诺氏疟原虫的人体自然感染

云南省墨江县1例“间日疟”患者的血片,经回顾性镜检,发现疟原虫形态特别,早期滋养体多核,红细胞内有多个虫体寄生,晚期滋养体有形成带状趋势。裂殖体和配子体与间日疟原虫相似。经分子生物学鉴定为诺氏疟原虫。     

人体自然感染诺氏疟原虫(Plasmodium knowlesi)--形态学描述、分子鉴定和msp-1片段表达

疟疾是威胁人类健康的重要传染病之一。全球每年有3.5亿~5亿临床病例,100万~300万人死于该病,其中大部分是儿童。由于原虫及媒介均已对药物产生抗药性,疟疾控制面临重重困难。此外,在东南亚国家屡有报道灵长类疟原虫在人体寄生,这种情况有可能引发严重的公共卫生问题:首先,对感     

套式PCR扩增SSU rDNA特定片段检测云南金平县疟原虫感染的研究

目的：采用套式PCR系统诊断、鉴别人体疟原虫感染。方法：采用已建立的套式PCR系统扩增SSUrDNA特定片段检测云南金县恶性疟镜检阳性患者的6份血样，并设阳性与阴性对照。结果：间日疟原虫、恶性疟原虫和三日疟原虫感染血样中分别扩增出104bp、102bp和115bp预期大小的特定扩增带。正常人血、人源弓形虫、杜氏利什曼原虫DNA及灭菌双蒸水均未产生特异扩增带。6份血样中检出4份p.v.、p.f.和p.m.的混合感染1份p.v.和p.f.及1份p.f.和p.m.的混合感染。结论：该系统特异、灵敏、稳定，在诊断疟疾的同时可准确地判定混合感染，对疟疾的诊断、大规模流行病学研究及疫情监控等具有实际应用价值。     

恶性疟原虫海南株子孢子期SSU rRNA编码基因的克隆及序列分析

目的 克隆恶性疟原虫海南株子孢子期小亚基核糖体核糖核酸(SSU rRNA)编码基因片段,分析其序列特征.方法 根据恶性疟原虫基因库相关核酸序列设计1对引物,采用PCR方法从海南恶性疟患者血样核酸提取物中扩增出恶性疟原虫SSU rRNA基因片段,纯化后与pGEM-Teasy质粒连接,构建重组子并转化大肠杆菌JM109;阳性克隆经双酶切鉴定后,双脱氧末端终止法测定序列,采用BLAST软件分析其特征.结果 恶性疟原虫子孢子期SSU rRNA基因扩增片段大小约为347bp;阳性克隆重组质粒双酶切及PCR扩增均得到预期大小的片段;核酸序列测定显示插入的SSU rRNA基因扩增片段含有347个核苷酸,与GenBank中的恶性疟原虫3D7株相同序列进行比对,其同源性为100%,而与7G8株的同源性则为98.0%,其中第153位碱基发生了缺失,第184位碱基由C取代了T,而第188位T碱基与第189位A碱基为插入碱基,第243位碱基则由T取代了C.结论 成功克隆恶性疟原虫海南株孢子期SSU rRNA编码基因序列,该序列相对保守,不同地理株间存在单核苷酸多态性.     

感染人类的第五种疟原虫—猴诺氏疟原虫

猴诺氏疟原虫（Plasmodium Knowlesi）目前被公认是感染人类的第五种疟原虫,研究结果表明该猴疟不仅在自然的猴群中通过传疟按蚊相互传染,也可以通过传疟按蚊传染给人类,造成人-人和人-猴之间的传播,人类同样也可以通过血液进行传染。该猴疟原虫不论是感染人或猴之后红细胞内裂变周期均为24h,有报道被感染者血内原虫短期内达到高密度原虫血症并发肾功能衰竭而导致死亡心。     

