Fig. 2.

PCR amplification of samples negative with the HRP2 RDT which were positive by microscopy or by pLDH RDT. PCR was performed to test for Plasmodium DNA in samples that were negative by HRP2 RDT but positive by either microcopy or by pLDH RDT. PCR was performed using a tiered, 3-stage approach. Rows 2–4 of the flowchart reflect PCR performed in stages 1–3, respectively. The primers used at each stage are listed in the gray boxes to the right of the flowchart. If a sample was identified as positive by PCR at any stage, no further PCR testing was pursued. Samples positive by PCR for the hrp2 gene were considered P. falciparum isolates containing the hrp2 gene. Samples negative by PCR for the hrp2 gene and positive by PCR for Pf 18S rRNA were considered P. falciparum isolates lacking the hrp2 gene. Samples negative by PCR for the hrp2 gene and Pf 18S rRNA but positive by PCR for Pv, Pm or Po 18S rRNA were considered non-Pf malaria. Samples negative by PCR for the hrp2 gene, Pf 18S rRNA and Pv, Pm and Po 18S rRNA were considered negative for Plasmodium DNA. Pf Plasmodium falciparum, Pv P. vivax, Pm P. malariae, Po P. ovale