Effectiveness and safety of intermittent preventive treatment ...

The datasets that are generated in the current study will be available from the corresponding author on reasonable request and approval by the collaborating institutions and signing the data transfer agreement (DTA) from NIMR [ 78 ].

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Malaria intervention using IPTsc strategy may be integrated in the existing national school health programme. However, there is limited systematic evidence to assess the effectiveness and operational feasibility of this approach. School-aged children are easily accessible in most endemic malaria settings. The evidence from this study will guide the implementation of the strategy to provide complementary approach to reduce malaria related morbidity, anaemia and contribute to the overall burden reduction.

The trial is a phase IIIb, individual randomized, open label, controlled trial enrolling school children aged 5&#;15 years, who receive either DP or ASAQ or control (no drug), using a &#;balanced block design&#; with the &#;standard of care&#; arm as reference. The interventional treatments are given three times a year for the first year. A second non-interventional year will assess possible rebound effects. Sample size was estimated to school children (534 per group) from selected primary schools in an area with high malaria endemicity. Thick and thin blood smears (to measure malaria parasitaemia using microscope) were obtained prior to treatment at baseline, and will be obtained again at month 12 and 20 from all participants. Haemoglobin concentration using a haemoglobinometer (HemoCue AB, Sweden) will be measured four monthly. Finger-prick blood (dried bloodspot-DBS) prepared on Whatman 3 M filter paper, will be used for sub-microscopic malaria parasite detection usingPCR, detect markers of drug resistance (using next generation sequencing (NGS) technology), and malaria serological assays (using enzyme-linked immunosorbent assay, ELISA). To determine the benefit of IPTsc on cognitive and psychomotor ability test of everyday attention for children (TEA-Ch) and a &#;20 m Shuttle run&#; respectively, will be conducted at baseline, month 12 and 20. The primary endpoints are change in mean haemoglobin from baseline concentration and reduction in clinical malaria incidence at month 12 and 20 of follow up. Mixed design methods are used to assess the acceptability, cost-effectiveness and feasibility of IPTsc as part of a more comprehensive school children health package. Statistical analysis will be in the form of multilevel modelling, owing to repeated measurements and clustering effect of participants.

In high transmission settings, up to 70% of school-aged children harbour malaria parasites without showing any clinical symptoms. Thus, epidemiologically, school aged children act as a substantial reservoir for malaria transmission. Asymptomatic Plasmodium infections induce inflammation leading to iron deficiency anaemia. Consequently, anaemia retards child growth, predisposes children to other diseases and reduces cognitive potential that could lead to poor academic performance. School aged children become increasingly more vulnerable as compared to those aged less than five years due to delayed acquisition of protective immunity. None of the existing Intermittent Preventive Treatment (IPT) strategies is targeting school-aged children. Here, we describe the study protocol of a clinical trial conducted in north-eastern Tanzania to expand the IPT by assessing the effectiveness and safety of two antimalarial drugs, Dihydroartemisinin-Piperaquine (DP) and Artesunate-Amodiaquine (ASAQ) in preventing malaria related morbidities in school-aged children (IPTsc) living in a high endemic area.

We hypothesize that, IPTsc with either DP or ASAQ will improve Hb concentration and reduce malaria incidence, with subsequent improvement in cognitive levels in school-aged children. We further hypothesize that, all study drugs (DP and ASAQ) are effective and safe with tolerable minor side effects.

The fact that artemisinin partner drug have noted antagonistic effect on parasite resistance cannot be ignored [ 31 , 32 ]. Suggesting IPTsc using a drug that is a first choice in the treatment guideline may not be a good approach. In this study, we chose DP based on its prophylactic effect and it is an alternative to first line treatment of uncomplicated malaria in Tanzania. We also chose ASAQ which has been noticed to have increased sensitivity in areas where it was banned in the early s as it has been in Tanzania [ 32 ]. Nevertheless, the evidence on effective malaria preventive strategies in school aged children still need to be strengthened. Therefore, this study will provide evidence on effective malaria preventive strategies to guide the formulation of policy on targeted malaria control interventions among school aged children.

Indeed, a study in Ugandan school children showed Dihydroartemisinin-Piperaquine (DP) was highly effective in eliminating asymptomatic infections when administered as a full course and was superior for preventing new infections compared to SP + Amodiaquine (SP-AQ) [ 26 ]. Several other studies conducted in African children for IPTsc strategy using DP alone [ 3 ] or in combinations such as SP + Piperaquine (PQ) in the Democratic Republic of Congo [ 27 ], DP, SP + AQ and SP + PQ in Senegal [ 28 ] and The Gambia [ 29 ], have demonstrated the safety and efficacy of these drugs in reducing malaria incidence, anaemia and the feasibility of conducting school based IPT programs. A community based Ghanaian IPT study in under-five children using ASAQ given every 4 months, showed a reduction from 25.0% malaria prevalence at baseline to 3.0% at one year evaluation (protective efficacy 88.0%) and decreased anaemia prevalence from 27.6% to 16.8% [ 30 ]. In all these studies, study drugs were effective and safe for IPT in children.

The WHO malaria treatment guideline [ 24 ], stipulates that a 3-day course of the artemisinin component of ACTs covers two asexual cycles, ensuring that only a small fraction of parasites remain for clearance by the partner drug, thus reducing the potential development of resistance to the partner drug. It is argued that clearing, otherwise untreated, asymptomatic infections may provide a window for haematological recovery by decreasing the rate of destruction and removal of parasitised red blood cells and improving the erythrocyte production rate in the bone marrow [ 8 , 25 ]. A recent review on ACT impact on gametocytes argued that, if transmission is largely driven by asymptomatic individuals (by definition not seeking treatment), then including these asymptomatically infected individuals in treatment campaigns may have a much larger impact on malaria transmission than the choice of ACT for first-line treatment [ 4 ]. In addition, some groups working on school children recommended use of ACTs for IPT in endemic settings with high SP resistance [ 26 ].

A malaria related anaemia and without concurrent fever is highly related to gametocytaemia [ 19 , 20 ]. It has been pointed that reduced haemoglobin (Hb) concentrations are often a consequence of prolonged duration of infections or recurrent malaria episodes [ 21 , 22 ], both of which have been associated with increased gametocyte production [ 19 ]. Low Hb concentrations and reticulocytosis directly stimulate gametocyte production [ 4 ]. Artemisinins are highly effective against multiple stages of Plasmodium targeting both asexual and sexual stages resulting in a substantial parasite biomass reduction [ 4 , 23 ].

Overall, the mainstay for malaria control include the use of insecticide-treated nets (ITNs), indoor residual spraying (IRS), prompt diagnosis and treatment with an effective antimalarial drug ie. Artemisinin combination therapies (ACTs) and intermittent preventive treatment during pregnancy using Sulfadoxine-Pyrimethamine (SP) during pregnancy (IPTp-SP) and Seasonal Malaria Chemoprevention in children under the age of five (SMC) in (sub-) Sahel region [ 18 ]. The IPTp as well as SMC have been implemented in several sub-Saharan countries. However, there has been no targeted interventions on school aged children. The importance of malaria burden in school aged children is not well addressed, and consequently adequate support for school-based malaria control interventions is lacking.

The WHO malaria report , shows the African region had the largest burden of malaria morbidity, with 213 million cases (93% globally) in [ 1 ]. Though, the report did not categorise malaria burden by age groups, in areas of high transmission (which are mostly in Africa), the main burden of malaria, including nearly all malaria deaths, is in young children [ 2 ]. In high transmission settings, up to 70% of school-aged children harbour malaria parasites [ 3 ] without showing any clinical symptoms (asymptomatic), thus, acting as a relevant reservoir for malaria transmission [ [4] , [5] , [6] , [7] ]. A study done in Kenyan school children showed a baseline prevalence of anaemia and Plasmodium falciparum infections of 22% and 42% respectively, and were both associated to academic performance [ 8 ]. In Muheza, Tanzania, children aged 5&#;14 years had a P. falciparum infection prevalence of 39% that was stable throughout the year [ 9 ]. Asymptomatic Plasmodium infections induce inflammation leading to iron deficiency anaemia [ 10 ]. Consequently, anaemia retards child's growth, predisposes children to other diseases and reduces cognitive potential that could lead to poor academic performance [ 7 , [11] , [12] , [13] , [14] ]. If malaria burden decreases due to malaria control activities, school aged children become increasingly more vulnerable as compared to those aged less than five years due to delayed acquisition of protective immunity [ 2 ]. In addition, co-morbidities with soil-transmitted helminths (STH), schistosomiasis and malnutrition complicate the problem further as they equally contribute to nutritional deficiencies, anaemia and cognitive impairment [ [15] , [16] , [17] ].

