海南省恶性疟原虫氯喹抗性相关基因pfcrt多态性分析

目的了解海南省恶性疟原虫产生氯喹抗性是否与pfcrt基因中的关键性点突变有关。方法对45份采自海南省恶性疟患者血样，采用套式PCR法分别扩增pfcrt基因中含有第76位和220位氨基酸的多态性片段，并对扩增产物进行限制性内切酶酶切分析，观察是否存在突变位点。结果45个样本中，pfcrt基因发生K76T点突变的样本有28个，其中20个是抗性株，8个是敏感株；全部样本的pfcrt基因第220位氨基酸均发生A220S点突变。结论我国海南株恶性疟原虫产生氯喹抗性与发生在pfcrt基因中的K76T点突变有一定的关联。     

云南恶性疟原虫氯喹抗药性基因76位点突变研究

目的研究云南恶性疟原虫氯喹抗药性基因（pfcrt）76位点突变的情况,以及与抗药性表现型的关系. 方法应用PCR和限制性酶切片段长度分析方法,检测现症病人干滤纸血样的恶性疟原虫pfcrt基因点突变. 结果 云南省恶性疟原虫pfcrt基因76位点的突变型很高,占85.0%（51/60）;野生型和混合型较少,分别占8.3%（5/60）和6.7%（4/60）.体内法测定的氯喹抗性和敏感样本均有pfcrt76突变型;体外法测定的17份氯喹抗性样本中,有13份带有pfcrt76突变型. 结论 云南省恶性疟原虫pfcrt基因氨基酸编码76位点突变频度很高.体内和体外法测定的氯喹抗性表现与pfcrt76突变型有较高的一致性.     

用pfcrt点突变基因检测技术检测恶性疟原虫氯喹抗药性的初步研究

目的　建立一种恶性疟原虫氯喹抗药性基因pfcrt点突变的检测方法,以判断是否存在氯喹抗药性。方法　根据恶性疟原虫pfcrt基因序列设计巢式PCR引物,以恶性疟原虫DNA为模板扩增出一条包含第76位密码子的DNA片段;扩增产物经限制性内切酶Apo I消化,用琼脂糖凝胶电泳观察恶性疟原虫pfcrt等位基因是否为突变型。结果　31份样本经巢式PCR扩增均出现14 0 bp左右的特异性片段。酶切消化后,9份滤纸血样本中有4例出现1条14 0 bp左右的片段,为突变型pfcrt等位基因,其余5份出现97bp与4 8bp两种酶切片段,为野生型pfcrt等位基因;2 2份血涂片样本中有10份突变型,突变率为4 5 .16 %。14例突变样本中,有1例体内实验氯喹治疗有效。结论　巢式PCR- RFL P法可以快速、高效的检测恶性疟原虫pfcrt基因76位密码子的点突变,并且能够初步应用于恶性疟原虫氯喹抗药性的鉴别。     

海南省恶性疟原虫氯喹抗性相关基因pfcrt多态性分析

目的了解海南省恶性疟原虫产生氯喹抗性是否与pfcrt基因中的关键性点突变有关。方法对45份采自海南省恶性疟患者血样,采用套式PCR法分别扩增pfcrt基因中含有第76位和220位氨基酸的多态性片段,并对扩增产物进行限制性内切酶酶切分析,观察是否存在突变位点。结果45个样本中,pfcrt基因发生K76T点突变的样本有28个,其中20个是抗性株,8个是敏感株;全部样本的pfcrt基因第220位氨基酸均发生A220S点突变。结论我国海南株恶性疟原虫产生氯喹抗性与发生在pfcrt基因中的K76T点突变有一定的关联。     

云南恶性疟原虫氯喹抗药性基因76位点突变研究

目的研究云南恶性疟原虫氯喹抗药性基因(pfcrt)76位点突变的情况,以及与抗药性表现型的关系。方法应用PCR和限制性酶切片段长度分析方法,检测现症病人干滤纸血样的恶性疟原虫pfcrt基因点突变。结果云南省恶性疟原虫pfcrt基因76位点的突变型很高,占85.0%(51/60);野生型和混合型较少,分别占8.3%(5/60)和6.7%(4/60)。体内法测定的氯喹抗性和敏感样本均有pfcrt76突变型;体外法测定的17份氯喹抗性样本中,有13份带有pf-crt76突变型。结论云南省恶性疟原虫pfcrt基因氨基酸编码76位点突变频度很高。体内和体外法测定的氯喹抗性表现与pfcrt76突变型有较高的一致性。     

