| Part 1 Study Plasmodium falciparum resistance to antimalarial drugs as well as the molecular mechanisms of resistance about the samples of year 2008 and 2012Objective:To measure the drug sensitivity of 2008 and 2012 plasmodium falciparum which are sing-infected isolates,and obtain the IC50 values to quinine、 naphthoquine phosphate、 pyronaridine phosphate、Artesunate and dihydroartemisinin.and the molecular mechanism of resistance.Methods:1、the Plasmodium falciparum were cultured in vitro, then after synchronizing the life cycle of P.falciparum,we do drug-resistance assay with quinine、 naphthoquine phosphate、 pyronaridine phosphate、Artesunate and dihydroartemisinin, then finally obtain the IC50 values.2、the samples were amplified by Nest-PCR DNA, whole gene sequences of K13 gene 2181bp, the fragments 590bp,960bp of pfindrl gene and pfnhe-1 gene fragments. After sequencing, the samples and the standard strain 3D7 were compared to obtain the gene mutation.Results:1、Resistant Results1. 1、The IC50 of year 2008 samples to quinine、naphthoquine phosphate、pyronaridine phosphate、Artesunate and dihydroartemisinin are 304.1±106.1 nM、9.245±4.726nM、 12.51±3.866nM、4.411±1.145nM.3.347、0.789 nM...The ICso of year 2012 Plasmodium falciparum to quinine、naphthoquine phosphate、pyronaridine phosphate、Artesunate and dihydroartemisinin are 441.5±147.7nM、±9.176±5.377nM、12.97±6.647nM、6.231±3.385nM、 4.896±1.926nM, Compare the IC50 value for five drugs of strains of 2008 and 2012,2008 and 2012 QN ICso value (P= 0.0083<0..05), DHAIC50 value (P= 0.0083<0.05) P values were less than 0.05. The PND IC50 value (p= 0.8702), NQ ICso value (p= 0.9714), AS ICso value (p= 0.0605) three drugs between the two years in P values were greater than 0.05.1.2、According to the sensitivity and resistance criteria, in the 28 strains,1/28 strains is sensitive to quinine, sensitivity decreased by 50%. Followed by sensitive strainsto naphthoquine phosphate is 15/28, sensitivity fell 35.71%. Phosphate Pyronaridine sensitive strains is 13/28 strain, sensitivity decreased 17.85%. Artesunate and dihydroartemisinin sensitive strain were 23/28,22/28, the sensitivity decreased to 3.57%,0.1.3、we analysis the ross-resistance to Five antimalarial,we found NQ and PND (p<0.0001), NQ and AS (p= 0.008), QN and AS (p= 0.0004), QN and DHA (p= 0.0002), AS and DHA (p= 0.0006). P values were less than 0.05.2、Molecular assay results:2.1、K13 gene:the 28 DNA samples were used for amplification of K13 gene the full-length 2181bp fragment. We analyzed F446I, C469Y, Y493H, R539T, P553L, R574L, C580Y. and 143 NN into the case. In 28 samples, and 446、539% 143 site mutation was found and Three loci mutation rate in 2008 is respectively 46.66%、0、73.33%.Three loci mutation rates In 2012 respectively were 46.15%,、7.69%、69.23%。The total mutation rate were 46.42%、3.57%、71.42%. compare mutation point mutation rate in K13 gene of 2008 and 2012 strains,F446I (p= 1.000), R539T (p= 0.464),143NN (p= 1.000), P values are greater than 0.05.Pfmdr1:DNA sample of 28 isolates are used for the amplification of pfmdrl gene 590bp and 960 bp fragments. We analyzed N86Y, N184F, S1034C, N1042D, F1226Y and D1246Y. In 28 samples, only the mutations of 184 and 1226 site are found, mutations were not found in other sites, two loci mutation rates were 33.33%,13.33% In 2008. and the mutation rates were 7.69%,46.15% in 2012, the total mutation rate was 21.42%,28.57%. compare the mutation rate of mutation point in pfindrl gene of 2008 and 2012 strains,Y184F (p= 0.173), F1226Y (p= 0.096), P values are greater than 0.05.2.3% Pfnhe-1:28 DNA samples are used for pfnhe-1 amplification. In this experiment, We have sequenced the ms4760 fragment of pfnhe-1 gene and found 6 ms4760 microsatellite profiles. The most prevalent ms4760 profile was ms4760-7, with the frequency of 75%. There are samples showed ms4760-7 genotype, respectively, the probability of subtype 2008 and 2012 was 66.66%,84.61%. The other subtypes of ms4760-1, ms4760-3, ms4760-5, ms4760-6, ms4760-7, ms4760-9. Compare the pfnhe-1 gene haplotype of 2008 and 2012 strains 1 (p= 1.000),3 (p= 0.464),5 (p= 1.000),6 (p= 0.484),7 (p= 0.396),9 (p = 1.000), P values are greater than 0.05.3、the correlation between the results of in vitro assays and molecular measurement results:3.1、compare AS IC50、DHAIC50 of K13 gene 446 sites mutant and not mutant strains, P values were(P= 0.0506, P= 0.0252), compare AS IC50、DHA IC50 of K13 gene 143 sites mutant and not mutant strains,P values were(P= 0.1530, P= 0.2803).3.2、comparer the four groups QNIC50, AS IC50, DHA IC50, NQIC50 of pfmdrl gene 184 sites mutant and not mutant strains, P values were (P= 0.4212, P= 0.9703, P= 0.8061, P= 