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).     

海南省乐东县恶性疟原虫对氯喹抗性的变化

目的：监测海南省停用氯喹后抗氯喹恶性疟原虫对氯喹抗性的消长。方法：选择恶性疟原虫对氯喹有高度抗性且恶性疟发病率较高的海南省乐东县为观察点,采用ＷＨＯ标准体外微量法和体内四周法,间隔一定时间检测一次。结果：停用氯喹１８年,体外法,抗性率由１９８１年的９７．９％下降至１９９７年的２６．７％（Ｐ＜０．００１）,完全抑制裂殖体形成的平均药浓度由１０．４６±７．１４ｐｍｏｌ／μｌ血降至１．６３±１．４７ｐｍｏｌ／μｌ血（Ｐ＜０．００１）;体内法,抗性率由１９８１年的８４．２％降为１９９７年的１８．４％（Ｐ＜０．００１）,ＲⅢ占抗性病例的比例由５３．１％降为１４．３％。结论：海南省停用氯喹后恶性疟原虫对氯喹逐渐恢复了敏感性     

云南省恶性疟原虫对氯喹抗性的变化

目的了解云南恶性疟原虫对氯喹抗性的变化. 方法从1981年起,在滇西、滇南、滇东南监测点选择符合条件的恶性疟病人,取静脉血,采用WHO推荐的体外微量法测定恶性疟原虫对氯喹的敏感性,并将相隔8～10年的测定结果进行比较. 结果滇西恶性疟原虫对氯喹抗性率从1982年的100%下降到2003年的83.3%,ID50 从240.0 nmol/L降到123.0 nmol/L,MIC从1 171.6 nmol/L降至325.9 nmol/L,分别下降13.0%、48.8%和72.2%.滇南恶性疟原虫对氯喹抗性率从1981年的97.4%降到2004年的69.0%,ID50从170.0 nmol/L降至58.6 nmol/L,MIC从1 108.0 nmol/L降至312.6 nmol/L,分别下降29.1%、65.5%和71.8%;滇东南抗性率从1993年的78.9%下降至2003年的53.3%,ID50从136.0 nmol/L降至75.5 nmol/L,MIC从452.0 nmol/L 降至238.3 nmol/L,分别下降32.4%、44.5%和47.3%. 结论云南恶性疟原虫对氯喹仍具有抗性,但抗性程度明显下降;中越边境恶性疟的抗性程度低于中老、中缅边境地区.目前仍不宜用氯喹治疗当地恶性疟.     

红河州蔓耗地区恶性疟原虫对氯喹的敏感性试验

为进一步评估红河流域恶性疟原虫对氯喹的抗性程度,１９９５～１９９６年在蔓耗地区应用ＷＨＯ体内测试法,观察恶性疟现症病人３２例,结果抗性率为９０．６３％,Ｓ３例ＲⅠ１例,ＲⅡ１５例、ＲⅢ７例,表明当地恶性疟原虫对氯喹的抗性程度恶性。提示在尽快改善氯喹恶性疟患者治疗的同时,亟需采取包括控制媒介在内的综合性防治对策,才会使恶性疟得到有效控制。     

云南省恶性疟原虫对氯喹抗性的变化

目的了解云南恶性疟原虫对氯喹抗性的变化。方法从1981年起,在滇西、滇南、滇东南监测点选择符合条件的恶性疟病人,取静脉血,采用WHO推荐的体外微量法测定恶性疟原虫对氯喹的敏感性,并将相隔8~10年的测定结果进行比较。结果滇西恶性疟原虫对氯喹抗性率从1982年的100%下降到2003年的83.3%,ID50从240.0nmol/L降到123.0nmol/L,MIC从1171.6nmol/L降至325.9nmol/L,分别下降13.0%、48.8%和72.2%。滇南恶性疟原虫对氯喹抗性率从1981年的97.4%降到2004年的69.0%,ID50从170.0nmol/L降至58.6nmol/L,MIC从1108.0nmol/L降至312.6nmol/L,分别下降29.1%、65.5%和71.8%;滇东南抗性率从1993年的78.9%下降至2003年的53.3%,ID50从136.0nmol/L降至75.5nmol/L,MIC从452.0nmol/L降至238.3nmol/L,分别下降32.4%、44.5%和47.3%。结论云南恶性疟原虫对氯喹仍具有抗性,但抗性程度明显下降;中越边境恶性疟的抗性程度低于中老、中缅边境地区。目前仍不宜用氯喹治疗当地恶性疟。     

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。     

恶性疟原虫对氯喹抗性及其逆转剂的研究进展

恶性疟原虫对氯喹抗性的出现和广泛传播迫使人类调整治疗疟疾的用药策略并寻找更加有效的新型抗疟药。然而,在一些贫困的疟疾流行区,氯喹仍被用于治疗恶性疟。了解氯喹抗性机制、探索逆转其抗性的方法,将使氯喹这一价廉高效的抗疟药继续发挥作用。抗性逆转剂的研究和发展为上述目标提供了线索,当与氯喹合用时它能够部分恢复氯喹对氯喹抗性株的作用。为此,本文对恶性疟原虫氯喹抗性机制及其逆转剂的研究进展作一综述。     

