A case-control approach to the identification of polymorphisms associated with severe malaria in Kilifi, Kenya

Projects:

Locations: Kenya (KE)

About this study

Malaria is a leading cause of death in much of sub-Saharan Africa (World Health Organization, 2008). Host genetic factors are thought to be important modifiers of severe and fatal malaria (Mackinnon MJ et al, 2005), yet little is known about which genes are involved or the degree of protection that they might confer. 

Summary

This study was undertaken a case–control study of severe P. falciparum malaria in a rural area on the coast of Kenya to investigate the genetics of malaria resistance. Cases were children younger than 14 years who were admitted from within a defined study area to Kilifi District Hospital between June 1995 and February 2008. Controls consisted of children born consecutively within the same study area as cases who were recruited at 3-12 months of age into a genetic cohort study (the Kilifi Genetic Birth Cohort Study) between August 2006 and July 2008.

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 and genome-wide SNP typing is planned for the future.

Single- and multi-locus analysis will be conducted using multivariate logistic regression, accounting for the effects of a range of confounders, to assess the relationship between genetic markers and well-defined severe malaria including sub-phenotypes such as severe malarial anaemia, cerebral malaria and respiratory distress.

Study site description

The KEMRI-Wellcome Trust Research Programme recruited severe malaria cases and healthy controls as part of on-going epidemiological studies of severe malaria at Kilifi District Hospital, Kenya. Kilifi District Hospital is situated in a rural area on the Kenyan Coast and has a catchment population of roughly of 500,000 people. The local economy is predominantly rural, being based on subsistence farming of maize, cassava, cashew nuts, and coconuts as well as dairy cows and goats. Two large agricultural estates, two research institutes and several tourist hotels contribute to local employment. The majority of the population fall within the Chonyi, Giriama and Kauma sub-divisions of the Mijikenda ethno-linguistic group.

Malaria transmission in Kilifi is seasonal, generally coinciding with the long and short rains in October and May, respectively.  Recent years have seen a significant decline in the rate of transmission from meso-endemic in the 1990’s to hypo-endemic transmission today. P. falciparum is the cause of the vast majority of malaria infections. Malaria is predominantly transmitted by mosquitoes of the species Anopheles gambiae (Mbogo CN et al, 1993). The overall annual entomological inoculation rate (EIR) has been estimated at 1-100 (Mbogo CN et al, 1993; Mbogo CM et al, 2003).

Methods

An unmatched case-control study was conducted in Kilifi District. Cases of severe malaria, resident within a defined study area, were recruited through a process of systematic surveillance of the paediatric wards at Kilifi District Hospital between June 1995 and February 2008. Cases consisted of children less than 14 years of age who were admitted to the high dependency ward with a primary diagnosis of severe malaria (defined by the presence of P. falciparum parasitaemia complicated by one or more of the following features: severe anaemia (Hb <5g/dl), convulsions before/during admission, cerebral malaria (Blantyre Coma Score ≤3) or respiratory distress).

Controls consisted of children born consecutively within the same study area as cases who are recruited at 3-12 months of age into a genetic cohort study (the Kilifi Genetic Birth Cohort Study) between August 2006 and July 2008. 

A standardised case report form (CRF) was created for the Consortial Project and used by all sites to collect standardised clinical data. The data collected in Kenya (and all other sites) were uploaded onto secure web-based software developed by MalariaGEN. Here, the integrity of the data was checked and data was standardized and amalgamated.

Genomic DNA was extracted from whole blood, in Kilifi, using Qiagen DNeasy Blood kits (http://www.qiagen.com/) [Qiagen, Crawley, UK].  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: 2740 Male: 1358 (49)

Female: 1235 (45)

Not recorded: 175 (6)

<1: 526 (19)

1-2: 609 (22)

2-5: 1144 (42)

5-15: 297 (11)

Not recorded: 1 (<1)

Chonyi: 621 (23)

Giriama: 1622 (59)

Kambe: 101 (4)

Kauma: 197 (7)

Other: 183 (7)

Not recorded: 16 (<1)

Healthy controls: 4183 Male: 2058 (49)

Female: 2022 (49)

Not recorded: 103 (2)

<1: 4078 (97)

1-2: 105 (3)

Chonyi: 1508 (36)

Giriama: 1930 (46)

Kauma: 465 (11)

Other: 273 (7)

Ethics

The study was approved by KEMRI Research Ethics Committee, Kilifi (proposal number: SCC1192).

Informed consent was obtained from parents or guardians of cases and mothers for the population controls. Population controls were collected by consenting women attending maternity units to give birth. DNA was extracted from cord blood samples.

Additional contributors

  • Alexander Macharia, Kemri-Wellcome Trust Research Programme, Kenya
  • Charles Newton, Kemri-Wellcome Trust Research Programme, Kenya
  • Dorcas Kamuya, Kemri-Wellcome Trust Research Programme, Kenya
  • Evasius Bauni, Kemri-Wellcome Trust Research Programme, Kenya
  • Jay Berkley, Kemri-Wellcome Trust Research Programme, Kenya
  • Sassy Molyneux, Kemri-Wellcome Trust Research Programme, Kenya
  • Sophie Uyoga, Kemri-Wellcome Trust Research Programme, Kenya
  • Vicki Marsh, Kemri-Wellcome Trust Research Programme, Kenya

Acknowledgements

We thank the patients and staff of the Kilifi District Hospital and the KEMRI-Wellcome Trust Programme, Kilifi for their help with this study. We particularly thank the members of the Genetics Laboratory for their help with sample processing.

This work was supported by funds from the MalariaGEN Consortium Project. Sample collection and processing was further supported through a Programme Grant (092654) to the Kilifi Programme from the Wellcome Trust. Thomas N Williams is funded through a Senior Research Fellowship from the Wellcome Trust (076934) and by the EVIMalR Consortium, a European Union Network 7 Network of Excellence. Carolyne Ndila is supported by funds from the Wellcome Trust (084538) and MalariaGEN consortium. This work is published with permission from the director of KEMRI.

Key People

Prof Tom Williams
Head of the Department of Epidemiology and Demography
KEMRI Wellcome Trust Research Programme, Kenya
Chair in Haemoglobinopathy Reseach
Imperial College London, UK