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Background Rapid diagnostic tests (RDTs) are widely used for malaria diagnosis of both symptomatic and asymptomatic infections. Although RDTs are a reliable and practical diagnostic tool, the sensitivity of histidine-rich protein 2 (HRP2)-based RDTs can be reduced if  pfhrp2  or  pfhrp3  ( pfhrp2/3 ) gene deletions exist in the  Plasmodium falciparum  parasite population. This study evaluated dried blood spot (DBS) samples collected from a national household survey to investigate the presence of  pfhrp2/3  deletions and the performance of the RDT used in the cross-sectional survey in a low transmission setting. Methods The 2015 Ethiopia Malaria Indicator Survey tested household members by RDT and collected DBS samples. DBS (n = 2648) from three regions in northern Ethiopia were tested by multiplex bead-based antigen detection assay after completion of the survey. The multiplex assay detected pan- Plasmodium  lactate dehydrogenase (LDH), pAldolase, and HRP2 antigens in samples. Samples suspected for  pfhrp2/3  gene deletions (pLDH and/or pAldolase positive but low or absent HRP2) were further investigated by molecular assays for gene deletions. Antigen results were also compared to each individual’s RDT results. Dose–response logistic regression models were fit to estimate RDT level of detection (LOD) antigen concentrations at which 50, 75, 90, and 95% of the RDTs returned a positive result during this survey. Results Out of 2,648 samples assayed, 29 were positive for pLDH or pAldolase antigens but low or absent for HRP2 signal, and 15 of these samples (51.7%) were successfully genotyped for  pfhrp2/3 . Of these 15  P. falciparum  infections, eight showed single deletions in  pfhrp3,  one showed a single  pfhrp2  deletion, and six were  pfhrp2/3  double-deletions. Six  pfhrp2  deletions were observed in Tigray and one in Amhara. Twenty-five were positive for HRP2 by the survey RDT while the more sensitive bead assay detected 30 HRP2-positive samples. A lower concentration of HRP2 antigen generated a positive test result by RDT compared to pLDH (95% LOD: 16.9 ng/mL vs. 319.2 ng/mL, respectively). Conclusions There is evidence of dual  pfhrp2/3  gene deletions in the Tigray and Amhara regions of Ethiopia in 2015. As the prevalence of malaria was very low (&lt; 2%), it is difficult to make strong conclusions on RDT performance, but these results challenge the utility of biomarkers in household surveys in very low transmission settings.

malaria journal

Investigation of Plasmodium falciparum pfhrp2 and pfhrp3 gene deletions and performance of a rapid diagnostic test for identifying asymptomatic malaria infection in northern Ethiopia, 2015