The present study provides recent data on the Plasmodium species circulating in Bangui, CAR, as well as on the presence of parasites with PfKelch13 mutations.
First, we demonstrate that both P. falciparum and P. ovale are circulating in Bangui. Although P. falciparum was found in all malaria-positive cases, this species was associated with P. ovale in 1.57% of malaria cases. This figure is higher than that of previous observations. A study carried out in 2010 in Bangui estimated a prevalence of 0.3% P. ovale . Of note, a co-infection of P. ovale, P. falciparum and P. malariae was observed in Rouen, France in 2017 in an imported malaria case in two children from the CAR . In the other imported malaria study, 4 cases (4/200, i.e. a prevalence of 2%) of P. ovale were diagnosed among Peruvian peace-keepers deployed in support of United Nations operations in the CAR from 2016-2017 after to return to Peru .
Since 2006–2007, resistance to artemisinin has emerged in Southeast Asia, along the Thai-Cambodian border. This resistance, first identified as an increase in parasite clearance times after treatment with artesunate monotherapy or with ACT, is now better understood. It involves early ring-stages that resist treatment by ceasing to grow when exposed to the drug. This phenomenon has been demonstrated by the development of a new in vitro test called the ring-stage survival assay (RSA). Resistant parasites show a proportion of >1% of parasites that survive after 72 h compared with susceptible parasites (<1%). Since 2014, these two phenotypes (clinical and in vitro) have been clearly associated with the presence of several non-synonymous mutations in the propeller domain of the PfKelch13 gene. The two main hotspots of emergence are located in the Greater Mekong subregion in Southeast Asia, where the parasites carrying the C580Y or the F446I mutations are now dominant . Other single point mutations (N458Y, Y493H, R539T, I543T, M476I, P553L and R561H) have also been validated as conferring resistance to artemisinin . In addition, some mutations (P441L, G449A, C469F, P527H, N537I, G538V, V568G, P574L, F673I and A675V) are candidates suspected to be associated with artemisinin resistance . In sub-Saharan Africa, validated or candidate mutations associated with resistance (R539T, P574L) have been observed in Angola, Equatorial Guinea and in Rwanda, whereas mutations potentially associated with artemisinin resistance (M476I) have been detected in Senegal [20, 21, 22, 23]. A particular case was the observation of a local mutation (M579I) in Equatorial Guinea . This mutation was shown to be associated with artemisinin resistance, while the A578S mutation, common in Africa, was not found to be associated with artemisinin resistance . Our study on samples collected in Bangui between 2017 and 2019 demonstrate the presence of one non-synonymous mutation (Y653N) and three synonymous mutations (frequency of 2.1%). This frequency of PfKelch13 mutants is similar to those observed in neighboring countries (Brazzaville Congo, certain regions of Cameroon), which showed frequencies of 1.57% and 2.9%, respectively [26, 27], confirming the absence of artemisinin resistance in Central Africa . The only previous data on PfKelch13 polymorphism performed in 2014 in the CAR (K13 artemisinin resistance multicenter resistance, KARMA study), revealed a 4.5% prevalence of non-synonymous mutations . The difference or the fluctuation in the frequency of non-synonymous mutations in the CAR in 2014 and that of Bangui in 2017–2019 is likely due to the representativeness of the samples tested, but also to the fact that mutants appear at random and disappear probably because they do not have a selective advantage compared with wild strains . The previously validated or candidate resistance-associated mutations were not detected in our study. Interestingly, the A578S codon, common in Africa, was not observed either. The sole non-synonymous mutation detected here has never been reported in other African countries. It remains to be seen if this mutation is associated with artemisinin resistance. It is possible that local mutant strains resistant to artemisinin can emerge in addition to the risk of spread of the resistant Southeast Asian strain, as observed previously with other strains resistant to chloroquine or sulfadoxine–pyrimethamine. The dynamics of resistance and its emergence are likely to be complex, particularly due to the interactions specific to each world (sub) region . Similar studies in Cameroon and Nigeria have shown large differences according to region and time period, which makes it difficult to determine the spatio-temporal dynamics on polymorphism [30, 31]. Some countries in this African subregion (Congo, Gabon, and DRC) have revealed the presence of parasites polymorphic for the A578S allele, others have detected novel alleles and still others, particularly in Benin, have not detected any polymorphism [32, 33].
If the Y653N mutation increases in frequency in subsequent studies, it will then be necessary to assess its in vivo impact on the parasite clearance half-life in patients treated with ACT and its susceptibility in vitro to confirm or disprove its association with artemisinin resistance.