合肥市输入性恶性疟原虫Pfcrt基因K76T位点突变研究

目的探讨合肥市输入性恶性疟原虫Pfcrt基因K76T位点的突变情况。方法采集2010年从非洲疟疾高流行区,务工回国输入性恶性疟病人血样11份,采用恶性疟原虫Pfcrt基因序列特异引物,以滤纸血片中的恶性疟原虫DNA为模板,进行巢氏PCR扩增基因,扩增产物经限制性内切酶酶切后鉴定。结果收集到11例滤纸血样,成功扩增6例,其中2例被酶切,为Pfcrt等位基因野生型;4例未被酶切,为Pfcrt等位基因突变型。结论合肥市输入性恶性疟中Pfcrt等位基因发生突变,应该加强输入性恶性疟的药物抗性监测。     

氯喹抗性相关基因pfmdrl两个突变点的初步研究

目的：对氯喹抗性相关基因pfmdrl两突变点进行研究。方法：用新颖的等位基因特异PCR和限制性酶切分析技术（AS－PCR／RFLP）检测云南恶性疟的与氯喹抗性相关的pfmdrl基因^Asn86^Tyr和^Asp1246^Try的突变点,了解其与氯喹抗性表现型的相关性。结果：在10个氯喹抗性分离株未检测pfmdrl基因86位氨基酸编码子的突变（^Asn86型）;而在9个氯喹抗性分离株中检测pfmdrl 基因86位氨基酸编码子由A变为T,即86^Try型,未检测到pfmdrl基因^Asn1246^Tyr的突变。结论,提示在云南恶性疟氯喹抗性流地区,氯喹抗性表现型与pfmdrl基因86^Tyr的位点突变符合性仅47．4％,似无明显关系;而基因^Asn 1246^Tyr的位点突变似乎是不存在, 这支持除^Asn86^Tyr突变外,其它基因可能参与了氯喹抗性。     

贵州省恶性疟原虫对氯喹敏感性的调查

用WHO推荐的体外微量法和体内法,测定望谟、荔波和三都等县恶性疟原虫对氯喹敏感性的结果表明,用体外微量法测试三县均对氯喹产生抗性;用体内法测试对氯喹均敏感,但周围血无性体消失时间较过去延长。     

ABCG8 T400K基因多态性与高脂血症的关系

目的探讨ABCG8 T400K基因多态性与高血脂症的相关性。方法采用酚-氯仿法提取370例高脂血症患者及303例健康志愿者DNA,用聚合酶链反应-限制性片断长度多态性(PCR-RFLP)技术检测ABCG8 T400K基因多态性,用χ2检验检测基因多态性与高脂血症的关系。结果ABCG8 T400K基因多态性与高脂血症患病风险间显著相关,ABCG8 400K等位基因为患高脂血症的高风险因素(OR=1.870,P=0.002)。结论ABCG8 T400K基因多态性与高脂血症患病风险间显著相关,ABCG8 400K等位基因携带者患高脂血症的风险较高。     

赤道几内亚Bioko岛的恶性疟原虫多药抗性基因(pfMDR-1)的研究

目的对赤道几内亚Bioko岛上恶性疟原虫多药抗性基因(pfMDR-1)进行分析,为Bioko岛的疟疾防控和治疗提供依据。方法2012年雨季期间采集的恶性疟原虫感染患者样本151份,用巢式PCR技术特异性扩增N86Y、E130K、Y184F、S1034C、N1042D、V1109I、D1246Y耐药分子标记的pfMDR-1基因片段,然后进行测序分析。结果虫株中共发现了4种不同的单倍型:YEY/SNVD、NEF/SNVD、YEF/SNVD和NEY/SNVD。91.39%(138/151)的样本发现了耐药性位点突变,包括3.31%(5/151)的86Y,29.80%的184F,和58.29%(88/151)的双重突变86Y/184F。结论结果表明赤道几内亚Bioko岛上的恶性疟原虫株存在较高比例耐药基因突变和耐药复合基因突变,为当地的抗疟疾选用药物提供指导。     

