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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
2 Eziefula AC Staedke SG Yeung S et al BMJ Open 20133e002759 doi101136bmjopen-2013-002759
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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
Eziefula AC Staedke SG Yeung S et al BMJ Open 20133e002759 doi101136bmjopen-2013-002759 3
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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
6 Eziefula AC Staedke SG Yeung S et al BMJ Open 20133e002759 doi101136bmjopen-2013-002759
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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
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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
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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
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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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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
2 Eziefula AC Staedke SG Yeung S et al BMJ Open 20133e002759 doi101136bmjopen-2013-002759
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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
Eziefula AC Staedke SG Yeung S et al BMJ Open 20133e002759 doi101136bmjopen-2013-002759 3
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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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Dose-finding trial for single-dose primaquine to block malaria transmission
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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
6 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
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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
Eziefula AC Staedke SG Yeung S et al BMJ Open 20133e002759 doi101136bmjopen-2013-002759 7
Dose-finding trial for single-dose primaquine to block malaria transmission
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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
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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
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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
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Dose-finding trial for single-dose primaquine to block malaria transmission
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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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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
2 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
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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
Eziefula AC Staedke SG Yeung S et al BMJ Open 20133e002759 doi101136bmjopen-2013-002759 3
Dose-finding trial for single-dose primaquine to block malaria transmission
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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
4 Eziefula AC Staedke SG Yeung S et al BMJ Open 20133e002759 doi101136bmjopen-2013-002759
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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
Eziefula AC Staedke SG Yeung S et al BMJ Open 20133e002759 doi101136bmjopen-2013-002759 5
Dose-finding trial for single-dose primaquine to block malaria transmission
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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
6 Eziefula AC Staedke SG Yeung S et al BMJ Open 20133e002759 doi101136bmjopen-2013-002759
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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
Eziefula AC Staedke SG Yeung S et al BMJ Open 20133e002759 doi101136bmjopen-2013-002759 7
Dose-finding trial for single-dose primaquine to block malaria transmission
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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
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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
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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
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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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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
Eziefula AC Staedke SG Yeung S et al BMJ Open 20133e002759 doi101136bmjopen-2013-002759 3
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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
4 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
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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
Eziefula AC Staedke SG Yeung S et al BMJ Open 20133e002759 doi101136bmjopen-2013-002759 5
Dose-finding trial for single-dose primaquine to block malaria transmission
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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
6 Eziefula AC Staedke SG Yeung S et al BMJ Open 20133e002759 doi101136bmjopen-2013-002759
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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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Dose-finding trial for single-dose primaquine to block malaria transmission
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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
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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
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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
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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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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
4 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
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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
Eziefula AC Staedke SG Yeung S et al BMJ Open 20133e002759 doi101136bmjopen-2013-002759 5
Dose-finding trial for single-dose primaquine to block malaria transmission
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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
6 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
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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
Eziefula AC Staedke SG Yeung S et al BMJ Open 20133e002759 doi101136bmjopen-2013-002759 7
Dose-finding trial for single-dose primaquine to block malaria transmission
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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
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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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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
Eziefula AC Staedke SG Yeung S et al BMJ Open 20133e002759 doi101136bmjopen-2013-002759 5
Dose-finding trial for single-dose primaquine to block malaria transmission
groupbmjcom on February 25 2014 - Published by bmjopenbmjcomDownloaded from
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
6 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
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
