多重巢式PCR检测恶性疟和间日疟原虫

目的建立鉴别诊断恶性疟原虫(P.f)和间日疟原虫(P.v)的多重巢式PCR法。方法针对P.f、P.v 18S rRNA基因设计外引物和内引物,优化引物浓度与退火温度,建立可扩增出两种疟原虫基因片段的多重巢式PCR,并检测54例疑似疟疾临床标本,以镜检法为金标准评价敏感性和特异性等指标。结果该方法可扩增出162 bp(P.f)和112 bp(P.v)基因片段,并能检出混合感染。该方法检测P.f,敏感性为87.50%、特异性为63.33%;检测P.v,敏感性为69.23%、特异性为68.29%。结论所建立的多重巢式PCR方法能可靠诊断疟疾并鉴别虫种,敏感性高,在混合感染的诊断方面具有优越性。     

湖北省首例输入性卵形疟原虫wallikeri亚种的病原学诊断分析

目的应用巢式PCR技术对1例罕见卵形疟原虫wallikeri亚种进行诊断和鉴定。方法采集初诊为输入性间日疟患者血样,进行疟疾快速诊断试剂盒、显微镜镜检和巢式PCR检测,并进行测序分析。结果该患者疟疾快速诊断试剂盒检测阴性;镜检查见寄生于红细胞内的疟原虫环状体和滋养体;采用卵形疟原虫wallikeri亚种特异性引物(rOVA1v/rOVA2v)进行巢式PCR,在760bp处有特异性扩增条带,测序后经Blast比对,与GenBank数据库中卵形疟原虫wallikeri亚种部分序列的一致性为99%。结论该患者经巢式PCR和序列分析确诊为湖北省首例卵形疟原虫wallikeri亚种感染。     

复合聚合酶链反应检测恶性疟原虫及混合感染

目的应用一种简易、快速的复合ＰＣＲ扩增系统检测恶性疟原虫及混合感染。方法以间日疟原虫（Ｐｖ）和恶性疟原虫（Ｐ．ｆ）小亚单位核糖体核糖核酸基因（ＳＳＵｒＤＮＡ）特定片段为靶基因，设计并合成引物，建立复合ＰＣＲ扩增系统。采用煮沸法快速制备ＤＮＡ模板研究本系统的敏感性和特异性，并用于临床血样的检测。结果从Ｐ．ｖ和Ｐ．ｆ感染血样中分别扩增出分子量大小为７０５ｂｐ和５７５ｂｐ的特定扩增带。而食蟹猴疟原虫、诺氏疟原虫及正常人血样均无扩增带出现。单独检测Ｐ．ｆ敏感度达到０４７×１０－６（约２虫／μｌ全血），在Ｐ．ｖ存在的情况下，检测Ｐ．ｆ敏感度为０４７×１０－５。检测１０４例疟区门诊患者冻存血标本，８４例与镜检法结果相同，并发现２４例混合感染和２例为镜检虫种鉴别失误。结论本系统敏感性高，特异性强，操作简单，并可在一次扩增中同时检出Ｐ．ｖ和Ｐ．ｆ，具有一定的推广应用价值。     

复合聚合酶链反应检测恶性疟原虫及混合感染

目的 应用一种简易、快速的复事ＰＣＲ扩增系统检测恶性疟的虫及混合感染。方法 以间日疟原虫（Ｐ．ｖ）和恶性疟原虫（Ｐ．ｆ）小亚单位核糖体核糖核酸基因（ＳＳＵｒＤＮＡ）特定片段为靶基因，设计并合成引物，建立复合ＰＣＲ护增系统。采用煮沸法快速制备ＤＮＡ模板研究本系统的敏感性和特异性，并用于临床血样的检测。结果 从Ｐ．ｖ和Ｐ．ｆ感染血样中分别扩增出分子量大小为７０５ｂｐ和５７５ｂｐ的特定扩增带。而食蟹猴疟     

单管-套式/多重聚合酶链反应在间日疟原虫基因型鉴定中的价值

目的对单管-套式多重聚合酶链反应(PCR)鉴定间日疟原虫基因型方法的应用价值进行研究。方法以间日疟原虫环子孢子蛋白(CSP)基因为模型，综合套式多重PCR、等位特异PCR和标签引物扩增等技术，使用Primer Premier5．0软件和NCBI的BLAST网络改进与优化引物设计，并用矩阵试验法优化反应条件和程序，同时，用单管-套式多重PCR和套式PCR对71份间日疟患者血标本的间日疟原虫基因型鉴定结果作比较。结果单管-套式多重PCR可同时扩增出3种不同长度的基因型/族特异片段，且基本消除了二聚体等噪音。用单管-套式多重PCR鉴定检测71份间日疟患者血标本，62份与套式PCR鉴定结果一致，并新发现6份不同基因型和PV-Ⅱ型的混合感染；还将2份套式PCR鉴定为热带族的血标本纠正为PV-Ⅱ型，1份阴性血样鉴定为温带族。结论研究建立的单管-套式/多重PCR鉴定间日疟原虫基因型的方法，简化了试验步骤，节省了试剂耗材，消除了PCR噪音，提高了检出间日疟原虫混合感染和低度感染的敏感性；是一种具有发展潜力的检测单核苷酸多态性和基因型鉴定的方法。     

