The clinical trial protocol was approved by the national Ethic Committee of Yaoundé, Cameroon, and by the Ethics Committee of the Technical University of Dresden, Medical Faculty, Germany. This study is registered with ClinicalTrials.gov as NCT00978172.
Young children between 10 and 20 kg of weight attending the Clinic of Centre Mère et Enfant de la Fondation Chantal Biya in Yaoundé, Cameroon, were considered for enrolment if they presented with acute uncomplicated P. falciparum malaria (according to WHO criteria). Inclusion criteria: count of asexual forms of P. falciparum between 2,000 (amended to 1,000 μl of blood) and 250,000 parasites, fever or history of fever, written informed consent of the guardian, and the ability of the child to take oral medication.
Exclusion criteria: children were excluded if they suffered from severe malaria (according to WHO criteria), or had a history or evidence of clinically significant neurological, psychiatric, cardiovascular, pulmonary, metabolic, gastrointestinal, oncologic or endocrine disease. In addition, children were excluded if they were treated with anti-malarial drugs within 7 days prior to diagnosis or if they had participated in any investigational drug trial within 30 days prior to enrolment. Further exclusion criteria were vomiting three or more times within 24 h of enrolment, more than three copious liquid stools within 24 h, parenteral treatment, allergy to artesunate or mefloquine, splenectomy, HIV infection, or renal impairment.
A full medical history and clinical examination was carried out after obtaining consent from the children's guardians. The neurological and neuropsychiatric assessment was performed before administration of the study drug and presented the baseline value.
According to the study protocol, examination visits took place on baseline, and days 4, 7 (±1), 28 (±2) and 63 (±4). Malaria blood smears were checked on each visit and PCR blood samples were taken at each visit. Haematological exams (haemoglobin, haematocrit, RBC, WBC) were carried out on baseline and days 4, 7, 28 and 63. A complete physical examination was carried out on baseline and day 63. Vital signs and symptoms of malaria were recorded during every visit. Adverse events could be reported any time, at the latest on the next visit.
Adverse events were defined and classified as "mild", "moderate" and "severe" according to ICH Harmonised Tripartite Guideline for Good Clinical Practice E6 (R1) . All adverse events were recorded. For evaluation they were divided into "drug-related" ("certain", "probably", "possibly" and "unlikely") and "not drug-related". A written statement by the investigator was required to justify why the neurological and neuropsychiatric adverse event was not drug-related.
The neurological and neuropsychiatric examination
The neurological and neuropsychiatric examination took place on the baseline visit and on days 7, 28 and 63. Performing the examination took approximately 30 minutes. Children were questioned and examined by the same investigator during all visits. Three experienced paediatricians of the Centre Mère et Enfant de la Foundation Chantal Biya were trained in the use of the questionnaire and in the standardized neurological assessment with a video recording of the examination and by examining volunteer patients according to the protocol. This training involved demonstrating the tests and then getting the investigator to perform and score the examination by consensus. During the study course, the training was repeated after 7 months.
Phrasing and questions were tested in about 15 patients and guardians in Yaounde, Cameroon prior to the beginning of the study to ensure, that the questions were well understood by the guardians to identify accurately the neuropsychological symptoms.
The neurological and neuropsychiatric examination included 23 questions to the guardian concerning his/her observations, questions to the child (older than 3.5 y as a rule), the investigators observations and twelve clinical assessments. The questionnaire was available in French and English, containing twenty-three prephrased questions covering dizziness, vertigo, headache, convulsions, paraesthesia (only >3.5 y), dysaesthesia (only >3.5 y), insomnia, nightmares (only >5 y), hyperactivity, anxiety, panic attacks, sadness, mood changes, confusion, aggressive behaviour, tension, visual hallucinations, acoustic hallucinations (only >3.5 y), hearing loss, dysarthria, word-finding disturbance (only >3.5 y), eating behaviour and swallowing disturbance. Questions were addressed to the guardian with specific questions to the child him/herself. To avoid misunderstanding or misinterpretation, all investigators used the same terms to ask those questions.
The neurological examination covered twelve items: tremor, dystonia and ataxia, hyperreflexia, hyporeflexia, clonus, dysdiadochokinesis, disturbed vision, nystagmus, double vision, acoustic acuity, forgetfulness, and word-finding disturbance. For this exam investigators were equipped with an examination set containing a description of the examination process, a reflex hammer, a lamp and a Lang Stereo Vision test (item disturbed vision), five coloured building blocks (item forgetfulness), a toy car (item word-finding disturbance), and a Denver Developmental test.
Tremor, dystonia and ataxia, hyperreflexia, hyporeflexia, clonus, dysdiadochokinesis, nystagmus, double vision and acoustic acuity were tested doing a standard paediatric neurological examination based on Touwen . Word-finding disturbance was tested by showing the child a toy car, a bird (or telephone), a key, a pen, a chair and asking to name the object. A score of "NOT present" for word-finding disturbance was given if the child could name all objects promptly.
Some exams/questions were age restricted to certain age limits or modified according to the age of the child. Item forgetfulness was tested in children younger than 3.5 years by presenting the child 3 coloured building blocks in a given order and letting the child repeat the placement after mixing the building blocks. Forgetfulness in children older than 3.5 years was tested by enumerating number groups. To begin the test, the child had to repeat two groups of two digits, then two groups of three digits, then two groups of four digits, then two groups of five digits.
During the baseline visit, it was recorded whether a 2-digit, 3-digit, 4-digit or 5-digit group could be repeated, and a score of one point per correct repeat was given. During follow-up visits, the forgetfulness was recorded as "present" if there was a decrease in the digit group that could be repeated. For example, if a child was able at baseline to repeat a 4-digit group but could not repeat a 3-digit group on day 28, this was marked as presence of forgetfulness. This procedure was chosen in accordance with tasks from cognitive tests such as Hamburg Wechsler Intelligence Test for Children  or Kaufman-Assessment Battery for Children .
Children could refuse to participate. If the exam could not be performed investigators scored this with "not done". Otherwise questions were scored with "yes (disorder present) " or "no (disorder not present) " at baseline. In the follow-up visits questions were scored with "no (disorder not present) " or "yes (disorder present, still the same)" or "yes (disorder present, improved)" or "yes (disorder present, worsened or new)" in which case an adverse event was reported. The option "not done" was chosen if the child was not cooperating. All children were followed up for fever and parasite clearance irrespective of their participation in neurological and neuropsychiatric examinations.
Only the appearance of new symptoms - occurring after baseline - was evaluated as an adverse event. If a child was already hyperactive before taking the study medication, this was not considered as an adverse event at the next study visit, if it was still present. But if hyperactivity appeared newly afterwards during the study course or worsened during two visits, it was reported as an adverse event.
The clinical neurological and neuropsychiatric examination is based on standard paediatric neurological examination (based on Touwen ); the questionnaire was adapted to symptoms recorded in adults during mefloquine treatment or prophylaxis. With respect to the absence of a standardized test for paediatric neurological and neuropsychiatric evaluation in this age group in a resource-poor setting, the Division of Child Neurology of the University Children's Hospital of Basel, Switzerland, developed the examination setting used in this study.