risk of or an absence of adverse effects but thattheir gametocytocidal efficacy is retained

METHODS AND ANALYSISStudy designThe study is a prospective randomised parallel armplacebo-controlled double-blinded clinical trial of redu-cing doses of primaquine administered with artemetherlumefantrine (AL) for the treatment of uncomplicatedclinical P falciparum malaria infection in children aged1ndash10 years of age The study uses a non-inferioritydesign to evaluate the efficacy and a superiority designto evaluate the safety of 01 and 04 mgkg primaquinecompared with 075 mgkg when added to AL

Study objectives1 To evaluate the efficacy of 01 04 and 075 mgkg

primaquine when administered together with thefifth dose of AL as measured by gametocyte preva-lence and density

2 To evaluate the safety of 01 04 and 075 mgkgprimaquine when administered together with the

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fifth dose of AL as measured by change in meanhaemoglobin prevalence of severe anaemia(haemoglobin lt5 gdl) and evidence of black urine(haemoglobinuria)

3 To assess the safety of different doses of 01 04 and075 mgkg primaquine when administered togetherwith the fifth dose of AL as measured by preva-lenceincidence of adverse events and tolerability

Participants and enrolmentThe study is conducted at Walukuba Health Centre IVin Walukuba subcounty Jinja district in eastern UgandaIn this area malaria transmission is year-round with twoseasonal peaks The entomological inoculation rate(EIR) was estimated at 7 infectious bites per person peryear in Walukuba37 Study participants are recruitedfrom children attending the Health Centre IV with sus-pected malaria (figure 1) Inclusion criteria are age1ndash10 years weight over 10 kg fever (tympanic tempera-ture gt38degC) or history of fever in the last 24 h P falcip-arum mono-infection with a parasite density lt5 00 000microl and normal G6PD enzyme function Exclusion cri-teria are evidence of severe illnessdanger signs knownallergy to study medications haemoglobin lt8 gdlstarted menstruation pregnancy or breastfeeding anti-malarials taken within the last 2 days primaquine takenwithin the last 4 weeks and blood transfusion within thelast 90 daysThe fluorescent spot test38 is used for G6PD screen-

ing This test has a cut-off of approximately 20 enzymefunction below that there is no fluorescence TheWHO classification defines severe G6PD deficiency as10 enzyme function39

Randomisation blinding and interventionAfter enrolment (day 0) participants are randomised toa treatment arm stratified by gender (figure 2) Thestudy pharmacist selects sequential opaque envelopes(from either the male or the female pile) Each enve-lope contains a predetermined treatment assignmentcode The study pharmacist is the only member of theclinic team not blinded to the treatment arm and is notinvolved in assessing patients or assigning outcomes Allstudy site staff who administer drugs assess patients andprocess laboratory samples do not have access to the ran-domisation code breakerAll participants receive a 3 day course of artemether

lumefantrine according to Ugandan national treatmentguidelines for uncomplicated malaria Participants are ran-domised to receive a placebo or a dose of 01 04 or075 mgkg primaquine in addition to the AL treatmentThe dose of primaquineplacebo is given at the same timeas the fifth dose of AL in the morning of day 2 To pre-serve the accuracy of lower weight-based doses all prima-quine doses are administered in aqueous solution andmeasured using a sterile syringe The placebo is aqueoussolution alone All doses including placebo are mixed withglucose-based syrup that masks the colour and taste ofprimaquine All treatments are directly observed A snackwith approximately 5 g of fat is administered prior to bothAL and primaquine administration to optimise absorptionof AL and minimise gastrointestinal side effects withprimaquine Participants are observed for 30 minutestreatment is readministered in any case of vomiting withinthis period Repeated vomiting (gt3 times) leads to exclu-sion from the study and treatment as complicated malariaaccording to national guidelines