细胞免疫在抗疟原虫感染中的作用

了解细胞免疫在抗疟原虫感染中的作用机制对研制疟疾疫苗和进行免疫预防有一定意义。在国外有关这方面的研究较多,并有一定进展,其研究结果主要有:(1) 疟疾保护性免疫的产生依赖巨噬细胞提呈抗原以及抗愿提呈后 T 淋巴细胞的激活和致敏。(2) T 淋巴细胞通过辅助 B 淋巴细胞和住驻 T 淋巴细胞而发生抗感染效应;T_h 和 T_s 淋巴细胞亚群的平衡协调对疟疾的保护性免疫颇为重要;用 T 淋巴细胞克隆技术已从恶性疟患者的外周血中筛选出对肿瘤细胞有杀伤作用的克隆 T 淋巴细胞。(3) 不同宿主对同种原虫易感性的差异与宿主固有的自然杀伤细胞(NK 细胞)的活性高低有关。(4) 干扰素可有效地预防动物宿主感染子孢子;氧代谢物、肿瘤坏死因子,嗜酸粒细胞阳离子蛋白等在体外对疟原虫均有杀伤作用;白细胞介素_2在体外可恢复感染疟原虫的宿主 T 淋巴细胞的增殖。     

细胞因子在疟原虫感染中的作用研究进展

细胞因子在疟疾感染免疫中的作用正不断得到阐明。其中,对TNF-a、INF-r、IL-12、IL-10、IL-4和TGF-β等的免疫保护与病理损伤机制的研究较为深入。这为疟疾疫苗研制、探索重症疟疾的预防控制方法提供了理论支持,但如何将模型研究结果运用于人体仍是亟待解决的问题。     

检测和鉴定疟原虫虫种PCR方法的比较

高灵敏度、准确、快速的疟原虫检测与鉴定对疟疾的防治起着至关重要的作用.随着分子实验技术的发展,在大规模流行病学调查中,PCR方法比传统的镜检方法更加高效、准确.该文搜集了部分用PCR方法检测、鉴定虫种的研究结果,综合比较了不同的PCR技术在实验方法、检测精度、现场检测等方面的优劣.     

637例发热病人疟原虫血片复检质量分析

目的了解宁波市江北区发热病人疟原虫血片质量,为镜检质量考核以及疟疾防治效果评价提供科学依据。方法对江北区各级医疗卫生单位已检血片进行随机抽样,抽取所有阳性血涂片和不少于10%的阴性血涂片进行复检。从血片制作、染色、清洁度方面对血片质量进行对比分析和评价。结果共抽查已检血片637张,血片制作、染色和清洁度总体合格率分别为86.03%、89.79%和89.64%,并有逐年增高的趋势。不同月份复检血片质量亦有显著性差异（P〈0.05）。血片质量缺陷以沉渣、厚膜制作不规范以及偏碱性为主。结论宁波市江北区疟原虫血片质量较好,基本达到考核标准。但仍需加强培训和指导,促进血片质量不断提高。     

Molecular Identification of a Case of Paragonimus pseudoheterotremus Infection in Thailand

