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Human genetic determinants of severe malaria in Nigeria

Location: Nigeria (NG).

Human

About this study

Malaria remains one of the leading causes of morbidity and mortality in malaria endemic regions of the world. The epidemiology of malaria in Nigeria is characterised by a spectrum of different malaria transmission patterns, ranging from unstable transmission in the North to stable intense transmission in the South. The main evidence for a major genetic component of disease susceptibility comes from the growing number of disease associations with specific SNP polymorphisms in recent years. A number of malaria candidate genes (May J et al, 2007; Clark TG et al, 2009; Lyke KE et al, 2011; Millimono TS et al, 2011; Schuldt K et al, 2011), and more recently genome-wide association studies (Jallow M et al, 2009), have identified genomic loci, which contribute to susceptibility and/or protection against severe malaria.

Summary

An unmatched case-control study was conducted during the May-September transmission season of 2007 with the aim of identifying human genetic determinants of susceptibility/resistance to severe malaria in Ibadan, Southwest Nigeria.

The inclusion criteria for recruitment were categorised according to the World Health Organization definitions. Severe malaria cases (including those with severe malarial anaemia and cerebral malaria) were recruited from three hospitals: the University College Hospital (a tertiary Institution) and two secondary level hospitals, Adeoyo Maternity Hospital and Oni Memorial Hospital in Ibadan, Nigeria. Asymptomatic controls comprised of clinically-well children recruited during a cross-sectional survey in September 2007 from communities in the areas surrounding the three hospitals from which cases were recruited.

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) will be included in our analysis. Single- and multi-locus analysis will be conducted using various multivariate logistic regression models to assess the relationship between these polymorphisms and well-defined clinical phenotypes.

Study site description

Malaria is endemic in Nigeria with seasonal transmission patterns, the peak transmission of which is during the rainy season (April-October) followed by the dry season (November-March). Average annual rainfall in Ibadan is 80 inches and it has a relative humidity typically above 80% during the wet season. The predominant species of malaria parasite is Plasmodium falciparum accounting for about 96% of malaria infections. The two main vector species in Nigeria are Anopheles gambiae and A. funestus, with an EIR of 24.7 infective bite per person per rainy season (Okwa OO et al, 2009).

The case-control study was conducted at three hospitals: the University College Hospital (a tertiary Institution) and two secondary level hospitals, Adeoyo Maternity Hospital and Oni Memorial Hospital in Ibadan, Nigeria. Ibadan city is located in the Southwest of Nigeria in the rainforest belt at 7.38 latitude, 3.8 longitude and at an elevation of 239m. The population of Ibadan is about 4 million. The secondary government hospitals cater for the middle and lower social class, and the UCH is a tertiary health care facility which caters for all classes and attends to more complicated and severe cases. The communities within the catchment areas of the three hospitals, with a population of about 200,000, are mainly traders, civil servants, academic and artisans. There are private health facilities and Mission-based facilities within the city and in the outlying communities, villages and markets. Patent medicine sellers provide health services to the people at cheaper rates but with the risk of being provided with fake, expired and adulterated drugs. The great majority of the population belongs to the Yoruba ethnic group.

Methods

An unmatched case-control study was conducted. Cases consist of children (aged 4 months – 11 years) presenting with severe malaria, cerebral malaria and/or severe malarial anaemia. Children were recruited from three hospitals in the city of Ibadan during the May-September transmission season of 2007. The inclusion criteria for recruitment were defined according to the World health Organization definitions. Severe malaria was defined by the presence of P. falciparum in the thick blood film with at least one of the following conditions: unrousable coma, BCS ≤2, clear cerebro-spinal fluid from lumbar puncture, extreme weakness/prostration (inability to sit or stand without support), convulsions (more than one episode within 24 hours), severe anaemia (haematocrit <15% or Hb <5 g/dl). Children with co-existing bacterial meningitis and other encephalitis (especially the locally prevalent viral encephalitides) were excluded from the study.

Controls comprised of children (aged 6 months – 12 years) recruited during a cross-sectional survey in September 2007 from communities in the areas surrounding the three hospitals from which cases were recruited. Clinically well children who had not been ill two weeks preceding recruitment, were recruited into the study.

A standardised case report form (CRF) was created for MalariaGEN CP1 and used by all sites to collect standardised clinical data. The data collected in Nigeria (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 Institute of Child Health, using Nucleon™ BACC2 Genomic DNA extraction kits® by Tepnel (Gen-Probe Life Sciences Ltd., Manchester, UK) using manufacturer’s instructions and quantified. 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.

Table 1: Breakdown of samples
Number Gender: n (%) Age in years: n (%) Ethnicity: n (%)
Malaria cases: 114 Male: 66 (58)

Female: 43 (38)

Not recorded: 5(4)

<1: 12 (11)

1-2: 27 (24)

2-5: 60 (53)

5-15: 15 (13)

Yoruba: 110 (96)

Other: 4 (4)

Healthy controls: 88 Male: 43 (49)

Female: 36 (41)

Not recorded: 9 (10)

<1: 15 (17)

1-2: 19 (22)

2-5: 32 (36)

5-15: 22 (25)

Yoruba: 86 (98)

Other: 2 (2)

Ethics

Ethical approval was obtained from the Joint University of Ibadan/ University College Hospital Ethical Review Committee (proposal number: ID UI/IRC/06/0034) and the Oyo State Ministry of Health Ethical Committee.

After a thorough explanation of the study and the procedures involved, consent (both verbal and written) was obtained from the mothers/care givers of the children recruited. Consent was also obtained from the Community head for the cross-sectional study.

Additional contributors

  • Adebola E Orimadegun, Institute of Child Health, University of Ibadan, Nigeria
  • Folakemi Anjola Amodu, Institute of Child Health, University of Ibadan, Nigeria
  • Olajumoke Oni, Institute of Child Health, University of Ibadan, Nigeria
  • Olayemi O Omotade, Institute of Child Health, University of Ibadan, Nigeria
  • Peter E Olumese, Global Malaria Programme, World Health Organization, Geneva
  • Subulade Olaniyan, Institute of Child Health, University of Ibadan, Nigeria

Acknowledgements

We acknowledge all the children who participated in these studies. We are grateful to the staff, the nurses and doctors of the hospitals, for their care of the patients. We are also grateful to the entire Malaria Research Team of the Institute of Child health, the field staff (Mojisola Oyeniyi, Anjola Amodu, Funmi Ajayi and Damilola Faseyitan) and laboratory staff (Subulade Olaniyan and Olajumoke Oni).

The research leading to these results received funding from the European Community under grant agreement LSHP-CT-2004-503578 and Seventh Framework Programme (FP7/2007-2013) under grant agreement N° 242095. Additional support for these studies was from a downstream laboratory support by MalariaGEN.

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