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。     

1例输入性罕见远期复发卵型疟原虫多重感染的实验室检测分析

目的 对1例疟疾复发患者通过实验室多种方法,并结合其临床症状,做出快速准确的诊断.方法 对患者进行血常规检查、疟疾快速诊断试剂盒(RDTs)检测和形态学检查,结合其临床症状进行初步诊断;再通过巢式PCR和一代测序的方法,诊断虫种并鉴定亚型.结果 通过血常规散点图和RDTs检测的结果,初步判断为疟原虫感染,且排除恶性疟疾...     

三例输入性卵形疟的实验室诊断

目的分析3例输入性疟疾的实验室诊断,减少卵形疟的误诊与漏诊。方法采用形态学检查、巢氏聚合酶链反应(PCR)及序列分析等方法确诊3例输入性疟疾病例。结果 3例患者形态学检查结果分别是未检出疟原虫(实验室初次检查)、检出卵形与恶性疟原虫和检出卵形疟原虫;巢氏PCR检测结果依次是卵形疟原虫感染、卵形与恶性疟原虫混合感染和卵形与间日疟原虫混合感染。结论需加强疟疾消除地区疟原虫形态学检查技术的培训和推广。应用分子生物学检查技术可减少输入性卵形疟的误诊与漏诊。     

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。     

加拿大报道来自印度旅行者发热病例1例

正77岁印度赴加拿大旅游者,抵达前2 d开始发热,后因发热16 d就医。体温最高39.2℃,贫血,血小板减少(81 000/μL),转氨酶轻度升高。6%红细胞中有环状体,疟疾快速诊断试验呈阴性。安大略湖公共卫生实验室重复血涂片检查,发现6.9%十字花型环状体滋养体,而疟疾快速诊断试验仍阴性,但通用疟原虫PCR检测阳性,提示血寄生虫含量0.1%;而特异性疟原虫PCR检测为阴性。微小巴贝西虫(Babesiamicroti)PCR     

三种实验室检测方法在疟疾检测中的应用比较

目的探讨三种实验室检测方法对疟疾检测的应用价值。方法分别采用传统镜检法、巢式聚合酶链反应(PCR)法及实时荧光(Real-Time)PCR法,对江西省2012年-2014年的61份疟疾疑似病例血样进行检测。结果巢式PCR法和Real-Time PCR法的结果一致,与镜检结果有17份样本不一致。结论巢式PCR法和Real-Time PCR法检测疟疾感染具高度敏感性和特异性,对疟疾鉴别诊断和明确诊断具有重要价值。     

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。     

肠道轮状病毒巢式PCR检测方法的建立及初步应用

目的 建立一种特异性和灵敏度高的检测婴幼儿腹泻粪便标本中肠道轮状病毒(RV)的巢式PCR方法,并探讨其在临床诊断中的初步应用价值。方法 根据RV保守区基因序列并利用Primer5.0软件设计巢式PCR两对引物,建立一种特异性和灵敏度高的检测RV的巢式PCR方法,并评价其特异度和敏感度。同时采用所建立的巢式PCR法对136份临床样品进行检测,并用单一的RT-PCR法、胶体金和基因序列法进行结果验证。结果 巢式PCR法只扩增检测出192 bp的RV特异性条带,对其他肠道病毒均呈阴性反应,特异性好;巢式PCR法最低能检测到8.705 fg/μL的RV;巢式PCR法与单一的RT-PCR法检测结果符合率为100%,但灵敏度低于胶体金法,差异有统计学意义(χ~2=2.615,P0.05);将巢式PCR法扩增的RV产物进行核苷酸正反向测序,经BLAST比对,RV与EU679386、DQ870492、AF531912等20多株已知序列的同源性为98%～100%。结论 建立了检测肠道病毒RV的巢式PCR法,具有特异性强、灵敏度高,且检测快速,值得临床推广应用。     

输血传播恶性疟疾实验室检测和溯源分析——附报告1例

目的通过对有输血史的恶性疟疾感染者进行检测和溯源分析,确定其传染源和传播方式。方法对患者和为其输血的8名献血者血样进行疟疾快速检测、血涂片镜检和Real-time PCR检测,并对阳性血样进行分子溯源和基因分型。结果患者与1名非洲返乡献血者在疟疾快速诊断试剂检测、血涂片镜检和Real-time PCR 3项检测中,均显示为恶性疟阳性。两者血样SSU rRNA基因序列两两比对后同源性为100%,Pf EMP-1基因分型显示受血者和献血者血样均为恶性疟原虫K1型和MD20型混合感染。确定该患者的恶性疟确因输入非洲返乡献血者的血液而感染。讨论由于我国未将疟疾筛查列入献血前检测,因此境外返乡人员在疟疾潜伏期或复发期内参加献血易引起输血传播疟疾。采供血机构需仔细征询既往病史和外出史,并加强宣教,将经血传播的疾病控制在源头。     