恶性疟原虫氯喹抗性的研究进展

恶性疟原虫氯喹抗药性的产生是一个多基因、多因素作用的复杂过程。该文通过对恶性疟原虫氯喹抗性产生机制以及与抗性相关的pfmdr1基因、pfcrt基因和cg2基因等的研究进展进行综述。探讨氯喹抗性产生的机制，为恶性疟原虫氯喹抗性机制的研究及新药设计提供参考。     

海南恶性疟原虫pfcrt等位基因多态性的单体型研究

目的 建立恶性疟原虫pfcrt（Plasmodium falciparum chloroquine resistant transporter）等位基因多态性的单体型的研究方法，比较我国海南省与东南亚及非洲地区恶性疟原虫的pfcrt等位基因多态性的单体型的特征。 方法 来自海南省19份恶性疟滤纸血样提取DNA，通过巢式PCR反应，扩增出包含第72～76、97位密码子的基因片段，根据限制性片段长度多态性（RFLP）分析结果，各选取6份突变型和野生型PCR产物进行DNA测序，检测第72～76、97位密码子序列，从而建立并分析我国海南省恶性疟pfcrt等位基因的单体型。 结果 19份滤纸血样本提取的DNA全部扩增出1条195 bp的片段，酶切消化后，有11份出现1条100 bp左右的酶切片段，为野生型pfcrt等位基因，其余8份为1条195 bp的片段，为突变型。基因序列分析发现，野生型pfcrt等位基因的第72～76位密码子单体型为CVMNK，突变型为CVIET，第97位密码子未发现突变。 结论 我国海南省氯喹抗药性恶性疟原虫pfcrt等位基因第72～76位密码子的单体型与东南亚和非洲地区抗氯喹恶性疟原虫相应单体型一致。     

云南边境地区氯喹及与青蒿琥酯伍用治疗前后恶性疟原虫pfcrt和pfmdr1抗药性有关基因点突变的变化特征

目的 了解氯喹单用及与青蒿琥脂伍用治疗恶性疟前后，pfcrt和pfmdr1抗药性有关基因的点突变变化特征。方法 使用PCR—RFLP技术检测基因点突变。结果 氯喹及与青蒿琥脂伍用治疗前后的所有样本都发现有恶性疟原虫pfcrt基因氨基酸编码76突变为苏氨酸的特征。但是，氯喹治疗前，50％pfmdr1基因氨基酸编码86为天冬酰氨酸(野生型)，而剩余的50％为野生型和突变型(苏氨酸)的特征。氯喹治疗后，在18个复燃的病例中，83．3％的pfmdr1基因86位点为野生型，剩余的16．7％是混合型。氯喹与青蒿琥脂伍用治疗前，3个样本携带混合型基因型，剩余的(86％)为野生型，但治疗后，所有样本只携带野生型。结论 这些结果可能支持这样的假说：pfcrt基因突变起主导作用，但pfmdr1基因突变增强了氯喹抗药性的效果。     

停用氯喹12年抗氯喹恶性疟原虫对氯喹抗性...

In view of the fact the resistance of Plasmodium falciparum to chloroquine occurred extensively in Hainan, a decision was made in 1979 that the use of chloroquine should be quit in the whole province. A longitudinal survey on chloroquine-sensitivity of P. falciparum was carried out during 1981-1991 to observe the variation in resistance of the parasite after the cessation of the chloroquine medication for every 2-3 years. A tendency of progressive decline of resistance was revealed. By using in vitro test, the rate of chloroquine-resistant P. falciparum dropped from 97.9% in 1981 to 60.9% in 1991 (P < 0.001). The mean dosage of chloroquine for complete inhibition of schizont formation declined from 10.46 pmol/microliters in 1981 to 3.02 pmol/microliters in 1991 (P < 0.001). The percentage of population requiring larger dosage (6.4 pmol/microliters to completely inhibit schizont formation declined from 83.3% in 1981 to 17.4% in 1991 (P < 0.001); whereas those requiring small dosage (1.6 pmol/microliters), increased from 4.2% in 1981 to 60.8% in 1991 (P < 0.001). In in vivo test, the rate of chloroquine-resistant P. falciparum decreased from 84.2% in 1981 to 40% in 1991 (P < 0.001). The proportion of RII plus RIII cases of the total resistant cases dropped from 59.4% in 1981 to 37.5% in 1991 (0.02 > P > 0.01).     