杭州市淡色库蚊对杀虫剂的抗性及抗性基因研究

目的了解杭州市淡色库蚊对杀虫剂的抗性及抗性基因,为蚊虫防制提供依据。方法浸液法和PCR技术。结果杭州市淡色库蚊对溴氰菊酯的抗性倍数为9．10倍,敌敌畏为4．52倍,三氯杀虫酯为2．77倍,残杀威为2．04倍,高效氯氰菊酯、丙烯菊酯未形成抗性;经PCR扩增,检测到11个与抗性相关的基因,其中P450类1个,钠离子通道基因2个,酯酶基因2个,乙酰胆碱酯酶受体基因1个,超氧化物歧化酶基因1个,其他相关基因4个。结论杭州市淡色库蚊对溴氰菊酯和敌敌畏产生了抗性;PCR检测,存在11个抗性相关基因。     

自然抗性相关巨噬细胞蛋白1基因多态性与肺结核易感性的研究

目的　探讨自然抗性相关巨噬细胞蛋白 1基因 (NRAMP1)多态性与中国汉族人群肺结核发病的关系。方法　采用以医院为基础的病例对照研究设计 ,用聚合酶链反应 限制性片段长度多态性分析 (PCR RFLP)法检测NRAMP1基因中INT4、D5 4 3N及 3′UTR 3个多态性位点的基因型 ,并对结核病相关危险因素进行问卷调查 ,应用SPSS软件进行单因素和多因素非条件logistic回归分析。2 0 0 1年 4月至 2 0 0 2年 6月选择 110例肺结核病例 ,平均年龄为 (2 8± 13)岁 ;对照组为 180名健康体检者 ,平均年龄为 (2 7± 9)岁。对NRAMP1基因各多态性位点进行单因素分析。结果　病例组D5 4 3NG/A及 3′UTRTGTG +/del基因型频率显著高于对照组 ,OR值 (95 %CI)分别为 2 2 2 (1 0 3～ 4 78)和 1 93(1 14～ 3 2 6 )。病例组和对照INT4各基因型频率比较差异无显著性。多因素分析调整暴露史和疫苗接种史 2个因素后 ,D5 4 3NG/A及 3′UTRTGTG +/del基因型仍与结核病显著相关 ,调整OR值 (95 %CI)分别为 3 0 4 (1 12～ 8 2 7)和 2 36 (1 2 0～ 4 6 4 ) ,而病例和对照组INT4位点多态性比较差异未见显著性。结论　NRAMP1基因D5 4 3N及 3′UTR位点多态性可能是汉族人群肺结核的易感因素。     

基于白纹伊蚊转录组的溴氰菊酯抗性相关基因分析及验证

目的 通过对白纹伊蚊溴氰菊酯抗性品系(DR)和敏感品系(DS)进行转录组分析,筛选可能与抗性相关的差异表达基因。方法 提取白纹伊蚊实验室敏感品系(DS品系)、溴氰菊酯抗性品系(DR品系)的总RNA进行高通量测序,对差异基因进行COG和KEGG的功能注释与分析,筛选出可能与抗性相关的差异表达基因,并进行实时定量PCR验证。结果 DR品系的LC₅₀为2 087.75μg/L,相较于DS品系,抗性倍数为109.8倍,达到了高度抗性标准。RNA-seq测序后基于FPKM值进行差异性分析结果显示,差异表达的基因共有551个,其中在DR品系中上调表达基因有278个,下调表达基因有273个。COG和KEGG功能富集结果显示,显著性富集的通路和蛋白质功能预测均主要与物质代谢相关。基于此进一步分析了代谢抗性相关基因,且10个目标基因的RNA-seq结果与实时定量PCR结果在DR和DS品系中表达趋势具有一致性。结论 白纹伊蚊可能通过上调表达细胞色素P450基因、谷胱甘肽S转移酶基因、羧酸酯酶、表皮蛋白等基因应对杀虫剂的选择压力。     

