Study design
This protocol is for a two-arm, parallel, double-blind, placebo-controlled, randomised trial in Zambia designed to evaluate the effect of various parasite genotypes on the efficacy of single-dose SP among asymptomatic children between 3-5 years of age.
Study Site and Recruitment
The trial will be conducted with the Tropical Disease Research Centre (TDRC) in the Nchelenge District. The area is situated in the marshlands of Luapula Province along the shores of Lake Mweru where Zambia shares an international border with the Democratic Republic of Congo, and malaria transmission is high, persistent, and perennial [20]. Nchelenge covers an area of 4,090 km2 and had just under 235,000 residents according to the 2022 census. For the PCPI trial, TDRC staff will work with four health clinics accessible by road and all within 30km of the field station. Health centres are as follows (estimated population in parentheses): Kashikishi (50,000), Nchelenge (16,000), Kafutuma (14,000), and Kabuta (30,000).
Each recruitment day, TDRC staff will identify potential participants and provide a brief explanation of the trial at the designated health facility. The parent/guardian will be provided with a high-level summary of the objectives and procedures. For parents/guardians who are interested, inclusion/exclusion criteria will be reviewed. If eligibility criteria are met, parents/guardians will be provided with a consent form to read, sign and date.
Frequency of genotypes
In Nchelenge, there are three prevalence data points of the dhps 540E mutation on which to base our assumptions. Our first data point comes from an observational study of pregnant women attending antenatal care in Nchelenge from 2013 to 2014 where 72.9% (70/96) of genotyped samples contained the dhps 540E mutation (including mixed dhps K540/540E) [22].More recently, analysis of a sub-sample of dried blood spots collected in 2020 as part of a large double-blinded randomised partially-placebo-controlled trial of asymptomatic pregnant women in Nchelenge (ASPIRE trial) found the prevalence of the dhps 540E including mixed dhps K540/540E was 66.8% (133/199). Analysis of 200 more DBS from the ASPIRE trial from early 2022 found a slightly higher prevalence of dhps 540E at 86.7% (130/150) including mixed dhps K540/540E.
Eligibility criteria
Trial staff will recruit children between 3 to 5 years old without symptoms of malaria. Parents/guardians of the participants will provide written, informed consent before any study procedure occurs and must meet eligibility criteria.
Inclusion criteria
To be included in the PCPI Zambian trial, children must: (i) be 3-5 years old; (ii) exhibit no symptoms of malaria; (iii) have parents/legal guardians willing to have their child participate in all follow-up visits and seek care from study staff; and (iv) reside in the study catchment area.
Exclusion criteria
Children are not eligible to participate in the PCPI Zambian trial if they: (i) have evidence of acute illness as determined by clinical examination; (ii) exhibit symptoms of malaria (axillary fever ≥ 37.5 °C and / or history of fever in past 48 hours); (iii) have known allergy to study medications; (iv) have received antimalarial treatment or azithromycin within 28 days prior to screening; (v) are concomitantly receiving co-trimoxazole (trimethoprim-sulfamethoxazole) for any or no indication; or (vi) are categorized as severely malnourished according to WHO child growth standards.
Group Allocation and Masking
Before the onset of the study, an independent statistician at the London School of Hygiene and Tropical Medicine (LSHTM) will generate a list of participant identification numbers that are randomly allocated to one of two treatment groups in a ratio of 2:1 and based on a random block length of 6, 12, or 18. The independent statistician will provide this list to the trial pharmacist who will then prepare a set of sequentially numbered, opaque envelopes that will contain the identification number that will be assigned to each participant and the investigational product corresponding to the treatment group. Tablets will be placed into resealable plastic pouches, one for each dosing day and stored at St Paul’s Mission Hospital in Nchelenge under temperature-controlled conditions. Neither the trial statistician nor the trial pharmacist will otherwise be involved in the study.
