High prevalence and extended deletions in Plasmodium falciparum hrp2/3 genomic loci in Ethiopia

Deletions in Plasmodium falciparum histidine rich protein 2( pfhrp 2) gene threaten the usefulness of the most widely used HRP2-based malaria rapid diagnostic tests (mRDTs) that cross react with its structural homologue, Pf HRP3. Parasites with deleted pfhrp 2/3 genes remain undetected and untreated due to ‘false-negative’ RDT results. As Ethiopia recently launched malaria elimination by 2030 in certain selected areas, the availability of RDTs and the scale of their use have rapidly increased in recent years. Thus, it is important to explore the presence and prevalence of deletion in the target genes, pfhrp 2 and pfhrp 3. From a total of 189 febrile patients visited Adama Malaria Diagnostic centre, sixty-four microscopically-and polymerase chain reaction (PCR)-confirmed P . falciparum clinical isolates were used to determine the frequency of pfhrp 2/3 gene deletions. Established PCR assays were applied to DNA extracted from blood spotted onto filter papers to amplify across pfhrp 2/3 exons and flanking regions. However, analysis of deletions in pfhrp 2, pfhrp 3 and flanking genomic regions was successful for 50 of the samples. The pfhrp2 gene deletion was fixed in the population with all 50(100%) isolates presenting a deletion variant. 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 Pf3D7 0831700 (MALPI.228) gene, upstream of pfhrp 2. Similarly, the pfhrp 3 gene was deleted in all isolates (100%), while 40% of the isolates had an extension of the deletion to the downstream flanking region that codes for Pf3D7 13272400 (MAL13PI.485).The pfhrp 3 deletion also extended upstream to Pf3D7 081372100 (MAL13PI.475) region in 49/50 (95%) of the isolates, exhibiting complete absence of the locus. Although all samples showed deletions of pfhrp 2 exon regions, amplification of an intron region was successful in five cases. Two different repeat motifs in the intron regions were observed in the samples tested. Pfhrp 2/3 gene deletions are fixed in Ethiopia and this will likely reduce the effectiveness of Pf HRP2-based mRDTs. It will be important to determine the sensitivity Pf HRP 2/3-based RDTs in these populations and conduct a countrywide survey to determine the extent of these deletions and its effect on routine RDT-based malaria diagnosis.