Paragonimiasis is an important food-borne parasitic zoonosis caused by infection with lung flukes of the genus Paragonimus. In Southeast Asia, Paragonimus heterotremus is the only proven causative pathogen. Recently, a new Paragonimus species, P. pseudoheterotremus, was found in Thailand. This species is genetically similar to P. heterotremus and is considered as a sister species. However, infectivity or pathogenicity of P. pseudoheterotremus to humans remains unclear. We report the first confirmed human pulmonary paragonimiasis case caused by P. pseudoheterotremus infection. After polymerase chain reaction/sequencing of the DNA extracted from Paragonimus eggs in the sputum of the patient, partial internal transcribed spacer 2 and cytochrome c oxidase subunit 1 sequences were approximately identical (98–100%) with those of P. pseudoheterotremus. For P. heterotremus, the partial internal transcribed spacer 2 sequence was approximately identical (99–100%), but the partial mitochondrial cytochrome c oxidase subunit 1 sequence showed a similarity of 90–95%.Paragonimiasis is a food-borne zoonosis caused by infection with lung flukes of the genus Paragonimus. Only 7 of approximately 40 species can infect humans: 2 species (Paragonimus kelikotti and P. mexicanus) in the Western Hemisphere, 2 (P. africanus and P. uterobilateralis) in Africa, and 3 (P. westeremani, P. skrjabini, and P. heterotremus) in Asia.^1–3 Among them, P. westermani is the major pathogenic fluke for human paragonimiasis in eastern Asia where 20.7 million people (20 million in China) were infected.⁴ In addition to P. westermani infection, sporadic cases of P. skrjabini infection have been reported from China and Japan (as P. miyazakii infection in Japan). From southwestern China to northeastern India including the Indochina Peninsula, P. heterotremus is the only proven species responsible for human infection.^1,2 In Thailand, although 6 Paragonimus species were registered in wild life and/or experimental animal infections, P. heterotremus is the only confirmed pathogen for human paragonimiasis by the recovery of worms from patients.⁵In 2007, a new Paragonimus species, P. pseudoheterotremus was described as the seventh species in Thailand mainly on the basis of the morphologic difference of metacercariae (P. pseudoheterotremus metacercariae were oval shape and smaller than those of P. heterotremus).⁶ Although ribosomal internal transcribed spacer 2 (ITS2) sequences of P. pseudoheterotremus and P. heterotremus were almost completely identical, (only one base difference), the partial sequence of mitochondrial cytochrome c oxidase subunit 1 (cox1) genes of these parasites were significantly different from each other, suggesting their sister species relationship.⁷ Although adult worms of P. pseudoheterotremus were obtained by experimental infection in a cat,⁶ the pathogenicity of this new species to humans remains unsolved. We report a case of human pulmonary paragonimiasis caused by P. pseudoheterotremus in Thailand. Diagnosis was confirmed by molecular evidence using Paragonimus eggs in the sputum of the patient.The study was approved by the Human Ethics Committee of Khon Kaen University (Reference no. {"type":"entrez-nucleotide","attrs":{"text":"HE551071","term_id":"288736612","term_text":"HE551071"}}HE551071). Oral informed consent was obtained from the patient. A 57 year-old man (monk) from Thailand was admitted to our hospital on July 1994 because of chronic productive cough with bloody sputum and dyspnea for the past eight months. He stated that one year before development of his respiratory symptoms, he occasionally ate mountainous crabs while staying in a cave as a part of his meditation training in Loei Province in northeastern Thailand.A chest radiograph showed a faint pulmonary nodule at the left upper lobe with minimal pleural effusion with pleural thickening of both lower lung fields and generalized thickening of lung markings. Bronchoscopic examination showed nodular obstruction at the posterior basal segment of the left lower lung field. Many Paragonimus sp. eggs were found in bronchoalveolar lavage fluid. Mycobacterium tuberculosis was not detected in bronchoalveolar lavage fluid. After bronchoscopy, Paragonimus eggs were detected in his sputum (Figure 1). The eggs (n = 5) had a mean ± SD length of 79.6 ± 5.4 μm and a mean ± SD width of 45.0 ± 3.0 μm. Eggs were slightly asymmetric in shape and had an operculum at one end but lacked a thickening at the abopercular pole. Paragonimus eggs in the sputum sample were kept at −70°C in the Department of Parasitology, Faculty of Medicine, Khon Kaen University, Thailand, until a polymerase chain reaction (PCR) and DNA sequencing were performed.Open in a separate windowFigure 1.Paragonimus pseudoheterotremus egg from the sputum of a patient, Thailand.He was treated with praziquantel, 25 mg/kg of body weight, 3 times/day for 2 consecutive days. Two days after treatment, Paragonimus eggs in sputum disappeared. His clinical symptoms and laboratory data markedly improved without any complication by two months after treatment.For accurate identification of the causative species of Paragonimus in this patient, a PCR/sequencing method was used for diagnosis. Genomic DNA was extracted from the eggs in the sputum. In brief, egg-containing sputum was homogenized in a disposable polypropylene pestle (Bellco Glass Inc., Vineland, NJ), and the DNA was extracted by using a NucleoSpin^® Tissue Kit (Macherey-Nagel GmbH and Co., Düren, Germany). DNA was eluted in 100 μL of distilled water and stored at −20°C until used.The ITS2 sequence was amplified by using species-specific primers PhITS2-F (5′-CTG TGT GAA TTA ATG TGA ACT GC-3′) and PhITS2-R (5′-AGT GAT ATG CTT AAG TTC AGC G-3′), which were designed from the known ITS2 sequence