Sample size calculation
A simulation approach to calculate the power to detect a significant difference in the duration of protection against new infection with sensitive and with resistant P. falciparum strains was used to inform the required sample size. For a given sample size and assumptions on the setting epidemiology of the trial setting and study design, we simulated 1000 datasets and then fitted a Bayesian model to each simulated dataset. Power was defined as the proportion of simulations that rejected the null hypothesis (i.e., no significant difference in the duration of SP protection against sensitive and resistant P. falciparum strains). Based on malaria surveillance data collected in Nchelenge, we assumed a P. falciparum slide prevalence of 37.8% as measured (TDRC data from International Centres of Excellence in Malaria Research for Southern Africa), and a loss to follow-up of 10% [21]. A sample size of 400 children in the SP arm and 200 in the artesunate (AS) control group would achieve a 89.1% power in estimating a significant difference in the mean duration of SP protection against dhps K540 and dhps 540E. Details on this modelling approach are described elsewhere [22], and an expanded description of this method applied to the PCPI study in Zambia can be found in Additional File 2.
Description of the intervention
Children will receive treatment according to their group assignment. After randomisation, children in the SP group (Group 1) will receive a 7-day course of placebo AS monotherapy, whereas children in the AS group (Group 2) will be given a 7-day course of active artesunate (Table 1). This will span day -7, -6, -5, -4, -3, -2, and -1. Then, on day 0, children who had been given placebo artesunate will receive active SP, whereas children in the AS group will receive placebo SP. Group 2 will reflect a cohort of children who are parasite-free at day 0 and allow for an accurate estimate of background incidence (reflecting transmission intensity) to which all groups will be exposed during follow up. This is due to artesunate’s very short half-life and hence provides no protection, despite clearing existing infections. We will look at parasite clearance among those who were qPCR-positive at day 0 and time to incident infection among qPCR-negative at day 0. This will inform parameters of models we will use in data analyses.
Table 1 Summary of treatment arms and number of children per group
SP consists of one tablet containing 250 mg of sulfadoxine and 12.5 mg of pyrimethamine (MA158 trade name, WHO prequalified product, Macleods Pharmaceuticals Ltd, Mumbai, India) given as a single oral dose for 1 day. The placebo tablets will also be manufactured by Macleods Pharmaceuticals and have same appearance as active SP. AS consists of one tablet containing 50 mg of artesunate given orally once daily for 7 consecutive days (MA044 trade name, WHO approved product, Guilin Pharmaceuticals Co., Ltd, Guilin, China). The placebo tablets will also be manufactured by Guilin Pharmaceuticals and have the same appearance as active AS. Information regarding the use of these medicines can be found in the patient information leaflets in Additional File 3.
Participant Follow-Up
All children in Groups 1 and 2 will be followed 71 days total, comprising 7 days prior to day 0, defined as the first day that the two chemo-prevention groups receive their SP, day 0 itself and 63 days (9-week period total) following day 0 (see activity timeline in Table 2). The study medications will be administered as directly observed therapy by PCPI clinical staff on all days of dosing at the home of each child. After dosing the child will be observed for 30 minutes before the study team departs to record drug-related adverse events (AEs) and to re-dose if necessary. Daily follow-up dosing visits will be held on days -6, -5, -4, -3, -2, -1 for all participants. Similar monitoring of AEs will be carried out on day 0 after dosing, and thereafter during scheduled visits on days 2, 5, 7, 14, 21, 28, 35, 42, 49, 56, and 63, and any unscheduled visit.
Thick blood smears and DBS will be prepared from pin-prick sampling during health facility visits on day 0 (pre-dose), 2, 5, 7, 14, 21, 28; and DBS only on days 42 and 63. Any child who is symptomatic at a scheduled or unscheduled visit from day -7 onward will provide a pin-prick of blood for a malaria rapid diagnostic test (mRDT) (SD Biosensor, Healthcare Private Limited, Gurugram, India). From day 0 onwards any child who is symptomatic at a scheduled or unscheduled visit will provide a pin-prick of blood for a blood film, and a DBS (Whatman 3MM CHR, Cytiva, Cardiff, UK). If mRDT-positive, a DBS will be collected on glass fiber filter paper (Fisherbrand, Fisher Scientific, Loughborough, UK) to test for presence of sulfadoxine. The child will be provided a full course of the first-line antimalarial treatment as rescue treatment as recommended by the national malaria case management guidelines and no longer sought for further blood collection in the study, unless the child again becomes febrile prior to the end of day 63, in which case the child will be tested by mRDT again and treated if positive. Any child suspected of developing a case of severe malaria will be referred promptly to the district-level reference facility hospital and be given treatment according to national guidelines. On any occasion that a child is found to be mRDT-positive, the participant will have haemoglobin levels checked. Any child with haemoglobin levels < 11 g/dL will receive iron supplements according to national guidelines. Follow up activities are shown in Table 2.
