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Background  Rapid diagnostic tests (RDTs) targeting histidine rich protein 2(HRP2) are widely used for diagnosis of  Plasmodium falciparum  infections. Besides PfHRP2, the PfHRP3 antigen contributes to the detection of  P. falciparum  infections in PfHRP2 RDTs. However, the performance HRP2-based RDT is affected by  pfhrp2/3  gene deletions resulting in false-negative test results. The objective of this study was to determine the presence and prevalence of  pfhrp2/3  gene deletions including the respective flanking regions among symptomatic patients in Assosa zone, Northwest Ethiopia.    Methods  A health-facility based cross-sectional study was conducted in febrile patients seeking a malaria diagnosis in 2018. Blood samples were collected by finger-prick for microscopic examination of blood smears, malaria RDT, and molecular analysis using dried blood spots (DBS) prepared on Whatman filter paper. A total of 218  P. falciparum  positive samples confirmed by quantitative PCR were included for molecular assay of  pfhrp2/3  target gene.    Results  Of 218  P. falciparum  positive samples, exon 2 deletions were observed in 17.9% of  pfhrp 2 gene and in 9.2% of  pfhrp 3 gene. A high proportion of deletions in short segments of  pfhrp2  exon1-2 (50%) was also detected while the deletions of the  pfhrp3  exon1-2 gene were 4.1%. The deletions were extended to the downstream and upstream of the flanking regions in  pfhrp2/3  gene (above 30%). Of eighty-six PfHRP2 RDT negative samples, thirty-six lacked  pfhrp2 exon 2 . Five PfHRP2 RDT negative samples had double deletions in  pfhrp2 exon 2  and  pfhrp3 exon2 . Of these double deletions, only two of the samples with a parasite density above 2000 parasite/µl were positive by the microscopy. Three samples with intact  pfhrp3 exon2  in the  pfhrp2 exon2  deleted parasite isolates were found to be positive by PfHRP2 RDT and microscopy with a parasite density above 10,000/µl.    Conclusion  This study confirms the presence of deletions of  pfhrp2/3  gene including the flanking regions.  Pfhrp2/3  gene deletions results in false-negative results undoubtedly affect the current malaria control and elimination effort in the country. However, further countrywide investigations are required to determine the magnitude of  pfhrp2/3  gene deletions and its consequences on routine malaria diagnosis.

malaria journal

Detection of high prevalence of Plasmodium falciparum histidine-rich protein 2/3 gene deletions in Assosa zone, Ethiopia: implication for malaria diagnosis