Molecular surveillance of pfhrp2 and pfhrp3 genes deletion in Plasmodium falciparum isolates and the implications for rapid diagnostic tests in Nigeria

Abstract Prompt diagnosis and appropriate treatment of malaria remain the hallmark for reducing malaria-related mortality in high transmission areas. Plasmodium falciparum histidine-rich protein2 ( PfHRP 2) based rapid diagnostic tests (RDT) play a vital role in prompt and accurate malaria diagnosis. However, pfhrp 2 gene deletion threatens the RDT test sensitivity. This study reports the presence of pfhrp 2 and pfhrp 3 genes deletion among parasite isolates in Nigeria. Febrile children were screened using histidine-rich protein (HRP2) specific RDT (SD-Bioline RDT) and microscopy for P. falciparum infections. All RDT negative samples were re-evaluated by polymerase chain reaction (PCR). The presence of parasite in RDT false negative cases and randomly selected RDT positive cases were validated using PCRs targeting glutamate-rich protein ( glurp ) and merozoite surface proteins ( msp-1 and msp-2 ). Thereafter, exon 2 of pfhrp 2 and pfhrp 3 were amplified, and Sanger sequenced. A total of 511 febrile children were enrolled out of which 309 (61%) were positive by RDT. The presence of pfhrp 2 and pfhrp 3 genes were analyzed in 66 PCR positive samples comprising of 31 RDT false negative and 35 RDT true positive randomly selected samples. The pfhrp 2 and pfhrp 3 genes failed to amplify in 17% (11/66) and 6% (4/66) samples, respectively. Seven of the eleven samples had only pfhrp 2 deletion while four had both pfhrp 2 and pfhrp 3 deletions. The absence of the pfhrp 2 gene may be responsible for the seven RDT false negative cases observed. Three RDT positive cases lacked pfhrp 2 whereas pfhrp 3 was absent in only four RDT false negative cases. The pfhrp 2 and pfhrp 3 amino acid repeat sequences were highly diverse. The P. falciparum isolates lacking pfhrp 2 and pfhrp 3 genes may be circulating and contributing to RDT false negativity in Nigeria. More studies in larger population and seasonally defined cases will be needed to determine the extent of pfhrp 2/3 genes deletion in different geographical areas of Nigeria.