江西口岸首例输入性卵形疟原虫wallikeri亚种感染的PCR鉴定及序列分析

目的对江西口岸1例输入性疟疾病例的血样本进行检测、确认。方法利用普通PCR检测全血样本中疟原虫18S r RNA基因,将得到的测序结果在NCBI数据库进行比对,并结合吉姆萨染色镜检、胶体金快速检测和荧光定量PCR加以验证。结果血涂片镜检并未发现疟原虫,但胶体金快速检测结果为阳性。普通PCR扩增到18S r RNA基因特异性片段,卵形疟原虫特异性引物扩增到阳性条带,而恶性疟、间日疟和三日疟原虫特异性引物没有扩增到目的条带。用卵形疟不同亚型引物特异性扩增发现卵形疟原虫wallikeri亚种阳性,序列与卵形疟原虫wallikeri亚种(Gen Bank号:KF219564.1和KF696359.1)18S r RNA同源性达99%。荧光定量PCR检测亦确认为卵形疟阳性。结论该病例确认为江西口岸首例输入性卵形疟wallikeri亚种感染。     

疟疾四重巢式PCR检测方法的建立

目的建立一种能同时检测4种疟疾的多重巢式PCR检测方法。方法以4种疟原虫为研究对象,提取全血样本核酸,采用疟原虫18SSU r RNA作为分子标记,合成通用引物和4种疟原虫特异性引物,建立和优化四重巢式PCR反应体系。结果建立的方法在原虫之间无交叉反应,检测敏感性可达102copies/μl,经对随机选取的口岸发热样本进行检测,35份血液样本中,检出恶性疟13例、间日疟7例、卵形疟1例、以及恶性疟和间日疟的混合感染2例,阳性率为65.7%;比镜检的阳性率54.3%高,尤其是检出混合感染病例比例高,并对检测结果进行测序验证。结论建立的四重巢式PCR能同时检测4种不同疟原虫引发的疟疾,具有较高的特异性和敏感度,适合于口岸输入性疟疾的快速检测。     

逆转录聚合酶链反应检测疟原虫混合感染的研究

〔目的〕建立在同一反应体系中能同时检测和鉴定恶性疟、间日疟的一步逆转录聚合酶链反应（RT-PCR）检测方法,并用于国境口岸归国劳务人员的疟疾检测。〔方法〕以疟原虫小亚单位核糖体核糖核酸基因为靶片段,设计恶性疟原虫和间日疟原虫的上游通用引物和各自的下游特异性引物,在同一反应体系中同时扩增恶性疟原虫和间日疟原虫的特异片段,对疟疾患者的PCR产物进行序列测定和分析。〔结果〕用建立的RT-PCR检测方法对广东口岸回国劳务人员中发现的2名疟疾患者的不同采样时间的全血进行检测,2006年10月和2007年1月采集的样本被分别扩增出预期大小为360bp和450bp特异扩增带,推测2名患者为恶性疟原虫和间日疟原虫的混合感染,其在入境时处于恶性疟的发作期和间日疟的潜伏期,均属于典型的集体输入性疟疾案例。〔结论〕RT-PCR检测疟原虫方法具有敏感性高、特异性强的特点,对国境口岸疟疾诊断、镜检质量控制和流行病学研究具有较大的实用价值。     

基因扩增检测四种感染人的疟原虫种类、数量的方法

目的建立对四种感染人的疟原虫种类、数量进行基因检测的方法。方法根据恶性疟、三日疟、卵形疟、间日疟的18SrRNA基因序列,设计属、种特异性引物和TaqMan探针,用荧光定量扩增反应确定标本中的基因拷贝数,用电泳区别各种疟原虫,并对2份疟疾血样进行检测。结果建立的检测方法对疟疾血样进行检测,荧光定量PCR具有良好的反应性,通过电泳能区分检测标本中的疟原虫株。结论建立的方法能够特异而灵敏地检测出标本中疟原虫的种类、数量,适合疟疾防治检测的需要。     

