Susceptibility Of Plasmodium Falciparum To Artemisinin Drugs And Association Withk13-propellerpolymorphisms At The China-Myanmar Border

Objective:This study aims to assess the efficacy of dihydroartemisinin/piperaquine(DP)for treating uncomplicated falciparum malaria at the China-Myanmar border area,and investigate polymorphisms in Plasmodium falciparum parasites from the China-Myanmar border area.To evaluate drug susceptibilities to artemisinin drugs by two methods in Plasmodium falciparum at the China-Myanmar border area and then related these finding to the observed alleles of the K-13 gene.Methods:1 A clinical trial was conducted in 8 suburban natural villages at the China-Myanmar border area from March 2012 to December 2013.The patients aged 1-60 years,infected by Plasmodium falciparum without serious complications(asexual parasite density at the time of admission was above 100/mL)were recruited for the research.The cases were given a 2-day course with DP tablets(each contain-ing 40 mg of dihydroartemisinin and 320 mg of piperaquine)with the total dosage varied based on weight of the patient.All cases were followed-up at Day-0,Day-1,Day-2,Day-3,Day-7,Day-14,Day-21,Day-28,and Day-42 for assessment of symp-toms,density of parasites,body temperature and side effects of the drug.The thera-peutic efficacy was assessed by using WHO classification of therapeutic response to the treatment of antimalarial drugs,including the time of fever subsidence,the clear-ance time of asexual parasites.2 Plasmodium falciparum isolates collected from the China-Myanmar border from 2007 to 2013 were used as a source of DNA.The full-length K13 gene was se-quenced in all samples using polymerase chain reaction and direct sequencing me-thods.3 Blood films from atients treated with dihydroartemisinin/piperaquine were analyzed by microscope three days after treatment and then divided into two groups based on detection of Plasmodium:Day-3 negative and Day-3 positive.To measure drug susceptibilities in vitro,survival rates of the ring stage(RSA)parasites and IC50 values for artesunate(AS),dihydroartemisinin(DHA)and artemisinin(ART)solved.These assays were performed on clinic isolates(both Day-3 negative and Day-3 posi-tive)and strain 3D7 parasites and include parasties with and without mutations in the K-13 gene.Results:1 Among the 109 cases enrolled,71 cases completed the 42-day fol-low-up.The mean time of fever subsidence was 29.8 ± 6.7 h,the median of clearance time of asexual parasites in blood was 36.9 h,the rate of fever subsidence and elimi-nation of asexual parasites by day-3 was 100%and 92.9%,and the rate of fever sub-sidence and elimination of asexual parasites by day-7 was 100%and 100%.Nine-teen cases had gametocytes before treatment.These were cleared with a rate at day-3,day-7 and day-14 of 89.5%,94.7%,100%,respectively.Only mild side effects were reported in the trial.2 N-insert and 16 point mutations(N11Y,K189T,E252Q,R225K,1352T,F446I,N458Y,C469Y,L492S,F495L,5397,P553L,P574L,580Y,A676D,and H719N)were with the NN-Insert mutation being the most frequently identified(in 59.6%of the samples).The F446Imutation was identified in 30.3%of the parasite isolates,while a mutation after the AA locus 440 was identified in 41.6%of the sam-ples.Most mutation after the AA locus 440 were accompanied by the NN-insert.Iso-lates without mutations in the K13 gene made up 44.0%of samples characterized in 2007 while only 9.8%of the samples from 2010-2013 had unchanged genes.H719N 3 ?(? 1.7%)?3 The IC50 values from Day-3 positive strains for AS,DHA,and ART 3.30 ±0.96 nM,2.85 ± 0.82 nM,5.62 ± 0.99 nM,respectively,while they were 3.53 ± 0.83 nM,3.09 ± 0.76 nM,5.80 ± 0.97 nM for Day-3 negative strains.Strain 3D7 para-sites had IC50 values for AS,DHA,and ART of 3.47 ± 0.84 nM,2.59 ± 0.97nM,and 5.55 ± 0.87 nM,repsectively.The median RSA was 12.8%,3.5%,and 0.8%for Day-3 positive,Day-3 negative,and 3D7 strains,respectively.There were no significant dif-ferences in IC50 values from patient strains and 3D7 parasites(P>0.05).Additionally,there were no significant differences in IC50 values of parasites with or without K-13 gene mutations.However,differences were noted in the RSA of patient strains and 3D7(P<0.05).Additionally,the RSA of Day-3 positive isolates was significantly higher than Day-3 negative parasites(P<0.05).Conclusions:1 The combination of dihydroartemisinin/piperaquine remains highly efficacious for treatment of uncomplicated falciparum malaria at the China-Myanmar border area.However,in some cases there was a noted delay of elimina-tion of asexual parasites,suggesting some parasites may have been partially refrac-tive to treatment.The mean time of fever subsidence was shorter than clearance time of asexual parasites in blood.2 The main mutations in the K13 gene were NN-Inser and mutations after the AA locus 440,and F446I was the dominant mutations at the China-Myanmar border area.The percentage of samples harboring unaltered K13 was found to be de-creasing in the region through time.3 Here we have found that in vitro IC50S values of drug usceptibility do not accu-rately reflect susceptibility of Plasmodium falciparum to artemisinin drugs in vivo,while RSA analysis is more predictive,suggesting that RSA is an important means of evaluating Plasmodium falciparum susceptibility to artemisinin drugs.Additionally,there were significant differences in the number and frequency of mutations in the K-13 gene from samples of parasites that were sensitive to artemisinin(Day-3 negative)or refractory(Day-3 positive)(P<0.05).