Plasmodium vivax relapse reservoirs in Ethiopia: microhaplotype sequencing informs recurrent infection origins

Determining the origin of recurrent Plasmodium vivax infections is critical for quantifying the public health burden of the hypnozoite reservoir, understanding transmission potential, and evaluating anti-relapse antimalarial efficacy. In Ethiopia, P. vivax is a major contributor to malaria burden, but the relative contribution of relapse to recurrent infections remains poorly quantified. We performed microhaplotype genotyping using a panel of 93 genome-wide markers and applied a probabilistic recurrence classification framework to recurrent P. vivax samples collected in two Ethiopian radical cure trials (IMPROV and OPRA). Genotyping was conducted on parasite isolates collected at enrolment and subsequent recurrences, with participants monitored over a 12-month follow-up period (IMPROV: 604 samples from 444 participants; OPRA: 621 samples from 342 participants). Recurrent infections were classified using a Bayesian hierarchical model implemented in Pv3Rs, which estimated marginal posterior probabilities of relapse, reinfection, or recrudescence based on genotyping data and background population dynamics. Priors were structured across biologically motivated time windows reflecting the natural history of P. vivax infection in Ethiopia and anticipated drug efficacy. Recurrences with a probability of relapse (p(L)) [≥] 0.75 were classified as relapses. In the absence of primaquine, probable relapse was the dominant classification for primary recurrent infections in both trials (75.5%; 102/135) and for consecutive recurrent infections within individuals (75.2%; 204/271). Primaquine substantially reduced the proportion of participants experiencing recurrent infections attributable to probable relapse, from 57.3% to 1.7% in IMPROV and from 60.2% to 9.5% in OPRA. Polyclonal infection at enrolment was independently associated with increased odds of recurrence and a higher rate of probable relapse in OPRA, with a consistent but non-significant trend observed in IMPROV. These findings provide direct molecular evidence that the hypnozoite reservoir is the dominant driver of recurrent P. vivax infections in Ethiopia in the absence of radical cure. The marked reduction in probable relapse following primaquine treatment highlights the critical importance of expanding access to safe and effective radical cure strategies in Ethiopia and other comparable high-transmission settings.