Plasmodium falciparum resistant to artemisinin and diagnostics have emerged in Ethiopia.

Diagnosis and treatment of Plasmodium falciparum infections are required for effective malaria control and are pre-requisites for malaria elimination efforts; hence we need to monitor emergence, evolution and spread of drug- and diagnostics-resistant parasites. We deep sequenced key drug-resistance mutations and 1,832 SNPs in the parasite genomes of 609 malaria cases collected during a diagnostic-resistance surveillance study in Ethiopia. We found that 8.0% (95% CI 7.0-9.0) of malaria cases were caused by P. falciparum carrying the candidate artemisinin partial-resistance kelch13 (K13) 622I mutation, which was less common in diagnostic-resistant parasites mediated by histidine-rich proteins 2 and 3 (pfhrp2/3) deletions than in wild-type parasites (P = 0.03). Identity-by-descent analyses showed that K13 622I parasites were significantly more related to each other than to wild type (P < 0.001), consistent with recent expansion and spread of this mutation. Pfhrp2/3-deleted parasites were also highly related, with evidence of clonal transmissions at the district level. Of concern, 8.2% of K13 622I parasites also carried the pfhrp2/3 deletions. Close monitoring of the spread of combined drug- and diagnostic-resistant parasites is needed.

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

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.