Sample collection, DNA extraction, and real-time PCR
At 3-month intervals from enrolment until 18 months old, participants were seen at the health center where they underwent a clinical check-up and blood sampling. From the blood samples taken at the clinic, 100 μl (2 spots, each 50 μl) was applied to Whatman FTA filter paper (Whatman plc, Maidstone, UK), air-dried, and placed in individually sealed plastic bags with a desiccant. The sample bags were then stored in dry condition at room temperature for about 5 years before the analyses. In total, there were 2799 DBS samples stored from this trial in 2008.The DNA extraction of Quantitative polymerase chain reaction (qPCR) was performed at laboratories of the College of Medicine, University of Malawi to amplify the LDH gene, a marker of All reactions were run in duplicate on the StepOneplus Real-Time PCR machine (Applied Biosystems, Foster City, CA, USA). The cycling conditions were 50 °C for 2 min, 95 °C for 10 min, and 45 cycles of 95 °C for 15 s and then 60 °C for 1 min. Four 