Human genetic determinants of severe malaria in Malawi

Projects:

Locations: Malawi (MW)

About this study

Plasmodium falciparum malaria has been identified as the cause of 30-40% of paediatric hospital attendances and up to 30% of in-hospital child deaths in Malawi. Parasitaemia is common in the population and is frequently asymptomatic, so that attributing an illness to malaria may often be erroneous. Nevertheless, responses to treatment and the findings of autopsy studies confirm that malaria imposes a heavy burden of both morbidity and mortality upon children in Malawi.

Asymptomatic parasitaemia and uncomplicated malarial febrile illness greatly outnumber episodes of severe and life-threatening malaria. The factors determining progression to severe disease remain unknown. Failure to obtain prompt treatment increases the risk of disease progression, but severe and complicated P. falciparum malaria may develop rapidly and cannot always be attributed to delayed treatment. The virulence of an infecting strain of P. falciparum, the frequency and inoculum-size of infective mosquito bites and the strength of the host’s existing specific anti-malarial immunity may each play a part in determining the severity of a malarial illness. It is also likely that host genetic factors contribute to determining the outcome of the parasite-host interactions that lead to disease.

Summary

As part of the Malaria Genomic Epidemiology Network, we set out to accurately identify children with severe P. falciparum malaria, to obtain samples of their DNA and to analyse these for genetic variants that might be associated with susceptibility or resistance to severe malaria.

A total of 1,815 children (aged 2 months - 14 years) admitted to the national hospital in Blantyre, Malawi, were enrolled into an unmatched case-control study during 1994-2007. All cases (children diagnosed with severe malaria) were followed-up according to a detailed protocol providing information on the clinical phenotype and outcome of the disease. Information on the presence or absence of retinopathy, a clinical feature that strengthens the diagnosis of cerebral malaria in a patient with coma and parasitaemia, was also collected. DNA was extracted from the buffy coat of venous blood collected from cases on admission to hospital. As a background comparator population, DNA from cord bloods was collected from 3,777 infants born in the same hospital during 2006-2007. DNA was also obtained from blood samples collected from the parents of a number of cases.

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 were generated for a number of malaria–associated single nucleotide polymorphisms (SNPs) and the appropriate data have 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.

These malaria associated SNPs will be analysed for an association with severe malaria and the sub-phenotypes cerebral malaria and severe malarial anaemia. We will also investigate the association of various haplotypes with these phenotypes. This will identify any genetic factors that are over- or under- represented among children with severe malaria, and that may therefore play a part in affecting a child’s susceptibility to severe P. falciparum infection.

Study site description

The Malawi-Liverpool Wellcome Clinical Research Programme (MLW) together with the Blantyre Malaria Project (BMP) recruited severe malaria cases, healthy controls and parents from the Queen Elizabeth Central Hospital (QECH) in Blantyre, Malawi. Blantyre, a compact, densely populated city of 500,000 people, is situated at 1000m above sea level. Daytime temperatures average around 25oC, with considerable variation throughout the year. The climate is characterised by a cool, dry season (May - September) and a hotter rainy season (October - April). Malaria transmission is year-round, with considerable seasonal fluctuation and most new infections occur during the hot and wet season. Over 90% of human malaria infections are due to P. falciparum, while 5-10% are P. malariae and 1-2% P. ovale. P. vivax is not encountered, owing to the almost universal presence of Duffy-negativity among the Malawian population. The entomological inoculation rate (EIR) for P. falciparum in the city of Blantyre is estimated to be around one infective bite/person/year, but a high proportion of families make regular visits to nearby rural areas where the EIR is estimated to be greater than 100.

Methods

An unmatched case-control study, including parents of cases, was conducted. Cases and controls were recruited from the Queen Elizabeth Central Hospital in Blantyre, between 1994 and 2007.

Cases consist of children (aged 2 months - 14 years) admitted to the national teaching hospital with signs of severe malaria, during the period of January to June each year. Severe malaria was defined by the presence of P. falciparum parasitaemia in the thick blood film and either severe anaemia (packed cell volume (PCV) of 15% at any time during admission) or cerebral malaria (Blantyre Coma Score of 2 on admission and for at least 2 hours thereafter, no evidence of meningitis, and no improvement after correction of hypoglycaemia or within 2 hours of a witnessed convulsion) (World Health Organization, 2008). From 1998 onwards, the presence or absence of malarial retinopathy was recorded for each patient. For those with cerebral malaria, this will allow more accurate classification.

Controls consist of cord blood samples taken from the Queen Elizabeth Central Hospital in Blantyre, between September 2006 and September 2007. Blood samples from the parents of a number of severe malaria cases were also collected.

A standardised case report form (CRF) was created for MalariaGEN Consortial Project 1 and used by all sites to collect standardised clinical data. The data collected in Malawi (and all other sites) were uploaded onto secure web-based software developed by MalariaGEN. Here, the integrity of the data was checked and data were standardised and amalgamated.

Genomic DNA was extracted from whole blood, at the MLW Laboratory in Blantyre, Malawi, using the Chelex®method according to manufacturer’s guidelines. 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 that appropriate clinical data were available. Baseline genotype data for 69 malaria-associated SNPs were 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: 1815 Male: 907 (49)

Female: 844 (46)

Not recorded: 86 (5)

<1: 213 (12)

1-2: 350 (19)

2-5: 922 (51)

5-15: 321 (18)

Not recorded: 9 (<1)

Malawi: 1815 (100)
Healthy controls: 3777 Male: 1781 (47)

Female: 1613 (43)

Not recorded: 385 (10)

<1: 3777 (100) Malawi: 3777 (100)

Ethics

The study was approved by the College of Medicine Research and Ethics Committee (COMREC), University of Malawi (proposal number: P.05/06/442).

All sample collections were subject to the informed consent of the parent or guardian of the child, and in the case of cord blood samples to the informed consent of the mother obtained prior to parturition.

Additional contributors

  • Ajib Phiri, Malawi-Liverpool-Wellcome Trust Clinical Research Programme and Queen Elizabeth Central Hospital, Malawi
  • Annie Munthali, Queen Elizabeth Central Hospital, Malawi
  • David Kachala, Malawi-Liverpool-Wellcome Trust Clinical Research Programme, Malawi
  • Labes Njiragoma, Queen Elizabeth Central Hospital, Malawi
  • Mike Moore, Malawi-Liverpool-Wellcome Trust Clinical Research Programme, Malawi
  • Neema Ntunthama, Malawi-Liverpool-Wellcome Trust Clinical Research Programme, Malawi
  • Paul Pensulo, Malawi-Liverpool-Wellcome Trust Clinical Research Programme, Queen Elizabeth Central Hospital, and Blantyre Malaria Project, University of Malawi, Malawi

Acknowledgements

We thank the children and their parents and guardians who participated in this study. The Obstetrics Dept of QECH permitted access to the labour ward so that our nurses could collect cord samples. The Department of Paediatrics has throughout hosted the clinical aspects of this study and provided space in the Paediatric Research Ward.

The Malawi-Liverpool-Wellcome Trust Programme and the Blantyre Malaria Project together provided clinical and laboratory support that made the study possible. These have been funded by The Wellcome Trust, UK, and the National Institutes of Health, USA, respectively.

Key People

Prof Malcolm Molyneux
Malawi-Liverpool-Wellcome Trust Clinical Research Programme