DETECTING HRP2 GENE DELETIONS FOR MALARIA CONTROL AND ELIMINATION IN UGANDA

Rapid diagnostic tests (RDTs) provide a great tool for malaria elimination, especially in low resource settings where microscopy or PCR are not available. The most widely used RDT in Africa detects the Plasmodium falciparum-specific antigen, HRP2. There is a new generation of RDTs targeting the same analyte but with significant improvement in limit of detection to support malaria elimination. However, these tests can generate false negative results in infections from parasites that have HRP2 gene deletion leading to variable RDT performance. To confirm false negatives on the High Sensitive Rapid Diagnostic Test (HS-RDT) as pfhrp2 and/or pfhrp3 gene deletions, a genus and species-specific quantitative PET-PCR and 4 pfhrp2 and pfhrp3 nested PCRs were established as reference assays. P. falciparum strains, with different pfhrp2 and pfhrp3 genetic profiles, were serially diluted to determine the sensitivity of each reference assay and the HS-RDT. PET-PCR and pfhrp2 exon 1-2 nested PCR detect 1.56 parasites/uL. Pfhrp2 exon 2, Pfhrp3 exon 1-2 and pfhrp3 exon 2 nested PCRs detect 25 parasites/uL, 50 parasites/uL, and 3.13 parasites/uL, respectively. 8 clinical samples negative on the HS-RDT from Uganda were evaluated on the reference assays. 1 clinical sample out of 8 was confirmed to have pfhrp2 and pfhrp3 genes deleted. Thus, we established a novel set of reference assays for the HS-RDT using a quantitative sample validation test, the PET-PCR, and pfhrp2 and pfhrp3 nested PCRs. We were also able to confirm one false negative HS-RDT result as a pfhrp2 and pfhrp3 gene deletion in Uganda.