Figure 1 Enrolment and

selection procedures

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Follow-up measurementsStudy participants are reviewed on days 0 1 2 3 7 10 1421 and 28 after enrolment or on any day of illness Oneach of the scheduled visit days they are assessed clinicallywith standardised adverse event recording and bloodsamples are taken for microscopical detection of asexualparasites and gametocytes molecular detection of gameto-cytes and haemoglobin measurements (table 1)Blood smears from all visits are Giemsa-stained and 100

microscopic fields are screened for asexual parasites ondays 0 1 2 3 7 10 14 21 and 28 Asexual parasites arecounted against 200 white blood cells (WBC) or if fewerthan 10 parasites are observed per 200 WBC against 500WBC Gametocytes are recorded if observed duringthis screening process On day 0 100 microscopic fieldsare reread for gametocytes specifically If gametocytes areobserved they are quantified against 500 WBC All micros-copy readings are performed by two independent micro-scopists if they disagree on prevalence or if density resultsdiffer by more than 25 a third reading is requestedGametocytes are quantified on days 0 2 3 7 10 and

14 using quantitative real-time nucleic acid sequence-based analysis (QT-NASBA) detecting and quantifyingPfs25 mRNA One hundred microlitres of finger prickblood is mixed with 900 microl L6 guanidine buffer (SevernBiotech UK) and stored at minus80degC until automaticnucleic acid extraction by MagNAPure (Roche) usingcommercial high-yield kits The Pfs25 QT-NASBA is spe-cific for mature gametocytes with a sensitivity of 001ndash01gametocytesmicrol of blood when 50 microl blood samples areused for RNA extraction40

Haemoglobin is measured on days 0 1 2 3 7 10 1421 and 28 using HemoCue 201+ photometers(HemoCue Angelholm Sweden) At each follow-up visitstudy staff assess participants in an objective manneraccording to a clinical record form and assessment for

adverse events is conducted in a prospective systematicfashion during all visits including the enrolment visit(eg vomiting post-AL) All data are double-entered inreal time

Safety considerationsA protocol was developed in order to standardise thedetection investigation and management of severe haem-olysis in this trial (figures 3 and 4) A Data SafetyMonitoring Board (DSMB) has been installed clinicallyrelevant haemolytic events hospital admissions bloodtransfusions and deaths are reported within 72 h to thisDSMB

Ethical considerationsThe study protocol and informed consent forms wereapproved by the Makerere University School ofMedicine Research Ethics Committee (protocol 2011ndash210) the Uganda National Council of Science andTechnology (protocol HS1056) and the London Schoolof Hygiene and Tropical Medicine research ethics com-mittee (protocol 5987) The Ugandan National DrugAuthority approved the protocol and importation ofprimaquine for the purposes of the study The DSMBand Trial Advisory Committee for the study agreed tomeet at predetermined stages of the study Before thestudy began local community stakeholders (includingvillage health team and local council members) inWalukuba were consulted and a community advisoryboard meeting was held

Sample sizeFor efficacy the sample size calculation is based onnon-inferiority of each of the two test dose arms to thecomparator arm the WHO-recommended dose ofprimaquine 075 mgkg The primary outcome

Figure 2 Participant flow

diagram

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measure is number of days to gametocyte clearanceThe addition of primaquine (075 mgkg) to ACT inTanzania reduced the time to gametocyte clearancefrom 286 to 63 days (SD 6 days)41 Allowing for a 10loss to follow-up a sample size of 120arm will provideover 80 power at the 005 significance level to detectnon-inferiority to the standard arm with a non-inferiority margin of 25 days which was considered tobe a clinically relevant reduction in gametocyte clear-ance time This sample size also allows for an analysisof superiority of the efficacy of the two test dose armsto placeboFor safety the sample size calculation is based on

superiority of each of the two test dose arms to the com-parator arm (075 mgkg) For this comparator armShekalaghe et al34 found an overall mean absolute dropin haemoglobin by day 7 after treatment of 06 gdl (SD15) Therefore with 80 power and at the 005 signifi-cance level a sample size of 99 would be required to

detect a difference in mean maximal drop in haemoglo-bin between treatment groups of 06 gdl