2021年深圳口岸首起聚集性输入性疟疾疫情的调查

目的 调查2021年深圳口岸首起聚集性输入性疟疾疫情,为输入性疟疾防控提供经验。方法 根据海关总署的布控指令,深圳海关对1架入境航班实施登临检疫,并对所有旅客进行流行病学调查,排查到的有症状旅客及来自疫区的重点旅客后,采集血液样本,用胶体金法、实时荧光PCR和涂片镜检法等检测疟原虫。结果 检疫查验发现航班上33名旅客有疟疾流行区旅行史,其中5人用胶体金法筛查为恶性疟阳性,并用实时荧光PCR方法确认为恶性疟病例,血涂片镜检在其中3人血样中发现恶性疟原虫。结论 在全球新冠肺炎疫情下应加强多病共防,防止疟疾输入再传播,巩固国内疟疾消除成果。     

疟疾的实时荧光PCR快速检测方法

目的:建立疟疾的实时荧光PCR检测方法并用于疟疾的快速检测。方法:设计了疟原虫通用型实时荧光PCR检测的引物和探针,建立了疟疾的实时荧光PCR检测方法。对40份疟疾镜检阳性样本和20份镜检阴性样本进行实时荧光PCR检测,并和巢式PCR方法检测结果进行了比较。结果:疟疾实时荧光PCR检测速度快,20分钟即可完成。40份镜检阳性样本的荧光PCR检测均为阳性,2份样本的巢式PCR结果为阴性;20份镜检阴性样本中有3份样本为实时荧光PCR阳性,其余17份样本为实时荧光PCR阴性,与疟疾巢式PCR检测结果相同。结论:该实时荧光PCR方法检测速度快,特异性强,准确性高,阳性率高,不易漏检,适用于疟疾的快速检验,这对防止该病在国境口岸的传入提供了技术依据。     

太原市1例输入性卵形疟的处置及分析

目的 对太原市1例输入性卵形疟病例进行流行病学调查、处置和分析，探讨赴疟疾流行区的出入境重点人群疟疾防控策略，为口岸疟疾防控工作提供参考。方法 对1例疑似疟疾病例进行流行病学调查，采集外周血制作血涂片镜检和血常规检测，用快速检测试剂条检测疟原虫抗原，实时荧光PCR方法对样本中疟原虫进行分型。结果 该病例有低热、头痛症状，有高疟区工作、多次罹患疟疾流行病学史，血涂片镜检可见疟原虫配子体、环状体，疟原虫抗原快速检测为阳性，荧光定量PCR检测结果为卵形疟。根据WS 259—2015《疟疾的诊断》，诊断为输入性卵形疟。结论新冠肺炎疫情常态化背景下，须重视疟疾高流行区出入境重点人群的疟疾防控与宣教工作，加强口岸多病共防，巩固消除疟疾成果，防止输入再传播。     

快速检测试剂条、镜检和PCR在疟疾诊断中的应用研究

目的采用快速检测试剂条（rapiddiagnostictest,RDT）、镜检及聚合酶链反应（polymerasechainreac-tion,PCR）,3种检测方法进行疟原虫检测,比较3种方法在疟疾诊断中的敏感性、特异性,为科学分析检测结果和探究RDT能否在基层替代疟原虫镜检提供依据。方法收集安徽省2012年1月～2013年3月确诊以及疑似病例抗凝血148份,血片148张,采用RDT进行检测,并与镜检法和PCR的结果对比分析其敏感性与特异性。结果148份血样采用RDT、镜检和PCR3种方法检测结果阳性率分别为43．24％、36．49％、37．84％,经配对x2检验RDT阳性率高于镜检和PCR;镜检和PCR均检出2例卵形疟,而RDT结果为阴性;以镜检为标准,对间日疟和恶性疟进行分析得出,RDT的敏感度为100％,特异度87％,阳性预测值81％,阴性预测值100％;以PCR为标准,对间日疟和恶性疟进行分析得出,RDT的灵敏度为100％,特异度89％,阳性预测值84％,阴性预测值100％。3种方法均检出间日疟16例,而对恶性疟分别为RDT检出48例,镜检检出36例,PCR检出38例。结论RDT对间日疟和恶性疟敏感性较高,可以覆盖全部阳性病例,在今后疟疾低流行时期RDT有望取代基层镜检对疟疾进行诊断。     

