The study was conducted at Aduku Health Centre, Apac District, Uganda. The district experiences perennial holoendemic malaria. The entomological inoculation rate in Apac, a measure of transmission intensity, was measured at 1,564 infectious bites per person per year [15
]. The study protocol was approved by the Makerere University Research and Ethics Committee, the Uganda National Council of Science and Technology, and the University of California San Francisco Committee for Human Research.
Consecutive patients presenting to the health center with symptoms suggestive of malaria and a positive screening thick blood smear were referred to study physicians for further assessment. Patients were enrolled if they fulfilled the following selection criteria: (1) age 6 mo to 10 y; (2) weight ≥ 5 kg; (3) history of fever in the last 24 h or axillary temperature ≥ 37.5 °C; (4) no history of serious side effects to study medications; (5) no evidence of a concomitant febrile illness; (6) provision of informed consent by a parent or guardian; (7) no danger signs or evidence of severe malaria; and (8) Plasmodium falciparum monoinfection with parasite density 2,000–200,000/μl of blood. Because laboratory results were generally not available until the following day, a patient could be excluded after randomization.
At enrollment, we asked children's parents or guardians about prior antimalarial therapy, use of other medications, and presence of common symptoms. Axillary temperature and weight were measured, and a physical examination was performed. A brief neurological assessment, consisting of simple clinical tests for fine finger dexterity (ability to pick up a small object), was undertaken. We also obtained blood by fingerprick for thick and thin blood smears, for hemoglobin assessment, and to store on filter paper for molecular analysis.
Patients were asked to return for follow-up on days 1, 2, 3, 7, 14, 21, 28, 35, and 42, and any other day that they felt ill. Follow-up evaluation consisted of a standardized history and physical examination, including neurological assessment on all days of follow-up. We obtained blood by fingerprick for thick blood smears and storage on filter paper on all follow-up days except day 1. Hemoglobin measurement was repeated on day 42 or the day of recurrent symptomatic malaria. If patients did not return for follow-up, they were visited at home.
Blood smears were stained with 2% Giemsa for 30 min. Parasite densities were determined from thick blood smears by counting the number of asexual parasites per 200 white blood cells (WBCs), or per 500 if the count was less than 10 parasites/200 WBCs, assuming a WBC count of 8,000/μl. A smear was considered negative if no parasites were seen after review of 100 high-power fields. We also assessed gametocytemia from thick blood smears. Thin blood smears were reviewed for non-falciparum infections. A second microscopist, who was unaware of the results of the first reading, re-read all slides. A third microscopist unaware of the first two readings resolved discrepant slides. Hemoglobin measurements were made using a portable spectrophotometer (HemoCue, http://www.hemocue.com
On day 0, patients were randomly assigned to receive AL or DP. A nurse administered study medications according to weight-based guidelines for administration of fractions of tablets. We administered all drugs orally as follows: AL (Coartem, Novartis, 20 mg artemether/120 mg lumefantrine tablets), administered according to weight as one (5–14 kg), two (15–24 kg), three (25–34 kg), or four (≥ 35 kg) tablets given twice daily for 3 d; DP (Duocotexin, Holley Pharm, 40 mg dihydroartemisinin/320 mg piperaquine tablets), targeting a total dose of 6.4 and 51.2 mg/kg of dihydroartemisinin and piperaquine, respectively, given in three equally divided daily doses to the nearest quarter tablet. We used a pill cutter to ensure that the tablet fractions were as close to the nearest quarter tablet as possible. Participants in the DP group also received placebo tablets administered in the evening over 3 d to simulate the AL dosing schedule. Study medications were administered with water, and patients were given a glass of milk after each dose of study medication.
All treatment was directly observed. Participants were given the option either to wait at the clinic for the evening dose (lunch was provided) or to leave the clinic and return in the evening (transport was provided). After each dose, children were observed for 30 min, and the dose was readministered if vomiting occurred. All patients were provided with a 3 d supply of acetaminophen for treatment of febrile symptoms. Children with hemoglobin of less than 10 g/dl were treated according to Integrated Management of Childhood Illness guidelines with ferrous sulfate for 14 d and antihelminthic treatment if appropriate. Households of all patients were given two long-lasting insecticide-treated bed nets (ITNs) (PermaNet, Vestergaard Frandsen, http://www.vestergaard-frandsen.com
) on the day of enrollment, with instructions for one net to be used by the study patient.
The objectives of the study were to compare the efficacy and safety of AL and DP for the treatment of uncomplicated falciparum malaria at a high transmission–intensity site in Uganda.
