Study design and duration
This surveillance study was a one-arm prospective evaluation of clinical and parasitological responses to the treatment of AL for uncomplicated malaria. The study was conducted between March 2021 and January 2022.
Study setting
This study was conducted in Shecha health centre, Arba Minch town. Arba Minch town is administratively located in Gamo Gofa zone of the Southern Nations, Nationalities and Peoples’ Region of Ethiopia. Arba Minch town, is located at about 500 km South of Addis Ababa, the capital city of Ethiopia. At an altitude of 1200–1400 m above sea level, the town has an average annual rain fall of 800–1000 mm and an average annual temperature of 29.0 °C. The administration of Arba Minch town is divided into four kifle-ketemas (sub-towns) -Shecha, Sikella, Abaya and Nechsar. The four kifle-ketemas totally comprise 11 kebeles, all of which are malarious areas.
Study population
The population consisted of outpatients over six months of age, with uncomplicated P. falciparum malaria attending the study health centre.
Sample size calculation
According to the working WHO protocol for anti-malarial TES, a minimum of 73 participants should be recruited to detect an expected failure rate of 5% at a confidence level of 95% and an estimate precision of 5% [8]. To account for follow-up and withdrawal losses, an additional 20% was added to the total. Following this calculation, 88 study participants were recruited for the study.
Eligibility criteria
Inclusion criteria were patient of six months or older, having a microscopically positive P. falciparum mono-infection, having an asexual parasitaemia level of 1000 to 200,000/µl, having an axillary temperature of 37.5 °C or higher at presentation or having a history of fever within the previous 24 h, being able and willing to comply with the study protocol and study visit schedule for the duration of the study, and being willing to sign an informed consent or assent (children under 12 years old) form.
The following exclusion criteria were considered for the study. General danger signs in children under the age of 5 years or signs of severe falciparum malaria; weight under 5 kg; haemoglobin < 8 g/dl; severe malnutrition; the presence of febrile conditions caused by diseases other than malaria or other known underlying chronic or severe diseases; regular medication, which may impair anti-malarial pharmacokinetics; a history of hypersensitivity reactions or contraindications to AL; pregnancy or breastfeeding; being unable to or unwilling to take contraceptives (for women of child-bearing age).
Sample collection
Thick and thin blood films were obtained and examined at screening on day 0 to confirm adherence to the inclusion and exclusion criteria. Thick blood films were also examined on days 2, 3, 7, 14, 21, 28, or any other day when the patient returned spontaneously, and parasitological reassessment was required. Specimens were labelled anonymously (study number and day of follow-up). Two to three drops of blood samples were collected on Whatman 903 filter paper during screening and each time treatment failures were encountered. Specimens were labeled anonymously (study number, day of follow-up), dried, kept in individual plastic bags with desiccant pouches and protected from light, humidity, and extreme temperature.
Microscopic analysis
Three blood slides per patient were obtained at enrolment: two thick blood films and one thin blood film. One thick blood film was then stained rapidly (10% Giemsa 15 min) for initial screening, while the others were retained. When the patient was subsequently enrolled, the second blood film was stained slowly (3% Giemsa for 45 min). This was used to calculate the parasite density, by counting the number of asexual parasites in a set number of white blood cells (typically 200) with a hand tally counter. The same technique was used to establish the parasite count on each subsequent blood film. A blood film was considered negative when examination of 1000 white blood cells (WBCs) showed no asexual parasites. The presence of gametocytes on an enrolment or follow-up slide was recorded. In addition, 100 fields of the second thick film were examined to exclude mixed infections; in case of any doubt, the thin film was examined. Parasite density was calculated as follows:
$${\text{Parasite density}}\, (\text{Per}\; \mu l) = ({{\text{parasites counted}} \mathord{\left/ {\vphantom {{\text{parasites counted}} {\text{WBCs counted}}}} \right. \kern-0pt} {\text{WBCs counted}}})\;8000$$
As a quality control measure, all slides were examined by two senior WHO-certified microscopists and during discrepancy a third microscopist read the result and the agreed results were final.
Haemoglobin measurement
Finger-pick blood samples were used to measure haemoglobin on day 0, day 14 and day 28. Haemoglobin was calculated by taking one third of the hematocrit level.
Treatment and followup
AL (20 mg/120 mg) [Batch No.: (10): HWE110049; Mfg: (11), 01/2020; Exp: (17):12/2021] manufactured by Ipca Laboratories Ltd (India) was obtained from the WHO Addis Ababa office. Participants were treated twice daily for three consecutive days with the standard six-dose regimen of AL (Additional file 1: Table S1). Drug dosage was determined according to the patient’s body weight. The first dose of the medicine was administered under the supervision of a well-trained clinician. The study patients were observed for 30 min after medicine administration for adverse events or vomiting. Any patient who vomited during this observation period were re-treated with the same dose of medicine and observed for an additional 30 min. The first doses of day-1 (2 d of treatment) and day-2 (3 d of treatment) were directly observed by the study staff, while evening doses were given each day to patients or guardians to be administered at home”. Patients were verbally monitored if they encountered any problem in taking the second dose and confirm taking of the drug. On each day, the first doses for children were given with milk biscuits. All participants were also advised to take the evening doses with food. Thereafter, patients were required to undergo regular clinical and parasitological reassessment as detailed above. Patients or guardians were advised to return on any day during the follow-up period if symptoms returned and not to wait for the next scheduled visit day.
