Human genetic determinants of severe malaria in Mali

Projects:

Locations: Mali (ML)

About this study

Malaria is a life-threatening parasitic disease transmitted by Anopheles mosquitoes. Despite major efforts to control the disease, it still persists as a major health burden, and caused over 780,000 deaths in 2009, mainly among children in sub-Sahara Africa. In Mali, there are over 800,000 recorded cases of malaria among its ~12 million people every year, and it accounts for 17% of child deaths (World Health Organization, accessed 12 Dec 2011). Malaria is a complex disease with many genetic and environmental determinants influencing the observed variation in response to infection, progression and severity. It has been estimated that 25% of the total variation in mild and severe malaria in a Kenyan cohort was explained by host genes (Campino S, Kwiatkowski D and Dessein A, 2006). The different geographical distributions of sickle-cell disease, α-thalassemia, glucose-6-phosphatase dehydrogenase (G6PD) deficiency, ovalocytosis, and the Duffy-negative blood group are examples of the general principle that different populations have evolved different genetic variants to protect against malaria (Mackinnon MJ et al, 2005).

Summary

A case-control study, including parents of cases, was conducted in Mali with the aim of identifying human genetic determinants associated with susceptibility or resistance to severe malaria and its sub-phenotypes: severe malarial anaemia, respiratory distress and cerebral malaria. We consider a cohort of over 800 individuals recruited in Bamako, predominantly from the Bambara ethnicity, which is under-represented in other genetic epidemiological studies in Western Africa.

Cases were children (aged 3 months-14 years) with signs of severe or uncomplicated malaria. Severe malaria cases were recruited from the paediatric ward at the reference hospital of Gabriel Touré in Bamako, the capital of Mali, between November 2006 and January 2008. A small number of uncomplicated malaria cases were also enrolled and matched to severe malaria cases by age (+/- 6 months), ethnicity and location and duration of residence. Severe malaria was defined by the presence of Plasmodium falciparum parasitaemia or positive dipstick of a rapid diagnostic test and one or more criteria included in the WHO 2002 standard definition for severe malaria. Controls were healthy children (aged 5 months-14 years) with no personal known history of severe malaria. Controls were individually matched to a severe malaria case by age (+/- 6 months), ethnicity, location and duration of residence.

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, baseline genotyping data 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) were included in our analysis.

Study site description

The Malaria Research and Training Centre (MRTC) in Mali recruited malaria cases, healthy controls and a small number of parents of index cases. Cases were enrolled at the Paediatric ward of the National Hospital Gabriel Toure in Bamako, the capital of Mali, where the MRTC team has been conducting studies on severe malaria since 1999. This hospital receives patients from across the country but the majority of patients come from the city of Bamako and the surrounding rural areas.  This catchment has a population of 1.8 million inhabitants and covers an area of 252 km2. Bamako had become a major market town. Economical activities are mainly trade, retailing and others services. The majority of people belong to the Bambara and Malinke ethnic groups.

The climate is tropical with a hot dry season and a wet rainy season that extends from June to October. The end of the wet season coincides with a peak in the incidence of severe malaria (Doumbo O, 1992). Most people live in traditional compounds with several nuclear households.

Five patterns of malaria transmission have been described in Mali:

  1. An area of long seasonal and intense malaria transmission between June and November in the south of Mali (corresponding with the Soudano-Guinean area where plasmodic index (parasite prevalence in children aged 2-9 years old) is higher than 75%);
  2. An area of short, seasonal, yet intense, malaria transmission from July to October (corresponding with the northern Savannah and the Sahel where plasmodic index varies from between 50 and 75%);
  3. An area of bimodal or multi-model transmission in the inner delta of the river Niger and areas of irrigated rice cultivation (where plasmodic index is <40%);
  4. Urban areas (where plasmodic index is <10%); and,
  5. An area of sporadic or epidemic malaria transmission in the sub-Saharan region, in the north of the country (where plasmodic index is <5%).

P. falciparum is the dominant parasite species (93.5%) and Anopheles gambiae s.l. and A. funestus are the main vectors. Entomologic Inoculation Rate (EIR) can be higher than 300 infective bites/person/year in areas of intense transmission and drops to almost zero during the dry season, specifically in the Sahel (Dicko A et al, 2005).

Methods

A matched case-control study, including parents of cases, was conducted between November 2006 and January 2008. Cases were children (aged 3 months-14 years) with signs of severe or uncomplicated malaria recruited from the paediatric ward at the reference hospital of Gabriel Touré in Bamako. A small number of uncomplicated malaria cases were also collected and matched to severe malaria cases by age (+/- 6 months), ethnicity and location and duration of residence.

All cases were children admitted to hospital with P. falciparum positive blood films and clinical features of severe malaria (Marsh K et al, 1995, World Health Organization, 1990).Severe malaria was defined by the presence of P. falciparum parasitemia or OptiMAL® test positive and one of the following: coma (Blantyre coma score < 3) without other obvious explanations (e.g. no evidence of pyogenic meningitis), anaemia (haemoglobin <5 g/dl or hematocrit < 15%) without obvious evidence of other causes (e.g. kwashiorkor).