Eziefula AC Staedke SG Yeung S et al BMJ Open 20133e002759 doi101136bmjopen-2013-002759 7
Dose-finding trial for single-dose primaquine to block malaria transmission
groupbmjcom on February 25 2014 - Published by bmjopenbmjcomDownloaded from
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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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
6 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
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
Eziefula AC Staedke SG Yeung S et al BMJ Open 20133e002759 doi101136bmjopen-2013-002759 7
Dose-finding trial for single-dose primaquine to block malaria transmission
groupbmjcom on February 25 2014 - Published by bmjopenbmjcomDownloaded from
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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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
Eziefula AC Staedke SG Yeung S et al BMJ Open 20133e002759 doi101136bmjopen-2013-002759 7
Dose-finding trial for single-dose primaquine to block malaria transmission
groupbmjcom on February 25 2014 - Published by bmjopenbmjcomDownloaded from
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
httpbmjopenbmjcomcontent33e002759fullhtmlUpdated information and services can be found at
These include
References httpbmjopenbmjcomcontent33e002759fullhtmlref-list-1
This article cites 59 articles 18 of which can be accessed free at
Open Access
httpcreativecommonsorglicensesby-nc20legalcodehttpcreativecommonsorglicensesby-nc20 and compliance with the license seeis properly cited the use is non commercial and is otherwise in distribution and reproduction in any medium provided the original workcommons attribution non-commercial license which permits use this is an open-access article distributed under the terms of the creative
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CollectionsTopic
(149 articles)Research methods (193 articles)Pharmacology and therapeutics
(204 articles)Infectious diseases (127 articles)Global health
(159 articles)Evidence based practice (635 articles)Epidemiology
Articles on similar topics can be found in the following collections
Notes
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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
httpbmjopenbmjcomcontent33e002759fullhtmlUpdated information and services can be found at
These include
References httpbmjopenbmjcomcontent33e002759fullhtmlref-list-1
This article cites 59 articles 18 of which can be accessed free at
Open Access
httpcreativecommonsorglicensesby-nc20legalcodehttpcreativecommonsorglicensesby-nc20 and compliance with the license seeis properly cited the use is non commercial and is otherwise in distribution and reproduction in any medium provided the original workcommons attribution non-commercial license which permits use this is an open-access article distributed under the terms of the creative
serviceEmail alerting
the box at the top right corner of the online articleReceive free email alerts when new articles cite this article Sign up in
httpgroupbmjcomgrouprights-licensingpermissionsTo request permissions go to
httpjournalsbmjcomcgireprintformTo order reprints go to
httpgroupbmjcomsubscribeTo subscribe to BMJ go to
groupbmjcom on February 25 2014 - Published by bmjopenbmjcomDownloaded from
CollectionsTopic
(149 articles)Research methods (193 articles)Pharmacology and therapeutics
(204 articles)Infectious diseases (127 articles)Global health
(159 articles)Evidence based practice (635 articles)Epidemiology
Articles on similar topics can be found in the following collections
Notes
httpgroupbmjcomgrouprights-licensingpermissionsTo request permissions go to
httpjournalsbmjcomcgireprintformTo order reprints go to
httpgroupbmjcomsubscribeTo subscribe to BMJ go to
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
httpbmjopenbmjcomcontent33e002759fullhtmlUpdated information and services can be found at
These include
References httpbmjopenbmjcomcontent33e002759fullhtmlref-list-1
This article cites 59 articles 18 of which can be accessed free at
Open Access
httpcreativecommonsorglicensesby-nc20legalcodehttpcreativecommonsorglicensesby-nc20 and compliance with the license seeis properly cited the use is non commercial and is otherwise in distribution and reproduction in any medium provided the original workcommons attribution non-commercial license which permits use this is an open-access article distributed under the terms of the creative
serviceEmail alerting
the box at the top right corner of the online articleReceive free email alerts when new articles cite this article Sign up in
httpgroupbmjcomgrouprights-licensingpermissionsTo request permissions go to
httpjournalsbmjcomcgireprintformTo order reprints go to
httpgroupbmjcomsubscribeTo subscribe to BMJ go to
groupbmjcom on February 25 2014 - Published by bmjopenbmjcomDownloaded from
CollectionsTopic
(149 articles)Research methods (193 articles)Pharmacology and therapeutics
(204 articles)Infectious diseases (127 articles)Global health
(159 articles)Evidence based practice (635 articles)Epidemiology
Articles on similar topics can be found in the following collections
Notes
httpgroupbmjcomgrouprights-licensingpermissionsTo request permissions go to
httpjournalsbmjcomcgireprintformTo order reprints go to
httpgroupbmjcomsubscribeTo subscribe to BMJ go to
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
httpbmjopenbmjcomcontent33e002759fullhtmlUpdated information and services can be found at
These include
References httpbmjopenbmjcomcontent33e002759fullhtmlref-list-1
This article cites 59 articles 18 of which can be accessed free at
Open Access
httpcreativecommonsorglicensesby-nc20legalcodehttpcreativecommonsorglicensesby-nc20 and compliance with the license seeis properly cited the use is non commercial and is otherwise in distribution and reproduction in any medium provided the original workcommons attribution non-commercial license which permits use this is an open-access article distributed under the terms of the creative
serviceEmail alerting
the box at the top right corner of the online articleReceive free email alerts when new articles cite this article Sign up in
httpgroupbmjcomgrouprights-licensingpermissionsTo request permissions go to
httpjournalsbmjcomcgireprintformTo order reprints go to
httpgroupbmjcomsubscribeTo subscribe to BMJ go to
groupbmjcom on February 25 2014 - Published by bmjopenbmjcomDownloaded from
CollectionsTopic
(149 articles)Research methods (193 articles)Pharmacology and therapeutics
(204 articles)Infectious diseases (127 articles)Global health
(159 articles)Evidence based practice (635 articles)Epidemiology
Articles on similar topics can be found in the following collections
Notes
httpgroupbmjcomgrouprights-licensingpermissionsTo request permissions go to
httpjournalsbmjcomcgireprintformTo order reprints go to
httpgroupbmjcomsubscribeTo subscribe to BMJ go to
groupbmjcom on February 25 2014 - Published by bmjopenbmjcomDownloaded from
CollectionsTopic
(149 articles)Research methods (193 articles)Pharmacology and therapeutics
(204 articles)Infectious diseases (127 articles)Global health
(159 articles)Evidence based practice (635 articles)Epidemiology
Articles on similar topics can be found in the following collections
Notes
httpgroupbmjcomgrouprights-licensingpermissionsTo request permissions go to
httpjournalsbmjcomcgireprintformTo order reprints go to
httpgroupbmjcomsubscribeTo subscribe to BMJ go to
groupbmjcom on February 25 2014 - Published by bmjopenbmjcomDownloaded from