Background Prompt diagnosis and appropriate treatment remain the hallmark needed to reduce malaria-related mortality in areas of high transmission. Rapid diagnostic tests (RDTs) that target the Pfhrp-2 gene, are essential in resource-limited settings where microscopy is not available. However, Pfhrp-2 gene deletion is implicated in limiting RDT sensitivity. Studies evaluating Pfhrp-2 and Pfhrp-3 deletion and the amino acid sequence diversity has not been investigated in Nigeria. We therefore hypothesised that malaria parasites in Nigeria are lacking Pfhrp-2/Pfhrp-3 genes with variable amino acid repeats sequences.

Methods The study was part of a prospective cohort study evaluating RDTs performance. We pooled 66 samples comprising false negatives (n=31) and true positives (n=35) to elucidate Pfhrp-2/Pfhrp-3 gene deletion, RDT cross-reactivity with Pfhrp-3 antigen and amino acid sequence diversity. The 18SrRNA, msp 1, msp2 and glurp genes were amplified to establish active Plasmodium falciparum infection and the exon-2 regions of Pfhrp-2 and Pfhrp-3 genes were amplified to determine the presence or absence of Pfhrp-2 and Pfhrp-3 genes. Isolates with conserved Pfhrp-2/Pfhrp-3 were sequenced.

Results All 66 samples were positive for 18SrRNA, msp1, msp2 and glurp, indicating active P. falciparum infection. However, 16.7% and 6.0% of the samples were lacking Pfhrp-2 and Pfhrp-3 genes. Of the false negative samples, 25.8% and 12.9% has Pfhrp-2 and Pfhrp-3 deletions. Three Pfhrp-3 conserved antigens cross reacted to give RDT positive results. An extensive diversity in the amino acid sequence was observed.

Conclusion Plasmodium falciparum parasites in Nigeria lack Pfhrp-2 and Pfhrp-3 genes. However, the proportion of deletions is low compared to reports from other malaria-endemic regions. In addition, a high amino acid tandem repeat was observed. A combination of pLDH and Pfhrp-2 based RDTs is recommended for accurate malaria diagnosis.