套式聚合酶链式反应在疟原虫检测和分型中的应用研究

目的比较疟疾病例样本套式PCR检测和传统镜检结果,探讨套式PCR在疟疾诊断中的应用价值。 方法采集疟疾患者血样,分别涂制厚、薄血膜用于常规镜检;抗凝血或滤纸血用于提取疟原虫DNA等。按要求设计属种等多套引物,应用套式PCR扩增感染人的4种疟原虫目的基因片段,将检测结果与镜检结果进行比较分析,并对目的片段进行测序鉴定等。 结果259份血样,套式PCR共检测出恶性疟187例、间日疟45例、卵形疟8例和三日疟1例,阴性10例。其中恶性疟阳性检出率最高(75.10%),间日疟阳性检出率次之(18.07%),还检测到混合感染血样8份(3.21%)。与GenBank标准序列对比显示,扩增片段大小及测序结果均完全正确,显示套式PCR在分型上比传统镜检更客观。套式PCR检测与传统镜检结果比较显示,前者阳性率(96.14%)明显高于后者(90.73%),并且差异具有统计学意义(χ²＝12.07,P<0.05)。 结论套式PCR法检测疟原虫具有较高敏感性和特异性,且适用于疟原虫混合感染,在疟疾病例诊断和分型方面均具有良好的应用前景。     

增强化学发光法标记的DNA探针诊断恶性疟疾

采用增强化学发光法（ＥＣＬ）标记的ＤＮＡ探针诊断恶性疟疾。结果显示，ＥＣＬ标记的探针能检出２５ｐｇ纯化的恶性疟ＤＮＡ或原虫率为０．００１％体外培养的恶性疟原虫血，并且和人白细胞ＤＮＡ无交叉反应。对１４６份疟疾病人血样进行检测，ＤＮＡ探钉法和常规镜检法有较好的相关性。对于镜检中确诊的９９例恶性疟，ＥＣＬ法检出９３例；对于４７例间日疟血样，ＥＣＬ法仅和１例有阳性反应；该探针和４８例正常人血均无交叉反应。结果表明，ＥＣＬ标记的探针具有较好的特异性和敏感性，可用于恶性疟疾的特异性诊断和大规模流行病学调查。     

套式聚合酶链式反应诊断三日疟原虫感染的临床应用

目的应用套式聚合酶链式反应(PCR)扩增小亚单位核糖体核糖核酸(SSUrRNA)基因诊断三日疟原虫感染,以减少三日疟的漏诊和误诊。 方法分别提取可疑三日疟患者抗凝血DNA和对照间日疟、恶性疟患者抗凝血DNA,以此为模板,用疟原虫属特异性引物进行第一轮扩增;然后以第一轮扩增产物为模板,用4种疟疾的种特异性引物进行第二轮扩增,比较扩增出的18 SSU rRNA基因片段的大小,并对目的片段进行测序鉴定。 结果经属特异性引物PCR扩增后,3个样本均出现大小约为1200bp的条带。经种特异性引物PCR扩增后,间日疟及恶性疟确诊样本均扩增出相应的120bp和205bp特异性条带;可疑患者样本仅在用三日疟原虫种特异性作引物时扩增出144bp特异条带,与理论值相符,与Genbank标准序列对比显示,扩增片段大小及测序结果均完全正确。证实患者感染三日疟原虫。 结论利用小亚单位核糖体核糖核酸基因片段三日疟种特异引物进行扩增可以用于诊断三日疟原虫感染。     

增强化学发光法标记的DNA探针诊断恶性疟疾

采用增强化学发光法（ＥＣＬ）标记的ＤＮＡ探针诊断恶性疟疾。结果显示，ＥＣＬ标记的探针能检出２５ｐｇ纯化的恶性疟ＤＮＡ或原虫率为０．００１％体外培养的恶性疟原虫血，并且和人白细胞ＤＮＡ无交叉反应。对１４６份疟疾病人血样进行检测，ＤＮＡ探针法和常规镜检法有较好的相关性。对于镜检中确诊的９９例恶性疟，ＥＣＬ法检出９３例；对于４７例间日疟血样，ＥＣＬ法仅和１例有阳性反应；该探针和４８例正常人血均无交叉反应     

