The development of artemisinin (ART)-resistant parasites in Southeast Asia (SEA) threatens malaria control globally. Mutations in the Kelch 13 (K13)-propeller domain have been useful in identifying ART resistance in SEA. ART combination therapy (ACT) remains highly efficacious in the treatment of uncomplicated malaria in Sub-Saharan Africa (SSA). However, it is crucial that the efficacy of ACT is closely monitored. Toward this effort, this study profiled the prevalence of K13 nonsynonymous mutations in different malaria ecological zones of Kenya and in different time periods, before (pre) and after (post) the introduction of ACT as the first-line treatment of malaria. Nineteen nonsynonymous mutations were present in the pre-ACT samples (N = 64) compared with 22 in the post-ACT samples (N = 251). Eight of these mutations were present in both pre- and post-ACT parasites. Interestingly, seven of the shared single-nucleotide polymorphisms were at higher frequencies in the pre-ACT than the post-ACT parasites. The A578S mutation reported in SSA and the V568G mutation reported in SEA were found in both pre- and post-ACT parasites, with their frequencies declining post-ACT. D584Y and R539K mutations were found only in post-ACT parasites; changes in these codons have also been reported in SEA with different amino acids. The N585K mutation described for the first time in this study was present only in post-ACT parasites, and it was the most prevalent mutation at a frequency of 5.2%. This study showed the type, prevalence, and frequency of K13 mutations that varied based on the malaria ecological zones and also between the pre- and post-ACT time periods.

Genetic analysis of molecular markers is critical in tracking the emergence and/or spread of artemisinin resistant parasites. Clinical isolates collected in western Kenya pre- and post- introduction of artemisinin combination therapies (ACTs) were genotyped at SNP positions in regions of strong selection signatures on chromosome 13 and 14, as described in Southeast Asia (SEA). Twenty five SNPs were genotyped using Sequenom MassArray and pfmdr1 gene copy number by real-time PCR. Parasite clearance half-life and in vitro drug sensitivity testing were performed using standard methods. One hundred twenty nine isolates were successfully analyzed. Fifteen SNPs were present in pre-ACTs isolates and six in post-ACTs. None of the SNPs showed association with parasite clearance half-life. Post-ACTs parasites had significantly higher pfmdr1 copy number compared to pre-ACTs. Seven of eight parasites with multiple pfmdr1 were post-ACTs. When in vitro IC₅₀s were compared for parasites with single vs. multiple gene copies, only amodiaquine and piperaquine reached statistical significance. Data showed SNPs on chromosome 13 and 14 had different frequency and trend in western Kenya parasites compared SEA. Increase in pfmdr1 gene copy is consistent with recent studies in African parasites. Data suggests genetic signature of artemisinin resistance in Africa might be different from SEA.

Sulphadoxine-pyrimethamine (SP), an antifolate, was replaced by artemether-lumefantrine as the first-line malaria drug treatment in Kenya in 2004 due to the wide spread of resistance. However, SP still remains the recommended drug for intermittent preventive treatment in pregnant women and infants (IPTP/I) owing to its safety profile. This study assessed the prevalence of mutations in dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes associated with SP resistance in samples collected in Kenya between 2008 and 2012.

In combination with antibiotics, quinine is recommended as the second-line treatment for uncomplicated malaria, an alternative first-line treatment for severe malaria, and for treatment of malaria in the first trimester of pregnancy. Quinine has been shown to have frequent clinical failures, and yet the mechanisms of action and resistance have not been fully elucidated. However, resistance is linked to polymorphisms in multiple genes, including multidrug resistance 1 (Pf mdr1 ), the chloroquine resistance transporter (Pf crt ), and the sodium/hydrogen exchanger gene (Pf nhe1 ). Here, we investigated the association between in vitro quinine susceptibility and genetic polymorphisms in Pf mdr1 codons 86 and 184, Pf crt codon 76, and Pf nhe1 ms4760 in 88 field isolates from western Kenya. In vitro activity was assessed based on the drug concentration that inhibited 50% of parasite growth (the IC ₅₀ ), and parasite genetic polymorphisms were determined from DNA sequencing. Data revealed there were significant associations between polymorphism in Pf mdr1 -86Y, Pf mdr1 -184F, or Pf crt -76T and quinine susceptibility ( P < 0.0001 for all three associations). Eighty-two percent of parasites resistant to quinine carried mutant alleles at these codons (Pf mdr1 -86Y, Pf mdr1 -184F, and Pf crt -76T), whereas 74% of parasites susceptible to quinine carried the wild-type allele (Pf mdr1 -N86, Pf mdr1 -Y184, and Pf crt -K76, respectively). In addition, quinine IC ₅₀ values for parasites with Pf nhe1 ms4760 3 DNNND repeats were significantly higher than for those with 1 or 2 repeats ( P = 0.033 and P = 0.0043, respectively). Clinical efficacy studies are now required to confirm the validity of these markers and the importance of parasite genetic background.