3.&#;Methods/design

3.1. General study design

This is a randomized, controlled, open label study assessing the effectiveness and safety of two antimalarial drug-combinations for IPTsc, namely DP and ASAQ by a 3-arm trial using a &#;balanced block design&#; with the &#;standard of care&#; arm as reference. Randomisation was done using available online randomisation service [33], where block size of six was assigned to ensure equal representation of each study arm. This setup, allowed the study team to conduct recruitment within a period of one month (at baseline). During enrolment, every six pupils from the same class used the same randomisation block to ensure balanced allocation per school and within class. Eligible school children, were randomized to receive either full course of DP or ASAQ or control (no drug), they will be followed up for a period of 20 months categorised in two years. The interventional treatments are given at four months intervals for the first year, a second non-interventional year will assess possible rebound effects (see ). Study arms are distributed to allow a head-to-head comparison and the establishment of the treatment's relative value according to a series of outcomes. All study-arms receive the recommended malaria control interventions e.g. bed nets and early diagnosis and care, which, also takes control of any confounding effect from ongoing interventions. In combination, other school health control interventions (i.e. against schistosomiasis and STH) are incorporated. Mixed design methods are used to assess the acceptability, cost-effectiveness and feasibility of this IPTsc as part of a more comprehensive school children health package. This approach has the advantage of testing two treatment options at the same time, maximizing the use of resources, and most likely, this will provide identification of antimalarial suitable for IPTsc.

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3.2. Study area and school selection

The study is conducted in Muheza District, Tanga, North-eastern Tanzania ( ), where, malaria transmission occurs throughout the year with two seasonal peaks following the long rainy season from July to August and the short rainy season from December to January [9]. Malariometric surveys conducted in  at Muheza showed mean malaria prevalence of 22% and 25% after the short and long rains, respectively, and was significantly higher among children aged 5&#;14 years old, compared to those <5 years old (38% vs. 18% and 39% vs. 34% after the short and long rains, respectively) [9]. It was from this Muheza data, that schools located in villages with high malaria prevalence were selected in descending order until the targeted sample size was reached. The existing pillars for malaria control in the area includes use of Long Lasting Insecticides Nets (LLINs) and prompt diagnosis and treatment with ACTs (mainly artemether-lumefantrine, AL) as the first line treatment for uncomplicated falciparum malaria [34]. Intervention on pregnant women (IPTp-SP) is implemented as a policy countrywide as per the WHO recommendations and national malaria treatment gudelines. However, evidence suggest that the strategy is seriously compromised due to the alarming level of high-grade SP resistance in some regions and especially in north Eastern Tanzania [35]. The high grade SP resistance harbouring K540E and A581G mutation (proxy for the sextuple mutants) in the Pfdhps gene have been described in the area [36].

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Current primary school enrolment at Muheza District is about 98% and the number of pupils ranges from 150 to per school. As of February , there were about 111 registered primary schools with about 90,160 enrolled pupils. In this study, the participants are recruited from seven selected primary schools located in villages with high malaria prevalence in Muheza district [9]. Study team make regular visits at selected schools where engagement with the Ministry of Health, Community Development, Gender, Elderly and Children (MoHCDGEC), the Ministry of Education Science and Technology and the President's Office Regional Administration and Local Government (PO-RALG) has been established. In addition, several other community-based studies have been conducted by the NIMR in Muheza and have established good cooperation with the local community leaders and community members. In this study, from each of the involved village; community health workers, school health teachers, local health facility health workers and village administrators are involved during surveys and in continued follow-up to capture adverse events and school absenteeism.

3.3. Randomisation

Randomisation was done using available online randomisation service [33], where block size of six was assigned to ensure equal representation of each study arm. The data manager generated the list of 300 blocks of 6 (i.e. allocations) and prepared the blocks in the envelopes that were provided to a study nurse. During enrolment, every six pupils from the same class used the same randomisation block to ensure balanced allocation per school and within class. Pupils deemed eligible by the study clinicians would pass through the study nurse for randomisation. Since the study is open label, the nurse would follow the list as provided by the data manager to openly allocate the pupils in respective study groups. The nurse would document the randomisation number and study group assigned on each pupils' case report form (CRF). The allocation paper from the envelope would also be pinned/stapled on the CRF's randomisation section. The number of blocks assigned per school were determined by the number of pupils who are class 5 or below who would possibly be recruited. Recruitment was done in a successive pattern, where schools were assigned numbers from the first to the last to be recruited i.e., school 1 (S1), school 2 (S2), school 3 (S3) etc. Then, each school received its randomisation blocks in a successive order, i.e. S1 received block number 1&#;35 because it had 210 possible eligible pupils (i.e.35x6), S2 received block number 36&#;80 because it had around 270 possible eligible pupils and so forth for S3, S4, S5, S6, and S7. This allowed the team to allocate time to recruit those who missed in the previous school but following their respective blocks assigned in that particular school. Thus, randomisation blocks were never mixed between schools. Eligible school children, were randomized to receive either full course of DP or ASAQ or control (no drug).

3.4. Sensitisation and recruitment

At different stages of study implementation, the study team liaised with the local government officials mainly the District Medical Officer (DMO), the Council of Health Management Team (CHMT), malaria and NTDs program officers, District Education Officer, Head of selected primary schools, school committees, village leadership and village health workers including other community stakeholders such as religious leaders and later parents or guardians of children in the selected schools for study briefing to attain support and local community engagement for the study.

We aimed to recruit a total of children (534 per study arm) aged 5&#;15 years who were class 5 or below accumulated from 6 to 12 primary schools selected. In each school all children of class 5 or below were regarded as possible participants. We excluded those in class 6 or 7 due to the fact that they will graduate before the study ends. During sensitisation meetings that were held at schools and included all parents or guardians and village leaders (typically a school meeting), interested parents or guardians were consented on their children participation to the study. Children were enrolled after obtaining a written consent from respective parents and or guardian. Full-enrolment was determined after all inclusion criteria have been met. Parents or guardian and pupils were informed of their right to withdraw from the study at any time. In order to avoid reporting bias or missing information, baseline data on social demographics and well-being of school children were obtained from parents or guardians who have signed consent, at this stage home visit was necessary for global positioning system (GPS) satellite location data and verification of social demographic data with regard to household characteristics. In case a parent or guardian missed a briefing meeting, he/she was followed at home for briefing and consent processes.

3.5. Selection and withdraw criteria

3.5.1. Inclusion criteria

The following inclusion criteria are adhered:

• a.

  Male and female primary school children in a selected school

• b.

  Includes parental/guardian informed consent.

• c.

  Assent by primary school children aged 11 years and above.

• d.

  Aged 5&#;15 years at the baseline assessment.

• e.

  Currently, lives within the pre-defined catchment area of Muheza District.

• f.

  Will remain within the same area throughout the study period (preferably pupils of class five and below).

3.5.2. Exclusion criteria

Participants with at least one of the following criteria are excluded:

• a.

  Pupils of class/grade 6 or 7

• b.

  Currently enrolled in another study or participated in another investigational drug study within the last 30 days.

• c.

  Known to have heart disease or a known cardiac ailment.

• d.

  Reports known hypersensitivity to the study drugs or any sulphonamides.

• e.

  Not willing to undergo all study procedures including physical examination and to provide blood samples as per this study protocol.

• f.

  Having clinical features of severe anaemia

• g.

  Has apparent severe infection or any condition that requires hospitalization

• h.

  Illness or conditions like haematologic, cardiac, renal, hepatic diseases which in the judgement of the investigator would subject the participant at undue risk or interfere with the results of the study, including known G6PD deficiency and sickle cell traits.

• i.

