Streamlined, PCR-based testing for pfhrp2- and pfhrp3-negative Plasmodium falciparum

Background: Rapid diagnostic tests (RDTs) that detect histidine-rich protein 2 (PfHRP2) are used throughout Africa for the diagnosis of Plasmodium falciparum malaria. However, recent reports indicate that parasites lacking the pfhrp2 and/or histidine-rich protein 3 (pfhrp3) genes, which produce antigens detected by these RDTs, are common in select regions of South America, Asia, and Africa. Proving the absence of a gene is challenging, and multiple PCR assays targeting these genes have been described. A detailed characterization and comparison of published assays is needed to facilitate robust and streamlined testing approaches. Results: Among six pfhrp2 and pfhrp3 PCR assays tested, the lower limit of detection ranged from 0.01 pg/mu L to 0.1 ng/mu L of P. falciparum 3D7 strain DNA, or approximately 0.4-4000 parasite genomes/mu L. By lowering the elongation temperature to 60 degrees C, a tenfold improvement in the limit of detection and/or darker bands for all exon 1 targets and for the first-round reaction of a single exon 2 target was achieved. Additionally, assays targeting exon 1 of either gene yielded spurious amplification of the paralogous gene. Using these data, an optimized testing algorithm for the detection of pfhrp2- and pfhrp3-negative P. falciparum is proposed. Conclusions: Surveillance of pfhrp2-and pfhrp3-negative P. falciparum requires careful laboratory workflows. PCR-based testing methods coupled with microscopy and/or antigen testing serve as useful tools to support policy development. Standardized approaches to the detection of pfhrp2-and pfhrp3-negative P. falciparum should inform efforts to define the impact of these parasites.