0.5379), comparer the four groups QNIC50, AS IC50, DHA IC50, NQIC50 of pfmdrl gene 1226 sites mutant and not mutant strains, (P values were P= 0.0029, P= 0.0390, P= 0.0175, P= 0.8323)3.3、compare IC50 value of the number of repetitions of pfnhe-1 gene fragment DNNND and NHNDNHNNDDD strains respectively, P values were (P= 0.6896, P= 0.7215)Conclusions:1、we compare the five drugs IC50 values of the samples between 2008 and 2012, it is showed that the two drugs QN、DHA had significant differences in sensitivity of two years, the sensitivity of the samples in 2012 is lower than in 2008. While it is recognized the sensitivity to three kinds of drugs PND、NQ、AS in two years had no significant difference.,2、the 28 samples has had a certain resistance to the two drugs quinine and naphthoquine phosphate, and the sensitivity fall to pyronaridine. It means that Plasmodium falciparum in 28 samples are sensitive to Artesunate and artemisinin、five kinds of antimalarial drugs:cross resistance between pyronaridine, naphthoquine, quinine, artesunate and dihydroartemisinin,The linear correlation was found between NQ and PND, NQ and AS, there are cross resistance between QN、AS、DHA (P<0.05)).4、it were not significant that the mutation rate of mutations site of K13, pfmdr1, pfnhe-1 gene in 2008 and 2012 strains.5、It were correlated that K13 gene mutant 446 points and the strains to the Dihydroartemisinin sensitivity, It were correlated that pfmdr1 gene mutant 1 226 points and the strains to quinine, artesunate, dihydroartemisinin sensitivity.1、we compare the five drugs IC50 values of the samples between 2008 and 2012, it is showed that the two drugs QN、DHA had significant differences in sensitivity of two years, the sensitivity of the samples in 2012 is lower than in 2008. While it is recognized the sensitivity to three kinds of drugs PND、NQ、AS in two years had no significant difference.,2、the 28 samples has had a certain resistance to the two drugs quinine and naphthoquine phosphate, and the sensitivity fall to pyronaridine. It means that Plasmodium falciparum in 28 samples are sensitive to Artesunate and artemisinin、five kinds of antimalarial drugs:cross resistance between pyronaridine, naphthoquine, quinine, artesunate and dihydroartemisinin,The linear correlation was found between NQ and PND, NQ and AS, there are cross resistance between QN、AS、DHA (P<0.05)).4、it were not significant that the mutation rate of mutations site of K13, pfmdr1, pfnhe-1 gene in 2008 and 2012 strains.5、It were correlated that K13 gene mutant 446 points and the strains to the Dihydroartemisinin sensitivity, It were correlated that pfmdr1 gene mutant 1 226 points and the strains to quinine, artesunate, dihydroartemisinin sensitivity.Part2 Continued drug screening experimentObjective:Plasmodium falciparum were cultured in vitro, when to meet the conditions of sustained drug pressure of four drugs,quinine、naphthoquine phosphate、pyronaridine phosphate and benflumetol. drug resistant strains were screened, and we can got IC50 value of the drug resistant strains in different drug concentration time,finaly obtained the relationship resistant strains in different concentration of drug and different the periodMethods:The samples using intermittent pressure method to filter out high resistant strains in the laboratory was 26, continuous drug quinine、naphthoquine phosphate、pyronaridine phosphate and benflumetol were put in the culture.Plasmodium falciparum were cultured in vitro with sustained drug. when reached a certain concentration that the strains all is death, the screening is over, then the survival parasite in the drug concentration were detected the drugs resistance.Results:1、samples from the laborator strains were 26 strains, One QN strains of 26 samples was screened out-A46. We study its resistance under the concentrations of OnM,400nM, 500nM; One NQ samples of 26 samples was screened out-F08B62. We study its resistance under the concentrations of OnM 15nM,20nM;One PND strains of 26 samples was screened out-F08B62. We study its resistance under the concentrations of OnM,15nM,25nM2、IC50 of A46-QN under the Three concentrations of OnM,400nM,500nM were 251.033±15.785、334.167±33.173、518.433±10.546;IC50 of F08B62 PND under the Three concentrations of OnM 15nM,25nM were 7.592±0.701、12.077±1.134、15.013±0.179; IC50 of F08B62NQ under the Three concentrations OnM,15nM,25nM were of 4.000±0.046, 7.803±0.777、12.463±1.140。Conclusions:the IC50 value of Three the concentration of quinine、naphthoquine phosphate、 pyronaridine phosphate increased with the increase of drug concentration. Three drug resistance is strengthened with increasing concentration. |
PDF Full Text Request