PLASMODIUM FALCIPARUM HISTIDINE-RICH PROTEIN GENE MUTATIONS IN NORTHERN GHANA: IMPLICATIONS FOR THE USE OF HRP2-BASED MALARIA DIAGNOSIS

Abstract

Human malaria is caused by five Plasmodium species transmitted by the female Anopheles mosquitoes. PCR assays, microscopy and RDT (most convenient), are malaria diagnostic approaches. RDT works on the principle of MAbs detecting antigens expressed by specific markers of Plasmodium spp. like the Pfhrp2/3 genes. This study sought to determine the rate of Pfhrp2/3 gene mutations in northern Ghana and the implications of these mutations for the use of PfHRP2-RDTs for malaria diagnosis in the region. Blood samples from 267 malaria patients diagnosed by microscopy were tested for malaria infection by PfHRP2/3-based RDT and PCR using whole blood and extracted DNA, respectively. Nested PCR was also used to investigate the presence/absence of the Pfhrp2/3 genes and genetic variation of nucleotide and in-silico translated amino acids done using GenTLE, MEGAX and popART software. In all, 160 (59.9%) of the total samples tested positive to P. falciparum-specific PCR with 158/267 (59.2 %) positive by PfHRP2-based RDT. A false RDT negative rate of 21.9% (35/160) was, however observed which can be attributed to several factors, including mutation or deletion of the target antigenic marker. PCR assay revealed that 19 (11.9 %) and 22 (13.8 %) samples lacked exon1−2 of Pfhrp2 and 3 genes, respectively with 17 (10.6%) samples lacking both genes. Twenty (20) of the false RDT negative samples lacked either or both exon1−2 of Pfhrp2 and 3 genes. Genetic variation studies revealed higher variability indices for both nucleotide sequences and amino acid residues of the sequenced exon1 of Pfhrp2 and 3 genes. Haplotype diversity was 0.925 (π=0.05273) and 1.000 (π=0.13517) for intron sequences of Pfhrp2 and 3, respectively. Isolates of this study formed a monophyletic clade but a paraphyletic clade with other isolates. This study revealed mutations to the Pfhrp2/3 genes and raise concerns about the reliability of PfHRP2/3-RDT for malaria diagnosis.