Patient withdrawal
The following general criteria were set forth to classify patients as withdrawn: withdrawal of consent; failure to complete treatment (due to: persistent vomiting of the treatment, failure to attend the scheduled visits during the first three days, or serious adverse events necessitating termination of treatment before the full course is completed); enrolment violation (i.e. severe malaria on day 0, erroneous inclusion of a patient who does not meet the inclusion criteria); voluntary protocol violation (i.e. self- or third-party administration of anti-malarial drug or antibiotics with anti-malarial activity); involuntary protocol violation (i.e. occurrence during follow-up of concomitant disease that would interfere with a clear classification of the treatment outcome, detection of a mono-infection with another malaria species during follow-up, or misclassification of a patient due to a laboratory error (parasitaemia), leading to administration of rescue treatment).
Classification of treatment outcomes
Treatment outcome of patients were classified according to the WHO guideline [8]. On the basis of an assessment of the parasitological and clinical outcome the protocol classifies treatment outcomes as early treatment failure (ETF), late clinical failure (LCF), late parasitological failure (LPF) or an adequate clinical and parasitological response (ACPR).
Early treatment failure
Development of danger signs or severe malaria on day 1, 2 or 3, in the presence of parasitaemia; parasitaemia on day 2 higher than on day 0, irrespective of axillary temperature; parasitaemia on day 3 with axillary temperature ≥ 37.5 °C; and parasitaemia on day 3 ≥ 25% of count on day 0.
Late clinical failure
Development of danger signs or severe malaria in the presence of parasitaemia on any day between day 4 and day 28 in patients who did not previously meet any of the criteria of early treatment failure; and presence of parasitaemia on any day between day 4 and day 28 with axillary temperature ≥ 37.5 °C in patients who did not previously meet any of the criteria of early treatment failure.
Late parasitological failure
Presence of parasitaemia on any day between day 7 and day 28 with axillary temperature < 37.5 °C in patients who did not previously meet any of the criteria of early treatment failure or late clinical failure.
Adequate clinical and parasitological response
Absence of parasitaemia on day 28, irrespective of axillary temperature, in patients who did not previously meet any of the criteria of ETF, LCF or LPF.
Safety endpoint
Safety was assessed by recording the nature and incidence of adverse events. Adverse events were assessed by direct interview. All patients were consistently asked about prior symptoms as well as new symptoms that had appeared since their last appointment. The incidence of any adverse event, independent of its relationship to the study drug, was used as a safety end-point. An adverse event was defined as any unfavourable, unintended sign, symptom, syndrome or disease that develops or worsens with the use of the study drug, regardless of whether it is related to the medicinal product.
Molecular genotyping
Dried blood spots were obtained for PCR analysis on day 0 and on days of treatment failure to differentiate a recrudescence (same parasite strain) from a newly acquired infection (different parasite strain). A nested PCR analysis was conducted on paired dried blood spots (day 0 and day of treatment failure) for treatment outcomes classified as late treatment failures (LCF or LPF). PCR correction was made based on fragment analysis on gel electrophoresis. The WHO recommends the use of three markers (msp1, msp2, and glurp) were to make primary end point analysis. The current study used two of the three markers, the most polymorphic msp2, and the less polymorphic msp1 (Additional file 2: Table S2 and Additional file 3: Table S3). The other highly polymorphic marker result was not included for technical and logistic reasons. PCR genotyping of the polymorphic genes was done at Ethiopian Public Health Institute (EPHI) according to the recommendation of the WHO.
Ethical consideration
The study was initiated after getting ethical clearance from the institutional ethical review board of the Scientific and Ethical Review Office of EPHI and Ethics Review Committee of the School of Pharmacy, Addis Ababa University. Letter of Permission to conduct the study was also obtained from Arbaminch Zuria district office and Shecha health centre. Patients were enrolled in the study after informed consent (parents or guardians for children). Additional assent was obtained for children from age 12 to 17 years.
Statistical analysis
Data entry and analysis were done using the WHO designed Excel spreadsheet. Efficacy data was analysed using the Kaplan-Meier (K-M) and per-protocol (PP) analysis methods. In PP analysis, the efficacy estimate (the proportion cured) of AL was derived by taking all participants followed until treatment failure and adequate response while excluding those who were withdrawn and lost to follow-up. In the K-M approach, patients without treatment failure during the study period and who did not complete follow-up due to withdrawal or lost to follow-up were included in the analysis until the last recorded visit when they were censored. In addition to the criteria for withdrawal, patients were withdrawn from the analysis when PCR results were unclassifiable.