Data analysisData will be double entered in Microsoft Access andimported into Stata V120 (Statacorp Ltd Texas USA)All efficacy analyses will be based on gametocyte detec-tion by Pfs25 QT-NASBA Gametocyte density on days 710 and 14 will be compared with the comparator arm(075 mg primaquinekg) by χ2 test The mean durationof gametocyte carriage and 95 CI will be estimated ineach treatment arm and compared with the comparatorarm using a previously validated mathematical model42

The area under the curve of gametocyte density overtime will be calculated using the method described byMendez et al43 For individuals who are gametocyte posi-tive at enrolment Kaplan-Meier survival analysis will beused to compare the decline in gametocyte prevalence

Table 1 Summary of outcome measures

Outcome measure Description

Efficacy

Primary Mean number of days to gametocyte clearance

(GCT)

Mean number of days per treatment arm for

gametocytes to become undetectable using

submicroscopic molecular testing methods (QT-NASBA)

Reappearance of gametocytes after day 14 will be

considered as re-infection and excluded

Secondary Mean (plusmnSD) area under the curve of gametocyte

density per day during 14 days of follow-up

Total number of gametocytes (measured by QT-NASBA)

seen over follow-up averaged per day of follow-up (days

0ndash14)

Density of gametocytes on days 7 10 and 14 Mean number of gametocytes (measured by

QT-NASBA) per treatment arm on days 7 10 and 14

Proportion () of participants with gametocytes on

each day of follow-up

For each treatment arm percentage of participants with

gametocytes (measured by QT-NASBA) on each day of

follow-up from days 0ndash14

Safety

Primary Mean (plusmn SD) maximal fall (plusmn) in Hb (haemoglobin

gdl) from enrolment to day 28 of follow-up

Mean maximal greatest negative difference in Hb

(measured by HemoCue) from enrolment value per

treatment arm over 28 days follow-up

Secondary Follow-up day of Hb nadir Mean day of follow-up (day 0ndash28) per treatment arm of

lowest Hb measurement (by HemoCue)

Maximal percentage fall in Hb level compared to

enrolment value

Size of maximal Hb drop (by HemoCue) during follow-up

(day 0ndash28) from enrolment value divided by enrolment

value 100

Percentage of participants with Hblt5 gdl during

follow-up

Percentage (number) per treatment arm during days

0ndash28

Requirement for blood transfusion Percentage (number) of children receiving blood

transfusion per treatment arm during days 0ndash28

Evidence of black urine Percentage (number) of children with documented black

dark urine with urine dipstick positive for Hb per

treatment arm during days 0ndash28

Incidence of serious adverse events by sign

symptom laboratory parameter and relationship to

taking study drug

Percentage (number) per treatment arm during days

0ndash28

Incidence of gastrointestinal symptoms after taking

study drug

Percentage (number) per treatment arm during days 2ndash7

GCT gametocyte clearance time Hb haemoglobin QT-NASBA quantitative real-time nucleic acid sequence-based analysis

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The primary safety outcome mean maximal fall inhaemoglobin concentration during 28 days of follow-upwill be assessed for each treatment arm Pair-wise com-parisons will be made between each of the treatmentarms and compared with the comparator arm usingunpaired t tests

DISCUSSIONIn the 2010 edition of the Guidelines for theTreatment of Malaria the WHO recommends thata single dose of 075 mgkg primaquine is added toACT in malaria elimination programmes and forepidemic control provided the risks of haemolysis in

G6PD-deficient patients are considered This guidancewas recently updated to recommend a lower dose of025 mgkg primaquine without G6PD testing for newmalaria elimination programmes and to prevent thespread of artemisinin resistance31 The revision wasbased largely on grey literature and historical datarather than on recent clinical trials and few of the dataare in the public domain44 There have been no formaldose-finding studies using contemporary tools and stan-dards for the measurement of drug efficacy and safetyfor the combination of ACTs and primaquine In thecurrent study we aim to provide these urgently neededefficacy data and provide safety data for individuals withnormal G6PD enzyme function

