About this study
Severe malaria is a life-threatening disease that kills over a million individuals every year, with 90% of the deaths occurring in sub-Sahara African children under the age of five (Greenwood B and Mutabingwa T, 2002). People living in an endemic area often become infected with Plasmodium falciparum malaria during childhood, but a small proportion of children experience severe complications, the clinical outcome depending on many factors, including the genetic make-up of the human host (Verra F, Mangano VD and Modiano D, 2009).
Genetic association studies can inform us on mechanisms of protective immunity against malaria at every stage of infection as well as on mechanisms of pathogenesis. The understanding of which is crucial for the development of effective vaccines and therapeutic measures.
In order to investigate the human genetic determinants of severe malaria in Burkina Faso, we set up a retrospective case-control study including children experiencing severe malaria (cases), children with an attack of uncomplicated malaria (mild malaria controls) and children with no signs and symptoms of malaria or other diseases (healthy controls).
Clinical data and DNA samples were contributed to the MalariaGEN Consortial Project 1 (CP1) along with those of 11 other case-control studies from a total of 11 malaria-endemic countries. As part of the sample handling process by the MalariaGEN Resource Centre, baseline genotyping data has was generated for a number of malaria–associated single nucleotide polymorphisms (SNPs) and the appropriate data has been returned to each site for site-specific analysis. A total of 69 SNPs at candidate genes (selection based on previous reports of association with severe malaria or on their likely biological role in malaria infection/disease) will be included in our analysis. Candidate genes include loci encoding erythrocyte factors (e.g. HBB, ABO, G6PD) as well as immunological factors (e.g. TNFA, TLR4, NOS2A) and factors involved in cyto-adherence (e.g. CD36, ICAM1).
The University of Rome together with Centre National de Recherche et de Formation sur le Paludisme and Centre Médicale St. Camille recruited malaria patients and healthy children from the capital of Burkina Faso, Ouagadougou, and surrounding rural zones in the province of Bazega, district of Sapone.
The study area lies in a plateau characterised by shrubby savannah vegetation. While in the city the type of occupation is diverse, in rural zones the main occupation is subsistence farming. Most people live in compounds housing multiple nuclear families often closely related. The healthcare system at the district level consists of 14 community clinics (CSPS) with a dispensary and a maternity unit, which among other activities provide EPI vaccination and malaria treatment. A medical centre with a surgical unit (CMA) is located in Sapone; the first line of referral. The highest levels of referral are sent to the national hospital (CHN) Yalgado Ouédraogo in Ouagadougou. The population of Ouagadougou is estimated around 1 million and that of the district of Sapone around 90,000 people. The great majority of the population belongs to the Mossi ethnic group.
The climate in this area is characteristic of the Sudanese savannah, with a dry season from November to May and a rainy season from June to October. Malaria is endemic with a marked seasonal pattern characterised by high transmission during the rainy season and low transmission during the dry season. Differences exist in malaria transmission levels between urban and rural areas: entomological inoculation rates vary from 1 to 10 per person per year in urban areas of Ouagadougou, and from 50 to 500 in the surrounding rural zones. P. falciparum is the predominant malaria parasite, accounting for more than 95% of infections in children under 5 years of age. The main malaria vectors are Anopheles gambiae, A. arabiensis, and A. funestus. The use of insecticide-treated nets was uncommon at the time of the study (about 1%) and the use of indoor residual spraying was non-existent with malaria control relying mainly on the treatment of clinical cases (Nebie I et al, 2008).
An unmatched case-control study was conducted. Severe malaria cases and mild malaria controls were children (aged 1 month – 15 years) of Mossi ethnicity, admitted to hospital with signs of severe and uncomplicated malaria, respectively. The children were recruited from patients admitted to the paediatric ward of three hospitals in Ouagadougou (Centre Hospitalier Universitaire Yalgado Ouédraogo, Centre Médical Paul VI and Centre Médical Saint Camille) during the high malaria transmission seasons of 1993-94.