Chloroquine resistance of Plasmodium falciparum malaria in Ethiopia and Eritrea

We report on the first 2 years of operation of a new strategy for treatment for P. falciparum malaria patients who were not cured by a standard course of chloroquine. Any such patient who returned to a malaria treatment and detection post within 2 weeks was treated daily under supervision with chloroquine. Patients whose parasitaemia had not decreased below 25% of the initial level by day 3 or cleared completely by day 7 were given sulphadoxine/pyrimethamine (Fansidar). Of 39 824 patients treated initially with chloroquine, 4% returned to the malaria post within 2 weeks of treatment; 87% of these were chloroquine resistant and treated with Fansidar and 28% of the returning patients were RIII resistant. Resistance was associated with geographical area, initial parasite density and age. Earlier studies had shown resistance to be confined to border areas, but we found that it was highest in the centre of the region, notably in the lowlands of the Shewa and Arsi provinces, and lowest in the west. Although imported cases have been held responsible for the development of resistance in border areas, other factors are likely to be important in the middle of the region. The implications of these findings for a treatment policy of P. falciparum malaria in the region are discussed.     

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。     

多重PCR技术检测恶性疟原虫抗药性相关分子标志的方法研究

目的 建立恶性疟原虫5个主要抗药性相关基因的单管多重PCR扩增方法，用于恶性疟原虫抗药性分子标志检测。 方法 依据各基因参考序列，运用Primer Premier 5.0和Oligo 6.0软件，设计5对特异性引物，采用Hot Start Taq DNA聚合酶，设置递增延伸温度，对恶性疟原虫标准株（3D7、Dd2和HB3）、分离株（FCC1/HN、 CMH/YN）、现场标本（来源于海南、 云南和缅甸）、 近缘虫种对照（间日疟原虫、 伯氏疟原虫、 食蟹猴疟原虫、 杜氏利什曼原虫和牛源隐孢子虫）和空白对照（以H2O为模板）进行5个抗药性相关基因（包括恶性疟原虫氯喹抗性转运蛋白基因Pfcrt、多药抗性基因Pfmdr1、二氢喋酸合成酶基因Pfdhps、二氢叶酸还原酶基因Pfdhfr和三磷酸腺苷酶第6亚基基因PfATPase6）的单管多重PCR扩增，2%琼脂糖凝胶电泳鉴定扩增结果，测定扩增产物序列，并与参考序列（3D7株）比对。 结果 经电泳，恶性疟原虫标准株、分离株和现场标本的多重PCR扩增产物均可见5条目标条带。测序结果与参考序列比对，高度同源，最低同源性为98.5%。模板DNA量达0.1 ng即满足扩增要求，近缘虫种对照和空白对照未见扩增产物。 结论 多重PCR技术实现了单管1次反应完成5个抗药性相关基因的扩增，该方法灵敏，特异性好，有助于提高恶性疟原虫抗药性分子标志的检测效率。     

Molecular analysis of chloroquine resistance in Plasmodium falciparum in Yunnan Province, China

Resistance of Plasmodium falciparum to chloroquine (CQ) is determined by the mutation at K76T of the P. falciparum chloroquine resistance transporter (pfcrt) gene and modified by other mutations in this gene and in the P. falciparum multidrug resistance 1 (pfmdr1) gene. To determine the extent of polymorphisms in these genes in field P. falciparum isolates from Yunnan province of China, we genotyped the pfcrt codon 76, pfmdr1 codons 86 and 1246. Our results showed that although CQ has been withdrawn from treating falciparum malaria for over two decades, 90.3% of the parasites still carried the pfcrt K76T mutation. In contrast, mutations at pfmdr1 codons 86 and 1246 were rare. Sequencing analysis of the pfcrt gene in 34 parasite field isolates revealed CVIET at positions 72-76 as the major type, consistent with the theory of Southeast Asian origin of CQ resistance in the parasite. In addition, two novel pfcrt haplotypes (75D/144Y/220A and 75E/144Y/220A) were identified. Real-time polymerase chain reaction was used to determine pfmdr1 gene amplification, which is associated with mefloquine resistance. Our result indicated that in agreement with that mefloquine has not been used in this area, most (>90%) of the parasites had one pfmdr1 copy. Genotyping at two hypervariable loci showed relatively low levels of genetic diversity of the parasite population. Meanwhile, 28.4% of cases were found to contain mixed clones, which favour genetic recombination. Furthermore, despite a unique history of antimalarial drugs in Yunnan, its geographical connections with three malarious countries facilitate gene flow among parasite populations and evolution of novel drug-resistant genotypes. Therefore, continuous surveillance of drug resistance in this area is necessary for timely adjustment of local drug policies and more effective malaria control.     

