About this study
Malaria remains one of the most widespread infectious diseases of humankind, causing debilitating illness in hundreds of millions of people, particularly in sub-Saharan African children under five years of age. The susceptibility of human populations to severe Plasmodium falciparum malaria has been associated with variations in more than 30 genes (Sinha S et al, 2008), some of which have exhibited differential association in distinct populations of the world (Serghides L et al, 2003).
Carriers of the sickle-cell trait (haemoglobin [Hb] type AS), have been shown to be less likely to experience severe, potentially fatal, malaria (Aidoo M et al, 2002). Studies by Mackinnon and colleagues in Kenya suggest that human genetics explain 25% of the variation in susceptibility to and manifestation of malaria but that HbS explains only 2% of the total variation (Mackinnon MJ et al, 2005). This implies the existence of many unknown protective genes, notably single nucleotide polymorphisms (SNPs), each individually having small population effects.
In order to understand the mechanisms of pathogenesis and protective immunity against malaria and inform future vaccine and anti-malaria therapies, we designed large case-control studies of severe malaria in children living in the Centre, Littoral and South West regions of Cameroon. Two studies were conducted; the first study between 2003/05 and the second in 2007/08. These studies aim to identify human genetic determinants of severe malaria and the sub-phenotypes cerebral malaria and/or severe malarial anaemia, hyperparasitaemia and respiratory distress as well as mild/uncomplicated malaria and anaemia.
Severe malaria cases were recruited from the paediatric wards of eight health facilities in Buea, Douala, Limbe and Yaounde. Most of the chosen health facilities were the main government institutions in the selected towns, also receiving patients from surrounding areas. Cases consisted of children (aged 1 month - 17 years) with cerebral malaria (CM) and/or severe malarial anaemia (SMA) or uncomplicated/mild malaria recruited according to WHO criteria. Two control categories were used; school children (afebrile and free from any obvious illness) and blood bank donors (asymptomatic, from the community).
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. Phenotypes that could potentially be tested include severe malaria and sub-phenotypes such as cerebral malaria, severe malarial anaemia, hyperparasitaemia and respiratory distress.
This study was conducted in four towns across three regions of Cameroon, namely: Yaounde in the Central region, Douala in the Littoral region and Buea and Limbe in the South Western region. The study sites included hospitals (Bota District Hospital - Limbe, Laquintinie Hospital - Douala, Mother and Child Hospital - Yaounde, Regional Hospital - Limbe and Regional Hospital Annex - Buea) and health centres (Bokova Health Centre, Mount Mary Health Centre - Buea and PMI Down Beach - Limbe). Except for Mount Mary, the chosen health facilities were the main government institutions in the selected towns, also receiving patients from surrounding areas. Controls were recruited from primary schools which included: Catholic School (CS) Buea Station, CS Great Soppo, CS Muea, Government School (GS) Bolifamba, GS Bonduma, Government Practising School (GPS) Molyko I and II, GPS Muea I and II, HOTPEC Primary School Mile 15 Buea, Oxford Primary School Muea and Government Bilingual Primary School Muea.
Although malaria is endemic throughout Cameroon, the country has very different geographical and epidemiologic strata that may alter the course of the infection. In general, malaria transmission is intense and perennial in the Central, Littoral (Coastal) and South Western regions, with peak periods corresponding to the rainy seasons (Mackinnon MJ et al, 2005). Furthermore, Bolifamba, Molyko and Muea are located at low altitude (400–650 m above sea level) while Bonduma, Great Soppo and Buea Town are situated at high altitude (900–1000 m above sea level).
The central region (Yaounde) is located within the rainforest belt of central Africa and has the Guinea-type equatorial climate. This is characterised by fairly constant temperatures (ranging from 17- 30oC [mean = 23.1oC]) (Manga L et al, 1997), abundant rainfall (1,500–2,000 mm), an average relative humidity index ranging from 85% to 90%, and four distinct seasons: two rainy seasons (March–May/June and September–November) and two dry seasons (December–February and June/July–August). Maximal transmission of malaria occurs during and immediately following the two rainy seasons (Quakyi IA et al, 2000). The Mother and Child Hospital is a referral hospital for children and mothers, located in the heart of the city of Yaounde. It also attracts patients from neighbouring villages such as Simbok and Etoa that are stable, rural, farming communities with fields irrigated by water from the Mefou and Biyeme Rivers (Quakyi IA et al, 2000). Inhabitants of this region are of the Ewondo tribe and part of the Bantu ethnic group.
The South Western and Littoral regions have a Cameroonian-type equatorial climate characterized by fairly constant temperatures and two seasons: a short dry season (November–March) and a long rainy season (March-November) with abundant precipitation (2,000–10,000 mm) (Manga L et al, 1997). In the Mt Cameroon region of the South West, the mean annual rainfall is 2625 mm, relative humidity is constantly high (75%–80%) and the temperature varies from 18oC in August to 35oC in March (Wanji, S et al, 2003). Human malaria is meso-endemic during the dry season but becomes hyper-endemic in the rainy season, with incidence peaking in July–October (Achidi EA et al, 2008). The prevalence of malaria parasitaemia in the low-altitude areas range from 30% in the dry season to 65% in the rainy season. P. falciparum accounts for up to 96% of malaria infections in this area, with Anopheles gambiae s. s. the dominant vector (Wanji, S et al, 2003). The great majority of the population in these regions belong to the Bantu and Semi-Bantu ethnic groups.
An unmatched case-control study was conducted between 2003/05 and 2007/08. The children were recruited from nine hospitals and health centres in Cameroon. Seven were in the South-West Region (e.g. the Mt Mary Hospital Buea and Bota district Hospital Limbe), one in the Littoral Region (Hospital Laquintinie de Douala) and one in the Cental Region (Mother and Child Hospital - Yaounde).