PCR扩增特定SSUrRNA片段检测疟原虫的研究进展

众所周知,疟疾的诊断迄今为止仍主要依靠血片镜检,该方法虽然简便、经济、适于基层;但费时费力,易于漏榆,特别对低原虫血症者,漏检率高,不利于快速诊断和大批标本检测,临床应用受到许多限制。因此,寻找高效、准确的诊断方法,成为疟疾诊断的关键所在。20世纪90年代后,聚合酶链反应(PCR)技术在疟疾的诊断上得到广泛的研究,并取得显著的成绩[1,2],不少结果显示已达到或超过镜检水平[3],而且适合快速诊断和大批标本检测[4]。其中,疟原虫小亚单位核糖体核糖核酸(Small subunit ribosomal RNA,SSUrRNA)特定片段的PCR扩增在疟疾诊断中的应用是目前最为活跃的研究方向之一,结果显示,它不仅在检测的敏感性和特异性方面较镜检大大提高,而且具有疟原虫种甚至株的鉴别能力[5],显示了良好的应用前景。笔者仅就PCR扩增SSUrRNA特定片段在疟疾诊断中的应用简介如下:     

Investigation of β-cyclodextrin–norfloxacin inclusion complexes. Part 1. Preparation,physicochemical and microbiological characterization

Malaria chemoprophylaxis concerns prescribing healthy individuals medication for an infection they have an unknown chance of getting. Sensible use of malaria chemoprophylaxis is a balance between the risk of infection and death, and the risk of side effects. The risk of infection can be broken down into the risk of being bitten by a malaria-infected mosquito and the risk of the malaria parasites being resistant to the drug used for prophylaxis. Our knowledge of these parameters is patchy. The risk of infection is not uniform at a given location and the standard of living will greatly influence risk. It is suggested that chemoprophylaxis should not be recommended in areas with less than ten reported cases of P. falciparum malaria per 1000 inhabitants per year. The resistance pattern is known to a certain extent but, for instance, diverging opinion of how much resistance to chloroquine there is in West Africa illustrates the lack of data. There is much debate on rare adverse events, which usually escape Phase III studies prior to registration and are only picked up by passive, postmarketing surveillance. The lessons over the past 20 years with the introduction of amodiaquine, pyrimethamine/dapsone (Maloprim^®, GlaxoSmithKline) and pyrimethamine/sulfadoxine (Fansidar^®, Roche), which were all withdrawn for prophylaxis after a few years, show how sensitive drugs for chemoprophylaxis are to side effects. Three levels of chemoprophylaxis are used: chloroquine in areas with sensitive P. falciparum, chloroquine plus proguanil in areas with low level chloroquine resistance, and atovaquone/proguanil (Malarone^®, GlaxoSmithKline), doxycycline or mefloquine (Lariam^®, Roche) in areas with extensive resistance against chloroquine and proguanil. Primaquine and the primaquone analog tafenoquine may be future alternatives but otherwise there are few new drugs for chemoprophylaxis on the horizon.     

Current developments in malaria transmission-blocking vaccines

Malaria is still a leading cause of morbidity and mortality in human populations. Problems, including drug-resistant parasites and insecticide resistant mosquitoes, ensure the continued hold of malaria in the tropics and sub-tropics. Each year around 100 million cases of malaria result in at least 50,000 deaths outside of sub-Saharan Africa; within sub-Saharan Africa itself, malaria causes around one million child deaths per year. New approaches for malaria control are badly needed and much effort has gone to develop malaria vaccines. In addition to giving personal protection, most such vaccines would also tend to reduce the transmission of malaria. One class of vaccine is being developed specifically for this purpose - the malaria transmission-blocking vaccines (TBVs). TBVs are based upon antigens expressed on the surface of the sexual and mosquito mid-gut stages of malaria parasites. These antigens are the targets of antibodies induced by vaccination of the host and ingested with the parasites in a mosquito blood meal. The antibodies act by inhibiting the parasite’s development within the mosquito itself and they thereby prevent the onward transmission of the parasites. TBVs could contribute to the total interruption of malaria transmission in many locations with relatively low transmission rates, mostly outside sub-Saharan Africa. Under almost all transmission rates, however, TBVs would help reduce malaria incidence and malaria-related morbidity and mortality. Promising recombinant TBV candidate antigens for the two main human malaria parasite species, Plasmodium falciparum and Plasmodium vivax, have been produced and tested in the laboratory; one has undergone early clinical trials.     

