A Research On Resistance Of Plasmodium Falciparum To Antimalarial Drugs In The Sino-Burmese Border Area

Part 1 A research on DHPS、DHFR、PFMRP1 gene polymorphism of plasmodium falciparums in the Sino-Burmese Border AreaObjective:To study the DHPS/DHFR/PFMRP1 in falciparum malaria endemic areas of Sino-Burmese border area with gene amplified technology, to analyze the status in quo of resistance gene point mutation in 2008 and 2012, and to compare the difference between them.Methods:We amplified DHPS/DHFR/PFMRP1 gene segment with Nest-PCR and sequenced them. And then we compared the results with standard strains-3D7. It showed that there are Genetic mutations. The results were statistically analyzed.Results:1. The 28 samples of DHPS gene were amplified. We get 319bp and 472bp fragments. We find mutation at locus 436,437,540 and 581. The mutant types are S436A, A437G, K540E/N and A581G. In the samples of 2008, the mutation rate of the 4 locus are 60%,100%,80%and 33.33%;in the samples of 2012, the mutation rate of the 4 locus are 30.77%,100%,61.54%and 69.23%.2. The 28 samples of DHFR gene were amplified. We get 646bp fragment. We find mutation at locus 51,59,108 and 164. The mutant types are N51I, C59R, S108N and I164L. In the samples of 2008, the mutation rate of the 4 locus are 53.33%,100%,100% and 80.33%;in the samples of 2012, the mutation rate of the 4 locus are 76.92%,100%, 100% and 61.54%.3. The 28 samples of PFMRP1 gene were amplified. We get 5469bp fragment. We find mutation at locus 191,325,437,785,876,1007 and 1390. The mutant types are H191Y, N325S, S437A, H785N, I876V, T1007M and F1390I. In the samples of 2008, the mutation rate of the 7 locus are 66.67%,6.67%,60%,13.33%,60%,26.67%and 20%;in the samples of 2012, the mutation rate of the 7 locus are 84.62%,15.38%, 69.23%,38.46%,53.85%,47.15%and 7.69%.Conclusions:There was no significant difference of mutation rate between the samples in 2008 and 2012 (P>0.05).Part 2 A study on measuring sensitivity of 4 kinds of antimalarial drugs (including pyrimethamine) in vitro, and the relevance with the DHFR geneObjective:To measure the drug sensitivity (include mefloquine, piperaquine, chloroquine, pyrimethamine and lumefantrine) of the samples which were treated by drugs respectively 3 days and still found in the affected patient’s peripheral blood smear.To analyze the difference of drug sensitivity between the year of 2008 and 2012.To analyze the status in quo of DHFR gene mutations to study the relevance between gene mutation and drug sensitivity.Methods:1. We synchronize the 15 samples in 2008 and 13 samples in 2012. Then we measured the drug sensitivity including mefloquine, piperaquine, chloroquine, pyrimethamine and lumefantrine.2. We amplified DHFR gene segment with Nest-PCR and sequenced them. Then we compared the results with standard strains-3D7 to obtain the gene mutation and the relevance between gene mutation and drug sensitivity.Results:1. IC50 to mefloquine, piperaquine, chloroquine, pyrimethamine and lumefantrine of the 15 samples in 2008 are 56.87±29.79nM,13.85±8.901nM,1265±845.2nM,3055± 2178nM and 4.669±2.147nM. And IC50to the same drugs of the 13 samples in 2012 are 58.09 ± 20.40nM,17.13±8.893nM,808.1±481.5nM,2079±943.2nM and 4.906± 2.430nM.2. We compared the IC50 to the 5 kinds of drugs in 2008 and 2012, each and every P >0.05, there is no statistically significant difference. So there is no significant difference of sensitivity to the 5 kinds of drugs of the two years.3. In the totally 28 samples, the highest rate of mutational sites of DHFR gene are 59 and 108.The mutation rates of them are both 100%. There is a statistically significant different between unmutated and mutated samples of pyrimethamine’s IC50 at the mutational site 51, P=0.0440<0.05.4. In the totally 28 samples, there are 2 samples of double mutation of DHFR gene (C59R and S108N),and their median of pyrimethamine’s IC50 are 1743nM; 6 samples of triple mutation(N51I, C59R and S108N), and their median of pyrimethamine’s IC50 are 2679nM; 8 samples of triple mutation(C59R、S108N、I164L), and their median of pyrimethamine’s IC50 are 1926nM; 