Study sites
The study was carried out in four health facilities across four Regions: Kibaha-Pwani, Mlimba- Morogoro, Mkuzi-Tanga, and Ujiji-Kigoma between April and August 2018 (Fig. 1). These facilities are among the eight National Malaria Control Programme (NMCP) sentinel sites for monitoring therapeutic efficacy of antimalarial drugs since 19971,17. In general, there is high to low malaria transmission in most parts of Mainland Tanzania. However, seasonal transmission peaks occur after the main long rainfall season between June and August with tendencies to shift between March and May in most areas depending on the timing of rainfalls. Throughout Tanzania, P. falciparum is the predominant malaria species and Anopheles gambiae complex and Anopheles funestus are considered the main vectors. The study sites have been fully described in previous studies which were conducted between 2011–2016 and showed sustained high efficacy of AL (> 96%)[18, 19].
Study design and participants
This was a single-arm prospective study for assessing the therapeutic efficacy and safety of AL for treatment of uncomplicated falciparum malaria according to the standard WHO protocol[17].
Study participants were recruited among patients presenting at the study sites for care. Inclusion criteria were as follows; children aged between six months and 10 years, fever (axillary temperature ≥ 37.5oC) and/or reported history of fever in the past 24 hours, mono-infection of P. falciparum detected by microscopy, parasitaemia between 250 and 200,000 asexual parasites/µl of blood, ability to swallow oral medications; ability and willingness to attend scheduled follow-up visits, informed consent provided by parent or guardian, and stable residence within the catchment area throughout the study period.
Initial screening utilized malaria rapid diagnostic tests (mRDTs) followed by microscopy to confirm eligibility of study participants. Exclusion criteria included patients with negative mRDT results and general danger signs or signs of severe falciparum malaria. Patients with mixed or mono-infections with another Plasmodium species, severe anaemia (Hb < 5 g/dL), presence of severe malnutrition (defined as a child who had symmetrical oedema involving at least the feet or mid-upper arm circumference < 110 mm) were excluded from the study. Other exclusion criteria included febrile conditions due to diseases other than malaria (e.g. measles, acute lower respiratory tract infection, severe diarrhoea with dehydration) or other known underlying chronic or severe diseases (e.g. cardiac, renal, and hepatic diseases, HIV/AIDS etc.), regular medications which may interfere with antimalarial pharmacokinetics, and history of hypersensitivity reactions or contraindications to any of the medicine(s) tested or used as alternative treatment(s). Excluded patients received appropriate treatment according to the national guidelines 21.
Sample size estimation
The sample size was determined based on WHO 2009 standard protocol22 with the assumption that 5% of the patients treated with AL were likely to have treatment failure. At a confidence level of 95% and an estimate precision of 5%; a minimum sample size was 73 patients at each site. With 20% increase to allow for the loss to follow-up and withdrawals during the 28-Day follow-up, 88 patients were targeted per site.
Treatment, laboratory procedures and follow-up
Patients enrolled in the study were treated with artemether-lumefantrine (AL, Coartem®, Beijing Novartis Pharma Ltd, Beijing China for Norvatis Pharma AG, Basle, Switzerland) obtained from WHO. This was a fixed combination of 20 mg of artemether and 120 mg lumefantrine in a tablet. The drugs were administered according to the recommended doses based on the weight of patient23. One tablet was given to children weighing 5-14kg; two tablets to children weighing 15–24 kg and three tablets to children weighing 25–35 kg. A full course of AL consisted of 6-doses given twice daily (8 hourly apart on day 0 and 12 hourly apart on days 1 and 2). Food was not provided with treatment; however, guardians were counselled to provide fatty food at home to optimise absorption of the drug. Patients were observed for 30 minutes to ensure that they did not vomit the study drugs. When vomiting occurred, a repeat dose was given after vomiting stopped. Any patient who persistently vomited the study medication was withdrawn and treated with parenteral quinine or injectable artesunate according to the national guidelines for management of complicated and severe malaria21. Paracetamol was given to all patients with body temperature greater than or equal to 38oC. All doses were administered orally under direct observation of a study nurse. Patients with persistent asexual parasitaemia on day 7 or who develop a recurrent parasitaemia after day 7 with no signs of severity were treated with an alternative ACT, either artesunate amodiaquine (ASAQ) or dihydroartemisinin piperaquine (DHAPQ). In case of development of any danger signs or signs of severe malaria at any point, rescue treatment consisted of parenteral artesunate was given and patients were withdrawn in the study. The procedures for blood smear staining, parasite counting, parasite density calculation, and quality control of blood slide readings are described in the WHO protocol20.
Patient follow-up
Follow-up was done for 28 days with scheduled visits on days 1, 2, 3, 7, 14, 21, and 28 or at any other day (unscheduled visits) when patients felt unwell. Parents/guardians were informed and encouraged to bring their children to the clinic whenever they were unwell without waiting for scheduled visits. Patients who could not come for their scheduled visit by mid-day were visited at home by a member of the study team and asked to come to the health centre. In case a patient travelled to other places and could not be traced for scheduled follow-up, he/she was classified as lost to follow-up and was withdrawn from the study. During the visits, both clinical and parasitological assessments were performed; and follow-up samples (blood slides and dried blood spots (DBS)) were also collected.
