Study Area and Population
The study was conducted at Serbo Health Center, Kersa district which is located in Jima zone, Oromia region, 323 km south west of Addis Ababa (Fig. 1) between November 2007 and January 2008. The district is malarious and covers 678.6 km square, with an altitude ranging between 1600-2400 above sea level. Malaria is the most prevalent seasonal disease in the area, accounting for 77.1% of all the reported diseases in the health center in 2006 and 2007. October to December is the peak transmission season. Both P. vivax and P. falciparum exist in the area with P. vivax prevailing all year, even in non-transmission (relapse) seasons.
Study subjects
The study subjects were recruited from febrile patients visiting Serbo Health Center based on WHO inclusion guideline for the assessment and monitoring of antimalarial drug efficacy for the treatment of uncomplicated falciparum malaria [2].
Subject inclusion criteria
The following inclusion criteria were used for the study: Mono-infection with P. falciparum, a parasitemia level of 1000 - 100,000/μl, absence of danger signs or signs of severe and complicated malaria according to the definition given by [2] presence of axillary temperature (> 37.5°C), absence of other concomitant infections like pneumonia which can cause fever, no use of antimalarial drug two weeks prior to the study and compliance for successive visits as per the protocol for uncomplicated malaria recommendations.
Exclusion criteria
Patients with: (i) severe signs of malaria according to WHO criteria, which included severe anaemia defined by haemoglobin < 5 g/dl, (ii) history of allergic reactions to the study drug Coartem® (iii) concomitant presence of febrile condition with the potential to confound study outcome (Example: acute respiratory infection (ARI), measles, severe diarrhea, etc.) (iv) severe malnutrition and (v) pregnant and lactating women were not included in the study [2].
Sample collection
Finger-prick blood samples were collected from consenting patients for malaria parasite identification and hemoglobin level measurement. Patients that satisfied the criteria, were enrolled to the study and followed up on days 1, 2, 3, 7, 14, 21, and 28 where finger-prick samples were taken for microscopic glass slides. A parallel drop of blood was collected on filter paper on day 0 during enrollment and on any unexpected visit. The filter paper was air dried and stored in a self sealing plastic bag with desiccators for further molecular analysis.
Sample size
Considering the very low or unknown proportion of clinical failure of artemether/lumefantrine resistance in Ethiopia, sample size was calculated with 10% precision, 95% confidence interval, 15% withdrawal and 28 day study period, 90 study participants were included in the study [2].
Microscopic diagnosis
Thick and fixed thin blood smears were stained with 10% Giemsa (pH 7.4) for ten min. Blood films were taken at least eight times for each patient during the study period (day 0, 2, 3, 7, 14, 21 and 28) and on any unexpected visit by the patient. These smears were used in species identification and thick blood films were used to determine parasite density (100 high power field (HPF) were the number of asexual parasites per HPF were recorded, according to the method described in the WHO Protocols [2–6]. A blood slide was considered negative when no parasites were seen after examining 100 fields. Parasite count was based on the number of asexual parasites observed against 200 leukocytes. This number was then multiplied by 40 to gain an approximate count per microlitre.
Slides were read by two senior microscopists from the Ethiopian Health and Nutrition Research Institute (EHNRI) and one from Serbo health center. In case of disagreement the majority result was taken. All slides were properly documented for quality control.
Hemoglobin measurement
Finger-pick blood sample was used to measure hemoglobin using a portable spectrophotometer (Haemocue).
Treatment and follow up schedule
Artemether-lumefantrine (Coartem®) was obtained from Novartis pharma AG, Basel, Switzerland, Bach No. T2005-59, through the WHO office, Addis Ababa. All eligible patients were treated with Coartem® at day 0. Dosing was a six dose regimen given twice daily for three days. Study medication was administered based on weight; the first dose was given under direct observation. The successive day's doses were given to the patient/guardian for self administration in front of health professionals in the area (Health extension workers). Patients were followed for 30 min post treatment and if vomiting occurred, a second full dose was administered. If repeated vomiting occurred, patients were withdrawn from the study. Patients were asked to return to the clinic on days 1, 2, 3, 7, 14, 21, and 28 or were highly encouraged to return whenever they did not feel well. Patients, who failed to come to the clinic at the scheduled time, were visited in their home on the same day and the necessary blood sample, reports of adverse effects and temperature data were collected.
Patient withdrawal
Patients were withdrawn from the study in case of (i) vomiting the drug twice, (ii) withdrawal of consent, (iii) onset of a serious febrile illness, (iv) intake of any drug with antimalarial properties, (v) missing repeated treatment doses, (vi) mixed species parasitemia or (vii) any protocol violation. Patients who missed follow-up visits and did not come on the successive day despite tracing were considered as lost to follow-up. Patients withdrawn and with complications were referred to the health centers for proper treatment. Patients withdrawn for the re-appearance of P. falciparum were treated with quinine and those infected with P. vivax were treated with chloroquine.
In vivo analysis and classification response
Patients were classified as early treatment failure (ETF), late clinical failure (LCF), late parasitological failure (LPF) or adequate clinical and parasitological response (ACPR) as per WHO definitions [2].
ETF
Development of danger signs for severe malaria on Day 1, Day 2 or Day 3, in the presence of parasitemia; parasitemia on Day 2 higher than Day 0 count irrespective of axillary temperature; parasitemia on Day 3 with axillary temperature ≥ 37.5°C; parasitemia on Day 3 ≥ 25% of count on Day 0.
LCF
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 >37.5°C (or history of fever) on any day from Day 4 to Day 28, without previously meeting any of the criteria of ETF.
LPF
Presence of parasitemia on any day from Day 7 to Day 28 and axillary temperature > 37.5°C, without previously meeting any of the criteria of early treatment failure or late clinical failure.
ACPR
Absence of parasitemia on Day 28 irrespective of axillary temperature without previously meeting any of the criteria of ETF, LTF or LPF.
Molecular analysis
Samples that were classified as ETF, LCF or LPF were genotyped to differentiate recrudescence from re-infection. Blood samples for PCR were collected on standard filter paper, air dried and stored in cool and dark boxes with desiccants. Genotypic analysis was performed at EHNRI as previously described for msp-1 [7] and msp-2 [8, 9]. Cases in which pre- and post-treatment genotypes were identical were considered as recrudescence; cases in which pre- and post-genotypes were different were considered as re-infection, mixed genotypes were classified as failures.
Ethical clearance
The study protocol was reviewed and approved by Ethical Review Committee of Ethiopian Health and Nutrition Research Institute and Nation Ethical Review Committee of Ethiopia.
Consent form
Prior to the trial, a consent form was signed by the patient or by the parent/guardian after being translated and read in the vernacular language that the patients or the carers understood.
Statistical Analysis
All the data from recruited patients were imported into an Excel spreadsheet and the WHO designed Excel data analysis program was used for analysis and SPSS 11 were used for descriptive statistics and comparing data [2].