恶性疟原虫抗氯喹株分离和培养建立虫库的实验研究

１９８３～１９９２年我们在位于北纬２１°～２４°．东径９８°～１０２°间的云南及相邻的缅甸等地的恶性疟病人中成功分离和培养恶性疟原虫株１２株。体外抗药性测定，原再被抑制的氯喹浓度为８～６４ｐｍｏｌ。１９９２年１１月～１９９３年３月复测．结果有５株原虫被抑制，氯喹浓度有自然降低现象，仍属抗氯喹恶性疟原虫株．为国内首次建立了抗氯喹恶性疟虫库．     

饮用水中抗生素抗性基因与重金属关系的研究进展

目的对饮用水中抗生素抗性基因(antibiotic resistance genes,ARGs)与重金属的关系进行综述。方法通过"ARGs"、"金属"、"饮用水"等关键词或主题,在Web of Science、PubMed、CNKI、万方数据库检索文献,将重复文献与非相关研究排除后,最终有11篇文献被纳入,且均为英文文献,其中近5年文献占81.8%。结果 ARGs与重金属之间存在相关性,且相关性与重金属的类型和浓度以及int I1有关。抗生素和重金属联合作用比二者单独作用对ARGs的作用更强。外排泵、共抗性、氧化应激反应等是重金属对ARGs产生影响的机制。结论饮用水中即使存在低浓度的重金属,也会增加ARGs的浓度及转移,从而增加饮用水中细菌耐药性的风险。     

Minority-variant pfcrt K76T mutations and chloroquine resistance, Malawi

Genotyping of the chloroquine-resistance biomarker pfcrt (Plasmodium falciparum chloroquine resistance transporter gene) suggests that, in the absence of chloroquine pressure, Plasmodium falciparum parasites in Malawi have reverted to chloroquine sensitivity. However, malaria infections in Africa are commonly polyclonal, and standard PCRs cannot detect minority genotypes if present in <20% of the parasites in an individual host. We have developed a multiple site-specific heteroduplex tracking assay (MSS-HTA) that can detect pfcrt 76T mutant parasites consisting of as little as 1% of the parasite population. In clinical samples, no pfcrt 76T was detected in 87 pregnant Malawian women by standard PCR. However, 22 (25%) contained minority-variant resistant genotypes detected by the MSS-HTA. These results were confirmed by subcloning and sequencing. This finding suggests that the chloroquine-resistant genotype remains common in Malawians and that PCR-undetectable drug-resistant genotypes may be present in disease-endemic populations. Surveillance for minority-variant drug resistant mutations may be useful in making antimalarial drug policy.     

Amodiaquine resistant Plasmodium falciparum malaria in vivo is associated with selection of pfcrt 76T and pfmdr1 86Y.

The choice of partner drug is critical for artemisinine-based combination therapy (ACT) to remain effective and amodiaquine (AQ) is one important candidate to evaluate. We treated 81 children <5 years with uncomplicated Plasmodium falciparum malaria with AQ alone and related the treatment outcome to the possible selection of pfcrt 76T, 152T, 163S, 326S, pfmdr1 86Y and pfmrp 191H, 437S in recurrent infections (recrudescenses and re-infections) and to the blood concentration of desethylamodiaquine (DEAQ). During 21 days follow-up 28 children had a recurrent infection (9 recrudescenses, 13 re-infections and 6 mixed). Neither genotyping of the polymorphisms before treatment nor DEAQ blood concentrations could predict treatment outcome. pfcrt 76T was however significantly selected for in recurrent infections (p=0.020). pfmdr1 86Y was also selected for, but only in recrudescent infections (p=0.048). The study showed high prevalence of AQ resistant parasites in vivo, which appeared to be associated to pfcrt 76T and pfmdr1 86Y.     

