日照市首例输入性卵形疟的实验室检测分析

目的 目的 对日照市首例输入性卵形疟病例进行实验室检测, 以明确诊断。方法 方法 收集患者流行病学资料和血样, 分别进行疟疾快速诊断检测 （RDT）、 疟原虫镜检及巢式PCR检测。结果 结果 患者从非洲刚果金务工返乡, 回居住地莒县陵 阳镇1个月后出现不规则发热、 乏力。RDT检测提示为非恶性疟原虫感染。血涂片镜下可见感染红细胞明显变形, 呈多 种不规则形状; 环状体粗大, 可见大滋养体期和裂殖体期疟原虫。巢式PCR扩增基因产物长度约800 bp, 与卵形疟原虫 相符。综合上述结果, 该病例诊断为单一卵形疟原虫感染。结论 结论 虽然镜检是疟疾诊断的金标准, 但卵形疟原虫在镜下 很难鉴别, 结合 PCR检测结果可明确诊断。     

乐山市1例输入性三日疟的实验室检测与诊断

目的对2016年乐山市1例输入性疑似三日疟疟疾病例进行实验室检测与诊断分析。方法收集该病例的临床资料,并进行流行病学调查。按照《疟疾的诊断》(WS259-2015)的要求,对疑似三日疟患者采集血样后制作血涂片染色镜检、进行疟疾快速诊断检测(RDT)和上送干血斑到四川省疾病预防控制中心进行巢式PCR检测并进行测序比对。结果该患者外周血涂片镜检厚血膜查见疟原虫,薄血膜中查见少量疟原虫,虫体分期主要为小滋养体期、大滋养体期和未成熟裂殖体期,其中大滋养体期、未成熟裂殖体中疟色素呈深褐色、粗大、沿边缘分布,同时被寄生的红细胞不涨大,镜检结果判定为三日疟原虫;RDT检测结果提示为感染除恶性疟以外的其他三种疟疾(三日疟、卵形疟、间日疟)的单项感染,四川省疾病预防控制中心对干血斑中的DNA使用巢式PCR进行检测,扩增出与三日疟原虫阳性对照一致的条带;将基因扩增产物送至成都市擎科生物股份有限公司测序并进行在线Blast序列分析比对,与三日疟原虫阳性对照的基因序列同源性为96%。结论根据实验室联合使用镜检、RDT和巢式PCR3种方法进行检测,结果为三日疟原虫,同时结合患者的流行病学史和临床症状确定该患者为境外输入性三日疟原虫感染病例。     

一个红细胞内同时寄生不同期间日疟原虫输入性病例1例

2012年对云南省腾冲市1例间日疟患者血样采用Care Start~(TM)疟疾快速诊断试剂盒、吉氏染色镜检和巢式PCR方法进行鉴定。Care Start~(TM)疟疾快速诊断试剂盒检测结果判定为除恶性疟原虫以外的间日疟原虫、三日疟原虫或卵形疟原虫感染。镜检结果显示,患者血样厚、薄血片中可见间日疟原虫环状体多核、多重感染现象,1个环状体有2个及以上核的占14.68%(188/1 280);同时寄生2个及以上环状体的红细胞占22.50%(288/1 280);1个红细胞内同时寄生环状体和滋养体、环状体和配子体。巢式PCR结果显示,患者血样为间日疟原虫特异性DNA片段阳性。结合检测结果、流行病学资料和临床表现,确诊该患者为输入性间日疟原虫感染病例,且一个红细胞内同时寄生不同期间日疟原虫。     

恶性疟原虫体外培养污染皮氏罗尔斯顿菌的检测

目的探讨恶性疟原虫体外培养过程中皮氏罗尔斯顿菌污染的检测及预防。方法体外培养恶性疟原虫,涂片、染色后镜检观察;提取被污染虫血的总DNA,采用真菌、细菌通用引物对保守序列进行PCR扩增和克隆测序,将测序结果在Gen Bank中比对。结果导致恶性疟原虫体外培养失败的污染物单个细胞和污染物聚集体在油镜下的形态分别与恶性疟原虫环状体和裂殖体相似,但其胞质内无空泡或无明显细胞核,且位于红细胞外侧;测序比对结果显示污染物与皮氏罗尔斯顿菌相似度99%。结论恶性疟原虫体外培养过程中污染的皮氏罗尔斯顿菌形态与恶性疟原虫环状体或裂殖体相似,可导致镜检误判甚至使培养失败。因此,应严格无菌操作以防疟原虫体外培养污染。     

