Molecular surveillance reveals the presence of pfhrp2 and pfhrp3 gene deletions in Plasmodium falciparum parasite populations in Uganda, 2017-2019

Abstract

Background: Histidine-rich protein-2 (HRP2)-based rapid diagnostic tests (RDTs) are the only RDTs recommended for malaria diagnosis in Uganda. However, the emergence of Plasmodium falciparum histidine rich protein 2 and 3 (pfhrp2 and pfhrp3) gene deletions threatens their usefulness as malaria diagnostic and surveillance tools. The pfhrp2 and pfhrp3 gene deletions surveillance was conducted in P. falciparum parasite populations in Uganda.

Methods: Three-hundred (n = 300) P. falciparum isolates collected from cross-sectional malaria surveys in symptomatic individuals in 48 districts of eastern and western Uganda were analysed for the presence of pfhrp2 and pfhrp3 genes. Presence of parasite DNA was confirmed by PCR amplification of the 18s rRNA gene, msp1 and msp2 single copy genes. Presence or absence of deletions was confirmed by amplification of exon1 and exon2 of pfhrp2 and pfhrp3 using gene specific PCR.

Results: Overall, pfhrp2 and pfhrp3 gene deletions were detected in 29/300 (9.7%, 95% CI 6.6-13.6%) parasite isolates. The pfhrp2 gene was deleted in 10/300 (3.3%, 95% CI 1.6-6.0%) isolates, pfhrp3 in 9/300 (3.0%, 95% CI 1.4-5.6%) while both pfhrp2 and pfhrp3 were deleted in 10/300 (3.3%, 95% CI 1.6-6.0%) parasite isolates. Proportion of pfhrp2/3 deletions was higher in the eastern 14.7% (95% CI 9.7-20.0%) compared to the western region 3.1% (95% CI 0.8-7.7%), p = 0.001. Geographical location was associated with gene deletions aOR 6.25 (2.02-23.55), p = 0.003.

Conclusions: This is the first large-scale survey reporting the presence of pfhrp2/3 gene deletions in P. falciparum isolates in Uganda. Roll out of RDTs for malaria diagnosis should take into consideration the existence of pfhrp2/3 gene deletions particularly in areas where they were detected. Periodic pfhrp2/3 surveys are recommended to inform future decisions for deployment of alternative RDTs.

Keywords: Deoxyribonucleic acid; Gene deletion; Histidine rich protein 2; Histidine rich protein 3; Malaria rapid diagnostic tests; Microscopy; Plasmodium falciparum.

Fig. 1

Geographical information system (GIS) mapping and geographical distribution of sites where Plasmodium falciparum isolates were collected. Showing the location of sites where study samples were collected across 48 districts in eastern and western regions of Uganda. RDT−/microscopy+ (indicated by red dots) are samples that were RDT negative but microscopy positive. RDT+/microscopy+ (indicated by green symbols) are samples that were positive on both RDTs and microscopy

Fig. 2

pfhrp2 and pfhrp3 study profile. RDT in this case means samples tested with HRP2 rapid diagnostic tests. PCR is the polymerase chain reaction for parasite detection and speciation. pfhrp2 and pfhrp3 PCRs are the polymerase chain reactions for amplification of exon 1 and exon 2 of P. falciparum histidine-rich protein 2 and histidine-rich protein 3 genes. pfhrp2/pfhrp3 PCR negative are samples in which pfhrp2/3 genes were missing despite presence of parasite DNA and msp1 and msp2 single copy genes. Low quality DNA means samples that were DNA PCR positive but could not amplify two single copy genes (msp1 and msp2) as indicator of quality of DNA

Fig. 3

Mapping the exact locations of pfhrp2 and pfhrp3 gene-deleted Plasmodium falciparum isolates. Exact location of collection sites for 29 gene-deleted P. falciparum parasites by latitude and longitudes coordinates. pfhrp2−/pfhrp3+ (indicated by red dots), pfhrp2+/pfhrp3− (indicated by green circles) and pfhrp2-/pfhrp3− (indicated by the purple hexagons)