of P. heterotremus (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AB308377","term_id":"148781861","term_text":"AB308377"}}AB308377). Because the ITS2 sequence of eggs from the patient was completely or almost completely identical with those of P. heterotremus and P. pseudoheterotremus, a partial fragment of cox1 gene was amplified by using the species-specific primers PphCOI-F (5′-CCG GGT TTG GTG TTG TG-3′) and PphCOI -R (5′-ACA ACG AAC CAA GTG TCA TG-3′), which were designed from a specific region of P. pseudoheterotremus cox1 gene (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"EF446315","term_id":"149789357","term_text":"EF446315"}}EF446315).The PCR was conducted by using a GeneAmp^® PCR System 9700 (Applied Biosystems, Singapore). The reaction was carried out in a 25-μL volume containing PCR buffer (60 mmol/L Tris sulfate, pH 8.4, 18 mmol/L ammonium sulfate, 1.5 mmol/L MgCl₂), 200 μmol/L of each deoxyribonucleotide triphosphate, 0.2 μmol/L of each primer, and 0.625 units of Taq DNA polymerase. The DNA template was initially denatured at 94°C for 5 minutes. The amplification procedure comprised 35 cycles at 95°C for 30 seconds (denaturation), 55°C for 30 seconds (annealing), and 72°C for 30 seconds (extension), with a final extension at 72°C for 10 minutes. The amplified product was subjected to electrophoresis on a 1.5% agarose gel; the cox1- and ITS2-fragments were then removed from the gel and purified for DNA sequencing, which was performed by using the MegaBACE™ 1000 DNA Analysis System (GE Healthcare, Piscataway, NJ).The ITS2 and cox1 gene sequences of the Paragonimus eggs from the patient were analyzed by BLAST-N search (National Center for Biotechnology Information, Bethesda, MD), and DNA alignment was performed by using ClustalW⁸ and the maximum composite likelihood tree using MEGA 4.⁹ The partial ITS2 sequence of Paragonimus sp. eggs from the patient was identical with that of P. pseudoheterotremus and various geographic isolates of P. heterotremus (identity = 99–100%). The partial cox1 gene sequence also showed extremely high (98–99%) sequence similarities with those of P. pseudoheterotremus. The homology with P. heterotremus was 90–95% (Figure 2). Phylogenetic analysis of the cox1 gene sequence (Figure 3) showed that the Paragonimus sp. from the patient was a member of the P. pseudoheterotremus clade, which is distinct from the P. heterotremus clade. From these results, the patient was given a diagnosis of infection with P. pseudoheterotremus. The partial ITS2 and cox1 sequences were deposited in GenBank under accession numbers {"type":"entrez-nucleotide","attrs":{"text":"JQ796814","term_id":"389889074","term_text":"JQ796814"}}JQ796814 and {"type":"entrez-nucleotide","attrs":{"text":"JQ796815","term_id":"389889075","term_text":"JQ796815"}}JQ796815, respectively.Open in a separate windowFigure 2.Alignment of partial cytochrome c oxidase subunit I (cox1) gene sequence of Paragonimus pseudoheterotremus eggs from a patient in Thailand (P. pseudoheterotremus THA patient) with those of P. pseudoheterotremus ({"type":"entrez-nucleotide","attrs":{"text":"EF014339","term_id":"122704923","term_text":"EF014339"}}EF014339 from Thailand) and Paragonimus heterotremus ({"type":"entrez-nucleotide","attrs":{"text":"AB325519","term_id":"148806834","term_text":"AB325519"}}AB325519 from India) sequences from a database. ISO 3166-1 alpha-3 codes were used as the country code.Open in a separate windowFigure 3.Neighbor-joining tree based on partial cytochrome c oxidase subunit I (cox1) gene sequences (342 bp). Bootstrap scores expressed as percentages of 1,000 replications are given at each node. Sequences of Paragonimus heterotremus, P. pseudoheterotremus, and P. westermani obtained from a DNA database are indicated with species accession no. country code (ISO 3166-1 alpha-3 codes).In Thailand, there were six species of Paragonimus (P. westermani, P. siamensis, P. heterotremus, P. bangkokensis, P. harinasutai, and P. macrorchis)¹⁰ until the recent discovery of P. pseudoheterotremus as the seventh species.⁶ The present results confirmed that P. pseudoheterotremus can be a pathogen to humans. For species identification of etiologic agents of paragonimiasis, detection of species-specific band by serum samples of patients in an enzyme-linked immunoelectrotransfer blot¹¹ or a binding inhibition enzyme-linked immunosorbent assay using homologous and heterologous antigens¹² have been developed. Whether such immunologic or molecular methods could be applicable for the discrimination of P. heterotremus and P. pseudoheterotremus should be explored.Paragonimus heterotremus is distributing widely from southwestern China to northeastern India, and confirmed cases of human infection have been reported in Thailand,^13,14 Vietnam,^15,16 Laos,^17,18 and India.¹⁹ Conversely, metacercariae of P. pseudoheterotremus were first found in freshwater crabs in Kanchanabhuri, Thailand, and adult worms were obtained by experimental infection in a cat.⁶ However, the natural definitive hosts and distribution or pathogenicity of this parasite to humans have not been elucidated. The present case is the first detection of human infection with P. pseudoheterotremus.Because P. pseudoheterotremus and P. heterotremus are difficult to discriminate on the basis of morphology of eggs,⁶ and because P. heterotremus is widely distributed in Southeast Asia, molecular diagnosis using sputum eggs is necessary for accurate identification of the pathogen of paragonimiasis in Southeast Asia. The present results also suggest possible intra-species variation among geographically different isolates of P. pseudoheterotremus. Large scale community-based surveys along the Mekong River Basin and neighboring countries are needed to identify the human pathogenic species.     

