We conducted the trial in Chulaimbo sub County Hospital, Lake Region of Kisumu County, Western Kenya. The area altitude is 1,131M above sea level with yearly rainfall between 1200 mm and 1300 mm. The humidity ranges between 50% - 68% with temperatures ranging between 20oC and 35oC. Chulaimbo Sub County is a malaria-endemic zone with stable Plasmodium falciparum transmission . Malaria infection accounts for 38% of all outpatient hospital visits and 40% of all admissions being children below five years and expectant mothers .
Children aged ≥6 and ≤60 months visiting the outpatient clinic of Chulaimbo Sub County Hospital with signs of uncomplicated malaria were recruited for the study in the month of May 2015 to November 2015. Inclusion criteria was children who were residents of the area, with a body weight of ≥5 kg, history of fever in the previous 24 hours or fever with temperature ≥37.5oC, infected only with Plasmodium falciparum and parasitemia in the range of 2,000 to 200,000 asexual parasites per microliter of blood, no general danger signs of severe and complicated malaria (prostration, breathing difficulties, severe anemia, convulsions and inability to drink or vomiting). Written consent was obtained from the parent or guardian of the child before recruitment into the study. Patients were excluded from the study if they were below 6 months and above 60 months, body weight less than 5 kg with history of fever for more than 24 hours with a temperature of above 37.5oC, multiple infections apart from malaria. Also, the inability to take the drug orally, having taken antimalarial chemotherapy in the past two weeks, evidence of liver disease or acute infection other than malaria and unwilling to participate were excluded from the study.
- This was a single-arm prospective in vivo study intended to assess the efficacy of
- Artemether-lumefantrine resistant after treatment of uncomplicated malaria using WHO
Sample size determination
The sample size determination was calculated using the formula n =Z2 p(1-P)/ e2 as described by Lwanga and Lemeshow .
Z = standard normal deviation of the required confidence.
n = the desired sample size
p = Proportion in the target population estimated to have characteristic being measured. According to KIAS, 2010 the prevalence of malaria stands at 40% or 0.4.
e = the level of statistical significance set.
Therefore substituting Z for 1.96, 0.38 for P and 0.1 for e, then the minimum sample size was
1.962 x 0.38 (1 -0.38) / 0.12 = 90.
Approximately, 0.05 ml of blood from a finger prick was collected; thick and thin smears were prepared on two different slides. One of the slides was stained with 10% Giemsa for 10-15 min and examined by microscopy to detect the presence of the malaria parasites and density estimated. The second thin film blood slide was stained with 3% Giemsa for 30-45 min and used to determine the species and presence of gametocytes. Parasitemia was measured by counting the number of asexual parasites against 200 leucocytes in thick blood films. Parasite density per µL of blood calculated by multiplying the total count by 40, assuming that 1µL of blood had a mean count of 8000 leucocytes . The blood slides were declared negative when the examination of 100 high power fields did not show the existence of any malaria parasite. For quality control, each slide was re-examined by a second laboratory technologist, and those with discrepant results were re-examined by a third technologist. Final parasitemia was calculated from the average between the two readings .
Treatment, clinical monitoring and follow-up
Treatment with AL was done for 3 days according to WHO weight-based regime . A fixed-dose combination of 20 mg of artemether and 120 mg lumefantrine per tablet, translating to one, two or three tablets per patient depending on the individual weight respectively was administered. A full course of AL consisted of 6 doses given twice daily (8 hours apart on day 0 and 12-hour on days 1 and 2) with no gametocide drug added. All drugs used for this study were provided by the Ministry of Health. Patients were observed for 20 minutes to make sure they did not vomit. If vomiting occurred, a repeat dose was given after the patient was in a state of swallowing. Any patient who persistently vomited was withdrawn and treated with parenteral artesunate or quinine according to the national guidelines for the management of severe malaria . Besides, paracetamol was given to all patients with body temperature ≥38oC. Patients were admitted at the health facilities for close monitoring, the drugs were administered orally at the health facility under direct observation of a nurses throughout the three days. The drug was crushed and mixed with spoonful of porridge and little sugar to minimize vomiting and was given to each child to gulp. No serious adverse effects was observed throughout the 28 days follow up.