Figure 4 Procedure for

management of haemolysis

Figure 3 Procedure for

investigation of suspected

haemolysis

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Relatively few trials have been designed specifically totest gametocytocidal drugs in vivo Standardised proto-cols and trial designs for assessing the efficacy of drugstargeted against asexual parasites45 46 are not suitable toassess gametocytocidal drugs where the main outcomeis transmission-blocking activity rather than clinical orparasitological cure There is no agreement on the besttools to quantify gametocyte carriage Many trials haveused microscopy to measure gametocytes26ndash28 47 48 whileit has been known for decades that microscopy is notori-ously insensitive for detecting gametocytes49

Gametocytes typically circulate at densities that are le1of asexual parasite densities16 50 Nevertheless gameto-cytes are often simply recorded while screening forasexual parasites If slides are specifically read for game-tocytes the number of microscopic fields that isscreened is mostly the same as that for asexual para-sites51 As a consequence gametocytes measured micro-scopically by routine underestimate the total gametocyteprevalence by up to 10-fold16 17 21 22 In the currentstudy gametocytes are quantified with the most widelyused quantitative molecular gametocyte detectionmethod QT-NASBA that has an estimated sensitivity of001ndash01 gametocytesmicrol blood in the blood sampletaken40 The use of this sensitive molecular method willincrease the power of our efficacy estimates since up to90 of symptomatic malaria patients may harbour (sub-microscopic) gametocyte densities prior to the initiationof treatment16

Gametocyte density is associated with the likelihood ofmosquito infection and some of the lowest gametocytedensities may therefore be unlikely to result in mosquitoinfections In general there are limitations to whichgametocyte prevalence or density can be used to predictmosquito infection rates The fitness or infectivity ofgametocytes is variable especially after treatment19 52 53

Very early studies demonstrated that primaquine mayrender gametocytes non-infectious several days beforethey are cleared from the circulation30 54 55 The onlyapproach to directly measure transmission-blockingpotential involves assessing the infectiousness of the par-ticipantrsquos blood to mosquitoes using the membranefeeding assay or direct skin feeding assays56 the latterbeing described by early malariologists57 58 Howeverthe capacity for mosquito feeding assays is not widelyavailable and repeated assessments of infectiousness onthe same patients have never been performed as part ofclinical trials This is partly because of ethical concernsrelated to repeated venous bleeding in young childrenand partly because of the complexity of mosquito hus-bandry when large numbers of mosquitoes are requiredfor robust transmission estimates59 In the absence ofbiomarkers using the prevalence and density of gameto-cytes after treatment is the most pragmatic approach toassess the transmission-blocking efficacy of drugs acrossa variety of malaria endemic settingsTo assess the safety of the 8-aminoquinoline drugs

there must be a clear definition of the risk of haemolysis

and how it should be measured31 60 The safety profilemay best be defined by the incidence of endpoints thatcould compromise health such as signs of severe haem-olysis and the need for interventions such as haematinicdrug administration hospitalisation or blood transfu-sion These events however are rare and changes inhaemoglobin concentration may be a more sensitiveprimary safety outcome for standard clinical trials In arecent Cochrane review of randomised controlled trialsof primaquinersquos efficacy only one trial25 was found tohave measured the haemoglobin concentration to assesssafety61 In this current study clinically relevant safetyendpoints have been selected and a standardised pro-cedure is in place for the investigation and managementof severe haemolysis A shortcoming of the current studyis that safety data are most urgently needed in the mostvulnerable group G6PD-deficient individuals Forethical reasons this group was excluded The authorsconsider that the priority is first to determine theminimal effective dose in a G6PD normal populationbefore G6PD-deficient individuals are exposed to thislow dose of primaquine to assess safetyThe ultimate evidence for a beneficial role of prima-

quine in reducing malaria transmission would comefrom trials assessing the effect of the drug on measuresof community-level transmission Once a safe and effica-cious dose of primaquine in combination with ACTs isestablished the next step involves designing these com-munity trials Treatment of symptomatic cases could playan important role in reducing the spread of (resistant)malaria strains from symptomatic patients62 Howeverbecause of the large pool of asymptomatic parasite car-riers in all endemic settings63 and their importance indefining transmission potential any effect of prima-quine on community-wide transmission will be limited ifadministration is restricted to symptomatic cases Otherstrategies such as pro-active screening and treatmentand (focal) mass drug administration may have a largerimpact in some settings64 This trial forms the startingpoint for defining the optimal dose of primaquine foruse in transmission-blocking interventions

