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Human genetic determinants of severe malaria in North East Tanzania

Location: Tanzania (TZ).

Human

About this study

Malaria is known to be a strong factor for selection of human genes that give protection against the disease. A quarter of the risk of severe malaria is determined by host genetic factors, and although we already know a number of human genes that appear to influence the risk of severe malaria, this represents only a small fraction of the total genetic component and only 2% is attributed to HbS (Mackinnon MJ et al, 2005). SNPs identified by the human genome HapMap project and improved technology of genome-wide association analysis will help to identify malaria resistance genes in a systematic and comprehensive manner which may assist in understanding the role and molecular mechanism of genes in malaria immunity (International HapMap Consortium, 2005).

Summary

A matched case-control study (children aged 3 months-10 years) was carried out in Muheza, Tanzania between 2006-2008 with the aim of identifying the genes involved in protection against severe malaria, and the sub-phenotypes acidosis, severe malarial anaemia, cerebral malaria, shock, respiratory distress and bacteraemia.

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.

In addition, conventional PCR and ELISA tests were conducted for alpha thalassaemia and antibody analysis, respectively. A number of regression models will be used to assess the association between genetic polymorphisms and malaria outcome.

Study site description

The Joint Malaria Programme together with the London School of Hygiene and Tropical Medicine recruited severe malaria cases and healthy participants from Teule Hospital and community, respectively, in Muheza, North East Tanzania. This hospital serves a rural population of approximately 277,000 with child mortality of 165/1,000. The hospital receivespatients from all over the Tanga region.

The study villages lie at an altitude of 199-300 metres. The area is dominated by Anopheles gambiae sensu stricto and Anopheles funestus (Maxwell CA et al, 2003) as the main vectors. Transmission of Plasmodium falciparum is intense (50-700 infected bites/person/year) and perennial, with two seasonal peaks (Maxwell CA et al, 2003). The community prevalence of P. falciparum in children aged 2-5 years in the study area was recorded as 88.2% in 2002 (Maxwell CA et al, 2003). The dominant climate is warm and wet. In most cases, there is not a big variation in temperature at the coast due to the influence of the Indian Ocean. However, during the hot season (December to March) the average temperature is approximately 30-32oC during the day and 26-29oC at night. During the cool season (May to October) temperatures are approximately 23-28oC in the day and 20-24oC at night. Another characteristic of the coastal climate is the high atmospheric humidity, which often goes up to 100% maximum and 65-70% minimum. Mean annual rainfall ranges from 600-800mm. The outstanding feature of the vegetation is its complexity. The coastal area is dominated by bush land, palm gardens, village cultivations and estates (mainly sisal). The upland plateaus are covered with bush land and shrub thickets interrupted by swampy low-lands and river swamps as well as village cultivations, estates and palm gardens.

The economy of Muheza depends on subsistence agriculture, livestock keeping and fishing. Food production to a large extent is undertaken by small holders, while cash crop production is carried out by both small holders and large scale farmers (public and private institutions). The leading and prominent food crops in terms of area coverage are maize, cassava, banana, pulses (mainly beans) and rice. Important cash crops include sisal, cotton, coffee, tea, cardamon, coconuts, tobacco and cashewnuts. Livestock reared are cattle, goats and sheep. Modern dairy farming and poultry keeping is not very common in the rural areas.

There are health facilities in most villages in Muheza, and a large part of the population has access to primary health facilities within a distance of 6kms. The main problems remain to be “shortage of medicines”, user charges and the poor state of health facilities. About half of all households use iron sheeting for roofing while the rest use grass, leaves or mud (National Bureau of Statistics – Tanzania, 2005). The majority of the population belongs to the Mzigua and Wasambaa ethnic groups.

Methods

A matched case-control study was conducted. The cases were retrospectively recruited from patients who had been hospitalized for severe febrile illness (SFI) at Teule hospital in Muheza, Tanga in 2005/2006. Cases were recruited as part of a study which was investigating reasons why some patients were suffering from severe illness due to common disease (Nadjm B et al, 2010). Cases consist of children (aged 3 months-10 years) who were admitted to hospital with signs of severe malaria. Consecutive daytime admissions were triaged for the need for emergency treatment and then screened for study eligibility.

