Low Prevalence of Plasmodium falciparum Histidine-Rich Protein 2 and 3 Gene Deletions-A Multiregional Study in Central and West Africa

Abstract

Plasmodium falciparum parasites carrying deletions of histidine-rich protein 2 and 3 genes, pfhrp2 and pfhrp3, respectively, are likely to escape detection via HRP2-based rapid diagnostic tests (RDTs) and, consequently, treatment, posing a major risk to both the health of the infected individual and malaria control efforts. This study assessed the frequency of pfhrp2- and pfhrp3-deleted strains at four different study sites in Central Africa (number of samples analyzed: Gabon N = 534 and the Republic of Congo N = 917) and West Africa (number of samples analyzed: Nigeria N = 466 and Benin N = 120) using a highly sensitive multiplex qPCR. We found low prevalences for pfhrp2 (1%, 0%, 0.03% and 0) and pfhrp3 single deletions (0%, 0%, 0.03% and 0%) at all study sites (Gabon, the Republic of Congo, Nigeria and Benin, respectively). Double-deleted P. falciparum were only found in Nigeria in 1.6% of all internally controlled samples. The results of this pilot investigation do not point towards a high risk for false-negative RDT results due to pfhrp2/pfhrp3 deletions in Central and West African regions. However, as this scenario can change rapidly, continuous monitoring is essential to ensure that RDTs remain a suitable tool for the malaria diagnostic strategy.

Keywords: Central Africa; Plasmodium falciparum; West Africa; histidine-rich protein 2 and 3; pfhrp2/pfhrp3 deletions; rapid diagnostic test.

Figure A1

Histograms showing the respective ΔC_q values of pfbtub–pfhrp2 or pfbtub–pfhrp3 for each study center.

Figure A2

Boxplot comparing P. falciparum/pfcytb C_q-values of RDT false-negative samples and RDT true-positive samples. Median and inter-quartile ranges (IQR) are displayed with whiskers in the style of Tukey (extends to the smallest/largest value no further than 1.5 × IQR (inter-quartile range) from the hinge with outliers depicted as circles); C_q—cycle of quantification.

Figure A3

Amplification curves of respective genes in the 4-plex qPCR from the 3D7 control and the single-deleted P. falciparum strains found in Gabon (1,2) and Nigeria (3,4).

Figure A4

Amplification curves of the respective genes in the 4-plex qPCR from the five double-deleted P. falciparum strains found in Nigeria.

Figure 1

Geographical overview of the study sites in West and Central Africa. Red dots represent sampling sites (1) Benin: Kpomasse, Ouidah; (2) Nigeria: Awka, Nnewi, Onitsha; (3) Gabon: Lambaréné; (4) Republic of Congo: Goma Tsé-Tsé District, Brazzaville. The map was created with the ggplot2 package V3.4.0 in R Version 4.2.2.

Figure 2

Venn diagram of concordant P. falciparum diagnostics. Concordance of positive test results between the 4-plex qPCR (P. falciparum detection via pfcytb), RDT and TBS microscopy in (A) Gabon and (B) the Republic of Congo. Only samples with available results for all three diagnostic tests (qPCR, TBS, RDT) were included. RDT: Malaria rapid diagnostic test, TBS: Thick blood smear microscopy.