Author affiliations1Faculty of Infectious and Tropical Diseases London School of Hygiene andTropical Medicine London UK2Faculty of Epidemiology and Population Health London School of Hygieneand Tropical Medicine London UK3Infectious Diseases Research Collaboration Kampala Uganda4Mahidol Oxford Tropical Medicine Research Unit (MORU) Faculty of TropicalMedicine Mahidol University Bangkok Thailand5Department of Medical Microbiology Radboud University Nijmegen MedicalCentre Nijmegen The Netherlands

Acknowledgements The authors would like to thank staff at the InfectiousDiseases Research Collaboration (IDRC) in Uganda notably Mr MosesKiggundu Dr Catherine Maiteki-Sebuguzi the members of the Programmefor Resistance Immunology Surveillance and Modelling of Malaria in Uganda(PRISM) and the ACT PRIME and PROCESS research teams and also MsCarolynne Stanley for logistical support at the London School of Hygiene andTropical Medicine

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Contributors ACE SGS SY NJW TB and CD have conceived and designedthe study and participated in logistical planning together with EW and MKEW provided the statistical support for the sample size estimates and thedesign of the statistical analysis TB provided the expertise forsubmicroscopic gametocyte measurement ACE organised the ethicalapplications community sensitisation and study implementation and wrotethe manuscript together with TB and CD All authors have read and approvedthe final manuscript

Funding This study is funded by ACErsquos Clinical Fellowship from the WellcomeTrust of Great Britain (grant 090558Z09Z)

Competing interests None

Ethics approval Makerere University School of Medicine Research EthicsCommittee (protocol 2011-210) the Uganda National Council of Science andTechnology (protocol HS1056) and the London School of Hygiene andTropical 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

REFERENCES1 Griffin JT Hollingsworth TD Okell LC et al Reducing Plasmodium

falciparum malaria transmission in Africa a model-based evaluationof intervention strategies PLoS Med 20107 doi101371journalpmed1000324 (published Online First Epub Date)

2 Dondorp AM Nosten F Yi P et al Artemisinin resistance inPlasmodium falciparum malaria N Engl J Med 2009361455ndash67

3 Amaratunga C Sreng S Suon S et al Artemisinin-resistantPlasmodium falciparum in Pursat province western Cambodia aparasite clearance rate study Lancet Infect Dis 201212851ndash8doi S1473-3099(12)70181-0 [pii] 101016S1473-3099(12)70181-0(published Online First Epub Date)

4 Stevenson J St Laurent B Lobo NF et al Novel vectors of malariaparasites in the Western highlands of Kenya Emerg Infect Dis2012181547ndash9

5 Riehle MM Guelbeogo WM Gneme A et al A cryptic subgroup ofAnopheles gambiae is highly susceptible to human malariaparasites Science 2011331596ndash8

6 Trung HD Bortel WV Sochantha T et al Behavioural heterogeneityof Anopheles species in ecologically different localities in SoutheastAsia a challenge for vector control Trop Med Int Health200510251ndash62

7 Dondorp AM Editorial commentary single-dose primaquine asgametocytocidal treatment in patients with uncomplicated falciparummalaria Clin Infect Dis 201356694ndash6

8 Alonso PL Brown G Arevalo-Herrera M et al A research agenda tounderpin malaria eradication PLoS Med 20118e1000406

9 Field JW Shute P The microscopic diagnosis of human malaria IIA morphological study of the erythrocytic parasites Studies from theInstitute for Medical Research Federated Malay States 1956

10 Smalley ME Abdalla S Brown J The distribution of Plasmodiumfalciparum in the peripheral blood and bone marrow of Gambianchildren Trans R Soc Trop Med Hyg 198175103ndash5

