Person:

Bei, Amy

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.

Background: The World Health Organization has recommended rapid diagnostic tests (RDTs) for use in the diagnosis of suspected malaria cases. In addition to providing quick and accurate detection of Plasmodium parasite proteins in the blood, these tests can be used as sources of DNA for further genetic studies. As sulfadoxine-pyrimethamine is used currently for intermittent presumptive treatment of pregnant women in both Senegal and in the Comoros Islands, resistance mutations in the dhfr and dhps genes were investigated using DNA extracted from RDTs. Methods: The proximal portion of the nitrocellulose membrane of discarded RDTs was used for DNA extraction. This genomic DNA was amplified using HRM to genotype the molecular markers involved in resistance to sulfadoxine-pyrimethamine: dhfr (51, 59, 108, and 164) and dhps (436, 437, 540, 581, and 613). Additionally, the msp1 and msp2 genes were amplified to determine the average clonality between Grande-Comore (Comoros) and Thiès (Senegal). Results: A total of 201 samples were successfully genotyped at all codons by HRM; whereas, in 200 msp1 and msp2 genes were successfully amplified and genotyped by nested PCR. A high prevalence of resistance mutations were observed in the dhfr gene at codons 51, 59, and 108 as well as in the dhps gene at codons 437 and 436. A novel mutant in dhps at codon positions 436Y/437A was observed. The dhfr I164L codon and dhps K540 and dhps A581G codons had 100 % wild type alleles in all samples. Conclusion: The utility of field-collected RDTs was validated as a source of DNA for genetic studies interrogating frequencies of drug resistance mutations, using two different molecular methods (PCR and High Resolution Melting). RDTs should not be discarded after use as they can be a valuable source of DNA for genetic and epidemiological studies in sites where filter paper or venous blood collected samples are nonexistent. Electronic supplementary material The online version of this article (doi:10.1186/s12936-015-0861-6) contains supplementary material, which is available to authorized users.