Background
Malaria caused an estimated 228 million new infections and 405,000 deaths globally in 2018 [
1]. In Kenya, there were over 15 million suspected and 1.5 million confirmed cases of malaria in 2018 [
1], with the burden highest in areas around Lake Victoria and the coastal region [
2]. Artemisinin-based combination therapy (ACT) is recommended by the Kenya Ministry of Health as first- and second-line treatments for uncomplicated malaria, and available data suggest that they remain highly effective in Kenya and throughout sub-Saharan Africa [
3,
4]. However, the emergence and spread of
Plasmodium falciparum resistance to artemisinin and partner drugs presents one of the greatest challenges to global malaria control and elimination efforts and warrants continuous surveillance of resistance in malaria endemic areas.
The World Health Organization (WHO) recommends routine surveillance for early detection of resistance or emergence of resistant parasites [
5]. While in vitro, ex vivo drug testing and in vivo therapeutic efficacy studies (TES) are important for assessing the effectiveness of anti-malarial drugs [
6], molecular surveillance using genetic markers associated with resistance provides a valuable tool for detecting and tracking resistance as well as providing an in-depth understanding of the development and spread of resistance. Earlier studies have shown that parasite resistance to anti-malarial drugs is often associated with single nucleotide polymorphisms (SNPs) or amplifications of the genes coding for drug target proteins or transporters [
7]. For example, SNPs at codons 86, 184, and 1246 in the
P. falciparum multidrug resistance 1 gene (
Pfmdr1) have been implicated to confer parasite resistance to multiple anti-malarial drugs, with individual polymorphisms leading to opposite effects on different drugs [
8]. Mutations at
Pfmdr1 86
Y and 1246
Y have been linked to decreased sensitivity to chloroquine and amodiaquine, but increased sensitivity to lumefantrine, mefloquine and artemisinin [
9‐
13], while mutations at 184
F have been associated with reduced susceptibility to lumefantrine [
14,
15]. Likewise, mutations at codons 72 to 76 of the
P. falciparum chloroquine resistance transporter gene (
Pfcrt) have been associated with resistance to chloroquine and amodiaquine [
16,
17], with the 76
T point mutation being the most predictive of chloroquine resistance. Amplification of the
plasmepsin2 gene (
Pfpm2) has been recently associated with piperaquine resistance [
18]. Additionally, polymorphisms in the
P. falciparum kelch 13 (
Pfk13) propeller region have been causally linked to artemisinin resistance in Southeast Asia [
19,
20] and serve as valuable molecular markers for tracking and monitoring artemisinin-resistant parasites.
Clinical trials performed in Africa have provided evidence for the selection of particular
Pfmdr1 alleles in patients with newly acquired or recurrent
P. falciparum infections within 28 or 42 days after ACT treatment [
21‐
25]. Additionally, genetic mutations in the
Pfk13 propeller region have been associated with delayed parasite clearance, which has been shown to lead to more treatment failures after ACT treatment [
26]. While previous studies performed in western Kenya have compared the prevalence of
Pfmdr1 and
Pfk13 molecular markers before and after ACT was introduced [
27‐
29] and assessed their association with parasite clearance rates [
27], there is limited information on the association between these markers and treatment outcomes. Understanding the relationship between molecular markers, parasite resistance and treatment failure is important for evidence-based decision making on anti-malarial drug policies.
The main objective of this study was to assess the frequency of molecular markers associated with resistance to anti-malarial drugs from parasite isolates collected during a TES conducted in Siaya, western Kenya in 2016–2017. This current manuscript reports the frequencies of SNPs in genes associated with resistance or tolerance to anti-malarial drugs including,
Pfcrt for chloroquine resistance [
30],
Pfmdr1 for lumefantrine tolerance [
31],
Pfk13 for artemisinin resistance [
19], and amplification of
Pfpm2 for piperaquine resistance [
18]. The association between these mutations with TES treatment outcome was also examined. Another manuscript in process details the efficacy and clinical endpoints [
32].