Cases were aged between 2 months and 13 years with a history of fever within the previous 48 hours, asexual P. falciparum parasitaemia and any of the following: more than 2 seizures in the previous 24hrs (information given by the mother); Blantyre coma score <3 (repeated if BCS<5 and convulsion within 1 hr or anticonvulsant given within 6 hrs); prostration (inability to sit unsupported or, if age<8months, inability to drink); respiratory distress (deep breathing or low chest wall indrawing or respiratory rate>70 bpm or O2 sat<90%); jaundice (identified by inspection of sclera); severe anaemia (haemoglobin <5g/dl), blood glucose <2.5mmol/l, blood lactate >5mmol/l.

Controls consist of children (aged 1-10 years), in good health (i.e. eating and drinking normally and playful), haemoglobin >8 g/dl and had no skin condition or other impediment to obtaining a venous blood sample. Controls were recruited between July 2007 and August 2008. These were individually matched to cases for tribal origin of at least one parent, electoral ward of residence and within 3 years of age to a case. Potentially eligible controls were initially identified by the health worker in the local primary care clinics. These children were later visited by research staff and the ethnicity match was confirmed on interview with one of the parents. Controls were excluded if they had been admitted to Teule hospital for severe malaria in the previous 12 months.

A standardised case report form (CRF) was created for CP1 and used by all sites to collect standardised clinical data. The data collected in Tanzania (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 using the Nucleon™ BACC2 Genomic DNA extraction kit® (Gen-Probe Life Sciences Ltd., Manchester, UK) using manufacturer’s instructions and quantified using spectrophotometer at the KCMC Biotechnology laboratory in Moshi, Tanzania. 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: 501 Male: 260 (52)

Female: 224 (45)

Not recorded: 18 (3)

<1: 121 (24)

1-2: 172 (34)

2-5: 188 (38)

5-15: 20 (4)

Mzigua: 132 (26)

Wasambaa: 100 (20)

Other: 269 (54)

Healthy controls: 504 Male: 222 (44)

Female: 267 (53)

Not recorded: 15 (3)

<1: 7 (1)

1-2: 119 (24)

2-5: 337 (67)

5-15: 51 (8)

Mzigua: 142 (29)

Wabondei: 61 (12)

Wasambaa: 101 (20)

Ethics

Ethical clearance was obtained from the ethical review board of the London School of Hygiene and Tropical Medicine and the Tanzanian National Medical Research Institute (Proposal number: ID 4093).

Written informed consent was obtained from adults and the parents or legal guardians of all children enrolled in this study. The details of the consenting procedure for cases have been detailed in Nadjm B et al (2010). Study information were read to the parents or legal guardians of all children recruited as controls. They were given the opportunity to ask questions and when they voluntarily agreed to participate in the study, were consented.

Additional contributors

  • Behzad Nadjm, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, UK
  • Caroline Maxwell, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, UK
  • Frank Mtei, Joint Malaria Programme, Kilimanjaro Christian Medical Centre, Tanzania
  • George Mtove, National Institute for Medical Research, Amani Centre, Tanga
  • Hannah Wangai, Joint Malaria Programme, Kilimanjaro Christian Medical Centre, Tanzania
  • Sarah Joseph, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, UK

Acknowledgements

All participants are highly acknowledged for their involvement. Special thanks to the District medical officers for the administration at their respective districts during data collection. Health workers of the health facilities and village leaders are thanked for facilitating in recruitment of controls. Joint Malaria Programme staffs in Moshi, Tanzania are also highly appreciated for facilitating data collection during field work.

The Core funding for the cases study was provided by European Commission (Europaid) grant code SANTE/2004/078-607. While the funding for recruitment of the controls was provided by MalariaGEN.

References

National Bureau of Statistics (NBS) [Tanzania] and ORC Macro, 2005. Tanzania Demographic and Health survey 2004-2005. Dar es Salaam, Tanzania: Bureau of Statistics and ORC Macro.

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