Objective:To investigate the distribution and patterns of pfcrt and pfmdr1 polymorphisms in Plasmodium falciparum(P.falciparum)isolates collected from the malaria endemic area of Thailand along Thai-Myanmar border.Met...Objective:To investigate the distribution and patterns of pfcrt and pfmdr1 polymorphisms in Plasmodium falciparum(P.falciparum)isolates collected from the malaria endemic area of Thailand along Thai-Myanmar border.Methods:Dried blood spot samples were collected from 172 falciparum malaria patients prior received treatment.The samples were extracted using chelex to obtain parasite DNA.PCR-RFLP was employed to detect pfert mutation at codons 76,220,271,326,3S6 and 371,and the pfmdr1mutation at codon 86.Pfmdr1 gene copy number was determined by SYBR Green 1 real-time PCR.Results:Mutant alleles of pfcrt and wild type allele of pfmdrl were found in almost all samples.Pfmdrl gene copy number in isolates collected from all areas ranged from 1.0 to S.0 copies and proportion of isolates carrying>1 gene copies was 38.1%.The distribution and patterns of pfcrt and pfmdrl mutations were similar in P.falciparum isolates from all areas.However,significant differences in both number of gfmdr1 copies and prevalence of isolates carrying>1 gene copies were observed among isolates collected from different areas.The median pfmdr1 copy number in P.falciparum collected from Kanchanaburi and Mae Hongson were 2.5 and 2.0,respectively and more than half of the isolates carried>1 gene copies.Conclusions:The observation of pfindr1 wild type and increasing of gene copy number may suggest declining of artesunate-mefloquine treatment efficacy in P.falciparum isolates in this border area.展开更多
Objective:To identify prevalence of chloroquine resistance point mutation at(Pfcrt,K76T)and(Pfindr1.N86Y) copy number variation.Methods:SYBR Green I based real time PCR was used.One hundred and thirty-three samples we...Objective:To identify prevalence of chloroquine resistance point mutation at(Pfcrt,K76T)and(Pfindr1.N86Y) copy number variation.Methods:SYBR Green I based real time PCR was used.One hundred and thirty-three samples were analyzed for(Pfcrt,K76T) and(Pfmdr1.N86Y) copy number from dried blood spot.Parasite DNA was extracted using high pure DNA preparation kit.The amplification of DNA was done by using AccuPower 2* GreenStar '' qPCR Master mix.For quantification purpose a new primer pair was designed for 178 base pair template from complete genome sequence of Plasmodium falciparum strain 3D7 at NCBI.Absolute quantification method was used to determine the Pfmdr1-N86 Y copy number variations.Standard curve was built from strain3D7 gDNA since it has single copy of Pfindr1 per haploid genome.The known positive controls with single and multi-copy number of Pfindr1 gene were included in each experiment.The copy number ratio of the samples to the standard calibrator was made to obtain the fold difference among the samples with respect to copy number variation.Results:Out of 133 samples 73(54.89%) were confirmed as mutant(Pfcrt,76T) and the remaining 60(45.11%) were genotyped as wild type(Pfcrt,K76).The(Pfindr1.N86Y) copy number variation was determined for 133 clinical samples.Out of these samples 61(45.86%)had single copy and the remaining 72(54.14%) had multi-copy numbers higher than 1.5 copies per genome.Thirty-four(25.56%) multi-copies were between 1.5 and 2.5 copies per genome while 38(28.57%) were more than 2.5 copies per genome.The minimum and maximum copies per genome were 0.474 and 4.741.respectively.Conclusions:The study showed high prevalence level and fixation of Pfcrt.76 T mutation after chloroquine withdrawal.The prevalence of Pfindr1 copy number variant suggested that the presence of modulating factor for emergence of Plasmodium falciparum strains with higher copy numbers.However,the prevalence level was not statistically significant.展开更多
基金Supported by the National Research University Project(NRU)of Thailand(Grant No.10/2555)Thammasat University(Grant for student No.33/2555)
文摘Objective:To investigate the distribution and patterns of pfcrt and pfmdr1 polymorphisms in Plasmodium falciparum(P.falciparum)isolates collected from the malaria endemic area of Thailand along Thai-Myanmar border.Methods:Dried blood spot samples were collected from 172 falciparum malaria patients prior received treatment.The samples were extracted using chelex to obtain parasite DNA.PCR-RFLP was employed to detect pfert mutation at codons 76,220,271,326,3S6 and 371,and the pfmdr1mutation at codon 86.Pfmdr1 gene copy number was determined by SYBR Green 1 real-time PCR.Results:Mutant alleles of pfcrt and wild type allele of pfmdrl were found in almost all samples.Pfmdrl gene copy number in isolates collected from all areas ranged from 1.0 to S.0 copies and proportion of isolates carrying>1 gene copies was 38.1%.The distribution and patterns of pfcrt and pfmdrl mutations were similar in P.falciparum isolates from all areas.However,significant differences in both number of gfmdr1 copies and prevalence of isolates carrying>1 gene copies were observed among isolates collected from different areas.The median pfmdr1 copy number in P.falciparum collected from Kanchanaburi and Mae Hongson were 2.5 and 2.0,respectively and more than half of the isolates carried>1 gene copies.Conclusions:The observation of pfindr1 wild type and increasing of gene copy number may suggest declining of artesunate-mefloquine treatment efficacy in P.falciparum isolates in this border area.
基金Supported by the Pan African University Institute of Basic Science Innovation and Technology(Grant No.132)
文摘Objective:To identify prevalence of chloroquine resistance point mutation at(Pfcrt,K76T)and(Pfindr1.N86Y) copy number variation.Methods:SYBR Green I based real time PCR was used.One hundred and thirty-three samples were analyzed for(Pfcrt,K76T) and(Pfmdr1.N86Y) copy number from dried blood spot.Parasite DNA was extracted using high pure DNA preparation kit.The amplification of DNA was done by using AccuPower 2* GreenStar '' qPCR Master mix.For quantification purpose a new primer pair was designed for 178 base pair template from complete genome sequence of Plasmodium falciparum strain 3D7 at NCBI.Absolute quantification method was used to determine the Pfmdr1-N86 Y copy number variations.Standard curve was built from strain3D7 gDNA since it has single copy of Pfindr1 per haploid genome.The known positive controls with single and multi-copy number of Pfindr1 gene were included in each experiment.The copy number ratio of the samples to the standard calibrator was made to obtain the fold difference among the samples with respect to copy number variation.Results:Out of 133 samples 73(54.89%) were confirmed as mutant(Pfcrt,76T) and the remaining 60(45.11%) were genotyped as wild type(Pfcrt,K76).The(Pfindr1.N86Y) copy number variation was determined for 133 clinical samples.Out of these samples 61(45.86%)had single copy and the remaining 72(54.14%) had multi-copy numbers higher than 1.5 copies per genome.Thirty-four(25.56%) multi-copies were between 1.5 and 2.5 copies per genome while 38(28.57%) were more than 2.5 copies per genome.The minimum and maximum copies per genome were 0.474 and 4.741.respectively.Conclusions:The study showed high prevalence level and fixation of Pfcrt.76 T mutation after chloroquine withdrawal.The prevalence of Pfindr1 copy number variant suggested that the presence of modulating factor for emergence of Plasmodium falciparum strains with higher copy numbers.However,the prevalence level was not statistically significant.