Table 2 Summary of activities and follow-up period by treatment group (days)
At the individual level, study completion will be considered achieved when a child has either reached day 63 in the follow-up period or, at any time prior if a symptomatic child is confirmed mRDT-positive, prompting the administration of antimalarial rescue treatment has been provided. At study level, clinical trial termination will be considered when the sample size has been achieved and all participants have completed their last visit.
Strategies for Retention
As part of pre-screening procedures, parents/guardians will be counselled on the importance of following the study visit calendar. They will also be assessed for the probability of travelling out of the catchment area of the study prior to delivery and, therefore, being at risk of not completing the trial. If the study team believes there is a risk, the child will not be included in the study. All parents/guardians will be reimbursed for compensation of time and transportation costs to and from the study site.
If parents/guardians are not at home or available during household scheduled follow-up visits or do not return for health facility scheduled follow-up visits with the child, they will be contacted to schedule the following visit. Children will be categorised as lost to follow-up if the parent/guardian repeatedly fails to return to the facility or be at home for more than three scheduled visits and is unable to be contacted by the study team.
Follow-up visits from day -7 to day 5 will have a 1-day window (+/-) that will be acceptable for the corresponding scheduled visit. From day 7 onward, a 3-day window (+/-) will be acceptable for the corresponding weekly visits. Visits outside of a 3-day window over the nine-week period will be recorded as an unscheduled visit and all procedures will be followed in the manner as a scheduled visit. A participant may miss some scheduled visits and remain in the study at the discretion of the investigators, although the participant will be considered lost to follow-up and no longer in the study seven days following the final scheduled visit, i.e., day 71, if no contact occurs during this period. All attempts made to contact a participant will be documented in the medical notes.
Participant Withdrawal
Every reasonable effort will be made to maintain protocol compliance and participation in the study. A withdrawal from the investigational product is defined as any participant who does not receive the complete treatment. A parent/guardian may withdraw their child from the study at any time for any reason as consent is entirely voluntary. A participant may also be withdrawn at any time at the discretion of the investigator for safety, behavioural, compliance, or administrative reasons.
Parents/guardians may decide to withdraw their child from the study at any time by simply informing a staff member of their desire to do so. Staff will notify the Principal Investigator who, in turn, will notify the Co-Chief Investigators. If the parents/guardians want to withdraw consent for use of data and/or samples already collected, and/or to withdraw consent for long term storage and future use of data and samples, the data and samples for their child will be removed and destroyed within one month of request. If withdrawal is the result of a Serious Adverse Event (SAE), the investigator will offer to arrange for appropriate management of the event and its cost. If parent/guardian withdraws a child prematurely for any reason, the child will not be re-entered into the trial. The participant ID number and treatment number will not be reused. An end of study form will be completed at the day 63 visit and for withdrawn participants prior to the last scheduled study visit. If consent is withdrawn by the parent/legal guardian for any reason, then the last visit prior to withdrawal of consent will be considered the final visit and documented accordingly. All study withdrawals will be recorded in the participant’s medical records and in the Case Report Form.
Adverse events
Most AEs and adverse drug reactions that occur in this study, whether they are serious or not, will be expected treatment-related side effects due to the drugs used in this study. The assignment of causality as unrelated, unlikely, possible, probable, or not assessable event will be made by the investigator responsible for the care of the participant. A data safety and monitoring board will be established for SAEs monitoring purposes.