  Body weight <14 kg

3.5.3. Withdrawal criteria

Study withdrawal is an event where a participant who is randomised to a treatment group but does not complete the study or study procedures including medication. If a participant gives informed consent and then does not enter the study, is classified as a screen failure. If a participant shifts to another school that is not involved in the study, s/he is regarded as lost to follow up. Every effort is made to follow-up participants who are withdrawn due to drug-related adverse events in order to determine the final outcome of the adverse event. Participants may choose to withdraw or be asked to withdraw for any one of the following reasons: Withdrawal of consent (at any stage), severe adverse event related to study drug, protocol deviation (including non-compliance), lost to follow-up, termination by sponsor and discretion of the investigator.

3.6. IPTsc intervention, drug administration and accountability

3.6.1. Investigational drugs

3.6.1.1. Dihydroartemisinin Piperaquine (DP)

A fixed-dose combination (FDC) of piperaquine with dihydroartemisinin was approved by the European Medicines Agency (EMA), and is also registered by the Tanzania Medicines and Medical Devices Authority (TMDA). The Tanzania's national malaria treatment and diagnostic guideline [34] considers DP as an alternative to first line ACT (i.e. AL) in treatment for uncomplicated malaria. This drug is safe and efficacious at 60&#;73.9 mg/kg dose in 3 daily doses against uncomplicated P. falciparum malaria [37,38]. The ED90 of PQ at 1.68 ± 0.21 mg/kg/day for P. berghei K-173, CT100 of 42h and has a long elimination half-life [39]. In this study, DP will be dosed using weight-based guidelines targeting a total dose of 6.4 mg/kg dihydroartemisinin and 51.2 mg/kg PQ [3] and will be given as per manufacturer's instruction. The DP drug (D-Artepp) manufactured by Guilin Pharmaceutical Co Ltd from China was donated for this study by Guilin pharmaceuticals Tanzania limited.

3.6.1.2. Artesunate Amodiaquine (ASAQ)

This drug is indicated for treatment of uncomplicated malaria [34]. A fixed dose combination is available and results in better treatment efficacy than loose tablets. Artesunate is a semi-synthetic derivative of artemisinin that is water-soluble quickly adsorbed orally, the highest concentration in blood is achieved within 1 h following an oral intake and the half-life is between 20 and 72 [40]. Artesunate, like other artemisinin derivatives, kills all erythrocytic stages of malaria parasites, including the ring stages and early schizonts, as well as the gametocytes responsible for continuing transmission, although it has only partial activity against the mature stages (stage V) gametocytes [24]. Amodiaquine is readily absorbed from the gastrointestinal tract and rapidly converted by the cytochrome P450 (CYP) enzyme CYP2C8 into N-desethylamodiaquine(DEAQ), which is the main metabolite of amodiaquine (AQ). While AQ is a prodrug which is rapidly eliminated, the elimination of DEAQ is slower, with a terminal half-life of 4&#;10 days [24]. Children will receive 10 mg/kg body weight of amodiaquine (AQ) and 4 mg/kg of artesunate (AS) daily (given as a single dose) over three days [30,41]. The WHO prequalified ASAQ (ASAQ Winthrop®) drug manufactured by Sanofi Pharmaceuticals were procured from HighChem Pharmaceuticals, a Sanofi agent/distributor in Kenya.

3.6.1.3. Treatment administration and assessment of compliance

Pupils who meet all inclusion criteria, are randomised to allocate them into three study groups and hence receive allocated study drugs. Children are observed for 30 min after ingesting the study drug. If a child vomits within this time period, he/she is given another course only to repeat once and the information is documented on the electronic CRF. If a child vomits the study drug for the second time, he or she is withdrawn from receiving the study drug. The study drugs are administered basing on participant's weight following manufacturer's instruction (package insert/leaflet). Both DP and ASAQ have a three days full dose course (administered once per day). Study nurses are responsible for dispensing the first day dose, while doing this they also train school health teachers for administration of the same drugs and documentation on accountability logs. School health teachers would administer subsequent doses (day 2 and 3) to participants receiving study drugs, while study nurses would supervise them. This will happen in all three rounds planned for IPTsc intervention. This ensures that all IPTsc study drugs are directly observed and compliance assessed, and a capacity is built among teachers. In case the IPTsc programme becomes a national policy, teachers would be key people to administer the drugs as it happens for MDA of NTDs that are delivered through schools. In all three days of drug administration, we also provide lunch to all pupils in the school regardless of their recruitment statuses. Lunch is usually composed of rice and beans, a staple food in Tanzania.

3.6.1.4. Medicines accountability

Study drugs are stored at adequate security and environment as described by the manufacturer. At the pharmacy temperature is monitored and there is a drug accountability form, where the pharmacist records drugs received (shipped in) from suppliers and also drugs given out to field nurses for a particular field activity. The field nurses account for drugs they dispense to eligible participants by filling a special drug dispensing log, following the randomisation list. The drug dispensing log records participants' study ID, participants&#; initials, and date dispensed, dose/number of tablets given, comments on whether a participant vomited or not. Appropriate standard operating procedures (SOPs) are adhered both by the pharmacist and the nurse dispensing drugs to study participants. The drug accountability form and the drug dispensing log will be used for drug reconciliation for accountability of all drugs received, dispensed or destroyed in case of expiration. All study drugs either received in from suppliers or sent to the field are verified by the investigator or designee.

3.6.2. Non-investigational drugs

3.6.2.1. Albendazole

An oral 400 mg treatment will be given at month 0, and 12 following the WHO guideline and the national NTD programme. The drug is provided after stool and urine samples have been collected at baseline and at month 12. All enrolled children receive the drug irrespective of their study group and or stool/urine analysis results. A single tablet of 400 mg is administered orally under direct observation. Children are required to chew it. In case one spits it another tablet is provided. Both study nurses and school health teachers administer the drugs to enrolled participants. Since the timing for annual MDA by NTD national-wise could overlap with that of the study; the study team works closely in liaison with the district NTD coordinator, to ensure schools in the study area are not provided the drug twice (i.e. as annual MDA by NTD and by study team) or before the team has collected stool and urine samples.

3.6.2.2. Praziquantel

This will be given at 40 mg/kg orally to children found with schistosomiasis infection. Basing on body weight, praziquantel will be provided as per study clinician's prescription.

3.6.3. Concomitant treatments

During enrolment and follow up, the administration of paracetamol (acetaminophen) is allowed for participants found with fever (>37.5 °C). This together with any other medication provided for treatment during visits, or taken by the participant during the trial period, are all recorded on the appropriate section of the CRF and adverse event (AE) form. Drugs with antimalarial activity (such as co-trimoxazole, macrolides, tetracycline or doxycycline) are also reported as concomitant.

3.7. Routine follow up visits

The study has 6 visits for surveys that are done after every 4 months where the first three visits includes study drug administration, while the successive visits are for assessment of rebound effect ( ). During these surveys we collect clinical samples as scheduled on . We also conduct monthly visits to schools and local health facilities to collect information on school attendance and reported illnesses during that month. Enrolled children were given special identity cards that show scheduled follow up visit to be conducted within schools in collaboration with teachers and local community health workers (CHW). In each school, the study team works closely with the Head teacher, 2 school health teachers and 2 CHWs. If a participant misses school on the day of follow up visit, the study team will do home visit for such a participant. During or in between follow up visits, teachers in each school are encouraged to contact the study team via whenever a study participant is absent or is sick. Any sick or unwell child together with his/her parent or guardian are advised to visit a nearby health facility with notification to the study team.

Table 1

Time points in monthsBaselineActual time linesMar/Apr
Aug *Jan Apr Aug Nov/Dec Eligibility screenXInformed consentXSocial-economic and demographicXHistory (symptoms)XXXXXXClinical physical examinationXXXXXXRandomize to study groupsXBlood slide for malaria parasitesXXXHaemoglobinXXXXXXStool and urine samplesXXXDried blood spot (DBS) from finger prickXXXXXXPCR for submicroscopic malaria infectionaXxxXXXDP and ASAQ drug resistance makersaXXXSerology for malariaaXXXSchool attendance monitoringXXXXXXSchool performanceXXXCognitive and psychomotor assessmentXXXStudy drugs administration as randomisedXXXAdherence assessmentXXXCost effectiveness and acceptability assessmentXData review and quality checkXXXXXXAdverse eventsXXXXXXSTH treatmentXXOpen in a separate window

3.8. Illness records and attendance tracking

Health workers at the health facilities in the study area are involved in the study for documenting events found to sick participants who happen to have attended at respective health facilities. All children presenting with any symptom at a health facility are checked for malaria using malaria rapid diagnostic test (mRDT). Case report forms with incentives are presented to health workers for this purpose. In addition, the study clinician would make weekly calls to each school confirming participant's attendance and remind teachers to document such occasion in a special attendance form. For children who missed school on any day of that week, the CHW is contacted to do a home visit to find out reasons for missing school if it was not reported at school. In case the reason may be a child was sick, information of the child's sickness and management thereof will be documented by the CHW in collaboration with the study clinician and the health workers at the health facility in which the child was attended.

