Pfhrp2/3 gene deletion and genetic variation in PfHRP2-based RDTs with P. falciparum positive samples from India and its implication on malaria control.

Abstract

Recent studies have documented Pfhrp2/3 gene deletion globally as one of the biological threats in the fight against malaria. For malaria diagnosis, PfHRP2 based RDTs are most widely used in India, and performance of these RDTs are affected by deleted Pfhrp2/3 gene in Plasmodium falciparum. This study was planned to confirm Pfhrp2/3 gene deletion incidences and genetic variation in PfHRP2-based RDT positive with P.falciparum malaria cases from India. Confirmed positive samples by PfHRP2-based RDTs as P. falciparum (n=240) from six different endemic regions of India were validated by PCR to assure the actual infection. Two hundred forty samples qualified for DNA intactness by single-copy genes were subjected to amplification for the Pfhrp2/3 gene and its neighbouring gene (downstream and upstream) by PCR genotyping. Genetic variation in samples was analysed post-sequencing using Mega X software. Statistical analysis was performed to validate the genetic variation using Mann-Whitney Test. RDT target region of Pfhrp2 gene (exon2) was found deleted in a single sample with presence of the Pfhrp3 exon2. Complete gene deletion of 4.2% was observed in the Pfhrp3 gene. Partial gene deletion was recorded for both pfhrp2 gene (exon2-0.4%, upstream 25.8% and downstream -9.1%) and Pfhrp3 gene (exon2-18.75%, upstream - 22.08% and downstream 13.3%). Eleven new unique types of amino acid repeat sequence and earlier reported amino acid repeat type was found in the Pfhrp2 gene, prompting high genetic variation. This study suggests that parasites lacking Pfhrp2/3 gene and its neighbouring gene (downstream and upstream) are present in malaria endemic areas of India, resulting in false positive results by RDT. Systematic countrywide monitoring for malaria control and elimination of malaria is warranted in this regard.