As the absence of gene amplification does not definitively prove the presence of a gene deletion, we also assessed expression of PfHRP2 in study samples. Unfortunately, adequate material was available for this analysis for only 39 of the 56 RDT-negative P. falciparum samples. For these assays recombinant PfHRP2 (Microcoat Biotechnologie GmbH, Bernried am Starnberger See, Germany) was used as a positive control and blood from persons not infected with malaria as a negative control. PfHRP2 levels was quantified using a bead-based immunoassay with a MAGPIX instrument (Luminex Corp., Austin, TX), as previously described [2628]. Briefly, the bead-based HRP2 immunoassay, which relies on antigen capture, is capable of detecting PfHRP2 at sub-picogram levels, allowing fast processing and screening of large numbers of samples. As in prior studies, the cut-off for a positive PfHRP2 antigen result was the mean plus three standard deviations based on a panel of 92 antigen negative blood samples [28].

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