12 samples of quadruple mutation(N51I, C59R, S108N and I164L) and their median of pyrimethamine’s IC50 are 3127nM. The IC50 of the quadruple mutation group is highest and double mutation group is lowest. There is no statistically significant different between each multiple mutation groups, P=0.1819>0.05.Conclusions:1. In the totally 28 samples,6 samples are sensitive to mefloquine. Its sensitivity decrease by 3.57%, meaning that mefloquine is sensitive to Plasmodium falciparum in this area.4 samples of the 28 are sensitive to piperaquine. Its sensitivity decrease by 21.43%, meaning that piperaquine is not very sensitive to Plasmodium falciparum in this area. The 28 samples are all high resistant to chloroquine. The sensitivity decrease by 100%. We can draw the conclusion that Plasmodium falciparum in this area are significantly resistant to chloroquine.2 samples of the 28 are sensitive to pyrimethamine. Its sensitivity decrease by 71.43%, meaning that Plasmodium falciparum in this area are resistant to pyrimethamine.7 samples of the 28 are sensitive to lumefantrine. Its sensitivity decrease by 42.86%, meaning that lumefantrine is not sensitive to Plasmodium falciparum in this area.2. There is no significant difference of drug sensitivity between the samples from the border area in Sino-burmese in 2008 and 2012.3. There is relevance between the 51 mutational sites of DHFR gene and the drug resistance of pyrimethamine.4. According to statistical analysis, there is no relevance between the multiple mutation and the drug resistance of pyrimethamine. The pyrimethamine’s IC50 of mutated samples is higher than the unmutated samples.Part 3 Continued administration of drugs with High concentrations screening testObjective:We practice continuous pressurized administration of mefloquine, artesunate, dihydroartemisinin and piperaquine on Plasmodium falciparum from falciparum malaria endemic areas to screen out the high resistant samples. We get the IC50 under different concentrations and we can explore the correlation of resistance and concentration. With the data, we can compare the difference of drug resistance caused by artemisinin-based drugs and long acting drugs.Methods:We apply 4 kinds of drugs (mefloquine, artesunate, dihydroartemisinin and piperaquine) for the 25 single infection samples which drugs’IC50 are 3 times than standard strain. We culture the Plasmodium falciparum samples in vitro with double times concentrations of IC50 or even higher, until the samples are all dead. Samples that live with high concentrations are screened out. And we measure their IC50.Results:1.1 AS sample (F08B32) was screened out. We study its drug resistance with the concentrations of 30nM,60nM and 90nM; 2 DHA samples (F08B41 and F08B62) were screened out. We study their drug resistance with the concentrations of 10nM,20nM and 40nM.1 MQ sample (F09N64) was screened ou. We study its resistance with the concentrations of OnM and 70nM. The sample was failed to revive with the concentration of70nM.2. The IC50 ofF08 B32 cultured with the AS concentrations of 30nM,60nM and 90nM are 22.690±2.325nm,39.170±2.461nm and 56.500±9.856nm. The IC50 of F08B41 cultured with the DHA concentration of 10nm,20nm and 40nm are 5.462± 0.666nm,8.304±0.225nm and 14.540±1.954nm. The IC50 of F08B62 cultured with the DHA concentration of 10nm,20nm and 40nm are 2.826±0.055nm,4.118±0.113nm and 8.140±0.072nm.3. We compare the sample of each concentration group. In the groups of F08B32 cultured with the AS concentrations of 30nM,60nM and 90nM, the IC50 values are statistically significant different, P=0.0014<0.05. In the groups of F08B41 cultured with the DHA concentrations of 10nm,20nm and 40nm, the IC50 values are statistically significant different, P=0.0002<0.05. In the groups of F08B62 cultured with the DHA concentrations of 10nm,20nm and 40nm, the IC50 values are statistically significant different, P<0.0001. In short, the IC50 values are increased with the increasing of drug concentration.Conclusions:The drug resistance of Plasmodium falciparum for AS and DHA is increased with the increasing of drug concentration.