pfcrt is more than the Plasmodium falciparum chloroquine resistance gene: a functional and evolutionary perspective

Genetic, physiological and pharmacological studies are gradually revealing the molecular basis of chloroquine resistance (CQR) in the malaria parasite, Plasmodium falciparum. Recent highlights include the discovery of a key gene associated with resistance, pfcrt (Plasmodium falciparum chloroquine resistance transporter; PfCRT), encoding a novel transporter, and the characterization of global selective sweeps of haplotypes containing a K76T amino acid change within this protein. Little is known about the cellular mechanism by which resistant parasites escape the effects of chloroquine (CQ), one of the most promising drugs ever deployed, due in part to an unresolved mechanism of action. The worldwide spread of CQR argues that investigations into these mechanisms are of little value. We propose, to the contrary, that the reconstruction of the evolutionary and molecular events underlying CQR is important at many levels, including: (i) its potential to assist in the development of rational approaches to thwart future drug resistances; (ii) the stimulation of the use of CQ-like compounds in drug combinations for new therapeutic approaches; and (iii) the consideration of how the CQ-selected genome will function as the context in which current and future drugs will act, particularly in light of the many reports of multidrug resistance. The purpose of this review is to highlight, discuss and in some cases challenge the interpretations of recent findings on CQR. We consider the natural function of the PfCRT protein, the role of multiple genes and “genetic background” in the CQR mechanism, and the evolution of CQR in parasite populations. Genetic transformation techniques are improving in P. falciparum and continue to provide important insight into CQR. Here, we also discuss more subtle, yet important pharmacological approaches that may have been overlooked in a traditional “gene for drug resistance” way of thinking.     

New haplotypes of the Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene among chloroquine-resistant parasite isolates

Mutations in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene were examined to assess their associations with chloroquine resistance in clinical samples from Armopa (Papua) and Papua New Guinea. In Papua, two of the five pfcrt haplotypes found were new: SVIET from Armopa and CVIKT from an isolate in Timika. There was also a strong association (P < 0.0001) between the pfcrt 76T allele and chloroquine resistance in 50 samples. In Papua New Guinea, mutations in the pfcrt gene were observed in 15 isolates with chloroquine minimum inhibitory concentrations (MICs) of 16-64 pmol, while the remaining six isolates, which had a wild-type pfcrt gene at codon 76, had MICs of 2-8 pmol. These observations confirm that mutations at codon 76 in the pfcrt gene are present in both in vivo and in vitro cases of chloroquine resistance, and that detection of the pfcrt 76T allele could predict potential chloroquine treatment failures.     

Distribution pattern of Plasmodium falciparum chloroquine transporter (pfcrt) gene haplotypes in Sri Lanka 1996-2006

Widespread antimalarial resistance has been a barrier to malaria elimination efforts in Sri Lanka. Analysis of genetic markers in historic parasites may uncover trends in the spread of resistance. We examined the frequency of Plasmodium falciparum chloroquine transporter (pfcrt; codons 72-76) haplotypes in Sri Lanka in 1996-1998 and 2004-2006 using a high-resolution melting assay. Among 59 samples from 1996 to 1998, we detected the SVMNT (86%), CVMNK (10%), and CVIET (2%) haplotypes, with a positive trend in SVMNT and a negative trend in CVMNK frequency (P = 0.004) over time. Among 24 samples from 2004 to 2006, we observed only the SVMNT haplotype. This finding indicates selection for the SVMNT haplotype over time and its possible fixation in the population.     

pfcrt Polymorphism and the spread of chloroquine resistance in Plasmodium falciparum populations across the Amazon Basin

The widespread occurrence of drug-resistant malaria parasites in South America presents a formidable obstacle to disease control in this region. To characterize parasite populations and the chloroquine-resistance profile of Plasmodium falciparum in the Amazon Basin, we analyzed a DNA segment of the pfcrt gene, spanning codons 72-76, and genotyped 15 microsatellite (MS) markers in 98 isolates from 6 areas of Brazil, Peru, and Colombia where malaria is endemic. The K76T mutation, which is critical for chloroquine resistance, was found in all isolates. Five pfcrt haplotypes (S[tct]MNT, S[agt]MNT, CMNT, CMET, and CIET) were observed, including 1 previously found in Asian/African isolates. MS genotyping showed relatively homogeneous genetic backgrounds among the isolates, with an average of 3.8 alleles per marker. Isolates with identical 15-loci MS haplotypes were found in different locations, suggesting relatively free gene flow across the Amazon Basin. Allopatric isolates carrying SMNT and CMNT haplotypes have similar genetic backgrounds, although parasites carrying the CIET haplotype have some exclusive MS alleles, suggesting that parasites with CIET alleles were likely to have been introduced into Brazil from Asia or Africa. This study provides the first evidence of the Asian pfcrt allele in Brazil and a detailed analysis of P. falciparum populations, with respect to pfcrt haplotypes, in the Amazon Basin.     