口岸适用性登革病毒检测模式的建立

目的优化我国口岸登革热检测模式。方法考核登革热快速检测试剂(胶体金法)和实时荧光定量PCR试剂(通用型及分型)的检测性能,再将两种方法联合应用于口岸登革热检测,在口岸现场完成胶体金快速筛查,在实验室完成确认。结果利用8份不同亚型的登革病毒阳性血清进行考核,胶体金法和实时荧光定量PCR的检测灵敏度均为100.00%。2019年1-6个月,在厦门口岸收集了108份入境发热旅客的血液样本,用实时荧光定量PCR方法从中检出25份登革病毒阳性,用胶体金法检出24份;胶体金法漏检1份,符合率为96.00%。结论初筛和确认相结合的检测方法能提高登革热疫情时登革病毒的检测效率,满足口岸登革病毒快速筛查的需求。     

弥勒市1例输入性间日疟的诊断与调查分析

目的 评价综合诊断技术对非典型形态疟原虫感染病例确诊、降低输入性病例误诊或漏诊率的重要性,巩固已取得的消除疟疾成果。方法 对2023年1月16日弥勒市某医院接诊1例间日疟患者进行流行病学调查;通过镜检、抗原胶体金法(RDT)和分子生物学方法(巢式PCR)检测,确诊为1例输入性间日疟感染病例。结果 该患者血涂片镜检厚血膜查见疟原虫,薄血膜中查见配子体皱缩成团,被寄生的红细胞不胀大甚至略缩小,颜色正常与周围正常红细胞一致,与WS 259—2015《疟疾的诊断》、《疟疾防治手册》中病原学检查描述间日疟形态不同。结论 用镜检、抗原胶体金法(RDT)和巢式PCR 3种方法,对非典型形态疟原虫感染病例进行确诊至关重要。     

湖南省郴州市2003-2012年疟疾流行特征及监测效果研究

目的 了解郴州市疟疾流行特征及监测效果,为疟疾防治和消除提供科学依据.方法 运用描述性流行病学方法对郴州市2003-2012年疟疾流行病学及监测资料进行统计分析.结果 郴州市2003-2012年共报告疟疾病例41例,年平均发病率为0.09/10万,死亡1例,无暴发疫情;其中间日疟24例（58.54％）,恶性疟15例（36.58％）,三日疟2例（4.88％）;本地病例4例（9.76％）,输入性病例37例（90.24％）,93.33％（14/15）的恶性疟病例均由非洲输入;发热患者血检107 975人次,阳性率为3.43/万,疟疾病例实验室检测率和确诊率均为100％;传疟媒介主要以中华按蚊为主（86.04％）,蚊密度高峰出现在7-8月.结论 郴州市基本消除疟疾后,疟疾疫情控制在较低水平,病例以输入性为主,在今后的疟疾防治和消除工作中,应加强流动人口监测和发热患者血检.     

