Background: Despite remarkable malaria reduction in recent years, malaria remains a public health problem in Ethiopia. With the introduction of rapid diagnostic tests (RDTs), malaria diagnosis has been transformed. However, the Plasmodium falciparum histidine rich protein 2 ( hrp 2) that is targeted by the most widely used RDTs is prone to genetic mutations and gene deletions as observed in recent years. Patients infected with P. falciparum malaria parasites with a deletion in hrp 2/3 gene locus would remain undetected and results in ‘false-negatives’, which are not treated. Undoubtedly, these untreated infected patients are at risk of developing complicated disease and may further fuel parasite transmission. Hence, molecular targeting of the region across exons and flanking genes has been used to provide greater confirmatory evidence of gene deletions. This study was initiated to determine pfhrp2 /3 gene deletions including the flanking regions.
Methods: A cross-sectional study was conducted to determine the prevalence of hrp 2/3 genes deletion.Finger-prick blood samples were collected from a total of 64 febrile patients attending Adama Malaria Diagnostic Centre in 2015. Thick and thin blood smears were prepared for microscopic slide readings, and parasitaemias were determined. Blood samples were spotted onto filter for parasite DNA extraction.
Results: From a total of 64 microscopically and PCR confirmed P. falciparum infections, 50 were successfully analyzed for deletion of pfhrp2 , pfhrp3 and flanking regions. Extensive deletions were observed in the pfhrp2 gene with all 50(100%) isolates presenting a deletion. This deletion extended downstream towards the Pf3D7 0831900 (MAL7PI.230) gene in 11/50 (22%) cases. In contrast, only 2/50 (4%) of samples had deletions for the upstream gene Pf3D7 0831700 (MALPI.228). Similarly, all isolates had deleted the pfhrp3 gene (100%) and this in 40% of isolates extended to the downstream flanking region Pf3D7 13272400 (MAL13PI.485) where 40% samples showed absence of this region. However, the deletion also extended toward the upstream region Pf3D7 081372100 (MAL13PI.475). The deleted pfhrp 3 genomic region also extended upstream to the region Pf3D7 081372100 (MAL13PI.475) with 49/50 (95%) of the isolates exhibiting absence of the locus.
Conclusion: As patient recruitment was not done on pfhrp 2/3-based RDTs, it is impossible to know if the isolates would test negative or positive in the absence of hrp 2/3 genes. Indeed, the sequence variation and high frequencies of deletion in pfhrp2 and pfhrp3 genes in Ethiopian isolates most likely will have a negative influence on the performance of currently used pfhrp2 RDTs. This study confirms the presence of that P. falciparum parasite population with extensive deletions of the pfhrp 2 and pfhrp3 genes in Ethiopia and this calls for a countrywide surveillance to determine the extent of these deletions and its effect on routine malaria diagnosis. Keywords: Plasmodium falciparum , Histidine rich protein 2/3, Deletion, RDTs, Microscopy, Ethiopia