Resistance occurs as a result of mutations in the target points in the parasite. Limited countries across South East Asia and malaria-endemic Africa have revealed evidence of low frequency ACTs linked mutations, with an initial indication of indigenous Pfk13 mutations in the East Africa region, speculating that the threat of independent acquisition of resistance should be taken seriously [24]. There is a critical need for augmented, uniform and prospective antimalarial resistance molecular surveillance across Africa. Investigation of the association between P. falciparum mutations and reduced susceptibility to ACTs through genome-wide association studies (GWAS) and gene manipulation studies, have shown a relationship between mutations in K13 and increased parasite survival in the in-vitro conditions [25].
Currently, mutations in Kelch13 propeller gene has been indicated as ACTs resistant molecular marker. Different mutations have been previously reported in Asia, America and Africa continents, with more prevalence recorded in the Asian continent [26]. The evolution and spread of mutant P. falciparum K13-mediated artemisinin (ART) resistance has led to extensive treatment failures all over Southeast Asia [27]. P. falciparum resistance to ACTs derivatives has been reported across Southeast Asia (SEA), having first confirmed a decade ago in western Cambodia [28, 29, 30].
The present study has reported the presence of Pfk13 polymorphisms at different loci. The mutations detected here includes R561H, R539T, N458Y, N431S and A671V. However, the frequencies of the mutations were low compared to those witnessed in ACTs resistant geographical locations. R561H Single Nucleotide Polymorphism hereby reported in one sample from Bonchari and Marani Sub Counties has previously been associated with reduced parasite clearance in South East Asia [31]. More over this mutation has been previously reported in Rwanda [32] and Tanzania [33], countries located in East Africa, thus raising concern on the probability of importation of ACTs resistant parasites as a result of human movements. Despite the fact, the mutation was only detected in one (1) sample, our data emphasize on the threat of the R561H mutation spreading in Kenya. Particularly, by comparing with the past great quantity of R561H mutation across the Myanmar and Thailand [34], the presence of the R561H variant in the study area points to the risk of ACTs resistance emergence.
Single Nucleotide Polymorphism (SNP) observed in locus R539T in the current study has also been reported in Kenya and Senegal. R539T was reported from a study conducted in Mbita district, Kenya [35]. This mutation was highly associated with in-vivo delayed parasite clearance among the patients from Mbita district. However, this mutation has not been previously associated with ACTs resistance in South East Asia.
The mutation reported in N458Y locus has also been reported elsewhere in Africa and other South Eastern Asian countries. But previously this mutation has not been validated as a candidate of resistance. However, it is associated with poor drug response by P. falciparum to ACTs [36]. The low frequencies of mutations associated with ACTs resistance reported here in comparison to South East Asia which has reported high frequency may be due to the fact ACTs usage in SEA started long time ago (1970s), compared to Kenya which adopted ACTs usage in 2004. The current study is the first report on the mutations associated with N431S and A671V, respectively. This is in tandem with other studies which have reported new mutations in P. falciparum clinical isolates [37].
More than 200 non-synonymous mutations have been recognized in K13 protein from P. falciparum [38]. However, fifty Pf-K13 mutations have been reported previously to be associated with ACTs resistance in South East Asia of which nine have been confirmed as resistant candidates while eleven are potentials for ACTs resistance. The other thirty K13 mutations have been reported from various locations in South Asia, however, they are not consistent with the clinical findings on ACTs failure. Out of these documented mutations, only nine have been authenticated as candidates for ACTs resistance under the ex-vivo conditions. The validated markers include; C580Y, Y493H F446I, M476IV, N458Y R539T, P553L, I543T, and R561H [39].
Surprisingly, previous studies have reported new non-validated mutations, which were present in patients who had poor recovery after treatment with ACTs [40]. This raises concern whether they have some roles they play in conferring resistance, and should hence be considered in the future as molecular markers of ACTs resistance. A mutation at codon A675V has been reported in Rwanda [41] and Uganda [42]. The same mutation at codon A675V had also previously been reported in SEA [43], an epicenter of the emergence and spread of ACTs resistance.