The criteria for inclusion followed the definitions stated by the World Health Organization (WHO). Severe malaria was defined by the presence of P. falciparum in the thick blood film associated with at least one of the following conditions: prostration (incapacity of child to sit without help in absence of coma), unrousable coma (Blantyre coma score ≤ 2), repeated generalised convulsions (more than two episodes in the preceding 24 hr), severe anaemia (haemoglobin <5 g/dl), hypoglycemia (<40 mg/dl), pulmonary edema/respiratory distress, spontaneous bleeding, and renal failure (plasma creatinine >3 mg/dl). Children with other detectable infections were not included in the study. Uncomplicated malaria was defined as a clinical illness characterised by an axillary temperature >37.5°C associated with a P. falciparum-positive thick blood film. Patients were treated according to WHO guidelines with a complete regimen of drugs that were provided free of charge as part of the study (Modiano D et al, 1998; Modiano D et al, 2001).
Healthy controls were children (aged 0-6 years) of Mossi ethnicity and no signs of severe or uncomplicated malaria or other diseases. They were recruited from rural villages 50 km south-west of Ouagadougou (district of Sapone, province of Bazega) during a cross-sectional survey conducted in August 2004. Children presenting with any clinical sign of malaria following direct physical examination were excluded from the study.
A standardised case report form (CRF) was created for the MalariaGEN Consortial Project 1 (CP1) and used by all sites to collect standardised clinical data. The data collected in Burkina Faso (and all other sites) was uploaded onto secure web-based software developed by MalariaGEN. Here, the integrity of the data was checked and data was standardised and amalgamated.
Genomic DNA was extracted from whole blood at the University of Rome La Sapienza using the Qiagen DNeasy Blood kits (http://www.qiagen.com/) [Qiagen, Crawley, UK] and Nucleon™ BACC2 Genomic DNA extraction kit (Gen-Probe Life Sciences Ltd., Manchester, UK) using manufacturer’s instructions. Aliquots of the DNA samples were shipped to the MalariaGEN Resource Centre in Oxford for further processing and quality control for quantity, quality (by genotyping) and confirming appropriate clinical data was available. Baseline genotype data for 69 malaria-associated SNPs was generated for all contributing samples; briefly, samples underwent a primer-extension pre-amplification (PEP) step (Xu K et al, 1993; Zhang L et al, 1992) prior to genotyping on the Sequenom® MassArray® platform. Following curation, the genotype data were returned to the PIs for local analyses.
|Number||Gender: n (%)||Age in years: n (%)||Ethnicity: n (%)|
Malaria cases: 983
420 severe malaria
563 uncomplicated malaria
|Male: 532 (54)
Female: 415 (42)
Not recorded: 34 (4)
|<1: 68 (7)
1-2: 189 (19)
2-5: 447 (45)
5-15: 273 (28)
Not recorded: 6 (1)
|Mossi: 983 (100)|
|Healthy controls: 816||Male: 407 (50)
Female: 391 (48)
Not recorded: 18 (2)
|<1: 161 (20)
1-2: 197 (24)
2-5: 453 (56)
5-15: 5 (<1)
|Mossi: 816 (100)|
The study was approved by the Health Research Ethical Committee of the Ministry of Health of Burkina Faso (proposal number: ID No 2007-048) and by the Ethical Committee of the University of Oxford.
For the recruitment of children with severe and uncomplicated malaria, informed consent was obtained from parents or legal guardians after admission to hospital. For the recruitment of healthy children informed consent was obtained prior to enrolment from a parent or legal guardian of each participating child.
- Amadou Tidiani Konate, Centre National de Recherche et de Formation sur le Paludisme, Burkina Faso
- Germana Bancone, University of Rome La Sapienza, Italy
- Issa Nebie Ouedraogo, Centre National de Recherche et de Formation sur le Paludisme, Burkina Faso
- Jaques Simpore, Centre Médicale St. Camille, Burkina Faso
We are indebted to the paediatric and laboratory staff of the Centre Hospitalier Universitaire Yalgado Ouédraogo, Centre Médical Paul VI and Centre Médical St Camille for skilled work and collaboration. We are grateful to the personnel of Centre National de Recherche et Formation sur le Paludisme. We would like to thank the the children and their parents for their participation in this study, and the local health authorities and the Ministry of Health of Burkina Faso for their cooperation.
The recruitment of children with severe and uncomplicated malaria was sponsored by the Italian Cooperation Programme in Burkina Faso and was supported by the World Health Organization, Division of Control of Tropical Diseases, and by the Fondazione Pasteur-Istituto Cenci Bolognetti of the University of Rome La Sapienza. The recruitment of healthy children was supported by Centre National de Recherche et de Formation sur le Paludisme (CNRFP).