Azithromycin disrupts apicoplast biogenesis in replicating and dormant liver stages of the relapsing malaria parasites Plasmodium vivax and Plasmodium cynomolgi

•A major obstacle to Plasmodium vivax eradication is its ability to cause relapse by the activation of hypnozoites.•The use of 8-aminoquinolines to kill hypnozoites is restricted by potential toxicity.•The apicoplast is the site of metabolic functions crucial to the survival of the malaria parasite.•This study presents evidence that azithromycin renders the hypnozoites unable to cause relapses by disrupting the apicoplast.•This work identifies apicoplast functions as potential drug targets in quiescent hypnozoites. The control and elimination of malaria caused by Plasmodium vivax is hampered by the threat of relapsed infection resulting from the activation of dormant hepatic hypnozoites. Currently, only the 8-aminoquinolines, primaquine and tafenoquine, have been approved for the elimination of hypnozoites, although their use is hampered by potential toxicity. Therefore, an alternative radical curative drug that safely eliminates hypnozoites is a pressing need. This study assessed the potential hypnozoiticidal activity of the antibiotic azithromycin, which is thought to exert antimalarial activity by inhibiting prokaryote-like ribosomal translation within the apicoplast, an indispensable organelle. The results show that azithromycin inhibited apicoplast development during liver-stage schizogony in P. vivax and Plasmodium cynomolgi, leading to impaired parasite maturation. More importantly, this study found that azithromycin is likely to impair the hypnozoite's apicoplast, resulting in the loss of this organelle. Subsequently, using a recently developed long-term hepatocyte culture system, this study found that this loss likely induces a delay in the hypnozoite activation rate, and that those parasites that do proceed to schizogony display liver-stage arrest prior to differentiating into hepatic merozoites, thus potentially preventing relapse. Overall, this work provides evidence for the potential use of azithromycin for the radical cure of relapsing malaria, and identifies apicoplast functions as potential drug targets in quiescent hypnozoites. [Display omitted]