Coartemether (artemether and lumefantrine): an oral antimalarial drug

Coartemether (Riamet^®, Coartem^®, Novartis), a tablet formulation of artemether and lumefantrine, is a well-tolerated, fast-acting and effective blood schizontocidal drug that serves primarily in the treatment of uncomplicated falciparum malaria that is resistant to other antimalarials. Initial clinical-parasitological response relies mainly on the artemether component, while lumefantrine effects radical cure. The absorption of lumefantrine is poor during the fasting state, the normal condition in acutely ill malaria patients, but with return to normal diet it becomes adequate. This highlights the need for an appropriate adjustment of the dose regimen. In the area where Plasmodium falciparum shows the highest degree of multidrug resistance worldwide, the best results (99% cure) were obtained with a six-dose regimen given over 5 days. Extensive cardiological investigations have demonstrated the high cardiac safety of coartemether.     

一种新的疟原虫浓集法

用人工感染约氏疟原虫的小白鼠血液和问日疟患者的静脉血作实验标本,在加有特定比重介质的微量血细胞压积管内离心。取离心后疟原虫浓集层涂片,姬姆萨染色后计算红细胞中疟原虫感染率。试验结果显示:加入该介质离心,能有效地浓集疟原虫,其效果比压积管直接离心浓集法好。     

High specificity of semi-nested multiplex PCR using dried blood spots on DNA Banking Card in comparison with frozen liquid blood for detection of Plasmodium falciparum and Plasmodium vivax

Background: Venipuncture sampling in test tubes for detecting malaria parasites using PCR assays possesses a number of limitations such as reluctance of patients, some difficulties in transportation of blood samples and freezing them for long time. To overcome the mentioned limitations, some approaches have been employed by a number of authors. This study was proposed to compare between DNA Banking Card (DBC) filter papers containing dried finger‐prick blood and venipunctured frozen liquid blood. Methods: A total of 75 specimens was prepared from the equal enrolled individuals using three blood storage approaches; making Geimsa‐stained thin and thick smears from each individual to determine the malaria‐positive or ‐negative specimens, spotting two to three drops of finger‐prick blood onto the DBC filter paper, and collecting a 2‐ml venous blood sample into EDTA‐contained test tube from each individual. A semi‐nested Multiplex PCRtechnique with DNA extracted from the two latter sets of specimens was used for plasmodia diagnosis. Results: DNA samples isolated from dried blood spotted on the DBC filter papers resulted in 32 (42.7%) positive and 43 (57.3%) negative cases comparable with the results outcome of frozen liquid blood with 35 (46.7%) positive and 40 (53.3%) negative cases. Statistical analysis revealed higher sensitivity for SnM‐PCR using DNA from liquid blood with 100% vs. dried blood spotted on DBC with 97% but higher specificity for the DBC with 100% vs. liquid blood with 95.2%. Conclusions: Based on the results obtained from this study to overcome the problems of venipuncture frozen liquid blood sampling, replacement of a reliable filter paper for preserving finger‐prick blood samples is a trustable and useful facilitator particularly in remote malaria‐endemic areas. J. Clin. Lab. Anal. 25:185–190, 2011. © 2011 Wiley‐Liss, Inc.     

斑点免疫金染色法检测恶性疟原虫循环抗原

应用胶体金探针建立了斑点免疫金染色法（Ｄｏｔ－ＩＧＳ）。以捕捉法Ｄｏｔ－ＩＧＳ检测３０份海南恶性疟病人全血，循环抗原阳性者２７份，阳性率为９０．０％；４０份其他寄生虫病人血样全为阴性。结果表明，捕捉法Ｄｏｔ－ＩＧＳ在敏感性和特异性方面都优于间接法Ｄｏｔ－ＩＧＳ和斑点酶联免疫吸附测定，是检测疟疾循环抗原的理想方法。     

间日疟原虫乳酸脱氢酶GST融合表达载体的构建及表达

目的构建间日疟原虫乳酸脱氢酶（PvLDH）融合表达载体,并表达PvLDH的GST融合蛋白。方法将PvLDH基因片段克隆到表达载体pGEX-4T-1中,构建PvLDH/pGEX-4T-1融合表达载体,IPTG诱导表达目的基因,SDS-PAGE电泳分析表达产物,Western blot检测其抗原性。结果成功构建了PvLDH/pGEX-4T-1融合表达系统,在大肠杆菌BL21中以包涵体形式高效表达,表达产物能与恶性疟原虫和间日疟原虫感染患者血清反应,而不与正常人血清反应。结论间日疟原虫LDH蛋白在大肠杆菌中获得高效表达,表达产物具有良好的抗原性。