11 Rogers NJ Hall BS Obiero J et al A model for sequestration of thetransmission stages of Plasmodium falciparum adhesion ofgametocyte-infected erythrocytes to human bone marrow cellsInfect Immun 2000683455ndash62

12 Thomson JGRA The structure and development of Plasmodiumfalciparum gametocytes in the internal organs and peripheralcirculation Trans R Soc Trop Med Hyg 19352931ndash4

13 Smalley ME Sinden RE Plasmodium falciparum gametocytes theirlongevity and infectivity Parasitology 1977741ndash8

14 Lensen A Bril A van de Vegte M et al Plasmodium falciparuminfectivity of cultured synchronized gametocytes to mosquitoesExp Parasitol 199991101ndash3

15 Adjalley SH Johnston GL Li T et al Quantitative assessment ofPlasmodium falciparum sexual development reveals potenttransmission-blocking activity by methylene blue Proc Natl Acad SciUSA 2011108E1214ndash23

16 Bousema T Drakeley C Epidemiology and infectivity of Plasmodiumfalciparum and Plasmodium vivax gametocytes in relation to malariacontrol and elimination Clin Microbiol Rev 201124377ndash410

17 Bousema JT Schneider P Gouagna LC et al Moderate effect ofartemisinin-based combination therapy on transmission ofPlasmodium falciparum J Infect Dis 20061931151ndash9

18 Ali E Mackinnon MJ Abdel-Muhsin AM et al Increased density butnot prevalence of gametocytes following drug treatment ofPlasmodium falciparum Trans R Soc Trop Med Hyg2006100176ndash83

19 Targett G Drakeley C Jawara M et al Artesunate reduces but doesnot prevent posttreatment transmission of Plasmodium falciparum toAnopheles gambiae J Infect Dis 20011831254ndash9

20 Drakeley CJ Jawara M Targett GA et al Addition of artesunate tochloroquine for treatment of Plasmodium falciparum malaria inGambian children causes a significant but short-lived reduction ininfectiousness for mosquitoes Trop Med Int Health 2004953ndash61

21 Schneider P Bousema T Omar S et al (Sub)microscopicPlasmodium falciparum gametocytaemia in Kenyan children aftertreatment with sulphadoxine-pyrimethamine monotherapy or incombination with artesunate Int J Parasitol 200636403ndash8

22 Mens PF Sawa P van Amsterdam SM et al A randomized trial tomonitor the efficacy and effectiveness by QT-NASBA ofartemether-lumefantrine versus dihydroartemisinin-piperaquine fortreatment and transmission control of uncomplicated Plasmodiumfalciparum malaria in western Kenya Malar J 20087237

23 Ouedraogo AL Bousema T Schneider P et al Substantialcontribution of submicroscopical Plasmodium falciparum gametocytecarriage to the infectious reservoir in an area of seasonaltransmission PLoS ONE 20094e8410

24 Tekwani BL Walker LA 8-Aminoquinolines future role asantiprotozoal drugs Curr Opin Infect Dis 200619623ndash31

25 Shekalaghe S Drakeley C Gosling R et al Primaquine clearssubmicroscopic Plasmodium falciparum gametocytes that persistafter treatment with sulphadoxine-pyrimethamine and artesunatePLoS ONE 20072e1023

26 Sutanto I Suprijanto S Kosasih A et al The effect of primaquine ongametocyte development and clearance in the treatment ofuncomplicated falciparum malaria with dihydroartemisinin-piperaquine in South Sumatra Western Indonesia an open-labelrandomized controlled trial Clin Infect Dis 201256685ndash93doi101093cidcis959 [published Online First Epub Date]

27 Smithuis F Kyaw MK Phe O et al Effectiveness of five artemisinincombination regimens with or without primaquine in uncomplicatedfalciparum malaria an open-label randomised trial Lancet Infect Dis201010673ndash81

28 Pukrittayakamee S Chotivanich K Chantra A et al Activities ofartesunate and primaquine against asexual- and sexual-stageparasites in falciparum malaria Antimicrob Agents Chemother2004481329ndash34

