This study provides evidence for a continuous selection of molecular markers associated with artemether-lumefantrine tolerance/resistance in the local P. falciparum population in Fukayosi village, Bagamoyo district, Tanzania, occurring after the introduction of this ACT as first-line treatment for uncomplicated malaria in 2006.
The results support previous observations of pfmdr1 N86 selection in Gabon, Kenya and Mozambique [19–21] as well as selection of both pfmdr1 N86 and 184F in Korogwe, Tanzania , and the pfmdr1 N86, 184F, D1246 haplotype in Mozambique  following wide scale deployment of artemether-lumefantrine. However, the present report adds substantially to the evidence base being more comprehensive both with regards to number of patients and genetic markers analysed, and importantly with a longer duration of follow-up.
Interestingly the selection of pfcrt K76 started already prior to the introduction of artemether-lumefantrine in Bagamoyo district. This probably represents an effect of the withdrawal of chloroquine as first-line treatment in 2001, consistent with observations from Malawi where withdrawal of chloroquine resulted in a fast re-expansion of a diverse chloroquine-susceptible pfcrt K76 population . Thus, the herein observed increase in pfcrt K76 may not necessarily only be due to the introduction of artemether-lumefantrine, but could also, at least partly, be explained by the withdrawal of chloroquine and/or other factors, such as parasite fitness and transmission intensity [24–26]. Conversely, no selection of pfmdr1 N86, 184F, D1246 occurred prior to introduction of artemether-lumefantrine in the study area. The selection of these SNPs seen after 2006 is therefore unlikely driven by the withdrawal of chloroquine.
There are evidences that exposure of artemether-lumefantrine is the main contributor behind the observed selection of pfmdr1 N86, 184F, D1246 SNPs and that it plays a role also for selection of pfcrt K76. These evidences include the previously reported specific lumefantrine-driven selection among re-infections during follow up after artemether-lumefantrine treatment [9, 10], in vitro findings  and a recent study conducted in Tanzania, which shows that the selection of N86, 184F and D1246 after artemether-lumefantrine treatment in vivo is significantly associated with the ability to withstand higher lumefantrine concentrations . In this context it is also worth noting that there are studies suggesting that both the artemisinin-derivatives and lumefantrine select for the same molecular markers [28, 29]. This, together with the recent evidence from Southeast Asia that P. falciparum is able to develop artemisinin tolerance/resistance, is of particular concern as it could result in an additive or even synergistic selection of molecular markers of anti-malarial drug resistance in the parasite population.
It is of note that the blood slide positivity rate in Study 5 was lower (17%) compared with the mean for all other studies combined (53%). This may be due to that Study 5 was conducted during October-January, when the malaria transmission is relatively low. However, this did not appear to have influenced the SNP prevalences. Furthermore, it is important to underline that clinical efficacy of artemether-lumefantrine remained high in the study area with PCR-corrected cure rate >95% in 2007 . Nevertheless, in an era when the number of malaria patients is slowly declining, standard in vivo trials are increasingly difficult and costly to conduct. In this context, molecular surveillance may play an important role to detect selection of genetic markers associated with ACT tolerance/resistance in the local P. falciparum population over time.