恶性疟血涂片见大滋养体1例

本文报告1例非洲输入性非典型恶性疟病例, 其血涂片显微镜下可见较多大滋养体期原虫, 部分虫体可见棕黄色 色素颗粒, 呈点状或团块状, 虫体形态与间日疟原虫、 卵形疟原虫相似, 经PCR基因检测确诊为恶性疟。非重症恶性疟病 例外周血中出现大滋养体期原虫较为罕见, 镜检时应注意与其他疟原虫鉴别。     

1例国内罕见的输入性卵形疟的实验室检测

目的对1例输入性疑似疟疾患者的血样进行实验室检测。方法制备疑似疟疾患者血样的血涂片,吉姆萨染色后进行疟原虫的镜检观察。利用实验室自行研制的疟原虫属特异性（通用型）和4种疟原虫种特异性的巢式PCR和实时荧光PCR检测方法,对该血样进行疟疾检测及分型。将PCR扩增片段进行序列测定,并与已知的卵形疟序列进行blast比对分析。结合血样的分子生物学检测结果,重新对镜检结果进行复核。结果血样初次镜检为疟疾阴性。使用疟原虫巢式PCR通用引物对血样的DNA进行PCR检测,扩增出了预期大小约240bp的条带;巢式PCR分型检测表明,血样仅扩增出预期大小约450bp的卵形疟条带,无对应大小的恶性疟、间日疟和三日疟扩增条带产生。疟原虫通用型和卵形疟特异性的荧光PCR检测结果均为典型的S形阳性曲线,卵形疟扩增片段的熔解温度为72.5℃。序列分析表明,扩增片段长度为434bp,blast比对发现去除引物后的393个碱基与GenBank DQ845247等卵形疟的SSU rRNA对应部分的基因序列同源性为100%。重新对血涂片进行镜检复核,结果在薄血膜中发现了卵形疟的环状滋养体,被寄生的红细胞为椭圆形,边缘呈伞矢状。结论使用巢式PCR、实时荧光PCR、序列分析和镜检等方法,证实该例输入性的疑似疟疾患者为卵形疟原虫感染。     

人恶性疟原虫培养物期特异蛋白质的合成和命运

作者用[~(35)S]-甲硫氨酸为标记物经同步培养分别获得恶性疟原虫环状体、滋养体和裂殖体。然后将这些不同生长期疟原虫制备成Triton-可溶蛋白制剂进行SDS-聚丙烯酰胺凝胶电泳分离,用放射自显影显示各期蛋白质的合成和命运。结果:在环状体、滋养体和裂殖体生长的每一阶段都合成了许多蛋白,并且从环状体到下一次红内期裂殖子这整个循环中许多蛋白质保持不变。至少15种高分子量为177,170,158,87,83,47KD_a的8种蛋白在裂殖体中出现而不是在裂殖子。分子量为240、203、106、80、35、19、15与14KD_a的8种蛋白在裂殖子中出现,但不在环状体到下一次裂殖子这一阶段中出现,有些蛋白合成     