During scheduled study visits, all children found with fever (temperature &#;37.5 °C) or report history of fever in the past 48 hours, regardless of study arm are tested for malaria using mRDT and get treated according to national standard treatment guideline (i.e. given AL if mRDT positive). However, if one is in the intervention arm (DP or ASAQ), s/he receives the respective study drug as long as one is diagnosed with non-severe malaria.

It should also be known that, not all villages in the study area have a health facility, in this case the CHW have been trained for early diagnosis and treatment of malaria using mRDT. They have been trained to dispense AL which is the first line treatment of uncomplicated malaria in Tanzania. If the illness is severe or is not malaria, they provide referral to a hospital facility for further management, where the study team will reimburse the treatment cost to parents or guardians. The study team does routine check up on quality of care and supplies provision. The CHW involved in the study are experienced on use of mRDT and malaria treatment as per national guideline, they have been trained not just in this trial but also other studies that NIMR has conducted in the study area in the past.

At the end of the study or at month 12 all documented cases will be pulled for malaria incidence calculation, this will be compared per study group in the analysis.

3.9. Strategies for retention

Although by nature of this study, we expect retention rate to be high. However, during enrolment, potential participants were asked of their availability in the study area for the entire study duration. In addition, children in their last years of school i.e. those in class six and seven were not recruited. The study team ensured participants have understood the informed consent and adhere to study procedures ( ). During follow up, school teachers and CHWs are incentivised in terms of communication allowance to help capture data on absentees and those who are sick in a real-time manner. Since the population is dynamic, in case, it happens that a participant shifts to another school not included in the study, he or she is considered a lost to follow up. However, in case it happens one has shifted to another school involved in the study, subsequent follow up will continue and his/her data will be included for analysis.

3.10. Safety assessment

Adverse events are collected from the time a participant has been enrolled to the study and throughout the duration of the study or until withdrawal. Study clinicians and or nurses ask participants a non-leading question in order to detect any AE encountered, this is documented in the CRF. The nature of each experience, date and time (whenever appropriate) of onset, duration, severity are recorded. Serious adverse events (SAE) or AEs not previously documented in the study will be recorded in the adverse experience section of the electronic CRF. The investigator reviews all documentation (e.g., hospital progress notes, laboratory, and diagnostics reports) relative to the event. The severity and relationship of an event to treatment is established by using clinical judgement. The intensity of each AE and SAE recorded in the CRF is assigned to one of the following categories: Mild: An event that is easily tolerated by the subject, causing minimal discomfort and not interfering with everyday activities. Moderate: An event that is sufficiently discomforting to interfere with normal everyday activities. Severe: An event that prevents normal everyday activities. Details of any corrective treatment will be recorded on the appropriate pages of electronic CRF.

Previously assessed AEs and designated as &#;continuing&#; are tracked by the study clinician through regular calls and at weekly interval to the parent or caregiver or class teacher or CHW in order to review and get event's outcome prior to next visit. All SAEs will be followed until resolution, until the condition stabilizes, until the event is otherwise explained, or until the subject is lost to follow-up. Once resolved, the AE will be updated on the site AE tracker. The investigator will ensure that follow-up includes any supplemental investigations as may be indicated to elucidate the nature and/or causality of the AE or SAE. This may include additional laboratory tests or investigations or consultation with other health care professionals or facilities. If an adverse experience changes in frequency or severity during a study period, a new record of the experience is started.

All AEs reported or detected will be summarised and accompany reports prepared for the sponsor and regulatory authorities. Separately, and per SOPs and or regulation, NIMR will provide the TMDA and MRCC with biannual reports of AEs observed incorporated on the six monthly reports. In contrast, SAEs will be reported to the sponsor immediately (within 24 h) from the time an investigator becomes aware. The SAE form will always be completed as thoroughly as possible with all available details of the event, signed by the investigator (or designee), and forwarded to the ethical and regulatory authorities and the sponsor without delay. If the investigator does not have all information regarding an SAE, he/she will not wait to receive additional information before notifying the Investigators, Sponsor and the Authorities (TMDA and NIMR-MRCC) of the event and completing the SAE form. The form will be updated when additional information is received. E-mails will be used to transmit the SAE form followed by or confirmation of receipt. All SAEs will be reported to local ethical and regulatory authorities (NIMR-MRCC and TMDA) within the time frame required, normally within 24 h for fatal ones, and within 14 days for non-fatal SAEs/SUSAR.

3.11. Cognitive and psychomotor assessments

To determine the benefit of IPTsc in school children on cognitive and psychomotor ability, selected children were assessed at baseline, and will be assessed again at month 12 and 20 for psychomotor, and cognitive impairments to determine risk factors and benefit of IPTsc interventions in averting the impairments. At baseline, we collected data on child's school performance for the past one year; this will be compared with that at month 12 and 20 of the study follow-up. For cognitive ability test, we adopted the method used by Clarke et al., in and on studies conducted in Kenya [8] and Mali [7], respectively. These were also used elsewhere [42] including Tanzania [16]. We, therefore, selected enrolled pupils from class 4 and 5, who we presume are old enough to understand the cognitive test instruction. We evaluated sustained attention using two code transmission tasks, adapted from the TEA-Ch (Test of Everyday Attention for Children) battery by Manly T. et al. [43,44] as described by Clarke et al., . Tests involved listening to a pre-recorded list of digits read aloud at the speed of one per second. Children were required to listen out a &#;code&#; [two consecutive occurrences of the number 5] and to write down the number (single-digit test) or two numbers (double-digit test) which immediately preceded the code. Cognitive evaluation was assessed at baseline and will be done again at month 12. Physical fitness was assessed using the 20mShuttle Run Test (20mSRT). During this test, children ran continuously between two lines apart turning when signalled to do so by recorded beeps and a &#;shuttle&#; was defined as a run between one line to another. The 20mSRT has 20 levels [16,45]. This will also be conducted again at month 12 and 20 of follow up.

3.12. Implementation research

To determine acceptability of the study strategy, including community and frontline caregivers' perceptions on the recommended drug combination usage. Qualitative research will be conducted at month 8 following the final dose of IPTsc. Respondents will include study participants/parents, teachers, other school staff, study personnel, policy-makers&#;/implementation specialists at the ministries responsible for health and education. Two focussed group discussions (FGD) per school (one for boys and one for girls) will be conducted to pupils receiving the intervention (about 10 randomly selected pupils distributed equally per intervention arm). Also in each school, in-depth interviews (IDI) will be conducted to two school health teachers and four randomly selected parents (two per study intervention arm). Also IDI will be conducted purposefully to district officials responsible for malaria, NTD, school health programs and other stakeholders. Questionnaire and interview topics will be adapted to the respondent type and include: socio-demographic details, experiences of IPT, perceptions of IPT, ideas about malaria and malaria prevention, potential bottlenecks in implementation of the IPT strategy following relevant models/framework [[46], [47], [48], [49]]. Data collection tools will be pre-tested with a number of respondents by the investigators and research assistant together to ensure comprehension and appropriateness. With the consent of participants, in-depth interviews and focus group discussions will be audio-recorded and transcribed verbatim (10% checked independently for accuracy). The Investigator and research assistant will also record observations of the administration of IPTsc as field notes.