Latest developments in the laboratory diagnosis of malaria

Malaria is the most important parasitic disease worldwide. With the advent of multidrug-resistant strains, it is highly important that the disease be diagnosed both early and accurately. For the diagnosis of malaria parasites, the thick blood film approach remains the gold standard. However, the use of that standard requires a microscope, stains, and a trained microscopist to interpret the films. The author describes the microscopical detection of the malaria parasite through the use of fluorochrome as well as the development of antigen detection tests to improve the laboratory diagnosis of malaria. Histidine-rich protein II (HRPII) is expressed by the asexual stages of Plasmodium falciparum. The detection of HRPII antigen appears to be a useful alternative diagnostic technique when microscopes are unavailable. However, a negative test result may indicate the presence of non-P falciparum malaria or that it is too early in the course of infection to detect parasites. One advantage of a parasite lactate dehydrogenase (pLDH) detection system is its ability to detect all 4 species of malaria and to diagnose both P. falciparum and P. vivax infections.     

Malaria prevalence and morbidity among children reporting at health facilities in Nouakchott, Mauritania

Although malaria has become a serious public health problem in Mauritania since the late 1990s, few documented data on its epidemiology exist. The objective of this study was to assess the morbidity of clinical malaria among children in Nouakchott. Three hundred and one febrile children, consulting at three health facilities of Nouakchott, were screened for malaria in 2009 (n=216) and 2010 (n=85). Plasmodium species identification and parasite density were determined by microscopic examination of Giemsa-stained thin and thick films and confirmed by rapid diagnostic test and nested PCR. Of 301 febrile children, 105 (34.9%) were malaria-positive by nested PCR and 87 (28.9%) by microscopy. Plasmodium vivax represented 97.1% (102/105) and P. falciparum accounted for 2.9% (3/105) of positive cases. All positive children under five years old were infected with P. vivax. The highest numbers of malaria positives were found during or shortly after the rainy season and the lowest during the dry season. Fifty-four of 105 (51.4%) malaria cases, all with P. vivax, had never travelled outside Nouakchott. Individuals belonging to the Moors ethnic group represented 97.0% of P. vivax cases. Results of the present study indicate that malaria is endemic in Nouakchott and that P. vivax is the principal causative agent. Regular surveillance is required to monitor malaria prevalence and incidence, and further measures are needed to counter the possible spread of malaria in the country.     

Demonstration by the polymerase chain reaction of mixed Plasmodium falciparum and P. vivax infections undetected by conventional microscopy.

Mixed malaria infections (Plasmodium falciparum and P. vivax) are suspected to occur at a greater frequency than is detected by conventional light microscopy. To determine this frequency we carried out a prospective 'blinded' comparison of diagnosis by conventional light microscopy and enzymatic amplification of the circumsporozoite gene extracted from dried spotted blood samples. Patients were previously healthy, active duty Thai soldiers assigned to a malaria risk area presenting with malaria. Microscopy (oil immersion objective at 1000 x magnification) involved examination of Giemsa-stained thick and thin blood films by an experienced microscopist. Whole blood samples (25 microliters) dried on filter paper were used for species-specific parasite deoxyribonucleic acid (DNA) amplification by the polymerase chain reaction (PCR) and hybridization with radiolabelled P. falciparum and P. vivax probes. Of 137 consecutive cases of malaria studied, 9% (3/32) of microscopically diagnosed P. falciparum infections and 5% (5/104) of microscopically diagnosed P. vivax infections were found to be mixed by the PCR/DNA probe systems, while 1 case was diagnosed as mixed by both microscopy and PCR. The possibility that malaria patients may have undetected mixed infections should be kept in mind because of the specific therapy required both for P. falciparum and for radical cure of P. vivax.     

Parasitological profile of the surveyed population for malaria in an endemic area in Faiyoum Governorate, Egypt

Egypt represents the only focus in the Mediterranean region where Plasmodium falciparum transmission still occurs. A longitudinal parasitological study has been implemented (September 1995 to December 1996) in Faiyoum, Egypt. A total of 9065 blood slides for malaria parasites were taken from all people in the study area as mass blood examination (MBE); those attending the malaria unit as passive case detection (PCD) as well as from neighborhood of the detected cases (NOD). They were stained by Giemsa stain and examined under standard conditions for positivity, parasite species and parasite density. Our results show that MBE detected 61.5% of malaria cases while 23.1% and 15.4% of the confirmed cases were detected through PCD and NOD respectively. The overall parasite rate was 5.7/1000 examined population. P. falciparum was the most predominant species (96.1%), followed by P. vivax (3.9%). The epidemiological factors causing the persistence of malaria transmission in the study area are discussed.     

