Emergence of pfhrp2 and pfhrp3 Gene Deletions in Plasmodium falciparum and Diagnostic Challenges in Bangladesh

Abstract Background Histidine-rich protein 2 (HRP2)-based rapid diagnostic tests (RDTs) have been a major tool in the malaria elimination efforts, but their use is complicated by false-negative results in regions where Plasmodium falciparum parasites have deletions in the pfhrp2 and pfhrp3 genes. But information about the prevalence and significance of these deletions is still limited in Bangladesh. Given the recent reports of diagnostic discordant P. falciparum infections from various locations in the malaria-endemic district of Bandarban, which has the highest malaria burden in Bangladesh, we sought to confirm pfhrp2 and pfhrp3 gene deletions in suspected samples. Methods A descriptive study was carried out in the laboratory with clinical samples of P. falciparum from Bandarban from 2023 to 2025. Twenty-two samples from symptomatic persons who were negative by routine HRP2-based RDTs, but positive by microscopy and Plasmodium species-specific polymerase chain reaction (PCR). Microscopy was used to determine parasite densities. The deletion of pfhrp2 , pfhrp3 and selected flanking loci were determined by conventional PCR and confirmed by probe-based real-time PCR. Results Microscopic examination revealed parasite densities ranging from 440 to 215,000 parasites/µL. Conventional PCR demonstrated pfhrp2 gene deletion in all isolates, with 20 (90.9%) of 22 also exhibiting pfhrp3 deletion, indicating a predominance of double-deletion parasites. Probe-based real-time PCR also confirmed pfhrp2 deletion in all isolates, while only two samples retained detectable pfhrp3 signals. Analysis of flanking regions revealed that genomic deletions extended beyond the target genes, with conserved loss patterns at the pfhrp2 locus. Conclusions This study shows significant pfhrp2 gene deletions and frequent co-deletion of pfhrp3 in P. falciparum isolates from Bandarban, with large genomic deletions in the flanking regions. These findings provide the most likely explanation for the observed false-negative HRP2-based RDT results and signal a potentially major threat to the current malaria diagnostic approaches in this high-endemic region. A targeted survey for pfhrp2/3 deletions is needed to assess the scale and scope of the threat in parallel with careful consideration for alternative interim and long-term diagnostic strategies to ensure accurate case detection and to support effective malaria elimination efforts in Bangladesh.