Plasmodium falciparum is evolving to escape malaria rapid diagnostic tests in Ethiopia

Abstract

In Africa, most rapid diagnostic tests (RDTs) for falciparum malaria recognize histidine-rich protein 2 antigen. Plasmodium falciparum parasites lacking histidine-rich protein 2 (pfhrp2) and 3 (pfhrp3) genes escape detection by these RDTs, but it is not known whether these deletions confer sufficient selective advantage to drive rapid population expansion. By studying blood samples from a cohort of 12,572 participants enroled in a prospective, cross-sectional survey along Ethiopia’s borders with Eritrea, Sudan and South Sudan using RDTs, PCR, an ultrasensitive bead-based immunoassay for antigen detection and next-generation sequencing, we estimate that histidine-rich protein 2-based RDTs would miss 9.7% (95% confidence interval 8.5–11.1) of P. falciparum malaria cases owing to pfhrp2 deletion. We applied a molecular inversion probe-targeted deep sequencing approach to identify distinct subtelomeric deletion patterns and well-established pfhrp3 deletions and to uncover recent expansion of a singular pfhrp2 deletion in all regions sampled. We propose a model in which pfhrp3 deletions have arisen independently multiple times, followed by strong positive selection for pfhrp2 deletion owing to RDT-based test-and-treatment. Existing diagnostic strategies need to be urgently reconsidered in Ethiopia, and improved surveillance for pfhrp2 deletion is needed throughout the Horn of Africa.

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

“…Furthermore, a multi-site study conducted according to WHO’s protocol on pfhrp2/3 deletion surveillance found evidence of high prevalence (9.7%) of pfhrp2 gene deletions in the Amhara and Tigray regions, specifically in areas bordering Sudan and Eritrea (2017–2018 WHO survey) [ 12 ]. This report adds to the growing literature that pfhrp2/3 deletions are of concern in Ethiopia [ 10 – 12 ], and more extensive surveys which are underway will help facilitate decisions regarding use of appropriate and effective RDTs in Ethiopia.…”

“…Additionally, of 79 PCR-confirmed P. falciparum samples collected from Djiboutian patients with suspected malaria in 2019, 87% had both pfhrp2 and pfhrp3 genes deleted [ 9 ]. Within Ethiopia, pfhrp2/3 deletions have been reported in multiple regions, including Tigray, Amhara, Oromia, and Benishangul-Gumuz Regions [ 10 – 12 ]. The highest prevalence estimate of pfhrp2 -deleted parasites causing false-negative RDT results published to date was in the Tigray Region (15%), which shares borders with Eritrea and Sudan [ 12 ].…”

“…Within Ethiopia, pfhrp2/3 deletions have been reported in multiple regions, including Tigray, Amhara, Oromia, and Benishangul-Gumuz Regions [ 10 – 12 ]. The highest prevalence estimate of pfhrp2 -deleted parasites causing false-negative RDT results published to date was in the Tigray Region (15%), which shares borders with Eritrea and Sudan [ 12 ].…”

“…The primary purpose of this research was to identify if participants in the Ethiopia MIS were infected with pfhrp2/3 deleted P. falciparum parasites in 2015 as multiple reports have shown the presence of P. falciparum isolates with deletions of pfhrp2 and/or pfhrp3 genes in Ethiopia recently [ 10 – 12 ]. A multiplex bead-based antigen detection assay was utilized to rapidly screen the large number of dried blood spot samples collected during the 2015 Ethiopia MIS for potential P. falciparum HRP2/3 gene deletions (samples with low or no HRP2 and presence of pan- Plasmodium LDH antigens) [ 16 ].…”

Dry season prevalence of Plasmodium falciparum in asymptomatic Gambian children, with a comparative evaluation of diagnostic methods

“…In terms of quanti cation, reliance on white blood cell counts to calculate parasite densities in thick lms can lead to inaccuracies as leucocyte counts may be raised or suppressed during infection. Moreover, although RDTs are relatively straightforward to administer and interpret, P. falciparum histidine-rich protein 2 (PfHRP2) mutations are leading to test failure for certain brands of RDT, leading to an overreliance on the less-sensitive pan-LDH and PfLDH antigens as diagnostic markers [40].…”