The circulation of K13 mutations have also been previously reported in Kenya but with limited studies [44]. A previous study conducted in different Malaria Transmission Areas of Kenya viz; Marigati, Kombewa, Kisumu, Kisii, Kericho and Malindi to ascertain the prevalence of K13 mutation during the pre-ACTs and post-ACTs eras, reported different polymorphisms at different locus [45]. The A578S and the V568G mutations reported in SEA were found in both pre-ACTs and post-ACTs parasites. D584Y and R539K mutations were found only in post-ACTs parasites. These mutations were also previously reported from clinical isolates from South East Asia [46], raising the question of the possibility of mediating resistance. The N585K mutation was described for the first time in this previous study among the post-ACTs parasites, and it was the most prevalent mutation at a frequency of 5.2%. However, the prevalence and type of mutations varied across the malaria ecological zones and between the pre- and post-ACT time periods. This study reported A578S in post-ACT parasites in two different study sites, Kombewa (4.3%) and Kisii (2.1%). Kombewa is situated in the holo-endemic lake region and Kisii is located in the highland epidemic region. The N585K allele was reported in only the post-ACTs era in the study areas, with the highest prevalence in Kombewa (10.6%) and Kisumu (9.8%). This mutation witnessed here might be under pressure for evolutionary through antimalarial drugs since the use of Artemether-Lumefantrine is high in Kombewa and Kisumu due to high malaria transmission [47].
Another study conducted at 4 islands in the Lake Victoria basin (Kibuogi, Ngodhe, Takawiri, and Mfangano) and the mainland of Mbita in Kenya reported different mutant alleles in the K13 propeller gene, with C580Y, Y493H and R539T being the most prevalent and significantly associated with in-vivo delayed parasite clearance. However, a new mutation of A578S was detected at Mfangano Island for two consecutive seasons. This mutation is closely related to the single nucleotide polymorphism C580Y detected from Cambodia, indicated to be conferring ACTs resistance [48]. Other mutations reported in this study included M442V, N554S, A569S, C439C, S477S, Y500Y, N531N and G538G. These mutations have not been previously associated with ACTs resistance.
A previous study on the Kenyan coast has reported limited P. falciparum K13 Artemisinin Resistance-Conferring Mutations over a 24-Year Analysis. The K189T mutation was the only polymorphism maintained at frequencies of 10%, while the rest of the observed alleles were rare, including codon A578S, with frequencies barely reaching 2% [49].
A report by the world health organization, 2021 has documented a 30-fold increase in the use of ACTs globally between 2006 and 2013 [50]. Thus, the augmented usage of artemisinin agents is expected to increase drug pressure, leading to resistance development. Consequently, irrational usage of ACTs coupled with the use of substandard drugs in the developing countries such as Kenya, may exacerbate the risk of resistance development. Bearing in mind that previous resistance to antimalarial agents was first detected in South East Asia and then later spread to Africa, it is possible that the artemisinin resistance documented in Cambodia may also spread through Myanmar via India to Africa by following the previous patterns [51]. This is likely to occur due to the increased international travel and migration, especially because Kenya serves as a transition point for travelers from Asia to Africa and South America.
After testing the departures of Nucleotide variability patterns of the sequence products from neutral expectations, the isolates from the current studies showed evidence of positive selection as highlighted by the negative values of the tests (Tajima’s D = − 1.72305; Fu and Li’s D of − 1.74248). Previous study has documented that indigenous populations and ecological level courses, such as drug pressure serve as essential mediators of resistance acquisition in the population [52]. The current study was unable to establish if the K13-propeller gene mutations detected in the P. falciparum clinical isolates from Kisii County resulted from local emergence because of ecological and population-level processes or through transfer because of global human travel or local emergence. In contrast to Africa and Kenya, where artemisinin agents are commonly used in the form of combinations, studies have indicated that more than 78% of artemisinin in South East Asia is used as monotherapies [53]. The use of artemisinin agents combined with their short half-lives may still protect the population from wide-spread emergence of resistant parasites.
The accumulation of data from Kenya will increase the understanding of the association between the K13 propeller gene and artemisinin resistance. Unfortunately, most of the molecular drug surveillance conducted in Kenya was performed in western and coastal regions. Thus no clear picture of the molecular data is available.