3.13. Laboratory methods

3.13.1. General overview

All samples including those taken at baseline will be processed and then archived in a central laboratory located at NIMR Tanga centre. Samples are labelled without revealing the randomisation group of a participant. Therefore, laboratory technicians are blinded on drug allocation of the participants. Thick and thin blood smears were obtained prior to treatment at baseline, and will be obtained again at month 12 and 20 from all participants. Malaria parasitaemia will be detected from blood slide after double reading by expert microscopists to verify the presence of P. falciparum and calculate the parasite density. Haemoglobin concentration was measured at recruitment and will be measured again during scheduled follow up visits using a haemoglobinometer (HemoCue AB, Sweden). Finger-prick blood (dried bloodspot-DBS) samples were collected at baseline and will be collected at every visit, being prepared on Whatman 3 M filter paper, air-dried and stored in plastic bags containing desiccant and archived at &#;20 freezer. These DBS samples will be used for sub microscopic parasite detection (by PCR), detect markers of drug resistance, malaria serological assays and future host-parasite genetic studies.

3.13.2. Blood slides for malaria parasite

Malaria parasitaemia will be checked prior to treatment at every visit in all participants. Thick and thin blood smears will be obtained to verify the presence of P. falciparum and to calculate the parasite density. Thick and thin blood films will be prepared, dried and stained with Giemsa stain according to SOPs. Parasite density will be calculated by counting the number of asexual parasites per 200 leukocytes in the thick blood film, based on an assumed WBC of /μl by light microscopy at × magnification. One hundred high-powered fields (HPF) will be examined (independent of presence or absence of asexual parasite stages). The parasite density per school will be calculated using the following formula:

Parasitedensity/μl=Numberofparasitescounted×8,000Numberofleukocytescounted

Two slides will be prepared for each participant, one will be read by two technicians and kept in the archives and a second will be retained for external quality control. The blades thick and thin smears reading will be performed by two independent technicians. If the discrepancy is greater than 15%, a technician will perform the third reading to decide between the two. A blood smear will be considered negative if no parasite is observed after travelling 100 microscopic fields.

3.13.3. Haemoglobin concentration

Haemoglobin concentration will be measured at recruitment and during scheduled follow up visits using a haemoglobinometer (HemoCue AB, Sweden).

3.13.4. Stool and urine samples

Stool and urine samples were collected at Month 0 and will be collected at 12 and 20 months. A stool sample will be used to determine prevalence (defined as adult worms or eggs) of STH and S. mansoni infection in school-aged children determined by duplicate Kato-Katz thick smears technique as reported elsewhere [50,51]. Stool samples will be labelled and stored in a refrigerator at 4 °C after collection until examined in the laboratory. Each child will be given a urine container and asked to collect 10 mL of the urine specimens. The samples will be visually examined for the presence of blood (macrohaematuria) followed by laboratory examination for schistosomiasis infection (S. haematobium) and presence of eggs as previously reported [50,51].

In this procedure, one stool and one urine samples will be collected. From the stool specimen, two slides of 25 mg will be processed according to duplicate Kato-Katz thick smears technique. The urine sample will be read on strip for search of microscopic haematuria. From each urine sample with (macroscopic or microscopic) haematuria, 2 slides will be made after filtration of 10 ml on filters. Each slide will be checked for the presence of eggs by microscopy and the number of eggs per gram (epg) in stool per 10 ml (ep10mL) urine will be determined.

3.13.5. Sample archiving

All samples will be processed in the laboratory following respective SOP and will then be archived in a central laboratory located at NIMR, Tanga centre.

3.13.6. Molecular analyses on sub-microscopic infection, drug resistance and serology

The occurrence of P. falciparum qPCR-positives will be measured in finger-prick blood samples from a random subset of school children at enrolment with an estimate of 22.5% (n = 120 from each study arm, in total n = 360). A similar random sub-sample at month 12 and 20 will be examined (n = ). DNA will be extracted from filter paper using the QIAamp® DNA Blood Mini Kit (QIAGEN) as per manufacturer's recommendation. A highly sensitive method based on Plasmodium species specific real time qPCR will be used to examine the samples [45,52].

All samples that are qPCR positive for P. falciparum at any given sampling point (month 0, 12 and 20) will be analysed for relevant genetic markers of drug resistance. All qPCR positive samples (depending on drug arm) will be examined for SNPs of relevance for Piperaquine (Pfmdr1), AQ (Pfmdr1 and Pfcrt) and artemisinin resistance (Pfkelch 13) gene mutations. Candidate molecular markers for PQ resistance; Plasmepsin 2-3 copy numbers will be analysed as reported elsewhere [53,54], plus any markers identified in the literature in the course of the project will also be examined. The detection of the targeted drug resistant genes will be carried out using a high-throughput next-generation sequencing (NGS) based on Illumina® platform [55]. After amplifying DNA sequences using multiplex PCRs, gene fragments will be sequenced using the Illumina Miseq® platform and each gene sequence will be indexed according to time and sampling site of origin using the platform. This will allow for simultaneous sequencing of at least samples in a Miseq assay, which will reduce workload and related costs.

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Antibody response to P. falciparum will be determined by ELISA using eluted dried blood from filter paper as described by Idris et al. [56] and Coran et al. [57]. A 3 mm disk will be punched from each dried blood spot and serum will be eluted in reconstitution buffer in 0.5 ml deep well plates (Corning Costar, PA, USA). The reconstituted blood spot solution, equivalent to a 1/200 dilution of serum, will be stored at 4 °C until used for antibody test. All sera will be tested for IgG antibodies by indirect quantitative ELISA to two recombinant blood-stage P. falciparum malaria antigens namely apical membrane antigen-1 (AMA-1) and merozoite surface protein-1 (MSP-119). Methods by Idris et al. [56] will be followed to further process and analyse serological assays.

3.14. Data management

This research generates qualitative and quantitative data (e.g. socio-demographic, clinical evaluation and laboratory data). Data is collected and managed through Research Electronic Data Capture software package (REDCap) which is hosted by the University of Antwerp. REDCap is a mature, secure web application for building and managing online surveys and databases housed by Vanderbilt University [58]. Android or iOS tablets are used to enable data collection offline or in remote places where internet connection is not possible. Hence, the electronic CRF from the REDCap database is uploaded to an electronic mobile device that is used to collect data and later uploaded to the main study on the central server located at the University of Antwerp (UA). Data from laboratory measurements are entered from the paper form into the tablet later and also uploaded to the server. Before data collection, measures such as piloting, pretesting and validation of tools were done to secure high data quality. In addition, REDCap provides data quality features that can be used to check for discrepancies in the data collected.

During the trial, all paper-based forms are kept in a locked cabinet with access restricted to authorized study staff. Computerized data system is locked with password only granted to authorized study staff. The computer servers are protected by a firewall against malware. The servers are backed up on a quasi-permanent base on a second server which ensures that all the data are secured in case of any unforeseen accident.

Measures are undertaken to ensure that all personal data collected during the study are appropriately protected. The collection of personal information is restricted to only the objectives of the trial, and only data relevant for the execution of the trial and interpretation of data is collected. Collected information is kept for a minimal time period, which is regulated by national or international laws. All personal data collected during the study is kept confidential.

The data transfer agreement has been signed by collaborating institutions and obtained approval from NIMR-MRCC, Tanzania.

3.15. Sample size calculation

Basing on the primary outcome, since change of Hb will be assessed per individual participant, we calculated sample size using a paired T-test, where, a sample size of participants (534 per study arm) was suitable to detect a change in Hb level of 0.2 g/dL (effect size based on previous study [27]), at a type one error of 0.05 (z1-α = 1.96), power of 90% (zβ = 1.282), assuming a standard deviation (SD) of 1.25 g/dL and a loss to follow-up of 30%. This was calculated using formula by Noordzij et al. [59] and an online calculator [60].

Basing on a previous study in the same area [9], primary schools were selected from villages with high malaria prevalence. In case a selected village had primary schools with less number of eligible pupils, then more schools in that village/area were added. Participants were individually randomized in blocks of six to equally allocate them into three study groups.

Again a sample size of 922 participants was deemed sufficient to detect 88 events with equal proportion allocation in three study groups (π1= π2 = 0.5), using a power of 90% (zβ = 1.282), type one error of 0.05 (zα/2 = 1.96), assuming intervention will be able to reduce malaria incidence rate by half [Hazard Ratio (HR) of 0.5] and 30% loss to follow-up. The probability of events being 0.2, determined at an incidence rate of 3.0 at site [9] [i.e. S1 = 0.3 and S2 = 0.15]. This sample size was calculated using the formula by Weaver et al. [61].