First evidence of pfcrt mutant Plasmodium falciparum in Madagascar

The island of Madagascar, lying in the Indian Ocean approximately 250 miles from the African coast, has so far remained one of the few areas in the world without noticeable Plasmodium falciparum high-grade chloroquine (CQ) resistance. Here we report genotyping data on pfcrt in Madagascar. The pfcrt K76T mutation, which is critical for resistance to CQ, was detected in six (3.3%) of 183 P. falciparum isolates screened, within the mutant haplotypes CVIET and CVIDT. This is the first observation of pfcrt mutant parasites on the island. The current massive distribution of CQ for in-home management of fever in children will promote the dissemination of these mutant CQ-resistant parasites. In this context, genotyping of pfcrt remains a useful tool for CQ resistance surveillance as the prevalence of pfcrt mutations is far from saturation in Madagascar.     

Novel Mutations in K13 Propeller Gene of Artemisinin-Resistant Plasmodium falciparum

We looked for mutations in the Plasmodium falciparum K13 propeller gene of an artemisinin-resistant parasite on islands in Lake Victoria, Kenya, where transmission in 2012–2013 was high. The 4 new types of nonsynonymous, and 5 of synonymous, mutations we detected among 539 samples analyzed provide clues to understanding artemisinin-resistant parasites.     

Correlation Between MTHFR Polymorphisms and Hepatocellular Carcinoma: A Meta-analysis

The correlation between methylenetetrahydrofolate reductase (MTHFR) polymorphisms and hepatocellular carcinoma (HCC) remains controversial. Therefore, we performed this study to better assess the relationship between MTHFR polymorphisms and the likelihood of HCC. A systematic research of PubMed, Medline, and Embase was performed to retrieve relevant articles. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. A total of 15 studies with 8,378 participants were analyzed. In overall analyses, a significant association with the likelihood of HCC was detected for the rs1801131 polymorphism with fixed-effect models in recessive comparison (P?=?0.002, OR = 0.62, 95% CI 0.43–0.82). However, no positive results were detected for the rs1801133 polymorphism in any comparison. Further subgroup analyses revealed that the rs1801131 polymorphism was significantly associated with the likelihood of HCC in Asians with both FEMs (recessive model: P?<?0.0001, OR = 0.42, 95% CI 0.29–0.62; allele model: P?=?0.004, OR =1.20, 95% CI 1.06–1.35) and random-effect models (recessive model: P?=?0.002, OR = 0.47, 95% CI 0.29–0.75). Nevertheless, we failed to detect any significant correlation between the rs1801133 polymorphism and HCC. In conclusion, our findings indicated that the rs1801131 polymorphism may serve as a genetic biomarker of HCC in Asians.     

对氧磷酶1基因Gln192Arg多态性与阿尔茨海默病相关的Meta分析

目的评价对氧磷酶1基因(PON1)Gln192Arg多态性与阿尔茨海默病(AD)的相关性。方法检索PubMed、EBSCO、中国学术期刊全文数据库(CNKI)、中国生物医学文献数据库(CBM)、万方数据库,收集PON1基因Gln192Arg多态性与AD相关性的文献。检索时间为建库至2011年9月。用Meta分析的方法检测基因型QR vsQQ、RR vs.QQ、QR+RR vs.QQ、和R vs Q在AD组与对照组中是否有差异。结果共纳入6篇文献,共有AD患者1270例,健康对照1335例。对总体人群进行Meta分析,未发现等位基因R与AD相关[OR=1.09,95%CI(0.84,1.42),P=0.51;]基因型QR vs QQ、RR vs.QQ、QR+RR vs.QQ在AD组与对照组中均无显著差异。结论本研究未鉴定出PON1基因Gln192Arg位点等位基因和基因型与AD相关。     

Genetic backgrounds of the Plasmodium falciparum chloroquine resistant transporter (pfcrt) alleles in Pakistan