An Autochthonous Case of Severe Plasmodium knowlesi Malaria in Thailand

A 58-year-old Thai man was infected with Plasmodium knowlesi in Chantaburi Province, eastern Thailand. In addition to pyrexia, the patient developed hypotension, renal failure, jaundice, and severe thrombocytopenia. The parasitemia at the time of admission was 16.67% or ∼503,400 parasites/μL. With artesunate treatment and supportive care, the patient recovered uneventfully. The occurrence of complicated knowlesi malaria in a low-endemic area underscores the risk of high morbidity from this simian malaria.     

基于凝集素基因对部分隐孢子虫种类的分子鉴定

目的 为了解隐孢子虫凝集素基因在不同分离株的序列差异及其遗传进化关系,以SSU rRNA基因作参照,评价Lectin基因位点作为隐孢子虫基因分型和进化关系分析的可行性。方法 采用巢式PCR,分别在Lectin和SSU rRNA位点处对本实验室分离保存的多个隐孢子虫分离株进行PCR的扩增。用Clustalx对扩增序列与参考序列进行比对,用MEGA5中的邻近法(Neighbor joining method NJ),进行进化树的构建。结果 基于Lectin基因位点,在C. parvum、C. hominis、C. cuniculus及其在SSU rRNA 处与人源C. hominis极为相近的horse genotype、驴源C. hominis均成功扩增出了大小在450 bp左右的目的 条带,并进行了进化树的分析,不同种类和同种不同动物来源的隐孢子虫分布在不同的分支上。结论 Lectin基因位点可很好的区分SSU rRNA序列极为相似的几种隐孢子虫,有望为人兽共患隐孢子虫分类和遗传研究提供新的基因靶标。     

