Deletion of Plasmodium falciparum Histidine-Rich Protein 2 (pfhrp2) and Histidine-Rich Protein 3 (pfhrp3) Genes in Colombian Parasites

Abstract

A number of studies have analyzed the performance of malaria rapid diagnostic tests (RDTs) in Colombia with discrepancies in performance being attributed to a combination of factors such as parasite levels, interpretation of RDT results and/or the handling and storage of RDT kits. However, some of the inconsistencies observed with results from Plasmodium falciparum histidine-rich protein 2 (PfHRP2)-based RDTs could also be explained by the deletion of the gene that encodes the protein, pfhrp2, and its structural homolog, pfhrp3, in some parasite isolates. Given that pfhrp2- and pfhrp3-negative P. falciparum isolates have been detected in the neighboring Peruvian and Brazilian Amazon regions, we hypothesized that parasites with deletions of pfhrp2 and pfhrp3 may also be present in Colombia. In this study we tested 100 historical samples collected between 1999 and 2009 from six Departments in Colombia for the presence of pfhrp2, pfhrp3 and their flanking genes. Seven neutral microsatellites were also used to determine the genetic background of these parasites. In total 18 of 100 parasite isolates were found to have deleted pfhrp2, a majority of which (14 of 18) were collected from Amazonas Department, which borders Peru and Brazil. pfhrp3 deletions were found in 52 of the100 samples collected from all regions of the country. pfhrp2 flanking genes PF3D7_0831900 and PF3D7_0831700 were deleted in 22 of 100 and in 1 of 100 samples, respectively. pfhrp3 flanking genes PF3D7_1372100 and PF3D7_1372400 were missing in 55 of 100 and in 57 of 100 samples. Structure analysis of microsatellite data indicated that Colombian samples tested in this study belonged to four clusters and they segregated mostly based on their geographic region. Most of the pfhrp2-deleted parasites were assigned to a single cluster and originated from Amazonas Department although a few pfhrp2-negative parasites originated from the other three clusters. The presence of a high proportion of pfhrp2-negative isolates in the Colombian Amazon may have implications for the use of PfHRP2-based RDTs in the region and may explain inconsistencies observed when PfHRP2-based tests and assays are performed.

Global analysis of Plasmodium falciparum histidine-rich protein-2 (pfhrp2) and pfhrp3 gene deletions using whole-genome sequencing data and meta-analysis

“…However, this approach may be compromised by the emergence of P. falciparum parasites with deleted pfhrp2. South America was the first region to report the occurrence of false negative RDT results due to this gene deletion (Gamboa et al, 2010;Akinyi et al, 2013;Abdallah et al, 2015;Akinyi Okoth et al, 2015;Murillo Solano et al, 2015;Rachid Viana et al, 2017). Similar evidence was followed by more recent studies from Africa (Dolo et al, 2012;Wurtz et al, 2013;Parr et al, 2016;Beshir et al, 2017) and Asia (Kumar et al, 2013;Li et al, 2015;Bharti et al, 2016).…”

“…Meta-analysis was then performed on the remaining ten studies. These studies consisted of data from Bolivia (Rachid Viana et al, 2017), Brazil (Rachid Viana et al, 2017), Colombia (Murillo Solano et al, 2015;Dorado et al, 2016), French Guiana (Trouvay et al, 2013), Guyana , Honduras (Abdallah et al, 2015), Peru (Gamboa et al, 2010), and Suriname (Akinyi Okoth et al, 2015) in South America, and from Eritrea (Menegon et al, 2017;Berhane et al, 2018), Ghana (Amoah et al, 2016), and Kenya (Beshir et al, 2017) in Africa .…”

“…Initial evidence of deletion was confirmed by PCR amplification of pfhrp2 and pfhrp3 and their flanking regions to infer the extent of the respective deletions. To test the quality of DNA in the samples and confirm the gene deletions, further PCR amplification was carried out in other genes, such as the merozoite surface protein-1 or -2 genes (Gamboa et al, 2010;Murillo Solano et al, 2015;Dorado et al, 2016;Beshir et al, 2017;Rachid Viana et al, 2017). However, the recommended antigen analysis based on re-testing the samples with a different pfhrp2-based RDT or using an enzyme-linked immunosorbent assay to quantify the levels of the pfhrp2 protein (Cheng et al, 2014) in a sample was only performed in three studies (Gamboa et al, 2010;Dorado et al, 2016;Rachid Viana et al, 2017).…”

A national health facility survey of malaria infection among febrile patients in Kenya, 2014

“…However, the associated risk of overtreatment of uninfected cases is considered more acceptable in malaria endemic zones than taking the risk of failing to detect cases. Recently, there has been reports of deletion of HRP2 gene which may lead to false RDT negative results [47, 48]. However, studies conducted in Africa showed HRP2 deletion in very small numbers of parasite isolates [49, 50], hence highly unlikely that the phenomenon may have influenced the results of this survey.…”

A novel metabarcoded DNA sequencing tool for the detection of Plasmodium species in malaria positive patients

“…The targeted antigens are abundant in the sexual and asexual stages of the parasites, while histidine-rich protein 2 (Pf-HRP2) based RDT is specific for P. falciparum and lactate dehydrogenase (Pf-LDH & Pv-LDH) specific for detecting both P. falciparum and P. vivax . Pan based RDT targets specific antigens including lactate dehydrogenase (P-LDH) and aldolase proteins found in all Plasmodium species (Akinyi Okoth et al, 2015; Murillo Solano et al, 2015). Several factors potentially affect the accuracy and false-negative results of RDT including the interpretation of the test strip colour change, the density of malaria infection in the host, improper storage/handling of the kit and poor test performance (Echeverry et al, 2016).…”