The Plasmodium falciparum in vitro culture system is critical for genotypic and phenotypic analyses of the parasites. For genotypic analysis, the genomic DNA can be obtained directly from the patient blood sample or from culture adapted parasites whereas for phenotypic analysis, immediate ex vivo or in vitro culture adapted parasites are used. However, parasite biology studies have not investigated whether culture adaptation process affects genotypic and/or phenotypic characteristics of the parasites in short- or long-term cultures. Here, we set out to study the dynamics and stability of parasite genetic and phenotypic profiles as field isolate parasites were adapted in continuous cultures. Parasites collected from three different patients presenting with uncomplicated malaria were adapted and maintained in drug-free continuous cultures. Aliquots from the continuous cultures were collected every 24–48 hours for analyses. Each aliquot was treated as a separate parasite sample. For genetic analysis, microsatellite (MS) typing and single nucleotide polymorphism (SNP) analyses of 23 drug resistance markers were done. The 50% inhibitory concentrations (IC₅₀) for some of the samples were also established for four antimalarial drugs. Samples from each patient (parasite-line) were compared as they were passed through the continuous culture. Data revealed genotypic and phenotypic profiles for the three parasite-lines fluctuated from one generation to the next with no specific pattern or periodicity. With few exceptions, multilocus analysis revealed samples from each parasite-line had high genetic diversity with unique haplotypes. Interestingly, changes in MS and SNP profiles occurred simultaneously. The difference in the IC₅₀s of samples in each parasite-line reached statistical significance. However, phenotypic changes did not correspond or correlate to genotypic changes. Our study revealed parasite genetic and phenotypic characteristics fluctuates in short- and long-term cultures, which indicates parasite genetic information obtained even in short cultures is likely to be different from the natural infection parasites.

There are concerns that resistance to artemisinin-based combination therapy might emerge in Kenya and sub-Saharan Africa (SSA) in the same pattern as was with chloroquine and sulfadoxine–pyrimethamine. Single nucleotide polymorphisms (SNPs) in critical alleles of pfmdr1 gene have been associated with resistance to artemisinin and its partner drugs. Microsatellite analysis of loci flanking genes associated with anti-malarial drug resistance has been used in defining the geographic origins, dissemination of resistant parasites and identifying regions in the genome that have been under selection. This study set out to investigate evidence of selective sweep and genetic lineages in pfmdr1 genotypes associated with the use of artemether–lumefantrine (AL), as the first-line treatment in Kenya. Parasites (n = 252) from different regions in Kenya were assayed for SNPs at codons 86, 184 and 1246 and typed for 7 neutral microsatellites and 13 microsatellites loci flanking (± 99 kb) pfmdr1 in Plasmodium falciparum infections. The data showed differential site and region specific prevalence of SNPs associated with drug resistance in the pfmdr1 gene. The prevalence of pfmdr1 N86, 184F, and D1246 in western Kenya (Kisumu, Kericho and Kisii) compared to the coast of Kenya (Malindi) was 92.9% vs. 66.7%, 53.5% vs. to 24.2% and 96% vs. to 87.9%, respectively. The NFD haplotype which is consistent with AL selection was at 51% in western Kenya compared to 25% in coastal Kenya. Selection pressures were observed to be different in different regions of Kenya, especially the western region compared to the coastal region. The data showed independent genetic lineages for all the pfmdr1 alleles. The evidence of soft sweeps in pfmdr1 observed varied in direction from one region to another. This is challenging for malaria control programs in SSA which clearly indicate effective malaria control policies should be based on the region and not at a country wide level.