Discussion
The use of ACT was recommended by the WHO in 2005 as a first-line treatment for falciparum malaria in all malaria-endemic countries [
47]. This was due to the emergence and rapid spread of parasite resistance to previously recommended drugs, such as chloroquine and sulfadoxine-pyrimethamine [
22]. ACT consists of an artemisinin component, which rapidly clears most parasites, and a longer acting partner drug, which eliminates remaining parasites and limits selection of drug resistance [
47]. With the ongoing challenges related to the emergence of artemisinin resistance in multiple countries in Southeast Asia, including Cambodia, Thailand, Myanmar, and Laos [
20,
27,
42,
43] and the threat of resistance emerging in sub-Saharan Africa [
44], there is need for continued surveillance to monitor the efficacy of ACT and the genetic markers associated with anti-malarial drug resistance in malaria-endemic areas.
In this study, no
Pfk13 propeller region mutations previously validated to be correlated with artemisinin resistance were detected. These findings are consistent with previous studies conducted in western Kenya [
29,
48] and coastal Kenya [
49], which did not report any
Pfk13 propeller region mutations that have been associated with artemisinin resistance. The absence of these
Pfk13 mutations associated with artemisinin resistance in this study, as well as in other African countries [
50], suggests that artemisinin resistance may not have emerged on the continent or spread from Southeast Asia.
Other non-synonymous mutations in the
Pfk13 propeller region, which have not been associated with resistance were detected; these were 522
C and 578
S. While a slightly higher proportion of the recurrent samples harboured the 578
S mutant allele, at 2.3% compared with 1.9% in day-0 samples, these findings were seen without any impact on clinical resistance to artemisinin as observed by absence of parasites on day 3 [
32]. Additionally, there was no statistically significant association between these observed mutations with either recrudescence or recurrent infections. The lack of association between the 578
S polymorphism and a resistant phenotype is consistent with previous studies conducted in western Kenya and in four other African countries [
27,
48] as well as multi-site studies in Southeast Asia and Africa that showed no association with drug resistance in patients treated with ACT [
51]. Despite the lack of association of these mutations with a resistant phenotype, there is a need for further studies in a large population to assess whether
Pfk13 propeller region mutations are relevant in determining artemisinin resistance in parasite isolates in sub-Saharan Africa. It is possible that the actual SNP(s) that confer resistance differ from one geographic location to another depending on several factors, including parasite genetics, malaria transmission intensity, treatment seeking behaviour, and adherence to treatment guidelines. In multiple countries in Southeast Asia, where ACT resistance emerged, use of artemisinin monotherapy and sub-standard drugs is rampant and may partially explain why ACT resistance has emerged there but not in other areas [
52].
The overall proportion of
Pfmdr1 86
Y mutant alleles in the current study was significantly lower (< 5%) than in a previous study conducted in western Kenya, which reported a prevalence of 69% in samples collected in 2010 [
44]. The selection of
Pfmdr1 N86 wild type allele is likely due to the withdrawal of chloroquine and the widespread use of AL as the first-line anti-malarial treatment in Kenya, which could have promoted the selection of the wild-type sequences at this allele as observed in another study in western Kenya [
27] and in other countries in Africa [
20,
27,
53,
54]. The increasing levels of
Pfmdr1 N86 wild type allele could also suggest decreasing sensitivity to lumefantrine [
24,
55] and artemisinin [
7,
9,
11] as observed in previous studies. Additional studies are needed to assess the effect of N86 wild type allele on ACT susceptibility given the increasing proportion of samples harbouring this wild type allele over time in western Kenya.