Plasmodium knowlesi in a traveller returning to New Zealand

The recent discovery that Plasmodium knowlesi causes malaria in human populations, established it as the fifth species of plasmodium that may do so. A case of P. knowlesi malaria is described in a helicopter pilot from New Zealand, who became ill after returning from recurring visits to Malaysian Borneo in June 2010. His P. knowlesi infection was not detected using microscopic examination and a rapid diagnostic test for malaria, but was confirmed by both PCR (polymerase chain reaction) and sequence analysis showing homology with the ribosomal RNA gene for P. knowlesi. He responded rapidly to treatment with artemether & lumefantrine combination. The evolution of a rapid diagnostic kit to diagnose P. knowlesi is needed, for early identification and appropriate anti-malarial therapy of suspect cases are both critical in the prevention of the potentially life-threatening disease through P. knowlesi. Clinicians need to consider knowlesi infection in the differential diagnosis in recent-onset febrile travellers to areas of forestation in Southeast Asia.     

Evaluation of Recombinant Plasmodium knowlesi Merozoite Surface Protein-133 for Detection of Human Malaria

Plasmodium knowlesi is now known as the fifth Plasmodium species that can cause human malaria. The Plasmodium merozoite surface protein (MSP) has been reported to be potential target for vaccination and diagnosis of malaria. MSP-1₃₃ has been shown to be immunogenic and its T cell epitopes could mediate cellular immune protection. However, limited studies have focused on P. knowlesi MSP-1₃₃. In this study, an approximately 28-kDa recombinant P. knowlesi MSP-1₃₃ (pkMSP-1₃₃) was expressed by using an Escherichia coli system. The purified pkMSP-1₃₃ reacted with serum samples of patients infected with P. knowlesi (31 of 31, 100%) and non-P. knowlesi malaria (27 of 28, 96.43%) by Western blotting. The pkMSP-1₃₃ also reacted with P. knowlesi (25 of 31, 80.65%) and non-P. knowlesi malaria sera (20 of 28, 71.43%) in an enzyme-linked immunosorbent assay (ELISA). Most of the non-malarial infection (49 of 52 in by Western blotting and 46 of 52 in the ELISA) and healthy donor serum samples (65 of 65 by Western blotting and ELISA) did not react with recombinant pkMSP-1₃₃.     

Naturally Acquired Antibodies to Plasmodium vivax Duffy Binding Protein (DBP) in Rural Brazilian Amazon

Duffy binding protein (DBP), a leading malaria vaccine candidate, plays a critical role in Plasmodium vivax erythrocyte invasion. Sixty-eight of 366 (18.6%) subjects had IgG anti-DBP antibodies by enzyme-linked immunosorbent assay (ELISA) in a community-based cross-sectional survey in the Brazilian Amazon Basin. Despite continuous exposure to low-level malaria transmission, the overall seroprevalence decreased to 9.0% when the population was reexamined 12 months later. Antibodies from 16 of 50 (36.0%) subjects who were ELISA-positive at the baseline were able to inhibit erythrocyte binding to at least one of two DBP variants tested. Most (13 of 16) of these subjects still had inhibitory antibodies when reevaluated 12 months later. Cumulative exposure to malaria was the strongest predictor of DBP seropositivity identified by multiple logistic regression models in this population. The poor antibody recognition of DBP elicited by natural exposure to P. vivax in Amazonian populations represents a challenge to be addressed by vaccine development strategies.