29 Alving AS Johnson CF Tarlov AR et al Mitigation of thehaemolytic effect of primaquine and enhancement of its actionagainst exoerythrocytic forms of the Chesson strain of Piasmodiumvivax by intermittent regimens of drug administration a preliminaryreport Bull World Health Organ 196022621ndash31

30 Burgess RW Bray RS The effect of a single dose of primaquine onthe gametocytes gametogony and sporogony of Laveraniafalciparum Bull World Health Organ 196124451ndash6

31 White NJ Qiao LG Qi G et al Rationale for recommending a lowerdose of primaquine as a Plasmodium falciparum gametocytocide inpopulations where G6PD deficiency is common Malar J201211418

32 Charles LJ Observations on the haemolytic effect of primaquine in100 Ghanaian children Ann Trop Med Parasitol 196054460ndash70

33 Reeve PA Toaliu H Kaneko A et al Acute intravascularhaemolysis in Vanuatu following a single dose of primaquine inindividuals with glucose-6-phosphate dehydrogenase deficiencyJ Trop Med Hyg 199295349ndash51

34 Shekalaghe SA ter Braak R Daou M et al In Tanzania hemolysisafter a single dose of primaquine coadministered with an artemisininis not restricted to glucose-6-phosphate dehydrogenase-deficient(G6PD A-) individuals Antimicrob Agents Chemother2010541762ndash8

35 Song J Socheat D Tan B et al Rapid and effective malaria controlin Cambodia through mass administration of artemisinin-piperaquineMalar J 2010957

36 El-Sayed B El-Zaki SE Babiker H et al A randomized open-labeltrial of artesunate-sulfadoxine-pyrimethamine with or withoutprimaquine for elimination of sub-microscopic P falciparumparasitaemia and gametocyte carriage in eastern Sudan PLoS One20072e1311

37 Okello PE Van Bortel W Byaruhanga AM et al Variation in malariatransmission intensity in seven sites throughout Uganda Am J TropMed Hyg 200675219ndash25

38 Beutler E Mitchell M Special modifications of the fluorescentscreening method for glucose-6-phosphate dehydrogenasedeficiency Blood 196832816ndash18

8 Eziefula AC Staedke SG Yeung S et al BMJ Open 20133e002759 doi101136bmjopen-2013-002759

Dose-finding trial for single-dose primaquine to block malaria transmission

groupbmjcom on February 25 2014 - Published by bmjopenbmjcomDownloaded from

39 Beutler E Duparc S Group GPDW Glucose-6-phosphatedehydrogenase deficiency and antimalarial drug development Am JTrop Med Hyg 200777779ndash89

40 Schneider P Schoone G Schallig H et al Quantification ofPlasmodium falciparum gametocytes in differential stages ofdevelopment by quantitative nucleic acid sequence-basedamplification Mol Biochem Parasitol 200413735ndash41

41 Bousema T Okell L Shekalaghe S et al Revisiting the circulationtime of Plasmodium falciparum gametocytes molecular detectionmethods to estimate the duration of gametocyte carriage and theeffect of gametocytocidal drugs Malar J 20109136

42 Okell LC Drakeley CJ Bousema T et al Modelling the impact ofartemisinin combination therapy and long-acting treatments on malariatransmission intensity PLoS Med 20085e226 discussion e26

43 Mendez F Munoz A Plowe CV Use of area under the curve tocharacterize transmission potential after antimalarial treatment Am JTrop Med Hyg 200675640ndash4

44 von Seidlein L Mini primaquine Controversy and uncertainty surroundWHO guidelines for the antimalarial primaquine PLoS Blogs 2012

45 Laufer MK Monitoring antimalarial drug efficacy current challengesCurr Infect Dis Rep 20091159ndash65

46 Organization WH Methods and techniques for clinical trials onantimalarial drug efficacy genotyping to identify parasitepopulations Geneva World Health Organization 2007