Controls were healthy children (aged 5 months-14 years) with no personal history of severe malaria. Controls were individually matched to index cases by age (+/- 6 months), ethnicity and location and duration of residence. To select controls, the research team visited the home of cases the day after enrollment and sought an unrelated child whose age, ethnicity and residence duration matched that of the index case. Blood samples from a small number of parents of severe malaria cases were also collected to aid with haplotype construction.

A standardised case report form (CRF) was created for Consortial Project 1 (CP1) and used by all sites to collect standardised clinical data. The data collected in Mali (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 at the MRTC main campus from whole blood using the 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 PI’s for local analyses.

Table 1: Breakdown of samples
Number Gender: n (%) Age in years: n (%) Ethnicity: n (%)
Malaria cases: 510

Male: 279 (54)

Female: 221 (43)

Not recorded: 13 (3)

<1: 60 (12)

1-2: 106 (21)

2-5: 234 (46)

5-15: 110 (21)

Bambara: 216 (42)

Malinke:  59 (12)

Other: 235 (46)

Healthy controls: 389

Male: 183 (47)

Female: 186 (48)

Not recorded: 21 (5)

<1: 25 (6)

1-2: 82 (21)

2-5: 192 (49)

5-15: 88 (23)

Not recorded: 2 (1)

Bambara: 180 (46)

Malinke:  46 (12)

Peulh: 23 (6)

Sarakole: 21 (5)

Other: 119 (31)

 

Ethics

This study has been approved by the Ethical Committee of the Faculty of Medicine, Pharmacy and Odonto-Stomatology (FMPOS), University of Bamako, Mali (Proposal number: ID No/06-18bis/FMPOS).

Written informed consent was obtained from adults and the parents or legal guardians of all cases enrolled in this study. For the recruitment of healthy children, informed consent was obtained prior to enrolment from a parent/guardian. During the search for matched healthy controls, compensation in the form of provision of one time treatment of all medical conditions encountered in all children living in the same compound was offered.

Additional contributors

  • Abdoulaye Barry, Malaria Research and Training Centre, University of Bamako; and Centre Hospitalier Universitaire Gabriel Touré, Mali
  • Abdourahmane Sall, Malaria Research and Training Centre, University of Bamako; and Centre Hospitalier Universitaire Gabriel Touré, Mali
  • Amadou Abathina, Malaria Research and Training Centre, University of Bamako, Mali
  • Amadou Niangaly, Malaria Research and Training Centre, University of Bamako, Mali
  • Awa Dembele, Malaria Research and Training Centre, University of Bamako; and Centre Hospitalier Universitaire Gabriel Touré, Mali
  • Belco Poudiougou, Malaria Research and Training Centre, University of Bamako, Mali
  • Elizabeth Diarra, Malaria Research and Training Centre, University of Bamako; and Centre Hospitalier Universitaire Gabriel Touré, Mali
  • Kariatou Bamba, Malaria Research and Training Centre, University of Bamako; and Centre Hospitalier Universitaire Gabriel Touré, Mali
  • Salimata Konate, Malaria Research and Training Centre, University of Bamako; and Centre Hospitalier Universitaire Gabriel Touré, Mali
  • Sibiry Sissoko, Malaria Research and Training Centre, University of Bamako; and Centre Hospitalier Universitaire Gabriel Touré, Mali

Acknowledgements

We acknowledge all our study participants, especially the parents and the children who participated in these studies. Our gratitude goes to the people and the health authorities of the Bamako districts and surrounding for their support. Our appreciation and gratitude goes to the study team at the Hospital Gabriel Touré for Medical Research, especially Kama Keita and Saran Kaba. Finally, we wish to warmly thank the study team and staff of Gabriel Touré, particularly Pr Mamadou Marouf Keita, Dr Boubacar Togo, Dr Broulaye Traore and Pr Mariam Sylla for their important contribution.

This study was supported by the MalariaGEN Consortial sub-grant from welcome Trust award and The Bill & Melinda Gates Foundation, through the Foundation for the National Institutes of Health as part of the Grand Challenges in Global Health initiative.

Reference

Doumbo O. Malaria epidemiology in Mali, chloroquine resistance study permethrin impregnated curtains trials as malaria control strategy associated with systematic treatment of fever cases. Sci. biol. (Parasitologie). 1992: 245.

Key People

Prof Mahamadou Thera
Malaria Research and Training Centre, University of Science, Techniques and Technologies of Bamako, Mali
Prof Ogobara Doumbo
Malaria Research and Training Centre, University of Science, Techniques and Technologies of Bamako, Mali
Ousmane Boubacar Toure
Malaria Research and Training Centre, University of Science, Techniques and Technologies of Bamako, Mali