恶性疟原虫二氢乳清酸脱氢酶抑制剂体外诱导恶性疟原虫耐药的实验研究

目的分析恶性疟原虫二氢乳清酸脱氢酶(Plasmodium falciparum dihydroorotate dehydrogenase,PfDHODH)抑制剂(二氢噻吩酮类化合物,编号50,以下简称PfDHODH抑制剂50)对体外培养恶性疟原虫的作用特点及其诱导耐药的可能机制。方法恶性疟原虫氯喹敏感株(3D7株)和氯喹抗性株(Dd2株)同步化培养后分为不加药对照组、环状体期加药组和大滋养体期加药组,药物终浓度为80 nmol/L。分别在同步化后0 h(环状体期)、24 h(大滋养体期)、42 h涂薄血膜片镜检;通过逐步加大药物浓度的方法,体外诱导产生耐药虫株,3个月后经有限稀释培养,获得单克隆耐药虫株。采用SYBR GreenⅠ染料法检测各耐药虫株对PfDHODH抑制剂50、氯喹和青蒿素的半数抑制浓度(IC_(50));PCR扩增各耐药虫株Pfdhodh基因并测序,分析其突变情况。结果与不加药对照组相比,环状体期加药组恶性疟原虫从滋养体到裂殖体的发育受到明显抑制,大滋养体期加药组恶性疟原虫呈现明显的空泡化,核质密度大大降低。通过体外诱导并经有限稀释培养,获得44株PfDHODH抑制剂50的单克隆耐药虫株,其中,母本为Dd2、3D7的耐药虫株分别为24和20株,它们对PfDHODH抑制剂50的IC_(50)分别为(2.284±0.096)和(0.678±0.018)μmol/L,较母本虫株的(0.018±0.002)和(0.015±0.002)μmol/L分别提高了近130倍和50倍;对氯喹和青蒿素的IC_(50)分别为(0.011±0.002)、(0.014±0.004)和(0.013±0.003)、(0.012±0.001)μmol/L;与母本Dd2虫株相比,Dd2耐药虫株对氯喹的IC_(50)从(0.072±0.002)μmol/L下降为(0.011±0.002)μmol/L。测序分析结果显示,23株Dd2来源的耐药虫株PfDHODH蛋白氨基酸序列发生了G181D的点突变,另有1株除G181D的点突变外,还产生了K32N的点突变;3D7来源的耐药虫株未发现相应突变。结论体外诱导获得PfDHODH抑制剂50的单克隆耐药虫株,G181D的点突变可能是导致恶性疟原虫高水平耐受PfDHODH抑制剂50的重要分子机制。     

枸橼酸钠抗凝剂对疟原虫生长活性的影响

目的　了解枸橼酸钠抗凝剂对疟原虫生长活性的影响。　方法　将疟原虫经抗凝剂(ACD,CD和SC)处理后以感染率为指标检测抗凝剂对虫体的影响。未同步化的恶性疟原虫分别使用3种不同浓度抗凝剂于37℃作用3h。处理后的红细胞与正常虫体混合,同时处理后的虫体与正常红细胞混合,随后观察两种培养混合物的感染率以确定抗凝剂作用的靶细胞。同上处理期同步化的疟原虫(环状体,滋养体和裂殖体)以观察抗凝剂作用的期特异性。将伯氏疟原虫用抗凝剂处理后接种小鼠,通过感染率的变化观察抗凝剂对鼠疟的影响。　结果　 3种抗凝剂均可抑制疟原虫的生长,其中ACD影响最甚。以抗凝剂分别处理红细胞和疟原虫,结果表明抗凝剂作用于虫体而非红细胞。处理同步化的虫体表明抗凝剂对裂殖体的抑制最为显著。同样处理伯氏疟原虫后接种小鼠进一步验证了抗凝剂对恶性疟原虫作用的抑制性效应。　结论　ACD对疟原虫的抑制性效应最为明显,SC是疟原虫试验中枸橼酸钠抗凝剂的首选。     

存在于感染裂殖体而非裂殖子的红细胞上的诺氏疟原虫变异抗原

诺氏疟原虫在红细胞内从环状体发育到裂殖体,使细胞膜发生明显改变。有人用裂殖体感染细胞凝集试验(SICA)检测有些红     

N1-acetyl-N2-formyl-5-methoxykynuramine modulates the cell cycle of malaria parasites

We previously reported that intraerythrocytic malaria parasites have their development synchronized by melatonin and other products of tryptophan catabolism (i.e. serotonin, N-acetylserotonin and tryptamine). Here, we show that N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK), a product of melatonin degradation, synchronizes Plasmodium chabaudi and Plasmodium falciparum. The synchronization is abrogated with a melatonin receptor antagonist, luzindole. We established quantitatively that a differential AFMK production occurred within the intraerythrocytic stages of rodent malaria parasite Plasmodium chabaudi (ring, trophozoite and schizont), when the infected erythrocytes were previously incubated with melatonin. Measurement of AFMK formation in P. chabaudi after incubation with melatonin at a concentration of 500 nmol/L revealed the following values for AFMK production: ring 0.1 +/- 0.1 nmol/L, trophozoite 22.9 +/- 0.5 nmol/L, schizont 29 +/- 5 nmol/L. Confocal and spectrofluorophotometer experiments with isolated parasites and infected-RBC, loaded with calcium indicator Fluo-4 showed that AFMK elicits an increase in the cytosol calcium concentration in these parasites. Our data suggest that AFMK could have an important role in modulating the cell cycle of malaria parasites mainly in the late stages (trophozoite and schizont).     

