In 2006, Senegal introduced ACT (artemether-lumefantrine, AL, and artesunate-amodiaquine, ASAQ) as first-line and dihydroartemisinin-piperaquine (DHA-PQ) as second-line in the treatment of uncomplicated P. falciparum malaria. To date, prompt management of the malaria cases using these antimalarial drugs have contributed significantly to a reduction in malaria-related morbidity and mortality26, confirming that AL, ASAQ and DHA-PQ remain effective and well tolerated in Senegal27–30. However, 1.2% of AL and 2.5% of DHA-PQ treatment failures have been reported in Senegal in 202031. These results suggest that monitoring the efficacy of these antimalarial drugs is essential for malaria control, as there is no other alternative drug treatment available32. An important tool for monitoring antimalarial drug resistance in field isolates is the use of ex vivo assays. They complement clinical drug studies, allowing researchers to measure parasite responses to different drugs individually, without patient-related factors. Importantly, ex vivo monitoring of malaria parasite drug responses plays an important role in early detection of reduced parasite drug susceptibility and treatment failure8. In this study, the ex vivo SYBR™ Green assay was used to test samples collected during the seasonal period of 2018 in Thiès, Senegal. Six drugs (amodiaquine, chloroquine, lumefantrine, mefloquine, piperaquine and quinine), combined as partner drug in ACT with the exception of chloroquine and quinine, were evaluated.
Our results showed that the IC50 values of chloroquine were slightly higher (GM = 26.9 nM) than those previously reported in Thiès in 2013 (GM = 18.29 nM)33, but lower than those reported in Dakar in 2014–2015 (GM = 62.2 nM)33,34, suggesting a decrease in parasite susceptibility to chloroquine and consistent with the high chloroquine resistance rate found in Dakar in 2015 (46.9%). The higher level of chloroquine susceptibility in Thiès compared to Dakar is not surprising, as Dakar is a known centre of chloroquine resistance35. No cross-resistance between chloroquine and amodiaquine was observed, but association between the pfcrt K76T and pfmdr1 Y184F mutations ex vivo susceptibility to chloroquine was detected.
Conversely, we found increased susceptibility to amodiaquine in Thiès (GM = 4.6 nM) with an IC50 < 60 nM according to the WHO guideline. The GM varied from 13.84 nM in 2012 to 6.48 nM in 201333. Here, no ex vivo resistant parasite was found to amodiaquine, contrasting with data from Dakar in 2015, where 28.1% of isolates were associated with in vitro monodesethylamodiaquine resistance34. We noticed that the GM of isolates with the pfmdr1 V798I mutation was higher than that of isolates with the wild-type allele.
Similarly to amodiaquine, we observed an increase in ex vivo susceptibility to lumefantrine in Thiès (from 173.4 nM in 2012, to 113.2 nM in 2013 and 87.2 nM in 2018)33. These values were very high in comparison with Dakar in 2015 (mean = 3.5 nM) and Ghana in 2016–2018 (mean = 2.7 nM)37. However, no AL treatment failure was reported in a clinical trial conducted in Thiès from 2012 to 201438.
The GM for piperaquine was estimated to 19.4nM. This is slighly higher than that reported in Pikine in 2014 (15.28 nM)39, but lower than that reported in Dakar in 2014 (36.5 nM). A mean of 4.6 nM was reported in a study conducted in Ghana37. Further studies are needed to better estimate piperaquine ex vivo susceptibility, as the lack of resistance cut-offs makes it impossible to estimate the resistance rates.
Mutations in pfcrt M74I, N75E, K76T, R371I are associated with chloroquine resistance in P. falciparum. The mutations at codons 76 of the pfcrt gene and pfmdr1 86 were found to be highly associated with resistance to chloroquine40–42, and again observed in this study. We detected that 16% of the tested isolates were resistant to chloroquine (i.e., IC50 > 100 nM), a value similar to the one observed from a study performed in Thiès in 2010 (23% of isolates exhibited chloroquine resistance)43. Similar trends are also observed in other regions of Senegal44 .
The study presented here has several limitations. First, our work included only 34 samples. Second, despite this small sample size, only 13 isolates from the ex vivo SYBR™ Green test were successufully assayed. Third, only 28 out of the 34 samples were successfully sequenced. Fourth, the study was conducted at only one site in Senegal (Thiès).