25 Artemisinin (ART) combination therapies were introduced on malaria endemic Bioko 26 Island in 2004 through Bioko Island Malaria Control Project. Recently, ART-resistant 27 Plasmodium falciparum strain with Kelch13 (K13) propeller M579I mutation 28 originating from Equatorial Guinea was observed as an increased parasite clearance 29 time on day 3 after dihydroartemisinin-Piperaquine (DHA-PIP) treatment (D3 30 positivity). Here, we surveyed DHA-PIP effectiveness and molecular markers of drug 31 resistance at D3 after DHA-PIP treatment on Bioko Island from 2014 to 2017. Among 32 the 371 uncomplicated P. falciparum patients, 86.3% (320/471) were successfully 33 followed up at D3. 5.9% (19/320) of patients showed D3 positivity. K13 and MDR1 34 gene were successfully sequenced from 46 patients collected at D0 (baseline 35 population) and 19 D3-positivity patients. Five non-synonymous K13 mutations 36 (H136N; K189N; K248N; K326E; K332N) were found. There was no statistical 37 difference in the frequency of these K13 mutations between baseline population and 38 D3-positivity samples (p>0.05). Additionally, none of the K13 propeller 39 polymorphisms known to be involved in ART-resistance in Asia or Africa were 40 detected. For MDR1 gene, 38.5% (25/65) carried N86Y mutation; 73.8% (48/65) the 41 Y184F mutation. Parasites surviving DHA-PIP at D3 post-treatment were 42significantly more likely than the baseline population to carry the N86Y (p <0.05).
43These results suggest that K13 is not the best predictive molecular marker for ART 44 resistance in Africa. More isolates from cases with delayed parasite clearance after 45 DHA-PIP treatment indicated that in vitro and in vivo monitoring for ART derivatives 46 and ACT partner drugs should be regularly performed on Bioko Island, Equatorial 47 Guinea.
48