Cambodian Plasmodium vivax parasites with reduced hemoglobin digestion display delayed clearance upon artesunate treatment

Artemisinin-based combination therapies are the frontline drugs for the treatment of malaria infections but, for Plasmodium falciparum, the efficacy of artemisinin is threatened by the spread of resistance. P. vivax is the second most common cause of human malaria but we have little information on its susceptibility to artemisinin due to the lack of in vitro cultures. Here, we analyze 161 P. vivax infections from Cambodian patients treated with 2 mg/kg/day of artesunate for seven days. All infections were successfully cleared by day 3. However, one third of the infections displayed a slow clearance after treatment, with nine infections (5.7%) with a parasite clearance time greater than 5 hours, meeting the WHO definition of artemisinin resistance. We observed no significant association between slow clearance and either patient- or infection characteristics (including stage composition). We used RNA-seq to characterize the gene expression of parasites from 15 fast- and 16 slow-clearing infections at baseline and 1, 2 and 4 hours after treatment. While fast-clearing parasites showed significant changes in gene expression immediately upon treatment, slow-clearing parasites displayed a significantly delayed gene expression response, with a downregulation of many genes associated with hemoglobin endocytosis and digestion. Overall, our results indicate that some Cambodian P. vivax parasites clear slowly after artesunate treatment, possibly due to a downregulation of hemoglobin metabolism that may reduce the efficiency of the artesunate. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSThe WHO treatment guidelines recommend artemisinin-combination therapy (ACT) for treatment of blood-stage infections caused by Plasmodium vivax in all areas (with chloroquine recommended only in areas where P. vivax are still chloroquine-sensitive). In P. falciparum, partial resistance to artemisinin derivatives is defined in vivo as either detected parasitemia on day 3 post treatment or as a half-life of the parasite clearance slope of [≥] 5 hours. We searched Pubmed for studies containing the terms "vivax" AND "clearance" AND ("artesunate" OR "dihydroartemisinin" OR "artemether" OR "artemisinin") published between 1990 and February 2025, with no language restrictions. Our search retrieved 102 studies for which title and abstracts were screened to identify 21 studies reporting outcomes of P. vivax treatment with an artemisinin derivative. While all these studies concluded that artemisinin derivatives provided rapid clearance of P. vivax parasites, two studies reported a low frequency of day 3 positivity following artesunate-amodiaquine treatment (2.6% in Brazil) or dihydroartemisinin-piperaquine (0.6% in Indonesia). No study reported clearance slope half-life [≥] 5 hours. Added value of this studyThis study used a cohort of Cambodian patients infected by P. vivax to rigorously examine the efficacy of artesunate monotherapy at clearing blood stage infections. Our study showed significant variations in clearance rates among infections, with 5.7% of the infections with a clearance slope half-life [≥] 5 hours, meeting the criteria for artemisinin partial resistance used for P. falciparum. Variations in clearance rate upon artesunate treatment were not associated with patient or infection characteristics. Gene expression analyses revealed that the slow-clearing parasites down-regulated upon treatment many genes involved in hemoglobin endocytosis and digestion, possibly resulting in a lesser activation of artesunate. Implications of all the available evidenceOur results confirm that 2 mg/kg of artesunate per day for seven days is effective at clearing P. vivax blood stage infections. However, a subset of the P. vivax parasites displayed a slow clearance following artesunate treatment meeting artemisinin partial resistance definition in P. falciparum. Gene expression analyses suggest that metabolic variations may underlie slow clearance. Increased monitoring of treatment efficacy and drug resistance in P. vivax is therefore recommended.