巢式聚合酶链式反应检测脑脊液标本中病原体的方法评价

目的探讨巢式聚合酶链式反应(PCR)检测脑脊液标本中病原体的可行性,为疾病的快速临床诊断提供参考。方法源自细菌16S rRNA基因序列设计的巢式PCR技术方法,包括2对引物,2次PCR扩增,第1对引物首次PCR扩增脑脊液标本提取的核酸DNA,第2对引物再次PCR扩增,其DNA模板为第1轮PCR扩增产物。将第2轮PCR扩增产物进行测序,对序列进行比对分析,从而确定感染的病原体。使用DNA微量分光光度测定布鲁氏菌纯菌核酸DNA,将DNA进行倍比稀释,用于巢式PCR敏感性测试。结果巢式PCR能够检测的最低限约为1个核酸DNA拷贝数。应用巢式PCR检测40份临床患者的脑脊液标本,扩增结果表明有37份标本获得约1 460 bp的预期扩增条带(不同细菌扩增片段有差异),测序比对结果显示,检出脑膜炎奈瑟菌7份、产碱假单胞菌1份、草假单胞菌22份、嗜麦芽窄食单胞菌2份、肺炎链球菌1份、未知细菌性病原体4份、未检出3份。结论巢式PCR能够快速检测与鉴定脑脊液标本中的细菌性病原体。     

Various pfcrt and pfmdr1 Genotypes of Plasmodium falciparum Cocirculate with P. malariae, P. ovale spp., and P. vivax in Northern Angola

Artemisinin-based combination therapy for malaria has become widely available across Africa. Populations of Plasmodium falciparum that were previously dominated by chloroquine (CQ)-resistant genotypes are now under different drug selection pressures. P. malariae, P. ovale curtisi, and P. ovale wallikeri are sympatric with P. falciparum across the continent and are frequently present as coinfections. The prevalence of human Plasmodium species was determined by PCR using DNA from blood spots collected during a cross-sectional survey in northern Angola. P. falciparum was genotyped at resistance-associated loci in pfcrt and pfmdr1 by real-time PCR or by direct sequencing of amplicons. Of the 3,316 samples collected, 541 (16.3%) contained Plasmodium species infections; 477 (88.2%) of these were P. falciparum alone, 6.5% were P. falciparum and P. malariae together, and 1.1% were P. vivax alone. The majority of the remainder (3.7%) harbored P. ovale curtisi or P. ovale wallikeri alone or in combination with other species. Of 430 P. falciparum isolates genotyped for pfcrt, 61.6% carried the wild-type allele CVMNK at codons 72 to 76, either alone or in combination with the resistant allele CVIET. No other pfcrt allele was found. Wild-type alleles dominated at codons 86, 184, 1034, 1042, and 1246 of the pfmdr1 locus among the sequenced isolates. In contrast to previous studies, P. falciparum in the study area comprises an approximately equal mix of genotypes associated with CQ sensitivity and with CQ resistance, suggesting either lower drug pressure due to poor access to treatment in rural areas or a rapid impact of the policy change away from the use of standard monotherapies.     

An 8-year survey on the occurrence of imported malaria in a nonendemic area by microscopy and molecular assays

Our study aimed to describe the occurrence of imported malaria in a nonendemic area (Parma, Italy) during the period 2000 to 2007, comparing the data obtained by microscopy and molecular assays targeting plasmodial 18S subunit rRNA gene. The prevalence of imported malaria in Parma was 21.8% by microscopy and 22.7% by polymerase chain reaction (PCR). Plasmodium falciparum accounted for 81.1% of the cases, followed by Plasmodium ovale (8.8%), Plasmodium vivax (3.8%), and Plasmodium malariae (1.9%). Mixed infections accounted for 4.4% of the cases. In this study, PCRs proved to be more sensitive and specific than microscopy and changed the picture of malaria epidemiology in Parma, detecting additional cases of malaria undiagnosed by microscopy and allowing speciation of plasmodia in cases misidentified by microscopy. Generally, imported malaria cases reflect the number of immigrants who visit their native countries, in particular, West Africa, explaining the increased prevalence of P. ovale cases among non-P. falciparum infections in Parma.     

