1256 individuals were enrolled in this study at different sites: 586 healthy blood donors from the ethnic group of the Kinh at the Than Hung Dao Hospital in Hanoi as an extension of the study described in , 199 Central Africans at the Albert Schweitzer Hospital in Lambarènè  and 265 Brazilians of Caucasian origins at the Hospital de Clinicas in Curitiba , 108 Kaingang and 98 Guarani were recruited in their villages. Kaingang and Guarani are the two major Amerindian tribes living in Southern Brazil but despite living side by side for centuries the two groups differ in many aspects of culture and genetics; e. g. HLA type .
The Brazilians of European descent were either patients enrolled in a hepatitis C treatment study (n = 101) or healthy controls (n = 164) . No patients had a history of alcohol or drug abuse. Patients were included into the study when HCV antibodies and HCV RNA was detected in serum; the presence of HBV and HIV antibodies was an exclusion criterion. From all patients biopsies were taken and liver histology was assessed blindly by two pathologists and staging of fibrosis.
Central Africans individuals were affected either by mild (n = 100) or severe malaria (n = 99) . Mild malaria was characterised by a parasitaemia < 50,000 parasites/μl, haemoglobin > 80 g/L, glucose > 50 mg/dL, lactate < 3 mmol/L, leukocytes < 12/nL, platelets > 50/nL, no schizontaemia, and no signs of severe malaria such as cerebral malaria or other infections. The severely infected children presented with severe anaemia or hyperparasitaemia as the main complications of clinical disease. Appropriate treatment with sulfadoxine/pyrimethamine was given to mild cases. Severe cases were treated with intravenous quinine plus clindamycin.
Amerindians were healthy subjects from a human genetics study .
The genetic analysis was approved by the relevant ethic committees of the involved institutions: the Institutional Review Board of the Tran Hung Dao Hospital, Hanoi, Vietnam; the ethics committee of the Medical Faculty in Tübingen, Germany, the ethics committee of the Hospital de Clínicas in Curitiba, Brazil and the ethics committees of the International Foundation for the Albert Schweitzer Hospital in Lambaréné, Gabon.
Blood from all the participants was collected in EDTA tubes and plasma was separated, aliquoted and stored at -20°C for later use. Genomic DNA was extracted from blood samples from using the QIAamp DNA blood kit (Qiagen, Hilden, Germany), as described in the user manual.
Polymerase chain reaction (PCR)
Genomic DNA was used as template for PCR. A single fragment of 480 bp from the IFNA2 promoter was amplified using the following primer sets: IFN-F: 5'-TTTCAAAAAAGTTGCTCTAAG-3', IFN-R: 5'-GTAGATGTTGCAGATGCTGCT-3' (Operon, Germany).
All amplifications were initiated with a denaturing step of 94°C for 5 min, followed by 42 cycles of 94°C for 30 sec, 45°C for 30 sec annealing time and 72°C for 1 minute elongation. The reaction was stopped after a final elongation step of 72°C for 10 min. A PTC-200 Peltier Termal Cycler (Bio-Rad, Munich, Germany) was used to amplify all the samples.
After purification, the respective PCR products were sequenced with the same primers described above using BigDye terminator chemistry on an ABI PRISM 3100 Genetic analyzer (Applied Biosystems, Foster City, USA). The sequences were analyzed for polymorphisms in the promoter of the IFNA2 gene after DNA alignment of the sequenced products using the Bioedit alignment program (North Carolina State University, USA). The quality of the sequencing was assessed by visual inspection with reference to the electropherograms.
IFNA2 allele frequencies and genotype distributions for each population as well as Hardy-Weinberg exact test were calculated using the Genepop version 3.4 http://genepop.curtin.edu.au/.
Differences in allele frequencies and genotype distributions between populations were calculated using Fisher's exact test using STATA http://www.stata.com/. P-value < 0.05 was considered to be significant.