The present study revealed a high proportion of the 184
F mutation in the
Pfmdr1 gene with a proportion of 59.7, 62.1, and 66.6% for day-0, recurrent infections and recrudescent parasites, respectively. These results are consistent with a recent study conducted in western Kenya which reported a proportion of 65% after introduction of ACT [
27]. The proportion of this mutant allele increased over time in comparison to a previous cross-sectional study in Siaya County, Kenya, which reported a prevalence of 23.3% in samples collected in 2010 [
44]. The data suggest that the 184
F mutant is possibly being selected for by ACT at the population level. However, there was no statistically significant association between the mutations with treatment failure (recrudescence) in both treatment arms in the current study. This is consistent with another study conducted in Senegal which did not find any association between
Pfmdr1 184
F mutants with susceptibility to various anti-malarial drugs in vitro [
56]. A clinical trial assessing anti-malarial drug levels in patients has, however, associated
Pfmdr1 184
F mutants with reduced susceptibility to lumefantrine [
14]. The role of this mutation as a marker of resistance in the current study could be unclear given that mutations were also present in the majority of successfully treated patients in both treatment arms. The discrepancy in conclusions from different studies regarding the role of 184
F mutation in lumefantrine and other various anti-malarial drugs could be due to different study designs (i.e., in vitro studies, cross-sectional studies over time at population level, or clinical trials testing drug levels in patients), making it difficult to compare the data across different studies.
For codon 1246 of
Pfmdr1, the proportion of mutant 1246
Y alleles was reduced compared to previous studies in western Kenya, which reported prevalence of 40 and 16.5% [
27,
44] compared to 9.3, 5.3, and 8.3% for day-0, recurrent infections and recrudescent samples, respectively in the current study. The decrease of the mutant alleles may be due to withdrawal of chloroquine and could also suggest decreased sensitivity to lumefantrine and artemisinin. In previous studies, the changes in lumefantrine sensitivity have been associated with polymorphisms in the
Pfmdr1 gene [
24,
31]. For example, Tanzanian parasites having the
Pfmdr1 N
FD (N86, 184
F, D1246) haplotype were able to withstand lumefantrine blood concentrations 15-fold higher than parasites with the
YY
Y (86
Y, Y184, 1246
Y) haplotype [
14]. In addition, in Uganda, AL was demonstrated to select for haplotypes with N86 in combination with 184
F and D1246, or both [
57].
In this study, a very high proportion (> 95%) of parasites harboured
Pfcrt wild haplotype (CVMNK). This may be a result of the withdrawal of chloroquine and the introduction of ACT in 2006 in Kenya, which may have promoted the re-emergence of chloroquine-sensitive isolates. This is consistent with other studies conducted in Kenya [
49,
58], Malawi [
59], Côte d’Ivoire [
60], and Tanzania [
8], which have reported re-emergence of chloroquine-susceptible parasites following years of discontinuation of chloroquine use. A small proportion of parasites (< 5%) contained a mixed (N75
D) or mutated (75
E) nucleotide at codon 75, yielding haplotypes CV
IDT, CVM
DT, and CV
IET. Prior literature suggests a stepwise mutation mechanism exists at codon 75, mutating from N75 to 75
D to 75
E [
61]. Although CV
IET is historically associated with chloroquine resistance [
62], variable levels of chloroquine resistance have been noted in other
Pfcrt isoforms, including a mixed mutation (N/
D) at codon 75 [
63]. Population-representative studies should be conducted with a larger number of samples from different regions to confirm the decrease of chloroquine-resistant parasites in Kenya. If the proportion of chloroquine-resistant parasites decreases at the national level to an undetectable level of
Pfcrt mutants, a reintroduction of chloroquine in combination with other anti-malarial drugs for malaria treatment and prophylaxis may be considered in the context of ongoing molecular monitoring.
This study had several limitations. The study utilized samples obtained from children enrolled in a TES, which is not a population representative sample. Population representative studies are needed in order for the findings to be generalizable. Additionally, the sample size was limited to blood samples collected in the TES and was not powered to test for the association between parasite genotypes and treatment outcomes [
46]. Because genotyping data are being collected as part of TESs conducted throughout the region and continent, metanalyses from similar studies across geographic areas may better assess the association between molecular markers and treatment outcome.
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