2017-2020年江西省境外输入性恶性疟原虫的抗药性基因多态性研究

目的 了解2017—2020年江西省境外输入性恶性疟原虫的抗药性情况,为临床用药提供技术支撑。方法 收集2017—2020年江西省输入性恶性疟病例的血液样本,采用巢式PCR方法,对样本抗药性基因进行扩增并测序。结果 共收集到输入性恶性疟病例血液样本85份,均为非洲输入。测序分析得出,恶性疟原虫氯喹抗性转运蛋白基因和多药抗性基因1突变比例较低,为16.67%(14/84)和1.18%(1/85),而二氢叶酸还原酶基因突变比例很高,为96.39%(80/83),未发现Kelch螺旋体蛋白基因的突变。结论 近几年江西省境外输入性恶性疟病例均来自非洲,且恶性疟原虫对氯喹的抗药性较低,可以结合临床实验,考虑用氯喹联合其他药物治疗非洲输入恶性疟病例的可能方案;对乙胺嘧啶抗药性较高,未发现青蒿素抗性株,但因为近2年受新型冠状病毒感染疫情影响,样本量少,不能排除江西省无青蒿素抗性株,需继续监测。     

2例输入性卵形疟散点图异常对疟原虫的筛查作用

目的 该文报道上海市公共卫生临床中心2014年9月份收治的两例境外输入性卵形疟患者的实验室诊断,并简单探讨自动血细胞分析仪散点图异常信息对疟疾筛查的意义。方法 2例患者抗凝外周血标本采用SYSMEX XT-4000i血细胞分析仪进行血细胞分析,同时制备厚薄血片显微镜镜检查找疟原虫虫体。结果 2例患者的外周血中找到卵形疟疟原虫虫体,血细胞分析的散点图出现异常,中性粒细胞与嗜酸性粒细胞散点图之间的间距缩小或消失。结论 血细胞分析仪特征性的散点图异常在筛查卵形疟原虫上有一定的提示价值,与显微镜镜检相结合更可促进疟原虫形态学的诊断。     

Evaluating the efficacy of rapid diagnostic tests for imported malaria in high income countries: A systematic review

BackgroundMalaria is a life-threatening disease. Prior to the pandemic, over a million people annually from non-endemic, high income countries such as Europe and North America visited countries with a risk of malaria transmission. Emergency care nurses in non-endemic countries frequently encounter returning travellers, presenting with symptoms suggestive of malaria. While rapid diagnostic tests are used in countries with endemic malaria, in countries such as the United Kingdom diagnosis is undertaken by microscopy and three negative tests are required to exclude.QuestionAre rapid diagnostic tests effective for diagnosing imported malaria in non-endemic, high income countries?MethodA systematic review of published research (January 2009 – November 2020) comparing rapid diagnostic tests with microscopy.ResultsFourteen studies were included, conducted in five countries with 14 different RDTs evaluated. Mean sensitivity and specificity for Plasmodium Falciparum was 91.8% and 97.7% and Plasmodium Vivax 81.6% and 99.2%. Higher sensitivities were related to higher parasite densities.ConclusionsInternational travel will return post-pandemic and rapid, accurate and cost-efficient tests will be required. The rapid diagnostic tests in these studies showed significant variation and were not as accurate as microscopy. Consequently, it cannot be recommended that rapid diagnostic tests replace the gold standard of microscopy. Further research is required.     

Comparison of Molecular,Microscopic, and Culture Methods for Diagnosis of Cutaneous Leishmaniasis

Cutaneous leishmaniasis (CL) is endemic in the northwest of Isfahan province, Iran. Increase in the incidence of the disease in Kashan has made it necessary to find out the best method for diagnosis and molecular characterization of Leishmania species. In the present study, 130 patients suspected to cutaneous leishmaniosis referred to health care centers of Kashan were examined. Serosity of lesion was collected for smear preparation and cultured in Novy‐Nicolle‐McNeal medium. DNA was extracted from serosity, and Leishmania species was determined by polymerase chain reaction (PCR) and nested PCR using kinetoplast DNA (kDNA) specific primers. The diagnostic criteria of CL were based on the observation of amastigotes in the smear, promastigotes in culture, presence of expected bands in PCR, or nested PCR. Of 130 specimens, 87 (66.9%), 72 (56.2%), 98 (75.4 %), 96 (73.8%), and 99 (76.2%) were positive for microscopic culture, PCR, nested PCR, and combined PCR and microscopy (proposed method), respectively. Sensitivity, specificity, positive and negative predictive values of PCR were 99%, 100%, 100%, 96.9%, respectively, for microscopy 87.9%, 100%, 100%, 72.1%, for culture 72.7%, 100%, 100%, 53.4 %, and for nested PCR 97%, 100%, 100%, 91.2%, respectively. Based on the results of the study, kDNA‐PCR was the most sensitive method for diagnosis of CL.