Intrahost modeling of artemisinin resistance in Plasmodium falciparum

Artemisinin-resistant Plasmodium falciparum malaria has emerged in western Cambodia. Resistance is characterized by prolonged in vivo parasite clearance times (PCTs) following artesunate treatment. The biological basis is unclear. The hypothesis that delayed parasite clearance results from a stage-specific reduction in artemisinin sensitivity of the circulating young asexual parasite ring stages was examined. A mathematical model was developed, describing the intrahost parasite stage-specific pharmacokinetic-pharmacodynamic relationships. Model parameters were estimated using detailed pharmacokinetic and parasite clearance data from 39 patients with uncomplicated falciparum malaria treated with artesunate from Pailin (western Cambodia) where artemisinin resistance was evident and 40 patients from Wang Pha (northwestern Thailand) where efficacy was preserved. The mathematical model reproduced the observed parasite clearance for each patient with an accurate goodness of fit (rmsd: 0.03-0.67 in log(10) scale). The parameter sets that provided the best fits with the observed in vivo data consist of a highly conserved concentration-effect relationship for the trophozoite and schizont parasite stages, but a variable relationship for the ring stages. The model-derived assessment suggests that the efficacy of artesunate on ring stage parasites is reduced significantly in Pailin. This result supports the hypothesis that artemisinin resistance mainly reflects reduced ring-stage susceptibility and predicts that doubling the frequency of dosing will accelerate clearance of artemisinin-resistant parasites.     

Increased phagocytosis of Plasmodium falciparum-infected erythrocytes with haemoglobin E by peripheral blood monocytes

Erythrocytes from subjects with homozygous and heterozygous haemoglobin E (HbE) infected with Plasmodium falciparum in vitro were phagocytosed to a greater extent by normal human monocytes than infected erythrocytes from normal subjects. Susceptibility to phagocytosis was maximal when the parasites developed to trophozoite and schizont stages in both normal and patients' erythrocytes. The increased susceptibility of P. falciparum-infected HbE erythrocytes to phagocytosis by monocytes may play a role in protection against malaria.     

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 SSU r DNA特定片段检测云南金平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 10 4 bp、10 2 bp和 115 bp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。     

Mode of action of iron (III) chelators as antimalarials: II. Evidence for differential effects on parasite iron-dependent nucleic acid synthesis

Iron chelation treatment of red blood cells infected with Plasmodium falciparum selectively intervenes with iron-dependent metabolism of malaria parasites and inhibits their development. Highly permeant hydroxamate iron chelator RSFileum2 affects all parasite stages when cultures are continuously exposed to drug, but affects primarily ring stages when assessed for irreversible effects, ie, sustained inhibition remaining after drug removal. On the other hand, the hydrophilic and poorly permeant desferrioxamine (DFO) affects primarily trophozoite/schizont stages when tested either in the continuous mode or irreversible mode. Unlike parasites, mammalian cells subjected to similar drug treatment show complete growth recovery once drugs are removed. Our studies indicate that parasites display a limited capacity to recover from intracellular iron depletion evoked by iron chelators. Based on these findings we provide a working model in which the irreversible effects of RSFs on rings are explained by the absence of pathways for iron acquisition/utilization by early forms of parasites. Trophozoite/schizonts can partially recover from RSFileum2 treatments, but show no DNA synthesis following DFO treatment even after drug removal and iron replenishment by permeant iron carriers. At trophozoite stage, the parasite uses a limited pathway for refurnishing its iron-containing enzymes, thus overcoming iron deprivation caused by permeant RSFileum2, but not by DFO because this latter drug is not easily removable from parasites. Their DNA synthesis is blocked by the hydroxamate iron chelators probably by affecting synthesis of ribonucleotide reductase (RNRase). Presumably in parasites, prolonged repression of the enzyme leads also to irreversible loss of activity. The action profiles of RSFileum2 and DFO presented in this study have implications for improved chemotherapeutic performance by combined drug treatment and future drug design based on specific intervention at parasite DNA synthesis.     