3.16. Outcome measures

3.16.1. Primary endpoints

The primary endpoints are grouped as follows:

• 1.

  • a.

    change from baseline in mean Hb concentration at month 12 and 20 of follow-up.

  • b.

    and prevalence of anaemia at baseline and at month 12 and 20 of follow up.

  Impact of IPTsc on Anaemia:

• 2.

  • a.

    the clinical malaria incidence from month 0 till months 12 and 20 of follow up. This will be measured from the number of cases (malaria illness) accrued from baseline to month 12 and 20 of follow up.

  Impact of IPTsc on clinical malaria incidence:

3.16.2. Secondary endpoints

• a.

  Prevalence of asymptomatic malaria infections at month 0, 12 and 20 of follow up.

• b.

  Prevalence of PCR confirmed sub-microscopic parasitaemia at months 0, 12 and 20 of follow up.

• c.

  Improvement in the cognitive and psychomotor test scores evaluated at baseline and at month 12.

• d.

  Improvement in school attendance, at month 12 and 20 compared to baseline.

• e.

  Proportion of participants completing dose of given study drugs.

• f.

  Implementation cost of delivering IPTsc using DP and ASAQ in school aged children

• g.

  Relative risk (RR), for all adverse events categorised to severity at month 12 and 20.

3.16.3. Explorative endpoints

• a.

  Prevalence of STH, schistosomiasis at baseline and month 12 of follow up.

• b.

  Proportion of school children with malnutrition at month 0, 12 and 20 through WHO's BMI z-score

• c.

  Prevalence of validated common P. falciparum polymorphisms known to be associated with drug sensitivity at baseline, at months 12 and 20.

• d.

  Proportion of children seropositive for P. falciparum apical membrane antigen (AMA-1) and merozoite surface protein 1 (MSP-119) at baseline, at month 12 and 20.

• e.

  Change in serum antibody responses to P. falciparum AMA-1 and MSP-119 at baseline, at month 12 and 20.

3.17. Quality assurance

The study is done in accordance with the principles of the Declaration of Helsinki and the International Council for Harmonisation Good Clinical Practice guidelines (ICH-GCP). The quality assurance of records and data are guaranteed. The study team were trained about the study protocol prior to start of the trial. The study clinicians and nurses complete CRF at each school visit. The investigators or designee carry out 100% Source Data Verification during the school visits and on follow-up to guarantee the best conduct of the study. Accuracy of the data, is ensured by contra-checking a completed CRF by another investigator or designee. The consistence of data is checked by the clinician, who, cross-checks the reports of the school teachers and of parents/guardians, and the medical records documented at the health facility. The investigators, have frequent contacts with the local health facilities, CHWs and the school teachers. To ensure quality integrity of the study drugs under investigation and avoid harm to participant health and safety [62,63], the study team ensured that drugs procured are WHO prequalified [64] and are on the list of registered drugs by the TMDA [64]. The drugs were then procured from authorised respective manufacturing company agents in Tanzania (for DP) and in East Africa for (ASAQ). In addition, the study obtained regulatory approval from the TMDA and ethical approval from the NIMR-MRCC, Tanzania.

3.18. Statistical analysis

3.18.1. General analysis

The impact of IPTsc using DP or ASAQ versus controls will all be analysed using multilevel analysis, modelling Hb concentration versus time including hierarchical random effects to attain the primary objective. Moreover, the model will include random effects (intercept and slope) for the individual, in control of lost to follow up and any biased results. The impact of IPTsc on clinical malaria incidence will be calculated as incidence rate ratios that will be compared across the study arms/groups. Survival analysis will be used to compare malaria cases detected during intervention at month 12 and at month 20 of follow-up that includes evaluation of any rebound effect observed one year after the last malaria preventive treatment between ASAQ and DP versus the control group. To attain secondary objectives, prevalence ratios, and mean differences will be calculated. We will also assess cluster effect per school and per village or hamlet and adapt the precision of the estimates accordingly. Risk factors such as age and bed net use associated with malaria attacks will be assessed using Cox regression, all significant risks will be included in a multilevel analysis model. A per-protocol analysis including all children who have completed the three IPTsc rounds will be done. Also, intention to treat analysis will be presented. Data will be analysed using STATA or R statistical software.

3.18.2. Qualitative analyses

Transcripts and field notes will be imported into QSR NVivo 11 qualitative data analysis software for analysis using an inductive and deductive approach. Coding will occur in parallel with data collection, using a codebook based on initial research questions and with additional codes added as themes emerge from the data.

3.18.3. Cost-effectiveness analysis

The effectiveness of implementation of IPTsc is a major determinant of its health impact under operational conditions. In this aspect, cost-effectiveness analysis is an important tool to support and guide policy decision on deployment of new interventions. This will be estimated by assessing the implementation cost (FTE, transport, price scenarios, etc.), the study impact as well as possible synergies with other school health intervention programs [65]. To quantify the investment needs and financial liabilities for implementation of the IPTsc intervention, we will design an initial economic model based on preliminary cost effectiveness findings from the study. Therefore, basing on these findings, the study will come up with possible options for funding and implementation at a national or international level.

3.18.4. Safety analysis

Side-effects as well as all non-serious AEs and SAEs following the administration of any study drug will be recorded starting day 0. All events will be graded by severity and relationship to the study treatment. The number and proportion of patients experiencing any AE, any SAE, and any drug-related SAE will be compared between treatment groups and control using Fisher's exact test. The risk of experiencing an adverse event will then be estimated and compared with control using χ2 tests. Since all children are recruited simultaneously, there will be no interim analysis.

3.19. Dissemination of results, authorship and publications

A kick-off meeting involving representatives from the Ministries of health (MoHCDGEC), education and local govenments (PO-RALG) was conducted at the beginning of the preparatory phase in September where, general strategic issues and contents of the study were specified including justification, procedures, risks and benefits of the trial. These meetings will continue on regular basis or as need arise throughout the trial to review and address trial implementation challenges and progress.

The study strategy and its possible implications will be discussed with the several cross-cutting stakeholders involved in school health at Ministerial level, NMCP, Regional and District implementers, teachers and community before the start of the trial. On completion of the study, results will be presented to the communities where the trial was undertaken, to local health authorities (at regional and district level) and to the NMCP as well as in the international and local conferences (oral and posters), peer reviewed scientific journal and press. Policy briefs will be prepared and published in public domain and media for consumption by the policy makers to support its subsequent implementation, as appropriate. Drug resistance data from the trial and related studies will be submitted to Worldwide Antimalarial Resistance Network (WWARN).

Authors will include all investigators that have participated and contributed in the trial. Authorship and publications emanating from this trial will depend on the NIMR publication policy and the principles for authorship criteria of the International Committee of Medical Journal Editors. The manufacturer of the trial medication will be provided with a draft of the manuscript but will have no role in review, data interpretation, or writing of the article.

3.20. Ethical approval and consent to participate

All research activities are conducted in accordance with the standards and codes of conduct accepted by the ICH guidelines. The study obtained ethical clearance from NIMR-MRCC with approval number NIMR/HQ/R.8a/Vol.IX/ and NIMR/HQ/R.8c/Vol.I/668 (for amendment) also NIMR/HQ/R.8c/Vol.I/ for ethical clearance extension. We obtained regulatory approval from the TMDA with approval number TFDA/CTR//07. Prior to start of the trial, permission was obtained from the local governments (PO-RALG), the DMO/CHMT, Village administration, school committee and school parents' or guardian's meetings. No real or perceived coercion to participate was done. Written informed consent was obtained from the parents/guardians for all children before enrollment. An assent was obtained from children who were 11-years-old or older.

The consent forms were translated from English into Swahili, a language spoken by almost all study participants. Written informed consent from parents or guardians and assent were obtained from each participant, two copies were signed by respondents, one copy was given to study participant and the other retained by study team. Whenever a parent or guardian was non-literate, the consent was read to them in most cases in the presence of a literate witness. The participants and parents or guardians were asked questions on the study information (the purpose of the study, the procedures to be followed, and the risks and benefits of participation) to test for comprehension of their understanding of the study. The study team also stressed that participation is voluntary, that any participant may withdraw from the study at any time, and that neither refusal to participate nor withdrawal from the study will have any adverse consequences on future healthcare provided at the study area or elsewhere or even jeopardize their relation with school teachers. Confidentiality was also explained in details to participants and their parents or guardians.

