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

Abstract

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 (< 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.

The potential of addressing asymptomatic malaria in the context of malaria elimination in Ethiopia: Scoping review

“…16 (94%) of the included studies were cross-sectional studies and one randomized controlled trial (RCT). As a result, nothing was known, about, how much of those no measured fever after the time of diagnosis, was developed the clinical malaria, after a certain period of time [ [12] , [13] , [14] , 17 , 19 , [21] , [22] , [23] , [24] , [25] , [26] , [27] , [28] ]. In addition, Microscopy, RDT and PCR were used as the main diagnostic tools in this review.…”

Performance of antigen detection for HRP2-based malaria rapid diagnostic tests in community surveys: Tanzania, July–November 2017

“…The most appropriate comparator to POC RDT result is a blood specimen from the same person which is assayed for Plasmodium antigens with a more sensitive antigen assay. This has been done successfully many times by post hoc evaluation of RDT performance through quantitative detection of Plasmodium antigens by laboratory assays [16,[24][25][26]. Beyond positive/negative results, RDT analytical sensitivity estimates have also been generated for the current best-in-class RDTs and have estimated reliable HRP2 detection in the range of 1.0 ng/mL [27].…”

Deletion patterns, genetic variability and protein structure of pfhrp2 and pfhrp3: implications for malaria rapid diagnostic test in Amhara region, Ethiopia

“…The differences between reported prevalence could be related to the association between deletion frequency and low malaria transmission, where it is less probable to find a mixed infection of parasites with and without pfhrp2 that masks the deletion [ 20 ]. Another explanation for the wide range of deletion prevalence reported could be the differences in genotyping approach, for example, a recent study in Ethiopia only analysed samples with low or without HRP2 signal by multiplex bead assay [ 47 ]. That makes it essential to generalized technical strategies across studies [ 48 ].…”