Plasmodium vivax malaria

We report 11 cases of severe Plasmodium vivax malaria in Bikaner (western India). Patients exhibited cerebral malaria, renal failure, circulatory collapse, severe anemia, hemoglobinurea, abnormal bleeding, acute respiratory distress syndrome, and jaundice. Peripheral blood microscopy, parasite antigen-based assays, and parasite 18s rRNA gene-based polymerase chain reaction showed the presence of P. vivax and absence of P. falciparum.     

2011 年全国输入性疟疾病例流行病学分析

目的阐明2011年我国输入性疟疾病例的流行病学特征,为输入性疟疾的监测、传播风险评估和科学防控提供依据。方法利用中国疾病预防控制中心疾病监测信息报告管理系统,回顾性收集2011年全国疟疾病例个案信息,对国外输入病例的感染疟原虫种类、国内分布、输自国家、国籍、人群特征、发病诊断间隔时间等进行描述性统计分析,采用Fisher确切概率法和Wileoxon秩和检验法进行统计检验。结果2011年全国共报告疟疾病例4402例,国外输入病例2716例;病例输自非洲国家1315例（恶性疟占79．70％）,输自东南亚及其他地区1401例（间日疟占73．59％）,其感染疟原虫种类不同（X^2=1057．04,P〈0．001）;主要以出国务工的男性青壮年为主;自非洲输入病例发病无明显季节性,自东南亚地区输入病例发病集中在5～6月（X^2=120．00,P〈0．001）;发病一诊断间隔时间中位数为3．67d。结论2011年我国疟疾发病以国外输入病例为主,各地均应加强对自非洲输入性疟疾尤其是恶性疟的监测和管理,在云南中缅边境地区应重点关注输入性间日疟的传播风险监测。     

Malaria relapse and recrudescence among travellers to the tropics

This study describes 14 cases of relapse and recrudescence of malaria, treated between 1991 and 2003. In that period, 146 patients were hospitalized in the Clinic of the Institute in Gdynia: 20 women and 126 men. In 103 cases the disease was caused by Plasmodium falciparum, in 31 cases by Plasmodium vivax, in 5 cases by Plasmodium malariae, and in 2 cases by Plasmodium ovale. Five patients were found to have mixed infections, with either P. falciparum and P. vivax or P. falciparum and P. ovale. Relapses in patients previously treated in the country or abroad accounted for 9.6% of all the treated cases of malaria. Recrudescences and relapses were diagnosed of both the tropical malaria (6 cases), and the tertian malaria caused by P. vivax (4 cases). Moreover, in 4 patients diagnosis was made of secondary malaria due to P. vivax infection, while the primary attack was caused by invasion of P. falciparum. Also discussed was the issue of drug-resistance of plasmodia and the resulting problems with the treatment.     

Phylogenetic analysis of CSP and MSP-9 gene sequences demonstrates the close relationship of Plasmodium coatneyi to Plasmodium knowlesi.

Plasmodium coatneyi is a simian malaria parasite with various biological features similar to the human malaria P. falciparum and potential as a model for severe cases of malaria. We have characterized two single-copy genes from P. coatneyi, the circumsporozoite protein and merozoite surface protein-9 genes, and determined its phylogenetic relationship among Plasmodium species. This study demonstrates that while it has similarities to P. falciparum, P. coatneyi belongs to a distant clade including several simian malaria parasites and the human malaria P. vivax. P. coatneyi forms a monophyletic group with P. knowlesi, demonstrating their close relationship despite some very distinctive biological characteristics.