Deletion patterns, genetic variability and protein structure of pfhrp2 and pfhrp3: implications for malaria rapid diagnostic test in Amhara region, Ethiopia

Abstract

Background: Although rapid diagnostic tests (RDTs) play a key role in malaria-control strategies, their efficacy has been threatened by deletion and genetic variability of the genes pfhrp2/3. This study aims to characterize the deletion, genetic patterns and diversity of these genes and their implication for malaria RDT effectiveness, as well as their genetic evolution in the Amhara region of Ethiopia.

Methods: The study included 354 isolates from symptomatic patients from the Amhara region of Ethiopia who tested positive by microscopy. Exon 1-2 and exon 2 of genes pfhrp2 and -3 were amplified, and exon 2 was sequenced to analyse the genetic diversity, phylogenetic relationship and epitope availability.

Results: The deletion frequency in exon 1-2 and exon 2 was 22 and 4.6% for pfhrp2, and 68 and 18% for pfhrp3, respectively. Double deletion frequency for pfhrp2 and pfhrp3 was 1.4%. High genetic diversity, lack of clustering by phylogenetic analysis and evidence of positive selection suggested a diversifying selection for both genes. The amino-acid sequences, classified into different haplotypes, varied widely in terms of frequency of repeats, with novel amino-acid changes. Aminoacidic repetition type 2 and type 7 were the most frequent in all the sequences. The most frequent epitopes among protein sequences were those recognized by MAbs 3A4 and C1-13.

Conclusion: Deletions and high amino acidic variation in pfhrp2 and pfhrp3 suggest their possible impact on RDT use in the Amhara region, and the high genetic diversity of these genes could be associated with a diversifying selection in Ethiopia. Surveillance of these genes is, therefore, essential to ensure the effectiveness of public health interventions in this region.

Keywords: Deletions; Ethiopia; Malaria; Plasmodium falciparum; Rapid diagnostic test; pfhrp2.

Fig. 1

Map of collection sites in Ethiopia. The red dots correspond to the location of the four cities’ health centres where samples were collected

Fig. 2

Methodological flow scheme. Microscopy diagnosis was confirmed by nested multiplex PCR, distinguishing P. falciparum, Plasmodium vivax, Plasmodium Ovale, and Plasmodium malaria. Four independent PCRs were run for the P. falciparum samples to detect deletions in exon 1–2 and exon 2 of pfhrp2 and pfhrp3. The deletion of any exon was confirmed by an absence of amplification after three PCR repetitions and the confirmation of maintained DNA quality. Samples that could not be confirmed for DNA quality were excluded from the analysis for that gene. Subsequent data analysis for the combined results was performed only with those samples included in the independent analysis for both genes. Additionally, a sub-sample lacking the deletion for exon 2 of both genes was sequenced and the genetic diversity and variation in amino-acid sequences analysed. Finally, a sub-sample of these sequences was used to predict the protein structure model and to locate the epitopes in the structure

Fig. 3

Summary of results of molecular analysis of pfhrp2 and pfhrp3. Figure represents the different results in the independent experiments for exon 1–2 and 2 of both genes. Deletions samples: samples that did not amplify in the first PCR for the exon. Confirmed deletions: samples that did not amplify in three independent PCR experiments for the same specific gene, process called ‘repetition pfhrp2/3 PCR’ in the figure. Presence: samples that amplified at least one for a specific exon

Fig. 4

Gel images of the full-length PCR of pfhrp2 and pfhrp3. Electrophoresis gel showed the result of the amplification for PCR combining exon 1–2 and exon 2, the expected fragment was around 900 base pairs as it could be observed in the positives controls placed at the end of the gel: 3D7, Dd2 and HB3. Negative controls for the first and second PCR were placed in the first two wells of the gel

Fig. 5

Bayesian inference phylogenetic tree for pfhrp2 (a) and pfhrp3 (b) sequences. The nucleotide sequences of exon 2 for both genes, with fragments ranging from 300 to 700 base pairs, were analysed. This analysis combined nucleotide sequences from this article, represented with a dot (n = 95 for pfhrp2 and n = 79 for pfhrp3) and sequences from bordering countries published in previous studies, represented with dots with different shapes (n = 33 for pfhrp2 and n = 33 for pfhrp3). Coloured dots indicate the geographical origin of the samples. Phylogenetic trees were performed using Bayesian inference

Fig. 6

Secondary structure, solvent surface area and mapping of epitopes in the PfHRP2 protein structure models. Model of structure was assessed by RSMD measured with Å. Solvent surface area is coloured gradually according to electrostatic potential from − 5 kT/e (red) to + 5 kT/e (blue). NA: Not applicable due to absence of epitope in the sequence

Fig. 7

Secondary structure, solvent surface area and mapping of epitopes in the PfHRP3 protein structure models. Model of structure was assessed RSMD measured with Å. Solvent surface area is coloured gradually according to electrostatic potential from − 5 kT/e (red) to + 5 kT/e (blue). NA: Not applicable due to absence of epitope in the sequence