Therapeutic efficacy of artemether–lumefantrine for the treatment of uncomplicated Plasmodium falciparum malaria in Enfranze, north-west Ethiopia

Study area

The study was conducted at Enfranze Health Centre. Enfranze is a sub-district, located
in North Gondar administrative zone, Amhara Region, 675 km north of Addis Ababa and
60 km from Gondar town and at an elevation of 1,500 m above sea level. This area is
malaria-endemic with a total population of about 45,686 (municipality report) and
the majority of the population depends on subsistence farming.

Study design and period

The design was a one-arm, prospective evaluation of the clinical and parasitological
response to directly observed treatment for uncomplicated P. falciparum malaria conducted between January and May 2013.

Study subjects

The study subjects were recruited among febrile patients attending Enfranze Health
Centre using inclusion criteria as defined in the WHO guidelines for assessing the
therapeutic efficacy of anti-malarial drugs against P. falciparum malaria 21].

Inclusion criteria and exclusion criteria

The following inclusion criteria were used for the study: mono-infection with P. falciparum, above 6 months of age, a parasitaemia of 1,000—100,000/?l, weight 5 kg, presence
of axillary temperature (?37.5°C) and no use of anti-malarial drugs 2 weeks prior
to enrollment into the study. Patients with danger signs of severe and complicated
malaria according to WHO criteria (including severe anaemia defined as haemoglobin
5 g/dl), history of allergic reactions to the study drug AL, mixed infection with
another Plasmodium species, concomitant presence of febrile conditions with the potential to confound
study outcome (e.g. acute respiratory infection, severe diarrhoea or other known underlying
chronic or severe diseases (e.g. cardiac, renal or hepatic diseases, HIV/AIDS), severe
malnutrition (defined as a child whose growth standard is below ?3 z-score, has symmetrical
oedema involving at least the feet or has a mid-upper arm circumference 110 mm for
6 month–18 years of age children and has a mid-upper arm circumference 170 mm, BMI
16 with or has a mid-upper arm circumference 180 mm with recent weight loss or underlying
chronic illness for adults), as well as pregnant and lactating women, were not included
in the study.

Sample size and sampling technique

The sample size was determined using a single population proportion formula according
to the WHO guidelines: assuming a maximum of 25% clinical failures, 10% precision,
and a confidence level of 95% with up to 10% losses to follow up a sample size of
80 was calculated 21].

Data collection procedures

A rapid screening procedure was used in an outpatient setting to identify patients
who meet enrolment criteria. The typical screening data set included age, sex, temperature,
body weight, pregnancy test, initial blood slide examination and haemoglobin. All
patients meeting the basic enrolment criteria during the screening procedure were
evaluated in greater depth by a member of the study team. Physical examination was
performed at baseline (day 0 before dosing) and on days 1, 2, 3, 7, 14, 21 and 28.
Body weight was determined on day 0 using a weight scale. The screening weight was
used to calculate the dose (number of tablets) to be administered. Axillary temperature
was measured at baseline (day 0 before dosing) and on days 1, 2, 3, 7, 14, 21 and
28. Female patients of child-bearing age (12–49 years) were asked to provide a urine
sample for pregnancy testing before enrolment in the study and if sexually active
were provided with condoms for the duration of the study.

Sample collection and processing

Finger-prick blood samples were collected from consenting patients for malaria parasite
identification and haemoglobin level measurement. Patients that satisfied the criteria
were enrolled into 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. Another drop of blood
was collected on Whatman 903
^®
filter paper on day 0 during enrollment and in case of recurrent parasitaemia. The
filter paper was air dried and stored in a self-sealing plastic bag with desiccators
for further molecular analysis.

Microscopic diagnosis of malaria parasites

Thick and thin blood smears were prepared and stained with 10% Giemsa (pH 7.4) for
10 min and read by two senior microscopists. Blood films were taken at least eight
times for each patient during the study period (day 0, 1, 2, 3, 7, 14, 21 and 28)
and during any unscheduled visit. A blood film was considered negative when no parasites
were seen after examining 100 high power fields on the thick film. Parasites were
counted on thick films relative to 200 leukocytes by two microscopists blinded to
each other’s results. Blood smears with discordant results (differences between the
two microscopists in species diagnosis, or differences in parasite density of 50%)
were re-examined by a third, independent microscopist, and parasite density was calculated
by averaging the two closest counts.

Genotyping of malaria parasites

In order to differentiate a recrudescence from a newly acquired infection, blood spots
were collected from all patients at day 0 (before drug intake) and in case of LPF
on Whatman filter paper and sent to Medical University of Vienna for genotyping of
merozoite surface protein 1 (MSP1), merozoite surface protein 2 (MSP2) and glutamate-rich
protein (GLURP). To exclude mixed infections or infections with other human malaria
parasites the samples were analysed with nested PCR for species classification as
reported previously 22], 23]. Afterwards P. falciparum monoinfections were genotyped. Gene loci—glurp, msp1 and msp2—of these samples were compared by PCR as described previously 24].

Haematological assessment

Finger-prick blood samples were used to measure haemoglobin. Due to limited resources
the actual haemoglobin concentration should be measured by hemocue. However, this
study assessed the haematocrit value only. In healthy persons, the haematocrit (expressed
as a percentage) is roughly three times the haemoglobin concentration (expressed in
grams per decilitre). This ratio is maintained in normocytic anaemia, but in most
of the tropical forms of chronic anaemia the ratio is 3.3:1.

Treatment and follow-up of patients

All eligible patients were treated with AL (Coartem
^®
) (Novartis Pharmaceutical Corporation, Suffern, New York, USA for Novartis Pharma
AG, Basel, Switzerland, and Bach No. F-2832) twice daily on days 0, 1, and 2. Study
participants were advised to take the study drug with milk to improve absorption.
Study medication was administered based on weight; the first and each morning dose
were directly observed by the study staff 25]. The evening doses were given to the patient/guardian for self-administration in
the presence of 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 health centre on days 1, 2, 3, 7, 14, 21, and 28 or whenever they did not feel
well. Patients withdrawn or with complications were referred to the health centre
for proper treatment. Patients experiencing a reemergence of P. falciparum parasitaemia were treated with quinine.

In vivo analysis and classification response

Patients were classified as early treatment failure (ETF), late clinical failure (LCF),
and late parasitological failure (LPF, adequate clinical and parasitological response
(ACPR) as per WHO definition 21].

Data analysis

After checking for completeness all data were imported into Excel and Kaplan–Meier
survival analysis was used for analysing primary (ETF, LCF, LPF, and ACPR) and secondary
(PCT, FCT, GCT) outcomes, Cox regression was used to identify predictor variables
of secondary outcomes. P values 0.05 were considered statistically significant.

Ethical consideration

The study protocol was reviewed and approved by the Ethical Review Committee of the
School of Biomedical and Laboratory Sciences, College of Medicine and Health Sciences,
University of Gondar. Written informed consent was obtained from all study participants
or their legal representatives after being translated and read in the vernacular language.