Sample collection and examination
Samples were collected through a finger prick for collection of blood sample for mRDT and collection of thick and thin blood smears for detection of malaria parasites by microscopy. From each patient, dried blood spots (DBS) on Whatman III filter papers were collected for laboratory analysis of malaria parasites including P. falciparum diversity, molecular markers of antimalarial resistance and distinguishing recrudescent from new infections by PCR genotyping. Collected blood slides were stained with 3% Giemsa for 30–45 minutes and examined by microscopy to detect presence of malaria parasites and the level of parasitaemia, Plasmodium species, and presence of gametocytes. Parasitaemia was measured by counting the number of asexual parasites against 200 white blood cells (WBCs) in thick blood films and detection of the different parasite species was done on thin films24. Parasite density, expressed as the number of asexual parasites per µl of blood, was calculated by dividing the number of asexual parasites by the number of white blood cells counted and then multiplying by an assumed white blood cell density (typically 6000 per µl)24. A blood slide was declared negative when examination of 100 high power fields did not reveal the presence of any malaria parasite. For quality control, each slide was re-examined by a second microscopist and those with discrepancy were re-examined by the third microscopist. Further disagreement was resolved by a team of three microscopists who examined the same slide at the same time. Final parasitaemia was calculated as the average between the two closest readings.
Sample processing, parasite genotyping and molecular analysis
Parasite deoxyribonucleic acid (DNA) was extracted from DBS using QIAamp DNA blood mid kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. Genotype analysis was conducted in order to differentiate a recrudescent (treatment failure/same parasite strain) from a newly acquired infection (reinfection/different parasite strain) among recurrent parasitemia found during follow-up. This analysis was based on the extensive diversity in the following P. falciparum genes: merozoite surface proteins 1 and 2 (msp1 and msp2), and glutamate rich protein (glurp) genes[20].
Distinguishing recrudescent from new infections was done by genotyping three length-polymorphic genetic markers (msp1 and msp2, and glurp) using gel electrophoresis detection based on the WHO protocol[21, 22]. Recrudescence was determined by comparing samples that contained at least one matching allelic band, indicating fragment size similarities qualifying as a match for each marker, with a difference of 10 bp for msp1 and msp2 and 50 bp for glurp., from paired samples collected on day of enrolment (D0) and day of recurrent infection (R0). A reinfection was defined as the absence of any matching allelic band in at least one marker in the paired blood samples. Msp2 was run for all samples, then glurp for those matching at msp2, and msp1 for those matching at both msp2 and glurp. The classification of a recrudescence was based on 3/3 algorithm, I,e match when all markers had genotyping results and in case of non-determinant results in any marker, results for available markers were used to assess [22]. We also assessed presence of recrudescence, by using a recent 2/3 algorithm as recommended by WHO in 2021[23].The genotyping data used for molecular correction has been included as a supplemental file( Supplementary Table 1).
Outcome classification
The primary end point was parasitological cure on day 28 as per WHO protocol of 2009[22] while secondary end points included occurrence and severity of adverse events. Treatment outcomes were classified as 1). Early treatment failure (ETF) if the patient had presence of parasitaemia and danger signs on day 1, 2 and 3 or persistence of parasitaemia till day 3.2). Late clinical failure (LCF) was defined as presence of danger signs with parasitaemia between day 4 and 28 to a patient who didn’t qualify as early treatment failure. 3). Late parasitological failure (LPF); a patient who had parasitaemia between day 7 and 28 and was not classified as early treatment failure. 4). Adequate clinical and parasitological response (ACPR) was defined as absence of parasitaemia to a patient who wasn’t classified as early, late clinical, or late parasitological failure. 5)Lost to follow-up occurred when despite all reasonable efforts, an enrolled patient does not attend the scheduled visits and cannot be found, the patient was withdrawn from the study. 6) Withdrawal; due to consent withdrawal, failure to complete treatment and protocol violation .
Ethical Considerations
Ethical clearance was obtained from the Medical Research Coordinating Committee (MRCC) of the National Institute for Medical Research with number NIMR/HQ/R.8a/Vol.IX/2687. Permission to conduct the study at the health facilities was sought in writing from the relevant regional and district medical authorities. Oral and written informed consent was obtained from parents or guardians of all eligible patients before they were screened for possible inclusion into the study.
Data management and analysis
The first data entry was performed at the study sites and followed by second data entry, which was centrally done at Muhimbili University of Health and Allied Sciences (MUHAS) after the end of data collection. The data was entered into a Microsoft Access database, and later validated, cleaned, and analysed using STATA for Windows, version 13 (STATA Corporation, TX-USA). Continuous variables were summarised using mean, median, standard deviation, and interquartile range while categorical variables were summarised using percentages. Treatment outcomes (primary and secondary) outcomes were analysed based on the WHO protocol and presented by site. Kaplan–Meier analysis (curves) were used to present the time to parasite infections based on PCR un-corrected and corrected. Individuals whose PCR results could not be resolved were reported as non-determined and excluded from the analysis of PCR-corrected treatment outcome.