Study area and study design
The study was conducted to evaluate the levels of anti-malarial drug resistance on a molecular basis in Bourasso village, Kossi District, Burkina Faso, approximately 30 km from the district town Nouna. The village is located within the area of the Nouna Health and Demographic Surveillance System (HDSS), which included almost 80,000 individuals under constant demographic surveillance. The HDSS population was used as the sample frame for various epidemiological and clinical studies [5, 6]. The under-five mortality rate in this area has dropped from about 40 per 1,000 person-years in the mid-1990s to below 30 per 1,000 in 2007 . Malaria is hyper- to holo-endemic in this area with a transmission peak at the end of the rainy season (June to October) and reduced transmission during the dry season (November to May) . The dry season consists of a cold period from November to February and a very hot phase between March and May. Bourasso had 2,263 inhabitants of different ethnic groups in March 2011 who mainly live from subsistence farming and cattle keeping. A first cross-sectional survey, which was already described elsewhere [13, 18, 19], was carried out in October 2000 and served as a baseline for this study. Starting in 2009 until 2012 the survey was repeated both in October and April at the end of the rainy and dry season respectively.
A random list of all households in the village was generated using the data from the HDSS. All household members of the first 50–100 households were invited to participate until the target of ~250 participants was reached (inclusion criterion above six months of age). Sample size was determined with the aim of ~50 parasite positive isolates available for drug resistance testing per cycle. For the dry season an overall parasite prevalence of ~15% (no previous data available) was assumed, which later had to be updated. The assumed parasite prevalence for the rainy season was 80% as it was reported in previous studies .
Written informed consent was taken from all patients prior to enrolment. Anthropometry for children was performed by skilled health care personnel. Each patient was examined by a physician including recent history of drugs taken. A blood sample (maximum 5 ml) was taken by a skilled provider. Thick and thin blood smears were prepared and parasite density counted against 200 WBC . One drop of blood was applied to filter paper (either GenoCard™, Hain Lifesciences, Germany or Whatman™ 3MM chromatography paper, Brentfort, UK). The filter papers were air-dried, stored in plastic bags and transported to the Parasitology Laboratory at Heidelberg University Hospital for molecular analysis. The blood smears were fixed in methanol and stained with Giemsa solution to be analysed on the spot. Treatment according to MOH guidelines was provided without charge. Patients whose blood smears were positive for Plasmodium parasites were treated with amodiaquine-artesunate. The blood sample was divided into blood for culture and serum for pharmacological analysis of anti-malarial drugs, then frozen and transported to Heidelberg for further experiments. Ethical approval was obtained by the ethical review board of Heidelberg University Hospital and the Institutional Review Board in Nouna.
The genomic DNA was extracted from the filter papers using the Chelex-100 method . The different Plasmodium species were analysed by microscopy and species-specific nested-PCR , the positivity for Plasmodium falciparum was the basis for the calculated PCR prevalence.
Graphs were created with Sigma-Plot 11 (Systat Software, Chicago, USA). Statistical analysis was performed using STATA 11 program (Stata Corporation, Duxbury, USA). A t-test for proportions was used to assess the significance of differences between parasite prevalences and species distributions in dry and wet season of this study. A Mann–Whitney Rank Sum Test was carried out to compare the parasite densities excluding all zero counts. To evaluate the joint influence of season, age, and parasite density on symptomatic disease/fever a logistic regression analysis was performed.