Widespread pfhrp2/3 deletions and HRP2-based false-negative results in southern Ethiopia
Authors: Tafa Bacha , Dugassa Sisay , Mekasha Sindew , Dufera Boja , Ademu Aderaw , Gidisa Bedasa , Mandefro Aynalem , Tassew Geremew , Golassa Lemu 
Published: October 2023

Abstract
Background
Rapid diagnostic tests (RDTs) have played a significant role in expanding case management in peripheral healthcare systems. Histidine-rich protein-2 (HRP2) antigen detection (RDT) is predominantly used to diagnose P. falciparum. However, the evolution and spread of P. falciparum parasite strains that have deleted HRP2/3 genes, causing false-negative results, have been reported. This study assessed the diagnostic performance of HRP2-detecting RDTs for P. falciparum cases and the prevalence of pfhrp2/3 deletions among symptomatic patients seeking malaria diagnosis in selected health facilities in southern Ethiopia.
Methodology:
A multi-health facilities-based cross-sectional study was conducted on self-presented febrile patients seeking treatment in southern Ethiopia from September to July 2021. A purposive sampling strategy was used to enroll patients with microscopically confirmed P. falciparum. Dried blood spot samples were collected from microscopy-positive P. falciparum patients for further molecular analysis. DNA was extracted using gene aid kits and a nested PCR assay. Exon 2 of the hrp2 and hrp3 genes, which is the main protein coding region, was used to confirm its deletion.
Results
Of the 3,510 participants enrolled in this study, 50.3% were male and their mean age was 22.45 years. Of the total febrile patients screened for malaria infections, 33.4% (1174/3510) had malaria, as determined by smear microscopy. Of these, P. falciparum, P. vivax, and mixed infections accounted for 53.6%, 39.8%, and 6.5%, respectively. Of all malaria-positive cases as determined by microscopy, 21.1% (77/363) were tested negative by HRP2-based RDTs and hence regarded as false-negative cases. The sensitivity of PfHRP2 RDT compared with microscopy and PCR was 79% (95% CI: 74.22% − 82.88%) and 76.5% (95% CI: 72.0% − 81.0%), respectively. Parasite DNA was extracted from 363 dried blood spots, of which the presence of P. falciparum DNA was confirmed in two hundred seventy-nine (279/363. 76.8%) of the samples. Of the 279 P. falciparum confirmed samples, single-copy gene msp-2 amplifications were successful in 249 (89.2%) and were subjected to genotyping of hrp2/3 genes deletions. Deletions spanning exon 2 of hrp2, exon 2 of hrp3, and double deletions (hrp2/3) accounted for 68 (27.3%), 76 (30.5%), and 33 (13.2%), respectively. While the HRP2 RDT false-negative due to the pfhrp2 exon-2 deletion is 27.3% (68/249), the population-level prevalence estimates of pfhrp-2 exon-2 deletion leading to HRP2 RDT false negative was 18.7% (68/363). The overall prevalence of any pfhrp2/3 gene deletions in symptomatic P. falciparum patients across health facilities was estimated to be 144 (57.8%), leading to false negative PfHRP2 RDT results.
Conclusion
Because the magnitude of pfhrp2/3 gene deletions exceeds the threshold recommended by the WHO (> 5%), the findings of this study promote the initiation of non-HRP2-based RDTs as an alternative measure to curb the grave consequences associated with the continued use of HRP-2-based RDTs in the study area in particular and in Ethiopia in general.