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Background  Usage of chloroquine was discontinued from the treatment of  Plasmodium falciparum  infection in almost all endemic regions because of global spread of resistant parasites. Since the first report in Malawi, numerous epidemiological studies have demonstrated that the discontinuance led to re-emergence of chloroquine-susceptible  P. falciparum , suggesting a possible role in future malaria control. However, most studies were cross-sectional, with few studies looking at the persistence of chloroquine recovery in long term. This study fills the gap by providing, for a period of at least 6 years, proof of persistent re-emergence/stable recovery of susceptible parasite populations using both molecular and phenotypic methods.    Methods  Ex vivo drug-susceptibility assays to chloroquine (n = 319) and lumefantrine (n = 335) were performed from 2013 to 2018 in Gulu, Northern Uganda, where chloroquine had been removed from the official malaria treatment regimen since 2006. Genotyping of  pfcrt  and  pfmdr1  was also performed.    Results  Chloroquine resistance (≥ 100 nM) was observed in only 3 (1.3%) samples. Average IC 50  values for chloroquine were persistently low throughout the study period (17.4–24.9 nM). Parasites harbouring  pfcrt  K76 alleles showed significantly lower IC 50 s to chloroquine than the parasites harbouring K76T alleles (21.4 nM vs. 43.1 nM, p-value = 3.9 × 10 −8 ). Prevalence of K76 alleles gradually increased from 71% in 2013 to 100% in 2018.    Conclusion  This study found evidence of stable persistence of chloroquine susceptibility with the fixation of  pfcrt  K76 in Northern Uganda after discontinuation of chloroquine in the region. Accumulation of similar evidence in other endemic areas in Uganda could open channels for possible future re-use of chloroquine as an option for malaria treatment or prevention.

Recovery and stable persistence of chloroquine sensitivity in Plasmodium falciparum parasites after its discontinued use in Northern Uganda