Plasmodium falciparum pfhrp2 and pfhrp3 Gene Deletions and Relatedness to Other Global Isolates, Djibouti, 2019-2020

Abstract

Deletions of pfhrp2 and paralogue pfhrp3 (pfhrp2/3) genes threaten Plasmodium falciparum diagnosis by rapid diagnostic test. We examined 1,002 samples from suspected malaria patients in Djibouti City, Djibouti, to investigate pfhrp2/3 deletions. We performed assays for Plasmodium antigen carriage, pfhrp2/3 genotyping, and sequencing for 7 neutral microsatellites to assess relatedness. By PCR assay, 311 (31.0%) samples tested positive for P. falciparum infection, and 296 (95.2%) were successfully genotyped; 37 (12.5%) samples were pfhrp2+/pfhrp3+, 51 (17.2%) were pfhrp2+/pfhrp3-, 5 (1.7%) were pfhrp2-/pfhrp3+, and 203 (68.6%) were pfhrp2-/pfhrp3-. Histidine-rich protein 2/3 antigen concentrations were reduced with corresponding gene deletions. Djibouti P. falciparum is closely related to Ethiopia and Eritrea parasites (pairwise G_ST 0.68 [Ethiopia] and 0.77 [Eritrea]). P. falciparum with deletions in pfhrp2/3 genes were highly prevalent in Djibouti City in 2019-2020; they appear to have arisen de novo within the Horn of Africa and have not been imported.

Keywords: Djibouti; Plasmodium falciparum; malaria; parasite relatedness; parasites; pfhrp2; pfhrp3; vector-borne infections.

Figure 1

Flow diagram for reporting pfhrp2 and pfhrp3 genotype for all specimens in study of Plasmodium falciparum parasites with pfhrp2 and pfhrp3 deletions, Djibouti, 2019–2020. Terminal boxes display number of samples successfully genotyped for pfhrp2/3.

Figure 2

Distributions of HRP2/HRP3 antigen concentrations by pfhrp2 and pfhrp3 genotype for specimens in study of Plasmodium falciparum parasites with pfhrp2 and pfhrp3 deletions, Djibouti, 2019–2020. A) Individual antigen concentrations for all 296 samples successfully genotyped for pfhrp2/3. Dashed line denotes the assay level of quantitation. B) Smoothed kernel density plots for log-transformed HRP2/3 concentration by the four pfhrp2/3 genotypes. HRP2/3, histidine-rich protein 2/3.

Figure 3

Relatedness of Plasmodium falciparum parasites from Djibouti, 2019–2020, with different pfhrp2 and pfhrp3 genotypes. Cluster PC analysis shown for 7 neutral microsatellite data for monogenomic infections by subpopulations: pfhrp2+/pfhrp3+ (n = 16), pfhrp2+/pfhrp3– (n = 15), pfhrp2–/pfhrp3+ (n = 4), pfhrp2–/pfhrp3– (n = 17). Plot shown with PC1 on x-axis and PC2 on y-axis with 95% confidence ellipses. PC, principal component.

Figure 4

Relatedness of Plasmodium falciparum parasites from Djibouti, 2019–2020 with other global isolates. A) Cluster PC analysis shown for neutral microsatellite data for monogenomic infections by collection from different countries: Angola (n = 32), Costa Rica (n = 14), Djibouti (n = 52), Eritrea (n = 187), Ethiopia (n = 20), Guyana (n = 27), Haiti (n = 86), Peru (n = 18), Rwanda (n = 42), Sudan (n = 37), Suriname (n = 44), Uganda (n = 25). B) Cluster PC analysis shown for neutral microsatellite data for monogenomic infections containing pfhrp2 deletions by collection from different countries: Djibouti (n = 21), Eritrea (n = 43), Peru (n = 18), Ethiopia (n = 8), Sudan (n = 4), and Suriname (n = 1). Plots shown with PC1 on x-axis and PC2 on y-axis and 95% confidence ellipses. PC, principal component.