Cases included febrile children (aged 1 month-17 years) with an axillary temperature ≥ 37.5oC (measured within 24hrs of admission), with symptomatic malaria parasitaemia infection and without any other disease with signs/symptoms similar to malaria.
The criteria for diagnosis of severe malaria were those included in the standard WHO definition for 2000 i.e. the presence of asexual parasitaemia and at least one of the following: cerebral malaria (impaired consciousness or unarousable coma [(Blantyre coma score ≤2, corrected for hypoglycaemia (blood glucose <2.2 mmol/l or <40 mg/dl)] and no record of recent severe head trauma, neurological disease or any other cause of coma), severe malarial anaemia (haemoglobin <5g/dl (or haematocrit <15%), be fully conscious, no cases of severe bleeding or observed convulsions), convulsions before/during admission, respiratory distress (presence of alar flaring, intercostals or subcostal chest recession, use of accessory muscles of respiration, or abnormally deep respiration), hypoglycaemia (blood glucose <2.2mmol/l); hyperpyrexia (axillary temperature ≥40oC), hyperparasitaemia (>250,000 parasites/µl) as well as uncomplicated malaria (fully conscious with haemoglobin ≥8g/dl and no signs of severity and/or evidence of vital organ dysfunction).
Controls consist of apparently healthy children (aged 1-14 years, afebrile and free from any obvious illness, though a fraction had asymptomatic parasitaemia) and asymptomatic adults (aged 17-52 years from the community). Children were recruited from primary schools located in the South-West Region of Cameroon (Buea Metropolis) between 2004-2005 and 2007-2008. Children with parasitaemia and a temperature of 37.5°C or above were not recruited as controls. Adults were identified from a blood bank in the Centre region of Cameroon (Mother and Child Hospital - Yaounde) between July and August 2007.
A standard case report form (CRF), created by MalariaGEN was used to collect standardised clinical data. The relevant data fields as per the MalariaGEN CP1 CRF were extracted from CRFs of the studies conducted prior to MalariaGEN (ie 2003/05 study conducted in south western region of Cameroon). The data collected in Cameroon (and all other sites) were uploaded onto the MalariaGEN central repository via secure web-based software, Topheno, developed by MalariaGEN. Here, the integrity of the data was checked and data was standardised and amalgamated.
Genomic DNA was extracted from whole blood or packed cells at the Malaria Research Laboratory, University of Buea using the Promega Wizard (Promega Corporation, Madison, USA) or Nucleon™ BACC Genomic DNA Extraction (Gen-Probe Life Sciences, Manchester, UK) kits 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.
|Number||Gender: n (%)||Age in years: n (%)||Ethnicity: n (%)|
|Malaria cases: 914||
Male: 430 (47)
Female: 389 (42)
Not recorded: 97 (11)
<1: 179 (19)
1-2: 220 (24)
2-5: 280 (31)
5-15: 135 (15)
>15: 1 (<1)
Not recorded: 99 (11)
Bantu: 348 (38)
Semi-Bantu: 384 (42)
Other: 102 (11)
Not recorded: 80 (9)
|Healthy controls: 914||
Male: 592 (65)
Female: 305 (33)
Not recorded: 17 (2)
<1: 0 (0)
1-2: 2 (>1)
2-5: 66 (7)
5-15: 391 (43)
>15: 372 (41)
Not recorded: 83 (9)
Bantu: 377 (41)
Semi-Bantu: 395 (42)
Other: 62 (7)
Not recorded: 96 (10)
This study was reviewed by the scientific and ethical review board of the University of Buea (proposal number: ID D7.1.A/MPH/SWP/PDPH/PS.CH/2340/811) and the South West Regional Delegation of Public Health. Authorization to conduct the surveys in primary schools was obtained from the Regional Delegation of Basic Education or the Catholic Education Secretariat.
For the cases and blood bank donors, informed consent was obtained from each case or their caregiver following a clear explanation of the content of the information sheet. Only subjects/caregivers who returned signed consent forms were enrolled into the study.
Written informed consent was obtained from the parents/guardians of healthy school children. Information sheets and consent forms were sent to parents/guardians of each child through head teachers in the schools and only those who returned signed consent forms were enrolled into the study.
- Andre Ndi Ngwai, Department of Medical Laboratory Sciences, University of Buea, Cameroon
- Judith Anchang-Kimbi, Department of Zoology & Animal Physiology, University of Buea, Cameroon
- Mbunwe Eric, Department of Medical Laboratory Sciences, University of Buea, Cameroon
- Njua Clarisse Yafi, Department of Biochemistry & Molecular Biology, University of Buea, Cameroon
- Richard Besingi, Department of Medical Laboratory Sciences, University of Buea, Cameroon
- Regina Ngoh Mugri, Department of Medical Laboratory Sciences, University of Buea, Cameroon
- Vincent Titanji, Department of Biochemistry & Molecular Biology, University of Buea, Cameroon
We remain particularly indebted to the parents/guardians who assented to the participation of their children in the study as well as the hospital/school staff and their collaborators for their immense help in participant recruitment and sample collection. Special thanks also go to the University of Buea for the facilities placed at our disposal during the study period.
The study received financial support from the Malaria Immunology & Pathogenesis Network (MIMPAC) and a capacity-building grant (A11034) awarded by the Multilateral Initiative on Malaria Special Programme for Research and Training in Tropical Diseases (MIM/TDR) as well as the Malaria Genomic Epidemiology Network (MalariaGEN) Grand Challenge Initiative grants to Dr Eric Achidi & Professor Dominic Kwiatkowski, respectively.