Artemether–lumefantrine (AL) became the first-line treatment for uncomplicated malaria in Kenya in 2006. Studies have shown AL selects for SNPs in pfcrt and pfmdr1 genes in recurring parasites compared to the baseline infections. The genotypes associated with AL selection are K76 in pfcrt and N86, 184F and D1246 in pfmdr1. To assess the temporal change of these genotypes in western Kenya, 47 parasite isolates collected before (pre-ACT; 1995–2003) and 745 after (post-ACT; 2008–2014) introduction of AL were analyzed. In addition, the associations of parasite haplotype against the IC50 of artemether and lumefantrine, and clearance rates were determined. Parasite genomic DNA collected between 1995 and 2014 was analyzed by sequencing or PCR-based single-base extension on Sequenom MassARRAY. IC50s were determined for a subset of the samples. One hundred eighteen samples from 2013 to 2014 were from an efficacy trial of which 68 had clearance half-lives. Data revealed there were significant differences between pre-ACT and post-ACT genotypes at the four codons (chi-square analysis; p < 0.0001). The prevalence of pfcrt K76 and N86 increased from 6.4% in 1995–1996 to 93.2% in 2014 and 0.0% in 2002–2003 to 92.4% in 2014 respectively. Analysis of parasites carrying pure alleles of K + NFD or T + YYY haplotypes revealed that 100.0% of the pre-ACT parasites carried T + YYY and 99.3% of post-ACT parasites carried K + NFD. There was significant correlation (p = 0.04) between lumefantrine IC50 and polymorphism at pfmdr1 codon 184. There was no difference in parasite clearance half-lives based on genetic haplotype profiles. This study shows there is a significant change in parasite genotype, with key molecular determinants of AL selection almost reaching saturation. The implications of these findings are not clear since AL remains highly efficacious. However, there is need to closely monitor parasite genotypic, phenotypic and clinical dynamics in response to continued use of AL in western Kenya.

BackgroundSulphadoxine-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.MethodsField isolates collected from Kisumu, Kisii, Kericho and Malindi district hospitals were assessed for genetic polymorphism at various loci within Pfdhfr and Pfdhps genes by sequencing.ResultsAmong the Pfdhfr mutations, codons N51I, C59R, S108N showed highest prevalence in all the field sites at 95.5%, 84.1% and 98.6% respectively. Pfdhfr S108N prevalence was highest in Kisii at 100%. A temporal trend analysis showed steady prevalence of mutations over time except for codon Pfdhps 581 which showed an increase in mixed genotypes. Triple Pfdhfr N51I/C59R/S108N and double Pfdhps A437G/ K540E had high prevalence rates of 86.6% and 87.9% respectively. The Pfdhfr/Pfdhps quintuple, N51I/C59R/S108N/A437G/K540E mutant which has been shown to be the most clinically relevant marker for SP resistance was observed in 75.7% of the samples.ConclusionSP resistance is still persistently high in western Kenya, which is likely due to fixation of key mutations in the Pfdhfr and Pfdhps genes as well as drug pressure from other antifolate drugs being used for the treatment of malaria and other infections. In addition, there is emergence and increasing prevalence of new mutations in Kenyan parasite population. Since SP is used for IPTP/I, molecular surveillance and in vitro susceptibility assays must be sustained to provide information on the emergence and spread of SP resistance.

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.

Genetically determined artemisinin resistance in Plasmodium falciparum has been described in Southeast Asia. The relevance of recently described Kelch 13-propeller mutations for artemisinin resistance in Sub-Saharan Africa parasites is still unknown. Southeast Asia parasites have low genetic diversity compared to Sub-Saharan Africa, where parasites are highly genetically diverse. This study attempted to elucidate whether genetics provides a basis for discovering molecular markers in response to artemisinin drug treatment in P. falciparum in Kenya. The genetic diversity of parasites collected pre- and post- introduction of artemisinin combination therapy (ACT) in western Kenya was determined. A panel of 12 microsatellites and 91 single nucleotide polymorphisms (SNPs) distributed across the P. falciparum genome were genotyped. Parasite clearance rates were obtained for the post-ACT parasites. The 12 microsatellites were highly polymorphic with post-ACT parasites being significantly more diverse compared to pre-ACT (p < 0.0001). The median clearance half-life was 2.55 hours for the post-ACT parasites. Based on SNP analysis, 15 of 90 post-ACT parasites were single-clone infections. Analysis revealed 3 SNPs that might have some causal association with parasite clearance rates. Further, genetic analysis using Bayesian tree revealed parasites with similar clearance phenotypes were more closely genetically related. With further studies, SNPs described here and genetically determined response to artemisinin treatment might be useful in tracking artemisinin resistance in Kenya.

BackgroundThere 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.MethodsThis 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.ResultsThe 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.ConclusionSelection 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.Electronic supplementary materialThe online version of this article (10.1186/s12936-018-2534-8) contains supplementary material, which is available to authorized users.

No abstract

No abstract

Aims This study evaluated the antimicrobial effect of coriander oil (CO) and isothiocyanates (ITC; Allyl, Benzyl and Phenyl-A-, B-, P-ITC) on clinical oral isolate Streptococcus mutans. We evaluated inhibition of biofilm, metabolic activity and exopolysaccharide formation. Methods The composition of CO was determined by gas chromatography- mass spectrometry (GC-MS). CO and ITC were dissolved in 0.1% Tween 80. Different concentrations (0.0039-1%) of CO, ITC and CO-ITC were made in Tryptic soy broth (TSB) and with sucrose (TSBS). Broth dilution method was used to determine the minimum inhibitory concentration (MIC). Minimum biofilm inhibitory concentrations (MBIC) were established by staining biofilm with crystal violet. Checkerboard assay was used to evaluate combination effects. An XTT assay was used to determine the metabolic activity and a sulphuric acid-phenol assay for inhibition of S. mutans exopolysaccharide production. Results CO was dominated by Linalool (65.5%) followed by 2-bornanone (6.16%) and Gamma-terpinene (4.31%). The least was Terpinen-4-ol (0.13%). The MIC of CO was 0.00195% and < 0.00195% for ITCs. MBIC for B-ITC was at < 0.0039%, while with CO-ITC at 0.00195% (p < 0.05). The MBC of CO was 0.125% and for CO-B-ITC MBIC was < 0.00195%. The B-ITC MBC was > 0.25%. Strong S. mutans exopolysaccharide inhibition was observed with ITC and CO at 0.0039%. CO reduced S. mutans metabolic activity at 0.125% and ITC at 0.0078%. Conclusion and clinical relevance: CO and ITC are promising agents in prevention of both periodontal disease and dental caries. Further data is required to warrant development of products to help mitigate dental caries and periodontal disease.