Chloroquine (CQ) resistance in Plasmodium falciparum has been associated with point mutations in the P. falciparum CQ resistance transporter gene (pfcrt). Previous studies have shown 4-5 independent origins for CQ resistant pfcrt alleles globally, two in South America, one each in Southeast Asia, Papua New Guinea (PNG) and Philippines. In Asia, at least two different alleles corresponding to amino acids 72-76 (CVIET and SVMNT) have been found. The CVIET allele originated in Southeast Asia and then spread to Asia and Africa as well. The SVMNT allele, originating from PNG, has been found in India. This study was undertaken to investigate the genetic background of the CQ resistant pfcrt haplotypes in Pakistan. We genotyped microsatellite markers surrounding the pfcrt gene (six different markers at -12.3, -4.8, -1, 1.5, 3.9, 18.8 kb) in 114 clinical isolates of P. falciparum collected from different regions in Pakistan. Microsatellite analysis showed a significant reduction in genetic variation among the mutant SVMNT pfcrt alleles when compared to wild type alleles. The predominant SVMNT haplotype found in this study shared the same microsatellite haplotype found in both PNG and India. Two isolates with CVIET haplotypes showed similar microsatellite background to those found in Africa and Asia. In conclusion, this study suggests that CQ resistant SVMNT haplotypes in India and Pakistan have a common ancestral origin similar to that of Papua New Guinean isolates.     

Prevalence of pfcrt point mutations and level of chloroquine resistance in Plasmodium falciparum isolates from Africa.

The development in Plasmodium falciparum of the resistance to chloroquine (CQ) constitutes a public health priority, due to its direct influence in childhood mortality. The molecular basis for CQ resistance (CQR) is still unclear but, recently, a new relevant gene, named pfcrt, with several point mutations was identified in P. falciparum. Two mutations, K76T and A220S, have been considered crucial for CQR in further studies, making the pfcrt a good candidate as determinant for CQR in P. falciparum. To contribute to this topic, we have undertaken a molecular screening on 164 P. falciparum isolates from Africa: 120 isolates were Italian imported malaria cases, 27 and 17 isolates were from a school-children survey from Congo and Tanzania, respectively. In vitro tests (pLDH and WHO-Mark III tests) for CQ sensitivity have been also carried out on 28 plasmodial isolates and results compared to those obtained by molecular analysis in the same isolates. The SVIET pfcrt haplotype has been identified in the samples from Congo, and this is the first time that this haplotype is detected in Africa. Our results give further evidence to the reliability of the 76T (and the linked 74I-75E) pfcrt point mutation as molecular marker for CQR.     

A point mutation in codon 76 of pfcrt of P. falciparum is positively selected for by Chloroquine treatment in Tanzania.

This study was undertaken to validate the relevance of Chloroquine (CQ) resistance markers pfcrt(76) and pfmdr1(86) in an endemic area in Tanzania. After treatment with CQ, recrudescence was distinguished from new infection by msp2 genotyping, and the number of concurrent infections was also determined. The rate of children with recrudescent parasites at day 7 and/or day 14 amounted to a parasitological failure rate of 22.4% using PCR. The mean multiplicity of infection at day 0 was 3.2 (n=71). The allelic frequencies of the mutated pfcrt(76) and pfmdr1(86) were estimated to be 92 and 77%, respectively. Both values exceeded by far the observed frequency of 14% of recrudescent parasites as calculated on the whole analysed parasite population taking multiple infections into account. Although neither mutant allele is of predictive value for parasitological resistance, there is evidence for a role of pfcrt(76) in CQ resistance in the natural parasite population. All wild-type pfcrt(76) alleles were eliminated before day 3, after the onset of CQ treatment and no recrudescent parasite with the wild-type allele was observed at later time points. The discrepancy between the rate of resistant parasites (14%) and the frequency of the mutant pfcrt(76) allele (92%), however, indicates that other polymorphisms and other factors must be involved in CQ resistance. No selective elimination of the pfmdr1(86) wild-type allele was observed.