47 Chomcharn Y Surathin K Bunnag D et al Effect of a single dose ofprimaquine on a Thai strain of Plasmodium falciparum SoutheastAsian J Trop Med Public Health 198011408ndash12

48 Kaneko A Kamei K Suzuki T et al Gametocytocidal effect ofprimaquine in a chemotherapeutic malaria control trial in NorthSumatra Indonesia Southeast Asian J Trop Med Public Health198920351ndash9

49 Boudin C Olivier M Molez JF et al High human malarial infectivityto laboratory-bred Anopheles gambiae in a village in Burkina FasoAm J Trop Med Hyg 199348700ndash6

50 Talman AM Domarle O McKenzie FE et al Gametocytogenesisthe puberty of Plasmodium falciparum Malar J 2004324

51 Kilian AH Metzger WG Mutschelknauss EJ et al Reliability ofmalaria microscopy in epidemiological studies results of qualitycontrol Trop Med Int Health 200053ndash8

52 Hallett RL Dunyo S Ord R et al Chloroquinesulphadoxine-pyrimethamine for Gambian children with malaria transmission to

mosquitoes of multidrug-resistant Plasmodium falciparum PLoS ClinTrials 20061e15

53 Fofana BDA Sagara I Dao A et al Impact of artemisinin-basedcombination therapy on malaria transmission in Mali 5th MIMPan-African Malaria Conference Nairobi Kenya 2009

54 Gunders AE The effect of a single dose of pyrimethamine andprimaquine in combination upon gametocytes and sporogony ofLaverania falcipara (Plasmodium falciparum) in Liberia Bull WorldHealth Organ 196124650ndash3

55 Jeffery GM Young MD Eyles DE The treatment of Plasmodiumfalciparum infection with chloroquine with a note on infectivity tomosquitoes of primaquine- and pyrimethamine-treated cases Am JHyg 1956641ndash11

56 Bousema T Churcher TS Morlais I et al Can field-based mosquitofeeding assays be used for evaluating transmission-blockinginterventions Trends Parasitol20132953ndash9

57 Mackerras MJ Ercole QN Observations on the action ofquinine atebrin and plasmoquine on the gametocytes ofPlasmodium falciparum Trans R Soc Trop Med Hyg194942455ndash63

58 Rieckmann KH McNamara JV Frischer H et al Gametocytocidaland sporontocidal effects of primaquine and of sulfadiazine withpyrimethamine in a chloroquine-resistant strain of Plasmodiumfalciparum Bull World Health Organ 196838625ndash32

59 Bousema T Dinglasan RR Morlais I et al Mosquito feeding assaysto determine the infectiousness of naturally infected Plasmodiumfalciparum gametocyte carriers PLoS ONE 20127e42821

60 Eziefula AC Gosling R Hwang J et al Rationale for short courseprimaquine in Africa to interrupt malaria transmission Malar J201211360

61 Graves PM Gelband H Garner P Primaquine for reducingPlasmodium falciparum transmission Cochrane Database Syst Rev20129CD008152

62 Dondorp AM Fairhurst RM Slutsker L et al The threat ofartemisinin-resistant malaria New Eng J Med 20113651073ndash5

63 Okell LC Bousema T Griffin JT et al Factors determining theoccurrence of submicroscopic malaria infections and their relevancefor control Nat Commun 201231237

64 Okell LC Griffin JT Kleinschmidt I et al The potential contributionof mass treatment to the control of Plasmodium falciparum malariaPLoS ONE 20116e20179

Eziefula AC Staedke SG Yeung S et al BMJ Open 20133e002759 doi101136bmjopen-2013-002759 9

Dose-finding trial for single-dose primaquine to block malaria transmission

groupbmjcom on February 25 2014 - Published by bmjopenbmjcomDownloaded from

doi 101136bmjopen-2013-002759 2013 3 BMJ Open

Alice Chijioke Eziefula Sarah G Staedke Shunmay Yeung et al Ugandauncomplicated falciparum malaria inclearance of gametocytes in children with efficacy and safety of primaquine fordouble-blinded trial of the dose-dependent Study protocol for a randomised controlled

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