Study site and design
The study was conducted in Seacha area, Arba Minch Zuria District, located in the Gamo Gofa zone of the Southern Nations, Nationalities, and Peoples’ Region of Ethiopia (Fig. 1). The study area is located about 500 km south of Addis Ababa in the Greater Rift Valley, at latitude 6° 01′ 59.99′′ N and longitude 37° 32′ 60.00′′ E, at an elevation of 1285 m above sea level. The average annual temperature is 29.7 °C and the average annual rainfall is 700 mm. Malaria transmissions in the study area is highly seasonal and markedly unstable with an entomological inoculation rate of 71.1 infectious bites per person per year [18]. Plasmodium vivax is the predominant parasite species and Anopheles arabiensis is the major vector. Based on the current malaria stratification and mapping of the country [19], this study area is considered an area of moderate (altitude range from 1000 to 1750 m) P. vivax transmission.
Study population, inclusion, and exclusion criteria
Febrile patients visiting the outpatient department of the Seacha Health Centre who fulfilled the inclusion criteria set by the WHO protocol for the assessment of the therapeutic efficacy of anti-malarial drugs were eligible for inclusion [20]. Inclusion criteria were age 6 months or older, fever (axillary temperature ≥ 37.5° C) or a history of fever in the preceding 24 h, microscopically confirmed P. vivax mono-infection, with an asexual parasitaemia of 250 parasites/μL or above and living within the facility catchment area (i.e. < 5–10 km radius of the health centre). Participants were excluded if they were pregnant, breast-feeding, had mixed species infection (P. falciparum and P. vivax), had a day 0 haemoglobin < 5.0 g/dL, were unable to take oral medication or had repeated vomiting, had known hypersensitivity to the study drugs, had evidence of severe malaria, heart or liver diseases, had severe malnutrition, or had evidence of a non-malarial febrile illness (i.e. otitis media, tonsillitis, measles, acute lower respiratory tract infection, severe diarrhoea with dehydration). Primaquine therapy was not provided until the day of first recurrence or day 42.
Sample size determination
The sample size was determined according to the WHO protocol [20]; using the single population proportion formula and calculated assuming a 5% margin of error, 95% confidence interval (CI). A minimum sample size of 73 was calculated, with a 20% increase to allow for loss to follow-up and withdrawal during the 42-day follow-up period, at least 88 patients were to be recruited.
Treatment and follow-up
Eligible patients were treated with a full three-day course of DHA–PPQ. DHA–PPQ (DHA 40 mg, PPQ 320 mg per tablet, Guillin Pharmaceuticals China; Bach no. SQ200201) was provided by the Ethiopian Federal Ministry of Health (FMoH) through WHO support. Daily drug dosage was based on body weight as follows: half a tablet, 5 to < 8 kg; three-quarters of a tablet, 8 to < 14 kg; one tablet, 14 to < 25 kg; two tablets, 25 to < 36 kg; three tablets, 36 to < 60 kg, four tablets 60 to < 80 kg and five tablets, > 80 kg.
All medication doses were given under the direct supervision of the study team. Medication given to young children was crushed, mixed with water, and administered as a suspension. Patients were observed for thirty minutes to 1 h after each dose to monitor for vomiting or other side effects. If vomiting occurred within 30 min after administration, the full dose was re-administered. If vomiting occurred between 30 min and 1 h after administration, half of the dose was re-administered. Patients who vomited a second time were withdrawn from the study and received parenteral therapy according to national guidelines. Standard dose primaquine (0.25 mg/kg daily for 2 weeks) was given to patients at the end of the follow-up period or at recurrence.
Patients were followed-up daily for the first 3 days after the first dose (day 0) and then weekly on day 7, 14, 21, 28, 35, and 42. Clinical and laboratory evaluations were undertaken during each follow-up visit. Patients were also assessed on an unscheduled visit if symptoms occurred. Adverse events and severe adverse events were defined according to the WHO protocol for monitoring the therapeutic efficacy of anti-malarial drugs and were monitored at each follow‐up visit [20]. Fever clearance and parasite clearance were assessed over the first seven days. Late clinical failure (LCF) and Late parasitological failure (LPF) was defined as the reappearance of parasitaemia between day 4 and day 28 (or 42) with fever and without, respectively, and without previously meeting any of the criteria for ETF or LCF,
Laboratory procedures
Thick and thin blood smears were prepared on a single slide, for parasite detection and species identification, respectively. Two smears were prepared from all participants at all follow-up visits. The first slide was prepared by staining with 10% Giemsa for 10–15 min for initial screening. The second slide was stained using 3% Giemsa for 30 min and read by two independent laboratory technicians from the health centre; in case of discrepancy between the first and second readings, a third reading was performed. Parasite densities were recorded for all positive slides. The number of asexual parasites was counted per 200 white blood cells (WBC) and parasitaemia was estimated assuming WBC counts of 8000/µl. Gametocytes were counted against 500 WBCs. Before any blood smear was interpreted as negative, two hundred oil-immersion high power fields on the thick film were read [21]. To ensure microscopy quality, all slides were cross-checked by a WHO-accredited microscopist at the Adama Malaria Control and Monitoring Centre. Haemoglobin concentrations were measured using a portable spectrophotometer (HemoCue®, Angelholm, Sweden), on days 0, 14, 28, and 42. Female study participants aged 12 years and older were screened for pregnancy on enrolment.
Statistical analysis
Study endpoints were categorized into primary and secondary endpoints. The primary outcome was the day 42 overall efficacy of DHA–PPQ stated as polymerase chain reaction (PCR)-uncorrected proportion with adequate clinical and parasitological response (ACPR). Secondary endpoints were the incidence of adverse events, fever and parasite clearance, and haemoglobin levels across the follow-up period. Data were double-entered into the WHO Excel spreadsheet designed for therapeutic efficacy data. Data were also entered into IBM SPSS (version 24) software to calculate descriptive statistics (mean, standard deviations, and percentages). Based on WHO guidelines, the adequate clinical and parasitological response on day 42 was calculated using the Kaplan–Meier cumulative risk method [20].
Ethical approval
The study was approved by the Institutional Review Board (IRB) of the Ethiopian Public Health Institute (EPHI). Written informed consent was obtained from all of the study participants ≥ 18 years of age or by parents or guardians of children less than 18 years of age, with children 12–17 years of age providing written informed assent.