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Human genetic determinants of severe malaria in Kumasi, Ghana
Project: Consortial Project 1

Location: Ghana (GH).

Human

About this study

Severe malaria, caused by infection with Plasmodium falciparum, remains one of the major health problems in sub-Saharan Africa (World Health Organization, 2000). Severe falciparum malaria is a complex clinical syndrome comprising a number of life threatening conditions including severe malarial anaemia, cerebral malaria, metabolic acidosis, respiratory distress and other, less frequent complications (Marsh K et al, 1995). In previous studies, carriers of the sickle cell haemoglobin locus (HbS) were found to be protected from all major forms of severe malaria, whereas protection for carriers of the haemoglobin C (HbC) variant was restricted to cerebral malaria (May J et al, 2007). This underscores the role of genetics in determining the severity of malaria.

Summary

The aim of this prospective study is to identify human genetic determinants of severe malaria and the sub-phenotypes severe malarial anaemia, respiratory distress and hyperlactataemia.

Two studies (case-control and family trio) were conducted simultaneously between August 2006 and December 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.

Study site description

The studies were conducted in the Kumasi Metropolitan District of the Ashanti Region of Ghana where Kumasi is the capital city. Kumasi is located in the transitional forest zone. It is between 6.35o–6.4o latitude and 1.3o –1.35o longitude, at an elevation which ranges between 250–300 metres above sea level, with an area of about 254km2. The unique centrality of the city as a traversing point from all parts of the country makes it particular attractive to migrants.

It has been projected to have a population of 1,625,180 based on a growth rate of 5.4% pa and this accounts for just under a third (32.4%) of the region’s population with the majority engaged in small businesses. The major ethnic groups are the Asantes but almost all other tribes are present due to the location of the Region. Houses in the metropolis can be classified into about five types. These include: single storey, traditional compound houses, multi-storey compound houses, government–built detached or semi-detached low-income households, large single household houses built on relatively large plots and blocks of flats. The rich cultural heritage of the people of Kumasi is visible in Akwasidae festival, funerals, child-naming ceremonies, communal spirit and religion. The traditional religious practices are still upheld through the pouring of libation, marriage rites and rites of passage.

Komfo Anokye Teaching Hospital (KATH), which is one of the two national autonomous hospitals, is located within the district. With over 1000 beds, it is the second largest hospital in Ghana. It is a major referral centre for the North and Middle belt of Ghana. The Paediatric Directorate is divided into neonatal and children’s ward. The Paediatric Emergency Unit is the first point of call for all childhood illnesses reporting to the hospital.

The Metropolis falls within the wet sub-equatorial climatic zone. The average minimum temperature is about 21.5oC and a maximum average temperature of 30.7oC. The average humidity is about 84.2% at 09:00 GMT and 60% at 15:00 GMT. Rainfall is highest in June (214.3mm) and September (165.2mm). The city falls within the moist semi-deciduous South-East Ecological Zone.

Transmission of malaria in the area is intense and perennial with some seasonal variations. Most of the transmission (91.4%) occurs during bedtime hours of 21;00 to 06:00h. Annual Biting Rates (ABRs) and Annual Entomological Inoculation Rates (AEIRs) in one study were reported to be 11,643 and 866, respectively. The main malaria vectors are Anopheles gambiae and A. funestus, and the main parasite species are Plasmodium falciparum, P. malariae, and P. ovale (Abonuusum A et al, 2011).

Methods

A prospective, unmatched case-control study was conducted between August 2006 and December 2008 recruiting cases admitted to the Paediatric Emergency Unit of Komfo Anokye Teaching Hospital.

Cases consist of children (aged 3 months – 12 years) with severe malaria. Criteria for diagnosis of severe malaria were those included in the standard WHO definition i.e. the presence of asexual parasitaemia and at least one of the following: cerebral malaria (Blantyre coma score of 3 or below and coma persists for more than 30 minutes after fits have ceased); repeated or prolonged generalized convulsions; severe malarial anaemia (haemoglobin <5g/dl); respiratory distress (presence of alar flaring, intercostals or subcostal chest recession, use of accessory muscles of respiration, or abnormally deep respiration); hypoglycaemia (blood glucose <2.2mM/l); circulatory collapse (systolic blood pressure<50mmHg); renal failure (urine output less than 12ml/kg/24 hours or serum creatinine >3.0mg/dl); hyperpyrexia (anxillary temperature >39oC , hyperlactataemia (blood lactate >2.0mmol/l and impaired consciousness.

Controls consisted of cord blood samples collected from the labour ward of the same hospital at the same time as cases were collected. Blood samples were also collected from the biological parents of children with severe malaria.

A standardised case report form (CRF) was created by MalariaGEN and used by all sites to collect standardised clinical data. Data was uploaded onto the MalariaGEN central repository via 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 the whole blood samples in Kumasi 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 PIs for local analyses.

Table 1: Breakdown of samples
Number Gender: n (%) Age in years: n (%) Ethnicity: n (%)
Malaria cases: 1923 Male: 954 (50)

Female: 846 (44)

Not recorded: 125 (6)

 <1: 434 (23)

1-2: 476 (25)

2-5: 713 (37)

5-15: 296 (15)

Not recorded: 4 (<1)

 Akans (Ashanti Eastern): 1038 (54)

Frafra Nankana Grushie Kusasi: 188 (10)

Notherner: 186 (10)

Other: 511 (27)
Healthy controls: 2326 Male: 1160 (50)

Female: 1079 (46)

Not recorded: 88 (4)

 <1: 2326 (100) Akans (Ashanti Eastern): 1526 (67)

Akans (Ashanti Eastern) Mixed: 140 (6)

Frafra Nankana Grushie Kusasi: 108 (5)

Other: 520 (23)

Ethics

Ethical clearance was obtained from the Committee on Human Research, Publications and Ethics of KATH / Kwame Nkrumah University of Science and Technology, Kumasi (proposal numbers: ID GHS-ERC-03/9/06, CHRPF/07/01/06, CHRPE SMS UST/24/05/2007).

For the recruitment of children with severe and uncomplicated malaria, written informed consent was obtained from the parents or legal guardians after admission to hospital. For the recruitment of healthy controls informed consent was obtained prior to enrolment from a parent or legal guardian of each participating child.

Additional contributors

  • Alex Osei Akoto, Kwame Nkrumah University of Science and Technology, and Komfo Anokye Teaching Hospital, Ghana
  • Alex Owusu Ofori, Komfo Anokye Teaching Hospital, Ghana
  • Daniel Ansong, Kwame Nkrumah University of Science and Technology, and Komfo Anokye Teaching Hospital, Ghana
  • David Sambian, Komfo Anokye Teaching Hospital, Ghana
  • Emmanuel Asafo- Agyei, Komfo Anokye Teaching Hospital, Ghana
  • Justice Sylverken, Komfo Anokye Teaching Hospital, Ghana
  • Sampson Antwi, Kwame Nkrumah University of Science and Technology, and Komfo Anokye Teaching Hospital, Ghana

Acknowledgements

We acknowledge all our study participants, especially the parents for guidance of the children who participated in these studies. We are also grateful to all Staff of the Paediatric Directorate, the laboratory data staff and data entry persons for their support during the period of study. To the field workers who dedicated their time to following up on cases we say big thank you.

The main source of funding was from MalariaGEN Consortial sub-grant award and additional downstream laboratory support by MalariaGEN.

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