恶性疟原虫海南分离株pfcrt基因同氯喹抗性的相关性分析

目的探讨恶性疟原虫海南株pfcrt基因多态性同氯喹抗性的关系。方法采集确诊恶性疟患者血样42份，应用套式PCR方法体外扩增pfcrt基因含有编码第76和356位氨基酸的基因片段，并对扩增产物进行限制性酶切分析。结果1）76位点：22个氯喹抗性虫株中有18个发生K76T突变型（81．82％），20个氯喹敏感虫株中野生型和突变型各占50％；2）356位点：所有42个样本的356位点均为野生型。结论我国海南省恶性疟原虫pfort基因的K76T突变与氯喹抗性存在一定关联，而356位点可能与氯喹抗性无关。     

中缅边境恶性疟原虫Pfcrt基因76位点突变研究

目的 检测分析中缅边境恶性疟原虫氯喹抗性基因(pfcrt)及其76位点氨基酸的突变情况。方法 采用巢式PCR方法扩增含76位点的pfcrt基因,并对扩增产物进行限制性内切酶及测序分析。结果 对恶性疟现症病人血样作pfcrt基因的巢式PCR扩增,目的基因片段(145 bp)检出率为74.05%(117/158),对PCR扩增阳性的产物进行RELP分析,检查突变型酶切片段,突变率为95.73%(112/117)。 结论 恶性疟原虫pfcrt基因可以作为一个分子标记用于监测中缅边境地区恶性疟原虫的氯喹抗性。     

The T76 mutation in the pfcrt gene of Plasmodium falciparum and clinical chloroquine resistance phenotypes in Papua, Indonesia.

The T76 mutation in the pfcrt gene has been linked to chloroquine (CQ) resistance in Plasmodium falciparum. PCR-based analysis of pfcrt alleles was performed on pre-treatment samples from 107 individuals who had P. falciparum infections and lived in Papua, Indonesia. The results of a 28-day, in-vivo test revealed clinical resistance to CQ in 79 (74%) of the samples. The crude sensitivity of the pfcrt T76 assay for detecting the CQ-resistant infections in the samples was 96% and the crude specificity 52%. Discordance between pfcrt genotype and in-vivo phenotype was analysed either by genotyping of the merozoite surface protein-2 (to distinguish re-infection from recrudescence) or by amplification of the P. falciparum-specific small-subunit ribosomal RNA (ssrRNA) gene, using nested PCR (to detect any sub-patent but resistant parasites in infections misclassified as sensitive by the in-vivo test). When adjusting for the results of these analyses, the sensitivity and specificity of the pfcrt T76 assay for detecting the CQ-resistant infections became 93% and 82%, respectively. Overall, the present results indicate that the pfcrt T76 assay may be used to forecast therapeutic failure caused by CQ resistance. Validation requires exploration of the phenotype classifications based on the results of in-vivo tests, using genetic analyses that distinguish re-infection from recrudescence and detect microscopically subpatent parasitaemias.     

氯喹抗药性的分子药理学作用研究进展

氯喹是治疗和预防疟疾最有价值的药物。它的药理学作用靶点是血阶段疟原虫的食物消化泡。其作用过程普遍认为氯喹与疟原虫消化血红蛋白后所释放的血红素结合 ,形成一种复合物—氯高铁血红素 ( Hemin) ,因此阻止了疟原虫消化血红蛋白 ,形成无毒的疟色素 ( Hemozoin) [1,2 ]。通过这样的过程 ,产生一种游离基团 ,破坏疟原虫食物消化泡内的各种酶 ,如磷酸脂酶、蛋白酶和胞膜 [3 ,4 ]。然而 ,抗氯喹恶性疟原虫是如何改变了这一过程却知之甚少。随着分子生物学技术的不断发展 ,对其药理学作用的认识也不断加深。本文对氯喹抗药性的分子药理学作…