Treatment outcomes were classified according to 2006 World Health Organization (WHO) guidelines as early treatment failure (ETF; danger signs or complicated malaria or failure to adequately respond to therapy days 0–3); late clinical failure (LCF; danger signs or complicated malaria or fever and parasitemia on days 4–42 without previously meeting criteria for ETF or LPF); late parasitological failure (LPF; asymptomatic parasitemia days 7–42 without previously meeting criteria for ETF or LCF); or adequate clinical and parasitological response (absence of parasitemia on day 42 without previously meeting criteria for ETF, LCF, or LPF) [16
]. Patients were treated with quinine sulfate (10 mg/kg three times daily for 7 d) on the day that they fulfilled criteria for early treatment failure or late clinical failure. Patients with late parasitological failure were followed, and were given quinine only if they developed fever with parasitemia or remained parasitemic on the last day of follow-up. Patients were excluded from further follow-up after enrollment if any of the following occurred: (1) use of antimalarial drugs outside of the study protocol; (2) withdrawal of consent; (3) loss to follow-up; (4) protocol violation; or (5) death due to a nonmalarial illness.
The primary efficacy outcomes were the 28- and 42-d risks of early treatment failure or recurrent parasitemia (LCF or LPF), unadjusted and adjusted by genotyping. Secondary efficacy outcomes included prevalence of fever and parasitemia during the first 3 d of follow-up, change in mean hemoglobin from day 0 to day 42 or day of repeat therapy, and prevalence of gametocytemia (presence of gametocytes on thick smears) during follow-up in participants lacking gametocytes at enrollment.
Molecular genotyping techniques were used to distinguish recrudescent from new infections for all patients with LCF or LPF response. Briefly, parasite DNA was isolated from filter paper blood samples collected at enrollment and on the day of recurrent parasitemia using chelex extraction. Paired samples were genotyped in a stepwise fashion using merozoite surface protein (msp)-2, msp-1,
and four microsatellites [17
]. If, for any of the six loci, an allele was not shared between day 0 and day of recurrence, the infection was classified as a new infection. If at least one allele was shared between day 0 and day of recurrence at all six loci, the infection was classified as a possible recrudescence. The term “possible recrudescence” was used because the complexity of infection (number of infecting parasite strains) was very high in our setting, making it difficult to distinguish definitively a true recrudescence from a new infection.
Secondary safety outcomes included risks of serious adverse events and common adverse events of any severity. An adverse event was defined as any untoward medical occurrence, irrespective of its suspected relationship to the study medications [18
]. At each follow-up visit, patients were assessed for any new or worsening event. All events were graded by severity (none, mild, moderate, severe, life-threatening) and relationship to study treatment (none, unlikely, possible, probable, or definite) using guidelines from the World Health Organization (Toxicity Grading Scale for Determining the Severity of Adverse Events) [19
]and the United States National Institutes of Health, Division of Microbiology and Infectious Diseases (Pediatric Toxicity Tables, May 2001) [20
]. A serious adverse event was defined as any adverse experience that resulted in death, life-threatening experience, inpatient hospitalization, persistent or significant disability or incapacity, or specific medical or surgical intervention to prevent serious outcome.
We calculated sample size to test the hypothesis that the risk of recurrent parasitemia after 42 d would differ between the two treatment groups. Based on previous data, the risk of recurrent parasitemia (unadjusted by genotyping) after 42 d was estimated to be 50% after treatment with AL [3
]. Using this estimate, we calculated that 200 patients (allowing for 10% loss to follow-up) would need to be enrolled in each treatment arm to detect a 15% risk difference between the treatment groups with a two-sided type I error of 0.05 and power of 80%.
Randomization: Sequence Generation, Allocation Concealment, Implementation
A randomization list was computer generated by an off-site investigator without the use of blocking or stratification. Sequentially numbered, sealed envelopes containing the treatment group assignments were prepared from the randomization list. The study doctors assigned treatment numbers sequentially and the study nurse allocated treatment by opening the envelope corresponding to the treatment number. The randomization codes were secured in a locked cabinet accessible only by the study nurse. Participants were enrolled by the study physicians, and treatments were assigned and administered by the study nurse.
Only the study nurse was aware of treatment assignments. All other study personnel, including the study physicians and laboratory personnel involved in assessing outcomes, were blinded to the treatment assignments. Patients were not informed of their treatment regimen, but the color of the two study medications was not the same (DP and placebo tablets were light blue; AL tablets were light yellow).
Data were entered and verified using Epi Info version 6.04 and analyzed using STATA version 8.0 (STATA, http://www.stata.com
). Efficacy and safety data were evaluated using a modified intention-to-treat analysis which included all patients who fulfilled enrollment criteria. Patients who were randomized to therapy but not enrolled in the study due to laboratory results available on day 1 were not included in the analysis. Risks of recurrent parasitemia at 28 and 42 d of follow-up (adjusted and unadjusted by genotyping) were estimated using the Kaplan-Meier product limit formula. Data were censored for patients who did not complete follow-up and for new infections when estimating outcomes adjusted by genotyping. Patients with LCF or LPF due to non-falciparum species were censored as non-failures at the time they were classified as LCF or LPF. The Z
-test was used to compare the Kaplan-Meier estimates of treatment efficacy at fixed points in time between the treatment groups. Confidence intervals around the difference between Kaplan-Meier estimates were calculated using normal approximation and Greenwood's estimates of standard errors. Categorical variables were compared using Chi-squared or Fisher exact test and continuous variables were compared using the independent samples t
-test. All reported p
-values are two sided without adjustment for multiple testing and were considered statistically significant if below 0.05.