The study team did their best to ensure participants who chose to be a part of the study feel adequately informed of its purpose, nature, procedures, risks, hazards and benefits. To achieve this, trial staff worked closely with local government officials, health officials and community members and religious leaders to incorporate a nuanced understanding of local customs, beliefs and perceptions into the informed consent process.

A copy of the information sheet and the written consent form in English and in Swahili are available for review by the Editor-in-Chief of the Journal.

3.21. Timeline

The study is planned for a period of 20 months; these includes 6 visits of 4 months intervals. In between visits there are monthly visits for supervision and collection of attendance and illness information at each school. The study field work started on March 23rd, and completed field baseline activities on May 10th, . Other follow up timelines are as described in .

3.22. Protocol amendments

If protocol amendments are needed, approval is obtained from all parties including UA, NIMR, MRCC and TFDA.

We show that prices could fall for most essential medicines in the UK and South Africa, and for nearly half of essential medicines in India. This suggests that even for old and widely used medicines, continued efforts should be made to encourage competitive supply.

The methodology presented in this study can be used to reliably estimate the generic price that can be achieved if profit margins are competitive, for a wide range of medicines.

We searched the PubMed database and grey literature using the term (cost* OR price*) AND (manufacture OR production) AND essential AND (medicine* OR drug*). While the costs of production for a few products have previously been analysed, we did not find any studies on production costs for all medicines on the WHO Essential Medicines List, or any similarly broad group of products.

The price of the raw medicine substance, or active pharmaceutical ingredient (API), is generally the most significant component of pharmaceutical cost of production. 9 This analysis used data of API exported from India to estimate generic prices for all medicines in solid oral dosage forms included in the EML. We have previously undertaken analyses of production costs for viral hepatitis, 10 11 tuberculosis (TB) 12 and cancer drugs, 13 14 using similar methods.

The WHO Model List of Essential Medicines (EML) was created in to support national health systems in prioritising drugs for procurement, and includes medicines that are needed to &#;satisfy priority health needs of the population&#;, 7 based on disease burden, efficacy, safety and cost-effectiveness compared with other medicines in the same therapeutic group. 8

Recent high-level groups have recommended greater transparency in drug pricing. 1 2 Data on the costs of production for medicines are not publicly available, and health systems have limited negotiating power when a medicine is sold in a monopoly situation. However, prices can fall substantially when generic competition is enabled, achieving, for example, price reductions of 99% in first-line HIV/AIDS medicines. 6

Lack of access to affordable medicines continues to represent a major global health burden. 1 2 A recent analysis found that, based on per-capita pharmaceutical expenditure, a basket of 201 essential medicines was unaffordable in nearly all low-income countries and 13 middle-income countries. 2 An earlier estimate put the number of people lacking regular access to essential medicines at one-third of the global population. 3 In low-income and middle-income countries (LMICs), only 58% of essential medicines are available in the public sector, and 67% in the private sector, according to surveys of pharmacies. 4 Medicines account for a quarter of all health expenditures globally, 2 and 100% of health expenditures for about half of households in LMICs. 5

We compared estimated generic prices to global lowest current prices of medicines for which there are global market-managing initiatives, such as the Global Fund. Details on price sources are available in the online supplementary appendix .

Lowest currently available prices (LCP) were collected from publicly accessible databases. For South Africa, we used public procurement prices published by the National Department of Health. For Tamil Nadu, India, prices were extracted from a list of a medicine tenders, or, if not available from that source, from an online database of Indian Maximum Retail Prices. For the UK, prices were extracted from the British National Formulary (BNF) and the drugs and pharmaceutical electronic market information tool (eMit), with the lower of the two used to represent the UK price. The BNF reports &#;indicative&#; prices, while eMit prices represent actual government purchases. In addition, the availability of more than one manufacturer in the UK was noted, as an indicator for generic availability and competition. Details on price sources and exchange rates use are available in the online supplementary appendix .

The average profit margin for the pharmaceutical industry in India was 8.8% in and 15.4% in . We assumed a profit margin of 10%. Tax rates in India range from 18.5% to 34.6%; we assumed a midpoint value of 26.6% tax charged on net profits. The final price estimation algorithm is shown in figure 1 . References are mentioned in the online supplementary appendix .

Per-unit conversion costs reported in analyses in the last 10 years (operating cost including depreciated capital cost) ranged from US$0. to US$0.013 per tablet. The estimate of conversion cost by Chaudhuri and West (US$0.01 per tablet) included costs of environmental protection and compliance with current Good Manufacturing Practice (cGMP) standards. 16 The costs attributed to cGMP compliance are similar to costs estimated in an Indian government white paper (see online supplementary appendix ). We therefore used a conversion cost of US$0.01 per tablet in this study.

A number of sources were used to inform an assumed per-unit cost for converting raw API and excipients into an FPP (&#;conversion cost&#;). We reviewed reports of capital and operating costs for pharmaceutical tablet formulating plants and contacted large generic manufacturers for confidential estimates. In addition, we identified the product with the lowest price in the UK, South Africa and India: with API cost and profit margins approaching zero for the cheapest products, the total per-unit cost can be seen as a high estimate of the conversion cost. Estimated conversion costs per tablet extracted from these sources are summarised in table 1 , with details and full references available in the online supplementary appendix .

Typical costs of excipients used in tablets and typical proportion of the FPP made up by each excipient were combined, and excipients were assumed to represent 50% of total tablet weight. This yielded an estimated average excipient cost of US$2.63 per kilogram of FPP (see online supplementary appendix ).

Linear regression was used to estimate the per-kilogram price on 1 July of exported API. API price per dose was calculated, and multiplied by a &#;salt-factor&#; for drugs whose dose is expressed in the EML in terms of the active molecule, but for which the API is manufactured in a salt form (eg, an &#;erythromycin 250 mg&#; tablet contains 360 mg of erythromycin estolate). Stata/IC V.14.0 for Mac was used for statistical analysis of the API dataset.

Data on price per kilogram of API exported from India was collected from an online database (infodriveindia.com) that collects data published pursuant to Indian customs regulations. Prices for exported goods in the database are given in both Indian rupees and US dollars, with the US dollar value calculated based on the exchange rate on the day of the transaction. Search terms used and full exclusion criteria are available in the online supplementary appendix . The default timeframe for API shipments used in the database search was 1 July to 1 July , and was extended backwards by 2 years until at least 100 records were available, up to a maximum 6-year timeframe.

We assumed API procurement and formulation of finished pharmaceutical product (FPP) in India due to its large generic industry and historical importance in treatment of the HIV epidemic. 6 The main alternative country to consider as a focus for the analysis was China; for pharmaceutical manufacture, India and China are similar in cost of labour, infrastructure and tax. However, Indian manufacturers have more experience with the WHO prequalification programme (an initiative that certifies the quality of medicines), which currently lists 335 products manufactured in India, but only 23 manufactured in China. 15

Additional references for this section are given in an online supplementary appendix . Medicines with solid oral formulations from the EML were included (see online supplementary appendix for excluded formulations). All monetary values are expressed in US dollars.

Large price variations were seen for many medicines that are presently under patent protection, or were under protection until recently. In many cases, generic medicines had relatively consistent prices, while for some there was notable variation in prices ( figure 3 ). The therapeutic group with most price variation was antiretroviral medicines.

In India, 118 of 298 (40%) comparable prices were above estimated generic price. The items with highest Indian price to estimated price ratios in were zidovudine 250 mg (45x), praziquantel 150 mg (15.5x), capecitabine 150 mg (13.8x), efavirenz/emtricitabine/tenofovir 600/200/300 mg FDC (10.7x) and efavirenz 200 mg (10.5x). Among prices collected from the database of tenders, LCP to estimated price ratios ranged from 0.04 to 4.3. Among private market prices, lowest Indian price to estimated price ratios ranged from 0.03 to 45.1.