Outcome measures
The aim of the PCPI study is to measure the chemoprevention efficacy of SP and quantify the effect on both parasite clearance and protection from infection of the parasite genotypes associated with SP resistance in participants who receive SP chemoprevention. Primary and secondary endpoints of interest can be found in tables 3 and 4 respectively.
Table 3 Study primary endpoints
Table 4 Study secondary endpoints
Data collection procedures
Data will be collected using REDCap (Research Electronic Data Capture), a web-based application that is GCP-compliant (good clinical practice) for building and managing online research surveys and databases. Only the Zambian investigators, Co-Chief Investigators, and the data managers will have access to this database. Data will be collected using REDCap on electronic tablets, verified by study team leads and thereafter transmitted at the end of each day to LSTHM servers for storage and back up.
Consent forms will be stored in a secure cabinet. At the end of the study, all documents with names or addresses will be destroyed by shredding. Data from the web-based REDCap will be used for analysis and preparation of reports for the DSMB. Access is controlled by a firewall policy based on the host address and application. Activity on LSHTM servers is fully audited recording both login details and file system access. Access will be limited to essential research personnel.
At enrolment, the study staff will administer a household and caregiver questionnaire integrated in REDCap. Questionnaire domains include sociodemographic and socioeconomic data. Follow-up data will be collected using REDCap according to the schedule outlined in Table 2.
Data analysis and laboratory methods
The deterministic version of the stochastic model used for power calculations will be used to analyse the data (using the probabilistic programming language Stan in R). Specifically, the model will be used to estimate (i) the mean duration of protection against each genotype dhps K540 and dhps 540E (and difference between the two), (ii) the protective efficacy provided by SP against each genotype (over time following treatment), (iii) the background incidence of malaria, and (iv) the underlying frequency of mutant strain (dhps 540E). By disaggregating the impact of these factors, protective efficacy of each drug can be estimated and applied to different settings with varying levels of transmission and resistance. Details of the model structure can be found elsewhere [22, 23].
Malaria microscopy slides will be prepared on days 0, 2, 5, 7, 14, 21, 28 for retrospective analysis only, but will be read for children who present with symptoms and are mRDT-positive. The procedures for slide preparation and reading will be detailed in the operations manual/laboratory standard operating procedures. Two independent readers will record their Plasmodium species and parasite counts. If results are discrepant, a third tie-breaking reader will determine the final reported observation using methods previously described [24].
DBS will be collected on filter papers with a pin-prick of blood at the same time-points for parasite detection using qPCR to carry out genotyping to determine the prevalence of mutations related to resistance to SP and other drugs, and on occasions when participants is mRDT-positive, to test for the presence of sulfadoxine in the subject plasma. DBS will be prepared, desiccated and securely stored under ambient temperature as described in the manual/laboratory SOPs. The use of microscopy and qPCR will allow distinguishing between recrudescent and new infections on paired samples.
Malaria parasite counting will be done as per WHO protocol as well as using a duplex qPCR employing hydrolysis probes to human and P. falciparum amplicons will be used to quantify parasite density in DNA extracted from blood spots as described elsewhere [25, 26]. The proportion of patients with parasitaemia will be presented, as well as the proportion of patients with parasites containing the dhps mutations of interest (in Zambia these are mainly the dhps K540E and dhps A581G). For qPCR positive samples, dhps and dhfr genotypes will be determined using a high-throughput pipeline established using next-generation targeted sequencing technology established at the Centre for Translational Medicine and Parasitology, University of Copenhagen [27].
Ethical considerations
The investigators will ensure that this study is conducted according to the protocol and procedures that meet the principles of the current, 7th revision of the Declaration of Helsinki 2013. 12.2. International Conference on Harmonisation guidelines for GCP in clinical studies. The study protocol and any subsequent amendments will have been approved by the following four institutions before participants are enrolled: the LSHTM Ethics Committee (UK); the Tropical Disease Research Centre Research Ethics Committee, the National Health Research Authority, and the Zambian Medical Regulatory Authority (Zambia); and the WHO Ethics Committee (Switzerland).