Analysis of pfhrp2 genetic diversity in Senegal and implications for use of rapid diagnostic tests

Abstract

Background: The Senegalese National Malaria Control Programme has recommended use of rapid diagnostic tests (RDTs) that target the histidine-rich protein 2 (HRP2), specific to Plasmodium falciparum, to diagnose malaria cases. The target antigen has been shown to be polymorphic, which may explain the variability in HRP2-based RDT results reported in field studies. The genetic diversity of the pfhrp2 gene has not been investigated in depth in many African countries. The goal of this study is to determine the extent of polymorphism in pfhrp2 among Senegal, Mali and Uganda parasite populations, and discuss the implications of these findings on the utility of RDTs that are based on HRP2 detection.

Methods: Sequencing data from the pfhrp2 locus were used to analyze the genetic diversity of this gene among three populations, with different transmission dynamics and malaria parasite ecologies. Nucleotide diversity (π) and non-synonymous nucleotide diversity (πNS) were studied in the pfhrp2 gene from isolates obtained in Senegal. Amino acid repeat length polymorphisms in the PfHRP2 antigen were characterized and parameters of genetic diversity, such as frequency and correlation between repeats in these populations, were assessed.

Results: The diversity survey of the pfhrp2 gene identified 29 SNPs as well as insertion and deletion polymorphisms within a 918 bp region. The Senegal pfhrp2 exhibited a substantial level of diversity [π = 0.00559 and πNS = 0.014111 (πS = 0.0291627)], similar to several polymorphic genes, such as msp1, involved in immune responses, and the gene encoding the SURFIN polymorphic antigen, which are surface exposed parasite proteins. Extensive repeat length polymorphisms in PfHRP2, as well as similar patterns in the number, organization and the type of predicted amino acid repeats were observed among the three populations, characterized by an occurrence of Type 2, Type 4 and Type 7 repeats.

Conclusions: These results warrant deeper monitoring of the RDT target antigen diversity and emphasize that development of other essential genes as a target for diagnostic tools is critical.

Figure 1

Frequency of polymorphism occurrence in thepfhrp2gene from Senegal, Mali, and Uganda isolates. Non-synonymous, synonymous, coding indels polymorphisms were observed. Synonymous and non-synonymous SNPs are the more common polymorphism in the three populations. Synonymous SNPs are higher than non-synonymous SNPs, showing variability between countries. Intronic indels and coding insertion were not observed in Mali.

Figure 2

PCR re-sequencing. To confirm the frameshifting polymorphisms in isolates SenTh113.09 and SenV042.05, PCR amplification and re-sequencing of the region surrounding the polymorphic positions corresponding to aa571 and aa574 in the coding sequence were performed. A: Alignment of the re-sequenced SenV042.1.05 PfHRP2 sequences with 3D7 isolate. The two sequences share 91.1% identity. B: Alignment of the re-sequenced SenTh113.1.09 PfHRP2 sequence with 3D7 isolate showed 79.7% identity. These results suggest that errors in Illumina sequencing or GATK’s Unified Genotyper produced a false call in these two cases.

Figure 3

Nucleotide diversity (π) and non-synonymous polymorphism (π_NS). A: Comparison of the nucleotide (π) of pfhrp2 in the Senegalese population, to other P. falciparum genes. All π values were rank in the P. falciparum genome π = 0.0000 to π = 0.07. The diversity π pfhrp2 = 0.00559 for Senegal falls within regions containing polymorphic genes such as msp1 or msp6.B: The non-synonymous polymorphism (π_NS = 0.014111) for pfhrp2 gene showed that 89% of genes have a lower π_NS than pfhrp2.

Figure 4

Variation in the number of types repeats. Differences in the median number of repeats of each type are shown. There is a significant difference in the median of the Type 4 repeat (P value = 0.0067) and the Type 7 repeat (P value = 0.0287) within the three African populations (S: Senegal, M: Mali, U: Uganda) of P. falciparum; P value <0.0001). Population comparison using Kruskall Wallis in a non-parametric way shows a significant difference in the median number of the Type 4 and the Type 7 repeats. The frequency and distribution of amino acids repeats shows inter and intra-species variation in the PfHRP2 sequence.