恶性疟原虫药物抗性相关分子标记研究进展

药物治疗是疟疾防治的重要手段,但是却面临着抗疟药抗性的威胁。近年来,随着分子生物学的发展,越来越多的分子标记用于疟原虫抗药性监测。本文就恶性疟原虫常见抗药性相关分子标记的研究进展作一综述。     

2012年和2018年我国输入性恶性疟原虫氯喹抗性基因多态性分析

目的对2012年和2018年我国输入性恶性疟原虫样本氯喹抗性分子标记位点基因多态性进行检测,分析恶性疟原虫氯喹抗性转运蛋白基因(Plasmodium falciparum chloroquine re sistant transporter,Pfcrt)第72~76位密码子抗性相关位点突变类型,并分析不同输入来源样本的特异性。方法收集2012年和2018年国家疟疾诊断参比实验室674例输入性恶性疟病例滤纸血样本,以样本中恶性疟原虫7号染色体上Pfcrt基因第72~76位点为扩增片段,采用巢式PCR法进行扩增并测序,对目的产物片段测序结果、地理分布等特征进行统计分析。结果2012年和2018年我国674例输入性恶性疟病例中,95.5%(644/674)来自非洲,其余4.5%(30/674)来自东南亚和大洋洲(巴布亚新几内亚);非洲又以西非和中非为主(占非洲样本的80.4%,518/644)。共检测到C72S、M74I、N75E、K76T 4个位点突变和5种单体型类型(CVMNK、CVIET、SVMNT和两种混合型),其中CVMNK与CVIET为非洲和东南亚地区恶性疟原虫共有的单体型类型,SVMNT仅在东南亚(缅甸)和巴布亚新几内亚输入样本中检测出;2种混合型为CVMNK/CVIET和CVMNK/SVMNT,前者在非洲和东南亚输入样本中分布,后者仅在东南亚缅甸来源样本中检出。自非洲输入的样本野生型较多,占77.7%(478/615);而自东南亚和巴布亚新几内亚输入的样本中,抗性分子标记样本占68.0%(17/25),两者差异有统计学意义(χ^2=28.5,P<0.05)。非洲不同地区来源样本中,抗性基因比例和野生型构成差异有统计学意义(P<0.01),西非野生型所占比例最低。结论2012年和2018年我国674例输入性恶性疟病例样本中,自东南亚输入的恶性疟原虫Pfcrt基因第72~76位点抗性基因比例和分子多态性均较非洲来源样本高。     

Molecular assessment of Plasmodium falciparum resistance to antimalarial drugs in China

Objective  In China, Chloroquine (CQ) and sulfadoxine–pyrimethamine (SP) were abandoned for the treatment of falciparum malaria 20 years ago due to resistance. Subsequent field studies showed a trend of declining CQ and SP resistance in the country. The main purpose of this study was to analyse the molecular markers of antimalarial resistance and thereby to assess the possibility of reintroduction of CQ or SP for falciparum malaria treatment.
Methods  Plasmodium falciparum field isolates were collected in 2006–2007 from Hainan and Yunnan provinces, China. Nested PCR-sequencing assays were applied to analyse the SNPs in four genes: P. falciparum chloroquine resistance transporter ( pfcrt ) gene, multi-drug resistance 1 ( pfmdr1 ) gene, dihydrofolate reductase ( dhfr ) gene and dihydropteroate synthetase ( dhps ) gene.
Results  We found the widespread presence of point mutations in the dhfr and dhps genes which are associated with SP treatment failure. The molecular analyses also showed the fairly high prevalence of point mutation in the pfcrt gene which is linked to CQ resistance.
Conclusion  The results of the present study indicate that CQ and SP should not be reintroduced for falciparum malaria treatment in the near future in China.     

缅甸拉咱市氯喹治疗间日疟的敏感性评价

缅甸拉咱市单纯间日疟原虫感染者48例分为氯喹A方案组（26例）和B方案组（22例）,分别采用成人总剂量1 200 mg （第1天顿服600 mg,第2、3天300 mg/d）和1 500 mg（第1天顿服750 mg,第2、3天375 mg/d）的三天疗法治疗。于服药当天、服药后第1、2、3、7、14、21和28天采集指血或耳垂血制作厚、薄血涂片检查疟原虫,测量体温和观察药物不良反应。氯喹治疗后,两组病例血内无性体原虫3 d内全部阴转。第28天随访治愈率为100%。结果表明,缅甸拉咱市的间日疟病例对氯喹治疗均敏感,该边境地区间日疟病例可采用氯喹进行临床治疗。     

中国中部疟疾流行区间日疟原虫对氯喹敏感性的体外检测

目的检测我国间日疟原虫对氯喹的敏感性。方法采用新建立的间日疟原虫药物敏感性体外微量测定技术,对中国中部间日疟流行区门诊确诊的间日疟病例进行药物敏感性测定。结果在完成药物测定的24例间日疟病例中,间日疟原虫对氯喹的IC50几何均数为63.23nmol/L,95%可信区间为26.23~152.40nmol/L,其中至少有6例显示对氯喹产生了抗性。结论在中国中部间日疟流行地区可能已出现间日疟原虫抗氯喹株。