On completion of the dose, after three days, the patients were allowed to go home and follow-up visits were done on day 7, 14, 21 and 28 or at any time the patient felt unwell. Parents/guardians were informed and encouraged to bring their children back to the clinic whenever they were unwell without waiting for scheduled visits. Parents who did not show up during their scheduled visit by mid-day of the same day they were visited at home by a member of a study team and asked to come to the health facility. If a patient could not be traced for scheduled follow-up, he/she was classified as lost to follow-up. During the visits, both clinical and parasitological assessment were performed. Patients with recurrent infections occurring on day 7 and afterward were treated with artesunate or quinine (tablets, injection/intravenous) based on clinical presentation according to WHO protocol .
Treatment outcome classification
The clinical outcome was corrected with the parasitological cure on day 28 based on WHO in-vivo drug trial protocol of 2009 . Treatment outcome was classified as either early treatment failure (ETF) which refer to danger signs for severe malaria on day 1, 2 or 3, in the presence of parasitemia; parasitemia on day 2 being higher than on day 0 count irrespective of axillary temperature; parasitemia on day 3 with axillary temperature ≥37.5oC; parasitemia on day 3 ≥ 25% of count on day 0. Late clinical failure (LCF) which include the development of danger signs for severe malaria after day 3 in the presence of parasitemia, without previously meeting any of the criteria of ETF; presence of parasitemia and axillary temperature ≥37.5oC or history of fever on any day from day 4 to day 28, without previously meeting any of the criteria of ETF. Late parasitological failure (LPF). This refers to the presence of parasitemia on any day from day 7 to day 28 and axillary temperature < 37.5oC, without previously meeting any of the criteria of early treatment failure or late clinical failure. Adequate clinical and parasitological response (ACPR). This is the absence of parasitemia on day 28 irrespective of axillary temperature without previously meeting any of the criteria of ETF, LTF or LPF.
DNA extraction using chelex method
The DNA extraction from dried blood spots (DBS) was done as described by Walsh et al.  while extraction from DBS as explained by Warhurst et al.  using QIAamp DNA Mini kit (QIAGEN Inc., Germany) according to the manufacturer’s instructions. Briefly, each dried filter paper was cut into small pieces and soaked in Saponin-phosphate buffered saline (PBS) overnight at 4oC. This was followed by washing with 1 x PBS and incubated for 30 minutes. The brown solution from the tube was discarded and 50 microliters of the stock 20% solution and 150 microliters of DNAse free water were added followed by vigorous vortexing. The tubes were then heated at 100oC and centrifuged at 10,000 g for two minutes. Lastly, the supernatant was transferred to a new tube, spun again, a final transfer done and the DNA stored at -20oC.
Merozoites surface antigens screening
During the study, patients with recurrent parasitemia on day 7 onwards, a sample was collected on filter paper. Genotypes from day 0 and day of recurrent parasites was compared to differentiate recrudescence from the new infection. However, there was no re-infection from the follow up of the children who completed the study up to day 28.
Amplification of Pf18sRNA gene
In order to confirm Plasmodium falciparum species infections, amplification of 18s RNA gene was done. Primers pairs designed by  of 18R-18F were used; (5’-CTGAGTCGAATGAACTAGCT-3’) and (5’-CCATTTTACTCGCAATAACG-3’) respectively. The PCR reaction included MgCl2, 400nM, 200 nM of primers, 1U of Taq Polymerase and 1µL of DNA template. Amplification was done using PCR that included: the initial denaturation at 94oC for 3 minutes, followed by 30 cycles of denaturation at 94oC for 1 minute, annealing at 55oC for 2 minutes and extension at 72oC for 2 minutes, and the final extension of 72oC for 10 minutes the reaction was held at 4oC.