In the UK, prices were above estimated generic price in 214 of 277 (77%) comparable cases, and more than three times above estimated price in 47% of cases. LCP were 0.2&#;387 times the estimated price where multiple suppliers existed, and 3.5&#; where only one supplier was available. The items with highest LCP to estimated price ratio in the UK were daclatasvir 30 mg (x), daclatasvir 60 mg (x), sofosbuvir 400 mg (958x), ledipasvir/sofosbuvir 90/400 mg FDC (593x) and dexamethasone 1.5 mg (387x). Prices were available only in the BNF in 109 cases, only in eMIT in 5 cases. The eMIT price was lower than BNF price in 185 of 209 cases where a price was available from both sources.

Estimated generic prices ranged from US$0.011 pertablet (glyceryl trinitrate 500 μg) to US$1.447 per tablet (darunavir 800 mg), and were heavily skewed towards lower prices in this range. There were 186 estimated generic prices <2.5 cents per tablet, versus only 51 prices above 10 cents per tablet. There was a strong correlation between the estimated generic prices with current global lowest prices for medicines used for HIV, TB and malaria ( figure 2 ). Current lowest UK prices were median 171% above estimated generic price (IQR 4%&#;%), South African prices were median 39% above estimated generic price (IQR &#;24% to 183%) and Indian prices were median 40% below estimated generic price (IQR &#;70% to 59%).

Average API price per kilogram was in the range of US$1&#;US$10/kg for 5 medicines (zinc sulfate, paracetamol, metformin, acetylsalicylic acid, niclosamide), US$10&#;US$100/kg for 43 medicines, US$100&#;US$/kg for 58 medicines, US$&#;US$10 000 for 19 medicines and were >US$10 000/kg for 3 medicines (anastrozole, methotrexate, entecavir).

There were sufficient API data for 148 medicines on the EML (including 20 fixed-dose combinations or medicines listed as combinations), and for 375 finished pharmaceutical products overall when including various dose forms. For 49 medicines on the EML, production costs could not be calculated due to a lack of data on exported API. Lowest current prices were identified for 73% of items in the UK, 47% in South Africa and 75% in India.

Discussion

This study calculated production costs and estimated generic prices for 148 medicines on the WHO EML, showing that most essential medicines can be manufactured at low cost. Calculation was possible for 148/197 (75%) of the medicines meeting inclusion criteria. Despite most medicines on the EML being off-patent, 214 of 277 comparable prices in the UK, 142 of 212 comparable prices in South Africa and 118 of 298 comparable prices in India exceeded the price that would be expected based on cost of production and a 10% profit margin.

This study used prices of actual, recent sales of API exported from India as the main data input for price estimation. For most medicines, at least 100 export shipments of API were available. Prices of API were generally stable over time, or had a slight decreasing trend (see online supplementary appendix). The estimation formula accounted for capital and operating expenses including labour costs, land and utilities costs, costs of running equipment, costs associated with environmental protection and compliance with cGMP standards, taxation and a profit margin. Validation exercises demonstrated a good ability of the estimation algorithm to predict current lowest global prices for treatments of HIV, TB and malaria&#;diseases with large international treatment programmes (figure 2). Lowest current prices in India were higher than the estimated generic price in 118 cases and lower in 180 cases, and median 40% lower than estimated generic price. This distribution suggests that the algorithm used to calculate minimum costs of production errs on the conservative side (table 1 and online supplementary appendix).

Most of the high-priced medicines in India were found only in the private market price source, and not the (Tamil Nadu) government tender list, suggesting a lack of availability in public facilities. Over 75% of health expenditure is out-of-pocket in India, of which the majority is spent on medicines.17 While we found Indian prices to be below our estimated generic price in many cases, Indian prices were mostly government tender prices, which are likely to be significantly lower than the private market prices more often experienced by those needing medicines in India. Further analysis of the Indian market would be necessary to determine prices available to various facilities, provinces and patient groups.

Generic competition achieved massive price reductions for antiretroviral drugs in the early s.6 However, non-communicable diseases (NCDs) now represent a larger disease burden than communicable disease, and similar price reductions in medicines for cancer, type 2 diabetes and anticoagulation may be valuable in enabling wider treatment of NCDs in LMICs.18

To protect the right to health, all countries need to ensure affordable access to medicines. This requires avoiding and tackling monopolies, including through legislation, as well as encouraging competition, ensuring a robust supply chain and monitoring shortages and stock-outs. Mechanisms to overcome intellectual property restrictions in the public interest include governments issuing compulsory licences and originator companies offering voluntary licences. Both are legal mechanisms that allow the use of patented products (including production, import and use) before patent expiry.1 One successful  example of voluntary licensing is embodied in the Medicines Patent Pool, which negotiates licences with originator companies, and then sub-licenses production rights to generic manufacturers in resource-limited countries.

The World Trade Organization&#;s Agreement on Trade-Related Aspects of Intellectual Property Rights (TRIPS), which sets minimum requirements and limitations on intellectual property laws globally, additionally provides (in article 31bis) for compulsory licences predominantly for export&#;meaning that compulsory licences can be granted in countries with large manufacturing capacity (such as India) in order to supply other countries where there is a pressing need.19

The TRIPS agreement does not prevent individual patients importing medicines for personal use, regardless of patent status, although national legislation must also permit importation for personal use to make this a viable route for access, and this is not uniformly true. This route is being used by patients to access affordable HIV pre-exposure prophylaxis and hepatitis C drugs, with evidence of good clinical outcomes.20 21 However, personal-use importation as a method to overcome to access barriers requires the patient population to be organised and assertive, have a high level of access to information and financial resources and cannot be considered a feasible large-scale solution.

Comparative clinical and cost-effectiveness is assessed both by national regulators such as the UK&#;s National Institute for Health and Care Excellence and the WHO Expert Committee reviewing proposed additions to the EML. Cost-effectiveness calculation depends on the efficacy and available price for the drug and the comparator. Where a novel drug is being compared with a drug that is near patent expiry, a cost estimation exercise can help anticipate the generic price of the latter. Recent examples where this analysis could have been applied include novel oral anticoagulants compared with warfarin, dasatinib as a second-line after imatinib for chronic myeloid leukaemia and dolutegravir compared with efavirenz for the treatment of HIV. Generic market entry of comparators should trigger recalculation of cost-effectiveness for drugs whose assessment depended on comparison with an earlier, higher non-generic price.

Estimation of production cost can improve transparency in pricing negotiations, and there are precedents using cost of production in price control mechanisms. In India, a formula based on costs of manufacture was used to set ceiling prices for &#;scheduled&#; medicines from until , when legislation changed.22&#;24 In South African government tenders, manufacturers are requested to provide in their bids a breakdown of drug price into API, formulation, packaging, logistics, and &#;gross margin&#; components.25 China, Iran, Bangladesh and Pakistan use similar mechanisms.26

Further research based on pricing data for API exported from India may be limited by a recent change in Indian law, removing the requirement for daily publication of customs data.25 Expanded international price comparisons may identify cases where resource-limited health systems face excessive generic prices.

Limitations

This analysis was limited by the inability to include an estimate for the costs of product development, bioequivalence studies, registration costs and costs of litigation, due to a lack of published data. This is balanced against numerous factors that may have contributed to overestimation of the API costs incurred by Indian manufacturers, and thus overestimation of profitable generic prices: API prices in export data likely include a profit margin for the API producer, paid by the manufacturer of the FPP&#;while if API were manufactured in-house by the producer of the FPP, this intermediate profit margin, as well as transport costs and duties would be avoided. Direct assessment of API adherence to stringent regulatory authority standards was not possible from the export data. However, the sources consulted for the assumed conversion cost included quality assessment of API purchased from the assumed third-party supplier in this cost.

Depending on the country in question, as many as 15% of medicines on the current EML may be under patent protection.27 We undertook our analysis before the list was published; patented medicines added in that could otherwise have been included were dolutegravir, raltegravir, velpatasvir, nilotinib and dasatinib.

Tamil Nadu state tender prices are likely to be lower than the prices normally encountered by patients in India, where most medicine purchases are out of pocket.17 Similarly, in the UK and South Africa the price sources represented hospital purchases and may thus represent the lower range of prices (for the UK, in a minority of cases BNF &#;indicative prices&#; were used instead, which are in general derived from prices paid by pharmacies).

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