A novel real-time PCR assay for the detection of Plasmodium falciparum and Plasmodium vivax malaria in low parasitized individuals

The rapid, accurate diagnosis of Plasmodium spp. is essential for the effective control of malaria, especially in asymptomatic infections. In this study, we developed a sensitive, genus-specific, real-time quantitative PCR assay. It was compared with the microscopic examination of Giemsa-stained blood smears and two different molecular diagnostic techniques: nested PCR and multiplex PCR. For the effective quantitative detection of malaria parasites, all reagents were designed with a lyophilized format in one tube. Plasmodium was detected successfully in all 112 clinically suspected malaria patients, including 32 individuals with low parasitemia (1-100 parasites/μl). The sensitivity threshold was 0.2 parasites/μl and no PCR-positive reaction occurred when malaria parasites were not present. This may be a useful method for detecting malaria parasites in endemic areas.     

Possibility of false-positive detection for sporozoites in mosquitos (Diptera: Culicidae) by nested polymerase chain reaction using Plasmodium yoelii genomic DNA

Anopheles stephensi Liston and An. saperoi Bohart and Ingram infected with the rodent malaria parasite Plasmodium yoelii nigeriense. They were examined 12 and 19 days after blood feeding for sporozoites in head with anterior thorax (HT) and oocysts in abdomen with posterior thorax (AB) by light microscopy and by the nested polymerase chain reaction (nested PCR-based on the amplification of the sequences of the small subunit ribosomal RNA gene). The detection rate of parasite DNA by nested PCR in HT samples 12 days after blood feeding was similar to that by microscopic method. However, in HT samples 19 days after blood feeding, the rate by the PCR method was higher than that by the microscopic method. The incidence of sporozoites in salivary glands of infected mosquitos for 12 days after blood sucking was examined by the PCR method. Parasite DNA in HT of Aedes albopictus Skuse (a non vector for the rodent malaria) as well as An. stephensi and An. saperoi was detected for up to 4 days after feeding on mouse with the rodent malaria parasites. The results indicate that when the PCR method is used for detection of sporozoites of human malaria in mosquitos collected in the field, there are possibilities of including false-positive data for mosquitos that have just or recently fed on human blood infected with malaria (erythrocytic form).     

Isolation and characterization of parasite-inhibitory Plasmodium falciparum monoclonal antibodies

Three mouse hybridomas were isolated that produced IgM monoclonal antibodies (Mab) which reacted with erythrocytic stages of Plasmodium falciparum and inhibited the invasion of erythrocytes in vitro. Those Mab, initially identified by an ELISA screening of hybridoma culture medium, exhibited a strong binding to trophozoite and schizont but not to ring or merozoite stage parasites or to erythrocytes in an indirect immunofluorescence assay. All inhibited the parasite's ability to infect erythrocytes in an in vitro invasion inhibition assay. Western blot analysis of the binding of the Mab to SDS-PAGE-separated parasite antigens isolated from the ring, trophozoite, schizont or spontaneously released merozoite stages, indicated that two of the Mab bound to a Mr 105,000 antigen in trophozoites and schizonts while the third Mab did not. All three Mab also bound to Mr 30,000-40,000 antigens in all stages, however, in all instances binding to these antigens was enhanced in merozoites. It was further observed that the two Mabs that bound to a Mr 105,000 antigen: exhibited a markedly reduced binding to the Mr 105,000 antigen in merozoite preparations; exhibited different relative intensities of binding to the trophozoite and schizont antigens; both bound to the same Mr 105,000 antigen as demonstrated through Western blot analysis of antigens separated by two-dimensional gel electrophoresis. The findings suggest that the inhibitory Mab bound to different epitopes of the same antigen and that the antigen may either be processed or degraded at about the time of merozoite release and erythrocyte invasion.